Philippe Callac
email: callac@bordeaux.inra.fr

Jacques Guinberteau

Cyril Férandon

Gérard Barroso

Cyril Ferandon

Gerard Barroso

AN ASIAN COMMERCIAL STRAIN OF AGROCYBE CHAXINGU AND A EUROPEAN WILD STRAIN OF AGROCYBE CYLINDRACEA EXHIBITING MORPHOLOGICAL DIFFERENCE AND HIGH GENETIC DIVERGENCE ARE INTERFERTILE

Keywords: Agrocybe chaxingu, Agrocybe cylindracea, interfertility, sporophores, molecular markers

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Agrocybe cylindracea (DC.: Fr) Singer, syn. A. aegerita (Brig.) Singer (Basidiomycota, Agaricales) is a species of mushroom cultivated in Europe since antiquity, but sporadically cultivated today. In contrast, in Asia, mushrooms called A. cylindracea but also A. chaxingu Huang are largely cultivated today. As published in the Edible Fungal Flora of China [START_REF]Edible fungal flora of China[END_REF], the diagnosis of A. chaxingu does not allow a clear distinction between this species and A. cylindracea.

During the last thirty years, based on its ability to easily achieve a complete life-cycle in laboratory conditions, A. cylindracea was largely studied as a laboratory model of mushroomproducing Basidiomycete. Numerous reports on its genetics and molecular biology were produced. Particularly, several reports have focused on the mitochondrial genes and genome of A. cylindracea wild-type strains. In 1997, the sequence and secondary structure of the A. cylindracea mitochondrial SSU-rDNA was described [START_REF] Gonzalez | DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita[END_REF]. This led to the characterization of variable domains (V4, V6 and V9) of the SSU-rRNA showing species-specific length variations within the genus Agrocybe, including European wild-type strains of A. cylindracea and Asian cultivated strains of A. chaxingu [START_REF] Gonzalez | Sequence and secondary structure of the mitochondrial smallsubunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe[END_REF]. In the same way, a comparative analysis of the mitochondrial cob gene of A. cylindracea and A. chaxingu strains revealed important genetic variations between the two species and the presence of non-orthologous group I introns in this gene [START_REF] Mouhamadou | Unusual accumulation of polymorphic microsatellite loci in a specific region of the mitochondrial genome of two mushroom-forming Agrocybe species[END_REF][START_REF] Mouhamadou | The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi[END_REF][START_REF] Mouhamadou | Molecular evolution of a mitochondrial polB gene, encoding a family B DNA polymerase, towards the elimination from Agrocybe mitochondrial genomes[END_REF]. More recently, the study of Uhart et al. [START_REF] Uhart | Evolution of mitochondrial SSU-rDNA variable domain sequences and rRNA secondary structures, and phylogeny of the Agrocybe aegerita multispecies complex[END_REF], mainly based on comparison of mitochondrial ribosomal SSU-rDNA variable domains, confirmed the strong genetic divergence between the European wild specimens of A. cylindracea and strains cultivated in Asia under the name A. chaxingu. At that time, these two entities were considered as belonging to a species complex but, taking in account some inter-sterility (not reported in details) between the two entities, the authors suggested that these strains could finally belong to two distinct species. However, in another paper, based on morphological comparisons, Uhart & Alberto [START_REF] Uhart | Morphologic characterization of Agrocybe cylindracea (Basidiomycetes, Agaricales) from America, Europe and Asia[END_REF] suggested that these two species should be finally synonymised but that the type specimen would have to be examined first. Here, we intend to complete previous studies about the genetic divergence between the two entities and to test the inter-fertility between two strains already studied in the papers cited above: a European wild specimen of A. cylindracea and a strain cultivated in Asia under the name A. chaxingu. We expect to clarify the situation not only at the taxonomic level but also to obtain a better understanding of species circumscription and of their geographical range in order to help manage their genetic resources and future breeding programs.

MATERIALS AND METHODS

Strains.

The European wild-type strain Agrocybe cylindracea (= Agrocybe aegerita) SM47 from South-West of France and the industrially cultivated Agrocybe chaxingu SM960903 from Eastern Asia were previously described [START_REF] Gonzalez | Sequence and secondary structure of the mitochondrial smallsubunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe[END_REF][START_REF] Mouhamadou | Unusual accumulation of polymorphic microsatellite loci in a specific region of the mitochondrial genome of two mushroom-forming Agrocybe species[END_REF][START_REF] Mouhamadou | The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi[END_REF][START_REF] Mouhamadou | Molecular evolution of a mitochondrial polB gene, encoding a family B DNA polymerase, towards the elimination from Agrocybe mitochondrial genomes[END_REF][START_REF] Uhart | Evolution of mitochondrial SSU-rDNA variable domain sequences and rRNA secondary structures, and phylogeny of the Agrocybe aegerita multispecies complex[END_REF]. Both strains are permanently deposited in the collection of the Laboratory (INRA UR 1264, Villenave d'Ornon, France).

In vitro DNA manipulation and sequencing. Sequences used as molecular markers (nuclear ribosomal unit, mitochondrial cob gene) were obtained by conventional procedures from cloned PCR products.

Total DNA of fungal strains were extracted from 0.1 g of dried carpophores or 0.2g of fresh mycelium, after grinding in liquid nitrogen to obtain a fine powder. Nucleic acids were extracted according to the N-cethyl-NNN-trimethyl ammonium bromide (CTAB) procedure adapted for small quantities of basidiomycete mycelia [START_REF] Barroso | A miniprep method for RFLP analysis and dsRNAs detection perfected in the cultivated fungus Agrocybe aegerita. In: Science and Cultivation of Edible Fungi Elliot[END_REF]. DNA (OD 260 ) was quantified using a NanoDrop spectrophotometer (NanoDrop ND-1000, Nanodrop technologie, DE, USA), diluted in deionized sterilized Milli-Q water (Milli-Q water system production, Millipore, Saint-Quentin en Yveline, France) and stored at -20°C.

PCR amplifications were carried out using the Go Taq polymerase from Promega Corp. (Madison, Wis, USA) and respective primer pairs synthesized by Eurofins MWG Operon (Germany).

The primers used to amplify the nuclear ribosomal unit were the conventional ITS1 (5'TCCGTAGGTGAACCTGCGG3') and ITS4 (5'GGAAGTAAAAGTCGTAACAAGG3') described by [START_REF] White | PCR Protocols: A Guide to Methods and Applications[END_REF]. A CAPS test was developed by using the HaeII restriction enzyme from Promega Corp. (Madison, Wis, USA), according to the manufacturer's recommendations.

The primer pairs used to verify, by PCR, the presence of the A. chaxingu (E1C/E2C) and A. cylindracea (EU1A/ER1A and EU2A/ER2A) introns have been previously described [START_REF] Mouhamadou | The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi[END_REF].

E1C (nt532 5′CGTAATTACTAATTTATTATC3′ nt552); E2C (nt1811 5′ACAAAAGGTAATAAATAGTG3′ nt1792); EU1A (nt267 5′ACCAAATGTAGATTTTGCA3′ nt285); ER1A (nt2976 5′CTTCCAATATGTATATATAC3′ nt2957); EU2A (nt3448 5′TTATATTCCAGCAGATCC3′ nt3465); ER2A (nt4675 5′TAGTTTGTTAGGAATAGATC3′ nt4656).

The sizes of the V4, V6 and V9 variable domains of the mitochondrial SSU-rDNA were verified by electrophoresis analyses of the specific PCR products in 1.2 % agarose gels. Specific primers were as previously described [START_REF] Gonzalez | Sequence and secondary structure of the mitochondrial smallsubunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe[END_REF][START_REF] Uhart | Evolution of mitochondrial SSU-rDNA variable domain sequences and rRNA secondary structures, and phylogeny of the Agrocybe aegerita multispecies complex[END_REF].

PCR were performed in a Programmable Thermal Cycler PTC 200 (MJ Research Inc., Watertown, Mass., USA). Each reaction contained 10 to 100 ng of fungal genomic DNA, 1 M of both primers, 200 of each dNTP, 1 unit of Taq DNA polymerase, in a final volume of 50 l of the appropriate buffer. Reactions were run for 30 cycles at 95 ° C for 30s, then two degrees below the lowest Tm of both oligonucleotides for 30s, 72° C for 1 to 2 min, and one final cycle at 72° C for 5 min. An aliquot of 10 µl of each PCR product was analysed by agarose (1%, w/v) gel electrophoresis containing 200ng/ml of ethidium bromide, in TEB buffer [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF].

