De L'université De Montpellier

Présentée Par

Ixchel Campos Avelar

Sous La

Sabine Schorr-Galindo

Olivier Puel

Angel Medina Vaya

Professor, Mirna Leonor Suárez Quiroz

Ixchel Campos-Avelar

Alexandre Colas De La Noue

Noel Durand

Blandine Fay

Véronique Martinez

Angélique Fontana

Caroline Strub

Noel Durand

Guillaume Cazals

Aurélie Ducasse

Monique Royer

Stéphane Cociancich

Jean Benoit Morel

Ixchel Campos

N Durand

Alexandre Colas De

La Noue

Lucile Pellan

Stéphane Conciancich

Jean-Benoît Morel

Florentin Constancias

Noël Durand

Sabine Galindo

THÈSE POUR OBTENIR LE GRADE DE DOCTEUR

Keywords: Fungal phytopathogens, Streptomyces, biocontrol, mycotoxins, detoxification, clustering analysis

Wheat is the main cereal crop consumed in Europe. Its contamination with fungal pathogens is one of the main threats, leading to massive yield losses and the potential occurrence of mycotoxins. Puccinia triticina causes leaf rust, Magnaporthe oryzae causes wheat blast, Septoria nodorum blotch and Septoria tritici blotch are caused by Parastagonospora nodorum and Zymoseptoria tritici, respectively, Fusarium head blight is caused by Fusarium graminearum and Fusarium verticillioides emerges as a wheat pathogen. Aspergillus flavus and Penicillium verrucosum, develop mostly during grain storage. The decrease on chemical fungicides has motivated the search for alternatives for fungal management. Streptomyces genus are promising as biocontrol agents (BCAs) due to their wide production of bioactive secondary metabolites and enzymes, but also due to their capacity to detoxify mycotoxins. In this study, the impact of Streptomyces isolates on fungal growth and mycotoxin accumulation was evaluated. Maximal decreases of fungal growth varied from 66% (F. graminearum) down to 10% of the control (P. nodorum). Mycotoxin accumulation was decreased from 35% of the control (DON), to a complete elimination (AFB1), while some isolates caused an overaccumulation. Bacterial Cell Free Extracts (CFEs) were in general less efficient at decreasing fungal growth, and DON, AFB1 and OTA accumulation. Sometimes causing a strong overproduction (at least 8 times more fumonisins than the control). This highlights the importance of assessing the impact on mycotoxins when evaluating BCAs. OTA was strongly degraded by bacterial isolates mainly in liquid medium (down to 38% of the control on average), while AFB1 was degraded in solid and liquid medium and by bacterial CFEs (33%, 43% and 69% of the control on average, respectively). Moreover, the evaluation of AFB1 residual genotoxicity indicated an effective detoxification down to 27% of the control. Clustering analysis and Pearson correlations allowed to classify the bacterial isolates according to their performances and to evoke possible antagonistic mechanisms, such as the production of antifungal compounds, mycotoxin degrading enzymes and potential inhibitors of mycotoxin biosynthesis. Bacteria colonized wheat leaves during in planta assays and remained viable after 1 week. However, only S. griseoviridis from Mycostop®, protected wheat against the tested fungi. This work presents an integrated approach for the screening of potential BCAs, focusing on the global analysis of the results to elucidate antagonistic mechanisms and to identify the most promising isolates.

ACKNOWLEDGMENTS

First of all, I would like to thank the Agropolis foundation for the investment on this research project. As well as the Mexican Council for Science and Technology (Conacyt) that granted me the PhD scholarship.

I would also like to thank the jury members that have kindly accepted to evaluate this work: Pr. Florence Mathieu, Dr. Olivier Puel, Dr. Angel Medina, Pr. Mirna Leonor Suárez Quiroz, Pr. Florence Richard-Forget. I cannot wait to exchange with you during my thesis defense. I hope you will find pleasure at reading this work I would also like to acknowledge the members of my PhD Committee: Jean Michel Salmon, Frédéric Carlin, Noel Durand, Stéphane Cociancich, Monique Royer and Jean Benoit Morel, who each year were present to assess the progress of my research. Thank you for your constructive feedback and your kindness, and for always caring about my wellbeing and my mental sanity when I would try to accomplish too much in too little time.

My deepest gratitude is extended to the QualiSud research team. Thank you for welcoming me among you and for all you have taught me during all these years, since my first internship until the end of my PhD. Pr. Sabine Galindo: you are an inspiration, thank you for your patience and your kindness, and also thank you for the evenings spent verifying this manuscript. You are a great leader and it was a pleasure having you as my thesis director. Dr. Angelique Fontana and Dr. Caroline Strub, thank you for always taking the time to resolve my questions and for your scientific advices. And of course, for the time you have dedicated to verify this manuscript and many scientific productions. Noel Durand and Blandine Fay: thank you for having analyzed near a thousand samples! and for your analytical insights and explanations. Finally, to my supervisor, Dr. Alexandre Colas de la Noue: I don't even know where to begin. For starters, I think you deserve a big THANK YOU for your patience, because you needed a lot, especially during the last rush. Thank you for always encouraging me to think further and to be meticulous and thorough, and for always taking the time necessary to resolve my many questions. I think of you as one of the most intelligent people around me, so I hope I was able to catch some of your critical thinking. Thank you also for all the laughs and the good mood. And especially, thank you for all your help, the achievement of this thesis would not have been possible without you.

I will also like to thank the research partners at PHIM: Dr. Jean Benoit Morel, Dr. Monique Royer, Dr. Stéphane Cociancich and Aurélie Ducasse, for their support during the evaluation of phytopathogenic fungi and especially for carrying out the in planta assays.

Thank you to all the people that I met at the laboratory and that in one way or another helped me and made my journey unforgettable:

-Caroline Cayzac and Luc Bidel, for not only lending us the plate reader but also for staying until late at night helping us analyze our samples. -Catherine Houeix, for helping me order the huge amount of supplies I needed for my experiments.

-Rémy Marcouire, for fixing all the things we broke and for the unconditional smiles and the good mood. -Nicolas Jouy, for the multiple cakes you shared with us for always saying hello in Spanish every time you saw me. -Charles Cunault, for your help with statistical analysis and R.

-Christophe Jourdan, for having tried very hard to sequence my bacterial isolates and for the molecular biology explanations, but also for the good laughs and the teasing. -Francis Courtois and Ziya Gunata, for your interest in the progress of my research.

To the interns that helped me accomplish the numerous experiments needed for this work: Jiahui Sun, Rana Osman, Ulrick Valentiner-Branth, Nata Paye.

To my fellow PhD students, Reem Al-Riachy, thank you for always making me laugh and for all the encouragement and help during the difficult days, you have a very special place in my hearth. Claudia Lopez, you are the definition of quality > quantity, because we shared very little time in the laboratory and still we managed to become good friends, thank you for that. Lucile Pellan, since we started I have always looked up to you, you are an inspiration and an amazing scientist. Thank you for motivating me to be a better PhD student. To Asma Chelaghema. I wish all the best to you and your newly created family, and a successful scientific venture. And last but not least, to Veronique Martinez, my favorite lab technician, thank you for your support, both scientific and emotional, as well as for your good mood and for being an exceptional laboratory partner.

To the friends I made in France: Nuria, Daniel, Gustavo, Valentina, Alejandro, Clément, Aurore, Caroline, Agustina, Anthony, Natalia, Elvira, Alicia Alejandra (yes, I know you hate when I call you that XD), Gabriel, Claudia and Reem again hehe, Thank you for all the good times, the "apéros", the incessant laughing, the all-night conversations…for knowing when no to ask me anymore how was the thesis manuscript going >:/ hahaha. Morgane and David, for offering we a wonderful writer's retreat when I felt unmotivated. You all made me feel at home so far away from home.

To my family in Mexico, thank you for always checking on me and giving me your support and good vibes. To my aunt Liduvina, for letting me stay with you so I could go to the University and to my aunts Sofia and Silvia for all the affective phone calls. To my cousin Yuyis, for being the sister I never had. You all inspire my strength. I kept going thanks to you. I thank God you are all ok. I hope I will see you soon.

To my mother Lucia, for always helping me realize my dreams, sometimes sacrificing your own. You have always put me first, I hope I made you proud, and made you feel like it was worth it. Thank you for never making me feel bad for being so far away for so many years, even when you needed me the most. This thesis, and all I have ever achieved in my life is thanks to your support. You taught me to relativize, to relax and enjoy each step of the journey. Thank you for comforting me and making me laugh it out when I felt that I could not keep going.

To my father, wherever you are, thank you for inspiring in me the love for learning new things. You made me the person I am today and I know you would have loved to hear all my stories about my bacteria fighting the mean toxic fungi. Until we see each other again.

And finally, to my dog, my loyal companion during the many sleepless nights, my escape to this pandemic awkwardness. Thanks to her for making me feel happy since the moment I open my eyes. I never felt alone as long as she was with me, and all my stress and burdens disappeared just by going out for a walk. Odyssée…you are also my family. List of tables from publications I.

List of figures from bibliographical synthesis and unpublished results

Minimizing ochratoxin A contamination through the use of Actinobacteria and their active molecules Table 1. Summary of specific features observed during the clustering analysis of the impact of the screened actinobacteria and their cell free extracts (CFEs) on P. verrucosum growth and ochratoxin A (OTA) specific production……………………………………………………………………………………………………………….117

Table S1. Values of the Pearson correlation between the measured parameters during the screening of the effect of actinobacteria and their CFEs on P. verrucosum growth and OTA production………..130

II.

Aspergillus flavus growth inhibition and Aflatoxin B1 decontamination by Streptomyces isolates and their metabolites Table 1. Summary of specific features observed during the clustering analysis of the impact of the screened Streptomyces isolates and their cell free extracts (CFEs) on Aspergillus flavus growth and aflatoxin B1 specific production (AFB1sp), together with their ability to degrade AFB1…………………….149

General introduction

Cereals are the main staple food for most communities around the world, they represent an important source of energy, fiber, proteins and lipids. Maize, wheat, barley, rice and oat are the main cereals consumed worldwide. Cereals crops suffer from a series of diseases provoked by insects, bacteria and fungi. Fungal contamination in particular leads to massive loses on yield and grain quality, and to the potential occurrence of toxic fungal metabolites that can remain active throughout the production chain, down to the finished products. These toxic metabolites, called mycotoxins, constitute a critical hazard due to their detrimental impact on human and animal health.

As good agricultural practices are insufficient to mitigate plant diseases and mycotoxins risk, the use of chemical fungicides is nowadays the main tool to counter fungal development.

However, their negative impacts on the environment and on human health, as well as the pathogenic resistance they generate, have urged the scientific community to develop new strategies for crop disease management. For instance, biocontrol by beneficial microorganisms represents an environmental-friendly alternative to chemical treatments.

Streptomyces are soil bacteria with great potential for agriculture due to their capacity to produce a wide variety of secondary metabolites, including antimicrobial agents and enzymes.

In addition, they apply a series of antagonistic mechanisms that confer them the ability to combat pathogenic fungi. Furthermore, Streptomyces beneficial impacts on plant development have been largely documented, due to their capacity to stimulate plant defense mechanisms, and to promote their growth and fitness. Moreover, their production of enzymes confers them potential as mycotoxin detoxification tools. For these main reasons, Streptomyces genus constitutes a promising strategy to handle fungal contamination and mycotoxin risk.

The study on the biocontrol of fungal wheat pathogens by Streptomyces isolates achieved during this thesis is presented in two main sections:

The first section includes a comprehensive bibliographical study, which starts by highlighting the nutritional and economical importance of cereal crops. Followed by a description of phytopathogenic and/or mycotoxinogenic fungi, affecting wheat in the field and during storage, targeted during this work. For mycotoxinogenic fungi, a description of their main mycotoxins' biosynthesis, toxicity and regulation is presented. Later, the principal means for control of plant molds during pre and post-harvest stages are detailed. Decontamination of mycotoxins by chemical, physical and biological methods is described next. Furthermore, a portrayal of actinobacteria, particularly Streptomyces, main characteristics and antagonistic mechanisms deployed during the biocontrol of plant pathogens is presented, together with a description of the principles of biocontrol agents' selection and formulation. The last part of this section includes details about the documented potential of Streptomyces strains as detoxification tools.

The second main section contains the experimental work, where the obtained results were organized in the form of scientific publications, a complementary research section and a general results synthesis and comparison section.

First, the description of the Streptomyces collection constitution, preliminary identification and conservation is detailed. As well as the results of the in vitro preliminary evaluation of their plant growth promoting traits.

The second segment comprises the work conducted on storage pathogenic fungi and their mycotoxins, namely Penicillium verrucosum, producer of ochratoxin A, and Aspergillus flavus, producer of aflatoxins. Streptomyces isolates were screened for their in vitro capacity to decrease fungal growth and mycotoxin production during dual culture assays. Then, the impact of their cell free extracts (CFEs) on the same parameters was also evaluated.

Degradation capacity of Streptomyces isolates and their CFEs was further assessed. For aflatoxin B1, degradation assays were followed by the evaluation of the residual toxicity. Next, a complementary study on the stimulation of Streptomyces' ochratoxin A degrading capacities is also described. Finally, a comparison between the results observed for both storage pathogens is detailed.

The next experimental section presents the evaluation of Streptomyces isolates against phytopathogenic fungi, starting by an in vitro assessment of the bacterial antifungal activities.

Then, active isolates were selected to perform an in planta verification of their biocontrol potential against three wheat diseases, under greenhouse conditions. Their impact on the expression on plant defense genes was also measured. Finally, their installation on the surface of wheat leaves was verified by Scanning Electron Microscopy (SEM).

The last experimental procedures consist on a study on the impact of Streptomyces and their CFEs on Fusarium graminearum and Fusarium verticillioides growth and mycotoxin production.

The experimental section concludes with a synthesis depicting the Clustering and correlation analysis of all the outcomes obtained for the different models evaluated, their biological interpretations and the identification of the most outstanding isolates.

A

Wheat phytopathogenic non-mycotoxinogenic fungi

As mentioned before, wheat is affected by a variety of diseases that cause important yield losses each year. Main diseases include the rusts, blotches and head blight/scab. The aspect of the lesions they provoke is illustrated in Figure 3. Rust fungi are obligated biotrophs, which depend on the living plan host to acquire the necessary nutriments for their growth and reproduction. There exist three wheat rust diseases: stem rust, stripe rust and leaf rust, caused by the Puccinia genus, namely P. graminis, P. striiformis and P. triticina, respectively [START_REF] Kolmer | Diseases Which Challenge Global Wheat Production-The Wheat Rusts[END_REF]. Strategies to control wheat rust infection include agricultural practices Figure 3. Authors illustration of the aspect of wheat lesions originated by the main foliar phytopathogenic fungi.

Puccinia triticina

Generalities

Puccinia triticina, the causal agent of rust disease of wheat, is mainly present in areas with temperate climate and high humidity, however, it has shown high adaptability to a large range of climates, which makes it the most common and widespread rust of wheat around the world [START_REF] Kolmer | Diseases Which Challenge Global Wheat Production-The Wheat Rusts[END_REF]). The decrease on yield caused by this disease is the result of a reduction in kernel weight and on the number of grains per head. Losses from this infection are calculated at hundreds of millions of dollars in the USA. The most economical way of reducing yield losses due to rust is the use of genetically resistant wheat varieties. Resistance to P. triticina is conditioned by 68 Leaf rust (Lr) genes being frequently used in wheat cultivars around the globe. Nevertheless, the use of resistant cultivars has caused the selection of virulent leaf rust races that are no longer affected by resistance genes [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF][START_REF] Kolmer | Diseases Which Challenge Global Wheat Production-The Wheat Rusts[END_REF].

Life cycle and pathogenesis

The infection by P. triticina starts by an uredinial stage of small round, brown to orange pustules on the leaf surface. These uredinia are capable of producing thousands of spores each day, that can be disseminated by the wind through hundreds of kilometers. Then, the urediniospores germinate to produce infection structures comprising appressoria, germ tube and penetration peg, that facilitate the penetration of the plant host stomata (Fig. 4). The uptake of nutrients from plant cells without killing them is achieved thanks to the production of specialized structures: the substomatal vesicle and haustoria. As uredinia maturate, the formation of teliospores begins. The production of pycniospores and aeciospores, related to sexual reproduction, occurs only on alternate hosts, not on wheat [START_REF] Kolmer | Diseases Which Challenge Global Wheat Production-The Wheat Rusts[END_REF]. Figure 5 illustrates the life cycle of P.

striformis, the causal agent of stripe rust.

Zymoseptoria tritici

Generalities

Zymoseptoria tritici (teleomorph Mycosphaerella graminicola) causes Septoria tritici blotch (STB), the main leaf disease striking wheat in temperate regions. STB is nowadays the most important wheat disease in Europe,

where it causes loses of about 800 to 2400 million of euros each year [START_REF] Fones | The Impact of Septoria tritici Blotch Disease on Wheat: An EU Perspective[END_REF]. Chemical control is the main mechanism for STB control in Europe, together with the use of genetically resistant varieties [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF]. It is estimated that about 70% of the volume of fungicides used in Europe is destined to control STB [START_REF] Fones | The Impact of Septoria tritici Blotch Disease on Wheat: An EU Perspective[END_REF]. STB occurs mainly in cool wet weather, under optimal temperatures between 20 and 25°C [START_REF] Tiley | Zymoseptoria tritici[END_REF].

Figure 6. SEM observation of Z. tritici mycelium entering the stomata of a wheat leaf [START_REF] Ponomarenko | Septoria Tritici Blotch (STB) of Wheat Septoria Tritici Blotch (STB) of Wheat[END_REF].

Life cycle and pathogenesis

STB is characterized by necrotic lesions on leaves and steam, the primary entrance site on the leaf is through the stomata (Fig. 6), which eliminates the need for specialized penetration structures such as an appressorium [START_REF] Brennan | A Review of the Known Unknowns in the Early Stages of Septoria Tritici Blotch Disease of Wheat[END_REF][START_REF] Ponomarenko | Septoria Tritici Blotch (STB) of Wheat Septoria Tritici Blotch (STB) of Wheat[END_REF]. Z. tritici has a hemibiotrophic lifestyle, it starts as a biotroph (latent phase) taking nutrients from the apoplast around living cells, then it kills the surrounding host cells and becomes necrotrophic. During the asymptomatic stage, the fungus produces cellulases, peptidases and xylanases which may help to degrade host cell wall components to release nutrients with minimal damage. It also produces effectors to inactivate the plant immune response and to protect its cell wall from host chitinases [START_REF] Kettles | Dissecting the Molecular Interactions between Wheat and the Fungal Pathogen Zymoseptoria tritici[END_REF][START_REF] Sánchez-Vallet | Is Zymoseptoria tritici a Hemibiotroph?[END_REF]. The switch from the biotrophic to the necrotrophic phase is still unknown, but it coincides with an abrupt increase in fungal growth rate and biomass [START_REF] Rudd | Transcriptome and Metabolite Profiling of the Infection Cycle of Zymoseptoria tritici on Wheat Reveals a Biphasic Interaction with Plant Immunity Involving Differential Pathogen Chromosomal Contributions and a Variation on the Hemibiotrophic Lifestyle Def[END_REF]. STB disease cycle is illustrated on Figure 7. Infection is initiated by air-borne ascospores and rain dispersal of conidia and is carried out following five phases [START_REF] Ponomarenko | Septoria Tritici Blotch (STB) of Wheat Septoria Tritici Blotch (STB) of Wheat[END_REF]):

1. Initial growth of the hyphae on the leaf surface (0-24 hours).

2. Host penetration via the stomata (24-48 hours).

3. Hyphae proliferation in the mesophyll tissue, nutrient uptake from the apoplast (2-12 days).

4. Change to necrotrophic growth, appearance of lesions on the leaf surface (12-14 days).

5. Further colonization of mesophyll tissue, formation of pycnidia with conidia (14-28 days).

To date, 21 different genetic loci of qualitative genes for resistance to STB have been identified [START_REF] Kettles | Dissecting the Molecular Interactions between Wheat and the Fungal Pathogen Zymoseptoria tritici[END_REF]. As well as 89 quantitative trait loci [START_REF] Tiley | Zymoseptoria tritici[END_REF]). In the field, resistance genes lose effectivity rapidly due to fast genetic changes in the pathogen.

Besides, wheat cultivars resistant in one region frequently show susceptibility in another [START_REF] Ponomarenko | Septoria Tritici Blotch (STB) of Wheat Septoria Tritici Blotch (STB) of Wheat[END_REF]. Z. tritici is commonly found concomitantly with Parastagonospora nodorum [START_REF] Duba | A Review of the Interactions between Wheat and Wheat Pathogens: Zymoseptoria tritici, Fusarium spp. and Parastagonospora nodorum[END_REF][START_REF] Willocquet | An Outlook on Wheat Health in Europe from a Network of Field Experiments[END_REF]).

Figure 7. Lifecycle of the wheat pathogen Zymoseptoria tritici [START_REF] Ponomarenko | Septoria Tritici Blotch (STB) of Wheat Septoria Tritici Blotch (STB) of Wheat[END_REF]).

Parastagonospora nodorum

Generalities

Parastagonospora nodorum (anamorph Stagonospora and teleomorph Phaeosphaeria) is a necrotrophic fungus and the causal agent of Septoria nodorum blotch (SNB) in wheat. SNB symptoms are visible at the leaves, stems, nodes, glumes and awns. They appear as small darkbrown lesions with a yellow halo, resulting from diffusible toxins produced by the pathogen [START_REF] Mehra | Septoria Nodorum Blotch of Wheat[END_REF], and as a discoloration of the head during glume blotch [START_REF] Solomon | Stagonospora nodorum: Cause of Stagonospora Nodorum Blotch of Wheat[END_REF]. Infected glumes result in shrunken kernels with low quality and quantity of grains, which lead to reduced yield loses going up to 20-50% [START_REF] Duba | A Review of the Interactions between Wheat and Wheat Pathogens: Zymoseptoria tritici, Fusarium spp. and Parastagonospora nodorum[END_REF]. P. nodorum is mainly present in Australia, Brazil and USA, but SNB cases have been documented in France and in the Scandinavian countries. Disease incidence can be reduced by good agricultural practices, such as crop rotation and tillage. However, effective management is currently based principally on chemical control and the use of wheat resistant varieties [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF][START_REF] Mehra | Septoria Nodorum Blotch of Wheat[END_REF].

Life cycle and pathogenesis

Host colonization by P. nodorum is light dependent [START_REF] Oliver | Stagonospora nodorum: From Pathology to Genomics and Host Resistance[END_REF]) and starts when hyphae growing from germinating spores penetrate through the leaf cuticle, and opportunistically through stomata, which causes the site of leaf infection to turn yellow.

Within the chlorotic areas, small regions of necrosis are often observed and pycnidia will begin to form in the lesion in about a week [START_REF] Solomon | Stagonospora nodorum: Cause of Stagonospora Nodorum Blotch of Wheat[END_REF]. During the first stage of infection, the pathogen produces toxic necrotrophic effectors that kill host tissue during colonization, such as ToxA, Tox1 and Tox2 [START_REF] Mehra | Septoria Nodorum Blotch of Wheat[END_REF][START_REF] Oliver | Stagonospora nodorum: From Pathology to Genomics and Host Resistance[END_REF]. The production of cell wall-degrading enzymes such as xylanases, glycanases, glucanases and cellulases, induce the host tissue necrosis as well, and is one of the characteristics that differentiates P. nodorum from Z. tritici [START_REF] Duba | A Review of the Interactions between Wheat and Wheat Pathogens: Zymoseptoria tritici, Fusarium spp. and Parastagonospora nodorum[END_REF][START_REF] Solomon | Stagonospora nodorum: Cause of Stagonospora Nodorum Blotch of Wheat[END_REF]. Pathogenic inoculum can originate from various sources, illustrated in Figure 8. Overwintering: the pathogen pseudothecia and/or pycnidia spend the winter in wheat debris. Primary infection: the main inoculum source consists on windborne ascospores released from pseudothecia, or splash-dispersed conidia released from pycnidia. Secondary spread: vertical pathogenic dispersion develops as conidia goes from lower canopy to upper, and then to glumes. Seedborne infection: seedlings can be infected by the dormant mycelium on contaminated seeds from a previous season where glume blotch occurred. Both conidia and ascospores can germinate and cause infection between 5 and 35°C (optimal: 15 to 25°C), favored by rainstorms [START_REF] Mehra | Septoria Nodorum Blotch of Wheat[END_REF].

Figure 8. Disease cycle of Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum [START_REF] Mehra | Septoria Nodorum Blotch of Wheat[END_REF]) (Original drawing by Hermod Karlsen).

Magnaporthe oryzae

Generalities

Wheat blast (WB) is a disease caused by the fungus Magnaporthe oryzae Triticum pathotype (MoT), which is a seed-borne pathogen. M. oryzae was mainly known as a rice pathogen but it was discovered as a wheat pathogen in Brazil in 1985. It was subsequently disseminated to Bolivia, Argentina, Paraguay and more recently to Bangladesh (March 2016), which represents a risk of dissemination through Asia, the world's greatest wheat producer [START_REF] Sadat | Wheat Blast: A New Fungal Inhabitant to Bangladesh Threatening World Wheat Production[END_REF]. Yield losses of 40%-100% have been reported in the affected countries.

Warm and humid environments favor fungal development. The optimal temperature ranges between 25 and 30 °C, with spike wetness lasting between 25 and 40 hours, both factors favor wheat blast intensity [START_REF] Cruz | Wheat Blast Disease: Danger on the Move[END_REF]. Nowadays, the main fighting strategies are based on plant genetic resistance and the use of chemical fungicides [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF]).

Furthermore, MoT resistance genes appear to be rare in wheat. Until now, only four have been identified: Rmg2, Rmg3, Rmg7 and Rmg8, which conferred resistance against early 90's

MoT strains. Unfortunately, their effectivity has decreased against recent isolates. Currently, the best sources of resistance are cultivars containing a 2NS translocation segment from

Aegilops ventricosa [START_REF] Cruz | Wheat Blast Disease: Danger on the Move[END_REF]. Concerning pesticides, an early application might reduce MoT inoculum from basal leaves and subsequently lower the risk of head blast.

Another management strategy is the treatment of seeds with chemical fungicides [START_REF] Cruz | Magnaporthe oryzae Conidia on Basal Wheat Leaves as a Potential Source of Wheat Blast Inoculum[END_REF].

Life cycle and pathogenesis

WB is mainly a head disease, but lesions can be observed on leaves, where the pathogen produces high amounts of conidia increment the risk of spike blast development [START_REF] Cruz | Magnaporthe oryzae Conidia on Basal Wheat Leaves as a Potential Source of Wheat Blast Inoculum[END_REF]. Symptoms on heads go from small elliptical lesions to complete bleaching and empty spikes, as the pathogen can block the translocation of photosynthates and kill the upper parts of the spike, causing partial or total spike sterility [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF]). Thus, MoT can be transmitted from spikes to seeds, and from infected seeds to seedlings [START_REF] Cruz | Wheat Blast Disease: Danger on the Move[END_REF]. Seeds produced by infected spikes are usually small, wrinkled, deformed and have low test-weight [START_REF] Cruz | Magnaporthe oryzae Conidia on Basal Wheat Leaves as a Potential Source of Wheat Blast Inoculum[END_REF]. M. oryzae produces both conidia (asexual cycle) and ascospores (sexual cycle) (Fig. 9). Fully fertile strains are self-sterile hermaphrodites, mating occurs by alternate alleles MAT1-1 and MAT1-2. Conidia are pyriform-shaped, hyaline to pale graycolored, and are considered the main source of inoculum, as they can remain on crop residue and alternate hosts [START_REF] Xue | Fungal Pathogenesis in the Rice Blast Fungus Magnaporthe grisea[END_REF]. M. oryzae is an hemibiotroph, as in initiates as a biotroph and then, upon causing the host cells death, it switches to a necrotrophic lifestyle [START_REF] Syed Ab Rahman | Emerging Microbial Biocontrol Strategies for Plant Pathogens[END_REF]. Both conidia and ascospores germinate and form appressoria and a penetration peg to penetrate the host leaf surface and develop invasive hyphae, which cause the development of lesions [START_REF] Cruz | Wheat Blast Disease: Danger on the Move[END_REF][START_REF] Fernandez | Rise of a Cereal Killer: The Biology of Magnaporthe oryzae Biotrophic Growth[END_REF].

Figure 9. Life cycle of Magnaporthe oryzae [START_REF] Xue | Fungal Pathogenesis in the Rice Blast Fungus Magnaporthe grisea[END_REF].

Wheat is one of the most important staple foods worldwide. Its consistent production has to be guaranteed in order to supply the future generations. Plant pathogenic non-mycotoxinogenic fungi of wheat cause important yield losses and the decrease on grain quality. Fungal pathogens have distinctive lifecycles, which determine the infecting mechanism and the repercussion on plant's health. Main fungal management strategies include plant resistance and the use of chemical fungicides.

Climate change is provoking the spread of fungal diseases to previously unaffected regions. Main mycotoxins found in food and feed are aflatoxins, ochratoxins, deoxynivalenol, fumonisins and patulin (Mannaa and Kim 2017b).

Wheat mycotoxinogenic fungi

In order to manage mycotoxin risk, most countries have established maximum limits for mycotoxin content in food, based on exposure data and toxicity assessments [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]. The established limits vary from one country to another, and although an homogenization of regulations would be preferable, limitations have to be adapted to ensure that they do not jeopardize the limited food supply of developing countries, neither make basic commodities excessively expensive (CAST 2003). Limitations for raw materials are usually less strict, as processing usually inactivate a fraction of the mycotoxins. However, some of them are resistant to thermal processes [START_REF] Bullerman | Stability of Mycotoxins during Food Processing[END_REF][START_REF] Raters | Thermal Stability of Aflatoxin B1 and Ochratoxin A[END_REF].

Environmental and molecular factors influencing mycotoxin production

The presence of mycotoxinogenic fungi in a food matrix does not automatically indicates the presence of mycotoxins. Inversely, the absence of toxinogenic fungi does not guarantee that the commodity is free of mycotoxins, as toxins may persist long after the fungi have lost viability [START_REF] Pitt | Mycotoxins and Toxigenic Fungi[END_REF]. Indeed, the conditions that favor fungal growth are not always conductive for mycotoxin production, and optimal conditions variate greatly for each fungal specie and mycotoxin. Several factors can affect fungal development and mycotoxin production, such as temperature, water availability, pH, light, nature of substrate and the presence of reactive oxygen species (ROS), as well as the interaction with other microorganisms and with the food matrix (Mannaa and Kim 2017b;[START_REF] Reverberi | Natural Functions of Mycotoxins and Control of Their Biosynthesis in Fungi[END_REF]) (Fig. 10), which makes difficult to define a particular set of factors that will promote mycotoxin appearance.

For instance, temperature and water availability can compromise fungi's ability to produce hydrolytic enzymes, such as β-D-galactosidase, which are fundamental for the fungal establishment on substrates [START_REF] Keller | PH Regulation of Sterigmatocystin and Aflatoxin Biosynthesis in Aspergillus Spp[END_REF]. Studies have shown that a low temperature stress provokes the increase on toxin production on certain Fusarium spp., which could be used by the fungus to increase competitiveness (Mannaa and Kim 2017b). Additionally, pH was found to greatly impact aflatoxin and fumonisin production and stability. In fungi, transitions from conidia to hyphal growth, and to mycelial growth to conidiogenesis are characterized by an oxidative burst, a factor also associated with the beginning of aflatoxin biosynthesis. In fact, oxidant stressors were found to stimulate aflatoxin production, while the addition of antioxidants inhibit aflatoxin synthesis without affecting fungal growth. The same phenomenon was observed for other mycotoxins such as trichothecenes, ochratoxin A and patulin. A regulation of mycotoxin biosynthetic genes is behind these effects [START_REF] Reverberi | Natural Functions of Mycotoxins and Control of Their Biosynthesis in Fungi[END_REF]. Light is also a generator of ROS. This was found to have an impact in mycotoxin production by Penicillium verrucosum, which can switch from producing ochratoxin A to producing citrinin when exposed to light [START_REF] Stoll | Comparative Proteome Analysis of Penicillium Verrucosum Grown under Light of Short Wavelength Shows an Induction of Stress-Related Proteins Associated with Modified Mycotoxin Biosynthesis[END_REF]. Nitrogen and carbon sources are known regulators of aflatoxin and ochratoxin biosynthesis. For example, lactose increases ochratoxin production, whilst glucose causes a repressive effect. Furthermore, the addition of suboptimal concentrations of fungicides can increase mycotoxin biosynthesis [START_REF] Cendoya | Effect of Fungicides Commonly Used for Fusarium Head Blight Management on Growth and Fumonisin Production by Fusarium proliferatum[END_REF][START_REF] Ferrigo | Fusarium Toxins in Cereals: Occurrence, Legislation, Factors Promoting the Appearance and Their Management[END_REF]. Plant organic compounds, such as methyl salicylate and oxylipins were found to modulate mycotoxin biosynthesis in pathogenic fungi [START_REF] Gao | Host-Derived Lipids and Oxylipins Are Crucial Signals in Modulating Mycotoxin Production by Fungi[END_REF]. Finally, global factors defining climate change (high temperature, water stress and elevated CO2) can also have an impact on the amount of mycotoxins produced by fungi during crop infection [START_REF] Medina | Effect of Climate Change on Aspergillus flavus and Aflatoxin B1 Production[END_REF].

The described factors, affecting the activation of secondary metabolism genes in fungi, are handled molecularly by a series of global regulators. Among them, the most studied have been the velvet protein complex VelB/VeA/LaeA, involved on sexual development and secondary metabolites production in response to light; PacC, the key factor for pH regulation; CBC, which regulates the response to redox status and iron stresses; CreA, implicated in the regulation of carbon metabolism; and AreA and AreB, involved in nitrogen metabolism [START_REF] Chen | Fusarium graminearum Trichothecene Mycotoxins: Biosynthesis, Regulation, and Management[END_REF][START_REF] Edwards | Zearalenone Risk in European Wheat[END_REF][START_REF] Gallo | Recent Advances on the Molecular Aspects of Ochratoxin A Biosynthesis[END_REF].

Figure 10. Factors having an impact on mycotoxin production (Modified from Manna and Kim 2017).

Biological function of mycotoxins

The role of mycotoxins remains enigmatic. Several years ago, Janzen hypothesized that toxin-producing fungi compete with large organisms for resources such as fruit or seeds by making them unpalatable [START_REF] Janzen | Why Fruits Rot, Seeds Mold, and Meat Spoils[END_REF]. Nowadays, proclamations about their use by the fungus to infect the plant host (virulence factors) or to adapt to hostile environments (defense mechanism) are the most widespread. Laboratory and field experiments have demonstrated that trichothecenes play an important role in wheat head blight caused by Fusarium graminearum, while fumonisins are not necessary for ear rot development caused by F.

verticillioides [START_REF] Desjardins | FUM1 -A Gene Required for Fumonisin Biosynthesis but Not for Maize Ear Rot and Ear Infection by Gibberella moniliformis in Field Tests[END_REF]. However, they seem to lower maize defenses during infection by inhibiting the β-1,3-glucanase activity (Galeana-Sánchez et al. 2017). As mentioned above, mycotoxins production is triggered by the presence of ROS, and recent research has suggested that they may contribute to the detoxification of fungal cells [START_REF] Reverberi | Natural Functions of Mycotoxins and Control of Their Biosynthesis in Fungi[END_REF]. For instance, aflatoxin production has been linked to the regulation of oxidative stress responses, decreasing oxidative stress in A. flavus [START_REF] Fountain | Transcriptional Responses of Toxigenic and Atoxigenic Isolates of Aspergillus flavus to Oxidative Stress in Aflatoxin-Conducive and Non-Conducive Media[END_REF].

Moreover, OTA production by P. verrucosum and P. nordicum was suggested to contribute to their adaptation to NaCl rich environments, by ensuring chloride homeostasis in the cell through the excretion of OTA, that carries a chlorine in its molecule [START_REF] Gallo | Recent Advances on the Molecular Aspects of Ochratoxin A Biosynthesis[END_REF]. In fact, NaCl rich environments lead to a shift production from citrinin to OTA [START_REF] Schmidt-Heydt | Oxidative Stress Induces the Biosynthesis of Citrinin by Penicillium verrucosum at the Expense of Ochratoxin[END_REF]. Alternatively, mycotoxins represent an important part of microbial interactions, where they may protect the producing fungi from competing or invading microbes. For example, fusaric acid produced by Fusarium spp., was found to interfere with the quorum sensing mechanism of the bacterial biocontrol agent (BCA) Pseudomonas chloraphis [START_REF] Van Rij | Influence of Fusaric Acid on Phenazine-1-Carboxamide Synthesis and Gene Expression of Pseudomonas chlororaphis Strain PCL1391[END_REF]). In the same way, patulin and penicillic acid produced by Penicillium inhibited quorum sensing and blocked chitinase production from bacteria [START_REF] Rasmussen | Identity and Effects of Quorum-Sensing Inhibitors Produced by Penicillium Species[END_REF]. Zearalenone, fumonisin, penicillic acid, citrinin and patuline also inhibited the quorum sensing in Chromobacterium violaceum, producer of the antibiotic violacein (Bacon, Hinton, and Mitchell 2017). Lastly, studies indicate that mycotoxins may be synthesized in response to microbial signals, while also serving as interspecies signals themselves [START_REF] Venkatesh | Mycotoxins in Conversation with Bacteria and Fungi[END_REF].

