Marie Fiers

Véronique Edel-Hermann

Catherine Chatot

Yves Le Hingrat

Claude Alabouvette

Christian Steinberg

Subtilis Jensen

; Mahmoud

Streptomyces Scabiei

S Acidiscabiei

Streptomyces Wanner

Karima Bouchek-Mechiche

Potato soil-borne diseases -A review

Keywords: pomme de terre, tubercule, défaut superficiel, microorganisme, stress environnemental, pratique culturale, communauté microbienne, Rhizoctonia solani potato, tuber, blemish, microorganism, environmental stress, cultural practice, microbial community, Rhizoctonia solani Clavibacter michiganensis var. sepedonicum Ring rot Narrow: potato, sugar beet, tomato, eggplant X X X Worldwide Clostridium spp. Bacterial soft rot Wide: animals and plants X Worldwide 33 P. atrosepticum, P. carotovorum, Dickeya spp. Black leg, soft rot Pectobacterium spp. and P. carotovorum: Wide: potato, rapeseed, sugar beet, chicory witloof, carrots, radish, weeds P potato tomato, cabbage, weeds Dickeya spp.: potato, ornementals, maize, chicory witloof, tomato, weeds Worldwide 21, 29, 33 Ralstonia solanacearum Thecaphora smut Seed tuber, soil, infested plant parts

de tâtonnements et de découvertes. Je tiens à remercier toutes celles et ceux qui ont pris part à cette aventure.

La science ouvre à l'esprit humain une voie infinie, et le lance, par une série d'étapes sans nombre, sur l'Asymptote de la Vérité.

Paul Bert (1933Bert ( -1986) ) secrétaires, Catherine, Fabienne, Sylvie et Stéphanie pour leur efficacité et leur bonne humeur.

Je voudrais remercier aussi l'équipe 3, Sébastien Aimé pour ses petites blagues au café, Fabiola Bastian, gracias Mamita pour ton grain de folie, bisoutitos ti!, Elodie Gautheron, pour les petites pauses quand mon bureau se trouvait sur son chemin, Johann Leplat, Julie Laurent et Marion Bégin; Grégory Girardot qui a partagé mon bureau pendant son stage. Un merci particulier à Chantal Olivain pour m'avoir fait confiance pour donner des TP à l'IUT de Génie Biologique. Ce fut une expérience très enrichissante.

La vie au labo aurait été moins drôle sans Céline Janvier, ma "maîtresse" de stage du tout début qui m'a appris beaucoup et avec qui on a vraiment bien rigolé. Merci à tous les stagiaires, thésards et permanents, Abdel, Amandine, Noémie, Mélanie, Sam, Fafa, Thérèse, Francky, Florence, pour les déjeuners à la cantine et les sorties le week-end.

Enfin merci à tous ceux qui comptent pour moi, tous mes amis, en particulier les Boubous dont j'ai fait la connaissance au milieu de ma thèse et avec qui les mardi soirs et les week-ends n'ont plus jamais été comme avant. Merci à ma famille, mes parents et ma soeur que j'aime. Merci à mes grand-mères qui ont toujours été fières de moi et à mes grand-pères qui, j'en suis sûre, l'auraient été. Nicolas, merci d'être à mes côtés.

Abstract

The visual quality of fresh potatoes, Solanum tuberosum, became a dominant criterion and a significative economical issue in potato market. According the vegetative reproduction of this species, requirements for visual quality are also needed for potato seed tubers. As an organ for reserve and propagation, the tuber grows underground and is in contact with soil-borne microorganisms, making it potentially exposed to blemishes, for the majority of which the origin is still unclear. The objective of this work is to make an inventory of those tuber blemishes, to characterize them and determine their causes. After the establishment of consensual nomenclature and classification of the blemishes, two hypotheses were formulated:

(1) blemishes are due to pathogenic attacks and/or (2) they result from a response of the plants to environmental stresses. The assessment of the first hypothesis allowed identifying a wide diversity of microorganisms living on the blemished tuber surface. Their pathogenicity was tested by several biological assays that allowed producing blemishes on progeny tubers and fulfilling the Koch's postulates for the fungus Rhizoctonia solani causing sclerotia. For many other blemishes no clear relationship was established between a microorganism and a blemish. A study of the microbial structure of the geocaulosphere of tubers blemished or not, showed that bacterial and fungal communities adopted different dynamics during the growing season and according to the sanitary status of the seed tuber, but no causality link could have been drawn. On the other hand, an increase of R. solani population around blemished tubers was observed. The diversity of strains of R. solani originating from France and from Europe and associated to the blemished tubers was characterized. The phylogenetic relationships between the strains were independent of the geographical origin and of the host cultivar, thus the existence of frequent genetic events and genetic mixing between the populations of R. solani was suggested. Concerning the potential implication of different abiotic factors, a survey conducted with farmers showed the implication of soil pH, some cultural practices, including the choice of the susceptible cultivars and meteorological conditions on the occurrence of some blemishes. This work made clear the blemish nomenclature, confirmed the implication of R. solani in the occurrence of some blemishes and suggested new hypotheses concerning the occurrence of blemishes as a plant response to a stressful environment. Thus, a path was opened toward the resolution of the issue asked by all the potato community, responsible for the marketing of a mass consumption fresh product and answering to market requirements related to visual and culinary qualities and to environmental friendly modes of production. America where Andeans people used to eat it since millennia. Recent evidence suggests that potato was domesticated 10,000 years ago near Titicaca Lake, in an area that is now the border between Bolivia and Peru, and where the greatest diversity of wild species is still to be found. First record of potato in Europe dates back in 1573 in Spain but it is only in the early seventeenth century that it becomes an important food and came back across the Atlantic ocean to North America [START_REF] Stevenson | Compendium of potato diseases[END_REF].

List of tables

List of figures

Potato crop is the fourth main food crop in the world after maize, rice and wheat with 325 million tons produced in 2007 (Table 0.1). This is also the world's number one non-grain food commodity and is integrated in the international food system. Despite potato trading is not as well developed as cereal trading on the global markets, efforts are made to promote potato spread all over the world. The year 2008 was declared international year of the potato by the FAO (Food and Agriculture Organization). Indeed, potato can help fulfilling the first ONU's millennium development goal that aims at eradicating extreme poverty and hunger in the world.

The potato produces more nutritious food more quickly, on less land, and in harsher climates than any other major crop. Up to 85 percent of the plant is edible human food, compared to around 50 % in cereals. Potatoes have good gustative and nutritional qualities; they are rich in carbohydrates, making them a good source of energy. A single medium-sized potato contains about half the vitamin C recommended daily intake -and contains a fifth of the recommended daily value of potassium (FAO, 2008). 1) (FAO, 2008), under temperate, subtropical and tropical conditions. This crop is from an agronomical point of view well suited to places where land is limited and labour can be abundant, conditions that characterize much of the developing world, although it is essentially a "cool weather crop". For that reason, potatoes are planted in early spring in temperate zones and late winter in warmer regions, and grown during the coolest months of the year in hot tropical climates. In some sub-tropical highlands, mild temperatures and high solar radiation allow farmers to grow potatoes throughout the year, and harvest tubers within 90 days of planting. In temperate climates, such as in northern Europe, it can take up to 150 days (FAO, 2008). As well as being a staple food, potato is also processed into French fries, crisps and is used for dried products and starch production. In some countries, potato is still used for animal feeding but this use is decreasing. In many countries in Asia, Africa, Central and South America, there is a need for high and stable potato production to meet increasing food demands from human population growth during the current period of environmental (including climate) change (Vreugdenhil, 2007).