For DNA sequencing, PCR products were purified with the Wizard SV gel and PCR Clean-Up System (Promega Corp.Madison, WI, USA) before they were sequenced by the primer walking methods using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Courtaboeuf, France) and the corresponding primers used for the PCR reaction. Sequence reactions were carried out according to the supplier's recommendations, in a final volume of 10 l containing 100 ng of PCR product and 0.5 µM of the specific primer. Sequence reactions were carried out in a thermocycler by applying an initial denaturation step at 95°C for 1 minute; 27 cycles with each composed of the three steps: 96°C for 10 s, 50°C for 5 s, 60°C for 4 min. The reaction products were ethanol precipitated, dried, and then separated by capillary electrophoresis (on an automated sequencer ABI 3130x1, ABI Prism Corp., France) at the genomic platform of the University Bordeaux Segalen (France). Sequencing profiles were edited and corrected using the BioEdit sequence alignment editor v7.0.9 free software (Ibis Biosciences Carlsbad, CA, USA).

Fruiting test, spore germination, and monospore culture. Polypropylene bags containing 250 g of chopped humidified wheat straw + 25 g of oat flour + 50 ml of glucose solution (15g / l), were sterilized in an autoclave. Malt agar medium colonized by mycelium was used to inoculate the bags: the culture medium of two Petri dishes of 9 cm in diameter were deposited at the top of each bag. Incubation lasted 15 days at 25 ° C; then, the temperature was lowered to 18 ° C. After 20 days, bags were opened and fruiting began after one week. Sporeprints were recovered from mature sporocarps on tracing paper. Spore suspensions at different dilution were spread on complete yeast medium [START_REF] Raper | Genetic analysis of the life cycle of Agaricus bisporus[END_REF]. Eight days later, spores began to give rise to young mycelia that were isolated. Single spore isolates (SSIs) were subcultivated individualy on malt agar medium.

Mating tests. Small pieces of agar culture of two different SSIs were placed at about 2 cm from each other in Petri dish containing malt agar medium. The inoculated mycelia were allowed to grow together at 25°C. Positive mating was indicated by the formation of aerial fluffy mycelium at the junction line between two monokaryons. The fluffy mycelium appearing at the junction line is a presumed dikaryon resulting from a plasmogamy between the two sexually compatible monokaryotic SSIs. For each confrontation two replicates were made. The fluffy mycelium was isolated and subcultured on malt agar medium.

Cytological method for positive mating confirmation. A simple method was used to confirm the positive mating. SSIs of Agrocybe are monokaryotic and without clamped hyphae, while fertile dikaryons exhibit clamp connections. For each positive mating, a fragment of mycelium from the junction zone of any replicate was examined under microscope (x100, congo red staining). Observation of clamp connections confirmed the dikaryotic status of the mated mycelium.

RESULTS

Morphological comparison of the parental strains. Ten culture bags were performed for each strain. Strain SM47 of A. cylindracea began to fruit 35 days after the inoculation of the substrate, while strain SM960903 of A. chaxingu began one week later in all replicates. Morphological differences between the sporophores of the two strains were noticeable (Fig. 1). These differences include cap color that was much more palish for SM47, or the longer stipe of SM960903.

However, these differences likely characterize the strains and not the species in which these criteria have shown to be highly variable and also sensitive to environmental conditions. However differences were also noted for two other traits that may be taxonomically pertinent: the color of the stipe and the aspect and behavior of the partial veil: (i) the color of the stipe was light brown in A. chaxingu SM960903 as in other strains of A. chaxingu that we have cultivated (data not reported here) along with the white color of tiny scales that make them more visible. In A. cylindracea, the stipe was almost uniformly white; (ii) the partial veil is larger and thicker in SM960903 with the underside layer breaking of in crown wheel before it comes off the cap. Moreover, it fits in the upper stipe; the hymenial cavity is very narrow; the cap opening occurs lately and often after spore maturity. At the opening of the cap, we put some bags on the outside and saw the large membranous ring floating in the wind, likely facilitating the spread of spores that had settled on it. In A. cylindracea, the cap opened earlier and the partial veil contracted, forming a shorter and thicker annulus that was not covered by spores at this time. In other respects, we did not observe significant difference in spore sizes between the two specimens. They were on average 10.2 × 5.9 μm (n = 30) for SM47 and 10.4 × 5.9 (n = 30) for SM 960903.

Genetic divergence between the parental strains of A. cylindracea SM47 and A. chaxingu SM960903. The genetic variation between the parental strain A. cylindracea SM47 and A. chaxingu SM960903 was studied using three different molecular markers: (i) the nuclear ribosomal unit, (ii) the variable domains V4, V6 and V9 of the mitochondrial SSU-rDNA encoding the 16S RNA of the small sub-unit of the mito-ribosome, and (iii) the molecular organization of the cob gene which encodes the unique mitochondrial protein of the Complex III of the respiratory chain. All these markers revealed high divergence between the two strains and it is to be noticed that the specific feature of each strains for all these markers were shared with all the studied European A. cylindracea strains (10 different strains) for A. cylindracea SM47 and with three additional Asian A. chaxingu strains for A. chaxingu SM960903 (data not shown).

In details, the nuclear ribosomal region of the A. cylindracea SM47 strain, corresponding to the ITS1/ITS4 PCR product had a size of 637 nt, while that of A. chaxingu SM 960903 had a size of 652 nt. When aligned (Clustal W), the two sequences possessed 67 polymorphic sites (distributed in 39 non contiguous loci), i.e. showing a percentage of nt identity slightly lower than 90 %. When cleaved by the restriction endonuclease HaeIII, the ITS1/ITS4 PCR product of A. cylindracea generates three fragments of size 341 nt, 180 nt and 116 nt. On the contrary, the HaeIII restriction profile of the ITS1/ITS4 PCR product of A. chaxingu is constituted by four fragments of size 341 nt, 201 nt, 84 nt and a small one of 26 nt not detectable in agarose gel electrophoresis. From this, the use of the HaeIII restriction endonuclease allowed the development of a CAPS test to easily discriminate both strains by agarose (1.2 %) gel electrophoresis analysis of the HaeIII-digested PCR product generated by the ITS1/IIT4 couple of primers. The amplified nuclear ribosomal region A. cylindracea strain had a profile with 3 fragments (341, 180 and 116 nt), the A. chaxingu one possess the same large fragment of 341 nt accompanied by two fragment of 201 nt and 84 nt. The length variations between the two polymorphic fragments of A. cylindracea (180 nt and 116 nt) and A. chaxingu (201 nt and 84 nt) were easily detectable by agarose gel electrophoresis analysis.

As previously reported, there were also important genetic variations between mitochondrial genes of A. aegerita SM47 and A. chaxingu SM960903 strains.

The length of the V4 domain varied from 170 nt in A. cylindracea to 281 nt in A. chaxingu; the length of the V6 domain varied from 172 nt in A. cylindracea to 158 nt in A. chaxingu and the length of the V9 domain varied from 221 nt in A. cylindracea to 246 nt in A. chaxingu. These size variations were mainly due to insertion/deletions events of large sequences occurring in these variables domains [START_REF] Gonzalez | Sequence and secondary structure of the mitochondrial smallsubunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe[END_REF].

In the same way, the previously established [START_REF] Mouhamadou | The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi[END_REF] molecular organization of the cob gene, shown in figure 2, was verified in the parental A. cylindracea SM47 and A. chaxingu SM960903 strains by PCR with specific primer pairs located in the flanking exon sequences of each gene. As shown in Figure 2, the two strains differed by the presence/absence of three non-orthologous large group I introns: two in A. cylindracea and one in A. chaxingu. Intra-stock and inter-stock mating tests. Parental strains SM47 of A. cylindracea and SM960903 of A. chaxingu exhibited hyphal clamp connections, in agreement with their dikaryotic status. Spores from SM47 easily germinated, while only few spores of SM960903 germinated. For SM47, 60 SSIs were isolated, but only 15 SSIs for SM960903. All the SSIs were monokaryotic and usable for intra-stock mating tests, except for two SSIs of SM960903 for which clamped hyphae were detected. For SM47, 12 SSIs chosen at random were mated in all pairwise combinations and four groups of incompatibility were recovered based of the detection of hyphal clamp connexions: all the confrontations between group I (1 SSIs) and group II (4 SSIs), or between group III (2 SSIs) and group IV (5 SSIs) were positive and produced clamped mycelia. These data agreed with the heterothallic life cycle and the bifactorial system of sexual incompatibility already described in A. cylindracea [START_REF] Barroso | Genetic evidence for nonrandom sorting of mitochondria in the basidiomycete Agrocybe aegerita[END_REF]. For SM960903, 12 SSIs were confronted in all pairwise combinations but only few positive reactions were observed, so, the inter-compatibility groups remain fully identified.