2.1.3.2 Wheat phytopathogenic fungi: The Fusarium genus Among phytopathogenic fungi producing mycotoxins, the genus Fusarium is one of the main groups that contaminate a wide diversity of crops in various climatic zones around the world. In addition to affecting crop yields, these fungi produce several mycotoxins endangering human and animal health [START_REF] Summerell | Resolving Fusarium: Current Status of the Genus[END_REF]. Fusarium spp. are mainly considered as field fungi, however, they are adapted to a wide range of habitats so they can proliferate and produce mycotoxins on stored moist grain under inadequate storage conditions [START_REF] Samapundo | The Influence of Modified Atmospheres and Their Interaction with Water Activity on the Radial Growth and Fumonisin B1 Production of Fusarium verticillioides and F. proliferatum on Corn. Part I: The Effect of Initial Headspace Carbon Dioxide Concentration[END_REF]. Fusarium head blight/scab (FHB) disease, caused mainly by Fusarium graminearum, provokes the premature senescence of wheat heads, which leads to a decrease on grain yield and quality, compromising the harvest and marketability. FHB comes with the presence of mycotoxins called trichothecenes, such as deoxynivalenol (DON), which represents a primary food safety risk. Fusarium verticillioides is mainly a maize pathogen, however, its emergence as a contaminant of wheat has been assessed, together with the presence of fumonisins [START_REF] Deepa | Fusarium verticillioides, a Globally Important Pathogen of Agriculture and Livestock[END_REF]. Indeed, recently published results revealed the presence of F. verticillioides on winter wheat in Finland [START_REF] Gagkaeva | Emergence of Fusarium verticillioides in Finland[END_REF]. The expansion on the hosts targeted by fungal pathogens could be influenced by the impacts of climate change [START_REF] Magan | Possible Climate-Change Effects on Mycotoxin Contamination of Food Crops Pre-and Postharvest[END_REF]. Genetic resistance, fungicides, crop rotation and tillage of the infected crop residues are the principal strategies to the control of Fusarium diseases, together with the elimination of damaged grains to reduce mycotoxin risk [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF]).

Fusarium graminearum

Generalities

Fusarium graminearum (teleomorph Gibberella zeae) (Fig. 11) is the main causal agent of Fusarium head blight (FHB) in several regions of the world, notably in North America, Europe and Asia. FHB is favored by moderate temperatures and high humidity. Rainfall during and after flowering also promotes FHB development [START_REF] Ferrigo | Fusarium Toxins in Cereals: Occurrence, Legislation, Factors Promoting the Appearance and Their Management[END_REF]. F.

graminearum is known to produce the mycotoxins deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN/ZEA/ZON) [START_REF] Pitt | Mycotoxins and Toxigenic Fungi[END_REF]. The optimal temperature for F. graminearum proliferation is 20-25°C, with an optimal and minimal ɑw of 0.98-0.995 and 0.90-0.91, respectively.

Germination occurs at an optimal ɑw of 0.88 (Mannaa and Kim 2017b).

A minimal pH of around 3 is necessary for its growth [START_REF] Pitt | Mycotoxins and Toxigenic Fungi[END_REF].

FHB infection appears as dark brown, water-soaked spots and later a reddish mold develops on the kernels, cob, husks and shank [START_REF] Nourozian | Biological Control of Fusarium graminearum on Wheat by Antagonistic Bacteria[END_REF].

Life cycle and pathogenesis

F. graminearum can remain as a saprophyte on crop residues. It then produces ascospores that get dispersed through the air and constitute the main inoculum source during flowering (anthesis). Macronidia, produced during the asexual phase can be water splashed onto adjacent plant spikes [START_REF] Haile | Fusarium Head Blight in Durumwheat: Recent Status, Breeding Directions, and Future Research Prospects[END_REF].

The disease cycle of F. graminearum in wheat is illustrated in Figure 12. The whole process of wheat head colonization and bleaching takes around 14 days [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF].

The infected pale yellow spikelets are easily detectable among the bright green healthy ones.

If the weather is humid, F. graminearum can infect the entire head and produce a pinkish mycelium. This pinkish crust on the surface of the grain is the reason why this disease was named "scab" (CAST 2003). Fusarium-damaged wheat is typically characterized by thin or shrunken chalk-like kernels. Infected grains are poorly filled so they have a low kernel weight.

Genetic resistance is a cost effective and environmentally safe way of managing FHB.

However, there is no bread or durum wheat cultivar currently available on the market that is the

F. ON),
. The e with an ectively. has complete resistance or immunity. The main quantitative trait locus (QLT) of bread wheat resistance for FHB is Fhb1, which contribute 25 to 42% of the resistance [START_REF] Haile | Fusarium Head Blight in Durumwheat: Recent Status, Breeding Directions, and Future Research Prospects[END_REF].

F. graminearum Infection starts as a biotroph by the direct entry into open florets by two routes, the first is spore landing on the emerged silks that then infect the ear by the silk channel, and the second is through wounds caused by birds, insects or extreme weather. As infection advances through the xylem and pith, no disease symptoms are observed [START_REF] Figueroa | A Review of Wheat Diseases-a Field Perspective[END_REF]. However, during this stage, various Tri genes, responsible for trichothecene production are activated, which has been found to aid plant infection. Indeed, DON is the only mycotoxin shown to be a virulence factor in wheat [START_REF] Trail | For Blighted Waves of Grain: Fusarium graminearum in the Postgenomics Era[END_REF]. Symptoms start to appear when the fungal hyphae enter the wheat cells and start provoking the death of the plant tissues which leads to the necrotrophic phase of the infection. Symptoms at this stage include water soaking, mainly of the chlorenchyma, followed by the bleaching of the colonized tissue [START_REF] Trail | For Blighted Waves of Grain: Fusarium graminearum in the Postgenomics Era[END_REF].

Figure 12. Fusarium graminearum life cycle [START_REF] Trail | For Blighted Waves of Grain: Fusarium graminearum in the Postgenomics Era[END_REF]). Tri11,Tri3,Tri1,and Tri8,generate deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-ADON) (Fig. 14) [START_REF] Boenisch | Structural Reorganization of the Fungal Endoplasmic Reticulum upon Induction of Mycotoxin Biosynthesis[END_REF]. The reason why Tri14 is required for DON synthesis in planta remains unknown [START_REF] Brown | Gain and Loss of a Transcription Factor That Regulates Late Trichothecene Biosynthetic Pathway Genes in Fusarium[END_REF]) but its deletion reduced DON production and F. graminearum ZEN is an estrogenic mycotoxin produced primarily by Fusarium graminearum and F. culmorum when they develop in substrates with high humidity and low temperatures. ZEN is a phenolic resorcyclic acid lactone (Fig. 15) that resembles the natural estrogen 17β-estradiol, which confers it the capacity to bind to estrogen receptors, hence its estrogenic potential [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]. It has been detected frequently in different cereals, such as wheat, barley, maize, sorghum, rye, and rice [START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]). It has also been detected in Figure 14. Primary and secondary metabolism pathways in Fusarium graminearum [START_REF] Boenisch | Structural Reorganization of the Fungal Endoplasmic Reticulum upon Induction of Mycotoxin Biosynthesis[END_REF]. processed products like bread, chocolate, flour, malt and milk [START_REF] Mahato | Occurrence, Impact on Agriculture, Human Health, and Management Strategies of Zearalenone in Food and Feed: A Review[END_REF]. Optimal production of ZEN has been observed at 25°C, 16% moisture and neutral pH. ZEN is known to be heat stable up to 150°C and to resist food and feed processing [START_REF] Rai | Occurrence and Toxicity of a Fusarium Mycotoxin, Zearalenone[END_REF]. 2.1.3.2.1.2 Toxicity of DON and ZEN

The ingestion of trichothecenes in high doses can cause immune response inhibition, nausea and vomiting (Mannaa and Kim 2017b), as well as reduced growth, anorexia and changes in brain neurochemicals [START_REF] Ferrigo | Fusarium Toxins in Cereals: Occurrence, Legislation, Factors Promoting the Appearance and Their Management[END_REF]. At the molecular level, DON disturbs standard cell function by obstructing protein synthesis and activating protein degradation in the proteasome, leading to translation inhibition [START_REF] Smith | In Vitro Co-Culture Models to Evaluate Acute Cytotoxicity of Individual and Combined Mycotoxin Exposures on Caco-2, THP-1 and HepaRG Human Cell Lines[END_REF].

ZEN is of major interest because despite its low acute toxicity, it has proven to be hepatotoxic, immunotoxic and carcinogenic to several mammalian species [START_REF] Ferrigo | Fusarium Toxins in Cereals: Occurrence, Legislation, Factors Promoting the Appearance and Their Management[END_REF]. Effects of ZEN involve hormonal changes, as it binds to estrogen receptors. However, mortality is not a concern due to this mycotoxin [START_REF] Richard | Some Major Mycotoxins and Their Mycotoxicoses-An Overview[END_REF]. In humans, ZEN occasionally causes hypoestrogenic syndromes and its suspected to provoke precocious puberty development in girls [START_REF] Smith | In Vitro Co-Culture Models to Evaluate Acute Cytotoxicity of Individual and Combined Mycotoxin Exposures on Caco-2, THP-1 and HepaRG Human Cell Lines[END_REF]. Additionally, ZEN causes altered progesterone level, decreased sperm count and serum testosterone level, and infertility. Its involvement in the occurrence of esophageal cancer and breast cancer has been reported around the world [START_REF] Rai | Occurrence and Toxicity of a Fusarium Mycotoxin, Zearalenone[END_REF]. ZEN has been categorized as a Group 3 carcinogen by the International Agency for Research on Cancer (IARC) due to the lack of evidence on its carcinogenicity to humans [START_REF] Mahato | Occurrence, Impact on Agriculture, Human Health, and Management Strategies of Zearalenone in Food and Feed: A Review[END_REF]).

Regulation of deoxynivalenol and zearalenone

DON limits for unprocessed durum wheat, oats and maize is 1750 µg/kg. For cereals intended for human consumption, cereal flour and pasta the limit was established at 750 µg/kg. Bread, pastries, biscuits, cereal snacks and breakfast cereals have a limit of 500 µg/kg.

Lastly, DON maximal limit for foods destined to babies, infants and young children is 200 µg/kg.

ZEN limit for unprocessed cereals is of 100 µg/kg, and of 75 µg/kg for cereals intended for direct human consumption and cereal flour. The limit for unprocessed maize, as well as maize intended for direct human consumption and derived products is 200 µg/kg. Bread, pastries, biscuits, snacks, breakfast cereals derived from cereals and maize must not exceed 50 µg/kg of ZEN. Cereal-based and maize-based foods for babies, infants and young children must remain under 20 µg/kg (European Commission 2006).

Fusarium verticillioides

Generalities

Fusarium ear rot (FER) in maize is caused mainly by Fusarium verticillioides and Fusarium proliferatum. F. verticillioides is mainly a maize pathogen, however it has been found on wheat [START_REF] Cendoya | Fumonisins and Fumonisin-Producing Fusarium Occurrence in Wheat and Wheat by Products: A Review[END_REF]) such as in its stalks [START_REF] Palazzini | Biocontrol and Population Dynamics of Fusarium Spp. On Wheat Stubble in Argentina[END_REF]) and husks [START_REF] Kuzdraliński | The Composition of Fusarium Species in Wheat Husks and Grains in South-Eastern Poland[END_REF]. Fusarium verticillioides (previously F. moniliforme) can grow between 4 and 37°C with an optimal temperature of 30°C. Regarding humidity, this pathogen requires a minimal ɑw of 0.90 (Mannaa and Kim 2017b). Fungal contamination by F.

verticillioides occurs in the field, seemingly under drought stress followed by warm, wet weather [START_REF] Richard | Some Major Mycotoxins and Their Mycotoxicoses-An Overview[END_REF]. It can infect the roots, stalk, ears and kernels. In addition to causing plant diseases, F. verticillioides is capable of producing fumonisins. To date, 28 fumonisin analogues have been identified. However, only 3 occur abundantly and have elevated toxicity, namely, fumonisin B1 (FB1), B2 and B3 [START_REF] Deepa | Fusarium verticillioides, a Globally Important Pathogen of Agriculture and Livestock[END_REF].

Life cycle and pathogenesis

F. verticillioides colonizes residues in the soil, it can be found in corn seeds and can infect the seedlings. However, the most common infection pathway is the entrance of airborne micronidia by the silks or through existent wounds (CAST 2003). Figure 16 [START_REF] Bezuidenhout | Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme[END_REF][START_REF] Bezuidenhout | Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme[END_REF]. They consist in diesters of propane-1,2,3-tricarboxylic acid and long-chain aminopolyol backbones (Fig. 17). Their structure is similar to those of the sphingoid bases sphinganine (SA) and sphingosine (SO) [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]. The principal fumonisins are FB1, FB2 and FB3 and they are mainly found on maize. Although their occurrence has been documented also in barley, wheat, sorghum and rice [START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]. Fungal proliferation can continue after harvest, under poor storage conditions, which provokes mycotoxin accumulation on stored moist grains (Mannaa and Kim 2017b). An ɑw of 0.93 is needed for fumonisin B1 production. Temperature must remain between 10 and 37°C, with an optimal range between 15 and 30°C.

Fumonisins are synthesized through the condensation of the amino acid alanine to an acetate derived precursor [START_REF] Sweeney | Mycotoxin Production by Aspergillus, Fusarium and Penicillium Species[END_REF]. At the end of their biosynthesis, fumonisins' constitution is as follows: the 18-carbon backbone from C-3 to C-20 is derived from acetate, and C-1 and C-2, as well as the C-2 amino, are derived from alanine. The two methyl groups at C-12 and C-16 are derived from methionine. The hydroxyl at C-3 is derived from acetate, while those at C-5, C-10, C-14, and C-15 are derived from molecular oxygen. The two tricarballylic esters at C-14 and C-15 are likely derived from the citric acid cycle. These transformations consisting on several hydroxylations and esterifications are regulated by a series of enzymes such as reductases (Fum13p), oxygenases (Fum6p) and transferases (Fum14p) (Fig. 18) [START_REF] Du | Biosynthesis of Sphinganine-Analog Mycotoxins[END_REF]).

Figure 18. A proposed pathway for fumonisins biosynthesis [START_REF] Du | Biosynthesis of Sphinganine-Analog Mycotoxins[END_REF]).

Toxicity of fumonisins

Fumonisins are competitive inhibitors of ceramide synthase. They obstruct the biosynthesis of complex sphingolipids and promote the accumulation of sphinganine, which is cytotoxic at high concentrations. This provokes an alteration of the cell morphology and its interactions with other cells, the conduct of cell-surface proteins, the activity of protein kinases, the metabolism of other lipids, and cell growth and viability [START_REF] Merrill | Fumonisins: Fungal Toxins That Shed Light on Sphingolipid Function[END_REF]. Fumonisins cause leukoencephalomalacia (softening of the white matter in the brain) in horses and lung edema in swine [START_REF] Richard | Some Major Mycotoxins and Their Mycotoxicoses-An Overview[END_REF]. In rodents, FB1 is hepatocarcinogenic and nephrotoxic [START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]. Fumonisins are classified by IARC as a class 2B possible human carcinogens [START_REF] Iarc | Some Naturally Occurring Substances: Food Items and Constituents[END_REF]. There is evidence linking the presence of Fusarium verticillioides in corn to the high prevalence of human esophageal cancer in South Africa and China (CAST 2003). Recent studies suggest that exposure to fumonisins could be related to stunting in children [START_REF] Cendoya | Fumonisins and Fumonisin-Producing Fusarium Occurrence in Wheat and Wheat by Products: A Review[END_REF]). The addition of β-chloroalanine, which inhibits sphinganine formation (by inhibiting serine palmitoyl transferase), prevented partially the effects of fumonisin on cell growth and cell death [START_REF] Yoo | Elevated Sphingoid Bases and Complex Sphingolipid Depletion as Contributing Factors in Fumonisin-Induced Cytotoxicity[END_REF].

Regulation of fumonisins

The presence of fumonisins is only regulated for maize products despite the fact that they can be found on wheat. The limit for fumonisins (sum of B1 and B2) in unprocessed maize is 2000 µg/kg. The limit decreases to 1000 µg/kg for flour, meal, grits, germ and refined oil.

Foods for direct human consumption must remain under 400 µg/kg. Lastly, the maximum limit for processed maize-based foods for babies, infants and young children is 200 µg/kg (European Commission 2006).

Some pathogenic fungi found in the field are capable of producing mycotoxins.

These fungal toxic metabolites have functions that seem to go from the competition against other microorganisms, through fungal protection, to abiotic stresses, and until plant infection factors. Several parameters affect mycotoxin production such as temperature, humidity and pH. Due to their toxic effects, the presence of main mycotoxins on food and feed is regulated in most countries, especially for cereals. To avoid their development on the seeds, good storage conditions have to be implemented, such as reducing the humidity content and the temperature [START_REF] Bothast | Fungal Deterioration and Related Phenomena in Cereals, Legumes and Oilseeds[END_REF]. Factors affecting fungal growth and mycotoxin production during storage include: water activity, temperature, pH, light and oxygen and carbon dioxide amount, among others [START_REF] Atanda | Fungi and Mycotoxins in Stored Foods[END_REF]. Optimizing storage conditions is fundamental, otherwise, after extensive periods of time, initial fungal inoculum and mycotoxin amount could increment considerably and make the product unfit for consumption. Crop losses of 20% to 50% were recorded in developing countries due to inadequate storage practices [START_REF] Agriopoulou | Advances in Occurrence, Importance, and Mycotoxin Control Strategies: Prevention and Detoxification in Foods[END_REF].

Penicillium verrucosum

Fungi of Penicillium genus can develop over a wide range of temperatures, but they are more abundant in temperate to cold climates. They are mostly associated with storage rather that preharvest contamination (CAST 2003). Penicillium verrucosum (Fig. 19)

is the main ochratoxin producing fungus present on stored grain in Northern Europe, it can also produce citrinin under certain conditions, such as oxidative stress [START_REF] Schmidt-Heydt | Oxidative Stress Induces the Biosynthesis of Citrinin by Penicillium verrucosum at the Expense of Ochratoxin[END_REF]. As a saprophytic fungus, P.

verrucosum develops mostly during food and feed storage. It develops at a wide range of temperatures (0-35°C) and at an optimal ɑw of 0.95, the minimal being 0.80 (Mannaa and Kim 2017b). It also grows over a pH range going from 2.1 to 10, with an optimal between 6 and 7 [START_REF] Pitt | Mycotoxins and Toxigenic Fungi[END_REF]. Schlösser and Prange 2019). OTA is an ubiquitous mycotoxin, as it can be found in several food products including cereals, coffee, nuts, grapes, chocolate, meat and spices, as well as in some beverages such as wines, beers and juices [START_REF] Eskola | Worldwide Contamination of Food-Crops with Mycotoxins: Validity of the Widely Cited 'FAO Estimate' of 25%[END_REF][START_REF] Geisen | Aspects of the Occurrence, Genetics, and Regulation of Biosynthesis of the Three Food Relevant Penicillium Mycotoxins: Ochratoxin A, Citrinin, and Patulin[END_REF][START_REF] Petzinger | Total Syntheses of Xiamycins A, C, F, H and Oridamycin A and Preliminary Evaluation of Their Anti-Fungal Properties[END_REF]. Humidity, pH and temperature are crucial factors affecting OTA production (Mannaa and Kim 2017b). OTA optimal production by P. verrucosum occurs at 25°C

but it can also be produced between 5 and 10°C at its optimal ɑw (0.90-0.95). A minimal ɑw of 0.83-0.85 is necessary for OTA production.

OTA biosynthesis originates from acetate and malonate by the formation of a polyketide dihydroisocoumarin moiety by a polyketide synthase (PKS), that then is linked via amide bond to the amino acid phenylalanine synthesized via the shikimic acid pathway. A nonribosomal peptide synthase (NRPS) is essential for this step, and the resulting molecule is ochratoxin B (OTB). Finally, a halogenation step is required to add the chlorine atom to the molecule via a halogenase (HAL), which yields ochratoxin A (OTA) (Fig. 22). The process is likely to involve other enzymes such as oxidases, esterases, transporters and transcription factors [START_REF] Gallo | Recent Advances on the Molecular Aspects of Ochratoxin A Biosynthesis[END_REF].

Citrinin (Fig. 21) is a yellow mycotoxin produced by some species of

Penicillium and Aspergillus, including ochratoxin producer

Penicillium verrucosum which has the ability to switch between OTA and citrinin under light induced stress conditions [START_REF] Stoll | Comparative Proteome Analysis of Penicillium Verrucosum Grown under Light of Short Wavelength Shows an Induction of Stress-Related Proteins Associated with Modified Mycotoxin Biosynthesis[END_REF].

The occurrence of this mycotoxin has been documented in rice, fruits, cheese and dried fruits [START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]. OTA is suspected to be the causative agent of the Balkan Endemic Nephropathy (BEN) originally described in 1956 as a pathology in Balkan areas where the presence of OTAcontaminated food was frequently reported [START_REF] Duarte | A Review on Ochratoxin A Occurrence and Effects of Processing of Cereal and Cereal Derived Food Products[END_REF]. Because of its analogy to the structure of the amino acid Phenylalanine, OTA is a competitive inhibitor of tRNA phenylalanine synthetases which causes a cease in protein synthesis. Furthermore, OTA provokes the occurrence of DNA adducts and indirect oxidative DNA damage [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF].

OTA was classified as genotoxic and possible human carcinogen, as well as hepatoxic and nephrotoxic (it affects mainly the kidney) and also teratogenic (Pfohl-Leszkowicz and Manderville 2007). Besides, it has recently been classified as immunosuppressant [START_REF] Petzinger | Total Syntheses of Xiamycins A, C, F, H and Oridamycin A and Preliminary Evaluation of Their Anti-Fungal Properties[END_REF]. In the last two decades, European countries most affected by OTA contamination have been Germany, Italy, Switzerland, Sweden, the UK and Czech Republic.

OTA presence can be detected in blood, urine and milk samples. Its rapid transportation through the body and bio-accumulation is due to its ability to bind to proteins, such as albumin Ochratoxin has three main forms A, B and C. OTB being a non-chlorinated form of OTA and OTC being an ethyl ester of OTA. The toxicity of OTA is given by its isocoumarin moiety.

OTB has been proved to be less toxic thanks to the rapidity of its elimination, since it has less affinity for plasma proteins. However, OTC remains almost as toxic as OTA, as it becomes available in the bloodstream [START_REF] Abrunhosa | Biodegradation of Ochratoxin a for Food and Feed Decontamination[END_REF]. Another known metabolite is OTα, which results from the hydrolysis of the amide bond, which also releases phenylalanine. OTα has been found to be about 100 times less toxic than OTA, with a 10-times shorter elimination half-life, and even if the isocoumarin is present, it seems to need the presence of phenylalanine to have a toxic effect [START_REF] Heussner | Comparative Ochratoxin Toxicity: A Review of the Available Data[END_REF].

As OTA, citrinin causes kidney damage in laboratory animals and its interaction with OTA allegedly causes a synergic effect (CAST 2003). The cellular mechanism of toxicity caused by citrinin is associated to a modification in mitochondrial functions by an alteration of enzymes and the redox chain, which induces oxidative stress [START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]).

Regulation of ochratoxin A and citrinin

The maximal residual level for OTA in cereals has been settled at 5 µg/kg by the European Legislation (European Commission 2006). However, many countries lack of ochratoxin limits regulation, including the USA, Australia, New Zealand, Japan, Mexico and South Africa [START_REF] Heussner | Comparative Ochratoxin Toxicity: A Review of the Available Data[END_REF].

EU limits for OTA were established at 0.5 µg/kg for processed cereal-based foods and baby foods; 2 µg/kg for wine, grape juice, grape nectar and grape must intended for direct human consumption; 3 µg/kg for products derived from unprocessed cereals; 5 µg/kg for unprocessed cereal, roasted coffee beans and ground roasted coffee beans and ground roasted coffee; 10 µg/kg for dried vine fruit and soluble coffee; 15 µg/kg for certain spices; 20 µg/kg for liquorice root for herbal infusion and 80 µg/kg for liquorice extract. Regarding animal feed, OTA maximal limits are 50 µg/kg for feed materials, 50 µg/kg for complementary and complete feeding stuffs for pigs and 100 µg/kg for poultry. The USA Food Drug Administration (FDA) does not settle limits for this toxin (European Commission 2006;[START_REF] Smith | Natural Co-Occurrence of Mycotoxins in Foods and Feeds and Their in Vitro Combined Toxicological Effects[END_REF].

Until recently, there was no legislation regarding citrinin presence in foodstuffs. The parasiticus can produce also AFG1 and AFG2 [START_REF] Verheecke | Microbial Degradation of Aflatoxin B1: Current Status and Future Advances[END_REF]. The M aflatoxins were isolated for the first time from the milk of lactating animals fed aflatoxin preparations; hence, the M designation (CAST 2003). Aflatoxins contaminate various agriculture and food products. The crops usually affected by aflatoxins are wheat, corn, groundnuts and tree nuts, as well as on soya, rice, coffee and cacao [START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF].

They can also be found on spices such as paprika, cumin and pepper [START_REF] El Mahgubi | Distribution and Toxigenicity of Aspergillus Section flavi in Spices Marketed in Morocco[END_REF].

The optimal temperature for aflatoxin production by Aspergillus flavus is 28°C, minimal and optimal ɑw are 0.84 and 0.99, respectively (Mannaa and Kim 2017b).

The aflatoxin biosynthesis pathway of Aspergillus parasiticus has been elucidated [START_REF] Yu | Completed Sequence of Aflatoxin Pathway Gene Cluster in Aspergillus parasiticus[END_REF] and it is highly homologous with that of A. flavus, as 25 genes within the cluster are the same [START_REF] Al-Saad | Impact of Bacterial Biocontrol Agents on Aflatoxin Biosynthetic Genes, AflD and AflR Expression, and Phenotypic Aflatoxin B1 Production by Aspergillus flavus under Different Environmental and Nutritional Regimes[END_REF]. Aflatoxins biosynthesis is coded by an 80 kb long DNA sequence containing 30 putative genes. Figure 25 by Verheecke et al. 2015 presents a synthesis of the aflatoxin biosynthesis pathway focusing on structural genes denominated aflD (early), aflM (medium) and aflP (late). The gene aflD encodes a reductase enzyme implicated in the transformation of norsolorinic acid to averantin; aflM is needed for the conversion of versicolorin A to demethylsterigmatocystin; and aflP encodes a methyltransferase converting sterigmatocystin to O-methylsterigmatocystin. Two cluster specific regulators are also known: aflR, which encodes a transcription activator that binds a consensus sequence in the promoter regions of aflatoxin structural genes, and AflS is a potential co-activator of AflR. In addition to AflS and AflR, the clustered genes are also regulated by specific transcriptional regulators such as LaeA or Ap-1 [START_REF] Verheecke | Reduction of Aflatoxin Production by Aspergillus flavus and Aspergillus parasiticus in Interaction with Streptomyces[END_REF].

Toxicity of aflatoxins

Aflatoxins are the most significant and dangerous mycotoxins due to their genotoxic, mutagenic and carcinogenic effects (Mannaa and Kim 2017b). Aflatoxin B1 is classified as a class I carcinogen by the IARC (IARC 1993). In addition, aflatoxins have oncogenic and immunosuppressive properties (Edite Bezerra da [START_REF] Bezerra Da Rocha | Mycotoxins and Their Effects on Human and Animal Health[END_REF]. The furofuran ring is mainly responsible for the toxic and carcinogenic activity upon activation of the C8-C9 double bond to 8-9 epoxide by the cytochrome P450 in animals [START_REF] Guengerich | Activation and Detoxication of Aflatoxin B1[END_REF]). However, the lactone ring seems to also contribute to the toxicity [START_REF] Lee | Role of Lactone Ring of Aflatoxin B1 in Toxicity and Mutagenicity[END_REF]. Initially, AFB1 is converted by P450 cytochromes to the highly reactive aflatoxin B1-8,9-epoxide [START_REF] Pitt | Mycotoxins and Toxigenic Fungi[END_REF]. The epoxidation of AFB1 allows its binding to N7-guanine and, followed by Guanine to Tyrosine transversions, to the DNA molecule. This mechanism is behind the carcinogenic effect. Also, epoxidized aflatoxins form Schiff bases with cellular and microsomal proteins, leading to acute toxicity [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]). Acute intoxication with aflatoxins provoke hemorrhagic necrosis and fatty acid infiltration in liver cells, and in some cases death [START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]. AFB1 is easily transformed in aflatoxin M1 (AFM1) in mammals, which then is excreted through the milk. Moreover, AFM1 was found to be highly stable to pasteurization temperature. AFM1 is less hepatotoxic and immunotoxic than AFB1, however, its classified as a possible human carcinogen (group 2B) by the IARC (Gil-Serna et al. 2014).

Figure 25. Aflatoxin biosynthesis pathway with a focus on structural enzymes: early (AflD), medium (AflM) and late (AflP) and two cluster-specific regulators (AflR and AflS) in Aspergillus parasiticus (Verheecke et al. 2015).

xin biosynthesis pathway ay with a focus on structural enzymes: early (Af Af

Regulation of aflatoxins

Established limits for the sum of aflatoxins in the EU are 4 µg/kg for groundnuts (2 µg/kg for AFB1 alone), dried fruits and cereals intended for human consumption; 10 µg/kg (5 µg/kg for AFB1 alone) for tree nuts, dried fruits, maize and rice subjected to sorting or any other physical process before human consumption, as well as spices, almonds, dried figs, apricot kernels, hazelnuts, and brazil nuts intended for human consumption; 15 µg/kg (8 µg/kg for AFB1 alone) for peanuts, almonds, pistachios, apricot kernels, hazelnuts and Brazil nuts subjected to sorting or other physical treatment before human consumption (European Commission 2006). The FDA limit for the sum of aflatoxins is 20 µg/kg for the foodstuff described before [START_REF] Smith | Natural Co-Occurrence of Mycotoxins in Foods and Feeds and Their in Vitro Combined Toxicological Effects[END_REF].

Regarding animal feed, EU established a limit for AFB1 of 20 µg/kg for feed materials and 10 µg/kg for complementary and complete feed, the limit for compound feed for young animals was settled at 5µg/kg. The FDA in comparison, established an aflatoxins limit of 20 µg/kg for corn, peanut products and other feeds for immature and dairy animals; 100 µg/kg for corn and peanut products for breeding cattle, breeding swine and immature poultry; 200 µg/kg for fishing swine and 300 µg/kg for finishing beef cattle, as well as cottonseed meal for beef, cattle, swine or poultry (European Commission 2006;[START_REF] Smith | Natural Co-Occurrence of Mycotoxins in Foods and Feeds and Their in Vitro Combined Toxicological Effects[END_REF].

For milk and milk-based products, only AFM1 is considered, as AFB1 gets metabolized to AFM1 by dairy cattle and then excreted in the milk. The EU limit was established at 0.05 µg/kg, while the FDA established the limit at 0.5 µg/kg [START_REF] Smith | Natural Co-Occurrence of Mycotoxins in Foods and Feeds and Their in Vitro Combined Toxicological Effects[END_REF].

Fungal species from the Penicillium and Aspergillus genus are capable of developing under storage conditions and to provoke the accumulation of harmful mycotoxins in grains. Aflatoxins are considered the most hazardous mycotoxins found in stored cereals, followed by ochratoxin A. Their occurrence increases the health risk of cereals. Thus, maximal limits for their levels in foodstuff have been established.

Control of molds

Wheat disease management nowadays is primarily based on the use of chemical fungicides and resistant wheat cultivars [START_REF] Syed Ab Rahman | Emerging Microbial Biocontrol Strategies for Plant Pathogens[END_REF]. In spite of the protection measures currently implemented in farmer's fields, yield losses from wheat diseases in North Western Europe are estimated at approximatively 25% [START_REF] Willocquet | An Outlook on Wheat Health in Europe from a Network of Field Experiments[END_REF]. The implementation of good agricultural practices such as crop rotation and tillage help to reinforce the fungal control approaches mentioned before. Moreover, the use of biological control agents is an important strategy being developed to mitigate mold occurrence [START_REF] Syed Ab Rahman | Emerging Microbial Biocontrol Strategies for Plant Pathogens[END_REF]. Several factors affect fungal growth during farming, harvesting and storage, such as the presence of spores, plant damage, the presence of insects, drought stress, nutrients availability, pH, oxygen and CO2 levels. Knowledge and management of all the factors influencing fungal development allow to prevent pathogenic proliferation and mycotoxin occurrence [START_REF] Amézqueta | Ochratoxin A Decontamination: A Review[END_REF]).

Pre and post-harvest agricultural practices/management

The first line of defense against mold contamination starts by optimizing crop management, in order to reduce the initial appearance of pathogenic fungi. However, despite the actions taken at pre-harvest stages, fungal growth can occur during the storage of grains.

If contamination persists, it should be addressed during post-harvest stages, in order to prevent further pathogenic fungi development and its consequences, that go from grain deterioration to mycotoxin accumulation.

Pre-harvest prevention of fungal contamination

Damaged plants tend to be an easy target for fungal contamination, which is why crops should be protected against mechanical and insect damage since pre-harvest stages (Mannaa and Kim 2017a). Timely harvest, crop rotation, tillage, appropriate irrigation, weed control and fertilization are some of the actions that could help prevent the apparition of molds since the earliest stages. If possible, products must be collected when they are mature [START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF].

Products overripen and damaged must be discarded, as they could represent a source of contamination during storage. Additionally, inoculum sources, dirty farming materials and agricultural residues must be eliminated [START_REF] Amézqueta | Ochratoxin A Decontamination: A Review[END_REF]).

Post-harvest management of fungal development

Management of storage conditions constitute the principal post-harvest prevention method. When possible, after product collection, it should be fastly and homogeneously dried prior to storage, to prevent fungal development. Ideally, products should be turned over to facilitate their aeration, particularly under tropical conditions [START_REF] Amézqueta | Ochratoxin A Decontamination: A Review[END_REF]). During storage, reducing insect infestation is also essential, as besides causing grain damage, they produce CO2 and water, that increment seed respiration, humidity and temperature, which altogether favors fungal development. In addition, insect motion contributes to the dissemination of fungal spores [START_REF] Birck | Fungus and Mycotoxins in Wheat Grain at Post Harvest[END_REF]. Modification of the storage atmosphere by enriching the amount of CO2 and reducing that of O2 is a method of food preservation largely studied. It preserves the quality of foodstuffs and increases their storage life by reducing the respiration rate, which subsequently inhibits fungal activity. When the gas conditions are regulated during the whole storage period it creates a controlled atmosphere, which requires more technical factors (Mannaa and Kim 2017b). It has been demonstrated that these practices reduce the growth rate of most storage-related fungi, as well as their mycotoxin production (Cairns-Fuller, Aldred, and [START_REF] Cairns-Fuller | Water, Temperature and Gas Composition Interactions Affect Growth and Ochratoxin A Production by Isolates of Penicillium verrucosum on Wheat Grain[END_REF][START_REF] Giorni | Effect of Aw and CO2 Level on Aspergillus Flavus Growth and Aflatoxin Production in High Moisture Maize Post-Harvest[END_REF][START_REF] Samapundo | The Influence of Modified Atmospheres and Their Interaction with Water Activity on the Radial Growth and Fumonisin B1 Production of Fusarium verticillioides and F. proliferatum on Corn. Part I: The Effect of Initial Headspace Carbon Dioxide Concentration[END_REF]). These preservation effects are increased in combination with reduced ɑw and low storage temperatures [START_REF] Ellis | Control of Growth and Aflatoxin Production of Aspergillus flavus under Modified Atmosphere Packaging[END_REF]).

Chemical treatments

On the field

The implementation of good agricultural practices contributes to the reduction of fungal contamination; however, they do not completely eliminate this risk. For this reason, chemical fungicides constitute one of the main components in the Integrated Pest Management (IPM), due to their efficacity for pests and disease control. Main fungicides against plant pathogens are composed of azoles, strobilurins, also known as quinone outside inhibitors (QoIs), succinate dehydrogenase inhibitors (SDHIs) or demethylation inhibitors (DMIs), or a mixture of several of them. In addition to a direct antifungal effect, QoIs inhibit mitochondrial respiration, which increases flag leaf lifespan and consequently impedes fungal sporulation, as fungus requires senescent tissue to develop. However, resistance to this kind of fungicides by some wheat pathogens has become frequent [START_REF] Solomon | Stagonospora nodorum: Cause of Stagonospora Nodorum Blotch of Wheat[END_REF].