Over the last 10 years, world potato production has increased at an annual average rate of 4.5 percent especially in developing countries. Potato consumption is declining in Europe and is increasing in developing world with a global average of 31.3 kg per capita and per year. France is the tenth potato producer in the world with 6.3 million tons and French people consume about 40 kg of fresh potatoes and 25 kg of transformed products per capita and per year. Main French potato production areas are located in the North (Nord-Pas-de-Calais, Picardie and Champagne-Ardennes), West (Haute-Normandie and Bretagne) and Center (Centre) of France.

Total cultivated surface represents 170 000 ha in which 100 000 ha are used for fresh potatoes production, 25 000 ha for early potatoes, 15 000 ha for seed potatoes and 30 000 ha for starch production (FAO, 2008).

At the European level, France has the leadership for fresh potato production and is the first exporting country. The exports are made toward other European countries among which Spain, Italy, Belgium, United Kingdom, Germany, The Netherlands and Greece are the main partners [START_REF] Cnipt | Usages et attitudes des consommateurs à l'égard des pommes de terre Paris[END_REF]. France also imports 116 000 tons of fresh potatoes (mostly for processing) and a reduced volume of early potatoes coming from non EU-countries like Egypt, Israel and Morocco. Concerning seed potato production, France ranks at the third level after the Netherlands and Germany.

The annual export volumes are between 80,000 to 100,000 tons towards European Union (Spain, The Netherlands, Belgium, Portugal, Italy, Greece, Germany, United Kingdom, and central Europe). Other destinations are North Africa, i.e. Algeria, Egypt, Morocco, and Tunisia and the Middle East (FNPPPT and GNIS, 2007).

Professional seed organizations such as Bretagne Plants in Brittany, Comité Nord in the North and Grocep in the Center of France are also responsible for breeding new cultivars. Germicopa, located in Brittany, is one of the oldest European private potato breeder, seed producer and trader. All breeding companies select interesting traits of plants and aim at creating new hybrids that integrate as much positive traits as possible.

In the North-West zone of Europe, fresh potato consumption per capita has been progressively declining in the past twenty years. Recent studies about consumer's behavior concerning fresh potato acquisition showed that visual quality -shape and smooth aspect of the tubers -was the first choice criterion (59%) well before taste or culinary use. Soil particles on tuber surface may not stimulate the majority of the consumers to buy potatoes which look like dirty and have a poverty connotation.

However, contradictory is the situation in the organic market where soil aspect of tubers may represent the authentic and natural connotation of the product [START_REF] Rapoport | Perceptions et attitudes des consommateurs vis-à-vis de la pomme de terre[END_REF]CNIPT and TNS-Sofres, 2008). In order to stimulate interest for the fresh product and satisfy clients' expectation, fresh potato professionals have put forward several inciting actions like washing potatoes before being conditioned.

But this action has had a major drawback: it highlighted tuber blemishes previously hidden by the soil residues and lead to the downgrading of a significant part of the commodity.

Indeed, potatoes -whether they are food or seed-are submitted to numerous diseases caused by fungi, bacteria, nematodes or viruses. Most of them can affect all parts of the plant: foliage, berries tubers and root system, as well as other Solanaceous hosts. As a consequence, affected tubers used as seeds are a very powerful mean of further disease dispersal. Transportations of tubers between different countries are submitted to control and rules to avoid the spread of diseases.

Sanitary quality levels are established for both branches of the potato industry, fresh potatoes and seeds. In the certification scheme of seed potatoes, strict regulations for field and lot inspections are required for the sanitary quality depending on seed categories and the type of diseases. For example, tubers for basic seeds (class Elite) must have less than 5 % of their surface covered by symptoms of R. solani and if the disease percentage reaches 5 to 10 %, seeds are downgraded to lower categories (A or B). For quarantine diseases (EPPO, 2008b) such as cyst nematodes, wart, ring rot or brown rot, no symptoms nor parasitic agents are tolerated.

Because the potato crop is vegetatively propagated, the visual quality of tubers, intertwined with its sanitary status, is then a consecutive requirement for seed production since it is a pre-requisite for ware potato. Among superficial blemishes of the tuber, most of them are due to well known and studied pathogens but others, called atypical blemishes, have not yet known causes thus making cultural control very difficult and inefficient.

In order to help potato growers to better know the causes of these blemishes and find technical solutions for improving the potato quality, Bretagne Plants and Germicopa -To determine whether the blemishes are caused by microorganisms, abiotic factors, or both.

-To identify environmental friendly products controlling the blemishes and to optimize their use.

This manuscript presents the PhD work carried out to fulfill the two first objectives (Figure 0.1). The third one was treated by BBV.

A review of the well known pathogenic microorganisms of the potato is carried out.

Data about the most favorable development conditions, their ecology and the methods used to detect and quantify them are necessary to set up efficient methods for disease control. Moreover, since the use of pesticides is limited in order to prevent environmental pollutions, the need of alternative control methods is becoming stringent. A review of the current available bibliography is presented in the Chapter 1 of this manuscript. This chapter is to be submitted to Agronomy for Sustainable Development.

Two consecutive sampling campaigns were led to collect tubers presenting blemishes in the main French -seed as fresh -potato production areas. Typical and atypical blemishes on tuber surface were observed, named and classified in different categories. This classification is the subject of Chapter 2 and was submitted to Plant Pathology (Fiers et al., submitted). Concerning the determination of the blemish causes, two hypotheses were suggested. First, the blemishes have a pathogenic origin. This hypothesis was tested by isolations of microorganisms living on the tuber surface and pathogenicity tests. This study appears in chapter 3 and is published in European Journal of Plant Pathology (Fiers et al., 2010). In parallel, Chapter 4, submitted to FEMS Microbiology Ecology, treats of the effects of stresses caused by the microbial communities living in the nearest surroundings of the tubers, tested by molecular methods. As Rhizoctonia solani appeared as an indeniable component of the biotic factors, chapter 5 aims at characterizing at the intraspecific level the structure of the populations of R. solani in France, compared with those from other countries. The second hypothesis suggests that the blemishes result from environmental stresses leading to a reaction of the plant. A survey was carried out with several French potato producers to determine the soil traits of the fields and the cultural practices. The results are described and discussed in chapter 6. Eventually, the results of this work are discussed and prospects are suggested. Potato soil-borne diseases -A review

Introduction

Potato crop, the world's number one non-grain food commodity, is the fourth main food crop in the world after maize, rice and wheat, with 325 million tons produced in 2007. Potatoes are grown in more than 100 countries, mainly in Asia (135 million tons) and Europe (130 million tons) (FAO, 2008). They have good gustative and nutritional qualities and can be grown under various climates. This is the reason why FAO (Food and Agriculture Organization) has declared the year 2008 the international year of the potato Indeed, potato can help fulfilling the first ONU's millennium development goal that aims at eradicating extreme poverty and hunger in the world. However, potato (Solanum tuberosum) crop can suffer more than 40 pests and diseases caused by insects, nematodes, viruses, bacteria and fungi. Those pathogens are air-borne or soil-borne and cause damages on all parts of the plant. In this review, we will focus on soil-borne fungi, bacteria and nematodes (Table 1.1, Figure 1.1).

Diseases caused by viruses or viroïds provoke generally foliar symptoms: leaf distortion, mosaic, crinkling, leaf and vein necroses, dwarfing and leaf rolling. Only some viruses -tobacco rattle virus (TRV), potato mop-top virus (PMTV), potato virus Y (PVY ntn ) and tobacco necrosis virus (TNV) -can cause damages on tubers such as blemishes or rots in tuber flesh (Table 1.1). They will not be considered in this review because they are not originating from soil-borne pathogens but depend on aphids, fungi or nematodes to be transmitted.

Soil-borne diseases affecting potato crop can be divided into two groups depending on symptoms: symptoms damaging tubers and those damaging other parts of the plant [START_REF] Gudmestad | Management of soilborne diseases of potato[END_REF].