For the inter-stock mating tests, each strain of the seven SSIs of the SM47 offspring, including the single SSI of the group I and two SSIs of each of the three remaining incompatibility groups, were confronted with each of the 12 SSIs of the SM960903 offspring. Positive reactions were generally not clearly visible but rather ambiguous. After six weeks, mycelia at the junction line were examined and hyphal clamp connections were detected in all 84 (= 7 x 12) confrontations.

Isolation of 56 presumed hybrid mycelia exhibiting clamp connections.

For 48 confrontations performed between the 12 SSIs from SM960903 and 4 SSIs from SM47 (SM47-6, SM47-7, SM47-11, SM47-13) that belonged to each of the four incompatibility groups I, II, III and IV, hybrid dikaryotic mycelia were isolated from the junction line, and they were then subcultured for further genetic analyses and fruiting tests. In other respects, in the dishes of confrontation between SSIs from the two parental strains, we also tried to detect hyphal clamp connections in the growing area of each monokaryon: they were almost always detected in the area of monokaryons deriving from strain SM960903, but less frequently in the area of the monokaryons deriving from strain SM47. In four dishes corresponding to four different confrontations, hyphal clamps were detected in the areas of both monokaryons, fragments of mycelia were isolated from each of the two areas, additionally to the mycelium isolated from the junction line. Finally, a total of 56 hybrid mycelia were isolated.

Genetic confirmation of the hybridization. DNA of the 56 presumed hybrid dikaryotic mycelia (isolated from 48 strains collected on the junction lines and 8 strains coming from regions distant from these lines) were extracted. For each strain, the nuclear ribosomal unit was amplified as described in the Materials and Methods section by using the conventional ITS1/ITS4 couple of primers. The resulting PCR products were digested by HaeIII and the restriction fragments analysed by agarose gel electrophoresis. As expected, this CAPS marker revealed for each strain, a typical heteroallelic profile. In details, all the 48 presumed hybrids from the junction lines, as well as the 8 strains isolated from the areas of the confronted homokaryons, distant from these junction lines possessed both largest HaeIII restriction fragments characterizing each parental strain: the 342 nt fragment of A. Cylindracea and the 422 nt fragment of A. Chaxingu. This pattern confirms in all tested cases the presence of both parental nuclei. All these 56 isolates were therefore assumed to be dicaryotic. The presence of both parental nuclei in the 8 strains coming from regions distant from the junction lines suggests that, as deduced from the presence of clamp connections on these mycelia, a migration of the compatible nuclei has occurred in the recipient homokaryotic hyphae.

Cultivation and morphology of the hybrids. Among the 48 isolated hybrid strains, we chose four representatives as follow: all the parental homokaryons from SM960903 that were different and all the parental homokaryons from SM47 that belonged to the four different incompatibility groups. From the information we had, these four hybrids were potentially the most different that we could find: SM47-13 × SM960903-6, SM47-7 × SM960903-3, SM47-11 × SM960903-12, and SM47-6 × SM960903-2. All of them easily fruited and sporulated. Sporophores of three of them are showed on Fig. 3. The cap color at the disc was dark brown like the A. chaxingu parent or palish brown like the A. cylindracea parent; however, the cap color at the edges was always palish like the A. cylindracea parent. In all cases, the stipe had a white brown color like the A. chaxingu parent. The annulus was rather small and did not open lately as for the A. cylindracea parent but a crown wheel is more or less visible but less pronounced than in the A. chaxingu parent.

Molecular characterization of the hybrid sporophores. Sporophores produced by the four hybrids were molecularly characterized by nuclear and mitochondrial markers. The CAPS test described above allow to verify that the sporophores differentiated by the four fruiting hybrids possessed the heteroallelic pattern of the nuclear ribosomal units (restriction fragments of both parental strains), and consequently were also dikaryotic.

The molecular organization of the mitochondrial cob gene of a sporophore differentiated by each of the four fruiting hybrids was studied by PCR as described in the Materials and Methods section with three primer pairs allowing the evidence of each of the three nonorthologous group I introns to be detected: iAeI and iAeII carried by the A. cylindracea parental cob gene and iAchax carried by A. chaxingu. Interestingly, all the four sporophores possess only the A. chaxingu iAchax intron, This suggests that the molecular organization of the mitochondrial cob gene present in the differentiated sporophores was that of the parental A. chaxingu strain. This was also confirmed by amplification of the V4, V6 and V9 variable domains of the mitochondrial SSU-rDNA whose size corresponded to the A. chaxingu SSU-rDNA. As both genes (cob and SSU-rDNA) are located in distant regions of the mitochondrial genome (data not shown), the differentiated sporophores were assumed to possess the mitochondria of the parental A. chaxingu strain. This is in accordance with the previously reported uniparental heredity of mitochondria observed in sporophores of A. cylindracea [START_REF] Meinhardt | Mating-types of Agrocybe aegerita[END_REF].

DISCUSSION

Although the main difference reported in the literature between A. cylindracea and A. chaxingu is their natural habitat, our comparison between a wild type strain of A. cylindracea and a strain cultivated in Asia under the name A. chaxingu showed that the sporophores of the latter had a more colorful stipe and a partial veil that separated later from the cap, forming a wider ring covered with spores. Such differences are not so visible on dried specimens. On the other hand, we are not sure that the habitat is a strong criterion in this complex because, in Europe, A. cylindracea grows easily on various trees, including Acer negundo which was introduced from North America. Moreover, both strains were able to grow and form fruiting bodies on wheat straw. At the molecular level, the 39 differences between the two parental strains were detected. This is much greater than the number of polymorphic positions that can be found between strains belonging to the same species in the genus Agaricus, which rarely exceeds 2 or 3. Such a difference suggests that mutation rate would be higher in the ITS of Agrocybe and/or that reproductive barrier more easily takes place in Agaricus.

Intra-stock mating tests revealed a bifactorial system of sexual incompatibility for the strain of A. cylindracea; although the reproductive system of the other strain was not resolved. In inter-stock mating tests, monokaryons from the two parents were all sexually compatible, indicating that the mating type alleles of tested monokaryons of A. chaxingu differ from the alleles of the strain of A. cylindracea. Systematic detection of clamped mycelia in the area of the monokaryon of A. chaxingu suggests that nuclei from A. cylindracea may have migrated, dikaryotizing the monokaryon of A. chaxingu. Many hybrid strains were isolated from the interstock mating tests and confirmed cytologically and molecularly. Four representatives of these hybrids were cultivated on fruiting medium: they fruited easily and abundantly. In the four cases, sporophores were fertile with a veil similar to the veil of the A. cylindracea parent, but the stipe was colored similarly as A. chaxingu. Moreover we showed that the mitochondria were inherited from the A. chaxingu strain. Such a uniparental inheritance of the mitochondria is frequent in basidiomycota and especially in Agrocybe cylindracea [START_REF] Meinhardt | Mating-types of Agrocybe aegerita[END_REF]. Since we also noted that nuclei of the monokaryon of A. cylindracea apparently easily invaded the monokaryon of A. chaxingu and since the putative hybrids were isolated relatively late (6 weeks after the beginning of the co-culture), it is possible that most of the isolates were in fact dikarotized monokaryons of A. chaxingu, bearing the mitochondria of A. chaxingu. We finally conclude that both entities are completely interfertile although they highly diverge genetically. These data on one hand show that the two entities are conspecific, but, on the other hand, support the concept of a varietal rank for the Asian entity. The type specimen of the latter remains to be studied to determine if the commercial strains named A. chaxingu are truly related to this species. Morphological differences being not easy to detect on dried specimens, the best solution should be to compare DNA sequences. Such data would be useful but are not necessary to describe and to name a new variety. To describe such a variety, specimens with morphological and molecular characteristics similar to those of commercial strains used here, but of known origin would be more appropriate.

Figure 1 .

 1 Figure 1. Sporophores of the parental strains differentiated in cultivation. 1a: Agrocybe chaxingu strain SM960903, cap after opening; 1b: Agrocybe chaxingu strain SM960903 cap before opening; 1c. Agrocybe cylindracea strain SM 47 at various stages of maturation.

Figure 2 :

 2 Figure 2: Comparison of the molecular organization of the cob gene in A. chaxingu (GenBank Acc. N° AY772389) and A. aegerita (GenBank Acc. N° AY781064). The cob exonic sequences and the intronic heg are in blue and green boxes, respectively. The typical LAGLIDADG motif of each HE are indicated. From [5].

Figure 3 .

 3 Figure 3. Sporophores of hybrids between A. cylingracea and A. chaxingu. 3a: SM47-7 × SM960903-3; 3b: SM47-6 × SM960903-2; 3c: SM47-13 × SM960903-6.