Wheat rusts are controlled by triazoles (e.g. Tilt, Folicur, Proline, Popimax) and QoIs (e.g. Quadris, Headline). Some commercialized products against rust include both of them (e.g. Stratego, Quilt) [START_REF] Marsalis | Leaf, Stem and Stripe Rust Diseases of Wheat[END_REF]. STB is controlled by mixtures of azole (e.g. triticonazole) and SDHIs (e.g. chlorothalonil) [START_REF] Torriani | Zymoseptoria tritici: A Major Threat to Wheat Production, Integrated Approaches to Control[END_REF], as well as by DMIs [START_REF] Tiley | Zymoseptoria tritici[END_REF]. QoIs are now ineffective against STB due to a widespread resistance [START_REF] Ponomarenko | Septoria Tritici Blotch (STB) of Wheat Septoria Tritici Blotch (STB) of Wheat[END_REF][START_REF] Tiley | Zymoseptoria tritici[END_REF]. Foliar fungicide sprays are effective against SNB, the most generally recommended are DMIs (e.g. metaconazole and prothioconazole), QoIs (e.g. pyraclostrobin, azoxystrobin, and picoxystrobin) and combinations of both (e.g. trifloxystrobin + prothioconazole) [START_REF] Mehra | Septoria Nodorum Blotch of Wheat[END_REF]. Some protectant fungicides such as mancozeb and copper can prevent infection by SNB if they are applied at the right time [START_REF] Li | Malayamycin, a New Streptomycete Antifungal Compound, Specifically Inhibits Sporulation of Stagonospora nodorum (Berk) Castell and Germano, the Cause of Wheat Glume Blotch Disease[END_REF]. WB can, under some conditions, be controlled at the heading stage by fungicides combining triazols with QoIs (e.g. carbandazim and tebuconazole) [START_REF] Debnath | Morphological, Pathological and Cultural Characteristics of Magnaporthe oryzae Triticum Causing Blast of Wheat and Its Fungicidal Control[END_REF]. Another approach for the management of WB is the treatment of seeds by active ingredients such as iprodione, fluazinam, difeconazol, carboxin and thiram [START_REF] Maciel | Diseases Affecting Wheat: Wheat Blast[END_REF]. Several other substances have also been tested against WB, namely, jasmonic acid, deacetylated chitosan, potassium silicate and potassium phosphate, the last being effective to decrease mycelium growth [START_REF] Kumar | Wheat Blast[END_REF].

Main fungicidal treatments applied against FHB consist on DMIs (e.g. metconazole,

propiconazole, prothioconazole and tebuconazole) [START_REF] Ferrigo | Fusarium Toxins in Cereals: Occurrence, Legislation, Factors Promoting the Appearance and Their Management[END_REF]. A novel fungicide, phenamacril shows not only a suppression of FHB but also a decrease on DON production [START_REF] Chen | Fusarium graminearum Trichothecene Mycotoxins: Biosynthesis, Regulation, and Management[END_REF]. As Fusarium ear rot (FER) in wheat caused by F. verticillioides has only been described in the recent years [START_REF] Gagkaeva | Emergence of Fusarium verticillioides in Finland[END_REF], there is a lack of studies indicating the effectivity on chemical fungicides to control this disease on wheat. Triazole groups of fungicides used for FHB management were evaluated for their efficacy against F.

proliferatum and they were found to reduce fungal growth. However, some fungicide sublethal doses stimulated fumonisin production [START_REF] Cendoya | Effect of Fungicides Commonly Used for Fusarium Head Blight Management on Growth and Fumonisin Production by Fusarium proliferatum[END_REF].

During storage

Various organic acids have been applied for the control of fungi during grain storage, namely, propionic acid, formic acid and butyric acid. Potassium sorbate and potassium benzoate also showed an inhibitory effect on germination and growth of A. flavus and A.

parasiticus (Mannaa and Kim 2017a). Synthetic food grade antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone (THB), and propyl paraben (PP) are allowed for use as antimicrobial agents during storage of foodstuffs [START_REF] Nesci | Prevention of Aflatoxin Contamination in Stored Grains Using Chemical Strategies[END_REF]. Ozone is a powerful fungicide and insecticide, which makes it an alternative technology for the disinfection of wheat. One of the main advantages of ozonation is the absence of residues, as ozone rapidly decomposes to diatomic oxygen. Mycotoxin decontamination by ozone has also been documented [START_REF] Wu | Investigation of Gaseous Ozone as an Anti-Fungal Fumigant for Stored Wheat[END_REF]. The fumigant methyl bromide, used for many years to control fungal pathogens during storage of grains, was discontinued in 2015 as a part of the "Montreal protocol", due to its ozone depletion. This lead to its replacement by gaseous phosphine (PH3) [START_REF] Hocking | Effects of Phosphine Fumigation on Survival and Growth of Storage Fungi in Wheat[END_REF], which has been found useful to control the growth of A. flavus on maize [START_REF] Birck | Fungus and Mycotoxins in Wheat Grain at Post Harvest[END_REF].

Other alternatives such as chlorine dioxide (ClO2) have been recently researched [START_REF] Han | Effect of Gaseous Chlorine Dioxide Treatment on the Quality of Rice and Wheat Grain[END_REF].

Despite the fact that chemical fungicides have helped reach indispensable crop yields, their systematic use has provoked resistance phenomena among the pathogens, which then leads to higher dosages and higher costs every year [START_REF] Syed Ab Rahman | Emerging Microbial Biocontrol Strategies for Plant Pathogens[END_REF].

Furthermore, the use of chemical pesticides causes several undesirable side effects, such as acute and chronic toxicity in humans, food contamination, environmental pollution and the destruction of beneficial organisms, like pollinator species. To these effects is added the reduction of the diversity of beneficial microbial species in the soil, that can repress the competition against pathogens and facilitate their development [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]. Besides, when used to manage mycotoxinogenic fungi, some chemical fungicides were found to stimulate mycotoxin production, even when fungal development was inhibited [START_REF] Medina | Efficacy of Natamycin for Control of Growth and Ochratoxin A Production by Aspergillus carbonarius Strains under Different Environmental Conditions[END_REF]). Moreover, evidences indicate that pathogenic fungal exposure to sub-lethal fungicide concentrations can motivate mycotoxin biosynthesis, for example by triggering oxidative stress [START_REF] Ferrigo | Fusarium Toxins in Cereals: Occurrence, Legislation, Factors Promoting the Appearance and Their Management[END_REF]. The previous reasons have motivated the search for more environmentally friendly pathogen management strategies, among which biological control has demonstrated to be a promising alternative.

Biological control

Biological control or biocontrol is defined as the association of factors aiming to decrease the undesirable effects of detrimental organisms and promote the growth of beneficial organisms. It refers both to the macro and microorganisms themselves and to the natural substances obtained from them [START_REF] Syed Ab Rahman | Emerging Microbial Biocontrol Strategies for Plant Pathogens[END_REF]). The organisms used in biocontrol are called biocontrol agents (BCAs) while their derived genes, enzymes and metabolites, are defined as "biopesticides" or "biofertilizers", depending on their application [START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. The use of fungi and bacteria against plant pathogens offers a more ecological alternative to chemical pesticides, as they are naturally associated with plants, which implies a minor impact on native microbial equilibrium [START_REF] Knudsen | Ecological Complexity and the Success of Fungal Biological Control Agents[END_REF]. Besides, they are able to inhibit fungal development and, in some cases, improve plant defense and resistance against fungal diseases [START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. Good BCAs often exhibit more than one antagonistic mechanism [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF].

Most biocontrol research has focused on a limited number of bacterial (Bacillus, Burkholderia, Lysobacter, Panotea, Pseudomonas, Rhizobium and Streptomyces) and fungal (Ampelomyces, Coniothyrium, Dactylella, Gliocadium, Paecilomyces, and Trichoderma) genera, due to the simplicity of their culture [START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. The genera Pseudomonas spp., Bacillus, Rhizobium and Trichoderma in particular have been mostly used for the control of soil-borne plant pathogenic fungi [START_REF] Chet | Biological Control of Fungal Pathogens[END_REF]. Pseudomonas have been largely studied for their production of antibiotics. Bacillus are especially attractive for their practical use, as they produce stable endospores which can survive the heat and desiccation processes that can take place during the formulation of BCAs [START_REF] Legein | Modes of Action of Microbial Biocontrol in the Phyllosphere[END_REF].

Trichoderma are able to parasite plant pathogens in addition to their production of antimicrobial compounds (Handelsman' and Stabb 1996). Actinobacteria, especially

Streptomyces genus, represent promising biocontrol agents as they combine several of the advantages previously described for the most studied BCAs. Detailed information will be presented in the last chapter of this bibliographical synthesis.

Several strategies are implemented during pre and post-harvest stages to avoid the presence of pathogenic fungi. Pre-harvest preventive approaches include crop rotation, tillage and proper irrigation. Post-harvest measures are based on the optimization of storage conditions. Chemical fungicides are intensively employed in the field to control the fungal contamination, which has led to a pathogenic resistance phenomenon. Biocontrol with active microorganisms exhibiting a variety of antagonistic mechanisms and plant beneficial traits, presents a more ecological alternative to decrease fungal development.

Decontamination of mycotoxins

Several approaches are applied to monitor and avoid mycotoxins presence in the production chain, comprising filed, storage, transportation and processing. Unfortunately, mycotoxin occurrence cannot always be prevented. For this reason, detoxification methods are sometimes applied to food and feed in order to reduce mycotoxin risk to the lowest possible amount, and possibly salvage contaminated batches, as long as there is no alteration in the expected quality [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF]. Some physical methods such as separation, milling and dehulling allow mycotoxin decrease during industrial processes. Moreover, the use of adsorption agents has become popular since the EU authorized their use as feed additives [START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF]. The application of chemical treatments to food, exclusively with mycotoxin decontamination purposes is not authorized. However, some food preparation processes include the addition of chemical products that incidentally help to decrease mycotoxin risk. Lastly, biological detoxification has been achieved by several microorganisms either naturally present on food matrices or added for technological purposes (Gil-Serna et al. 2014).

Modified mycotoxins

In vivo decontamination of mycotoxins occurs when plants, animals and fungi use detoxifying mechanisms to protect themselves from mycotoxins as a part of their defense against xenobiotics. These mechanisms can lead to chemical alterations in the structure of mycotoxins, resulting in modified chromatographic profiles, epitope conformation or polarity [START_REF] Smith | Natural Co-Occurrence of Mycotoxins in Foods and Feeds and Their in Vitro Combined Toxicological Effects[END_REF]), which hinders their correct chromatographic identification. These modified forms may be produced via conjugation with sugars or other biological components, escaping in this manner conventional analytical methods, thus called "modified mycotoxins".

They may also evolve into non-extractable forms on account of a strong interaction with the matrix, becoming "hidden mycotoxins". In both cases, they represent a latent risk as even when their conjugated form is partially less toxic, like for the FB1-glucose reaction products [START_REF] Fernández-Surumay | Glucose Reaction with Fumonisin B1 Partially Its Toxicity in Swine[END_REF], they can turn bioavailable again during digestion [START_REF] Berthiller | Masked Mycotoxins: A Review[END_REF]. Some of the metabolism derivatives known include zearalenone-14-glucoside (ZEA14G), zearalenone-14-sulphate (ZEA14S), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), deoxynivalenol-3-glucoside (DON3G), 15-acetyldeoxynivalenol (15-ADON) and 3acetyldeoxynivalenol (3-ADON) [START_REF] Cirlini | Hyphenated Chromatographic Techniques for Structural Characterization and Determination of Masked Mycotoxins[END_REF].

As stated before, decrease on the mycotoxin amount not necessarily means detoxification, as mycotoxins could be present in masked or modified forms that are no longer detectable but could still be toxic or reconvert to its toxic form during digestion. Besides, industrial processes could also release modified mycotoxins and make them bioavailable [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]. In some other cases, mycotoxin degradation by-products remain toxic.

For example, yeasts biotransform ZEN to α-ZEL which is more estrogenic [START_REF] Böswald | Metabolism of the Fusarium Mycotoxins Zearalenone and Deoxynivalenol by Yeast Strains of Technological Relevance[END_REF].

This fact highlights the importance of the study of degradation mechanisms and the characterization of degradation by-products. Designation of mycotoxin transformations as detoxification must be first corroborated [START_REF] Berthiller | Masked Mycotoxins: A Review[END_REF]. Therefore, there are two ways of validating an effective detoxification of matrices and foodstuffs: either less toxic degradation by products are detected and identified or the reduction of toxicity is assessed by one or more toxicity bioassays [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF].

For the evaluation of mycotoxin decrease and the search of degradation by products, chromatographic techniques are most commonly applied, such as HPLC (High Performance Liquid Chromatography), LC-MS (Liquid Chromatography-Mass Spectrometry), GC-MS (Gas Chromatography-Mass Spectrometry), TLC (Thin Layer Chromatography), and NMR (Nuclear Magnetic Resonance Spectroscopy). As well as specific mycotoxin detection by Enzyme-Linked

Immunosorbent assay (ELISA) [START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF]).

Evaluation of residual toxicity

If a decontamination protocol is applied to food matrices, it is essential to control their residual toxicity. Various methodologies allow the identification of toxicity in vitro and in vivo.

The in vitro procedures determine cell viability, apoptosis/necrosis, DNA damage and oxidative damage using cell models corresponding to major organs targeted by mycotoxins [START_REF] Smith | Natural Co-Occurrence of Mycotoxins in Foods and Feeds and Their in Vitro Combined Toxicological Effects[END_REF]. Toxic effects of trichothecenes, aflatoxins and zearalenone are evaluated using various cell lines which include human intestinal cells (Caco-2), human leukaemia monocytes (THP-1), human hepatic cells (HepaRG), intestinal porcine epithelial cells (IPEC-1), chinese hamster ovary cells (CHO-K1) and monkey kidney epithelial cells (Vero) [START_REF] Smith | In Vitro Co-Culture Models to Evaluate Acute Cytotoxicity of Individual and Combined Mycotoxin Exposures on Caco-2, THP-1 and HepaRG Human Cell Lines[END_REF][START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF]. One of the most extensively used in vitro tests for measuring mutagenicity is the Ames test, which measures reverse mutations from histidine auxotrophy to prototrophy in several especially constructed mutants [START_REF] Quillardet | The SOS Chromotest: A Review[END_REF]. A complementary in vitro tool for the determination of genotoxicity of compounds, such as aflatoxins, is the SOS-chromotest, a colorimetric assay that measures the sfiA expression during the SOS repairing system in a mutant strain of E. coli, by assaying the β-galactosidase activity. The fusion of two operons, places lacZ (structural gene of β-galactosidase) under the control of the sfiA gene which ensures that the β-galactosidase activity is strictly dependent on sfiA expression. In addition, the strain was made genetically more susceptible to genotoxic agents and has a mutation that renders de cell envelope more permeable [START_REF] Krifaton | Analysis of Aflatoxin-B1-Degrading Microbes by Use of a Combined Toxicity-Profiling Method[END_REF][START_REF] Quillardet | The SOS Chromotest: A Review[END_REF]. The toxicity of other mycotoxins such as zearalenone and ochratoxin A can be assessed by using the bioluminescent bacterium Aliivibrio fisheri as its light emission decreases in the presence of toxic compounds [START_REF] Krifaton | Adaptation of Bacterial Biotests for Monitoring Mycotoxins[END_REF]. The nematode Caenorhabditis elegans is an in vivo approach used to evaluate the effect of diverse mycotoxins (e.g. AFB1, DON, FB1, ZEN) on growth, reproduction, lethality and lifespan [START_REF] Yang | Multi-Toxic Endpoints of the Foodborne Mycotoxins in Nematode Caenorhabditis elegans[END_REF]. Regarding the teratogenicity of OTA in vivo, the zebrafish embryo toxicity assay (ZETA) using the zebrafish Danio rerio can be employed to assess the lack of toxicity of the degradation by-products [START_REF] Haq | Teratogenicity of Ochratoxin a and the Degradation Product, Ochratoxin α, in the Zebrafish (Danio Rerio) Embryo Model of Vertebrate Development[END_REF]).

Physical methods for mycotoxin decontamination

2016).

Heat treatment remains one of the most useful processes to reduce mycotoxin amounts on the final product. However, the several mycotoxins are chemically and thermally stable. Heating process include cooking, frying, baking, roasting, extrusion and canning [START_REF] Bullerman | Stability of Mycotoxins during Food Processing[END_REF]. The addition of an alkaline treatment usually increases the efficacy of extrusion and heating. Interestingly, bread baking does not eliminate OTA, while biscuits baking decreased it by about two thirds. Industrial processes such as malting, extrusion and roasting can also contribute to reduce OTA amounts [START_REF] Abrunhosa | Biodegradation of Ochratoxin a for Food and Feed Decontamination[END_REF]. As fumonisin B1 is relatively heat stable, boiling does not eliminate it, and processes involving higher temperatures are needed, such as canning, extrusion or roasting. The last two treatments have been reported to reduce fumonisins by 34-95% in maize grits. DON is eliminated from bread, biscuits and cookies at variable percentages (24-71%) by baking [START_REF] Bullerman | Stability of Mycotoxins during Food Processing[END_REF].

Non-ionizing radiation such as solar, UV light and microwaves, as well as ionizing gamma radiation, can reduce fungal contamination in food and also partially decrease mycotoxin amount [START_REF] Agriopoulou | Advances in Occurrence, Importance, and Mycotoxin Control Strategies: Prevention and Detoxification in Foods[END_REF]. Furthermore, aflatoxins in cereals can be decreased up to 75% by photodegradation after 30 hours of direct sunlight. OTA and fumonisins can also be reduced by gamma radiation. Microwaves can partially degrade DON (around 40%) [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]. Cold plasma has strong antimicrobial effects. Hence, it allows the disinfection of fragile or temperature-sensitive surfaces. However, there is a lack of studies about the potential formation of toxic compounds in food matrices by this sterilization technique [START_REF] Agriopoulou | Advances in Occurrence, Importance, and Mycotoxin Control Strategies: Prevention and Detoxification in Foods[END_REF]). Nevertheless, its efficacy for destroying about half of the amount of aflatoxins, fumonisins, OTA, DON and ZEN has been documented [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].

Another physical method for removing mycotoxins from foodstuffs is the use of binding matrices that impede the passage of toxic molecules into the bloodstream [START_REF] Agriopoulou | Advances in Occurrence, Importance, and Mycotoxin Control Strategies: Prevention and Detoxification in Foods[END_REF]. Some examples include activated charcoal, aluminosilicates and bentonite, which can be added to animal feed as granules, powders and pellets. The use of resins for the elimination of mycotoxins in liquid products was also studied [START_REF] He | Patented Techniques for Detoxification of Mycotoxins in Feeds and Food Matrices[END_REF][START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]. Such techniques are currently being researched for their use as food additives for reduce the exposure to mycotoxins in highly affected regions [START_REF] Afriyie-Gyawu | NovaSil Clay Intervention in Ghanaians at High Risk for Aflatoxicosis. I. Study Design and Clinical Outcomes[END_REF][START_REF] Wang | Short-Term Safety Evaluation of Processed Calcium Montmorillonite Clay (NovaSil) in Humans[END_REF][START_REF] Wang | NovaSil Clay Intervention in Ghanaians at High Risk for Aflatoxicosis: II. Reduction in Biomarkers of Aflatoxin Exposure in Blood and Urine[END_REF].

Chemical methods for mycotoxin decontamination

During food processing, a number of steps may include chemical treatments, such as ammoniation and ozonation (CAST 2003), which transform the nutritional quality, texture and flavor of the matrix. It has been found that some chemicals involved in industrial processes are able to transform mycotoxins. Regardless, chemical treatments with the objective of decontamination or detoxification of mycotoxins are not authorized in the EU [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].

The majority of mycotoxins are resistant to weak acids. However, strong acids such as HCl are able to detoxify aflatoxins, but their application to food is not allowed. Furthermore, food-grade acids, such as acetic acid, citric acid and lactic acid were evaluated during cooking process, revealing that lactic acid was the most effective to detoxify AFB1 by transforming it into AFB2 and AFB2a (hemiacetal form) [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].

The efficacy of bases involved in food processing as a degradation technique variates according to the temperature, pressure, moisture, duration and substrate. For instance, aflatoxins are unstable to alkaline treatments, as their lactone ring is opened under these conditions, however this reaction is reversible. Moreover, the use of ammonia has proven useful for the partial degradation of aflatoxins, resulting in aflatoxin D1 and D2, which are less toxic. Other alkaline agents able to detoxify aflatoxins include NaOH, Ca(OH)2, Na2CO3, Na3PO4, methylamine, ethylene-diamine and ethanolamine [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].

Manufacturing of tortillas by alkaline cooking and steeping of the corn (nixtamalization) and further processing into tortilla chips and corn chips led to a reduction of aflatoxins by 52% in the tortillas, 85% in the tortilla chips and 79% in the corn chips [START_REF] Torres | Revising the Role of PH and Thermal Treatments in Aflatoxin Content Reduction during the Tortilla and Deep Frying Processes[END_REF]. OTA was also decomposed by ammoniation in maize, wheat and barley [START_REF] Chelkowski | Mycotoxins in Cereal Grain. Part IV. Inactivation of Ochratoxin A and Other Mycotoxins during Ammoniation[END_REF]). FB1 was reduced by 79% in wheat by ammoniation, but the procedure was ineffective in maize. Besides, toxicity of the products resulting of FB1 alkaline hydrolysis was variable, indicating the need of supplementary research in order to use this procedure for detoxifying food matrices [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]).

The addition of ozone (O3) as an oxidizing agent has given positive results for the degradation of aflatoxins, the putative mechanism involves the opening of the lactone ring, followed by the rearrangement of the aflatoxin molecule into derivatives such as ketones, aldehydes and organic acids. Indeed, the presence of a terminal double bond in the dihydrofuran ring of AFB1, AFG1 and AFM1 makes them more susceptible to the attack of oxidizing agents than AFB2, AFG2 and AFM2 [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]). Furthermore, OTA, FB1 and ZEN were also degraded after being in contact with 10% of O3 for 15s. However, degradation of FB1 yielded a 3-keto derivative that remained toxic during two bioassays [START_REF] Mckenzie | Oxidative Degradation and Detoxification of Mycotoxins Using a Novel Source of Ozone[END_REF]. Despite its promising results, ozone application for the decontamination of foods is not yet allowed in several countries, including the EU. Ozone application on food as an antimicrobial agent and for decontamination purposes has been approved by the FDA in United States since 1997 [START_REF] Suman | Last Decade Studies on Mycotoxins' Fate during Food Processing: An Overview[END_REF]. Furthermore, hydrogen peroxide (H2O2) has proven effective to detoxify aflatoxins and to decrease ZEN, which is also destroyed by oxidation with ammonium persulfate. Contrastingly, OTA was resistant to oxidation by H2O2 [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]. Aflatoxins B1, G1 and M1, as well as DON can be destroyed by the addition of sodium bisulfite (NaHSO3) in maize and dried fruits [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].

Biological and enzymatical methods of mycotoxin decontamination

Biological mycotoxin decontamination methods have proven to be more specialized and more environmentally friendly than physical and chemical methods, which tend to be time-consuming and have limited efficacity. Additionally, they may cause nutrient loss and require specialized equipment [START_REF] Agriopoulou | Advances in Occurrence, Importance, and Mycotoxin Control Strategies: Prevention and Detoxification in Foods[END_REF]. Prokaryotic and eukaryotic microorganisms from various sources, as soil, plants and seeds are capable of transforming mycotoxins, sometimes by using them as carbon sources [START_REF] He | Patented Techniques for Detoxification of Mycotoxins in Feeds and Food Matrices[END_REF].

Decontamination could be provoked either by an adsorption on the microorganism cell wall surface [START_REF] Haskard | Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria[END_REF][START_REF] Pierides | Ability of Dairy Strains of Lactic Acid Bacteria to Bind Aflatoxin M 1 in a Food Model[END_REF] or an irreversible enzymatic transformation, such as acetylation, glucosylation, ring cleavage, hydrolysis, deamination and decarboxylation [START_REF] Mccormick | Microbial Detoxification of Mycotoxins[END_REF]. Determining which of the two alternatives is behind an observed mycotoxin decrease is of the utmost importance, as bound mycotoxins could be released during the consumption of the final product. However, in many studies, the mechanisms involved during detoxification remain sometimes unexplored.

Certain microorganisms involved on fermentation processes are capable of detoxifying mycotoxins, yet, no strain addition has been authorized with the sole objective of degrading mycotoxins. Despite this, selecting starter cultures able to detoxify mycotoxins, without modifying the technology, could improve the safety of these aliments [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].

Some enzymes naturally present in the food matrices can detoxify them by degrading the mycotoxins during fermentation processes, however, better results are obtained by the addition of purified and/or molecular engineered enzymes. Unfortunately, not many enzymes have been identified, purified and characterized for this activity [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF][START_REF] Lyagin | Enzymes for Detoxification of Various Mycotoxins: Origins and Mechanisms of Catalytic Action[END_REF]. It should be noted that in the EU no enzyme has been authorized so far for the reduction of mycotoxin contamination in food. Nevertheless, few commercial feed additives are available, namely, Mycofix®, FUMzyme®, Biomin® BBSH 797 and Biomin® MTV [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF][START_REF] Lyagin | Enzymes for Detoxification of Various Mycotoxins: Origins and Mechanisms of Catalytic Action[END_REF]. A defining property of enzymes is their specificity, nevertheless, enzymes such as laccases and peroxidases modify a large number of substrates, making them able not only to destroy mycotoxins, but also important food components. Besides, some of them could cause an allergic reaction, so their allergenic potential has to be investigated before approval. So far, no allergic reaction has been reported towards currently used food enzymes. The most important limitation for the application of mycotoxin degrading enzymes in real matrices is the occurrence of matrix effects that reduce their effectiveness as moisture, acidity and texture have a great influence in enzymatic performance [START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF]). Nevertheless, enzymes potential is vast as they can be expressed by several industrial microorganisms (e.g. yeasts, lactic acid bacteria) and their versatility allows for them to be used free or immobilized, which present several advantages [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]).

Mechanisms of mycotoxins detoxification 2.3.2.1 Detoxification of DON and ZEN

The toxicity of trichothecenes comes essentially from the 12,13-epoxide ring.

However, the degree of toxicity is dependent on the presence of substituents (acetyl, acyl) on C-4 and C-15. For the non-acetylated trichothecenes (e.g. DON, NIV), de-epoxidation is the first step towards detoxification, while for the monoacetyl trichothecenes (e.g. (Carere et al. 2018a(Carere et al. , 2018b). An actinobacteria strain capable of degrading DON is Marmoricola sp., which was reported to degrade DON from wheat kernels after applying a bacterial cell suspension [START_REF] Ito | A Novel Actinomycete Derived from Wheat Heads Degrades Deoxynivalenol in the Grain of Wheat and Barley Affected by Fusarium Head Blight[END_REF]. Rumen or intestinal bacteria's ability to transform DON into DOM-1 was also reported. Same mechanism was exhibited by enterobacteria such as Serratia, Clostridium, Citrobacter and Enterococcus. In fact, DOM-1 is 55 times less toxic than DON [START_REF] Mccormick | Microbial Detoxification of Mycotoxins[END_REF]. In human liver, DON can be conjugated to glucuronides producing a non-toxic by-product, glucuronide conjugated-DON [START_REF] Smith | In Vitro Co-Culture Models to Evaluate Acute Cytotoxicity of Individual and Combined Mycotoxin Exposures on Caco-2, THP-1 and HepaRG Human Cell Lines[END_REF].

Enzymes such as amylases, glucanases and proteases are commonly used during beer brewing and in bakery, where DON contamination is usually observed. Thus, the addition of detoxifying enzymes could be compatible with these processes [START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].

Unfortunately, food suitable enzymes able to irreversibly detoxify DON are not available yet, although some promising candidates have been identified [START_REF] He | Bacterial Epimerization as a Route for Deoxynivalenol Detoxification: The Influence of Growth and Environmental Conditions[END_REF][START_REF] Ito | Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol[END_REF][START_REF] Zhang | Enzymatic Degradation of Deoxynivalenol by a Novel Bacterium, Pelagibacterium halotolerans ANSP101[END_REF].

ZEN toxicity derives from its lactone ring and the free C-4 hydroxyl group, which allows the binding to the estrogen receptor [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF] In vivo, ZEN gets reduced to α-and β-zearalenol (α-ZEL and β-ZEL), both by-products are estrogenic and α-ZEL is more estrogenic than ZEN. A subsequent reduction leads to the formation of α-and β-zearalanol (α-ZAL and β-ZAL), which are less estrogenic than ZEN [START_REF] Böswald | Metabolism of the Fusarium Mycotoxins Zearalenone and Deoxynivalenol by Yeast Strains of Technological Relevance[END_REF][START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF].

ZEN degradation by modification of the C-

Detoxification of fumonisins

Toxicity of fumonisins is given by two structural groups on its structure, the free primary amino group in C-2 and the two tricarballylic acids [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF].

Fumonisins' detoxification mechanism consists in a deesterification by a carboxylesterase, which releases the two tricarballylic acid moieties, resulting in hydrolysed FB1 (HFB1), also The N-acetylation of the amino group can also decrease FB1 toxicity [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF].

HFB1 is also produced by the addition of lime during nixtamalization [START_REF] Palencia | Total Fumonisins Are Reduced in Tortillas Using the Traditional Nixtamalization Method of Mayan Communities[END_REF].

However, biochemical studies showed that hydrolyzed fumonisins could be transformed to highly toxic derivatives in vivo [START_REF] Humpf | Acylation of Naturally Occurring and Synthetic 1-Deoxysphinganines by Ceramide Synthase: Formation of N-Palmitoyl-Aminopentol Produces a Toxic Metabolite of Hydrolyzed Fumonisin, AP1, and a New Category of Ceramide Synthase Inhibitor[END_REF]. Fumonisin detoxification has been observed by microorganisms such as Sphingomonas sp., Sphingopyxis sp., Exophiala sp, Bacillus and yeasts [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF]. Enzymes involved in the degradation by these microorganisms consist mainly on carboxylesterases, aminotransferases and, when the amino group is targeted, deaminases [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]). The carboxylesterase produced by Sphingopyxis sp. is commercialized as a feed additive (FUMzyme®) by Biomin (McCormick 2013).

Detoxification of ochratoxin A and citrinin

The toxicity of OTA is mainly attributed to its isocoumarin moiety, due to its Cl group, than to the phenylalanine moiety [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF]. Two pathways could be involved during microbial degradation of OTA. The first consists in the hydrolysis of the amide bond between the phenylalanine and the isocoumarin residue, which results in the formation of Phenylalanine and OTα [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]. The second, more hypothetical, involves the hydrolysis of the lactone ring, which results in an opened lactone form of OTA (OP-OTA) [START_REF] Abrunhosa | Biodegradation of Ochratoxin a for Food and Feed Decontamination[END_REF]. Soil bacteria Phenylobacterium immobile putative degradation mechanism consisted in (i) a dioxygenase attack on the phenylalanine moiety followed by (ii) a dehydrogenation to a catechol derivative that suffers a meta-ring cleavage and finally (iii) a hydroxylation step that releases OTα (Wegst and Lingens 1983).

Several protozoal, fungal, yeast and bacterial species were found able to degrade, transform or bind OTA [START_REF] Chen | Advances in Biodetoxification of Ochratoxin A-A Review of the Past Five Decades[END_REF]. Rumen protozoa of ruminants, are able to degrade OTA by hydrolysis of the amide bond, which makes them resistant to OTA toxic effects. The main mechanism involved in the decontamination by lactic acid bacteria and yeasts is the absorption of OTA to the cell walls, however some catabolism may be involved, as some authors reported reduced toxicity. OTA decrease by yeasts such as Saccharomyces cerevisiae has been documented during brewing, but the phenomenon was reversible, indicating a binding mechanism [START_REF] Petruzzi | In Vitro Removal of Ochratoxin A by Two Strains of Saccharomyces cerevisiae and Their Performances under Fermentative and Stressing Conditions[END_REF] [START_REF] Abrunhosa | Biodegradation of Ochratoxin a for Food and Feed Decontamination[END_REF].

The presence of an amide bond in the OTA molecule allows the hydrolytic action of enzymes such as carboxypeptidases, chymotrypsins, amidases, proteases and lipases [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]. Two classes of carboxypeptidases can be involved in OTA degradation, namely

Carboxypeptidase A (CPA) and Y (CPY) [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF]. Many proteases, such as Protease A from Aspergillus niger, were able to hydrolyze OTA to OTα [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]). Lipases, like the one produced by A. niger are also able to hydrolyze OTA and to produce the less toxic OTα [START_REF] Stander | Screening of Commercial Hydrolases for the Degradation of Ochratoxin A[END_REF]. Carboxypeptidase Y from Saccharomyces cerevisiae was found to be able to hydrolyze OTA, in addition to the yeast adsorption phenomenon [START_REF] Abrunhosa | Biodegradation of Ochratoxin a for Food and Feed Decontamination[END_REF].

Citrinin degradation was achieved by a solvent-tolerant bacteria Moraxella sp., which converted citrinin to decarboxycitrinin, which also results from a heat treatment of the mycotoxin [START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF]. By a mechanism of oxido-reduction, citrinin transforms into dihydrocitrinone in urin [START_REF] Li | Detoxification of Mycotoxins through Biotransformation[END_REF]. Moreover, degradation of this mycotoxin has been reported by Klebisella pneumoniae and Rhizobium borbori (Ben Taheur et al. 2019).

Detoxification of aflatoxins

The toxicity of AFB1 originates mainly from the lactone ring. Its cleavage leads to a nonfluorescent compound with 450 times reduced mutagenicity and 18 times reduced toxicity.

The difuran ring moiety, especially the double bond in the terminal furan ring, also contributes to the toxicity. Generally, two main detoxification pathways are observed: modification of the difuran ring or modification of the coumarin structure [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF].

Several, not food related, microorganisms have been screened for their ability to degrade aflatoxins, the first report published in 1966 by Ciegler and co-workers [START_REF] Ciegler | Microbial Detoxification of Aflatoxin[END_REF]. Several fungal species have been found to be able to transform AFB1 into less toxic AFB1 was also documented, several decontamination studies were performed on Lactic Acid Bacteria (LAB), however, as mentioned before, this reduction was mainly due to cell wall binding of the mycotoxin, which seems to be reversible [START_REF] Guan | Aflatoxin B 1 Degradation by Stenotrophomonas maltophilia and Other Microbes Selected Using Coumarin Medium[END_REF]). On the other hand, bacteria able to effectively degrade AFB1 include Rhodococcus erythropolis [START_REF] Alberts | Biological Degradation of Aflatoxin B1 by Rhodococcus erythropolis Cultures[END_REF][START_REF] Teniola | Degradation of Aflatoxin B1 by Cell-Free Extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. Nov. DSM44556T[END_REF], Mycobacterium fluoranthenivorans [START_REF] Teniola | Degradation of Aflatoxin B1 by Cell-Free Extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. Nov. DSM44556T[END_REF] and Nocardia corynebacterioides (previously Flavobacterium aurantiacum) [START_REF] Smiley | Preliminary Evidence That Degradation of Aflatoxin B1 Flavobacterium aurantiacum Is Enzymatic[END_REF]. As well as Streptomyces lividans and Streptomyces aureofaciens [START_REF] Eshelli | Metabolomics of the Bio-Degradation Process of Aflatoxin B1 by Actinomycetes at an Initial PH of 6.0[END_REF]. The mechanism of degradation of AFB1 and AFG1 was described by the actinobacteria Mycobacterium smegmatis. First, a reduction of the α,β-unsaturated double bond between C-Phanerochaete sordida by a manganese peroxidase, as well as from the "aflatoxindetoxifizyme" (ADTZ) produced by the fungus Armillariella tabescens, which was confirmed to be an oxidase and renamed aflatoxin oxidase (AFO). The mechanism proposed for the degradation by this enzyme consists in a AFB1 transformation into AFB1-8,9-epoxide, followed by an hydrolysis into a dihydrodiol-derivative [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]. Another metabolite, produced by a peroxidase from the rot fungus Pleurotus ostreatus, was dihydrohydroxyaflatoxin B1 (AFB2a), which has reduced mutagenicity [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF]. By-products of the modification of lactone ring in the coumarin moiety include aflatoxins D1, D2 and D3. This mechanism was observed for Pseudomonas putida [START_REF] Samuel | Degradation and Detoxification of Aflatoxin B1 by Pseudomonas putida[END_REF]. Aflatoxicol is another degradation product that results of the reduction of the C-3 keto on the cyclopentanone ring and was produced by Rhizopus sp. Hypomyces rosellus and

Cornybacterium rubrum (McCormick 2013

). An extracellular enzyme extracted from bacterium Myxococcus fulvus was designated as myxobacteria aflatoxin degradation enzyme (MADE) [START_REF] Zhao | Preparation, Purification and Characteristics of an Aflatoxin Degradation Enzyme from Myxococcus fulvus ANSM068[END_REF]. In several studies, degradation by-products were not identified but the reduction of toxicity was evaluated.