Diseases affecting stems or roots affect the crop development and may lead to a reduction of the yield (Table 1.1). Stem lesions can be watery and they develop to the stem pith with (stem rot) or without the formation of sclerotia (black leg, white mold). Other lesions can appear like more discrete light brown lesions but nevertheless they affect the yield of the crop (skin spot, stem canker). Soil-borne pathogens (Phoma leaf spot, Verticillium wilt) sometimes cause aerial symptoms like necroses or chloroses occasionally associated with wilting and rolling (bacterial ring rot). Finally root lesions, mainly caused by nematodes feeding on the roots lead to necroses or rots. Nematodes feeding sites are good entry points for other soil microorganisms, which increase the destruction of the roots. Among diseases affecting tubers, symptoms can be divided into three categories: galls, blemishes and rots (Table 1.1). Galls causes outgrowth and tuber deformation.

The most frequent galls are known as powdery scab, wart, common and netted scabs, root-knot nematode and false root-knot nematode.

Blemishes affect only the tuber skin but they became economically important since consumers' habits have changed and tubers are washed before selling. Blemishes can appear on the tuber surface as spots called black dot, black scurf, skin spot or powdery scab) or as areas of atypical appearance presenting a more or less pronounced scabby (common scab) or silver (silver scurf) aspect. Rots, which affect the tuber flesh more deeply, include different types such as dry rots, soft rots (charcoal rot, leak, bacterial soft rot, black leg, and stem rot), flesh discoloration (pink rot) or vascular ring discoloration (ring rot, brown rot, Verticillium wilt, Fusarium dry rots). Dry rot diseases also damage stored potatoes.

Potato becoming a more and more important foodstuff in the world, it is essential to control diseases, which cause direct yield losses and decrease of farmer's incomes due to downgrading the quality of affected tubers. Therefore, knowledge about the pathogens as well as factors influencing disease severity is needed to set up efficient control strategies. Before reviewing the different causes of occurrence and development of the main soil-borne potato diseases it is important to recall the concepts of soil inoculum potential and soil suppressiveness which describe the complex interactions between the soil, the pathogens and the plant.

Plant diseases result from the compatible interactions between a susceptible host plant and a pathogen. These direct interactions are important but should not outshadow the key role of environmental factors, which influence these interactions and thereby disease incidence or severity. In contrast to aerial diseases, the soilborne diseases are induced by pathogens which are embedded in the soil matrix.

Thus, the soil interferes in many ways in the relationships between and among microorganisms, pathogens and host plant. It can even modify the interaction among microorganisms themselves. In some soils, disease incidence or severity commonly remain low in spite of the presence of the pathogen, a susceptible host plant and favourable climatic conditions. They are called disease suppressive soils [START_REF] Messiha | Potato brown rot incidence and severity under different management and amendment regimes in different soil types[END_REF][START_REF] Steinberg | Soil suppressiveness to plant diseases[END_REF]. Soil suppressiveness to diseases depends on the pathogen itself -its inoculum density and its intrinsic aggressiveness -and also on different soil factors, including both biotic and abiotic components.

In the first part of this paper, the influence of abiotic factors on disease severity will be reviewed. Then the characteristics of the inoculum and its relationships with the rest of the microbiota will be considered. Finally, risk assessment models, decision support systems and control strategies based on collected data will be discussed. Soil abiotic components such as texture, organic matter content, pH as well as temperature and moisture greatly affect the behaviour of the pathogens and determine disease incidence or severity.

I. 1. Soil temperature

Temperature and moisture of the soil are obviously greatly dependent on the climatic conditions, but also of some cultural practices such as irrigation. Temperature is of major importance in disease development since it determines pathogen growth rate [START_REF] Baljeet | Influence of depth of planting on development of black scurf of potato (Rhizoctonia solani)[END_REF], kind of symptoms (Bouchek-Mechiche et al., 2000b) and geographical distribution of the diseases. Most of the potato pathogens can grow at soil temperatures between 10°C and 25 °C, the optim al potato growth temperatures (Table 1.2). However, gangrene, black scurf and powdery scab are favoured by mean temperatures below 15°C [START_REF] Baker | Types of Rhizoctonia solani diseases and their occurence[END_REF][START_REF] Gindrat | Storage rot of potato. II. Lesions development in relation to the parasitic species and temperature. Control measures[END_REF][START_REF] Harrison | Powdery scab disease of potato -A review[END_REF]; on the contrary, black dot, black leg, stem rot and charcoal rot are favoured by temperatures above 27°C. Similarly, sting and root-knot nematode s reproduce better between 25°C and 30°C to 35°C depending on the origin of th e populations. Colonization, sclerotia [START_REF] Davet | Recherches dur le Colletotrichum coccodes (Wallr.) Hugues. La phase non parasitaire[END_REF]Lees, 2003;Tsror, 2004 Fusarium spp. Fusarium dry rots 15-20 X (9.2 % whc) X (27.9 % whc)

Mycelial growth Tivoli, 1983;[START_REF] Kong | Study on biological characteristics of Fusarium solani on Vanilla planifolia root[END_REF] Helminthosporium solani Silver scurf 15-32 X X

(sporulation) [START_REF] Adams | The effect of irrigation on powdery scab and other tuber diseases of potatoes[END_REF]Errampalli, 2001 Macrophomina phaseolina Charcoal rot > 30 X (RH > 52%)

Mycelial growth Pycnidia production, mycelial growth Germination tube elongation [START_REF] Gindrat | Storage rot of potato. II. Lesions development in relation to the parasitic species and temperature. Control measures[END_REF][START_REF] Vishwa | Studies on the germination and longevity of pycnidiospores of Macrophomina phaseolina[END_REF][START_REF] Muthukrishnan | Factors affecting survival of Macrophomina phaseolina in soil[END_REF][START_REF] Somani | [END_REF][START_REF] Amadioha | Microbial deterioration of potato tubers (Solanum tuberosum L.) in Nigeria[END_REF][START_REF] Mehta | Influence of light and temperature on induction of pycnidia and germination of pycnidiospores[END_REF]Chowdary and Govindalah, 2007 Phoma andigena var. andina Phoma leaf spot Phoma spp. Gangrene 5-18; optimum: 10

X

Pycnidial and conidial productions

Pycnidial production [START_REF] Fox | Biology of potato gangrene[END_REF][START_REF] Gindrat | Storage rot of potato. II. Lesions development in relation to the parasitic species and temperature. Control measures[END_REF]Bång, 1989;[START_REF] Coelho | Sporulation of Phomopsia and Phoma on different culture media, temperature and luminosity conditions[END_REF][START_REF] Lo | Factors affecting pycnidial production and pycnidiospore germination of Phoma wasabiae, the causal agent of wasabi[END_REF] Polyscytalum pustulans Skin spot 15-30 X (waterlogged soil)

X

Sclerotia formation [START_REF] Baker | Types of Rhizoctonia solani diseases and their occurence[END_REF]Hide and Firmager, 1989;[START_REF] Xu | Biological characteristics of Rhizoctonia solani Kuhnzheng[END_REF][START_REF] Bakali | Black scurf of potato[END_REF][START_REF] Panka | Influence of micro irrigation on health status of chosen potato cultivars grown in very light soil[END_REF] Rosellinia spp.