Xavier Vitrac
email: xavier.vitrac@u-bordeaux2.fr

Anaïs Reignier

Caroline Henry-Vitrac

Nathalie Minvielle

Jean-Michel Mérillon

Jean-Michel Savoie

CHANGES IN ANTIOXIDANT ACTIVITIES AND COMPOUNDS DURING CULTIVATION OF SHIITAKE (LENTINULA EDODES)

Keywords: Lentinula edodes, Shiitake, Antioxidants, Ergothioneine, Phenolic compounds

Mushrooms contain a variety of secondary metabolites, including various phenolic compounds, which have been shown to act as excellent antioxidants. Recently, a specific antioxidant, ergothioneine (ERG) has been identified in various genera of mushrooms, including shiitake (Lentinula edodes). Shiitake is the second most highly consumed mushroom in the world, and present several functional properties, such as antitumor, hypocholesterolemic, antioxidant and antimicrobial potentials that have been intensively investigated. Although research was focused on its therapeutic effects, little information is available about the compounds responsible of the antioxidant properties. Our objectives were to identify changes in quality of shiitake as antioxidant source during cultivation cycles on oak woodships. The antioxidant activities and contents of potential antioxidant components, including total phenolic (TP) compounds and ERG, were analyzed in two parts of mushrooms (stipe and cap) from the successive flushes. In addition, the seasonal variations were studied. The Folin-Ciocalteu reagent was used to quantify the TP, and ERG was quantified by HPLC-DAD. The antioxidant activity was measured using the ORAC assay. Our analysis revealed only minor seasonal variations in TP and ERG contents as well as the ORAC value, excepted during the winter where these contents were 50% higher in the caps. Regarding the age of the culture, we observed a progressive decrease in the contents of TP and ERG all along the flushes, whatever the part of the mushroom. Surprisingly, the ORAC value increased in the whole mushrooms, whereas it decreased in the caps and strongly increased in the stipes. We concluded that the antioxidant activities could be largely dependent on other molecules than ERG, perhaps specific phenolic compounds. Having established the main variations in the antioxidant activity of shiitake, the chemical characteristics of the antioxidative components will be now further investigated. This study could provide valuable new opportunities for mushroom growers, since shiitake can serve as a good source of antioxidants in the human diet.

INTRODUCTION

Shiitake, Lentinula edodes (Berk) Pegler, is the second largest cultivated and most popular edible mushroom in the world, comprising about 25% worldwide production. Shiitake mushroom contains several therapeutic actions such as antioxidant, antitumoral and antimicrobial properties, carried by the diversity of its components. Shiitake mushrooms are a very good source of three key antioxidant minerals: manganese, selenium and zinc. They also contain some Section: Mycosourced molecules and nutritional quality 248 unusual phytonutrient antioxidants. One of the best studied is ergothioneine (ERG). This antioxidant is derived from the amino acid histidine, and acts as an antioxidant by scavenging most reactive oxygen species, chelating various divalent metallic cations and suppressing the oxidation of homoproteins. Polyphenolic compounds have also been detected in shiitake, and may contribute to the antioxidant potential of this mushroom.

There is a growing interest to measure the antioxidant capacity in foods, since the compounds exhibiting such properties could be isolated and used for the prevention of free radicals mediated pathologies, such as cardiovascular and neurodegenerative diseases. In this study, the antioxidant capacity (radical scavenging) of shiitake cultivated on oak sawdust from the successive flushes was investigated in two parts of mushrooms (stipe and cap) using the oxygen radical absorbance capacity assay (ORAC). In addition, total phenolic compounds (TP) and ERG were quantified in order to evaluate their contribution to the antioxidant capacity of mushrooms.

MATERIALS AND METHODS

Chemicals. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein, 2,2'azobis (2-methylpropionamidine) dihydrochloride (AAPH), ergothioneine, phosphate buffer and Folin-Ciocalteu reagent were purchased from Sigma-Aldrich (France). All the solvents were of HPLC grade (Scharleau).

Sample collection and preparation.

The Lentinula edodes strain 3706 (Mycelia) was cultivated by EuroShiitake Company in insulated green houses in a temperate region, Dordogne, France. The composition of the cultivation substrate was: 85 Kg of oak woodchips and sawdust, 10 kg of wheat bran, 1 kg of gypsum, water to reach 62-65% water content. Substrate sterilization was performed with live steam for 4 hours in a tanker. After cooling 4 % (w/w) of spawn were added aseptically and mixed in the tanker. Polyethylene bags were filled with 6 Kg of spawned substrate. Incubation was at 22 ± 2 °C for 3 to 4 weeks. After removing of the bags, browning was induced by and placing them on shelves in a greenhouse with temperature from 16 to 19 °C and 92-98 % relative humidity, for 3 to 4 weeks. Fruiting was induced by soaking the blocks into cooled tap water (12-15 °C) until they reached their initial weight (6 Kg). This was done after each flushed of harvest.

Mushrooms were harvested at the French commercial stage of development (before veil had broken). For the studies of seasonal variations, mushrooms were randomly harvested one day of collect in October, January, April and July (3 Kg). For the studies of culture cycles variations, mushrooms were collected in winter during 4 successive flushes.

Harvested mushrooms immediately placed at 4°C and then freeze within 4 hours. In the laboratory they were freeze-dried, ground to a fine powder and stored in the dark at room temperature prior to analysis. Mushroom extracts were performed as followed: 100 mg of dried powder were extracted with 5 ml of MeOH overnight. The extract was centrifuged at 3000 rpm for 10 min. and the supernatant was used for analysis. All the extractions were performed in triplicates.

Determination of antioxidant capacity. The ORAC assay was applied as described previously [START_REF] Dudonné | Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays[END_REF]. The reaction was carried out in 75 mM phosphate buffer (pH 7.4) in a 96-well plate. 30 µl of mushroom extract or pure phenolic compounds solutions and 180 µl of fluorescein solution (70 nM final concentration) were mixed and preincubated for 5 minutes at 37°C. 90 µl of APPH solution (12 mM final concentration) were then added and the fluorescence was recorded for 60 Section: Mycosourced molecules and nutritional quality 249 minutes at excitation and emission wavelengths of 485 nm and 520 nm respectively using a Fluostar Optima plate reader (BMG Labtech, Germany). A blank sample and six calibration solutions of Trolox (0.1 to 4 µM, final concentration) were also tested in each assay. All samples were analyzed in triplicate. Area under curve (AUC) was calculated for each sample by integrating the fluorescence curve. Net AUC was calculated by subtracting the AUC of the blank. Regression equation between net AUC and Trolox concentration was determined and ORAC values were expressed as equivalent concentration of Trolox per dry weight.

Determination of total phenolic content. The total phenolic concentration in mushroom extracts was determined according to the Folin-Ciocalteu method using gallic acid as standard. 20 µl solution of mushroom extract and 80 µl of sodium carbonate solution (7.5% in deionized water) were added to 100 µl of Folin-Ciocalteu reagent (diluted 10 fold in deionized water) in a 96-well plate. A blank sample and six calibration solutions of gallic acid (0.625 to 20 µg/ml, final concentration) were analyzed under the same conditions. After incubation for 30 min. at room temperature, the absorbance was measured at 760 nm using a Fluostar Optima plate reader (BMG Labtech, Germany). All determinations were carried out in triplicate and results were expressed as mg gallic acid equivalent/100 g of dry weight.

Determination of ergothionein content. The ergothionein content of mushroom extracts was analyzed by reverse-phase analytical HPLC using a Varian Prostar HPLC. The separation was performed using a Prontosil C18 reverse phase column (250 mm × 4 mm, 5 μm particle size), protected with a C18 guard column. Mushroom extracts were injected (100 µl) and the elution (1 ml/min) was performed using a solvent system comprising solvents A (0.1 % trifluoroacetic acid (TFA) in water) and B (0.1 % TFA in acetonitrile) mixed using a gradient starting with 0% B and linearly increasing to 72% B in 50 min. ERG was quantified by monitoring absorbance at 254 nm and comparing the peak area of the sample to peak areas obtained from different concentrations of the authentic standard.

RESULTS AND DISCUSSION

Seasonal variations. Variations in yields are commonly observed in mushroom farms during the year. They might be due to the outside climatic conditions having consequences on how are maintained the temperature and humidity in the cultivation rooms, and to variations in the quality of raw ingredients used to produce the substrate throughout the year. Several studies have shown that the antioxidant activity of mushrooms was correlated with the content of their phenolic compounds [START_REF] Choi | Influence of heat treatment on the antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes) mushroom[END_REF][START_REF] Zhang | Effects of Different Drying Methods and Extraction Condition on Antioxidant Properties of Shiitake (Lentinus edodes)[END_REF][4]. Thus, it was important to consider the total phenolic (TP) in relation to the antioxidant activity (ORAC) of methanolic mushroom extracts at various periods of the year.