A summary of biological detoxification of mycotoxins is presented in Table 1.

Mycotoxin decontamination can be achieved by some physical and chemical methods, as well as by biological degradation. Some mycotoxin transformations lead to the modification of the original structure creating a "modified form" that can be difficult to detect with standard analytical methods. Certain physical steps involved in cereal processing, such as sorting, dehulling and heating, facilitate mycotoxin decrease.

Additional mycotoxin decontamination is achieved by some chemical treatments like nixtamalization and ozonisation. Microorganisms and their enzymes have proven to be a more rapid, effective and ecological approach for mycotoxin degradation. Furthermore, the majority of the known biological degradation by-products, resulting mostly from hydrolysis and oxidation, have proven reduced toxicity. A combination of a mycotoxin decrease measurement, the identification of degradation by-products and the evaluation of residual toxicity is essential to guarantee that an effective detoxification has occurred.

Streptomyces genus

Streptomycetes are aerobic members of the order Actinomycetales within the classis Actinobacteria [START_REF] Anderson | The Taxonomy of Streptomyces and Related Genera[END_REF]. The term Streptomyces was conferred by Selman Waksman in 1942, who also discovered their ability to produce antibiotics, for which he received a Nobel Prize [START_REF] Chater | Streptomyces Inside-out: A New Perspective on the Bacteria That Provide Us with Antibiotics[END_REF]. Nowadays, Streptomyces (Fig. 28) comprise more than 570 different species, of which only 10 are pathogenic, causing mainly the common scab of potatoes, e.g. Streptomyces scabies and S. europaeiscabiei [START_REF] Chater | Recent Advances in Understanding Streptomyces[END_REF]. The rest are saprophytes and include many endophytes [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF][START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]. Streptomyces are the most important source of antibiotics for medical, veterinary and agricultural applications [START_REF] Chater | Streptomyces Inside-out: A New Perspective on the Bacteria That Provide Us with Antibiotics[END_REF]. Approximatively 60% of agricultural compounds of importance reported in the literature originated from Streptomyces species [START_REF] Sharma | Biocontrol Potential and Applications of Actinobacteria in Agriculture[END_REF][START_REF] Tanaka | Agroactive Compounds of Microbial Origin[END_REF], as well as over 80% of all clinical antibiotics of actinobacterial origin [START_REF] Barka | Taxonomy, Physiology, and Natural Products of Actinobacteria[END_REF]. In fact, the collateral risks associated with antibiotic production is one of the main obstacles to their utilization [START_REF] Doumbou | Actinomycetes, Promising Tools to Control Plant Diseases and to Promote Plant Growth[END_REF]. Several studies have documented the discovery of about 20 biosynthetic gene clusters in the genome of Streptomyces which could account for the production of secondary metabolites [START_REF] Law | Taxonomic and Characterization Methods of Streptomyces: A Review[END_REF]. The reduction of pathogen damage of plants by Streptomyces species has been demonstrated in several studies [START_REF] Caceres | Streptomyces roseolus, A Promising Biocontrol Agent against Aspergillus flavus, the Main Aflatoxin B1 Producer[END_REF]Colombo, Kunova, Pizzatti, et al. 2019;[START_REF] Danial | Efficacy of Metabolites of a Streptomyces Strain (AS1) to Control Growth and Mycotoxin Production by Penicillium verrucosum, Fusarium verticillioides and Aspergillus fumigatus in Culture[END_REF][START_REF] Law | The Potential of Streptomyces as Biocontrol Agents against the Rice Blast Fungus, Magnaporthe oryzae (Pyricularia oryzae)[END_REF][START_REF] Li | Suppression of Magnaporthe oryzae by Culture Filtrates of Streptomyces globisporus JK-1[END_REF][START_REF] Sharkawy | Combined Effects of Streptomyces Viridosporus and Trichoderma Harzianum on Controlling Wheat Leaf Rust Caused by Puccinia triticina[END_REF][START_REF] Zarandi | Biological Control of Rice Blast (Magnaporthe oryzae) by Use of Streptomyces sindeneusis Isolate 263 in Greenhouse[END_REF], which places them as a promising strategy to manage fungal contamination. The efficacy of Streptomyces as BCAs is achieved thanks to a series of mechanisms, such as the production of metabolites and enzymes, including antifungal and antimicrobial compounds, as well as their rhizosphere colonization capacities, and the resulting plant growth promotion [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]. Moreover, the capacity of Streptomyces to form spores confers them resistance to formulation processes and increases their stability during conservation [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]).

Streptomyces strains development and secondary metabolites production

The intricated life cycle of Streptomyces species involves two distinct filamentous cell forms: the growing (or vegetative) hyphae and the reproductive (or aerial) hyphae, which differentiate into long chains of spores. Vegetative mycelia are formed by branching hyphae that prowl for nutrients, grow and proliferate. Hyphae develop by tip extension generating a ramifying mycelial network of filamentous cells delimited by occasional cross-walls [START_REF] Anderson | The Taxonomy of Streptomyces and Related Genera[END_REF][START_REF] Bush | C-Di-GMP Signalling and the Regulation of Developmental Transitions in Streptomycetes[END_REF]. During sporulation, hyphal branches differentiate into specialized sporogenic structures called aerial hyphae. Aerial growth is accompanied by a change in the surface properties of the hyphae, which confers them a rodlet hydrophobic layer [START_REF] Claessen | Regulation of Streptomyces Development: Reach for the Sky![END_REF]. Following their erection, chromosome replication, segregation, cell division, etc., they give place to aerial hyphae divided into a long chain of spores that, once mature, are liberated and dispersed by the wind or animals. Spores allow extended survival of Streptomyces species in the soil in an active form as they are resistant to water and nutrient scarcity [START_REF] Law | Taxonomic and Characterization Methods of Streptomyces: A Review[END_REF]. Spores then form new vegetative mycelium under favorable conditions (Fig. 29) [START_REF] Bush | C-Di-GMP Signalling and the Regulation of Developmental Transitions in Streptomycetes[END_REF].

Figure 29. The life cycle of Streptomyces [START_REF] Bush | C-Di-GMP Signalling and the Regulation of Developmental Transitions in Streptomycetes[END_REF]

Metabolite production

Streptomyces differentiation between surface and submerged cultures entails a complex modification on their metabolism, which can have an impact on their production of bioactive compounds [START_REF] Manteca | Mycelium Differentiation and Antibiotic Production in Submerged Cultures of Streptomyces coelicolor[END_REF][START_REF] Manteca | Streptomyces Differentiation in Liquid Cultures as a Trigger of Secondary Metabolism[END_REF]. Spore formation, which occurs mainly on solid culture, is closely related to the production of secondary metabolites [START_REF] Bobek | A Waking Review: Old and Novel Insights into the Spore Germination in Streptomyces[END_REF][START_REF] Law | Taxonomic and Characterization Methods of Streptomyces: A Review[END_REF]. Variations on culture conditions such as temperature and pH, can also diversify the molecules produced by Streptomyces during their active growth [START_REF] Yu | Effect of Liquid Culture Requirements on Antifungal Antibiotic Production by Streptomyces rimosus MY02[END_REF]. Secondary metabolites production does not usually occur until one or more nutrients become scarce. Carbon sources, nitrogen sources, and phosphate limitation have been described as important elicitors of secondary metabolism [START_REF] Běhal | Bioactive Products from Streptomyces[END_REF].

Parasitism

Parasitism or hyperparasitism is the most direct form of antagonism and it occurs when the BCA uses the pathogen for its own benefit, usually as a source of nutriments. Between filamentous microorganisms, usually the antagonistic microorganism wraps around the pathogenic fungi hyphae (coiling), restraining its propagation, however internal parasitism and perforation (lysis) have also been reported (Handelsman' and Stabb 1996). Numerous studies have documented the ability of several Streptomyces spp., such as S. griseous and S. griseoviridis to parasite fungal pathogens resulting on growth decrease and malformation or lysis of the fungal mycelium [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF]). Ursan et al. observed evidence of hyperparasitism by Streptomyces on Rhizoctonia solani, when evaluating their efficacity as biocontrol agents against fungal pathogens [START_REF] Ursan | The Potential of New Streptomyces Isolates as Biocontrol Agents Against Fusarium spp[END_REF]).

Antibiosis

Streptomyces bacteria are largely studied because of their ability to produce a wide variety of enzymes and metabolites of medical and agricultural interest [START_REF] Sharma | Biocontrol Potential and Applications of Actinobacteria in Agriculture[END_REF]. These compounds interfere with the pathogen growth and/or activities, for example by disrupting its cell membranes [START_REF] Pellan | Commercial Biocontrol Agents Reveal Contrasting Comportments Against Two Mycotoxigenic Fungi in Cereals: Fusarium graminearum and Fusarium verticillioides[END_REF]. Some enzymes include chitinases, cellulases, glucanases, proteases and phospholipases that, in addition to degrading pathogenic cell walls, are able to inhibit spore adhesion and germination [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF]. Furthermore, Streptomyces strains are able to produce amylases, keratinases, peroxidases, pectinases, phytases and xylanases that allow them to convert complex nutrients into simple mineral forms [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]. Another protein produced by Streptomyces species is an alkaline protease inhibitor (API), which has the ability to inhibit the fungal serine alkaline protease, indispensable for fungal growth [START_REF] Doumbou | Actinomycetes, Promising Tools to Control Plant Diseases and to Promote Plant Growth[END_REF]).

Among the variety of metabolites produced by Streptomyces bacteria, several have antifungal properties, such as Polyoxin B and D (S. cacaoi) that interfere with the formation of fungal cell wall by inhibiting chitin synthase. As well as Validamycin A, a trehalase inhibitor, which affects only microbes as vertebrates do not depend on the hydrolysis of this disaccharide for their metabolism. Furthermore, Kasugamycin, produced by S. kasugaensis, interferes with protein biosynthesis on microorganisms. Another example is mildiomycin, an inhibitor of fungal protein biosynthesis [START_REF] Doumbou | Actinomycetes, Promising Tools to Control Plant Diseases and to Promote Plant Growth[END_REF][START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF].

Streptomyces bacteria can also produce polyketides that provoke the inhibition of fungal growth [START_REF] Sharma | Biocontrol Potential and Applications of Actinobacteria in Agriculture[END_REF]. Hydrogen cyanide (HCN), a microbial product, can also contribute to pathogen suppression by blocking the cytochrome oxidase pathway [START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. The importance of antibiotic production as an antagonistic mechanism for biocontrol has been assessed by producing mutant strains unable to generate antibiotics, which were then incapable of suppressing the pathogen [START_REF] Singh | Management of Plant Pathogens with Microorganisms[END_REF]). However, as mentioned before, antibiotic production can hinder the use of Streptomyces strains, so further research on antibiotic repression could open new perspectives, as long as the targeted strains possess alternative antagonistic mechanisms. In addition to antimicrobials, Streptomyces strains are able to produce anthelmintic compounds, such as avermectin, produced by Streptomyces avermitilis, which is lethal to nematodes [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF].

Several volatile organic compounds (VOCs) are also produced by Streptomyces genus, such as isoprene, acetone, 1-butanol, cyclopentanone, dimethyl disulphide and geosmin [START_REF] Schöller | Volatile Metabolites from Actinomycetes[END_REF]. The latter confers them their characteristic "earthy" odour and has shown the ability to promote selective growth of geosmin-utilizing bacteria [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]. VOCs are reported to have antifungal activity by suppressing fungal growth, causing morphological abnormalities and inhibiting spore germination in fungi. They could present an alternative to chemical fumigants such as methyl bromide or chloropicrin [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF], however their sometimes unpleasant smell could obstruct their utilization.

Competition

Antagonistic Streptomyces strains can compete against the pathogen for nutritive resources such as carbon, nitrogen, iron, water and air; as well as for space in the ecological niche [START_REF] Nguyen | Crop Molds and Mycotoxins: Alternative Management Using Biocontrol[END_REF]. Competition for nutriments is often achieved by the production of antibiotics or hydrolyzing enzymes, thus, competition and antibiosis are closely linked [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF]. Iron is an essential element for the development of microorganisms, and its availability in the soil and plant surfaces is limited, which creates a fierce competition [START_REF] Singh | Management of Plant Pathogens with Microorganisms[END_REF]. Thus, the production of siderophores (iron-binding ligands) by certain microorganisms is an effective antagonistic mechanism, as it allows them to compete for the iron in the environment [START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. Streptomyces can produce siderophores such as coelichelin and albomycin [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]. Siderophores produced by S. albovinaceous, S. griseous and S. virginiae may be involved in their antagonism against Moniliophthora perniciosa [START_REF] Macagnan | Production of Lytic Enzymes and Siderophores, and Inhibition of Germination of Basidiospores of Moniliophthora (Ex Crinipellis) Perniciosa by Phylloplane Actinomycetes[END_REF]. Moreover, siderophore production by some actinobacteria leads to the enrichment of other actinobacteria in the plant rhizosphere [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF], which may lead to the creation of disease suppressive soils for the long-term management of plant pathogens [START_REF] Kinkel | Streptomyces Competition and Co-Evolution in Relation to Plant Disease Suppression[END_REF]).

Plant colonization

Streptomyces strains as endophytes have been reported to protect plants against pathogens, aid in nutrient uptake, modify plant development and stimulate rhizobial associations [START_REF] Kinkel | Streptomyces Competition and Co-Evolution in Relation to Plant Disease Suppression[END_REF]. The ability of Streptomyces strains to colonize and proliferate in the plant roots, leaves, fruits, etc., confers them an additional advantage as it contributes to a selective adaptation. Also, it provides a supplementary protection from the pathogen attack on the infection site. However, it has been shown that an initial competitive leverage has to be granted, such as high initial cell numbers. Once the beneficial organism is installed, the antagonistic mechanism could involve competition and antibiosis, as well as the stimulation of the plant self-defenses [START_REF] Singh | Management of Plant Pathogens with Microorganisms[END_REF]. Streptomyces bacteria might enter plant tissues by natural openings such as stomatal apertures, lenticels, hydathodes, wounds, broken trichomes, and root hair cracks formed by lateral root emergence zones [START_REF] Sousa | Plant Growth Promotion by Streptomycetes: Ecophysiology, Mechanisms and Applications[END_REF]. In a study performed by Vergnes et al., Streptomyces spp. rapidly colonized Arabidopsis leaf surface, including the trichomes and stomata [START_REF] Vergnes | Phyllosphere Colonization by a Soil Streptomyces sp. Promotes Plant Defense Responses against Fungal Infection[END_REF]. Moreover, Streptomyces griseoviridis from the commercial biofungicide Mycostop® has been documented as a good root colonizer [START_REF] Doumbou | Actinomycetes, Promising Tools to Control Plant Diseases and to Promote Plant Growth[END_REF].

Several Streptomyces bacteria are well known Plant Growth-Promoting Rhizobacteria (PGPR), as they develop into the plant rhizosphere and have a beneficial impact on plant development and tolerance against diverse stimuli. Certain Streptomyces strains have the capacity to boost plant immune response, as well as to increase the seedling and root length [START_REF] Sharma | Biocontrol Potential and Applications of Actinobacteria in Agriculture[END_REF]. The plant growth-promoting effects related to Streptomyces-plant interactions can be divided into biofertilization, biostimulation and bioprotection.

Biofertilization improves the nutrient acquisition, biostimulation is associated with the production of metabolic compounds with phytohormonal activity and bioprotection consists in the minimization of the impact of pathogenic fungi [START_REF] Sousa | Plant Growth Promotion by Streptomycetes: Ecophysiology, Mechanisms and Applications[END_REF].

Actinobacteria can influence soil fertility by rendering nutriments available to the plant [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]. For example, by fixing the nitrogen from the atmosphere to the soil in the form of ammonium and nitrates, by solubilizing phosphorus into soluble forms, by fixing and reducing iron and by producing/stimulating the production of phytohormones such as Indole Acetic Acid (IAA), which regulates root growth [START_REF] Doumbou | Actinomycetes, Promising Tools to Control Plant Diseases and to Promote Plant Growth[END_REF][START_REF] Nguyen | Crop Molds and Mycotoxins: Alternative Management Using Biocontrol[END_REF]. Indeed, Streptomyces have been documented to produce phytohormones such as cytokinins, gibberellic acid and indole-3-acetic acid (IAA) [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF], which have beneficial impacts on plant growth and development by induction of cell elongation and division [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]. Biotic and abiotic stresses trigger ethylene production by plants, which decreases plant growth. Streptomyces are able to alleviate this stress as they produce an enzyme called 1-aminocyclopropane-1-carboxylate (ACC) deaminase that transforms ACC, the precursor of ethylene, into ammonia and α-ketobutyrate, enhancing plant growth [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF]).

Induction of host resistance

The induction of host resistance is the enhancement of the plant self-defense capacity as a result of its interaction with a microorganism [START_REF] Nguyen | Crop Molds and Mycotoxins: Alternative Management Using Biocontrol[END_REF]. Plant active defenses involve changes in membrane function, oxidative bursts, cell wall reinforcement and hypersensitive response (HR), which results in programmed cell death (PCD) (Syed Ab Rahman et al. 2018). There are two main pathways of induced resistance. The first, named systemic acquired resistance (SAR), is mediated by salicylic acid (SA), which is often produced by the plant under pathogenic infection; it usually leads to the expression of pathogenesis-related (PR) proteins. These PR proteins consist in a variety of enzymes, that may act directly to lyse invading cells, reinforce cell wall boundaries or induce localized cell death. The second pathway, triggered by BCAs, is referred as induced systemic resistance (ISR) and is mediated by jasmonic acid (JA) and ethylene (ET) [START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. ISR results in the thickening of plant cell walls and alteration of the plant metabolic responses, which leads to an increase on plant defense chemicals [START_REF] Singh | Management of Plant Pathogens with Microorganisms[END_REF]. The induction of plant resistance by Streptomyces bacteria differs from traditional ISR produced by Pseudomonas and Bacillus bacteria, which do not induce the transcription of PR proteins [START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. Indeed, Streptomyces-mediated ISR involves features of both ISR and SAR, as well as the cross-talk between a variety of phytohormone signaling pathways [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]).

Screening for Streptomyces strains as biocontrol agents

In vitro evaluation of BCAs is commonly employed during potential antagonistic screenings. However, the protective effect observed in vitro is not always correlated with the in planta efficacity [START_REF] Pliego | Screening for Candidate Bacterial Biocontrol Agents against Soilborne Fungal Plant Pathogens[END_REF]). Thus, complementing in vitro assays with in planta validation is essential to identify promising biocontrol candidates. Moreover, to improve the first stages of selection, media used for dual culture assays should simulate at best the possible conditions of the targeted environment (Colombo, Pizzatti, et al. 2019). The authors suggested a comprehensive workflow for the selection of Streptomyces strains as biocontrol agents.

Their proposed integrated approach, which steps are described below, facilitates the effective selection of BCAs whose effectivity persists to the field (Colombo, Kunova, Cortesi, et al. 2019):

• Streptomyces species identification. Bacterial species identification and characterization are essential in order to guarantee the safety of their application.

• In vitro antagonistic assays. This first step of interaction between Streptomyces strains and the pathogen allows to identify antifungal properties and certain mechanisms of action.

• Effect of culture media. Media composition has an impact on the production of secondary metabolites. Thus, ideally, in vitro bioassays should be performed on different media to allow the broadening of the number of interesting strains identified.

• Use of fermentation extracts. The use of crude extracts of culture allows the identification of the metabolites and enzymes involved on the antagonistic effects.

Further steps of purification can lead to the obtention of bioactive compounds.

• Evaluation of the antagonistic mechanism. This step is essential to better address the impact of BCAs and avoid the risk of dispersing dangerous metabolites in the environment.

• Plant colonization assessment. A verification of the colonization of the intended plant niche is preferable to predict a consistent protection.

• Influence of complex environmental conditions. Subjecting a potential BCA to the conditions expected in the field is convenient as the complexity of environmental factors often impacts the biocontrol efficacy of BCAs.

• Determining plant growth promotion properties. The interaction of the BCA with the plant can lead to a series of positive and/or negative reactions that impact plant development and fitness against the disease. Indeed, some Streptomyces strains can be pathogenic, such as Streptomyces scabies that causes potato scab.

• Application method. The delivery system must be selected carefully according to the targeted pathogen. Optimized formulation of a BCA is fundamental for its endurance and efficacity.

• Mycotoxin quantification. As discussed before, there is a lack of correlation between fungal development and mycotoxin production. An inhibition of fungal growth does not guarantee a detoxification result, in some cases a stimulation of mycotoxin production was observed. Moreover, some secondary metabolites can limit toxin accumulation without impacting fungal proliferation, which is an important factor often neglected during the selection of BCAs against mycotoxinogenic fungi.

Modes of application of Streptomyces as biocontrol agents

The differences commonly observed between in vitro and in planta efficacity of BCAs can be attributed to several factors encountered in the field, such as organic matter, pH, nutrients availability, and humidity of the soil. Which is why the application of a BCA has to focus on facilitating its installation and proliferation under environmental conditions [START_REF] Law | The Potential of Streptomyces as Biocontrol Agents against the Rice Blast Fungus, Magnaporthe oryzae (Pyricularia oryzae)[END_REF]. The application of a biocontrol agent has to be adapted to its nature (living microbes or microbial products), the site or application and the targeted mode of action [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF]). Products such as Actinovate® (Streptomyces lydicus strain WYEC108), sold in Canada and USA, and Mycostop® (Streptomyces griseoviridis strain K61), which is also sold in Canada and USA and the EU [START_REF] Lahdenperä | Mycostop -A Novel Biofungicide Based on Streptomyces Bacteria[END_REF][START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF][START_REF] Rey | Plenty Is No Plague: Streptomyces Symbiosis with Crops[END_REF] are sold as dried formulations containing spores and mycelia, which can be either suspended in liquid and sprayed onto crops (foliar spraying), folded into the soil prior to sowing (soil inoculation), or be used as a seed coating. Foliar spray is intended to control pathogens causing foliar diseases (recommended for Actinovate®) while soil inoculation is designed to manage soil or seed-borne pathogens (recommended for Actinovate® and Mycostop®), or when the BCA strain is particularly vulnerable to desiccation. Another way of application is directly onto the plants root by fluid drill inoculation or root transplant dip.

However, these methods are labor-intensive. For seed coating application, seeds are usually immersed in a BCA suspension and dried before germination [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]). The main advantage of this technique is that it delivers the beneficial organism to the immediate surroundings of the seed and rhizosphere, some disadvantages include shelf-life and storage conditions [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]. Figure 31 illustrates some BCA application techniques.

Figure 31. Methods of application of biocontrol agents [START_REF] Law | The Potential of Streptomyces as Biocontrol Agents against the Rice Blast Fungus, Magnaporthe oryzae (Pyricularia oryzae)[END_REF].

Other than the mode of application, BCA's efficacity, consistency and stability rely on a great manner on its formulation. This process consist in mixing the active ingredient (i.e. bacterial spores, mycelium, secondary metabolites, enzymes) with inert materials such as diluents and surfactants, sometimes amended with nutritional supplements, in order to make it easy to handle and to increase its stability during storage and field application [START_REF] Junaid | Commercial Biocontrol Agents and Their Mechanism of Action in the Management of Plant Pathogens[END_REF][START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF]. Indeed, when introduced to an ecosystem, a BCA encounters abiotic stresses such as desiccation, osmotic stress, UV radiation and extreme temperatures; as well as biotic stresses [START_REF] Legrand | Challenges Facing the Biological Control Strategies for the Management of Fusarium Head Blight of Cereals Caused by F. graminearum[END_REF]. Some authors have experimented with different formulations to protect and deliver efficient Streptomyces BCAs, such as talcum powder, alginate beads, clay pellets and chitosan polyphosphate beads [START_REF] Jobin | Streptomycete Spores Entrapped in Chitosan Beads as a Novel Biocontrol Tool against Common Scab of Potato[END_REF][START_REF] Sabaratnam | Formulation of a Streptomyces Biocontrol Agent for the Suppression of Rhizoctonia Damping-off in Tomato Transplants[END_REF][START_REF] Tamreihao | Biocontrol and Plant Growth Promoting Activities of a Streptomyces corchorusii Strain UCR3-16 and Preparation of Powder Formulation for Application as Biofertilizer Agents for Rice Plant[END_REF][START_REF] Zamoum | Development of Formulations Based on Streptomyces rochei Strain PTL2 Spores for Biocontrol of Rhizoctonia solani Damping-off of Tomato Seedlings[END_REF]. It seems to be a consensus about talcum being an effective and stable carrier.

Streptomyces against phytopathogenic fungi

The use of Streptomyces strains as an alternative to the management of phytopathogenic fungi has recently been explored. For this purpose, the application of Streptomyces isolates, of culture broths and of bacterial metabolites have has been evaluated.

El-Sharkawy et al. established the efficacy of

Steptomyces viridosporus to control leaf rust under greenhouse and field conditions, with disease reductions going up to 50% and a general increase on yield parameters (spike, grain and kernel weight). They also found that S. viridosporus efficacy increased in combination with Trichoderma harzianum [START_REF] Sharkawy | Combined Effects of Streptomyces Viridosporus and Trichoderma Harzianum on Controlling Wheat Leaf Rust Caused by Puccinia triticina[END_REF]. As wheat blast is an emerging disease, most biological control assays have focused on rice blast [START_REF] Law | The Potential of Streptomyces as Biocontrol Agents against the Rice Blast Fungus, Magnaporthe oryzae (Pyricularia oryzae)[END_REF]). However, results obtained could give some leads on the efficacity of Streptomyces as a managing tool for M. oryzae pv Triticum. For instance, greenhouse assays using spore suspension of Streptomyces sideneusis strain 263 on rice seedling-leaves, resulted in a strong inhibition of M. oryzae and a suppression of leaf symptoms [START_REF] Zarandi | Biological Control of Rice Blast (Magnaporthe oryzae) by Use of Streptomyces sindeneusis Isolate 263 in Greenhouse[END_REF]).

Yi et al. determined that fermentation broth of a Streptomyces spp. strain was able to decrease leaf rust by 80% [START_REF] Yi | Antifungal Activity of Streptomyces sp. against Puccinia Recondita Causing Wheat Leaf Rust[END_REF]). Furthermore, culture filtrates of Streptomyces globisporus JK-1 were found to inhibit M. oryzae development and conidial germination, their efficacity was even superior of that of tricyclazole, a commonly used chemical fungicide for the control of this fungus [START_REF] Li | Suppression of Magnaporthe oryzae by Culture Filtrates of Streptomyces globisporus JK-1[END_REF]. Additionally, Fermentation broth of Streptomyces strain BG2-53, with 96% homology to S. lipmanii, according to 16S rDNA sequence analysis, showed potent antifungal activity against M. oryzae on rice [START_REF] Lee | Streptomyces with Antifungal Activity against Rice Blast Causing Fungus, Magnaporthe grisea[END_REF].

Li et al. 2008 determined that malayamycin, an antifungal compound produced by

Streptomyces malaysiensis, inhibited Parastagonospora nodorum sporulation in vitro, as well as lesion size and pycnidiation in wheat seedlings [START_REF] Li | Malayamycin, a New Streptomycete Antifungal Compound, Specifically Inhibits Sporulation of Stagonospora nodorum (Berk) Castell and Germano, the Cause of Wheat Glume Blotch Disease[END_REF]). In addition, Phenamide, produced by a member of the Streptomyces albospinus cluster was also active against P. nodorum on assays conducted by [START_REF] Makkar | Phenamide, a Fungicidal Metabolite from Streptomyces albospinus A19301[END_REF]

Streptomyces against mycotoxinogenic fungi

When evaluating actinobacteria as biocontrol agents of mycotoxin producing fungi, is of the utmost importance to take into consideration not only the bacteria's capacity to reduce fungal germination, growth and colonization, but also their ability to decrease mycotoxin amount, either by degrading it or by obstructing its production. Indeed, is well known that some pathogens can increase their mycotoxin production under stressful conditions as a defense mechanism [START_REF] Medina | Biocontrol of Mycotoxins: Dynamics and Mechanisms of Action[END_REF], thus inhibition of fungal growth is not always correlated with a decrease on mycotoxin amount.

According to Jung et al., spores of Streptomyces sp. BN1 sprayed onto wheat heads helped to decrease FHB severity during greenhouse experiments [START_REF] Jung | Biological Efficacy of Streptomyces sp. Strain BN1 against the Cereal Head Blight Pathogen Fusarium graminearum[END_REF]. Moreover, on field experiments performed by Palazzini et al., the application of Streptomyces albidoflavus 87B during wheat anthesis decreased FHB severity between 29 and 39%, as well as DON amount between 69 and 85% [START_REF] Palazzini | An Integrated Dual Strategy to Control Fusarium graminearum Sensu Stricto by the Biocontrol Agent Streptomyces sp. RC 87B under Field Conditions[END_REF]. In a later study, the same team confirmed that S. albidoflavus 87B reduced on 25% FHB incidence, on 30% its severity and up to 51% the DON accumulation in durum wheat [START_REF] Palazzini | Biocontrol of Fusarium graminearum Sensu Stricto, Reduction of Deoxynivalenol Accumulation and Phytohormone Induction by Two Selected Antagonists[END_REF]). Additional research conducted by Ursan et al., who evaluated the antagonistic capacity of 60 strains of Streptomyces sp. against Fusarium culmorum and F. graminearum in vitro, showed that at least 30% of the evaluated strains were able to inhibit fungal growth. Some of the mechanisms applied by the bacterial strains were the production of hydrolytic enzymes, such as cellulase and chitinase, as well as mycoparasitism and lysis of fungal mycelium. Additionally, two of the evaluated strains decreased ZEN amount [START_REF] Ursan | The Potential of New Streptomyces Isolates as Biocontrol Agents Against Fusarium spp[END_REF]. A recent in vitro study by Colombo et al. demonstrated that four Streptomyces strains were capable of inhibiting F. graminearum growth up to 71% and DON up to 99% when inoculated on detached wheat grains three days prior to fungal contamination. The evaluated Streptomyces strains were ineffective when inoculated simultaneously with the pathogen, which suggest that their antagonistic potential is preventive rather than curative [START_REF] Colombo | Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by QPCR[END_REF]).

On in vitro experiments performed by Samsudin et al., a Streptomyces sp. was able to decrease by almost a half the growth of F. verticillioides by the diffusion of antifungal metabolites, and a slight decrease on FB1 production was also observed [START_REF] Samsudin | Efficacy of Potential Biocontrol Agents for Control of Fusarium verticillioides and Fumonisin B1 under Different Environmental Conditions[END_REF]). An in vitro study of fourteen Streptomyces strains against F. verticillioides showed fungal growth decreases going up to 77% by bacterial cells and up to 73% by bacterial CFEs.

Furthermore, a considerable reduction on FB1 and FB2 production (up to 88% and 98%, respectively) was also reported [START_REF] Nguyen | Biocontrol of Fusarium verticillioides Using Organic Amendments and Their Actinomycete Isolates[END_REF]. Streptomyces strain AS1 was able to inhibit in vitro growth of P. verrucosum and F. verticillioides by 90% and 51%, respectively, by hindering germination. Its antifungal activity was hypothesized to be due to the production of metabolites such as valinomycin and brevianamide F. The strain was also able to decrease OTA and fumonisins FB1 and FB2 production by both pathogens respectively [START_REF] Danial | Efficacy of Metabolites of a Streptomyces Strain (AS1) to Control Growth and Mycotoxin Production by Penicillium verrucosum, Fusarium verticillioides and Aspergillus fumigatus in Culture[END_REF]. In another study by Campos-Avelar et al., the growth of P. verrucosum was strongly decreased by Streptomyces isolates during in vitro assays, but this inhibition caused sometimes an overaccumulation of OTA. Additionnaly, bacterial isolates degraded the mycotoxin, mainly during their growth in liquid medium [START_REF] Campos-Avelar | Minimizing Ochratoxin a Contamination through the Use of Actinobacteria and Their Active Molecules[END_REF]).

An interesting approach presented by Verheecke et al. when evaluating the interaction between Streptomyces and Aspergillus flavus, was to assess the bacterial impact on mycotoxin biosynthesis gene cluster. Indeed, for aflatoxins, genes such as aflD, aflM, aflP (structural) and aflR/aflS (regulatory) can be interfered by actinobacteria, thus decreasing aflatoxin production. In addition, they suggested that aflM expression could be an aflatoxin indicator during Streptomyces interactions (Verheecke et al. 2015). However, in some studies, the addition of bacterial antagonists inhibited aflD and still increased AFB1 production [START_REF] Al-Saad | Impact of Bacterial Biocontrol Agents on Aflatoxin Biosynthetic Genes, AflD and AflR Expression, and Phenotypic Aflatoxin B1 Production by Aspergillus flavus under Different Environmental and Nutritional Regimes[END_REF]. Streptomyces metabolites such as aflastatins, blasticidins A (Fig 33) and S, and dioctatin A were found to inhibit aflatoxin production by interruption of norsoloniric acid and sterigmatocystin production, both intermediates of aflatoxin. The reduction of the mRNA levels of genes encoding for enzymes needed during aflatoxin biosynthesis and the regulation of the AflR protein were also reported. Concerning dioctatin A, fungal conidiation inhibition was observed on Aspergillus parasiticus, as well as a significant increase on its kojic acid production (Sakuda 2010).

Use of Streptomyces in biocontrol, risks and benefits

There are several compounds derived from Streptomyces bacteria with biological properties. For example, the first antibiotics employed for the control of pathogenic fungus and bacteria are cycloheximide and streptomycin, both produced by Streptomyces griseus.

The Streptomyces strains capable of producing geldanamycin are also viewed as promising BCAs. Furthermore, the ability of generate several antibiotic compounds increases the BCA's importance, as is the case for Streptomyces violaceusniger YCED9, which has been reported to produce nigericin, guanidylfungin A-like compound, and geldanamycin [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF]. The soil application of azalomycin produced by Streptomyces malaysiensis MJM1968 showed antifungal activity against Fusarium oxysporum, Rhizoctonia solani, Cladosporium cladosporioides, Fusarium chlamydosporum, Colletotrichum gloeosporioides, Alternaria mali, and Pestalotia spp [START_REF] Cheng | Azalomycin F Complex Is an Antifungal Substance Produced by Streptomyces malaysiensis MJM1968 Isolated from Agricultural Soil[END_REF]. A thiopeptide produced by the strain Streptomyces S4-7 was purified and tested against Fusarium, showing a potent inhibitory activity against fungal cell wall biogenesis [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]. Additionally, antibiotics produced by Streptomyces are also commercialized as bactericides and fungicides, namely Polyoxin D, Streptomycin, Kasugamycin, Blasticidin-S, Phytomycin, Validamycin, Polyoxorim and Natamycin [START_REF] Palaniyandi | Effects of Actinobacteria on Plant Disease Suppression and Growth Promotion[END_REF][START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF].

The direct use of bacterial strains has also been proven effective for the management of fungal pathogens. The strain Streptomyces BN1 was able to alleviate the reduction in seedling length caused by Fusarium when applied as a spore preparation to seeds. This strain also significantly decreased Fusarium head blight symptoms when sprayed onto wheat heads [START_REF] Jung | Biological Efficacy of Streptomyces sp. Strain BN1 against the Cereal Head Blight Pathogen Fusarium graminearum[END_REF]). In addition to the commercial biological fungicides Mycostop® and Actinovate®, some other Streptomyces-based products are authorized as "fertilizers", such as Micosat F® (CCS Aosta srl, Aosta, Italy), containing three different Streptomyces spp.; Forge SP® (Blacksmith Bioscience, Spring, TX, USA), containing S. nigrescens; and Mykorrhyza soluble 30G (Glückspilze, Innsbruck, Austria), containing S. griseus and S. lydicus [START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF].

The application of Streptomyces as biocontrol agents has various advantages compared to other largely studied microorganisms. For instance, Pseudomonas have been investigated as potential biocontrol agents, however, these strains only colonize wheat plants during the early stages of growth before being out-competed, and they are also sensitive to desiccation [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]). Thus, the benefit of using Streptomyces as BCAs include their saprophytic lifestyle, which allows them to colonize the mature roots of cereal crops [START_REF] Conn | Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism[END_REF], as well as their spore-forming capacity, which allows them to survive well under unfavorable conditions [START_REF] Law | Taxonomic and Characterization Methods of Streptomyces: A Review[END_REF].