Rosellinia black rot

Sclerotinia sclerotinium

White mold [START_REF]Working Party on Agricultural Quality Standards[END_REF][16][17][18][START_REF] Falloon | Powdery scab control[END_REF][20][21][22][23][24][25][26][27] No effect of RH

Sclerotia production

Mycelial growth, sclerotia production [START_REF] Chowdhury | Effect of culture media, light, temperature and pH on the mycelial growth and sclerotia formation of Sclerotium rolfsii Sacc[END_REF]Prithiviraj et al., 2000;[START_REF] Blum | Temperature, light and culture medium affecting the production of sclerotia of Sclerotium rolfsii and Sclerotinia sclerotiorum[END_REF][START_REF] Gupta | [END_REF] Spongospora subterranea Powdery scab Tuber galls: 12-15 Root gall: 17 X Constant dampness [START_REF] Harrison | Powdery scab disease of potato -A review[END_REF][START_REF] Graaf | Effect of soil inoculum level and environmental factors on potato powdery scab caused by Spongospora subterranea[END_REF][START_REF] Merz | Review: powdery scab of potato -increased knowledge of pathogen biology and disease epidemiology for effective disease management[END_REF] Synchytrium endobioticum Wart 12-18 X Hampson and Coombes, 1997;[START_REF] Stachewicz | Do temperature and rainfall limit the occurrence of potato wart in Germany?[END_REF] Thecaphora solani Thecaphora smut 5-20 X [START_REF] Eppo | Thecaphora solani[END_REF][START_REF] Sepulveda | Effect of different soil humidity on the development of potato smut (Angiosorus solani) in two potato varieties (Solanum tuberosum) under greenhouse conditions[END_REF][START_REF] Wale | Diseases, pests and disorders of potatoes[END_REF] Verticillium dahliae and V. alboatrum [START_REF] Jaggi | [END_REF][START_REF] Serfontein | Shekhawat and Perombelon[END_REF][START_REF] Vries | [END_REF][START_REF] Latour | Control of bacterial diseases of potato caused by Pectobacterium spp. (Erwinia carotovora)[END_REF]Hélias, 2008 Ralstonia solanacearum Brown rot 23 (temperate strains) 30-35 (tropical strains) X (whc 60%) [START_REF] Shekhawat | Factors affecting survival in soil and virulence of Pseudomonas solanacearum[END_REF][START_REF] Sunaina | Persistence of Ralstonia solanacearum in naturally infested soil under changing environment. Potato, global research & development[END_REF]Tomlinson et al., 2005 Common scab: et al., 2001;[START_REF] Chandel | Effect of tillage and water management on the population behaviour of root-knot nematodes Meloidogyne triticoryzae in rice crop[END_REF][START_REF] Pandey | Effect of soil moisture and hydrogen ion concentration on population dynamics of Meloidogyne incognita at Allahabad[END_REF]. Wu et al., 2006 Nacobbus aberrans False root-knot nematode 10-25; optimum: 20 High soil moisture due to abundant rainfalls, poor drainage, heavy soils or irrigation, influences disease development and the opening of the lenticels which are further entry points for soil-borne pathogens into the tuber [START_REF] Helias | Pectobacterium spp. and Dickeya spp. on potato: a new nomenclature for Erwinia spp., symptoms, epidemiology and disease prevention[END_REF]. Several diseases, especially bacterial diseases, are enhanced by high moisture content (Table 1.2), but few diseases are favoured by low levels of moisture. This is the case for black dot, some dry rots induced by Fusarium spp., stem rot, wart, common scab, and sting and root-knot nematodes. High soil moisture generally has indirect effects which might favour disease severity. This is the case of flooding that provokes oxygen depletion and CO 2 enrichment resulting in an increase of Spongospora subterranea (powdery scab) development [START_REF] Harrison | Powdery scab disease of potato -A review[END_REF]. In some cases, the influence of soil moisture on disease severity is not clearly demonstrated. Depending on the studies, black scurf, stem canker, silver scurf (Helminthosporium solani) and Thecaphora smut (T. solani) are either positively or negatively correlated with soil moisture [START_REF] Adams | The effect of irrigation on powdery scab and other tuber diseases of potatoes[END_REF]Hide and Firmager, 1989;[START_REF] Sepulveda | Effect of different soil humidity on the development of potato smut (Angiosorus solani) in two potato varieties (Solanum tuberosum) under greenhouse conditions[END_REF][START_REF] Bakali | Black scurf of potato[END_REF][START_REF] Wale | Diseases, pests and disorders of potatoes[END_REF]. Conversely, high relative humidity during storage of tubers has always a negative impact (Table 1.2).

I. 3. Soil texture

The soil texture described the relative percentage of sand, loam and clay contents. Most of fungal diseases are enhanced in light sandy soils (Table 1.3).

Conversely, it is generally accepted that clay soils favour bacterial activity [START_REF] Marshall | Clay mineralogy in relation to survival of soil bacteria[END_REF][START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF] explaining that clay or heavy soils are conducive to bacterial soil-borne diseases (ring rot, soft rot, brown rot and netted scab).

Concerning nematodes, no general rule can be drawn up as some species are more prevalent in heavy soils (root-knot nematodes) and other species in light soils (sting nematodes). Soil texture also influences soil structure, through the distribution of different pore sizes, determining the actual living space for bacteria, fungi and predators. It also influences the water activity; water retained in pores of narrow diameter being less available for organisms that water present in big pores. [START_REF] Combrink | The effect of calcium, phosphate and boron on the keeping quality and quality determining tuber characteristics of potatoes[END_REF][START_REF] Tivoli | [END_REF][START_REF] Tivoli | [END_REF][START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF] Helminthosporium solani Silver scurf X X [START_REF] Lennard | Factors affecting the development of silver scurf (Helminthosporium solani) on potato-tubers[END_REF]Lutomirska, 2004 Macrophomina phaseolina Charcoal rot X 6.5

Singh and Kaiser, 1994

Phoma andigena var. andina

Phoma leaf spot Phoma spp. Gangrene X 3.8 -5.6 2.9 -7.6 ‰ El [START_REF] Fahl | The effect of soil treatment with sulphur and lime on the incidence of potato diseases with special reference to blight[END_REF]Rudkievicz et al., 1983;[START_REF] Lutomirska | Influence of the soil type and date of irrigation on tuber infection with Rhizoctonia solani (Kuhn)[END_REF] Sclerotinia sclerotinium

White mold High

Sclerotium rolfsii

Stem rot X ~ 6.5 High nitrogen, organic carbon and low phosphorus and potassium levels [START_REF] Sheoraj | Influence of soil texture, temperature and relative humidity on development of collar rot of lentil[END_REF][START_REF] Banyal | Soil characteristics and their relation to the development of tomato collar rot caused by Sclerotium rolfsii[END_REF] Spongospora subterranea Powdery scab X organic or over irrigated soils X poorly drained soils 4. 7 -7.6 High aluminium level [START_REF] Zambolim | Powdery scab of potato[END_REF]Van de Graaf et al., 2005;[START_REF] Gilchrist | Effect on the powdery scab of four isolates of the fungus Trichoderma asperellum in three types of soils[END_REF][START_REF] Merz | Review: powdery scab of potato -increased knowledge of pathogen biology and disease epidemiology for effective disease management[END_REF] Synchytrium endobioticum Wart X variable [START_REF] Hampson | Pathogenesis of Synchytrium endobioticum. 5. Wart disease suppression in potato in soils amended with urea and or ammonium-nitrate in relation to soil pH[END_REF]Hampson and Coombes, 1997 Thecaphora solani Thecaphora smut

High salt level [START_REF] Eppo | Thecaphora solani[END_REF] Verticillium dahliae and V.

albo-atrum

Verticillium wilt X 6-9 High Ca, low K, Mg and total soil C level Low [START_REF] Baard | Effect of alternate drying and wetting of the soil fertilizer amendment and pH on the survival of micro sclerotia of Verticillium dahliae[END_REF][START_REF] Höper | Importance of physical and chemical soil properties in the suppressiveness of soils to plant diseases[END_REF]Davis, 2001

BACTERIA

Clavibacter michiganensis var. sepedonicum

Ring rot X et al., 1974;Lücke, 1975;[START_REF] Lambert | Relationship of calcium to potato scab[END_REF]