Figure 1 shows that the amounts of total phenolic compounds in methanolic extracts are in the range of 0.8-1.5 % dry weight (dw) in the caps and 0.8-1.1 % dw in the stipes. There were minor seasonal variations in TP and ORAC values, but a significant difference between spring and summer was observed. The winter values in the caps were twice as high as those at spring.

Differences between the parts of mushrooms had previously been observed with other mushrooms [5] and should be taken into account for the extraction of antioxidants from mushrooms. On another hand, the level of variations in L. edodes composition and quality as source of bioactive molecules observed here in a same farm throughout a year of cultivation are significant and should affect the efficiency of mushrooms as source of antioxidant. That also stresses why it is so difficult to compare the data obtained by various authors on samples harvested at only one time under different cultivation conditions.

Section: Mycosourced molecules and nutritional quality 250 Effect of culture aging on mushroom contents and antioxidant activity. Changes in mushroom quality with fruiting body maturity stage are documented [5,6]. The changes between flushes of harvest that linked to culture aging are commonly suspected, but less documented.

Regarding the age of the culture, we observed a strong decrease in the contents of TP in the caps during the culture cycles, whereas those in the stipes were constant (Fig. 2). Surprisingly, the ORAC value increased of 30% in whole mushroom, whereas it decreased in the caps and strongly increased in the stipes. A recent study showed that ERG was responsible for the majority of the DPPH scavenging activity of shiitake mushrooms [7]. In this study, we investigated this hypothesis by analyzing the compounds in whole mushroom extracts during successive flushes using HPLC-DAD (280 nm). As shown in Fig. 3, the profiles of the different samples are similar but the intensity of several peaks increased or decreased during the culture cycle. Indeed, the ERG content decreased of 45% between flush 1 (36,4 mg/100 g) and 2 (19,7 mg/100 g), whereas the intensities of peaks A and B increased.

These results could explain the increase of the antioxidant activity during aging, and we suggest that the antioxidant activities of mushrooms could be largely dependent on other molecules than ERG, perhaps specific compounds which could be synthetized in the stipes during aging. Work is in progress to identify the compounds responsible for the antioxidant activity of aged cultures.

CONCLUSIONS

In this study, we have established the main variations in the antioxidant activity and selected compounds (ERG and TP) of shiitake during cultivation cycles on oak sawdust. We concluded that the antioxidant activities could be largely dependent on other molecules than ERG, perhaps Section: Mycosourced molecules and nutritional quality 252 specific phenolic compounds. The chemical characteristics of the antioxidative components will be now further investigated. This study could provide valuable new opportunities for mushroom growers, since shiitake can serve as a good source of antioxidants in the human diet.

Figure 1 :

 1 Figure 1: Effects of seasonal variations in the total phenolic contents (A) and antioxidant activities (B) of methanolic extracts from different parts of the mushroom.Results are expressed as means ± SD (n=3 extractions).

AB

  Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011Section: Mycosourced molecules and nutritional quality 251

Figure 2 :

 2 Figure 2: Effect of culture aging on total phenolic compounds (A) and antioxidant activity (B) in different parts of the mushroom.Results are expressed as means ± SD (n=3 extractions).

AB

  Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011

Figure 3 :

 3 Figure 3: HPLC chromatograms (280 nm) of whole mushroom methanolic extracts from successive culture cycles. ERG: ergothioneine.

ACKNOWLEDGEMENTS

We would like to thank the Regional Council of Aquitaine for financial support.


Jonathan Gaudin

Olivier Semetey

Xavier Foissac

Sandrine Eveillard

Phytoplasma titer in diseased lavender is not correlated to lavender tolerance to stolbur phytoplasma

Keywords: yellow decline of lavender, Q-PCR, stolbur phytoplasma, lavandin

Yellow decline of lavender is associated with stolbur phytoplasma. It is an economically important disease in south-eastern France, and there is no curative control method. In the past few years, susceptible and tolerant lavenders (Lavandula spp.) or lavandins (Lavandula latifolia x lavandula angustifolia) were proposed as disease management strategy to growers. However, the bases of tolerance were unknown. In order to establish these possible bases, we compared the titers of stolbur phytoplasma in the leaves and shoots of different lavenders and lavandins, either sensitive or tolerant. Results showed that symptom severity was not correlated to the tolerance status of all cultivars tested, and that symptom severity was not correlated to the phytoplasma titer.

Introduction

In south-eastern France, lavender essential oil production is endangered by lavender decline. Lavander (Lavandula angustifolia.) and lavandin (L. latifolia x L. angustifolia) cultures are affected by yellow decline ("deperissement jaune" in French). The disease is associated with the presence of stolbur phytoplasma, which is transmitted by Hyalesthes obsoletus [START_REF] Cousin | -Essais de traitement curatif du "dépérissement jaune" du lavandin[END_REF][START_REF] Cousin | Polymorphisme des microorganismes de type mycoplasme rencontrés dans les tubes criblés de lavandins "Abrial" atteints de "Dépérissement jaune[END_REF], Moreau et al., 1974, E. Boudon-Padieu, personal communication). Stolbur phytoplasma is also spread by vegetative propagation through lavender and lavandin nurseries.

Up to now, no curative methods exist againsts stolbur phytoplasma. The only way is to eliminate diseased plants, apply insecticide treatments against insect vectors and plant phytosanitary certified material. Control of phytoplasma infection with resistant plant has always been very limited. However, as alternatives to susceptible plants, tolerant lavenders(ins) were proposed to growers to reduce the damage caused by lavender decline. Up to now, little is known about the mechanism of this tolerance, which could involve interactions between the plant and the phytoplasma or interactions between the plant and the insect vector. In order to identify the bases of tolerance to the decline, we intended to quantify the stolbur phytoplasma in lavenders(ins).

Materials and methods

Lavenders and lavandins were sampled in fields in South-East France. Lavenders were classified as susceptible (Lavande Bleue, lavande fine, Carla, B7, Maillettes, Matherone) or tolerant (Rapido, Diva). Lavandins were classified as susceptible (Abrial) or tolerant (Grosso, Sumian). DNA extraction was done from 0.3-0.5 g of symptomatic leafy shoots with CTAB method and the final DNA pellet was resuspended in 50µl TE 1X (Murray and Thomson, 1980).

For setting an internal reference used in real-time PCR quantification, amplification of a 976 bp fragment of MAP gene was achieved using primers adkF2 (5'-GTTGGTCGCAGAATTTGTCC-3') and if1R2 (5'-CCAGAAACATAAGCGGTAATCGT-3') for 35 cycles (94°C 40 sec, 55°C 40 sec, 72°C 40 sec). MAP PCR product was cloned in pGEM-T easy plasmid (Promega) following the manufacturer instruction. Q-PCR was carried out using primers MapStol-F and MapStol-R with a method modified from Pelletier and colleagues [START_REF] Pelletier | Triplex real-time PCR assay for sensitive and simultaneous detection of grapevine phytoplasmas of the 16SrV and 16SrXII: a groups with an endogenous analytical control[END_REF]. The primers and probe for 'flavescence dorée' phytoplasma were removed, as well as the endogenous control targeting a grapevine tRNA which was replaced by a polyvalent endogenous control amplifying the COX gene [START_REF] Weller | Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay[END_REF]. Results were analyzed using MxPro QPCR software (Agilent). The number of phytoplasmas was calculated for all plants, and the difference between mean values were evaluated by Chi-square tests.

Results

Obtainment of the quantification standard. A fragment of the stolbur phytoplasma MAP gene used for the detection [START_REF] Pelletier | Triplex real-time PCR assay for sensitive and simultaneous detection of grapevine phytoplasmas of the 16SrV and 16SrXII: a groups with an endogenous analytical control[END_REF] was amplified from DNA extracted from infected lavenders(ins) using primers adkF2 and if1R2. The obtained product was cloned in pGEM-T Easy and sequenced. The plasmid was then quantified and diluted in order to obtain a range standard from 10 1 to 10 8 plasmids/µl. This standard was tested in duplicate in each Q-PCR assay.

Absence of correlation between phytoplasma titer and symptom severity (severity index). A total of 2,500 lavenders(ins), susceptible or tolerant, were collected in 2008, 2009 and 2010. Each plant was assigned a severity index (from 0: no symptoms, to 7: dead plant) and tested in Q-PCR assay 692 were positives for the presence of stolbur phytoplasma. Results showed that phytoplasma titer was not significantly correlated to the severity index.