There are some possible negative impacts of the use of Streptomyces strains as biocontrol agents. For instance, as the main antagonistic mechanism consist on antibiosis, pathogens could develop resistance to the antibiotics produced by the actinobacteria.

However, Streptomyces may also co-evolve with the pathogen and activate numerous putative antimicrobial biosynthetic gene clusters, resulting in the simultaneous production of various antibiotics with different modes of action, which could reduce the rate of evolution of pathogenic resistance [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]. Furthermore, these antibiotics may not be specific and affect non-targeted organisms, and they could even be detrimental to human health.

Nevertheless, some studies suggest that antibiotics are produced at very low quantities in the soil and that their role on pathogen inhibition is minor compared to space or resources competition. Also, several gene clusters encoding secondary metabolites may only be activated in the presence of pathogenic species, which gives them a certain selectivity [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]). Another possible negative impact is the transfer of genes from pathogenic Streptomyces species towards saprophytic species, this has been reported and should be studied carefully [START_REF] Doumbou | Actinomycetes, Promising Tools to Control Plant Diseases and to Promote Plant Growth[END_REF][START_REF] Vurukonda | Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. As Endophytes[END_REF]).

Screening for mycotoxin-biotransforming microorganisms

Mycotoxin-biotransforming microorganisms (MBMs) should be obtained from environmental sources or screened from a previous collection. Next step must consist on evaluating the detoxification abilities and the influencing factors of the selected microorganisms. Later, degradation mechanism and by-products should be investigated.

Afterwards, safety assessment of functional strains and biotransformation products should be performed. Finally, potential enzymes should be identified, isolated and/or cloned and expressed in order to be applied for detoxification purposes [START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF]. A workflow of this process is presented in Figure 34.

Use of Streptomyces as detoxifying tools

Due to the considerable number of secondary metabolites they produce, Streptomyces are not considered as GRAS (generally recognized as safe) microorganisms. However, it has already been reported that they are able to produce a variety of metabolites and enzymes with high potential for detoxification, which makes them promising sources for the isolation of compounds that could be later used for detoxification of food and feed.

Several Streptomycete strains studied by Palazzini et al. significantly decreased DON level on wheat grains and spikes in greenhouse experiments [START_REF] Palazzini | Potential Biocontrol Agents for Fusarium Head Blight and Deoxynivalenol Production in Wheat[END_REF]). Further work from the same research group, indicated strong inhibition of DON production in wheat spikes on the field by S. albidoflavus [START_REF] Palazzini | An Integrated Dual Strategy to Control Fusarium graminearum Sensu Stricto by the Biocontrol Agent Streptomyces sp. RC 87B under Field Conditions[END_REF][START_REF] Palazzini | Biocontrol of Fusarium graminearum Sensu Stricto, Reduction of Deoxynivalenol Accumulation and Phytohormone Induction by Two Selected Antagonists[END_REF]. Some in vitro studies have demonstrated the ability of streptomycetes to limit fumonisin accumulation. Strains isolated from organic amendments decreased significantly FB1 and FB2 production by F. verticillioides [START_REF] Nguyen | Biocontrol of Fusarium verticillioides Using Organic Amendments and Their Actinomycete Isolates[END_REF]. FB1 accumulation was also inhibited by treating milled maize with Streptomyces sp. [START_REF] Samsudin | Efficacy of Potential Biocontrol Agents for Control of Fusarium verticillioides and Fumonisin B1 under Different Environmental Conditions[END_REF]. A laccase produced by Streptomyces coelicolor was patented for its use to decontaminate AFB1 [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]. As mentioned before, the compounds aflastatin A, blasticidin A and dioctatin A, produced by Streptomyces sp., inhibit aflatoxin production in A. parasiticus [START_REF] Ren | Control of Aflatoxigenic Molds by Antagonistic Microorganisms: Inhibitory Behaviors, Bioactive Compounds, Related Mechanisms, and Influencing Factors[END_REF][START_REF] Tian | Natural Products for Preventing and Controlling Aflatoxin Contamination of Food[END_REF]. In a series of studies by Verheecke et al., they reported that several soil-born Streptomyces isolates were capable of degrading AFB1, and had a strong bioactivity against AFB1 and AFB2 production by A. flavus [START_REF] Verheecke | In Vitro Interaction of Actinomycetes Isolates with Aspergillus flavus: Impact on Aflatoxins B1 and B2 Production[END_REF]. Moreover, they repressed aflatoxin gene expression on A. parasiticus [START_REF] Verheecke | Streptomyces-Aspergillus flavus Interactions: Impact on Aflatoxin B Accumulation[END_REF]. Eshelli et al. optimized the AFB1 degradation conditions of R. erythropolis, S. lividans and S. aureofaciens. Subsequently, they identified a series of degradation by-products and proposed a plausible degradation mechanism [START_REF] Eshelli | Metabolomics of the Bio-Degradation Process of Aflatoxin B1 by Actinomycetes at an Initial PH of 6.0[END_REF]. Caceres et al. determined that S. roseolus was capable of down-regulate the AFB1 gene cluster in Aspergillus flavus [START_REF] Caceres | Streptomyces roseolus, A Promising Biocontrol Agent against Aspergillus flavus, the Main Aflatoxin B1 Producer[END_REF]. Streptomyces rimosus degraded zearalenone into 8'(S)-hydroxyzearalenone and 2,4-dimethoxyzearalenone (El- [START_REF] El-Sharkawy | Microbial Transformation of Zearalenone. 2. Reduction, Hydroxylation, and Methylation Products[END_REF]. A laccase, produced by S. coelicolor was also capable of degrading ZEN [START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF]). Harkai et al. documented the biodegradation of AFB1 and ZEN by several Streptomyces strains, and some of them eliminated the genotoxic and estrogenic effects of the mycotoxins, respectively [START_REF] Harkai | Biodegradation of Aflatoxin-B1 and Zearalenone by Streptomyces sp. Collection[END_REF]).

The Streptomyces genus has a vast potential as BCAs. They benefit from a series of mechanisms that allow them to fight against fungal pathogens but also to facilitate plant development and self defense. The use of Streptomyces bacteria as BCAs seems promising due to their technological properties, such as plant colonization and sporulation, which grants them an advantage compared to other potential biocontrol agents and facilitates their formulation and posterior application. Furthermore, Streptomyces represent a promising source of metabolites and enzymes with detoxifying properties.

Objectives of the study

The previous bibliographical synthesis, introduces the context involving the risk of wheat contamination by fungal pathogens and saprophytes, and the potential occurrence of harmful mycotoxins on derived products. The decrease on the use of chemical fungicides in the last decade has urged the search for alternative and most ecological control solutions, such as the use of beneficial microorganisms. This primary goal gave rise to the research project presented in this manuscript, focusing on a specific group of bacteria: actinobacteria.

The main purpose started as the screening of an actinobacteria collection for their antagonistic capacities in vitro against various phytopathogenic and/or mycotoxinogenic fungi of wheat. Additionally, in planta evaluation of the antagonistic capacities of selected actinobacteria were assessed for phytopathogens. For mycotoxinogenic fungi, we included a deeper comprehension of fungal-bacteria interactions and the resulting impact on mycotoxins accumulation.

The experimental approach was based on the following objectives:

1. Assessment of the impact of actinobacteria isolates on fungal growth in vitro.

2. Determination of the capacity of actinobacteria to decrease wheat foliar diseases under greenhouse conditions after foliar application. stage, an active production of enzymes and secondary metabolites takes place, such as antibiotics and melanoid pigments [START_REF] Barka | Taxonomy, Physiology, and Natural Products of Actinobacteria[END_REF].

Isolating actinobacteria from a similar environment to which their application is intended may help to maintain an equilibrium of the indigenous microflora and also facilitates their adaptation to the media [START_REF] Legrand | Challenges Facing the Biological Control Strategies for the Management of Fusarium Head Blight of Cereals Caused by F. graminearum[END_REF]. Several media were specifically created for the isolation and development of Streptomyces, and to facilitate the display of their typical morphological traits, such as powdery colonies with greyish colors, the characteristic "earthy"

odor and in some cases the production of pigments [START_REF] Dhanasekaran | Actinobacteria -Basics and Biotechnological Applications[END_REF][START_REF] Shirling | Methods for Characterization of Streptomyces Species[END_REF].

Some plant growth promoting traits exhibited by actinobacteria include iron and nitrogen fixation, phytohormone production and phosphate solubilization [START_REF] Anwar | Screening of Rhizospheric Actinomycetes for Various In-Vitro and in-Vivo Plant Growth Promoting (PGP) Traits and for Agroactive Compounds[END_REF]. They are also able to interfere with plant pathogens by producing degrading enzymes such as chitinases. Actinobacteria possessing these capacities constitute promising alternatives for plant fertilization and particularly for their application as biocontrol agents against pathogenic fungi causing plant diseases [START_REF] Passari | In Vitro and in Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants[END_REF].

Fifty-nine actinobacteria were isolated according to their morphology from soil samples collected at the Hérault region in France and from organic amendments. They were partially identified as Streptomyces by 16S rRNA sequencing. The resulting Streptomyces collection belonging to the UMR Qualisud at the University of Montpellier was created after spore production, enumeration and conservation. Afterwards, the Streptomyces isolates were evaluated in order to identify their plant growth promoting (PGP) traits and their chitinolytic activities.

Materials and methods

Actinobacteria isolation

Microorganisms present in soil samples collected at the Hérault department in France and in domestic organic amendments were cultured on PDA medium (Biokar BK095HA) during previous work [START_REF] Nguyen | Biocontrol of Fusarium verticillioides Using Organic Amendments and Their Actinomycete Isolates[END_REF]. Colonies having actinobacteria morphology were taken from the PDA plates and cultured on three media: ISP2 (4 g/L yeast extract, 10 g/L malt extract, 4 g/L dextrose, 20 g/L bacteriological agar, pH 7.2), ISP4 (10 g/L starch, 1 g/L K2HPO4, 1 g/L MgSO4, 2 g/L (NH4)2SO4, 1 g/L CaCO3, 1 mg/L FeSO4, 1 mg/L MgCl2, 1 mg/L ZnSO4, 18 g/L bacteriological agar, pH 7.2) and CYA (30 g/L sucrose, 5 g/L yeast extract, 1 g/L K2HPO4, 0.3 g/L NaNO3, 0.05 g/L KCl, 0.05 g/L MgSO4, 1 mg/L FeSO4, 1 mg/L ZnSO4, 0.5 mg/L CuSO4, 15 g/L agar, pH ~7.4) until the obtention of pure cultures.

Actinobacteria spore recovery and conservation assays

Four actinobacteria isolates having different morphological traits (IX06, IX23, IX35, IX43) were selected to evaluate their sporulation and stability during conservation. Isolates were cultured in the dark on ISP4 and CYA medium for 11 days at 28°C. Spores were scraped from the surface of Petri dish plates by adding 5 mL of distilled water + 0.01% Tween 20 and filtered through sterile cotton. Spore amount was determined on an optical microscope (Primo Star, Zeiss, Allemagne) using a Thoma cell counting chamber (Preciss, France).

Collected spores were preserved under three modalities: at -80°C with the addition of glycerol (25%), -80°C without glycerol and at 4°C. After 7, 14 and 22 days of conservation, spores were cultured again on ISP4 and CYA media to assess their viability by UFC counting. All tests were done in triplicate.

Actinobacteria spore production and enumeration

The fifty-nine actinobacteria isolates were cultured on ISP4 medium for 11 days at 28°C. Spores were collected as described above. Spore counting was performed using a Novocyte ACEA flux cytometer. Spore suspensions were aliquoted and conserved at -80°C without the addition of glycerol. Before each test, an aliquot of spores was unfrozen and homogenised at a concentration of 10 7 spores/mL.

Actinobacteria DNA extraction and 16S rRNA preliminary identification Actinobacteria were cultured on CYB medium for 5 days at 28°C and 180 rpm. For DNA extraction, 3 mL of culture were centrifugated 5 min at 10000 rpm. Supernatant was eliminated and the pellet was kept at -80°C overnight. Then, samples were lyophilized and kept under liquid nitrogen. Two ceramic beads were added to each tube. Samples were then ground in 3 cycles of 40 seconds using a FastPrep® instrument and kept for 1 minute in liquid nitrogen between each grinding.

Genomic DNA purification was performed using the Gram-positive DNA purification kit

MasterPure™ following the manufacturer's instructions. For this, 150 µL of TE buffer were added to each tube, then, samples were vortexed to resuspend the ground pellets. 1 µL of Ready-Lyse Lysozyme were added to each resuspended pellet. Samples were incubated overnight at 37°C. 1 µL of Proteinase K (50 µg/mL) were diluted into 150 µL of Gram-Positive Lysis Solution. 150 µL of the previous solution were added to each sample and mixed thoroughly. Followed by an incubation at 65-70°C for 15 minutes with a brief vortexing every 5 minutes. Samples were cooled to 37°C then placed on ice for 3-5 minutes before DNA precipitation.

DNA precipitation was conducted following manufacturer instructions as well. 175 µL of MPC Protein Precipitation Reagent were added to 300 µL of lysed sample, followed by vigorous vortexing for 10 seconds. Then samples were centrifuged at 4°C for 10 minutes at >10000×g in a microcentrifuge. Supernatant was then transferred to a clean microcentrifuge tube and pellet was discarded. 1 µL of RNase A (5 µg/mL) were added to each sample and mixed thoroughly, followed by an incubation at 37°C for 30 minutes. At the end of the incubation period, 500 µL of isopropanol were added to the supernatant and mixed by inversion for 30-40 minutes. After, tubes were centrifuged at 4°C for 10 minutes at 10000×g in a microcentrifuge. The supernatant was removed with a micropipette being cautious of not dislodging the DNA pellet. Then, the pellet was rinsed with 70% ethanol and centrifuged if dislodged. Finally, DNA was resuspended in 35 µL of ultrapure water.

Genomic DNA was quantified using a spectrophotometer-Nanodrop ND8000 (Thermo Fisher Scientific, Waltham, MA, USA). The 16S rRNA gene was amplified using the universal primers 27F/1492R. Amplification was accomplished by mixing 200 µL of AmpliTaq™ Gold

Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 1 µL of each primer at 10 µM and 1 µL of DNA template (~100 ng). The thermocycling program was started with initial denaturation at 95°C for 10 min and followed by 35 thermal cycles of 95°C for 30s, 52°C for 45s, and 52°C for 2 min. Finally, the final extension was done at 72°C for 10 min. The PCR products were separated by 1% agarose gel electrophoresis and the amplicon (~1.5 kb) was purified. DNA Sanger sequencing was carried out in the ISEM GenSeq platform (University of Montpellier, France). Obtained sequences were trimmed according to the quality scores on the chromatogram sequences. The forward and reverse sequences were aligned using the Geneious Prime (Version 2019.2) software with global alignment with free ends and gaps at 93% of similarity (5.0/-9.026168). The nucleotides obtained were subjected to BLAST analysis using the Geneious database.

Evaluation of PGPR properties of actinobacteria

In order to evaluate the PGPR capacities of the actinobacteria collection, different media for the identification of PGPR traits were prepared as described below. Actinobacteria were cultured on each specific media under the described conditions.

Chitinase activity

To determine the ability of actinobacteria isolates to produce chitinase, 10 µL of spore suspension at 10 7 spores/mL were inoculated on plates of colloidal chitin medium (15 g/L colloidal chitin at 10%, 0.5 g/L yeast extract, 1 g/L (NH₄)₂SO₄, 0.3 g/L MgSO₄, 1.36 g/L KH2PO4, 15 g/L bacteriological agar) for 5 days at 28°C. Actinobacteria exhibiting a clear halo around the colony were noted as positive.

Siderophore production

To detect the production of siderophores by the actinobacteria isolates, a CAS (Chrome Azurol Sulphonate) solution was prepared by mixing the three following solutions adapted from [START_REF] Payne | Detection, Isolation, and Characterization of Siderophores[END_REF]):

• Solution 1: 60 mg of CAS in 50 mL of distilled water

• Solution 2: 2.7 mg of FeCl3.6H2O in 10 mL of HCl 10 mM.

• Solution 3: 73 mg of HDTMA (hexadecyltrimethylammonium) in 40 mL of distilled water Actinobacteria were grown on ISP2 medium for 7 days at 180 rpm. Then, liquid cultures were centrifuged and 0.5 mL of supernatant were mixed with 0.5 mL of CAS solution. After 20 minutes, absorbance was measured at 630 nm. Siderophore production amount was calculated using the following equation:

%!"#!$&'()"*+")(!,-&.$!/*$,0 = /1) 2 1$03!455 1)
Where:

1) =!Absorbance at 630 nm of reference (uninoculated ISP2 + CAS solution)

1$ = Absorbance at 630 nm of the sample

Phosphate solubilization

To determine if actinobacteria were able to solubilize phosphate, 10 µL of a spore suspension at 10 7 spores/mL were cultured on PVK (Pikovskayas's) medium (0.5 g/L yeast extract, 10 g/L dextrose, 5 g/L Ca3(PO4)2, 0.5 g/L (NH₄)₂SO₄, 0.2 g/L KCl, 0.1 g/L MgSO₄, 0.1 mg/L MgSO₄, 0.1 mg/L FeSO₄, 15 g/L bacteriological agar) for 7 days at 28°C. A clear halo around the colony indicated a positive result.

Ammonia production

To verify the production of ammonia by the actinobacteria isolates, 20 µL of a spore suspension at 10 7 spores/mL were inoculated on peptone water (10 g/L peptone, 5 g/L sodium chloride) for 15 days at 180 rpm and 28°C. At the end of this period, 0.5 mL of Nesseler's reagent (3.5 g KI; 1.3 g HgI2; 70 mL H2O, 30 mL NaOH 30%) were added to the culture. A positive result was indicated by the development of a brown to yellow color.

Indole-3-acetic acid (IAA) production

500 µL of actinobacteria spore suspension 10 7 spores/mL were inoculated into flasks containing ISP2 medium, they were cultured at 28°C and 180 rpm for 7 days. 150 µL from the pre-culture were inoculated into flasks containing 100 mL of ISP2 medium with 0.2% Ltryptophan. After 14 days of culture at 28°C and 180 rpm, bacterial liquid cultures were centrifuged at 10000 rpm for 10 minutes. 1 mL of the supernatant was mixed with 2 mL of Salkowski's reagent (2 mL FeCl3 (0.5M) + 49 mL distilled water + 49 mL perchloric acid 70%)

and incubated for 25 minutes in the dark at 28°C. IAA production was observed as the development of a pink-red color in the tubes.

To quantify IAA production by the actinobacteria isolates, a standard curve of IAA was prepared on ISP2 medium. Absorbances were determined at 530 nm.

Results and discussion

Actinobacteria spore recollection and conservation assays

After the obtention of pure cultures of actinobacteria, four isolates presenting different morphological characteristics (IX06, IX23, IX35, IX43) were selected to perform sporulation and conservation assays on CYA and ISP4 media. Results indicate that both media allow a good sporulation yield between 1x10 8 and 5x10 9 spores/mL approximatively (Figure 36).

Conservation assays indicated a similar viability for actinobacteria spores regardless of the preservation method, time and medium of origin, with values fluctuating between 1.5x10 7 and 2.5x10 8 UFC/mL (data not shown). Based on these results, we decided selected ISP4 as the medium for spore production, and a conservation at -80°C without glycerol, to avoid the addition of carbon sources to the spore suspensions. Actinobacteria DNA extraction and 16S rRNA preliminary identification

The 16S sequencing indicated that the isolates from the collection belonged to the genus Streptomyces at 97%. As 16S identification is preliminary, several species can be assigned to the different RNA fragments, resulting in multiple possibilities of identification.

For instance, after partial BLAST analysis, isolate IX23 was assimilated to S. griseolus, S.

halstedi and eventually S. cyaneus, and isolate IX45 could be S. zaomyceticus or S. venezueleae.

However, isolate IX20 had numerous possibilities according to the 16S ribosomal RNA partial sequence, which include S. cyaneofuscatus, S. anulatus, and S. praecox, among 10 others. For this reason, a complete genome sequencing is ongoing. In order to provide a precise identification for further research.

Evaluation of PGPR properties of actinobacteria

Results indicate that forty-six isolates (78%) are capable of degrading chitin as they form clear halos around their colonies when grown on colloidal chitin medium (Fig. 38).

Several studies have determined the chitinolytic capacity of actinobacteria by growing them on colloidal chitin medium, and some of them validated the visual result by amplifying the chitinase gene [START_REF] Passari | In Vitro and in Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants[END_REF]. The observation of halos on PVK's medium allowed us to determine that 25 isolates (42%) were able to solubilize phosphate (Fig. 39). Seven isolates (12%) gave positive to ammonia production, as the culture medium turned brown to yellow upon addition of Nesseler's reagent. For comparison, around 50% of the actinobacteria isolates screened in the study of Passari et al. were capable of solubilizing phosphate, while all of them produced ammonia [START_REF] Passari | In Vitro and in Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants[END_REF].

Figure 38. Examples of halo formation by actinobacteria on chitinase degradation and phosphate solubilization assays.

Thirteen isolates (22%) presented positive results to IAA production with phytohormone concentrations going from 58 to 200 µg/mL (Table 2). This is comparable to the results obtained by Anwar et al. when screening rhizospheric actinobacteria. Indeed, their strains produced a maximal amount of IAA between 70 and 80 µg/mL, when cultured with a maximal concentration of 500 µg/mL of L-tryptophan [START_REF] Anwar | Screening of Rhizospheric Actinomycetes for Various In-Vitro and in-Vivo Plant Growth Promoting (PGP) Traits and for Agroactive Compounds[END_REF] As the addition to chemical products to stored food and feed is limited due to their undesirable effects, other alternatives for the control of fungal development and mycotoxin decontamination on food matrices have to be explored. The addition of mycotoxin degrading enzymes produced by Streptomyces constitutes a promising alternative.

This article presents the screening of a Streptomyces collection for their antifungal and antimycotoxinogenic capacities against Penicillium verrucosum, a ubiquitous fungus found regularly as a contaminant of cereals, and the main producer of ochratoxin A (OTA) among the Penicillium genus. It was found that most of the tested bacterial isolates were capable to inhibit fungal growth. However, fungal growth inhibition was often accompanied by a stimulation of OTA production, probably as a fungal defense mechanism. The evaluation of Streptomyces' cell free extracts allowed to determine that they may contain some constituent metabolites and/or active enzymes having an impact on mycotoxin accumulation.

An interesting observation of this work was the impact of the culture conditions on the capacity of bacterial cells to degrade OTA. Indeed, only five isolates were able to degrade the mycotoxin on solid medium, while almost all of them degraded it in liquid culture, some almost completely. This kind of differences were already described in the literature and reveal

Complementary research on the stimulation of actinobacteria

During the previously published work [START_REF] Campos-Avelar | Minimizing Ochratoxin a Contamination through the Use of Actinobacteria and Their Active Molecules[END_REF], it was determined that actinobacteria were able to decrease the amount of ochratoxin A produced by P.

verrucosum during dual cultures. Moreover, actinobacteria growing cells were able to degrade, at different rates, pure ochratoxin A present in liquid medium and, in rare cases, in solid medium. However, actinobacteria cell free extracts (CFEs) seemed unable to degrade the mycotoxin. This may indicate that OTA degrading enzymes are not constitutive, and are rather produced when actinobacteria are confronted with the mycotoxin and/or with the pathogen and its metabolites.

Indeed, results from several studies conducted on actinobacteria mixed cultures documented that a large fraction of actinobacteria biosynthetic genes, encoding for secondary metabolites, remains silent during mono cultures [START_REF] Marmann | Co-Cultivation -A Powerful Emerging Tool for Enhancing the Chemical Diversity of Microorganisms[END_REF]. Furthermore, when actinobacteria are subjected to co-cultivation with other microorganisms or their components, an elicitation of the secondary metabolism occurs [START_REF] Bertrand | Metabolite Induction via Microorganism Co-Culture: A Potential Way to Enhance Chemical Diversity for Drug Discovery[END_REF]. For instance, an enhanced production of antibiotics and enzymes was observed when cocultivating actinobacteria strains with other actinobacteria but also with other bacterial species and fungi. From an ecological approach, it could be a way for actinobacteria to protect themselves, to compete with other organisms and/or to better adapt to their environment [START_REF] Abdelmohsen | Elicitation of Secondary Metabolism in Actinomycetes[END_REF].

Furthermore, it has been reported that actinobacteria differential development in solid and liquid medium has an impact on their production of secondary metabolites [START_REF] Manteca | Streptomyces Differentiation in Liquid Cultures as a Trigger of Secondary Metabolism[END_REF]. This could give clues about the contrasting results on degradation capacity of actinobacteria grown under both conditions.

With this in mind, we decided to perform a series of complementary tests in an attempt to stimulate the secondary metabolism of actinobacteria to obtain active CFEs, capable of degrading OTA. For this, liquid cultures of actinobacteria were either supplemented with OTA alone, or with the full spectrum of fungal metabolites (fungal CFEs), or co-cultured with the fungi. After this, the resulting CFEs were evaluated for their ability to degrade OTA, as well as for their impact on fungal growth and mycotoxin production.

Materials and methods

Stimulation of actinobacteria during CFEs production

Five actinobacteria isolates having strong ochratoxin A degrading capacities in liquid medium were chosen for the CFEs stimulation assays (IX12, IX20, IX32, IX56 and MYC). 50 µL of actinobacteria (10 7 spores/mL) were inoculated into 50 mL of CYB medium (30 g/L sucrose, 5 g/L yeast extract, 1 g/L K2HPO4, 0.3 g/LNaNO3, 0.05 g/L KCl, 0.05 g/L MgSO4, 1 mg/L FeSO4, 1 mg/L ZnSO4, 0.5 mg/L CuSO4, pH ~7.4) modified following the 6 modalities below:

1. CYB (unmodified) 2. CYB + Acetonitrile (5.64 mL/L) 3. CYB + OTA (260 µg/L) in acetonitrile (5.64 mL/L) 4. CYB + CFEs of P. verrucosum's liquid culture (25%) 5. CYB + CFEs of P. verrucosum's solid culture (25%) 6. CYB + fungal inoculum of P. verrucosum (50 µL at 10 7 spores/mL) Modality 1 corresponded to the standard conditions previously described for the production of bacterial CFEs (Campos-Avelar et al. 2020). Modality 2 contained acetonitrile, in order to evaluate its impact on actinobacteria growth and metabolism, as the standard solution of OTA is diluted in this solvent. Modality 3 contained 260 µg/mL of a standard solution of OTA. For modalities 4 and 5, P. verrucosum was cultured in liquid and solid medium for 7 days at 25°C (180 rpm for liquid culture). Fungal metabolites for Modality 4 were retrieved after centrifugation of the liquid culture of P. verrucosum at 10000×g, and filtration of the supernatant (PES filter 0.22 µm). For Modality 5, an extraction step was included by cutting the P. verrucosum agar plate (15 g) into small pieces with a scalpel and putting it in a container with 50 mL of distilled water, under agitation at 250 rpm for 20 minutes. The water extract was finally recovered after separation by centrifugation and filtration as previously described for liquid fungal CFEs. To ensure homogeneous OTA concentration, the mycotoxin amount in fungal CFEs was measured by HPLC as described earlier [START_REF] Campos-Avelar | Minimizing Ochratoxin a Contamination through the Use of Actinobacteria and Their Active Molecules[END_REF] and adjusted at the same concentration as modality 3 (260 µg/mL). Modality 6 corresponds to a co-culture of bacterial and fungal cells at the same concentration (10 7 spores/mL) on unmodified CYB.

After 11 days at 25°C and 180 rpm, liquid cultures were centrifuged and filtered through a PES filter at 0.22 µm to eliminate bacterial cells and obtain the stimulated CFEs.

Degrading capacities of stimulated bacterial CFEs

OTA was added to 500 µL of the CFEs prepared under the 6 modalities (226 ng/mL final concentration), before incubation at 25°C and 180 rpm. After 72H, OTA was extracted and the residual concentration was determined by fluorescence HPLC as described in previous work [START_REF] Campos-Avelar | Minimizing Ochratoxin a Contamination through the Use of Actinobacteria and Their Active Molecules[END_REF].

Effect of stimulated bacterial

Results

Stimulation of actinobacteria during CFEs production

Bacterial CFEs were produced in liquid CYB prepared under 6 modalities by the addition of OTA, fungal CFEs, or by co-culture with the fungus. After 11 days, cultures were filtered and the residual amount of OTA in modalities 3, 4, 5 and 6 was measured by HPLC. Visual observations at the end of the culture period indicated that the addition of acetonitrile (Modality 2) or standard OTA solution (Modality 3) at the tested concentrations did not have a blatant impact on actinobacteria growth as compared to the standard culture (Modality 1).

Indeed, the resulting broths presented similar light brown color, apparent cell density and mycelia consistency. Contrasting results were observed for all actinobacteria isolates under modalities 4, 5 and 6, where the addition of colorless fungal metabolites and the bacteriafungus co-culture generated a dark red or brown broth (Fig. 40). The pH measure revealed that both kind of containers led to very different pH at the end of the culture, from approximatively neutral (pH~7, plastic containers) to more basic (pH~8.9, glass flasks). In order to verify this observation, an additional test was performed by comparing the two protocols with three isolates (IX12, IX56 and MYC). Once again, actinobacteria samples cultured in plastic containers had neutral pH (~7.4) and degraded almost completely the OTA, while actinobacteria cultured in glass flasks sealed with cotton all reached basic pH values (~9.1) and were unable to degrade the mycotoxin (Fig. 42). During the production of standard actinobacteria CFEs, cultures were carried out on glass flasks and the pH at the end of the culture was usually basic (data not shown). This suggests that the use of plastic containers modified somehow the bacterial development by acting on the culture parameters, such as the rate of aeration.

Regarding the remaining modalities in Figure 41, actinobacteria grown under modalities 4 and 5, containing P. verrucosum metabolites, were able to degrade the initial OTA added to the medium (260 ng/mL) whether the resulting pH was more neutral (Modality 5) or basic (Modality 4). However, the pH range was always lower than for Modality 3 where no degradation was observed. During the mixed fermentation in Modality 6, microscopical observations showed that fungal growth was scarce, while actinobacteria mycelia was abundant, as exemplified by the Figure 43 displaying the co-culture of P. verrucosum (red arrows) and actinobacteria isolate IX20.

Degrading capacities of stimulated bacterial CFEs

The resulting bacterial CFEs were evaluated for their OTA degrading capacities by incubating them with OTA (~226 ng/mL) for 72H. As a control, OTA was added to liquid CYB and cultured under the same conditions.

As shown by OTA dosage in Figure 44, the mycotoxin amount was not decreased after its culture with the CFEs under all modalities. Even the CFEs produced under modalities 4, 5 and 6 (where bacteria were able to degrade OTA during cell growth) were unable to degrade the mycotoxin afterwards, despite the pH of some of the samples being neutral. Culture: 72 hours at 25°C and 180 rpm.

Effect of stimulated bacterial CFEs on growth and OTA production by P. verrucosum

The "stimulated" bacterial CFEs were added at 25% to the growth medium of P.

verrucosum in order to evaluate their effect on fungal development and OTA production.

Results on fungal growth are presented in Figure 45. Globally, CFEs under modalities 1,2 and 3 did not have a significant impact on fungal growth. However, CFEs produced under modalities 4, 5 and 6 seemed to have stimulated the antifungal properties of all the isolates at different extents, except for IX12. CFEs of isolates IX32, IX56 and MYC were particularly active, a complete inhibition of fungal growth was caused by CFEs of isolates IX32 and IX56

under Modality 4 of stimulation.

The impact of CFEs on OTA production variated according to the bacterial isolate and the stimulation modality. Isolate IX12 had a slight impact under modalities 1 through 5 but caused the highest overproduction in modality 6. Isolates IX20 and IX32 caused an OTA overproduction in general, except under modality 4, where they reduced mycotoxin amount.

However, this decrease seems to follow the strong growth inhibition observed. A similar result was observed for isolate IX56, which caused also a mycotoxin decrease under modality 6. MYC decreased OTA amount under all modalities, except for modality 4.

Discussion

During this work we attempted to activate the Streptomyces secondary metabolism, by the hypothesis that OTA could act as a metabolism elicitor. OTA degrading capacity of actinobacteria' CFEs was particularly aimed in this complementary study, considering that previously published results were highly divergent regarding OTA degradation by bacterial cells and by their CFEs (Campos-Avelar et al. 2020).

As mentioned before, our approach included the addition of OTA during bacterial growth as, like other mycotoxins, OTA production by P. verrucosum may facilitate its competition against other microbes for space and nutrients [START_REF] Pfliegler | The Aspergilli and Their Mycotoxins: Metabolic Interactions With Plants and the Soil Biota[END_REF], and as such, it could act as signaling molecule for other microorganisms [START_REF] Venkatesh | Mycotoxins in Conversation with Bacteria and Fungi[END_REF]. We also stipulated that the previously observed OTA degradation by actinobacteria in liquid cultures (Campos-Avelar et al. 2020) was conducted by enzymes, and that the addition of the substrate (OTA) could further stimulate CFEs enzymatic activity. An example of this induction mechanism was observed for S. coelicolor, for which the addition of soil containing chitin activated the expression of chitinase genes [START_REF] Nazari | High Expression Levels of Chitinase Genes in Streptomyces coelicolor A3(2) Grown in Soil[END_REF]).

In addition to OTA, the impact of supplementing bacterial medium with fungal metabolites, on the bacterial CFEs degrading capacities was also evaluated. Nevertheless, in some cases, the activation of microbial metabolism requires cell to cell interactions, as the metabolites alone (cell free supernatants, killed cells, extracts) are not always capable of induce compound production [START_REF] Bertrand | Metabolite Induction via Microorganism Co-Culture: A Potential Way to Enhance Chemical Diversity for Drug Discovery[END_REF]. Indeed, co-cultivation or mixed fermentation allows to mimic the conditions encountered by microorganisms in complex ecosystems where the production of secondary metabolites is enhanced by competitive or antagonist relationships [START_REF] Marmann | Co-Cultivation -A Powerful Emerging Tool for Enhancing the Chemical Diversity of Microorganisms[END_REF]). To achieve this, a stimulating modality consisting in a co-culture of actinobacteria with P. verrucosum was also included in our study.

As acetonitrile was used as a solvent to bring OTA into the culture medium, it was included as a control to evaluate its potential influence on the growth and metabolism of actinobacteria.

However, some studies indicate that acetonitrile utilization by bacteria was only possible at very high pH (10) and moderate salinity [START_REF] Sorokin | Acetonitrile Degradation under Haloalkaline Conditions by Natronocella acetinitrilica Gen[END_REF]) so its utilization under the conditions of this study seems unlikely.

Visual observation of the resulting bacterial broths indicated that the addition of neither acetonitrile nor OTA had a visible impact on actinobacteria development, while bacterial growth with the addition of fungal metabolites and in co-culture, exhibited an intense brown pigmentation. In fact, the co-cultivation of microorganisms can trigger the production of pigments [START_REF] Bertrand | Metabolite Induction via Microorganism Co-Culture: A Potential Way to Enhance Chemical Diversity for Drug Discovery[END_REF], and some studies have found that a defense mechanism against degrading enzymes can be behind this effect [START_REF] Marmann | Co-Cultivation -A Powerful Emerging Tool for Enhancing the Chemical Diversity of Microorganisms[END_REF]. ). Thus, we can conclude that OTA does not trigger any pigment production, but that other P. verrucosum metabolites clearly impact the metabolism of the actinobacteria isolates in our study.

The dosage of the residual amount of OTA in the resulting broths indicated that actinobacteria were unable to degrade OTA during their growth in unmodified medium (Fig.

41, Modality 1), which differs from the results of previous work [START_REF] Campos-Avelar | Minimizing Ochratoxin a Contamination through the Use of Actinobacteria and Their Active Molecules[END_REF]).

The different nature of the containers between the precedent and the actual study led to a strong variation on pH values. Indeed, the results of this complementary assay seem to indicate that OTA degradation occurs mainly when the pH remains neutral. Two different hypotheses can be drawn from these findings:

1. Actinobacteria produce their degrading enzymes only when the growth is limited by stressful conditions (lack of aeration?) and before they reach basic pH 2. Enzymes are produced all along the actinobacteria growth, but they are not active at high pH To better understand the discrepancies observed, similar experiments could be performed in buffered medium to eliminate the influence of pH variation during the culture.

Moreover, the calibration of all bacterial CFEs at lower pH range might be interesting to verify how the pH range influences the degradation of OTA in the latter.