Ralstonia solanacearum

Brown rot X [START_REF] Messiha | Potato brown rot incidence and severity under different management and amendment regimes in different soil types[END_REF] X variable No ammonium intake Hsu, 1991;Schekhawat and Perombelon, 1991;[START_REF] Michel | Effect of a soil amendment on the survival of Ralstonia solanacearum in different soils[END_REF][START_REF] Yi | Control strategy of acidified nutrient solution on bacterial wilt of tomato plants[END_REF][START_REF] Keshwal | Effect of physical properties of soil on wilt incidence and population of Ralstonia solanacearum[END_REF][START_REF] Muller | Survival of Ralstonia solanacearum biovar 2 (race 3), the causal agent of potato brown rot, in soil and on several kinds of materials (microcosm)[END_REF] Streptomyces scabiei, S. acidiscabiei, S. europeiscabiei

Common and netted scab X 5.2 -7 Low Mn level Rudkievicz et al., 1983;[START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF][START_REF] Bukhalid | Plant pathogenicity in the genus Streptomyces[END_REF][START_REF] Milosevic | Effect of some soil reclamation measures on the disease severity in potato tubers caused by common scab (Streptomyces scabies Thaxt.). Radovi Poljoprivrednog Fakulteta Univerziteta u Sarajevu (Works of the Faculty of Agriculture University of Sarajevo)[END_REF][START_REF] Lazarovits | Edaphic soil levels of mineral nutrients, pH, organic matter, and cationic exchange capacity in the geocaulosphere associated with potato common scab[END_REF]

NEMATODES

Belonolaimus longicaudatus

Sting nematode X [START_REF] Mashela | Correlation of Belonolaimus longicaudatus, Hoplolaimus galeatus, and soil texture with yield of alyceclover (Alysicarpus spp.)[END_REF] Ditylenchus destructor Potato rot nematode Globodera pallida, Globodera rostochiensis Potato cyst nematode X 6.1 Low nitrogen level High [START_REF] Pelsmaeker | Nematodes in potato fields and the relation to some biotic and abiotic factors[END_REF][START_REF] Ruijter | Effects of potato-cyst nematodes (Globodera pallida) and soil pH on root growth, nutrient uptake and crop growth of potato[END_REF]Trifonova, 2001 Meloidogyne spp. Root-knot nematode X X 7.5 High [START_REF] Kumar | Influence of soil type on the interaction between root-knot nematode, reniform nematode and root-rot in eggplant[END_REF][START_REF] Kandji | Diversity of plant-parasitic nematodes and their relationships with some soil physico-chemical characteristics in improved fallows in western Kenya[END_REF][START_REF] Pandey | Effect of soil moisture and hydrogen ion concentration on population dynamics of Meloidogyne incognita at Allahabad[END_REF][START_REF] Melakeberhan | Effect of soil pH on the pathogenesis of Heterodera glycines and Meloidogyne incognita on Glycine max genotypes[END_REF] Nacobbus aberrans False root-knot nematode Paratrichodorus and Trichodorus spp.

Stubby-root nematode (TRV vector)

T. similis and P. pachydermus T. primitivus low High level of Fe [START_REF] Barbez | The distribution of virus-vector nematodes in seed-potato fields in Flanders (Belgium) and its relation to some biotic and abiotic factors[END_REF][START_REF] Spaull | Nematodes and nutrients: association between plantparasitic nematodes and soil chemicals[END_REF] Pratylenchus spp. 1.3). Soils with extreme pH values are often highly suppressive to several plant diseases [START_REF] Höper | Importance of physical and chemical soil properties in the suppressiveness of soils to plant diseases[END_REF]. However, pH fluctuations resulting from amendments influence pathogens and disease development.

Decreasing pH increases the availability of phosphorus, nitrogen and aluminium ions and decreases potato cyst nematode, brown rot and common scab damages respectively [START_REF] Mulder | Effects of soil characteristics on the relationship between potato cyst nematodes and yield. II. Acidity (soil pH)[END_REF][START_REF] Michel | Effect of a soil amendment on the survival of Ralstonia solanacearum in different soils[END_REF][START_REF] Ruijter | Effects of potato-cyst nematodes (Globodera pallida) and soil pH on root growth, nutrient uptake and crop growth of potato[END_REF][START_REF] Mizuno | Judging conducive soils from clay mineralogical properties and soil chemical method to suppress potato common scab[END_REF]. On the contrary, addition of urea in soil induces a very large increase in pH and a good control of Synchitrium endobioticum, the fungal pathogen causing wart [START_REF] Hampson | Pathogenesis of Synchytrium endobioticum. 5. Wart disease suppression in potato in soils amended with urea and or ammonium-nitrate in relation to soil pH[END_REF].

Soil organic matter is both the substrate for and the result of microbial activity. In addition, together with clay, organic matter affects soil structure and thus moisture content and aeration. The quantity of organic matter in a soil has an effect on the appearance and the development of diseases but its quality is also an important point which has been too poorly addressed [START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF].

I. 5. Soil organic matter

Soil organic matter is both the substrate for and the result of microbial activity.

In addition, together with clay, organic matter affects soil structure and thus moisture content and aeration. The quantity of organic matter in a soil has an effect on the appearance and the development of diseases but its quality is also an important point which has been too poorly addressed [START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF].

Most physico-chemical factors are not independent one from the others, which makes experiments and data interpretation very difficult. Soil texture can affect humidity, soil amendments impact on pH and all those factors influence availability of chemical elements. Thus, the pathogenic inoculum present either in the soil or on the tuber surface has to find the optimal climatic and edaphic conditions to develop.

II. Effects of biotic factors on the occurrence and development of soilborne potato diseases II. 1. Autecology of pathogens

Inoculum sources, survival and dissemination pathways

The survival of soil-borne pathogens during periods without potato crop depends on their ability to resist to unfavourable conditions. Most of them survive in soil under the form of resistant structures able to directly infect the new host crop.

Some pathogens can also survive as saprophytes on host crop residues or on alternative hosts during winter. Finally, inoculum can also be introduced in the field by the seeds; it is called seed-borne or tuber-borne inoculum. Inoculum sources are diverse and for any disease several inoculum sources can play a role (Table 1.4).

Soil-borne fungi produce different conservation structures. Fusarium spp. forms chlamydospores resistant to adverse conditions, Rhizoctonia solani, Verticillium spp., Sclerotinia sclerotinium overwinter as sclerotia. Bacteria can survive over winter with favourable moisture, temperature and soil type [START_REF] Ficke | Longevity of Pectobacterium carotovorum var. atrosepticum (van Hall) Dowson on seed material and in the soil[END_REF][START_REF] Bradbury | Erwinia carotovora var. atroseptica. [Descriptions of Fungi and Bacteria[END_REF][START_REF] Bignell | Thaxtomin biosynthesis: the path to plant pathogenicity in the genus Streptomyces[END_REF]. Nematodes can survive and persist in soil as protective cysts surrounding the eggs (Globodera spp.) or as juveniles in host roots (Meloidogyne spp.) [START_REF] Qian | Biology, identification and control of root knot nematode Meloidogyne hapla on Aconitum carmichaeli[END_REF][START_REF] Wharton | Water relations during desiccation of cysts of the potato-cyst nematode Globodera rostochiensis[END_REF].

In absence of resistant structures and of efficient saprophytic abilities, some pathogens need alternative hosts to survive in absence potatoes. These alternative hosts frequently belong to the Solanaceous family and act as a long term reservoir of the pathogen [START_REF] Chang | Local sources of Clavibacter michiganensis ssp. michiganensis in the development of bacterial canker on tomatoes[END_REF]Tomlinson et al., 2005).