The phytoplasma titer was measured in susceptible and tolerant lavenders(ins). Figure 1 shows the mean titer of phytoplasma in susceptible and tolerant lavandins in spring and autumn. There was a 2.4 fold and 1.2 fold differences in phytoplasma mean titer between susceptible and tolerant lavandins in spring and autumn respectively. However, these differences were not statistically significant (student impaired t-test). The same differences were observed between susceptible and tolerant lavenders in spring and autumn. The only significant difference in phytoplasma titer was observed for susceptible lavender between spring and autumn. Time course evolution of phytoplasma titer in lavenders(ins) of different susceptibility was studied. In one year, samples were collected every month on the same lavenders(ins) (from May to November 2010 except in August). Six different cultivars were analyzed: lavandins Abrial (susceptible, clonal), Grosso (tolerant, clonal), Blue-lavander (susceptible, populations), Rapido lavender (tolerant, controled population) and Diva (tolerant, clonal, in 2 different geographical locations). Quantification was achieved monthly.

Results showed that the phytoplasma titer evolution was not more rapid in susceptible plants as compared to tolerant plants. Most importantly, detection could become negative even when the plant sample had been previously tested positive, indicating the chronicity of the infection. Results concerning Diva lavender showed that disease evolution is dependant on the geographical location of the fields.

Discussion

Results showed that symptom severity was not correlated to lavender and lavandin susceptibility. Little differences in phytoplasma titer were observed between susceptible and tolerant lavenders(ins) in spring and autumn but they were not significant, only for susceptible lavenders. This might be due to temperature effect on the phytoplasma multiplication-rate.

The geographical location of the plants affected the disease development in Diva lavender, probably due to the influence of the climate on the stolbur phytoplasma multiplication, but also on the insect vector activity. Interactions between the plant and the insect vector could then be an important part of the tolerance mechanism of lavenders(ins) to stolbur phytoplasma.

Figure 1 .

 1 Figure 1. Histogram representing mean titer of phytoplasma (phytoplasma/µl) in susceptible (grey) or tolerant (black) lavandins (lavin), in spring and in autumn. Sampling was done in 2008-2009 and 2010.

Phytoplasma-plant interaction and control of phytoplasma diseases I

Acknowledgements

We greatly acknowledge the ministry of agriculture and France-Agrimer for their financial support.

We thank Marc Mauro, Stéphane Bourret and Marina Barbot for the work they have done during their training course.


est destinée au dépôt

Evolution temporelle des caractéristiques génétiques de l'inoculum aérien de Botrytis cinerea un champignon pathogène extrêmement polyphage

Christel Leyronas, Philippe C. Nicot, Fabien Halkett

To cite this version:

Christel Leyronas, Philippe C. Nicot, Fabien Halkett. Evolution temporelle des caractéristiques génétiques de l'inoculum aérien de Botrytis cinerea un champignon pathogène extrêmement polyphage.

01267798 P42-Evolution temporelle des caractéristiques génétiques de l'inoculum aérien de Botrytis cinerea un champignon pathogène extrêmement polyphage.

  Botrytis cinerea est un champignon Ascomycète très polyphage, à dissémination aérienne. Il provoque la pourriture grise sur de nombreuses espèces d'intérêt agronomique(vigne, laitue, tomate, etc.). Plusieurs groupes génétiques ont été décrits, adaptés notamment à différentes plantes hôtes(Fournier & Giraud, 2008). Dans cette étude nous avons cherché à décrire exhaustivement la diversité génétique chez cette espèce et prédire l'abondance des groupes génétiques en fonction des conditions climatiques. Nous avons développé une méthode de piégeage et de caractérisation moléculaire de l'inoculum de B. cinerea présent dans l'air et nous avons cherché à savoir si la structure génétique évoluait au cours du temps, en fonction de la saison et/ou de l'origine des masses d'air. La structuration génétique déterminée avec Geneland a fait apparaître huit clusters. Deux clusters (5 et 6) présentaient une diversité génétique plus faible. La saison ainsi que l'origine des masses d'air semblent avoir un impact sur la structuration de la population aérienne de B. cinerea. Les différences observées sont principalement dues aux fluctuations d'abondance du cluster 5 : largement majoritaire en été et en automne contrairement à l'hiver et au printemps (test exact Fischer P=0) ainsi que majoritairement présent quand les masses d'air venant du nord et du sud mais pas de l'ouest (test exact Fischer < 0,001).Des analyses complémentaires seront réalisées afin de déterminer quels sont les paramètres qui ont le plus d'impact sur la structuration génétique en essayant de dissocier paramètres biotiques (plantes hôtes) et abiotiques (climat local, origine des masses d'air).

	C. Leyronas 1 , P.C. Nicot 1 , F. Halkett 2
	1 : INRA, UR407 Pathologie Végétale, F-84140 Montfavet ; 2 : INRA, UMR 1136 Interactions Arbres-Microorganismes, F-54280 Champenoux
	Références bibliographiques
	Fournier E. et al., 2002. Molecular Ecology Notes 2: 253-255
	Fournier E. & Giraud T. 2008. J. Evol. Biol. 21: 122-132
	Walker A.S. et al., 2011. Phytopathology 101: 1433-1445
	Mots-clés

34. Réunion annuelle du Groupe d'Etude de Biologie et Génétique des Populations (Petit Pois Déridé 2012), Aug 2012, Avignon, France. 2012, Petit Pois Déridé 2012. Avignon, du 28 au 31 août 2012. Actes du colloque. Session 7 "Dispersion, épidémiologie et biologie des invasions". hal-

Pendant 84 journées réparties sur trois ans, les spores de B. cinerea en suspension dans l'air ont été collectées, puis quantifiées, sur le site de l'INRA d'Avignon. Pour 29 dates, des isolats ont été génotypés à l'aide de neuf marqueurs microsatellites

(Fournier et al., 2002)

. Pour chaque date la trajectoire de la masse d'air susceptible d'avoir amené l'inoculum sur le site de collecte a été modélisée à l'aide du logiciel Hysplit.

Sur les 683 souches génotypées, 67 présentaient un allèle privé sur le microsatellite 6, les classant dans l'espèce cryptique B. pseudocinerea

(Walker et al., 2011)

. Le pourcentage de souches de B. pseudocinerea variait selon les dates de 0 à 50% (ces souches ont été exclues dans les analyses ultérieures). Parmi les 616 souches de B. cinerea, 443 haplotypes distincts ont été déterminés. La diversité clonale (R) variait selon les dates de 0,67 à 1. champignon phytopathogène, groupe génétique, climat, saison, masse d'air

Actes du colloque


Vincent Segura
email: segura@orleans.inra.fr

Kévin Ader

Redouane El Malki

Violaine Murciano

Jean-Paul P Charpentier

A comparison of various

Introduction

The development and use of sustainable biofuels from renewable resources, such as lignocellulosic biomass, has become a priority in order to tackle the increasing demand for energy, together with the concerns about the negative effects of greenhouse gas emissions. Realizing this potential will require the simultaneous development of high yielding biomass production systems and bioconversion technologies that efficiently convert biomass into usable forms of energy, such as bioethanol [START_REF] Ragauskas | The path forward for biofuels and biomaterials[END_REF][START_REF] Rubin | Genomics of cellulosic biofuels[END_REF].

With a conservative average yield of ~10 dry Mg ha -1 year -1 of lignocellulosic biomass, short-rotation coppice poplar has a strong potential as a bioenergy feedstock [START_REF] Ragauskas | The path forward for biofuels and biomaterials[END_REF][START_REF] Sannigrahi | Poplar as a feedstock for biofuels: A review of compositional characteristics[END_REF]. Nevertheless, current poplar varieties have not been bred for this purpose, and thus a significant improvement in quantity and quality of biomass production is expected to be achieved by integrating biorefinery related selection criteria into poplar breeding programs. Biofuel yield results from both the amount of biomass to be converted into biofuel, and from the efficiency of the conversion. Wood chemical properties have been shown to significantly affect biomass saccharification, i.e. its conversion into simple fermentable sugars [START_REF] Davison | Variation of S/G ratio and lignin content in a Populus family influences the release of xylose by dilute acid hydrolysis[END_REF][START_REF] Studer | Lignin content in natural Populus variants affects sugar release[END_REF]. It is thus crucial to understand better the genetic architecture of wood chemical properties. IR spectroscopy is relevant for this purpose because it allows a high-throughput indirect characterization of the physical and chemical properties of the samples of interest, the number of which is typically very large in breeding programs. It has successfully been applied to predict lignin content [START_REF] Zhou | FTIR-ATR-based prediction and modelling of lignin and energy contents reveals independent intra-specific variation of these traits in bioenergy poplars[END_REF] and lignin monomer composition [START_REF] Robinson | Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling[END_REF] in hybrid poplars.