Regarding the impact of P. verrucosum metabolites (Modalities 4 and 5) on the production of CFEs, bacterial isolates were able to degrade the OTA during their growth, regardless of having a more neutral or basic pH (Fig. 41). The variation obtained in the pH range at the end of the culture of actinobacteria with fungal CFEs might originate either from a growth reduction due to fungal metabolites, or to the presence of organic acids produced by P. verrucosum [START_REF] Frisvad | Ochratoxin A Producing Penicillium verrucosum Isolates from Cereals Reveal Large AFLP Fingerprinting Variability[END_REF], acting as buffering agents. This result suggests that a compound, other than OTA, produced by the fungus was able to stimulate bacterial cells so they could degrade the OTA present in the medium. In fact, P. verrucosum, is capable of producing a wide variety of secondary metabolites (e.g. verrucine A, verrucolone, anacine), of which about 80% remain unknown [START_REF] Hautbergue | Evidencing 98 Secondary Metabolites of Penicillium verrucosum Using Substrate Isotopic Labeling and High-Resolution Mass Spectrometry[END_REF]. The screening of the influence of known P. verrucosum metabolites on Streptomyces' OTA degrading abilities could help in understanding the mechanism behind mechanism behind the observed stimulating effect.

A small amount of OTA was detected in mixed fermentation, which could be explained by the scarce fungal growth observed by microscopy. Nevertheless, mycotoxin inhibition or degradation phenomena cannot be excluded, as they have already been documented during fungal-bacterial interactions (El [START_REF] Khoury | Ability of Soil Isolated Actinobacterial Strains to Prevent, Bind and Biodegrade Ochratoxin A[END_REF][START_REF] Khoury | Ota Prevention and Detoxification by Actinobacterial Strains and Activated Carbon Fibers: Preliminary Results[END_REF]. For instance, Rodriguez et al. found that Brevibacterium sp. were able to completely degrade OTA by hydrolyzing it into OTα and phenylalanine during liquid cultures, probably by means of a carboxypeptidase [START_REF] Rodriguez | Degradation of Ochratoxin a by Brevibacterium Species[END_REF]. The observed degradation of OTA by bacteria, when exposed to fungal CFEs, also provides indications that such phenomenon could be involved.

The bacterial CFEs were unable to degrade OTA under any modality even when the pH values were neutral (Modalities 4, 5 and 6), which indicates that neither OTA, nor the whole spectrum of P. verrucosum fungal metabolites, nor the mixed culture granted the capacity to further degrade the mycotoxin. Our results differ from those of El Khoury and co-workers, as they found that actinobacteria strains were able to degrade OTA from about 20% to 50% while growing in liquid medium, and those strains' CFEs also decreased OTA. However, the observed degradation in CFEs were quite low (14%-22%) (El [START_REF] Khoury | Ability of Soil Isolated Actinobacterial Strains to Prevent, Bind and Biodegrade Ochratoxin A[END_REF]. For B. subtilis, cell free extracts were able to degrade 97.6% of 6 µg/mL OTA in two hours. However, intracellular supernatant was unable to degrade the mycotoxin [START_REF] Shi | Ochratoxin A Biocontrol and Biodegradation by Bacillus subtilis CW 14[END_REF]. The strong degradation observed in the presence of the cells for Modalities 4 and 5 along with the total absence of OTA degradation activity of the resulting supernatants points towards an intracellular degradation rather than a secretion of extracellular enzymes, independently of the pH.

Despite their lack of degrading activity, CFEs exhibited an impact on P. verrucosum development and OTA production. In particular, CFEs of modalities 4, 5 and 6, as some of them were able to almost completely inhibit fungal growth (IX32 and IX56, modality 4). This suggests that the presence of fungal metabolites, other than OTA, in the last three modalities seemingly stimulated the production of antifungal metabolites. As described by Bertrand et al., the production of antimicrobial compounds is commonly induced during microbial interactions [START_REF] Bertrand | Metabolite Induction via Microorganism Co-Culture: A Potential Way to Enhance Chemical Diversity for Drug Discovery[END_REF]. Furthermore, it was demonstrated that a slightly acidic pH (6) favors the production of antifungal antibiotics by Streptomyces rimosus [START_REF] Yu | Effect of Liquid Culture Requirements on Antifungal Antibiotic Production by Streptomyces rimosus MY02[END_REF]). In our study, the most active CFEs (IX32 and IX56) were produced under modalities 4, 5 and 6, whose pH was around 6-6.5, which could have promoted the production of antifungal compounds that later inhibited P. verrucosum growth. The impact on OTA production was variable, with some isolates causing an overproduction and others strongly decreasing the mycotoxin amount.

Moreover, the same isolate exhibited different effects depending on the stimulation modality, which evidences their sensitivity to the parameters evaluated. These results are of the utmost interest as they indicate that different isolates may have different antagonistic mechanisms and that their interactions with the fungi or its metabolites can lead to bioactive compounds for mycotoxin inhibition or stimulation.

Conclusion and perspectives

The different approaches evaluated during this complementary study for the stimulation of actinobacteria CFEs, were ineffective at conferring them OTA degradation capacities. Nevertheless, the presence of fungal metabolites greatly enhanced the degradation capacity by the bacteria itself. Moreover, some antifungal properties of actinobacteria CFEs were also enhanced by the presence of fungal metabolites or by the fungus itself during bacterial growth. Finally, an interesting phenomenon was elucidated concerning the impact of growth conditions on the capacity of actinobacteria to degrade OTA in liquid culture. It appears that peculiar growth conditions could possibly stress actinobacteria, modifying their development and allowing them to degrade/consume the OTA in the medium. Further studies under controlled aeration and pH could allow to validate this hypothesis and/or to elucidate new degradation mechanisms.

Aspergillus flavus growth inhibition and Aflatoxin B1 decontamination by

Streptomyces spp. and their metabolites

Article presentation

The Streptomyces collection previously screened against Penicillium verrucosum and OTA was this time evaluated against Aspergillus flavus and aflatoxins. Both pathogens occur mainly during grain storage and provoke the accumulation of mycotoxins. The purpose of this study was to identify the antifungal and detoxifying capacity of Streptomyces isolates, in order to set the basis for the potential identification and isolation of bioactive compounds for the detoxification of food matrices.

The conducted experiments were based on the approach previously described: in vitro dual culture assays; impact of CFEs on fungal growth and mycotoxin production; mycotoxin degradation by cells grown on artificially contaminated solid and liquid medium and degradation by bacterial metabolites (CFEs), as well as the search for degradation by-products.

An additional step of verification of the residual toxicity complemented this work.

Results showed that A. flavus growth was easily inhibited by most Streptomyces isolates.

The antifungal efficacy of their CFEs at the tested concentration was inferior, indicating either the involvement of a direct mechanism or the need to increase the concentration in order to have a similar impact. Moreover, thermally treated CFEs had a weak antifungal capacity, which evidences an enzymatic activity. Aflatoxin B1 and B2 production was highly decreased during dual cultures, suggesting a general detoxifying mechanism. CFEs caused also a mycotoxin general decrease, but to a lesser extent. However, some CFEs caused an overproduction of aflatoxins, which drives attention to the stimulating metabolites present in the bacterial extracts. Degradation assays confirmed the ability of bacterial cells to degrade AFB1 and also the degrading capacity of their CFEs. Residual toxicity assessment allowed us to validate an effective decrease on genotoxicity. These results lead to a promising research perspective for the production and isolation of aflatoxin degrading enzymes that could be applied in the food industry.

A comparison of the results obtained for both storage pathogens and both main mycotoxins is presented at the end of this section.

Article accepted for publication the 3 th May 2021

Comparison of the effect of Streptomyces strains on Penicillium verrucosum and Aspergillus flavus development and their main mycotoxins

As presented in the two previous sections, the impact of Streptomyces isolates on both storage pathogens evidenced several marked differences. Such as a general stimulation of OTA production on dual cultures in contrast with a general decrease on aflatoxins amount under the same test conditions. Another important disparity was the fact that OTA degradation was almost only possible during bacterial growth in liquid medium, while degradation of AFB1 by Streptomyces was relatively easy, not only in both culture conditions but also by constitutive bacterial extracellular metabolites. Complementary studies suggested that the tested Streptomyces isolates required stressful growth conditions in order to being able to degrade OTA.

The study of the origin of these differences seems interesting, and complementary research should be done to reach a better understanding of mycotoxin degrading mechanisms and its induction by external stimuli. Despite the observed heterogenicity, some isolates exhibited common antagonistic traits against both pathogens. Isolates exhibiting complementary activities represent interesting research prospects, as the presence of multiple mycotoxins is common in contaminated foodstuffs.

The following heatmaps regroup the effect of each Streptomyces isolate according to three parameters: fungal growth (Figure 47), mycotoxin specific production (Figure 48 isolates were inefficient at degrading AFB1 under the same conditions (Cluster I). Degradation of AFB1 by cells on liquid medium was accomplished by all isolates except 4 (first part of cluster I). On the other hand, about a half of bacterial isolates degraded OTA in liquid medium, and the observed degradations were remarkably stronger. Lastly, Streptomyces CFEs were unable of degrading OTA, while most of them degraded AFB1 at different rates. Cluster V contains the most efficient isolates according to their degradation capacities: IX20, IX25, IX45, IX52 and MYC, which were able to degrade both mycotoxins under both culture conditions, additionally, their CFEs were capable of degrading AFB1. Future research on these fourth isolates could lead to the identification and isolation of degrading enzymes with a broad spectrum of action. Moreover, pH variations could be tested in order to determine if the lack of OTA degradation by the bacterial CFEs could be due to their rather basic pH which may limit OTA degrading enzymes, while AFB1 degrading enzymes might not be affected at this pH range. 4.5 Biocontrol of wheat phytopathogens using Streptomyces spp.

Article presentation

Main phytopathogenic fungi affecting wheat development in the field include rusts, blotchs and blasts, produced by Puccinia triticina, Magnaporthe oryzae, Parastagonospora nodorum and Zymoseptoria tritici, respectively. Principal fungal control techniques currently applied include the use of resistant wheat varieties and the application of chemical fungicides.

However, both methods have increasing limitations and disadvantages, which urges the exploration of alternative management strategies, such as the biocontrol by beneficial microorganisms.

This section presents at first the observed in vitro antifungal activities of a collection of sixty Streptomyces isolates against three wheat foliar pathogens: M. oryzae, P. nodorum and Z. tritici. Different inhibition profiles were observed for the various bacterial isolates, in general, P. nodorum growth was more easily inhibited, followed by M. oryzae. Finally, Z. tritici exhibited the higher resistance.

Based on this first screening, twelve isolates were selected for further in planta testing against P. nodorum. Results showed that only four isolates depicted results significantly different from the control, two of them increasing disease symptoms and the other two decreasing them. These four isolates were later evaluated against P. triticina and Z. tritici.

Results indicated that only MYC had decreased the percentage of lesions caused by P. triticina, as well as the percentage of necrosis caused by Z. tritici. Unfortunately, in the case of Z. tritici, the decrease on plant disease was accompanied by an increased production of pycnidia, which is undesirable, as these structures facilitate fungal proliferation.

The expression of plant defense genes remained practically unchanged, which suggested that the observed protective effects were linked to a direct antagonistic mechanism, such as antibiosis or parasitism. The presence of fungal and bacterial mycelium in the surface of the wheat leaves was assessed by Scanning Electron Microscopy (SEM) and no parasitism was observed between the two microorganisms.

Article submitted to Biological Control on the 7 th of May 2021 can produce mycotoxins such as trichothecenes and zearalenone (ZEN), which can be found from the field to the final products derived from cereals [START_REF] Duverger | Influence of Culture Medium and Incubation Time on the Simultaneous Synthesis of Deoxynivalenol and Zearalenone by Fusarium graminearum[END_REF]). Among the trichothecenes, deoxynivalenol (DON) is the most common and its presence in food and feed represents a major risk, as it causes nausea, vomiting and the inhibition of the immune responses (Mannaa and Kim 2017b). F. graminearum can also produce acetylated DON variants such as 3-acetyl-ADON (3-ADON) or 15-acetyl-ADON (15-ADON) [START_REF] Haile | Fusarium Head Blight in Durumwheat: Recent Status, Breeding Directions, and Future Research Prospects[END_REF].

Different studies have reported that 15-ADON exhibits a higher intestinal toxicity than DON, while 3-ADON is less toxic [START_REF] Alizadeh | Deoxynivalenol and Its Modified Forms: Are There Major Differences?[END_REF][START_REF] Pinton | Toxicity of Deoxynivalenol and Its Acetylated Derivatives on the Intestine: Differential Effects on Morphology, Barrier Function, Tight Junction Proteins, and Mitogen-Activated Protein Kinases[END_REF]. Zearalenone is less toxic, but can lead to estrogenic disruptions and hormonal changes [START_REF] Richard | Some Major Mycotoxins and Their Mycotoxicoses-An Overview[END_REF]. Fusarium verticillioides is mainly a maize pathogen, causing Fusarium ear rot disease (FER). However, it has been recently found to affect wheat [START_REF] Gagkaeva | Emergence of Fusarium verticillioides in Finland[END_REF]. In maize, it causes important losses and produces mainly fumonisin B1 (FB1) and B2 (FB2). The latter are of major importance, due to their high occurrence and their severe toxic effects. Fumonisins are classified by the IARC as a class 2B possible human carcinogens [START_REF] Iarc | Some Naturally Occurring Substances: Food Items and Constituents[END_REF]. Maximal limits on food and feed have been established for DON, ZEN, and the sum of FB1 and FB2. In the case of foods intended for human consumption, limits were settled at 750 µg/kg, 75 µg/kg and 400 µg/kg, respectively. (European Commission 2006).

The use of chemical fungicides is nowadays the main strategy for the control of both Fusarium. However, they lead to fungal resistance and environmental pollution. Thus, new alternatives are currently researched [START_REF] Syed Ab Rahman | Emerging Microbial Biocontrol Strategies for Plant Pathogens[END_REF]. Biocontrol by beneficial microorganisms represents a promising alternative to manage fungal diseases [START_REF] Knudsen | Ecological Complexity and the Success of Fungal Biological Control Agents[END_REF]. Actinobacteria are soil microorganisms capable of producing a wide variety of secondary metabolites, including antifungal compounds and degrading enzymes [START_REF] Barka | Taxonomy, Physiology, and Natural Products of Actinobacteria[END_REF]. The genus Streptomyces are the most abundant and studied among actinobacteria.

The efficacity of Streptomyces as biological control agents to manage Fusarium has already been documented (Colombo, Kunova, Cortesi, et al. 2019). Mycostop®, formulated with spores of Streptomyces griseoviridis is already commercialized, and is preconized for the control of Fusarium and several other plant pathogens [START_REF] Lahdenperä | Mycostop -A Novel Biofungicide Based on Streptomyces Bacteria[END_REF]).

An aspect often neglected during the evaluation of potential biological agents is the impact on mycotoxin amount, as the reduction of fungal growth is not always accompanied by a decrease on mycotoxins accumulation [START_REF] Medina | Biocontrol of Mycotoxins: Dynamics and Mechanisms of Action[END_REF].

During our work, we evaluated a collection of 59 Streptomyces isolates and the commercial BCA Mycostop® as reference, for their in vitro antagonistic ability against F. graminearum and F. verticillioides. Their impact on fungal growth inhibition and on mycotoxin accumulation (DON, 15-ADON, zearalenone and fumonisins B1 and B2) were evaluated.

Additionally, ten bacterial isolates were selected to evaluate the impact of their cell free extracts (CFEs) on fungal growth and mycotoxin production.

Material and methods

Streptomyces isolates and fungal strains

Antagonistic in vitro evaluation

Dual culture assays were performed on Czapek Yeast Extract Agar medium (CYA: 30 g/L sucrose, 5 g/L yeast extract, 1 g/L K2HPO4, 0.3 g/L NaNO3, 0.05 g/L KCl, 0.05 g/L MgSO4, 1 mg/L FeSO4, 1 mg/L ZnSO4, 0.5 mg/L CuSO4, 15 g/L agar, pH ~7.4). For this, 10 µL of Streptomyces isolate at 10 7 spores/mL were inoculated on each side of the plate and allowed to grow at 25°C. On the third day, 10 µL of a pathogen spore suspension at 10 6 spores/mL were added to the center of the Petri dish. Then, plates were inoculated for 6 and 7 days for F. graminearum and F. verticillioides, respectively, before image analysis and mycotoxin extraction. As a control, the pathogen was inoculated on a CYA plate without bacteria. All tests were done in triplicate.

Impact of CFEs on fungal growth and mycotoxin production Ten Streptomyces isolates were selected, according to their dual culture assay results, to produce CFEs in order to evaluate the impact of their metabolites on F. graminearum and F. verticillioides growth and mycotoxin production. Bacteria were cultured in CYB medium for 5 days at 25°C and 180 rpm. Then, liquid cultures were centrifuged at 10,000×g for 5 min and filtered through a PES (Polyethersulfone) filter at 0.22 µm to eliminate bacterial cells. In order to identify a potential effect of actinobacteria extracellular enzymes, CFEs were also tested after a heat treatment at 100 •C for 10 min. The treated CFEs were added at 10% to CYA medium. Then, 10 µL of the pathogen spore suspension at 10 6 spores/mL were added to the center of the plate and left for 6 or 7 days at 25°C in the dark. As a control, the pathogen was inoculated on a CYA plate without CFEs. All tests were done in triplicate.

Fungal surface growth measurement and mycotoxin specific production calculation Surface growth of both Fusarium was measured using ImageJ software (1.52a, Wayne

Rasband National Institute of Health, Bethesda, MD, USA, 2018). Growth inhibition was established in comparison to the control without Streptomyces bacteria, which represented 100% of growth. Results are given in % of control.

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Mycotoxin specific production was calculated by dividing the mycotoxin amount in the whole sample by the area of the fungal colony. This calculation allows to compare the mycotoxin production for the same fungal surface between the control and the different treatments.

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Mycotoxin extraction and quantification

For mycotoxin extraction, the whole content of the Petri dish was transferred into a plastic container and the agar was cut into small pieces with a scalpel. After weighing the sample, 85 mL of solvent (acetonitrile/water/acetic acid, 79:20:10) were added to the container, followed by 20 min of agitation at 250 rpm. For trichothecenes, 500 µL of the mixture were diluted 1:50 with the mobile phase (acidified water, 0.5% acetic acid) before injection. For fumonisins, 500 µL of the mixture were diluted to 1:4 with the mobile phase.

After dilution, samples were thoroughly vortexed before filtering through a 0.45 µM CA (cellulose acetate) filter for the HPLC-MS/MS analysis.

Mycotoxin analysis

For both pathogens, mycotoxin detection and quantification were achieved using an Ultra High-Performance Liquid Chromatography (UHPLC, Shimadzu, Tokyo, Japan) coupled with a mass spectrometer (8040, Shimadzu, Tokyo, Japan). LC separation was performed using a Phenomenex Kinetex XB Column C18 (50 mm× 2 mm; 2.6 µm particles) at 50 •C, with an injection volume of 50 µL. Mobile phase composition was (A) 0.5% acetic acid in ultra-pure water and (B) 0.5% acetic acid in isopropanol (HPLC MS grade, Sigma, St Louis, MO, USA), and the mobile phase flow rate was 0.4 mL min-1. The mass spectrometer was operated in electrospray positive (ESI+) and negative (ESI-) ionization mode, and two multiple reaction monitoring (MRM) transitions for each analyte were monitored for quantification (Q) and qualification (q) (Table 4, Table 5). All data were analyzed using LabSolution Software (v5.91/2017, Shimadzu, Tokyo, Japan,2017). Limits of detection or quantification (LOD/LOQ in ng/mL, respectively) for each mycotoxin were: DON (4/14), 15-ADON (10/35), FB1 (0.03/0.1), and FB2 (0.01/0.05) [START_REF] Pellan | Commercial Biocontrol Agents Reveal Contrasting Comportments Against Two Mycotoxigenic Fungi in Cereals: Fusarium graminearum and Fusarium verticillioides[END_REF]).

Results

Antagonistic in vitro evaluation in dual culture

In vitro dual culture assays allowed to determine that F. verticillioides growth was more easily inhibited by Streptomyces isolates than F. graminearum, with a decrease going down to 27% of the control by isolate IX48 (mean=63%, median=67%) for F. verticillioides and to 66% of the control (mean=83%, median=84%) by isolate IX24 for F. graminearum (Fig. 51).

The in vitro growth of F. graminearum was faster and very invasive, filling the whole surface of the Petri dish in about 6 days, while F. verticillioides growth was slightly slower and less voluminous (Annex 1). This slower growth kinetics could contribute to the higher efficiency observed for F. verticillioides growth inhibition (Fig. 52). Cluster II is composed by Streptomyces isolates having the strongest growth inhibition capacities, and by the isolates causing the highest decrease on mycotoxins amount. Isolates in subcluster IIB were especially efficient at decreasing both F. verticillioides growth and fumonisin accumulation, particularly isolates IX11 and IX23. Most isolates in subcluster IIA also strongly decreased F. verticillioides growth, but the reduction of fumonisin accumulation was limited. In fact, isolates in subcluster IIA1 had either no impact on fumonisins or caused very slight overaccumulations.

Impact of cell free extracts on fungal growth and mycotoxin production

The impact of ten Streptomyces isolates cell free extracts (CFEs) on F. graminearum and F. verticillioides growth and mycotoxin production was also evaluated by adding them to the fungal growth medium (10% v/v).

Results show that fungal growth inhibition by CFEs was limited for F. graminearum, with a maximal growth decrease by the unheated CFEs to 86% of the control (mean=95%, median=96%) caused by isolate IX09 and to 80% of the control (mean=91%, median=89%) for the heated CFEs, caused by isolate IX49. Surprisingly, antifungal activity of the CFEs was slightly increased by the thermal treatment (Fig. 54, left). Concerning F. verticillioides, unheated CFEs were more effective at decreasing fungal growth down to 37% of the control (mean=61%, median=63%) by isolate IX09 as compared to a decrease down to 49% of the control (mean=71%, median=73%) by the heated CFE of isolate IX49 (Fig. 54, right).

DON and 15-ADON accumulation were decreased to a greater extent by unheated CFEs (DON mean accumulation = 32% of the control, 15-ADON = 26%) than by heated CFEs (DON = 42%, 15-ADON = 48%), with isolates IX09, IX23 and IX49 causing the strongest decreases. No ZEN production was detected on the CFEs assays. In contrast, accumulation of FB1 and FB2 was enhanced up to 800% of the control on average for both mycotoxins under both modalities.

In particular, unheated CFEs of isolate IX09, provoked one of the highest mycotoxin stimulations (about 13 times more than the control) along with its highest inhibition of fungal growth. The most important increase on fumonisins was caused by IX02 (about 15 times more than the control) (Fig. 54, right).

The Pearson correlation index in Figure 55 shows the correlations between the effect of Streptomyces isolates on the accumulation of mycotoxins for both Fusarium sp. during dual cultures and on bacterial CFEs assays.

In the case of F. graminearum, the effects of Streptomyces isolates on DON and 15-ADON accumulation during dual culture are strongly correlated (r = 0.79, p<0.001). Similar observations could be made for CFEs whether being heated (r = 0.77, p=.006) or not (r = 0.95, p<0.001). A moderate correlation was also observed between the effect of isolates in dual culture and their unheated CFEs on 15-ADON (r = 0.61, p=.045). Finally, unheated and heated CFEs remained correlated regarding their impact on DON (r = 0.84, p=.001). A slight correlation was observed between the effect on DON and ZEN on dual cultures (r = 0.34, p=.007) (data not shown).

The effect on fumonisins produced by F. verticillioides was more variable. For instance, the impact on FB1 and FB2 in dual cultures had a positive correlation (r = 0.79, p<.001), and a strong positive correlation was also observed between the impact of heated CFEs on both mycotoxins (r = 0.995, p<.001). The effects of dual culture and heated CFEs on FB1 were negatively correlated (r = -0.68, p=.02), and the same occurred with FB2 (r = -0.73, p=.01).

Discussion

During this study, we evaluated the impact of et al. 2018). An alkalinisation of the media by the Streptomyces isolates could explain the overproduction of ZEN observed in our assays, while DON production tends to decrease at higher pH [START_REF] Wu | Optimization for the Production of Deoxynivalenoland Zearalenone by Fusarium graminearum Using Response Surface Methodology[END_REF]. However, the impact on DON and ZEN has a slight positive correlation (r = 0.34, p=.007) so they do not seem to be affected in an opposite way by the bacteria.

The effect of Streptomyces isolates on fumonisins led to moderate decreases, with some isolates causing an overaccumulation (e.g. IX41, IX04 This type of mechanism could explain the decrease on fumonisin amount observed during our study. The observed FB1 and FB2 reductions during dual cultures could be caused by an inhibition of the mycotoxin biosynthetic pathway, or by a degradation of the toxic molecule once it has been produced by the fungi. However, the fact that bacterial metabolites alone (CFEs) strongly enhanced fumonisins production gives a clue that a potential degradation could occur in the dual culture assay, leading to an apparent reduction of fumonisin accumulation. Furthermore, the negative correlations observed between the impact of bacterial cells and CFEs on fumonisin accumulation, suggest a stimulating effect of Streptomyces metabolites that could be masked during dual cultures. Thus, the study of fumonisins degradation by the Streptomyces isolates seems necessary to validate this hypothesis. For instance, fumonisin degradation by the action of carboxylesterases and aminotransferases has been already documented for Sphingomonas sp. and Exophiala sp. [START_REF] Ji | Review on Biological Degradation of Mycotoxins[END_REF][START_REF] Mccormick | Microbial Detoxification of Mycotoxins[END_REF][START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF].

Our results show that CFEs were able to strongly decrease DON and 15-ADON amount without causing any overproduction. The thermal treatment did not eliminate this ability, which suggests the presence of heat-stable compounds [START_REF] Boukaew | Suppression of Rice Sheath Blight Disease Using a Heat Stable Culture Filtrate from Streptomyces philanthi RM-1-138[END_REF]. A hypothesis for the lack of ZEN observed in the CFEs assay, compared to the huge overaccumulation in dual culture, is that this mycotoxin may be produced by F. graminearum as a defense mechanism against the bacteria in dual culture [START_REF] Chen | Fusarium graminearum Trichothecene Mycotoxins: Biosynthesis, Regulation, and Management[END_REF], while the metabolites present in the CFEs do not trigger this response. The decrease on DON provoked by the bacterial CFEs could be due to the presence of mycotoxin production inhibitors or to a degradation by extracellular compounds. However, the great number of studies reporting microbial degradation of trichothecenes suggests that this explanation is highly probable, especially since trichothecenes can be detoxified by several mechanisms such as deepoxidation, oxidation or isomerization [START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF]. Indeed, DON degradation was documented for a series of Nocardioides strains. Additionally, degradation of DON in wheat and barley grain was assessed for the actinobacteria strain Marmoricola sp. Moreover, Eubacterium (Biomin® BBSH 797), has been developed into a commercial product to detoxify animal feed from trichothecenes [START_REF] Ito | A Novel Actinomycete Derived from Wheat Heads Degrades Deoxynivalenol in the Grain of Wheat and Barley Affected by Fusarium Head Blight[END_REF].

Regarding fumonisins, as mentioned before, a massive increase was caused by bacterial CFEs as compared to the control, probably as a fungal reaction to the stress caused The previous results and ours, evidence the interest of assessing the impact of BCAs on mycotoxin amount, since a reduction on disease severity is no always positively correlated to a reduction of mycotoxins production [START_REF] Colombo | Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by QPCR[END_REF].

The obtained correlations seem to indicate that, for each pathogen, the accumulation of the two mycotoxins was reduced simultaneously, which suggests that no reconversion or stimulation from one to the other occurred.

Globally, results indicate that several Streptomyces isolates in the collection have strong antagonistic properties against F. graminearum and F. verticillioides, such as IX11 and IX23, which not only inhibited both pathogenic fungi growth but also decreased their mycotoxin accumulation. However, additional research should be conducted to understand the triggering effect on fumonisin production caused by bacterial CFEs, which could lead to the identification of fumonisin biosynthesis regulators.

Conclusion and perspectives

Our work allowed to identify several Streptomyces isolates with in vitro antifungal properties against F. graminearum and F. verticillioides. Furthermore, several isolates were able to decrease mycotoxin amount, independently of fungal growth inhibition. This in vitro screening represents a first attempt on the identification of potential biocontrol agents.

Furthermore, in order elucidate the antagonistic mechanisms applied by Streptomyces during antagonistic assays, a selection of their CFEs was also evaluated. A concerning result obtained during these experiments was the massive increase on fumonisins production. Which raises an alert on the importance of always assessing the impact that potential BCAs have on mycotoxin amount. Complementary studies on the bacterial strains' ability to degrade the studied mycotoxins, especially fumonisins, could help elucidate the mechanism behind mycotoxin decrease. Furthermore, a more detailed analysis of the impact of CFEs on mycotoxin specific production could led to the identification of metabolites capable of regulating mycotoxin biosynthetic pathway.

In planta assays should be implemented in order to validate the observed antagonistic effects. An intermediate experiment performed on detached leaves or spikelets could represent a relatively fast an easy way to approach more natural conditions. Studies on the efficacity of Streptomyces as biological control agents will help improve the guidelines for the selection of potential antifungal microorganisms, by using integrated in vitro and in planta procedures. Furthermore, the comprehension of their antagonistic mechanisms and the optimization of their modes of application will increase their efficacy and guarantee their safe application, so they ultimately constitute a viable alternative to chemical fungicides.

Synthesis of the effect of Streptomyces isolates on all pathogenic models

The heatmap in Figure 56 summarizes the effect of all the Streptomyces isolates of the QualiSud collection (plus Mycostop®'s strain) on the growth of the seven fungal pathogens evaluated in this study. Two main Clusters are identified, I and II. The isolates with the strongest antifungal activities are assembled on Cluster II (e.g. IX23, IX49, IX50 and IX51). Red symbols indicate those isolates that were unable to degrade the chitin during our preliminary assays. As we can observe, they are mainly grouped on Cluster I, which exhibits inferior fungal inhibition properties. However, the chitinase activity is not directly correlated with an inhibition of the growth of the various fungi, as several isolates in this cluster have limited antifungal effect, even if their enzymatic activity was detected. As our procedure was only qualitative for chitinase activity, it is still possible that large variations in terms of production level occurred between the isolates. Although, this result does suggest that the degradation of the chitin on the fungal cell walls may be one of the mechanisms behind the antifungal activities observed, but not exclusively in the case of strong inhibitor isolates. In fact, the implementation of several mechanisms is well documented among Streptomyces strains [START_REF] Newitt | Biocontrol of Cereal Crop Diseases Using Streptomycetes[END_REF]. For the isolates without chitinolytic activity that still were capable of decreasing fungal growth (IX05 and IX31), two hypotheses can be drawn: the use of an alternative antifungal mechanism, such as antibiosis, or a stimulation of chitin production induced by the presence of chitin in the fungal walls [START_REF] Nazari | High Expression Levels of Chitinase Genes in Streptomyces coelicolor A3(2) Grown in Soil[END_REF].

Pearson correlation coefficients of the impact of Streptomyces isolates on fungal growth are presented in Figure 57 (top). Strong correlations were revealed between the effect of Streptomyces isolates on the growth of the four mycotoxinogenic fungi and M. oryzae, with coefficients ranging from 0.81 to 0.97 (p<.001), while P. nodorum and Z. tritici were slightly less correlated, as their coefficients ranked from 0.33 to 0.63 (p<.01 and p<.001). These results

suggest that there may be a common mechanism behind the inhibition of non-necrotrophic fungi, and that the interaction of Streptomyces isolates with the two necrotrophic fungal strains might be due to a different mode of action. Indeed, singular behaviors were recorded regarding the inhibition of this pathogens. Isolates from Cluster I, mostly inefficient on all other pathogens, were highly active against P. nodorum (i.e. IX39, IX42, IX44, IX41 and IX47) and Z. tritici (e.g. IX01, IX38, IX57, MYC). Necrotrophic fungi are able to resist mycotoxins, lytic enzymes, and reactive oxygen species, as a mechanism to counterattack host defenses The correlations of the effect of bacterial CFEs on the four mycotoxinogenic fungi growth were also calculated (Figure 57,bottom). Results indicate that the effects on A. flavus, F. graminearum and F. verticillioides were generally highly correlated, while those on P.

verrucosum were not. The difference between the impact of CFEs and dual cultures on P.

verrucosum growth suggests that its inhibition requires a potential induction of Streptomyces isolates by the fungi. Indeed, our complementary results confirmed this assumption, as bacterial CFEs produced by isolates stimulated by fungal metabolites exhibited strong antagonistic properties, while non-stimulated CFEs did not (see 4.2.2 Complementary results).

The correlations of the effects of Streptomyces isolates and their CFEs on the accumulation of the four mycotoxins targeted in this study were also calculated (Fig. 57).

Results revealed only one significant correlation in dual culture, between specific production of AFB1 and DON (r= 0.34, p<.05). Regarding CFEs, significant correlations were found almost exclusively between the effect of both CFE treatments (unheated vs heated) on the specific production of the same mycotoxin: r= 0.83 for OTA (p<0.001), r= 0.54 for AFB1 (p<0.001), r= 0.84 for DON (p=0.001) and r=0.67 for FB1 (p=.02).

Seemingly, the effects of Streptomyces isolates on mycotoxin accumulation during the growth of the various fungal pathogens are not conducted by a common mechanism. This could be expected, as the studied mycotoxins possess different structures and biosynthetic pathways [START_REF] Reverberi | Natural Functions of Mycotoxins and Control of Their Biosynthesis in Fungi[END_REF][START_REF] Vanhoutte | Biodegradation of Mycotoxins: Tales from Known and Unexplored Worlds[END_REF].

In conclusion, the capacity of Streptomyces isolates to decrease fungal growth seems to be linked to the production of chitinases, but not exclusively, as many isolates with chitinolytic activity were ineffective at decreasing fungal growth. This suggests that stronger inhibitors,

General conclusion and perspectives

The objective of our study was to evaluate a collection of Streptomyces isolates for their potential as antagonistic agents against various aspects of the control of fungal contamination from the field to the final products.

Initial studies were focused on the interactions of bacterial isolates with four mycotoxinogenic fungi, producers of four of the most prevalent mycotoxin groups: Fusarium Fifty-nine actinobacteria isolates were purified from culture plates, issued from preliminary studies of microbial isolation from crop soil and organic amendments. A strain of Streptomyces griseoviridis retrieved from the commercial product Mycostop® was added as a reference to constitute the collection of 60 actinobacteria evaluated during this work. A preliminary 16S identification allowed to determine that the isolates in the collection belonged all to the Streptomyces genus. A complete genome sequencing is currently been performed for most active isolates, in order to provide a precise identification for further research. Moreover, the obtention of the complete sequences will allow the application of genomic tools to compare the bacterial isolates exhibiting similar and contrasting features, in order to identify the genes implicated in each specific activity and elucidate the mechanisms of interaction.

The first approach to evaluate fungi-bacteria interactions was performed by in vitro dual culture assays of the sixty actinobacteria (including Mycostop®) against all fungal pathogens (except for Puccinia triticina, an obliged biotroph). This general assay allowed at first to identify their fungal growth inhibition capacities. Results indicated that fungal development inhibitions variated between each bacterial isolate and depended on the pathogen considered. However, growth inhibition of most pathogens remained highly correlated, suggesting that at least one common mechanism could be at play. The chitinolytic capacities exhibited by the bacterial isolates could be implied in such observation. That being said, one could note that the ability to degrade chitin was not always linked to a strong antifungal capacity. Thus, additional mechanisms are probably carried out by the stronger fungal growth

inhibitors. An interesting result was the broad antifungal spectrum of several isolates, such as IX23, IX49, IX50 and IX51, which were able to strongly inhibit the growth of all the studied fungi. Interestingly, the in vitro growth inhibition of P. nodorum and Z. tritici was less correlated to the inhibition of the other fungi. This could be explained by the fact that some Streptomyces isolates exhibited significant antifungal capacities only against one of these two pathogens and no other fungi. For instance, IX39, IX42, IX44, IX41 and IX47 inhibited only P. nodorum, while IX30, IX37, IX38, IX57, IX01 and MYC inhibited exclusively Z. tritici. This suggests the implication of particular mechanisms of interaction for these isolates, and these mechanisms seem to be different for each necrotrophic fungus. After this initial antifungal assessment, fungal pathogens were divided into storage pathogens and field pathogens to optimize the subsequent experimental approaches.

The evaluation of Streptomyces' ability to detoxify mycotoxins was conducted on the AFB1 producer A. flavus, and on the OTA producer P. verrucosum. In vitro assays were pursued by evaluating the impact of bacterial CFEs on fungal development and mycotoxin production, and through the assessment of their degradation abilities under various conditions. As actinobacteria cannot be applied directly into food matrices, the main purpose is to identify bacterial candidates for potential sources of detoxifying enzymes and metabolites. However, the use of CFEs was not as efficient, especially with P. verrucosum, which may indicate that both fungi react differently to the bacterial constitutive metabolites.