Fungal dissemination occurs frequently as spores (conidiospores, chlamydospores, pycnidiospores, sporangiospores, oospores and zoospores) or mycelium transported by water (rain, irrigation, and flow in soil), by soil adhering to farm equipment or introduced by contaminated seed tubers [START_REF] Zambolim | Powdery scab of potato[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Bae | Colonization dynamics and spatial progression of Verticillium dahliae in individual stems of two potato cultivars with differing responses to potato early dying[END_REF]. Moreover, some pathogens liberate mobile dissemination forms such as zoosporanges. Zoospores of P. erythroseptica, S. subterranea and S.

endobioticum are responsible for short distance dissemination of these pathogens [START_REF] Wharton | Seed treatment applicationtiming options for control of Fusarium decay and sprout rot of cut seedpieces[END_REF][START_REF] Merz | Review: powdery scab of potato -increased knowledge of pathogen biology and disease epidemiology for effective disease management[END_REF]. Adult nematodes such as P.

Chapter 1 -Potato soil-borne diseases penetrans are able to migrate on quite long distances better than do larvae [START_REF] Pudasaini | The influence of host and temperature on the vertical migration of Pratylenchus penetrans[END_REF].

Relationship between inoculum density and disease severity

Although there is not always a clear and linear relationship, the severity of the disease generally increases with an increasing level of inoculum (Table 1.4).

Sometimes a minimum inoculum threshold is needed to initiate the disease development. This is the case for potato cyst nematode (Samaliev et al., 1998).

Conversely, the disease severity of black dot does not increase any more beyond a maximum threshold of inoculum density [START_REF] Nitzan | Disease Potential of Soil-and Tuberborne Inocula of Colletotrichum coccodes and Black Dot Severity on Potato[END_REF]. In fact as stated above, the relationship between inoculum density and disease severity greatly depends on the environmental factors which determine the level of soil suppressiveness. et al., 1987;[START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Cullen | Use of quantitative molecular diagnostic assays to investigate Fusarium dry rot in potato stocks and soil[END_REF] Helminthosporium solani Silver scurf Seed tuber, soil Negative correlation [START_REF] Lennard | Factors affecting the development of silver scurf (Helminthosporium solani) on potato-tubers[END_REF][START_REF] Bains | Soil survival and thiabendazole sensitivity of Helminthosporium solani isolates from Alberta, Canada[END_REF]

3. Mechanisms of infection

Potato plants are essentially composed of cellulose, a very solid polymer and tubers are enveloped in a protective covering called periderm made of a suberin biopolymer providing the primary barrier against disease, insects, dehydratation, and physical intrusions for the potato tuber [START_REF] Lulai | Compendium of Potato Diseases[END_REF]. Soil-borne pathogens of potato have various ways to penetrate the host plant and break physical barriers. They enter the roots, young sprouts, underground stem, stolons or tubers. Some pathogens cannot infect intact tuber periderm or lenticels and penetrate through wounds [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Taylor | Differences in etiology affect mefenoxam efficacy and the control of pink rot and leak tuber diseases of potato[END_REF] whereas other pathogens can penetrate either directly by mechanical and/or enzymatic degradation of the host's cells or through natural openings (stomata, lenticels, eyes) (Table 1.5).

Once they have penetrated the host, pathogens colonize plant tissues. Fungi grow through the parenchyma of the cortex and often reach the vascular vessels. T. solani, S. endobioticum and Streptomyces spp. penetration provokes hypertrophy of the colonized tissues resulting in galls. They grow in the plant, induce cell death and feed on them saprophytically. They secrete phytotoxins -for example thaxtomin produced by Streptomyces spp. -inducing the formation of several layers of suberized corky cells, creating a large lesion firmly integrated within the tuber skin [START_REF] Stevenson | Compendium of potato diseases[END_REF]Mulder et al., 2008;[START_REF] Perez | Potato smut, Thecaphora solani[END_REF]. Compared to common scab, powdery scab pustules formation is a relatively short process, at the end of which a single wound-cork layer remains that covers the entire lesion. After hardening off, this layer can be easily removed from the lesion without any damage of the underlying tissues (Delleman et al., 2005). C. coccodes, H. solani, P. pustulans, R. solani, S.

subterranea and Streptomyces spp. are responsible for several superficial alterations called blemishes. Colonization by those pathogens is usually limited to superficial layers of tuber periderm [START_REF] Harrison | Powdery scab disease of potato -A review[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Cunha | Occurrence and epidemiological aspects of potato silver scurf in California[END_REF]Lehtonen et al., 2008a;[START_REF] Bignell | Thaxtomin biosynthesis: the path to plant pathogenicity in the genus Streptomyces[END_REF] but they can colonize other parts of the plant until they reach vascular system. Streptomyces spp. responsible for netted scab blemishes have pathogenic mechanisms that are assumed to not implicate thaxtomin but rather a necrotic protein [START_REF] Bouchek-Mechiche | Streptomyces turgidiscabies and Streptomyces reticuliscabiei: one genomic species, two pathogenic groups[END_REF].

Fungi and bacteria causing rots produce a wide range of hydrolytic enzymes such as cellulases, pectinases, xylanases and proteases [START_REF] Olivieri | Hydrolytic Activities of Fusarium solani and Fusarium solani f. sp eumartii Associated with the Infection Process of Potato Tubers[END_REF]. They are responsible for tissue maceration and cell death, after which the microorganisms have access to the nutritional resources of the dead plant tissues [START_REF] Amadioha | Interaction of hydrolytic enzymes produced by Rhizoctonia bataticola during rot development[END_REF][START_REF] Aveskamp | Biology and recent developments in the systematics of Phoma, a complex genus of major quarantine significance[END_REF]. Pectobacterium spp. develop an original pathogenic strategy based on quorum sensing, which utilise freely diffusible chemical signal molecules allowing pathogenic bacteria to synchronise the production of virulence factors and make the pathogenic attack more efficient [START_REF] Liu | Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen Pectobacterium atrosepticum[END_REF]. Finally, nematodes attacking potatoes can be classified in two categories: ectoparasites and endoparasites. Ectoparasites nematodes (B. longicaudatus, Paratrichodorus spp.

and Trichodorus spp.) are mobile and feed on potato roots in the area of cell division and elongation without penetrating the root [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Mugniéry | The canon of potato science: 15. Root-knot nematodes[END_REF].

The endoparasitic nematodes of potato, D. destructor and P. penetrans are migrating endoparasites; they feed from cell to cell into the host, whereas Globodera spp., Meloidogyne spp. and N. aberrans are sedentary endoparasites, they induce specialized feeding sites in plant roots. D. destructor and P. penetrans penetrate underground parts of the plant, feed on the cortical cells and migrate into the roots, destroying cell after cell. G. pallida, G. rostochiensis, Meloidogyne spp., and N.

aberrans develop feeding cavities in host root, causing galls [START_REF] Mugniéry | The canon of potato science: 15. Root-knot nematodes[END_REF]. [START_REF] Dillard | Survival of Colletotrichum coccodes in infected tomato tissue and in soil[END_REF]Cullen et al., 2002;[START_REF] Heilmann | Genetic variability in the potato pathogen Colletotrichum coccodes as determined by amplified fragment length polymorphism and vegetative compatibility group analyses[END_REF][START_REF] Erukhimovitch | Early and rapid detection of potato's fungal infection by Fourier transform infrared microscopy[END_REF][START_REF] Shcolnick | Additional vegetative compatibility groups in Colletotrichum coccodes subpopulations from Europe and Israel[END_REF]Nitzan et al., 2008 Fusarium spp. Fusarium dry rots 13 species, especially F. sambucinum and F. solani var. coeruleum (15 VCGs) Microconidia, chlamydospores and mycelium on plant debris