In the context of biofuels, calibrations for the saccharification of various biomasses have recently been reported (Sills and Gosset, 2012a;2012b), highlighting the potential interest of this technique for screening the saccharification potential of biomass samples in breeding programs.

In this context, we have recently started to use IR spectroscopy for predicting poplar biomass chemical properties and saccharification potential. With the aim of defining a relevant way of collecting IR spectra, the present study reports calibration models for 4 different conditions of spectra acquisition, including mid-infrared (MIR) vs. near-infrared (NIR).

Material and methods

The samples used in the present study consisted of 479 wood powders (50 µm -1 mm) that came from the milling of ~50 cm 1-year-old stems from a F1 mapping population tested in the nursery of INRA Orleans. A calibration set comprising 100 samples was selected from preliminary NIR spectra (8000-4000 cm -1

) collected in rotating cups with a spectrum 400 Perkin Elmer spectrophotometer. These samples were selected to be representative of the NIR spectral variability using the Kennard-Stone algorithm [START_REF] Kennard | Computer aided design of experiments[END_REF].

Four different conditions of acquisition of spectra were tested on this calibration set: (i) NIR (8000-4000 cm -1

) spectra averaged over 64 scans collected with a rotating system in a quartz cup (min 10 g of wood powder); (ii) NIR (8000-4000 cm -1 ) spectra averaged over 3 independent collections of scans in a glass vial (min 2 g of wood powder); (iii) NIR (8000-4000 cm -1 ) spectra from a single collection of scans in a glass vial (min 2 g of wood powder); and (iv) MIR (1800-900 cm -1

) spectra from a single collection of scans (several mg of wood powder).

The 100 samples from the calibration set were also phenotyped for 12 quantitative traits related to their chemical composition and saccharification potential (Table 1). For chemical composition, the amount of extractives and total (soluble and insoluble) lignins was assessed following standard protocols, and the amount of holocellulose (cellulose and hemicellulose) was deduced by difference, considering that the sum of these tree components was equal to 1 [START_REF] Carrier | Thermogravimetric analysis as a new method to determine the lignocellulosic composition of biomass[END_REF]. For saccharification potential, the amount of sugars and glucose was measured before and after enzymatic hydrolysis by a set of enzymes extracted and purified from the ascomycete fungus Trichoderma reesei using an automated assay [START_REF] Navarro | Automated assay for screening the enzymatic release of reducing sugars from micronized biomass[END_REF]. From these measured phenotypes, new phenotypes were calculated in order to get a better insight into the factors affecting saccharification potential (Table 1).

Prior to the calibration, the spectra were statistically pretreated in order to improve the signal quality. Statistical pretreatments consisted of normalization and/or derivation, yielding 8 modes for each acquisition condition: "raw", no pretreatment; "norm", normalization (centering and scaling); "der 1", first derivative using a 37 points Savitzky-Golay filter; "der 2", second derivative using a 61 points Savitzky-Golay filter; "norm_der1", normalization followed by first derivative; "norm_der2", normalization followed by second derivative; "der1_norm", first derivative followed by normalization; "der2_norm", second derivative followed by normalization. For each mode of each acquisition condition and each phenotype, the calibration procedure was carried out in 2 steps: (i) selection of relevant spectral bands from the IR spectra matrix in order to decrease its complexity using the Competitive Adaptive Reweighted Sampling (CARS) approach [START_REF] Li | Key wavelengths screening using competitive adaptive reweighted sampling method for multivariate calibration[END_REF]; (ii) on the resulting dataset, calibration model construction using partial least squares (PLS) regression in a 4-fold cross-validation scheme repeated 500 times (Monte Carlo Cross-Validation, MCCV), where the optimal number of latent variables was determined using a Wold's R criterion equal to one [START_REF] Wold | Model construction and evaluation when theoretical knowledge is scare. Theory and application of partial least squares[END_REF]. The best model among all 8 modes was determined for each combination of phenotype and acquisition condition on the basis of both the MCCV statistics (R² cv : cross-validation R²; RMSE cv : root mean square error of cross validation; and RPD cv : Ratio of standard error of performance in cross validation to standard deviation) and the number of latent variables. Afterwards, for each phenotype the best models of each acquisition condition were compared and ranked according to the same criteria (MCCV statistics and number of latent variables). All analyses were carried out in R (R Development Core Team, 2012).

Results and discussion

The best calibration models for each acquisition condition are presented in Tables 2 and3 for the phenotypes related to wood chemical composition and saccharification potential, respectively. Whatever the acquisition condition, the calibration models for the amount of extractives had RPD cv values close to 2. For lignins and holocellulose content, model quality was poorer, with RPD cv values ranging between 1.55 and 1.95. For saccharification potential related traits, the best calibration models were obtained whatever the acquisition condition for the soluble sugars (Soluble_Sug, Soluble_Gluc_ Soluble_Gluc_Prop), with RPDcv values ranging between 2.42 and 3.91, which was consistent with the results for the extractives. The calibration models for the proportion of solubilized glucose were of similar quality for all acquisition conditions, with RPD cv values higher than 2.5. For all the other phenotypes, the calibration model quality was in general poorer with RPD cv ranging between 1.28 and 2.20. Thanks to the prior spectral band selection with the CARS algorithm, all selected calibration models were characterized by a fairly low number of latent variables, ranging between 1 and 7.

For each phenotype, we then compared and ranked the selected models between the different acquisition conditions. A graphical representation of the rank frequencies per acquisition condition is presented in Figure 1. With a rank of 1 for half of the phenotypes ( 6) and of 2 for 4 phenotypes, an average spectrum over 3 acquisitions in a glass vial seemed to be the most promising approach for calibrating our phenotypes. It was substantially better than a single collection in a glass vial which reached the rank 1 only once and the rank 2 twice. The rotating cup system performed surprisingly worse than 3 acquisitions in glass vial, despite the fact it integrates spectra over multiple points. Nevertheless, this acquisition condition was, as expected, better than a single collection in a glass vial. The MIR condition consisted also of a single scan but it ranked better than a single scan in a glass vial, with a rank of 1 or 2 for half of the phenotypes. This acquisition condition had a comparable performance to the collection in a rotating cup, and thus it gave models of slightly worse quality than 3 acquisitions in vial, suggesting that in the case of MIR an average of 3 independent collections appears to be the most promising acquisition technique.

These comparisons should nevertheless be taken with care because the 12 phenotypes under study were not completely independent, with significant correlations between some of them (data not shown). It would thus be interesting to extend and confirm the present results with other phenotypes that would not be correlated with the ones used in the present study. However, the present results still allow us to conclude that, despite the fact it involves the advantage of analyzing only a small amount of material, spectral acquisition on a single point in a glass vial does not seem satisfactory for our purpose, and a spectrum averaged over multiple scans either in a rotating cup or preferably in a glass vial should be recommended.

Figures and tables

Figure 1 .

 1 Figure 1. Graphical representation of the frequency of calibration model ranks depending on the spectra acquisition condition over 12 phenotypes related to the chemical properties and saccharification potential of P. nigra wood.

Table 1 .

 1 List and definition of the 12 quantitative traits phenotyped on the 100 calibration samples of P. nigra wood.

	Trait Name	Trait Identifier	Formula
	Chemical properties
	Extractives content (%)	Extract
	Lignin content (%)	Lignin
	Holocellulose content (%)	Holocellulose	100 -(Extract + Lignin)
	Sacharification Potential
	Soluble Sugars
	Total Sugars (µmol /mg)	Soluble_Sug
	Glucose (µmol /mg)	Soluble_Gluc
	Proportion of glucose (%)	Soluble_Gluc_Prop	Soluble_Gluc / Soluble_Sug
	Solubilized Sugars
	Total Sugars (µmol /mg)	Solubilized_Sug
	Glucose (µmol /mg)	Solubilized_Sug
	Proportion of glucose (%)	Solubilized_Gluc_Prop	Solubilized_Gluc / Solubilized_Sug
	Hydrolized Sugars
	Total Sugars (µmol /mg)	Hydrolyzed_Sug	Solubilized_Sug -Soluble_Sug
	Glucose (µmol /mg)	Hydrolyzed_Gluc	Solubilized_Gluc -Soluble_Gluc
	Proportion of glucose (%)	Hydrolyzed_Gluc_Prop	Solubilized_Gluc_Prop -Soluble_Gluc_Prop

Table 2 .