Interestingly, the antifungal effect of CFEs produced in the presence of P. verrucosum metabolites was drastically increased for some isolates (e.g. IX32, IX56), while no activity was detected without activation. This finding opens new perspectives for the study of the stimulation of Streptomyces' metabolism and the production of active CFEs. Further studies on the impact of different concentrations of CFEs on fungal growth, together with the extraction of active compounds are strongly recommended.

The effects of Streptomyces isolates on OTA accumulation during dual cultures were diverse, with some isolates decreasing it while others enhanced it. By contrast, AFB1 accumulation was decreased by all bacterial isolates during dual cultures. Another important consideration is the observed increase on mycotoxin accumulation caused by certain bacterial CFEs, particularly for OTA, and mostly when the fungal growth was inhibited. The overproduction of OTA could be the result of a fungal defense mechanism that was revealed only occasionally and with few CFEs for A. flavus. However, a potential AFB1 overproduction might be hidden by the strong AFB1 degrading capacities of Streptomyces isolates on solid medium but also in their CFEs, while OTA degradation on solid medium and in CFEs was scarce.

Thus, even when the reduction of pathogenic development is desired, the impact on mycotoxin accumulation caused by the Streptomyces isolates must be considered.

Mycotoxin degrading assays revealed that OTA could be degraded by the bacterial isolates almost exclusively in liquid medium, while the culture conditions had few impacts on the isolates AFB1 degrading capacities. Besides, bacterial CFEs were unable to degrade OTA, but Antagonistic assays against Fusarium graminearum and Fusarium verticillioides showed that F. graminearum growth was hardly inhibited, while F. verticillioides inhibition was achieved by near half of the Streptomyces isolates. Furthermore, mycotoxin accumulation was hardly decreased by most isolates during dual cultures for both pathogens. A restricted number of isolates had strong inhibition properties but some of them also caused an important overaccumulation of the mycotoxins. In the case of ZEN, all bacterial isolates led to a huge overproduction by the fungi. The effect of bacterial CFEs on the growth of both fungi was similar to that of the bacterial cells, and thermal treatment decreased their efficiency for F. graminearum but not for F. verticillioides. This suggests that antifungal compounds are present in the CFEs and that some of them might be heat-stable. Interestingly, bacterial CFEs caused an important decrease on DON and 15-ADON as compared to the dual culture assay, without any mycotoxin stimulation. This was singular, as for P. verrucosum and A. flavus, the opposite was generally observed, with a higher decrease of mycotoxin accumulation in dual culture. Whether, the decrease of DON and 15-ADON is due to metabolites inhibiting mycotoxins biosynthesis, or to a degradation by Streptomyces enzymes remains to be elucidated.

Contrasting results were obtained for FB1 and FB2, for which specific production was stimulated from 4 to 15 times by all the bacterial CFEs, while it was generally decreased during the dual culture assay. The strong stimulation by the CFEs suggests that the reduction of FB1 and FB2 in dual culture could be due to a degradation mechanism rather than an inhibition of the fumonisins pathway. The hypothesis of an induced metabolism of the Streptomyces by the fungi cannot be ignored either. As already done for P. verrucosum, further studies on the induction of Streptomyces metabolism by F. graminearum and F. verticillioides metabolites seems to be an interesting approach for a deeper comprehension of the interactions occurring during bacteria-fungi confrontation. Moreover, it could be a promising strategy to promote the production of potential bioactive compounds.

The occurrence of single toxins in nature is uncommon. They appear mostly as a mix of produced by a consortium of fungi, which often cause additive and synergistic effects. For this reason, isolates capable of decreasing several mycotoxins are of great interest. Further research should include the evaluation of bacterial detoxifying properties of a combination of mycotoxins to get as close as possible to natural conditions. For this, degradation assays of artificially contaminated media with a mix of mycotoxins could be conceived.

In planta biocontrol potential was assessed by foliar application of selected Streptomyces isolates on wheat plants, which resulted in different levels of protection following the pathogen. SNB disease incidence was significantly decreased by two isolates: IX23 and MYC.

STB was decreased by MYC but an undesirable increment on Z. tritici pycnidia was also reported. Finally, a significant protective effect against P. triticina was only exhibited by MYC.

The evaluation of the influence of Streptomyces on the levels of expression of plant defense genes indicated that their biocontrol properties were not linked to an increase of plant immune response but to a more direct antifungal action. This hypothesis was reinforced by the observation of Streptomyces installation on the surface of wheat leaves. As the results of the in vitro and in planta assays on the selected isolates were not correlated, more isolates from the collection should be evaluated in planta, as other potentially bioactive isolates could have been neglected. For further research, the study of the efficiency of various modes of application (such as soil treatment or the addition of culture filtrates) and the persistence of the protective effect represent interesting perspectives. Due to the number of isolates, in planta evaluation can be time consuming and demand a lot of effort, so an intermediate in vitro assay using detached leaves or spikelets can be conducted, as they have shown to be accurate at predicting in planta protection.

Thanks to the identification of active isolates, further studies on the medium-scale culturing of promising candidates could be explored. Such as solid-state fermentation for the production of bacterial spores, and submerged culture for the obtention of secondary metabolites and enzymes. Additionally, the optimal culture conditions should be assessed, as well as the verification of the conservation of the antagonistic/detoxifying activities. These assays could eventually lead to the development of biological products.

In conclusion, this work presents a comprehensive approach for the screening of Streptomyces isolates, and suggests optimization steps to achieve an integral evaluation of their antifungal and/or detoxifying properties. The various assays processed simultaneously helped to understand the mechanisms involved in fungal growth inhibition and mycotoxin decrease. We highlighted the relevance of assessing the impact of fungi-bacteria interactions on mycotoxin accumulation, in order to avoid overproduction of toxic metabolites. We also

Figure 1 .

 1 Figure 1. Diagram of fungi/mycotoxins occurrence risks throughout a cereal industrialization chain.Figure 2. Author's illustration of the main cereal crops. ...................................................................... Figure 3. Authors illustration of the aspect of wheat lesions originated by the main foliar phytopathogenic fungi. .........................................................................................................................Figure 4. SEM observation of urediniospores of P. triticina gathered in the stomata of a wheat leaf.Figure 5. Life cycle of Puccinia striformis f. sp. tritici ............................................................................ Figure 6. SEM observation of Z. tritici mycelium entering the stomata of a wheat leaf. ..................... Figure 7. Lifecycle of the wheat pathogen Zymoseptoria tritici ............................................................ Figure 8. Disease cycle of Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum).Figure 9. Life cycle of Magnaporthe oryzae. ......................................................................................... Figure 10. Factors having an impact on mycotoxin production ............................................................ Figure 11. Author's illustration of microscopical observation of Fusarium graminearum perithecia. .Figure 12. Fusarium graminearum life cycle. ........................................................................................ Figure 13. Chemical structure of deoxynivalenol (DON). ...................................................................... Figure 14. Primary and secondary metabolism pathways in Fusarium graminearum ......................... Figure 15. Chemical structure of zearalenone (ZEN). ........................................................................... Figure 16. Fusarium verticillioides disease cycle, centered on the life of maize .................................. Figure 17. Chemical structure of fumonisin B1. .................................................................................... Figure 18. A proposed pathway for fumonisins biosynthesis ............................................................... Figure 19. Author's illustration of microscopical observation of P. verrucosum conidiophores. ......... Figure 20. Chemical structure of ochratoxin A. .................................................................................... Figure 21. Chemical structure of citrinin ............................................................................................... Figure 22. Critical steps of ochratoxin A biosynthesis .......................................................................... Figure 23. Author's illustration of microscopical observation of A. flavus conidiophores. ..................

Figure 2 .Figure 3 .

 23 Figure 1. Diagram of fungi/mycotoxins occurrence risks throughout a cereal industrialization chain.Figure 2. Author's illustration of the main cereal crops. ...................................................................... Figure 3. Authors illustration of the aspect of wheat lesions originated by the main foliar phytopathogenic fungi. .........................................................................................................................Figure 4. SEM observation of urediniospores of P. triticina gathered in the stomata of a wheat leaf.Figure 5. Life cycle of Puccinia striformis f. sp. tritici ............................................................................ Figure 6. SEM observation of Z. tritici mycelium entering the stomata of a wheat leaf. ..................... Figure 7. Lifecycle of the wheat pathogen Zymoseptoria tritici ............................................................ Figure 8. Disease cycle of Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum).Figure 9. Life cycle of Magnaporthe oryzae. ......................................................................................... Figure 10. Factors having an impact on mycotoxin production ............................................................ Figure 11. Author's illustration of microscopical observation of Fusarium graminearum perithecia. .Figure 12. Fusarium graminearum life cycle. ........................................................................................ Figure 13. Chemical structure of deoxynivalenol (DON). ...................................................................... Figure 14. Primary and secondary metabolism pathways in Fusarium graminearum ......................... Figure 15. Chemical structure of zearalenone (ZEN). ........................................................................... Figure 16. Fusarium verticillioides disease cycle, centered on the life of maize .................................. Figure 17. Chemical structure of fumonisin B1. .................................................................................... Figure 18. A proposed pathway for fumonisins biosynthesis ............................................................... Figure 19. Author's illustration of microscopical observation of P. verrucosum conidiophores. ......... Figure 20. Chemical structure of ochratoxin A. .................................................................................... Figure 21. Chemical structure of citrinin ............................................................................................... Figure 22. Critical steps of ochratoxin A biosynthesis .......................................................................... Figure 23. Author's illustration of microscopical observation of A. flavus conidiophores. ..................

Figure 4 .

 4 Figure 1. Diagram of fungi/mycotoxins occurrence risks throughout a cereal industrialization chain.Figure 2. Author's illustration of the main cereal crops. ...................................................................... Figure 3. Authors illustration of the aspect of wheat lesions originated by the main foliar phytopathogenic fungi. .........................................................................................................................Figure 4. SEM observation of urediniospores of P. triticina gathered in the stomata of a wheat leaf.Figure 5. Life cycle of Puccinia striformis f. sp. tritici ............................................................................ Figure 6. SEM observation of Z. tritici mycelium entering the stomata of a wheat leaf. ..................... Figure 7. Lifecycle of the wheat pathogen Zymoseptoria tritici ............................................................ Figure 8. Disease cycle of Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum).Figure 9. Life cycle of Magnaporthe oryzae. ......................................................................................... Figure 10. Factors having an impact on mycotoxin production ............................................................ Figure 11. Author's illustration of microscopical observation of Fusarium graminearum perithecia. .Figure 12. Fusarium graminearum life cycle. ........................................................................................ Figure 13. Chemical structure of deoxynivalenol (DON). ...................................................................... Figure 14. Primary and secondary metabolism pathways in Fusarium graminearum ......................... Figure 15. Chemical structure of zearalenone (ZEN). ........................................................................... Figure 16. Fusarium verticillioides disease cycle, centered on the life of maize .................................. Figure 17. Chemical structure of fumonisin B1. .................................................................................... Figure 18. A proposed pathway for fumonisins biosynthesis ............................................................... Figure 19. Author's illustration of microscopical observation of P. verrucosum conidiophores. ......... Figure 20. Chemical structure of ochratoxin A. .................................................................................... Figure 21. Chemical structure of citrinin ............................................................................................... Figure 22. Critical steps of ochratoxin A biosynthesis .......................................................................... Figure 23. Author's illustration of microscopical observation of A. flavus conidiophores. ..................

Figure 9 .

 9 Figure 1. Diagram of fungi/mycotoxins occurrence risks throughout a cereal industrialization chain.Figure 2. Author's illustration of the main cereal crops. ...................................................................... Figure 3. Authors illustration of the aspect of wheat lesions originated by the main foliar phytopathogenic fungi. .........................................................................................................................Figure 4. SEM observation of urediniospores of P. triticina gathered in the stomata of a wheat leaf.Figure 5. Life cycle of Puccinia striformis f. sp. tritici ............................................................................ Figure 6. SEM observation of Z. tritici mycelium entering the stomata of a wheat leaf. ..................... Figure 7. Lifecycle of the wheat pathogen Zymoseptoria tritici ............................................................ Figure 8. Disease cycle of Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum).Figure 9. Life cycle of Magnaporthe oryzae. ......................................................................................... Figure 10. Factors having an impact on mycotoxin production ............................................................ Figure 11. Author's illustration of microscopical observation of Fusarium graminearum perithecia. .Figure 12. Fusarium graminearum life cycle. ........................................................................................ Figure 13. Chemical structure of deoxynivalenol (DON). ...................................................................... Figure 14. Primary and secondary metabolism pathways in Fusarium graminearum ......................... Figure 15. Chemical structure of zearalenone (ZEN). ........................................................................... Figure 16. Fusarium verticillioides disease cycle, centered on the life of maize .................................. Figure 17. Chemical structure of fumonisin B1. .................................................................................... Figure 18. A proposed pathway for fumonisins biosynthesis ............................................................... Figure 19. Author's illustration of microscopical observation of P. verrucosum conidiophores. ......... Figure 20. Chemical structure of ochratoxin A. .................................................................................... Figure 21. Chemical structure of citrinin ............................................................................................... Figure 22. Critical steps of ochratoxin A biosynthesis .......................................................................... Figure 23. Author's illustration of microscopical observation of A. flavus conidiophores. ..................

Figure 12 .

 12 Figure 1. Diagram of fungi/mycotoxins occurrence risks throughout a cereal industrialization chain.Figure 2. Author's illustration of the main cereal crops. ...................................................................... Figure 3. Authors illustration of the aspect of wheat lesions originated by the main foliar phytopathogenic fungi. .........................................................................................................................Figure 4. SEM observation of urediniospores of P. triticina gathered in the stomata of a wheat leaf.Figure 5. Life cycle of Puccinia striformis f. sp. tritici ............................................................................ Figure 6. SEM observation of Z. tritici mycelium entering the stomata of a wheat leaf. ..................... Figure 7. Lifecycle of the wheat pathogen Zymoseptoria tritici ............................................................ Figure 8. Disease cycle of Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum).Figure 9. Life cycle of Magnaporthe oryzae. ......................................................................................... Figure 10. Factors having an impact on mycotoxin production ............................................................ Figure 11. Author's illustration of microscopical observation of Fusarium graminearum perithecia. .Figure 12. Fusarium graminearum life cycle. ........................................................................................ Figure 13. Chemical structure of deoxynivalenol (DON). ...................................................................... Figure 14. Primary and secondary metabolism pathways in Fusarium graminearum ......................... Figure 15. Chemical structure of zearalenone (ZEN). ........................................................................... Figure 16. Fusarium verticillioides disease cycle, centered on the life of maize .................................. Figure 17. Chemical structure of fumonisin B1. .................................................................................... Figure 18. A proposed pathway for fumonisins biosynthesis ............................................................... Figure 19. Author's illustration of microscopical observation of P. verrucosum conidiophores. ......... Figure 20. Chemical structure of ochratoxin A. .................................................................................... Figure 21. Chemical structure of citrinin ............................................................................................... Figure 22. Critical steps of ochratoxin A biosynthesis .......................................................................... Figure 23. Author's illustration of microscopical observation of A. flavus conidiophores. ..................

Figure 24 .

 24 Figure 24. Chemical structure of aflatoxin B1........................................................................................ Figure 25. Aflatoxin biosynthesis pathway and key genes in Aspergillus parasiticus. .......................... Figure 26. Summary of physical and chemical processes applied to food commodities being able to decrease mycotoxin amount. ................................................................................................................ Figure 27. Phylogenetic tree of Actinobacteria based on 1,500 nucleotides of 16S rRNA. .................. Figure 28. Left: solid culture of four Streptomyces strains showing their typical powdery morphology. Right: microscopical observation of Streptomyces mycelium............................................................... Figure 29. The life cycle of Streptomyces .............................................................................................. Figure 30. Antagonistic mechanisms exhibited by biocontrol agents .................................................. Figure 31. Methods of application of biocontrol agents....................................................................... Figure 32. Modification of UK-2A to fenpicoxamid and reconversion to UK-2A in plants and fungi ... Figure 33. Chemical structures of aflastatins and Blasticidin A. produced by Streptomyces sp. .......... Figure 34. Workflow of research on microbial detoxification of mycotoxins. ...................................... Figure 35. Workflow of the experimental approach followed during this work. ................................. Figure 36. Sporulation rates of four Streptomyces strains on CYA and ISP4 medium. ....................... Figure 37. Top: microscopical observation of individual and "chained" actinobacteria spores. Bottom: Examples of plots of "events" (bacterial spores passing through the laser beam) ............................ Figure 38. Examples of halo formation by actinobacteria on chitinase degradation and phosphate solubilization assays. ........................................................................................................................... Figure 39. Graphical abstract article Actinobacteria vs OTA............................................................... Figure 40. Example of liquidid culture of actinobacteria (IX32) under 6 modalities of "stimulation".134 Figure 41. Residual OTA on stimulated actinobacteria CFEs. ............................................................. Figure 42. Bar plot of the OTA degradation by three actinobacteria isolates during their development in liquid CYB, and dot plot of the resulting pH values ......................................................................... Figure 43. Microscopical observation of mixed culture of IX20 and P. verrucosum on CYB medium.Figure 44. OTA degradation by stimulated actinobacteria CFEs. ........................................................ Figure 45. Impact of 25% of actinobacteria "stimulated" CFEs on P. verrucosum growth on solid CYA. ............................................................................................................................................................. Figure 46. Impact of 25% of actinobacteria "stimulated" CFEs on P. verrucosum's OTA specific production. .......................................................................................................................................... Figure 47. Heatmap of the effect of Streptomyces isolates and their CFEs on Aspergillus flavus and Penicillium verrucosum growth. .......................................................................................................... Figure 48. Heatmap of the effect of Streptomyces isolates and their CFEs on AFB1 specific production by Aspergillus flavus and on OTA specific production by Penicillium verrucosum ............................. Figure 49. Heatmap of Streptomyces isolates and their CFEs ability to degrade AFB1 and OTA. ....... Figure 50. Pearson's correlation of the OTA and AFB1 degrading capacities of Streptomyces isolates and their CFEs.. .................................................................................................................................... Figure 51. Impact of Streptomyces isolates on fungal growth and mycotoxin accumulation by Fusarium graminearum (top) and Fusarium verticillioides (bottom left) during in vitro dual cultures. Bottom right: Amounts of mycotoxin specific production by the controls. ....................................... Figure 52. Effect of Streptomyces isolates on Fusarium graminearum and Fusarium verticillioides growth during in vitro dual cultures .................................................................................................... Figure 53. Summary heatmap of the effect of Streptomyces isolates on Fusarium graminearum and Fusarium verticillioides growth and mycotoxin accumulation. ........................................................... Figure 54. Impact of Streptomyces cell free extracts (CFEs) at 10% (v/v) on Fusarium graminearum and Fusarium verticillioides growth and mycotoxin production ......................................................... Figure 55. Pearson's correlation of the effects of Streptomyces isolates and their CFEs on DON and 15-ADON produced by F. graminearum and on FB1 and FB2 produced by Fusarium verticillioides.. .

Figure 44 .

 44 Figure 24. Chemical structure of aflatoxin B1........................................................................................ Figure 25. Aflatoxin biosynthesis pathway and key genes in Aspergillus parasiticus. .......................... Figure 26. Summary of physical and chemical processes applied to food commodities being able to decrease mycotoxin amount. ................................................................................................................ Figure 27. Phylogenetic tree of Actinobacteria based on 1,500 nucleotides of 16S rRNA. .................. Figure 28. Left: solid culture of four Streptomyces strains showing their typical powdery morphology. Right: microscopical observation of Streptomyces mycelium............................................................... Figure 29. The life cycle of Streptomyces .............................................................................................. Figure 30. Antagonistic mechanisms exhibited by biocontrol agents .................................................. Figure 31. Methods of application of biocontrol agents....................................................................... Figure 32. Modification of UK-2A to fenpicoxamid and reconversion to UK-2A in plants and fungi ... Figure 33. Chemical structures of aflastatins and Blasticidin A. produced by Streptomyces sp. .......... Figure 34. Workflow of research on microbial detoxification of mycotoxins. ...................................... Figure 35. Workflow of the experimental approach followed during this work. ................................. Figure 36. Sporulation rates of four Streptomyces strains on CYA and ISP4 medium. ....................... Figure 37. Top: microscopical observation of individual and "chained" actinobacteria spores. Bottom: Examples of plots of "events" (bacterial spores passing through the laser beam) ............................ Figure 38. Examples of halo formation by actinobacteria on chitinase degradation and phosphate solubilization assays. ........................................................................................................................... Figure 39. Graphical abstract article Actinobacteria vs OTA............................................................... Figure 40. Example of liquidid culture of actinobacteria (IX32) under 6 modalities of "stimulation".134 Figure 41. Residual OTA on stimulated actinobacteria CFEs. ............................................................. Figure 42. Bar plot of the OTA degradation by three actinobacteria isolates during their development in liquid CYB, and dot plot of the resulting pH values ......................................................................... Figure 43. Microscopical observation of mixed culture of IX20 and P. verrucosum on CYB medium.Figure 44. OTA degradation by stimulated actinobacteria CFEs. ........................................................ Figure 45. Impact of 25% of actinobacteria "stimulated" CFEs on P. verrucosum growth on solid CYA. ............................................................................................................................................................. Figure 46. Impact of 25% of actinobacteria "stimulated" CFEs on P. verrucosum's OTA specific production. .......................................................................................................................................... Figure 47. Heatmap of the effect of Streptomyces isolates and their CFEs on Aspergillus flavus and Penicillium verrucosum growth. .......................................................................................................... Figure 48. Heatmap of the effect of Streptomyces isolates and their CFEs on AFB1 specific production by Aspergillus flavus and on OTA specific production by Penicillium verrucosum ............................. Figure 49. Heatmap of Streptomyces isolates and their CFEs ability to degrade AFB1 and OTA. ....... Figure 50. Pearson's correlation of the OTA and AFB1 degrading capacities of Streptomyces isolates and their CFEs.. .................................................................................................................................... Figure 51. Impact of Streptomyces isolates on fungal growth and mycotoxin accumulation by Fusarium graminearum (top) and Fusarium verticillioides (bottom left) during in vitro dual cultures. Bottom right: Amounts of mycotoxin specific production by the controls. ....................................... Figure 52. Effect of Streptomyces isolates on Fusarium graminearum and Fusarium verticillioides growth during in vitro dual cultures .................................................................................................... Figure 53. Summary heatmap of the effect of Streptomyces isolates on Fusarium graminearum and Fusarium verticillioides growth and mycotoxin accumulation. ........................................................... Figure 54. Impact of Streptomyces cell free extracts (CFEs) at 10% (v/v) on Fusarium graminearum and Fusarium verticillioides growth and mycotoxin production ......................................................... Figure 55. Pearson's correlation of the effects of Streptomyces isolates and their CFEs on DON and 15-ADON produced by F. graminearum and on FB1 and FB2 produced by Fusarium verticillioides.. .

Figure 56 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 6 . 116 Figure 7 . 118 Figure S1 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 8 . 156 Figure S1 .Figure S2 .Figure S3 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 6 .Figure 7 .Figure 8 .

 56123461167118S11234568156S1S2S31234678 Figure 56. Summary heatmap of the effect of Streptomyces isolates on fungal growth of 7 pathogenic fungi. .................................................................................................................................................... 218 Figure 57. Pearson's correlation of the effects of Streptomyces isolates on fungal growth in dual culture (top) or by their CFEs (bottom). .......................................................................................................... 219 Figure 58. Pearson's correlation of the effects of Streptomyces isolates the accumulation of four mycotoxins (OTA, AFB1, DON, FB1) in dual culture (top) or by their CFEs (middle) ............................ 221 Figure S1. Kinetics of fungal growth and mycotoxin production of F. graminearum (up) and Fusarium verticillioides (bottom) growth …………………………………..…………………………………………………………………..257 Figure S2. Kinetics of fungal growth and mycotoxin production of Aspergillus flavus (up) and Penicillium verrucosum (bottom) growth …………………………………..…………………………………………………………………….258

  general conclusion resumes the principal results obtained during this work, highlighting the potential of Streptomyces as biocontrol agents and as a source of bioactive compounds for mycotoxin decontamination of food and feed. Future research perspectives are also suggested, with a focus on the elucidation of decontamination mechanisms displayed by Streptomyces genus and on the verification of the in planta protective effects. It is also suggested that the improvement of screening methodologies will accelerate the process for the development of novel agricultural products based on Streptomyces. The diagram on Figure 1 illustrates the context of the study, depicting the critical points where the presence of fungal pathogens represents a risk for cereal production and safety. Measures to avoid fungal contamination, which are currently applied and under development are indicated, particularly those where Streptomyces and their derived products could play an essential role.

Figure 1 .

 1 Figure 1. Diagram of fungi/mycotoxins occurrence risks throughout a cereal industrialization chain. Current and under development preventive/curative measures, with potential application of Streptomyces and their bioactive compounds, are indicated (icons taken from Freepik.com).

2. 1 Figure 2 .

 12 Figure 2. Author's illustration of the main cereal crops.

  Figure 2. Cont. Author's illustration of the main cereal crops.

Figure 4 .

 4 Figure 4. SEM observation of urediniospores of P. triticina gathered in the stomata of a wheat leaf (Photo taken during this study).

Figure 5 .

 5 Figure 5. Life cycle of Puccinia striformis f. sp. tritici (Schwessinger 2017). Original illustration from Jacolyn A. Morrison at the USDA-ARS Cereal Disease Laboratory, St Paul, MN, USA.

Figure

  Figure 11. Author's illustration of microscopical observation of Fusarium graminearum perithecia.

Figure 13 .

 13 Figure 13. Chemical structure of deoxynivalenol (DON)

Figure 15 .

 15 Figure 15. Chemical structure of zearalenone (ZEN).

Figure 17 .

 17 Figure 17. Chemical structure of fumonisin B1.

Figure 19 .

 19 Figure 19. Author's illustration of microscopical observation of P. verrucosum conidiophores.

Figure 20 .

 20 Figure 20. Chemical structure of ochratoxin A.

Figure 21

 21 Figure 21. Chemical structure of citrinin.

(

  [START_REF] Heussner | Comparative Ochratoxin Toxicity: A Review of the Available Data[END_REF][START_REF] Pfohl-Leszkowicz | Ochratoxin A: An Overview on Toxicity and Carcinogenicity in Animals and Humans[END_REF], which provokes an elevated elimination half-live in humans (35.5 days)[START_REF] Abrunhosa | Biodegradation of Ochratoxin a for Food and Feed Decontamination[END_REF].

Figure 22 .

 22 Figure 22. Critical steps of ochratoxin A biosynthesis (Chen et al. 2018).

  Figure 23. Author's illustration of microscopical observation of A. flavus conidiophores.

Figure 26 ,

 26 Figure 26, modified from Karlovsky et al. 2016, presents a summary of the main physical and chemical treatments used in food processing being able to mitigate mycotoxin risk.

Figure 26 .

 26 Figure 26. Summary of physical and chemical processes applied to food commodities being able to decrease mycotoxin amount. Modified from Karlovsky et al. 2016.

  known as aminopentol 1 (AP1). Next step consists on an oxidative deamination by an aminotransferase that produces 2-keto HFB1 (Loi et al. 2017), which can be further converted to hemiketal derivatives by cyclization of the hydroxyl at C-5 and the C-2 (McCormick 2013).

  metabolites. These species include Pleurotus ostreatus, Trametes versicolor, Rhizopus sp., Mucor sp.; some yeasts such as Trichosporon mycotoxinivorans, Saccharomyces cerevisiae, Trichoderma and Armillariella tabescens. Degradation by the latter originated a product that remained fluorescent, which suggests that the modification took place in the difuran rather than in the lactone fraction of the molecule (McCormick 2013). Bacterial ability to decrease

  Actinobacteria are Gram-positive bacteria with high G+C content in their DNA. They exhibit a wide variety of morphologies, from coccoid (Micrococcus) or rod-coccoid (e.g.Arthrobacter) to fragmenting hyphal forms (e.g. Nocardia) or permanent and highly differentiated branched mycelium (e.g. Streptomyces)[START_REF] Ventura | Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum[END_REF]). Actinobacteria are ubiquitously distributed in both aquatic and terrestrial ecosystems. In soil they develop particularly in the rhizosphere or inside plant roots[START_REF] Rey | Plenty Is No Plague: Streptomyces Symbiosis with Crops[END_REF]. MostActinobacteria are saprophytic and contribute to organic matter decomposition into humus[START_REF] Dhanasekaran | Actinobacteria -Basics and Biotechnological Applications[END_REF]. Filamentous actinobacteria develop by tip extension and branching of hyphae, constituting a vegetative mycelium. Then, in response to nutrient depletion, among other signals, they may form aerial mycelium carrying spores[START_REF] Colombo | Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by QPCR[END_REF]). Due to their extensive metabolism, these bacteria are of major importance for biotechnology, medicine, and agriculture. The genus Streptomyces accounts for over 95% of the Actinomycetales strains isolated from soil[START_REF] Barka | Taxonomy, Physiology, and Natural Products of Actinobacteria[END_REF]. Figure27depicts the phylogenetic tree of Actinobacteria.

Figure 27 .

 27 Figure27. Phylogenetic tree of Actinobacteria based on 1,500 nucleotides of 16S rRNA. Scale bar, 5 nucleotides[START_REF] Ventura | Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum[END_REF]).

Figure 28 .

 28 Figure 28. Left: solid culture of four Streptomyces strains showing their typical powdery morphology. Right: microscopical observation of Streptomyces mycelium.

  2.4.2.2 Antagonistic mechanisms deployed by Streptomyces as BCAsAs mentioned before, Streptomyces species are able to employ a series of direct and indirect mechanisms that allow them to combat pathogenic development, such as the production of antifungal molecules and the competition for the available nutriments. Direct antagonisms are linked to the physical contact of both microorganisms, while indirect antagonisms do not require sensing or targeting the pathogen by the BCA[START_REF] Pal | Biological Control of Plant Pathogens[END_REF]. The interaction with the plant host constitutes another mode of action of Streptomyces species, as it usually leads to an improvement of plant resistance and adaptation to biotic and abiotic factors. Generally, Streptomyces species with potential as BCAs employ more than one antagonistic mechanism[START_REF] Legein | Modes of Action of Microbial Biocontrol in the Phyllosphere[END_REF]) (Fig.30).

Figure 30 .

 30 Figure 30. Antagonistic mechanisms exhibited by biocontrol agents (Legein et al. 2020).

  . Another compound, Fenpicoxamid (Intrateq™ active), a derivative of the natural compound UK-2A, originally isolated from Streptomyces fermentation broths, is a fungicide with strong inhibitory capacity against Zymoseptoria tritici and Puccinia triticina growth on in vitro and greenhouse experiments, with effects equivalent to those of the commercial fungicide fluxapyroxad (Owen et al. 2017) (Fig. 32). Also, xiamycins, originated from Streptomyces species, demonstrated complete inhibition of Z. tritici growth on assays performed by Pfaffenbach et al. (Pfaffenbach et al. 2019). Antibiotics produced by Streptomyces spp. such as Blasticidin-S (S. griseochromogenes) and Kasugamycin (S. kasugaensis) have been used for the control of rice blast and further Studies on oligomycin A produced by S. diastatochromogenes demonstrated its ability to control this disease under greenhouse conditions (Law et al. 2017).

Figure 32 .

 32 Figure 32. Modification of UK-2A to fenpicoxamid and reconversion to UK-2A in plants and fungi (Leader et al. 2018).

Figure 33 .

 33 Figure 33. Chemical structures of aflastatins and Blasticidin A. produced by Streptomyces sp. (Sakuda 2010).

Figure 34 .

 34 Figure34. Workflow of research on microbial detoxification of mycotoxins[START_REF] Zhu | isolats préservée à l'UMR Qualisud. Une extraction du génome 16S a permis une identification préliminaire indiquant que les isolats de la collection appartiennent au genre Streptomyces. L'évaluation in vitro de leurs propriétés PGP (« plant growth promotion », en anglais) indique que plusieurs souches de la collection sont capables de solubiliser le phosphate, ainsi que de produire de la chitinase, des sidérophores[END_REF]).

3 . 4 .

 34 Evaluation of the effect of bacterial isolates on mycotoxin production by mycotoxinogenic fungi. Study of fungal development and mycotoxin accumulation in the presence of bacterial metabolites. 5. Evaluation of the mycotoxin degrading capacity of actinobacteria isolates and their metabolites. These various assays led to a series of research questions: I. Are the inhibitions observed in various fungi-actinobacteria interactions universal among mycotoxinogenic fungi focusing on: a. The growth inhibition b. The reduction of mycotoxin accumulation II. What are the mechanisms behind the variation on mycotoxin accumulation when actinobacteria are confronted with fungi? III. Does mycotoxin degradation by the actinobacteria isolates lead to an effective detoxification? IV. Can actinobacteria antifungal effects on wheat pathogenic fungi in vitro be transposed in planta on wheat leaves? The concomitant analysis of the results obtained for the different parameters by Clusterization and Pearson correlation allowed us to compare and regroup the evaluated isolates according to their similarities. The different models studied were analyzed thoroughly to highlight common and contrasting features between them. This allowed the identification of the potential antagonistic mechanisms involved, as well as the selection of promising actinobacteria isolates for further research. The workflow presented in figure 35 displays the experimental process carried out during this work. The obtained results are presented in six sections presented as scientific articles. One of these articles is already published, the second was recently accepted for publication, and another one has been submitted. Two additional sections include the synthesis and statistical comparison of the results obtained for the different models studied.

Figure 35 .

 35 Figure 35. Workflow of the experimental approach followed during this work. Orange boxes represent the implemented experimental protocols and green boxes indicate the main research questions.

Figure 36 .

 36 Figure 36. Sporulation rates of four Streptomyces strains on CYA and ISP4 medium after 11 days of culture at 28°C. Actinobacteria spore production and enumeration

Figure 37 .

 37 Figure 37. Top: microscopical observation of individual and "chained" actinobacteria spores. Bottom: Examples of plots of "events" (bacterial spores passing through the laser beam) obtained by the flux cytometer for isolates IX06, IX23, IX35 and IX43.

  Figure 39. Graphical abstract

  CFEs on growth and OTA production by P. verrucosum Stimulated CFEs (25%) were added to CYA medium before pouring the plates. Spores of P. verrucosum were inoculated at the center of the Petri dish (10 µL at 10 6 spores/mL) and incubated for 12 days at 25°C. Fungal growth was calculated by measuring the area of the fungal colony on ImageJ software (1.52a, Wayne Rasband National Institute of Health, Bethesda, MD, USA, 2018). OTA extraction and quantification was carried out as described earlier (Campos-Avelar et al. 2020).

Figure 41

 41 Figure 41 presents the residual OTA amount in the bacterial supernatants produced with the toxins or in co-culture with the fungi. Results indicate that actinobacteria were unable to degrade the OTA initially present in the liquid medium under modality 3 (CYB + OTA), which differs from the results obtained in our previous work (Campos-Avelar et al. 2020). The unique

Figure 41 .

 41 Figure 41. Example of liquid culture of actinobacteria (IX32) under 6 modalities of "stimulation". 1: CYB (unmodified), 2: CYB + Acetonitrile (5.64 mL/L), 3: CYB + OTA (260 µg/L) in acetonitrile (5.64 mL/L), 4: CYB + CFEs of P. verrucosum's liquid culture (25%), 5: CYB + CFEs of P. verrucosum's solid culture (25%), 6: CYB + fungal inoculum of P. verrucosum (50 µL at 10 7 spores/mL).

Figure 40 .

 40 Figure 40. Residual OTA after culturing actinobacteria under stimulating modalities: CYB + OTA (260 µg/L) in acetonitrile (5.64 mL/L), CYB + CFEs of P. verrucosum's liquid culture (25%), CYB + CFEs of P. verrucosum's solid culture (25%), CYB + fungal inoculum of P. verrucosum (50 µL at 10 7 spores/mL). 0D: OTA dosage at day 0. Control: OTA cultured under the same conditions without actinobacteria. ND: Not detected.

Figure 42 .

 42 Figure 42. Bar plot of the OTA degradation by three actinobacteria isolates during their development in liquid CYB, and dot plot of the resulting pH values. OD: OTA dosage at day 0. Control: CYB medium + OTA cultured under both conditions: P = plastic container, E = Erlenmeyer flask.

Figure 43 .

 43 Figure 43. Microscopical observation of mixed culture of IX20 and P. verrucosum on CYB medium after 11 days at 25°C and 180 rpm. Fungal mycelium is indicated by the red arrows.

Figure 44 .

 44 Figure 44. OTA degradation by stimulated actinobacteria's CFEs. Initial OTA = 226 ng/mL.

Figure 45 .