Wounds, enzymatic

Isolation and morphology , RT-PCR, PCR enzyme-linked immunosorbent assay, volatile profile [START_REF] Tivoli | [END_REF][START_REF] Ouellette | Volatile monitoring technique for disease detection in stored potatoes[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF]Oliveri et al., 2004;[START_REF] Cullen | Use of quantitative molecular diagnostic assays to investigate Fusarium dry rot in potato stocks and soil[END_REF][START_REF] Burlakoti | Real-time PCR quantification and mycotoxin production of Fusarium graminearum in wheat inoculated with isolates collected from potato, sugar beet, and wheat[END_REF][START_REF] El-Hassan | [END_REF][START_REF] Peters | Pathogenicity to potato tubers of Fusarium spp. isolated from potato, cereal and forage crops[END_REF][START_REF] Peters | [END_REF]Recep et al., 2009 Helminthosporium solani Silver scurf At least 4 years in the soil Enzymatic Classical detection methods, PCR [START_REF] Bains | Soil survival and thiabendazole sensitivity of Helminthosporium solani isolates from Alberta, Canada[END_REF]Errampalli et al., 2001;[START_REF] Martinez | Ultrastructure of the infection process of potato tuber by Helminthosporium solani, causal agent of potato silver scurf[END_REF]Geary et al [START_REF] Mcdonald | Differentiation of Phoma foveata from P-exigua using a RAPD generated PCR-RFLP marker[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Giebel | Pathogenesis of potato gangrene caused by Phoma exigua var. foveata: II. Activities of some hydrolases and dehydrogenases[END_REF][START_REF] Cullen | Development and validation of conventional and quantitative polymerase chain reaction assays for the detection of storage rot potato pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata[END_REF] Phytophthora erythroseptica Pink rot One species with few genetic variations Enzymatic [START_REF] Lucas | Immunochemical identification of a new molecular form of acid-phosphatase of host origin arising during infection of potato tubers by Phytophthora erythroseptica Pethybr[END_REF][START_REF] Peters | Influence of crop rotation and conservation tillage practices on the severity of soil-borne potato diseases in temperate humid agriculture[END_REF][START_REF] Peters | Limited genetic diversity in North American isolates of Phytophthora erythroseptica pathogenic to potato based on RAPD analysis[END_REF][START_REF] Cullen | Development and validation of conventional and quantitative polymerase chain reaction assays for the detection of storage rot potato pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata[END_REF]Taylor et al [START_REF] Tsror | A 9-year monitoring study of diseases on potato seed tubers imported to Israel[END_REF][START_REF] Gilligan | Inoculum density and spatial pattern of Rhizoctonia solani in field plots of Solanum tuberosum: Effects of cropping frequency[END_REF][START_REF] Carling | Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani[END_REF][START_REF] Lees | Development of conventional and quantitative real-time PCR assays for the detection and identification of Rhizoctonia solani AG-3 in potato and soil[END_REF][START_REF] Gvozdeva | Interaction of proteinases secreted by the fungal plant pathogen Rhizoctonia solani with natural proteinase inhibitors produced by plants[END_REF][START_REF] Hughes | Molecular diagnostics and their importance for Rhizoctonia solani control in potatoes[END_REF] [START_REF] Zambolim | Powdery scab of potato[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Graaf | Detection and quantification of Spongospora subterranea in soil, water and plant tissue samples using real-time PCR[END_REF][START_REF] Ward | A real-time PCR assay based method for routine diagnosis of Spongospora subterranea on potato tubers[END_REF][START_REF] Merz | Improved immunological detection of Spongospora subterranea[END_REF][START_REF] Qu | Detection and quantification of Spongospora subterranea f. sp subterranea by PCR in host tissue and naturally infested soils[END_REF][START_REF] Nakayama | Spongospora subterranea soil contamination and its relationship to severity of powdery scab on potatoes[END_REF] [START_REF] Nelson | Survival of Corynebacterium sepedonicum in potato stems and on surfaces held at freezing and above-freezing temperatures[END_REF][START_REF] Correll | Vegetative compatibility and pathogenicity of Verticillium albo-atrum[END_REF][START_REF] Joaquim | Vegetative compatibility and virulence of strains of Verticillium dahliae from soil and potato plants[END_REF][START_REF] Platt | Detection methods for Verticillium species in naturally infested and inoculated soils[END_REF][START_REF] Tsror | Control of Verticiilium dahliae prior to potato production by soil fumigation with chloropicrin[END_REF][START_REF] Strausbaugh | Assessment of vegetative compatibility of Verticillium dahliae tester strains and isolates from California potatoes[END_REF][START_REF] Zhang | Molecular detection of Verticillium albo-atrum by PCR based on its sequences[END_REF][START_REF] Atallah | Multiplex real-time quantitative PCR to detect and quantify Verticillium dahliae colonization in potato lines that differ in response to Verticillium wilt[END_REF]

BACTERIA

Clavibacter michiganensis var. sepedonicum

Ring rot

One species with few genetic variation ≈ 18 months in plain soil Enzymatic Immuno-fluorescence antibody, staining (IFAS), (ELISA), RT-PCR; LMW RNA profiles [START_REF] Nelson | Survival of Corynebacterium sepedonicum in potato stems and on surfaces held at freezing and above-freezing temperatures[END_REF][START_REF] Logan | Methods for detection of the blackleg ring rot and gangrene pathogens in potato nuclear stock mother tubers and plantlets[END_REF][START_REF] Eichenlaub | Ohazurike and Arinze[END_REF][START_REF] Palomo | Rapid identification of Clavibacter michiganensis subspecies sepedonicus based on the stable low molecular weight RNA (LMW RNA) profiles[END_REF][START_REF] Smith | Repetitive sequence-derived PCR profiling using the BOX-A1R primer for rapid identification of the plant pathogen Clavibacter michiganensis subspecies sepedonicus[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Vasinauskiene | Occurrence of Clavibacter michiganensis subsp. sepedonicus in Lithuania[END_REF][START_REF] Hukkanen | Epidemiology of Clavibacter michiganensis subsp sepedonicus in potato under European conditions: population development and yield reduction[END_REF]Gudmestad et [START_REF] Bradbury | Erwinia carotovora var. atroseptica. [Descriptions of Fungi and Bacteria[END_REF][START_REF] Ouellette | Volatile monitoring technique for disease detection in stored potatoes[END_REF][START_REF] Tsror | A 9-year monitoring study of diseases on potato seed tubers imported to Israel[END_REF]Hélias et al., 2000;[START_REF] Lazy | Pathogenic Prokaryotes[END_REF][START_REF] Atallah | A methodology to detect and quantify five pathogens causing potato tuber decay using real-time quantitative polymerase chain reaction[END_REF][START_REF] Latour | Control of bacterial diseases of potato caused by Pectobacterium spp. (Erwinia carotovora)[END_REF][START_REF] Pitman | Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand[END_REF]Hélias, 2008

Ralstonia solanacearum

Brown rot One species with several biovars (1, 2, and 2T) and races (1 and 3) attacking potato

Water, weeds, (soil ?) Enzymatic Isolation, PCR , immunofluorescence and fluorescent in-situ hybridisation (FISH) Hsu, 1991;[START_REF] Ronda | Evaluation of a PCR for detection of Ralstonia (Pseudomonas) solanacearum (race 3, biovar 2) and Clavibacter michiganensis subsp. sepedonicus and comparison with immuno-fluorescence microscopy, plating on semi-selective SMSA medium, pathogenicity test and fluorescent in-situ hybridisation[END_REF][START_REF] Rangaswami | Diseases of vegetables[END_REF][START_REF] Messiha | Potato brown rot incidence and severity under different management and amendment regimes in different soil types[END_REF][START_REF] Bignell | Thaxtomin biosynthesis: the path to plant pathogenicity in the genus Streptomyces[END_REF][START_REF] Nouri | Diversity of Ralstonia solanacearum causing potato bacterial wilt in Iran and the first record of phylotype II/biovar 2T strains outside South America[END_REF]Smith and