 2 Calibration models for the phenotypes related to chemical properties of P. nigra wood. For phenotype description see table 1. # comp: number of latent variables in the model; R² train : training R²; R² cv : cross-validation R²; RMSE cv root mean square error of cross validation; RPD cv : Ratio of standard error of performance in cross validation to standard deviation; # lambda: number of wave numbers selected by CARS. For the cross validation statistics, the average over 500 replications is indicated together with the standard deviation in parenthesis.

	Trait	Condition	Treatment	# comp	R	2 train	R	2 cv	RMSE cv	RPD cv	# lambda	rank
	Extract	NIR-COUP	der1_norm	7	0.85	0.79 (0.01)	1.25 (0.04)	2.20 (0.07)	37	1
		NIR-VIAL1	norm_der1	5	0.80	0.74 (0.02)	1.39 (0.04)	1.97 (0.06)	40	4
		NIR-VIAL3	norm_der2	3	0.79	0.75 (0.01)	1.36 (0.04)	2.02 (0.05)	20	2
		MIR	der2_norm	2	0.77	0.74 (0.01)	1.38 (0.02)	1.98 (0.03)	59	3
	Lignin	NIR-COUP	der2_norm	4	0.72	0.65 (0.02)	0.95 (0.03)	1.69 (0.05)	335	1
		NIR-VIAL1	der2_norm	4	0.71	0.62 (0.02)	0.98 (0.03)	1.64 (0.04)	204	4
		NIR-VIAL3	der2	3	0.66	0.62 (0.01)	0.98 (0.02)	1.63 (0.03)	37	3
		MIR	norm	6	0.73	0.63 (0.02)	0.97 (0.03)	1.66 (0.05)	205	2
	Holocellulose	NIR-COUP	der1	5	0.75	0.67 (0.02)	1.42 (0.04)	1.76 (0.05)	35	2
		NIR-VIAL1	norm_der1	6	0.81	0.73 (0.02)	1.28 (0.04)	1.95 (0.06)	40	1
		NIR-VIAL3	norm	5	0.70	0.66 (0.01)	1.45 (0.03)	1.71 (0.04)	79	3
		MIR	norm	1	0.60	0.58 (0.01)	1.61 (0.02)	1.55 (0.02)	12

Table 3 .

 3 Calibration models for the phenotypes related to saccharification potential of P. nigra wood. For phenotype description see table 1. # comp: number of latent variables in the model; R² train : training R²; R² cv : cross-validation R²; RMSE cv root mean square error of cross validation; RPD cv : Ratio of standard error of performance in cross validation to standard deviation; # lambda: number of wave numbers selected by CARS. For the cross validation statistics, the average over 500 replications is indicated together with the standard deviation in parenthesis.

	Trait	Condition	Treatment	# comp	R	2 train	R	2 cv	RMSE cv	RPD cv	# lambda	rank
	Soluble_Sug	NIR-COUP	der1_norm	7		0.93	0.89 (0.01)	0.02 (0.00)	3.06 (0.11)	759	4
		NIR-VIAL1	norm	4		0.90	0.89 (0.00)	0.03 (0.00)	3.00 (0.06)	39	3
		NIR-VIAL3	norm	3		0.91	0.90 (0.00)	0.02 (0.00)	3.15 (0.05)	10	2
		MIR	norm	5		0.95	0.93 (0.00)	0.02 (0.00)	3.91 (0.11)	50	1
	Soluble_Gluc	NIR-COUP	der1_norm	5		0.89	0.87 (0.01)	0.01 (0.00)	2.81 (0.06)	155	3
		NIR-VIAL1	der2	5		0.88	0.85 (0.01)	0.01 (0.00)	2.63 (0.07)	17	4
		NIR-VIAL3	norm_der1	5		0.90	0.88 (0.01)	0.01 (0.00)	2.92 (0.06)	11	2
		MIR	norm	6		0.94	0.92 (0.01)	0.00 (0.00)	3.50 (0.11)	98	1
	Soluble_Gluc_Prop	NIR-COUP	der2_norm	6		0.90	0.83 (0.01)	0.06 (0.00)	2.46 (0.09)	552	4
		NIR-VIAL1	der1	4		0.86	0.83 (0.01)	0.06 (0.00)	2.42 (0.06)	37	3
		NIR-VIAL3	der2_norm	6		0.89	0.85 (0.01)	0.05 (0.00)	2.57 (0.08)	182	2
		MIR	norm_der2	5		0.88	0.86 (0.01)	0.05 (0.00)	2.65 (0.08)	61	1
	Solubilized_Sug	NIR-COUP	norm_der1	5		0.75	0.69 (0.02)	0.04 (0.00)	1.82 (0.05)	17	3
		NIR-VIAL1	norm	6		0.76	0.69 (0.02)	0.04 (0.00)	1.81 (0.05)	22	4
		NIR-VIAL3	norm	6		0.78	0.73 (0.02)	0.04 (0.00)	1.94 (0.05)	48	1
		MIR	norm	5		0.75	0.70 (0.02)	0.04 (0.00)	1.84 (0.05)	20	2
	Solubilized_Sug	NIR-COUP	der2	6		0.76	0.68 (0.02)	0.03 (0.00)	1.79 (0.06)	46	2
		NIR-VIAL1	der1	4		0.71	0.66 (0.02)	0.03 (0.00)	1.72 (0.04)	34	3
		NIR-VIAL3	der2	6		0.82	0.74 (0.02)	0.02 (0.00)	1.97 (0.07)	90	1
		MIR	norm_der1	6		0.72	0.66 (0.02)	0.03 (0.00)	1.73 (0.04)	16	4
	Solubilized_Gluc_Prop	NIR-COUP	der2	5		0.89	0.86 (0.01)	0.03 (0.00)	2.65 (0.07)	66	3
		NIR-VIAL1	der2	3		0.87	0.84 (0.01)	0.04 (0.00)	2.55 (0.06)	59	4
		NIR-VIAL3	norm_der1	4		0.89	0.87 (0.01)	0.03 (0.00)	2.74 (0.07)	65	1
		MIR	norm	3		0.88	0.86 (0.01)	0.03 (0.00)	2.65 (0.06)	427	2
	Hydrolyzed_Sug	NIR-COUP	der1_norm	7		0.72	0.60 (0.03)	0.04 (0.00)	1.59 (0.05)	54	3
		NIR-VIAL1	norm_der2	6		0.73	0.62 (0.02)	0.04 (0.00)	1.64 (0.05)	50	2
		NIR-VIAL3	der1	7		0.72	0.63 (0.02)	0.04 (0.00)	1.66 (0.05)	26	1
		MIR	norm	3		0.49	0.39 (0.03)	0.05 (0.00)	1.28 (0.03)	459	4
	Hydrolyzed_Gluc	NIR-COUP	norm_der2	6		0.81	0.75 (0.02)	0.03 (0.00)	2.00 (0.07)	78	2
		NIR-VIAL1	der1_norm	5		0.78	0.73 (0.01)	0.03 (0.00)	1.92 (0.05)	23	3
		NIR-VIAL3	der2_norm	6		0.85	0.79 (0.01)	0.02 (0.00)	2.20 (0.07)	65	1
		MIR	norm_der1	3		0.74	0.71 (0.01)	0.03 (0.00)	1.86 (0.04)	32	4
	Hydrolyzed_Gluc_Prop	NIR-COUP	der2	4		0.69	0.63 (0.02)	0.05 (0.00)	1.65 (0.04)	56	3
		NIR-VIAL1	norm	6		0.71	0.64 (0.02)	0.04 (0.00)	1.68 (0.05)	22	2
		NIR-VIAL3	norm	5		0.74	0.68 (0.02)	0.04 (0.00)	1.77 (0.05)	15	1
		MIR	der1_norm	3		0.64	0.61 (0.01)	0.05 (0.00)	1.62 (0.03)	4	4

B e l l o n -M a u r e l V . , W i l l i a m s P . , D o w n e y G . , E d s

Acknowledgements

The present work was carried within the FUTUROL project funded by OSEO Innovation. We thank David Navarro and Jean-Guy Berrin for their contribution in the acquisition of phenotypes related to the saccharification potential.


Sara Pinosio

Fabio Marroni

Véronique V Jorge

Patricia P Faivre-Rampant

Nicoletta Felice

Eleonora Di Centa

Catherine Bastien

Federica Cattonaro

Michele Morgante

Sara Pinosio1

Fabio Marroni1

Veronique Jorge3

Patricia Faivre-Rampant4

Nicoletta Felice2

Eleonora Di Centa5

Catherine Bastien3

Federica Cattonaro5

Michele Morgante1

Genome-wide structural variation in poplar

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