 45 Figure 45. Impact of 25% of actinobacteria "stimulated" CFEs on P. verrucosum growth on solid CYA. Control: CYA medium without bacterial CFEs. Modality 1: CYB (unmodified), 2: CYB + Acetonitrile (5.64 mL/L), 3: CYB + OTA (260 µg/L) in acetonitrile (5.64 mL/L), 4: CYB + CFEs of P. verrucosum's liquid culture (25%), 5: CYB + CFEs of P. verrucosum's solid culture (25%), 6: CYB + fungal inoculum of P. verrucosum (50 µL at 10 7 spores/mL). NA: Not available data.

Figure 46 .

 46 Figure 46. Impact of 25% of actinobacteria "stimulated" CFEs on P. verrucosum's OTA specific production in solid CYA. Control: CYA medium without bacterial CFEs. Modality 1: CYB (unmodified), 2: CYB + Acetonitrile (5.64 mL/L), 3: CYB + OTA (260 µg/L) in acetonitrile (5.64 mL/L), 4: CYB + CFEs of P. verrucosum's liquid culture (25%), 5: CYB + CFEs of P. verrucosum's solid culture (25%), 6: CYB + fungal inoculum of P. verrucosum (50 µL at 10 7 spores/mL). NA: Not available data.

  In a study conducted by Traxler et al., color change in a co-culture of Streptomyces coelicolor with several other actinobacteria was caused by the production of pigmented antibiotics (Traxler et al. 2013). Co-cultures of Streptomyces albospinus and Phomopsis sp. lead to a morphology change by a color switch from clear to dark brown and the production of the antifungal compound amphotericin B (Chagas and Pupo 2018

  ), and mycotoxin degradation (Figure 49). Clear yellow indicates a strong activity (antifungal, detoxifying), while dark purple to black indicate a lack of activity or a mycotoxin boost, intermediate colors indicate moderate activities. Isolates are clustered according to the measured activities. For a more detailed explanation about heatmap construction and interpretation, please refer to previously published work (Campos-Avelar et al. 2020).As observed on the heatmap in Figure47, Streptomyces isolates with the strongest antifungal effect against A. flavus (A) were also capable of inhibiting P. verrucosum (P) development and therefore are grouped in Cluster II. As mentioned previously, this presents an important advantage considering that during fungal contamination of grains, both pathogens can occur simultaneously. Globally, CFEs were more efficient to control A. flavus, as P. verrucosum development was hardly affected. A decrease on CFEs antifungal capacities was evidenced for both pathogens.

Figure 47 .

 47 Figure 47. Heatmap of the effect of Streptomyces isolates and their CFEs on Aspergillus flavus (A) and Penicillium verrucosum (P) growth. Results are given in a range of colors according to their Zscore, where purple to black represents a lack of activity or an increase compared to the control, whereas yellow represents the strongest activity. Grey = missing value.

Figure 48

 48 Figure48illustrates the effect of the Streptomyces collection on mycotoxin specific production by both pathogens. During dual cultures, bacterial isolates were much more efficient at avoiding AFB1 accumulation than OTA accumulation. No isolate caused an overproduction of aflatoxin by A. flavus compared to the control, whereas a whole cluster (II) stimulated OTA production by P. verrucosum. Some isolates such as IX09, IX12, IX37 and IX43 (green arrows) were highly efficient at decreasing both pathogens' mycotoxin specific production, which makes them potential candidates for multitoxin inhibition/degradation assays. CFEs were more efficient at controlling AFB1 amount, compared to that of OTA, as for the latter several of them caused a boost of mycotoxin specific production, especially IX52 and IX09 (red arrows).

Figure 49

 49 Figure 49 presents the capacity of each Streptomyces isolate to degrade AFB1 and OTA, either during bacterial growth on solid and liquid medium or by contact with their CFEs. This heatmap has the most contrasting results. For instance, degradation of OTA during bacterial development on solid medium was only achieved by five isolates (Cluster V), while only 5

Figure 48 .

 48 Figure 48. Heatmap of the effect of Streptomyces isolates and their CFEs on AFB1 specific production by Aspergillus flavus and on OTA specific production by Penicillium verrucosum. Results are given in a range of colors according to their Z-score, where purple to black represents a lack of activity or an increase compared to the control, whereas yellow represents the strongest activity. Grey = missing value.

Figure 49 .

 49 Figure 49. Heatmap of Streptomyces isolates and their CFEs ability to degrade AFB1 and OTA. Results are given in a range of colors according to their Z-score, where purple to black represents a lack of activity or an increase compared to the control, whereas yellow represents the strongest activity. Grey = missing values.

Figure 50 .

 50 Figure 50. Pearson's correlation of the OTA and AFB1 degrading capacities of Streptomyces isolates and their CFEs. In the correlation scale, the 1 in blue indicates a strong positive correlation and the -1 in dark red is a strong negative correlation.

Fifty- nine

 nine Streptomyces isolates from Qualisud collection plus a Streptomyces griseoviridis strain isolated from the commercial product Mycostop® were employed during this work. Their conservation and spore recollection have already been described in previously published work (Campos-Avelar et al. 2020). F. graminearum isolate BRFM 1967 and F. verticillioides isolate BRFM 2251 were purchased from the International Centre of Microbiological Resources in Marseille (CIRM,University of Aix-Marseille, Marseille, France). Fusarium strains were maintained at 4°C in inclined PDA (Biokar BK095HA) tubes covered with paraffin oil. Fungal spores were produced by inoculating PDA plates and culturing them at 25°C for 7 days. Then, spores were scrapped from the surface by adding 5 mL of distilled sterile water and filtered through sterile cotton to eliminate fungal mycelium. Fungal spores were cultured, harvested and enumerated before each test.

Figure 51 .

 51 Figure 51. Impact of Streptomyces isolates on fungal growth and mycotoxin accumulation by Fusarium graminearum (top) and Fusarium verticillioides (bottom left) during in vitro dual cultures on CYA at 25°C for 6 and 7 days, respectively. Bottom right: Amounts of mycotoxin specific production by the controls.

  The heatmap presented in Figure53regroups the observed effects on fungal growth and mycotoxin production for each Streptomyces isolate during dual cultures. This representation allows to identify the particular impact of each isolate, as well as to group them according to their characteristics. A standardization was made to allow the comparison between the different parameters (growth, mycotoxin production), by converting the percentage values on z-scores separated in four categories: fungal growth, DON and 15-ADON, zearalenone and fumonisins. The obtained z-scores were then color coded, lower z-scores (clear yellow) correspond to a strong antagonistic activity, while weak antagonistic activities or isolates causing the strongest boost on mycotoxin production were colored dark purple to black. Intermediate colors represent moderate activities. Lastly, the similarity between Streptomyces isolates effects was calculated through the Euclidean distance.As observed in Figure53, bacterial isolates were separated in two main Clusters, I and II. Cluster I contains isolates with moderate to null effects on fungal growth, and also those isolates causing the highest stimulation of mycotoxin production. Subcluster IA1 provoked an overaccumulation of DON and 15-ADON, while subcluster IB1 caused an overproduction of fumonisins. Finally, subcluster IB2 caused the highest increase on ZEN production.

Figure 53 .

 53 Figure 53. Summary heatmap of the effect of Streptomyces isolates on Fusarium graminearum and Fusarium verticillioides growth and mycotoxin accumulation. The results are given in a range of colors according to their Z-score where purple to black indicated a weak activity or an increase as compared to the control and yellow indicated the strongest activity. Grey stars indicate the isolates selected for CFEs assay. A missing value is indicated in grey.

Figure 54 .

 54 Figure 54. Impact of Streptomyces cell free extracts (CFEs) at 10% (v/v) on Fusarium graminearum and Fusarium verticillioides growth and mycotoxin production on CYA at 25°C.

Figure 55 .

 55 Figure 55. Pearson's correlation of the effects of Streptomyces isolates and their CFEs on DON and 15-ADON produced by F. graminearum and on FB1 and FB2 produced by Fusarium verticillioides. In the correlation scale, the 1 in blue indicates a strong positive correlation and the -1 in dark red is a strong negative correlation.

  by the Streptomyces metabolites[START_REF] Adeline | Factors of the Fusarium verticillioides-Maize Environment Modulating Fumonisin Production[END_REF]. Similar fumonisin increases were documented during the application of sublethal doses of fungicides to F. proliferatum[START_REF] Cendoya | Effect of Fungicides Commonly Used for Fusarium Head Blight Management on Growth and Fumonisin Production by Fusarium proliferatum[END_REF]. It is therefore conceivable that the low concentration of bacterial CFEs used in this study (10% v/v) might have stimulated the production of fumonisins in a similar way. Further studies with different concentrations of bacterial CFEs will allow this hypothesis to be explored. Contrasting results were obtained by Dalié et al. when studying the antagonistic capacity of the lactic acid bacteria Pediococcus pentosaceus against F. verticillioides. They determined that co-inoculation with bacterial cells inhibited fungal growth and in parallel caused an increase on fumonisin production. Inversely, when they used bacterial CFEs, fumonisins biosynthesis was significantly decreased[START_REF] Dalié | Impact of Pediococcus pentosaceus Strain L006 and Its Metabolites on Fumonisin Biosynthesis by Fusarium verticillioides[END_REF].Furthermore, a study performed byDanial et al. depicted an important decrease on fumonisins when F. verticillioides was cultured with Streptomyces' CFEs[START_REF] Danial | Efficacy of Metabolites of a Streptomyces Strain (AS1) to Control Growth and Mycotoxin Production by Penicillium verrucosum, Fusarium verticillioides and Aspergillus fumigatus in Culture[END_REF]).

Figure 57 .

 57 Figure 57. Pearson's correlation of the effects of Streptomyces isolates on fungal growth in dual culture (top) or by their CFEs (bottom). Dual cultures were conducted against 7 fungi while CFEs were evaluated on 4. In the correlation scale, the 1 in blue indicates a strong positive correlation and the -1 in dark red is a strong negative correlation. CFE: Unheated cell free extracts. HCFE: Heated cell free extracts.

  such as isolates IX23, IX49, IX50 and IX51, might involve other antagonistic mechanisms, which makes them promising candidates for further research on antifungal properties. Moreover, correlations indicate the possible existence of two different mechanisms of interaction between Streptomyces and saprophytic/hemibiotrophic fungi and necrotrophic fungi. This is underlined by the singular interactions observed with isolates mostly inactive from Cluster I.Furthermore, the mechanisms behind mycotoxin decrease seem to be different for each mycotoxin.

Figure 58 .

 58 Figure 58. Pearson's correlation of the effects of Streptomyces isolates the accumulation of four mycotoxins (OTA, AFB1, DON, FB1) in dual culture (top) or by their CFEs (middle). In the correlation scale, the 1 in blue indicates a strong positive correlation and the -1 in dark red is a strong negative correlation.

  graminearum and Fusarium verticillioides, infecting wheat on the field and producing deoxynivalenol and fumonisins, respectively, and Aspergillus flavus and Penicillium verrucosum, developing mostly during the storage of grains and producers of aflatoxins and ochratoxin A, respectively. The impact of Streptomyces isolates on fungal growth and mycotoxin accumulation, as well as their ability to degrade mycotoxins revealed interesting mechanisms of decontamination, but it also evidenced some singular cases where mycotoxin specific production was enhanced. Afterwards, we focused on foliar phytopathogens of wheat, exhibiting different pathogenic mechanisms: Parastagonospora nodorum and Zymoseptoria tritici, which are necrotrophic, Magnaporthe oryzae, an hemibiotroph, and Puccinia triticina an obliged biotroph. A first assessment of the antifungal activity of the Streptomyces isolates against these phytopathogens was conducted (except for P. triticina), followed by in planta assays. The results revealed the lack of correlation between the antifungal capacities measured in vitro and the protective capacity once applied to the plant, despite the apparent leaf colonization by Streptomyces isolates. Overall, the designed experimental steps allowed us to answer to a series of research questions with gradual increasing complexity, and to identify new research perspectives. Clustering and Pearson correlation analyses allowed us to have a global view of the results and to compare the effect of all the isolates on each parameter measured. These statistical approaches facilitated the comprehension of the antagonistic mechanisms implemented by the Streptomyces isolates evaluated, and the identification of the most promising candidates for further research. The most relevant findings will be detailed in this conclusion, as well as some improvement points that will allow a deeper understanding of the interactions between Streptomyces and pathogenic fungi.

  they effectively degraded AFB1. Several hypotheses could have explained the previous result, such as an internal metabolization of OTA by the bacterial cells, or the stimulation of degradation mechanisms in the presence of fungal metabolites or even the influence of the pH. In our complementary study, the stimulation of bacterial CFEs by the addition of OTA or fungal metabolites during the growth of Streptomyces isolates did not confirm the theory of induced extracellular degrading enzymes. Indeed, despite the observed modification of bacterial metabolism (i.e. production of pigments), the resulting CFEs remained unable to degrade the mycotoxin. Nevertheless, bacterial cells' degradation capacities were stimulated by the fungal metabolites, which reinforces the hypothesis of an intracellular degradation of OTA. The role of the pH remains not fully elucidated but seemed correlated to a degradation of the toxins only when the pH remained around 7, perhaps linked to a slower growth rate of actinobacteria. Further studies implementing pH variations could help to get insight into OTA degradation mechanisms and to improve the degradation efficiency.For AFB1 a subsequent verification of the residual toxicity for the strongest degrading bacteria in liquid medium (IX20, IX45 and MYC) allowed to determine that these Streptomyces isolates effectively reduced the genotoxicity of AFB1. Streptomyces isolates that displayed no degradation capacities but decreased mycotoxin accumulation in dual cultures and by their CFEs, might produce active compounds able to inhibit mycotoxin biosynthetic pathway. This is the case for IX57, IX47, IX53, IX37, IX38 and IX16 for AFB1 and IX37, IX01, IX12, IX43 and IX09 for OTA. Further research could target the identification of active fractions and the isolation of enzymes and compounds capable of decreasing aflatoxin accumulation.

  made an emphasis on the lack of correlation between in vitro and in planta assays and the importance of considering the tripartite interaction BCA-Pathogen-Host by pursuing screenings up to in planta validation. The use of a large set of fungi and bacteria allowed us to analyze a great variety of scenarios and to identify not only Streptomyces isolates having a wide spectrum of activity, but also those exhibiting particular behaviors, encouraging additional research. To sum up, we validated the potential of Streptomyces isolates in the collection as promising sources of active enzymes and metabolites for the decontamination of mycotoxins in food matrices. Our results contribute to the search for more ecological solutions and to increase the availability of biological alternatives to substitute the chemical management of fungal pathogens and mycotoxins risk. Bacon, Charles W., Dorothy M. Hinton, and Trevor R. Mitchell. 2017. "Is Quorum Signaling by Mycotoxins a New Risk-Mitigating Strategy for Bacterial Biocontrol of Fusarium verticillioides and Other Endophytic Fungal Species?" Journal of Agricultural and Food Chemistry 65(33):7071-80.

Figure S2 .

 S2 Figure S2. Kinetics of fungal growth and mycotoxin production of Aspergillus flavus (up) and Penicillium verrucosum (bottom) grown on CYA medium at 25°C. Green arrow indicates the chosen incubation time for the antagonistic assays described in this work.

Table 2 .

 2 Evaluation of the residual toxicity of CYB supernatants issued of the degradation of AFB1 by Streptomyces isolates……………………………………………………………………………………………………………………..154

Table S1 .

 S1 Values of the Pearson correlation (r) between the measured parameters during the screening of the effect of Streptomyces isolates and Mycostop® and their CFEs on A. flavus growth and AFB1

	accumulation………………………………………………………………………………………………………………………………….171
	III.	Biocontrol of wheat phytopathogens using Streptomyces isolates

Table 1 .

 1 Primers used for plant gene expression analysis………………………………………………………………..187

	List of abbreviations
	3-ADON	3-adenosyldeoxynivalenol
	15-ADON	15-adenosyldeoxynivalenol
	AFB1	Aflatoxin B1
	AFB2	Aflatoxin B2
	AFM1	Aflatoxin M1
	AFG1	Aflatoxin G1
	ɑW	Water activity
	BCA	Biocontrol agent
	C-	Carbon
	CA	Cellulose acetate
	CAS	Chrome azurol sulphonate
	CFEs	Cell free extracts

  Mycotoxins are secondary metabolites of low molecular weight produced primarily by filamentous fungi, mainly of the genera Aspergillus, Fusarium and Penicillium. They are toxic to humans and animals, as they provoke a range of disorders going from gastroenteritis to cancer. Mycotoxins are one of the most toxic contaminants in the food chain. These metabolites are frequently found on food products such as cereals, fruits, nuts and meat, among others. According to the Rapid Alert for Food and Feed (RASFF), in the European Union their presence constitutes the major source of risk notifications (20%)[START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]).

	2.1.3.1 Mycotoxins

  During the infection process, Fusarium species can produce several trichothecene mycotoxins, such as DON and NIV[START_REF] Haile | Fusarium Head Blight in Durumwheat: Recent Status, Breeding Directions, and Future Research Prospects[END_REF]. The presence of DON and NIV has been reported in maize, oat, barley and wheat[START_REF] Gil-Serna | Mycotoxins: Toxicology[END_REF]).

	Studies of wheat and corn inoculated with F. graminearum on
	field conditions suggest that wheat can have elevated amounts
	of DON, but frequently little to none ZEN, whereas corn can be contaminated with both
	mycotoxins (CAST 2003). Trichothecenes are toxic sesquiterpenoid metabolites with a rigid
	tetracyclic ring (Loi et al. 2017), they are produced mainly by fungi of the genera Fusarium,
	Thrichoderma, Myrothecium and Phompsis. Hundreds of trichothecenes have been identified,
	however only few of them represent a risk as contaminants of food and feed, namely, DON,
	NIV and T-2. Trichothecene biosynthesis starts with the formation of the sesquiterpene
	trichodiene, which is then oxygenated, esterified, and cyclized (CAST 2003). DON (Fig. 13) is
	the most common trichothecene, it is produced mainly by Fusarium graminearum and F.
	culmorum. DON producing strains can generate different acetylated variants such as 3-acetyl-
	DON (3-ADON) and 15-acetyl-DON (15-ADON) (Haile et al. 2019). Optimal and minimal
	temperature for DON production in corn grains is 29-30°C and 11°C, respectively (Mannaa and
	Kim 2017b). If grains are stored under appropriate conditions, no further accumulation of DON
	is observed (Richard 2007).
	Trichothecenes biosynthesis precursors start on the mevalonate pathway, where the
	mevalonate pathway enzyme HMG-CoA reductase (Hmr1) catalyzes the synthesis of
	mevalonate, leading to farnesyl pyrophosphate (FPP), which is also used by essential
	metabolic pathways (e.g. biosynthesis of ubiquinone, steroids and terpenes). Then FPP is
	cyclized, catalyzed by the enzyme trichodiene synthase Tri5. Posterior oxygenation of the
	trichodiene product by Tri4 creates the toxic epoxide moiety in isotrichodermol and
	subsequent pathway intermediates. Further reactions catalyzed by Tri101,

2.1.3.2.1.1 Synthesis of DON and ZEN

  Fusarium verticillioides disease cycle, centered on the life of maize[START_REF] Blacutt | Fusarium Verticillioides: Advancements in Understanding the Toxicity, Virulence, and Niche Adaptations of a Model Mycotoxigenic Pathogen of Maize[END_REF]).

	capable of surviving and sporulating on crop residue, generating inoculum for subsequent
	infections (Blacutt et al. 2018).
	Figure 16. 2.1.3.2.2.1 Synthesis of fumonisins
	Fumonisins	are	non-fluorescent
	mycotoxins	primarily produced	by	F.
	verticillioides and F. proliferatum. Their structure
	was first elucidated by		describes
	the disease cycle of F. verticillioides around the life cycle of maize: A, seeds that germinate in
	contaminated soils could develop rot and seedling blight. B, endophytic colonization starts as
	asymptomatic. C, Stalk wounds from mechanical or insect damage become fungal entrances
	and may result in stalk rot. D, During the silk stage, fungus can colonize kernels via the stylar
	canal, generating the "starburst" pattern on kernels. E, Insects feeding on the kernels generate
	an entrance site for F. verticillioides stalk and ear rots. F. After harvest, the fungal pathogen is

  fumonisins, aflatoxins, DON and ZEN by around 70-80% on maize, wheat and barley are achieved by these processes[START_REF] He | Patented Techniques for Detoxification of Mycotoxins in Feeds and Food Matrices[END_REF][START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF]). However, sorting techniques are unable to decrease fumonisins contamination as Fusarium verticillioides infected kernels are frequently exempt of symptoms[START_REF] Karlovsky | Impact of Food Processing and Detoxification Treatments on Mycotoxin Contamination[END_REF].Milling and dehulling allow the reduction of mycotoxins present in the outer layer of the grain. Indeed, the distribution of the mycotoxins in the grain influences the efficacy of the detoxification by milling, as elucidated by Schwake-Anduschus et al., who revealed that ZEN is mainly concentrated on the fiber-rich parts of the wheat grain, whereas DON is distributed equally between all the grain fractions. Water soluble mycotoxins (e.g. fumonisins) can be partially eliminated by subjecting the grains to a washing or steeping process[START_REF] Schwake-Anduschus | Distribution of Deoxynivalenol, Zearalenone, and Their Respective Modified Analogues in Milling Fractions of Naturally Contaminated Wheat Grains[END_REF]. Sodium carbonate solutions are often used to improve the effectiveness of washing protocols for mycotoxins with less water solubility(Karlovsky et al.

	Physical approaches to decrease mycotoxin content on foodstuff include manual and
	mechanical sorting, density segregation, milling, steeping and extrusion, as well as binding
	matrices, thermal inactivation and irradiation (CAST 2003; Karlovsky et al. 2016). Technologies
	such as pulsed light exhibited high degrading percentages of zearalenone (84.5%),
	deoxynivalenol (72.5%), AFB1 (92.7%) and OTA (98.1%) in solution. But were only able to
	eliminate the toxicity for aflatoxin B1 (Moreau et al. 2013).
	Separation of damaged kernels based on weight, size, color and visible fungal growth

has proven to be useful to reduce aflatoxin contamination, as broken and damaged cereal kernels often carry the highest mycotoxin amounts. Flotation separation is achieved by using concentrated NaCl or sucrose solutions that cause the damaged kernels to float. For instance, reductions of

  . Reported fungal transformations of ZEN include reduction of carbonyl ketones, reduction of double bonds, hydroxylation, methylation, sulfation, glycosylation, and ring cleavage (McCormick 2013). The main detoxification method remains the lactone ring cleavage, which is catalyzed by esterases. The resulting hydroxyketones spontaneously decarboxylate, making the degradation irreversible.

  . Other yeasts such as Rhodotorula, Cryptococcus and Trichosporon mycotoxinivorans can hydrolyze the amide bond of OTA and produce OTα and phenylalanine (McCormick 2013). Several Aspergillus and Rhizopus species are able to detoxify OTA, as well as Botrytis cinerea and Pleurotus ostreatus

Table 1 .

 1 Main mycotoxins found in cereals and their biological detoxification mechanisms, resulting products and detoxifying microorganisms.

Table 1 .

 1 Continuation.

	Mycotoxin	Fungi	Source of toxicity Degradation mechanism	By-products	Structure	Examples of degrading microorganisms	References
	OTA	P. verrucosum	Isocoumarin moiety Hydrolysis amide bond			Rhodotorula sp.	(Stander et al.
		A. ochraceous A. carbonarius			Otα		Cryptococcus sp. Trichosporon mycotoxinivorans	2000) (Abrunhosa et al.
							Brevibacterium sp.	2010)
				Hydrolysis lactone ring	Phenylalanine OP-OTA		Aspergillus niger Rhizopus sp. Botrytis cinerea Pleurotus ostreatus Phenylobacterium immobile	(McCormick 2013) (Petruzzi et al. 2014) (Vanhoutte et al. 2016) (Loi et al. 2017)
								(Chen et al. 2018)
	Citrinin	P. verrucosum		Decarboxylation			Moraxella sp.
		P. expansum
					Decarboxycitrinin			(McCormick 2013)
	AFB1	A. flavus	Lactone ring	Modification of the
		A. parasiticus		coumarin
					AFD1
								(Ciegler et al. 1966)
								(Smiley and
								Draughon 2000)
					AFD2			(Teniola et al.
								2005)
								(McCormick 2013)
								(Samuel,
					AFD3			Sivaramakrishna,
								and Mehta 2014)
					Aflatoxicol		Rhizopus sp. Hypomyces rosellus Cornybacterium rubrum	(Eshelli et al. 2015) (Vanhoutte et al. 2016) (Loi et al. 2017)
			Double bond in	Modification of the furan			Pleurotus ostreatus
			terminal furan ring	ring	AFB2a		Armillariella tabescens

  Streptomyces genus as BCA and mycotoxin detoxifying agents

	2.4
	Trametes versicolor
	Mucor sp.
	Trichosporon mycotoxinivorans
	Saccharomyces cerevisiae
	Trichoderma sp.
	Rhodococcus erythropolis
	Mycobacterium fluoranthenivorans
	Nocardia corynebacterioides
	Streptomyces lividans
	Streptomyces aureofaciens
	Mycobacterium smegmatis
	Pseudomonas putida

Table 2 .

 2 , which is 4 times less than the concentration employed in this study. Results obtained by Passari et al. when investigating the PGPR properties of an endophytic actinobacteria collection indicated maximal IAA productions of about 30 µg/mL (Passari et al. 2015). Ranveer et al. estimated a IAA production of about 17 µg/mL by a Streptomyces strain (Ranveer and A. 2014). The IAA concentrations produced by Streptomyces anulatus and S. albidoflavus during a study by Kunova et al. were much lower, with about 4 µg/mL produced by both strains (Kunova et al. Quantitative estimation of IAA and siderophore production by the actinobacteria isolates exhibiting positive results. Common isolates between the two tests are indicated in bold.

	2016).

Our results also indicate that 15 of the tested isolates (25%) were able to produce siderophores with concentrations going from 4 to 82 percentage siderophore units (PSU).

Near 60 PSU were obtained by Kumar el al. during the siderophore production estimation of soil isolated bacteria

(S. et al. 2017)

. About 19% of the Streptomyces strains tested by Colombo et al. were capable of producing siderophores, as an orange halo was observed around the colonies on Chrome Azurol S agar overlay (O-CAS) medium

(Colombo, Kunova, Pizzatti, et al. 2019)

. All six actinobacteria isolates evaluated by Anwar et al. were capable of producing siderophores (orange halo formation)

[START_REF] Anwar | Screening of Rhizospheric Actinomycetes for Various In-Vitro and in-Vivo Plant Growth Promoting (PGP) Traits and for Agroactive Compounds[END_REF]

).

  4.6 Control of Fusarium and their mycotoxins by Streptomyces isolates 4.6.1 Introduction Fusarium graminearum causes the disease known as Fusarium head blight (FHB) on wheat, leading to important yield losses in Europe. Besides its pathogenicity, F. graminearum

Table 4 .

 4 MS/MS parameters for isotope-labelled internal standard (IS). MRM: Multiple reaction monitoring.

			IS Polarity	MRM	EC
			DON C13 -	370.3 > 59.0 35
			FB1 C13	+ 756.3 > 356.5 -47
	Table 5. MS/MS parameters for mycotoxins. Q: quantification; q: qualification.
	Mycotoxin Polarity	MRM Q	EC MRM Q	MRM q	EC MRM q	R 2 Calibration curve	IS
	FB1	+	722.35 > 334.5 -43 722.35 > 352.0	-44	0.9982	FB1 C13
	FB2	+	706.2 > 318.4		-37	706.2 > 336.4		-44	0.9980	FB1 C13
	DON	-	355.0 > 59.0		35	355.0 > 265.1		35	0.9998	DON C13
	Global analysis of Streptomyces isolates: clustering and Pearson correlation index
	Clustering and Pearson correlation were performed as detailed in previous published
	work (Campos-Avelar et al. 2020). Briefly, isolates were grouped according to their effect on
	fungal growth and mycotoxin accumulation, and illustrated in a color coded heatmap. Clear
	yellow indicates a strong activity and purple to black indicates a weak activity or an increase
	on mycotoxin accumulation. Furthermore, the Pearson correlations (r) between the different
	parameters evaluated were calculated. Heatmaps and Pearson correlation graphics were
	developed in Rstudio.

  The effect of Streptomyces isolates on DON, 15-ADON and ZEN produced by F. graminearum, and on FB1 and FB2 produced by F. verticillioides was also evaluated by comparing them to the mycotoxin accumulation of fungus alone. Mycotoxin accumulation was normalized to the fungus growth area (Fig.51, bottom right). Results showed that DON

	specific production was decreased down to 35% of the control (mean=89%, median=84%) by
	isolate IX11, but IX44 increased DON accumulation twice (208% of the control). A similar
	behavior was noted for 15-ADON with a reduction down to 20% of the control (mean=73%,
	median=67%), also by isolate IX11, and an accumulation of 184% caused by IX44 anew. Finally,
	zearalenone (ZEN) accumulation was highly increased by all bacterial isolates from 222% to
	4458% of the control (mean=1580%, median=1277%). Even isolate IX11, that showed the
	strongest reduction of DON and 15-ADON, caused an overaccumulation of zearalenone,
	although more limited than the other isolates (222%) (Fig. 51, top right). FB1 was diminished
	down to 15% of the control (mean=73%, median=71%) by IX27 while IX41 provoked an FB1
	overproduction of 143% (Fig. 50, bottom left). FB2, was strongly diminished down to 9% of the
	control (mean=55%, median=53%) by IX27 too. A slight increase (116% of the control) was
	caused by isolate IX04.

  Streptomyces isolates and their CFEs on fungal growth and mycotoxin accumulation by F. graminearum and F. verticillioides.Results indicate that fungal development was more easily inhibited for Fusarium verticillioides than for Fusarium graminearum during the in vitro dual culture assays. These results could be explained by the different growth rate of both pathogens. Indeed, microscopical observations by Pellan et al. revealed that F. graminearum produced rapidly long, dense and straight hyphae, while F. verticillioides developed fine, slightly branched hyphae[START_REF] Pellan | Commercial Biocontrol Agents Reveal Contrasting Comportments Against Two Mycotoxigenic Fungi in Cereals: Fusarium graminearum and Fusarium verticillioides[END_REF]. Moreover, the distant inhibition profiles observed for F. verticillioides suggest that this pathogen might be more sensitive to the production of diffusible antifungal compounds by the Streptomyces isolates[START_REF] Goudjal | Potential of Endophytic Streptomyces spp. for Biocontrol of Fusarium Root Rot Disease and Growth Promotion of Tomato Seedlings[END_REF][START_REF] Samsudin | Efficacy of Potential Biocontrol Agents for Control of Fusarium verticillioides and Fumonisin B1 under Different Environmental Conditions[END_REF]. . verticillioides growth inhibitions going up to 77% by bacterial cells and up to 73% by CFEs during an in vitro evaluation of Streptomyces isolates.The impact on DON, 15-ADON, amount was moderate during dual cultures, with some bacterial isolates causing strong decreases (e.g. IX11) and some others provoking an overaccumulation (e.g. IX44). In contrast, all the bacterial isolates evaluated caused a huge overproduction of ZEN. The work of Ursan et all. revealed contrasting results, as their Streptomyces strains were able to decrease the production of ZEN evaluated by HPTLC (Ursan

	The fungal growth inhibition percentages obtained in our study were inferior to the
	ones documented in the literature. For instance, Colombo et al. obtained growth inhibitions
	from 41% to 71% during dual cultures of endophytic Streptomyces against F. graminearum
	(Colombo et al. 2019). Another study by this research team reported inhibitions of 60% to 90%
	of F. graminearum and F. verticillioides growth, respectively (Colombo, et al. 2019). An
	evaluation of Streptomyces as biocontrol agents was conducted by Ursan el al., who analyzed
	60 bacterial strains against F. culmorum and F. graminearum. They obtained mean growth

Globally, bacterial CFEs' antifungal activity was similar to that of the bacteria themselves of both fungi. Moreover, thermal treatment did not completely eliminate their antifungal capacity. These results suggest that the tested bacterial isolates represent potential sources of heat-stable enzymes and extracellular antifungal compounds. Indeed, several bioactive molecules, such as allelochemicals, chitinases and volatile organic compounds (VOCs) with antifungal properties, have already been isolated from Streptomyces cultures. For this reason, the screening of crude extracts of Streptomyces isolates is essential for the subsequent identification and isolation of active compounds, as well as for the understanding of the antagonistic mechanisms

(Colombo et al. 2019)

. inhibitions of about 76% for F. graminearum. They hypothesized that the observed antifungal properties could be due to the synthesis of hydrolytic enzymes such as proteases and chitinases, produced by their Streptomyces strains

[START_REF] Ursan | The Potential of New Streptomyces Isolates as Biocontrol Agents Against Fusarium spp[END_REF]

.

Nguyen et al. reported F

fois plus de fumonisines que témoin). Ceci met en évidence l'importance d'évaluer l'impact sur les mycotoxines lors des évaluations des BCAs. L'OTA a été fortement dégradée par des isolats bactériens, principalement en milieu liquide (réduction jusqu'à 38% du contrôle en moyenne), tandis que l'AFB1 a été dégradée en milieu solide, liquide, et par les surnageants (33%, 43% et 69% du contrôle en moyenne, respectivement). De plus, l'évaluation de la toxicité résiduelle de l'AFB1 montrait une détoxification efficace, réduction jusqu'à 27% du contrôle. L'analyse du Clustering et des corrélations de Pearson a permis de classifier les isolats bactériens en fonction de leurs performances et d'évoquer des possibles mécanismes antagonistes, tels que la production des composés antifongiques, des enzymes dégradatrices de mycotoxines et des potentiels inhibiteurs de la biosynthèse des mycotoxines. Les bactéries ont colonisé les feuilles du blé lors des essais in planta et sont restées viables pendant une semaine. Néanmoins, seulement S. griseoviridis du Mycostop®, a protégé le blé contre les champignons testés. Ce travail présente une approche intégrée pour le criblage des potentiels BCAs, ciblant l'analyse globale des résultats pour élucider les mécanismes antagonistes et identifier les isolats les plus prometteurs.Mots clés : phytopathogènes fongiques, Streptomyces, biocontrôle, mycotoxines, détoxification, clusterisation.

and C-6 by a F420-H2 dependent reductase takes place, followed by a spontaneous breakdown of the product. The F420 cofactor does not occur in microorganisms applied in food processing but seem to be widespread among the Actinomycetales family[START_REF] Lapalikar | F 420H2-Dependent Degradation of Aflatoxin and Other Furanocoumarins Is Widespread throughout the Actinomycetales[END_REF][START_REF] Taylor | Identification and Characterization of Two Families of F420H2-Dependent Reductases from Mycobacteria That Catalyse Aflatoxin Degradation[END_REF]).Several enzymes have been studied for their capacity to degrade aflatoxins, such as the laccase enzyme produced by Trametes versicolor which degraded AFB1 by 87%[START_REF] Alberts | Degradation of Aflatoxin B1 by Fungal Laccase Enzymes[END_REF]. Degradation of AFB1 into AFB1-8,9-dihydrodiol was observed from the white rot fungi

Experimental work

Fungal cell walls are conformed of chitin, so bacteria capable to degrading this polymer have an advantage during antagonistic confrontations. In the same way, siderophore production allows them to sequester iron, rendering it unavailable for the fungi. PGPR properties such as phosphate solubilization, ammonia production and the production of the phytohormone IAA, confer them potential as plant development facilitators [START_REF] Anwar | Screening of Rhizospheric Actinomycetes for Various In-Vitro and in-Vivo Plant Growth Promoting (PGP) Traits and for Agroactive Compounds[END_REF]Colombo, Kunova, Pizzatti, et al. 2019;[START_REF] Kunova | Selection of Streptomyces against Soil Borne Fungal Pathogens by a Standardized Dual Culture Assay and Evaluation of Their Effects on Seed Germination and Plant Growth[END_REF][START_REF] Passari | In Vitro and in Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants[END_REF].

Table 3 shows a summary of the actinobacteria isolates exhibiting at least one positive result to the PGPR assays. Table 3. Positive of actinobacteria isolates to the PGPR assays. Only isolates having at least one positive result were presented.

Actinobacteria isolate

Chitinase activity

Siderophore production

Phosphate solubilization Ammonia production

Conclusion and perspectives

The conducted experimental protocols allowed the creation and conservation and preliminary characterization of the Qualisud actinobacteria collection. 16S identification indicates that the collected isolates belong to the Streptomyces genus. Additionally, a complete genome sequencing is currently being performed on thirty isolates having showed promising results to the assays in the next chapters of this work. Moreover, the various positive results of the PGPR evaluations show a promising BCA potential for the screened isolates under the perspective of soil treatments. These results constitute the first step towards the comprehension of antagonistic mechanisms established by actinobacteria against wheat fungal pathogens (i.e. chitinase production).

( [START_REF] Karlsson | Necrotrophic Mycoparasites and Their Genomes[END_REF]. These resistances may have modified the profile of bacterial isolates being able to prevent their growth.

Supplementary material