Mechanical and enzymatic

Soil extraction and, morphological identification, allele-specific PCR [START_REF] Wharton | Water relations during desiccation of cysts of the potato-cyst nematode Globodera rostochiensis[END_REF][START_REF] Moxnes | The population dynamics of potato cyst nematodes[END_REF][START_REF] Achenbach | Using SNP markers to dissect linkage disequilibrium at a major quantitative trait locus for resistance to the potato cyst nematode Globodera pallida on potato chromosome V[END_REF][START_REF] Reid | PCR Detection of Potato Cyst Nematode[END_REF][START_REF] Rehman | Identification and Characterization of the Most Abundant Cellulases in Stylet Secretions from Globodera rostochiensis[END_REF]

Mechanical and enzymatic

Morphometrics, host range, biochemical and molecular (RFLP) analysis [START_REF] Hlaoua | Effect of Meloidogyne incognita on the potato crop[END_REF][START_REF] Melakeberhan | Integrated approaches to understanding and managing Meloidogyne hapla populations' parasitic variability[END_REF][START_REF] Mugniéry | The canon of potato science: 15. Root-knot nematodes[END_REF]Dietrich and Sommer, 2009;[START_REF] Ozarslandan | First Report of Columbia Root-Knot Nematode (Meloidogyne chitwoodi) in Potato in Turkey[END_REF] Nacobbus

Enzymatic

Morphometric and molecular (PCR-RFLP) analysis [START_REF] Brown | Species of Pratylenchus associated with Solanum tuberosum cv Superior in Ohio[END_REF][START_REF] Saeed | Effect of initial nematode population density on the interaction of Pratylenchus penetrans and Verticillium dahliae on 'Russet burbank' potato[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF]Mugniery and Phillips, 2007 a: VCG: vegetative compatibility group; AG: anastomosis group. b: RT-PCR: reverse transcriptase polymerase chain reaction; Q-PCR: quantitative PCR; FT-IR: Fourier transformed infra red spectroscopy

4. Genetic variability

A soil-borne disease can be caused by several species of pathogens belonging to a single genus, by one species, or even by a subgroup of a species.

Each species or sub-species is adapted to particular conditions or variety.

Knowledge of the genetic diversity of pathogens is useful for precise diagnosis and control of potato diseases.

Since Erwinia has been divided into 2 different genera, Pectobacterium and Dickeya [START_REF] Helias | Pectobacterium spp. and Dickeya spp. on potato: a new nomenclature for Erwinia spp., symptoms, epidemiology and disease prevention[END_REF] [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Prescott | La classe des Clostridia[END_REF].

Within a same species, the pathogen may belong to different groups with various genetic, pathogenic and physiological traits leading to the characterization of races, biovars and, recently, genomovars -strains which are phylogenetically differentiable, but are phenotypically indistinguishable -, phylotypes and sequevars -one or several strains with a given sequence - [START_REF] Nouri | Diversity of Ralstonia solanacearum causing potato bacterial wilt in Iran and the first record of phylotype II/biovar 2T strains outside South America[END_REF]. Fungi without sexual reproductive stage, such as Colletotrichum spp., Fusarium spp., or Verticillium spp., are classified in vegetative compatibility groups (VCGs). Within a VCG, hyphae belonging to different isolates can anastomose and form stable heterokaryons, whereas hyphae from isolates belonging to different VCGs can not. This mechanism is the only known mechanism of genetic exchange between individuals of asexual fungi [START_REF] Hiemstra | Vegetative compatibility groups in Verticillium dahliae isolates from the Netherlands as compared to VCG diversity in Europe and in the USA[END_REF]. Hyphal anastomosis is also used to categorize the isolates of R. solani into anastomosis groups (AG). Presently 13 AGs have been described, several of which being divided into subgroups. Individual AGs are not strictly associated with a specific host or family of hosts, for example AG 1 with rice and AG 8 with cereals. AG 3 isolates, and more specifically isolates from the AG 3 PT subgroup, are often associated with potato diseases [START_REF] Kuninaga | Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3[END_REF][START_REF] Carling | Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani[END_REF]. However it was shown, in Great Britain and France, that AG 2-1 and AG 5 can cause disease in potato crops but with a much lower incidence than AG 3 PT [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Woodhall | Characterization of Rhizoctonia solani from potato in Great Britain[END_REF].

As a result of the genetic evolution of pathogens, new pathotypes are regularly discovered. Conversely, some populations such as P. erythroseptica and C. michiganensis subsp. sepedonicus vary slightly in pathogenicity and in genetic diversity suggesting a relatively recent introduction of a small founding population of the pathogen [START_REF] Smith | Repetitive sequence-derived PCR profiling using the BOX-A1R primer for rapid identification of the plant pathogen Clavibacter michiganensis subspecies sepedonicus[END_REF][START_REF] Peters | Limited genetic diversity in North American isolates of Phytophthora erythroseptica pathogenic to potato based on RAPD analysis[END_REF]. Genetic evolution can be achieved by vertical or horizontal gene transfer. Meloidogyne populations originally did not possess the cell-wall-degrading enzymes required to invade host roots.

Although the mechanism of horizontal gene transfer remains largely elusive, it has been speculated that a gene coding for a cell-wall-degrading enzyme was horizontally transferred from a rhizobial bacterium to the nematode and was kept in the genome of the nematode by strong selection pressures representing important initial steps facilitating the invasion of plants by nematodes [START_REF] Dieterich | How to become a parasite -lessons from the genomes of nematodes[END_REF]. By genetic evolution, pathogens can adapt to the different environmental conditions they are submitted to. This enables them to skirt control measures and continuously forced farmers to use new control methods.

5. Diagnosis and detection methods

Rapid detection of plant parasitic pathogens enables to set up adapted control measures and to avoid disease expansion and yield losses, even if the infestation level is low. Classical detection methods begin with visual observation and characterization of symptoms followed by identification using morphologic traits for nematodes [START_REF] Crow | Effects of potato-cotton cropping systems and nematicides on plant-parasitic nematodes and crop yields[END_REF][START_REF] Riga | First report of the stubby-root nematode, Paratrichodorus teres, from potato in the Columbia basin of Washington State[END_REF][START_REF] Melakeberhan | Integrated approaches to understanding and managing Meloidogyne hapla populations' parasitic variability[END_REF][START_REF] Mugniéry | The canon of potato science: 15. Root-knot nematodes[END_REF] or isolation on selective media for fungi and bacteria. Carbon source utilisation, sugar degradation and production of specific enzymes allow the biochemical identification of bacteria (Flores-Gonzalez et al., 2008;[START_REF] Pitman | Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand[END_REF]. However, these classical methods are often not accurate enough to distinguish different strains or pathovar of the same species. [START_REF] Abeln | Genetic differentiation of Phoma exigua varieties by means of AFLP fingerprints[END_REF][START_REF] Cullen | Development and validation of conventional and quantitative polymerase chain reaction assays for the detection of storage rot potato pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata[END_REF]Flores-Gonzalez et al., 2008;[START_REF] Pitman | Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand[END_REF].

Fluorescent in-situ hybridisation (FISH) or stable low-molecular-weight (LMW) DNA profiles were developed to detect R. solanacearum and C. michiganensis var.

sepedonicum, respectively [START_REF] Ronda | Evaluation of a PCR for detection of Ralstonia (Pseudomonas) solanacearum (race 3, biovar 2) and Clavibacter michiganensis subsp. sepedonicus and comparison with immuno-fluorescence microscopy, plating on semi-selective SMSA medium, pathogenicity test and fluorescent in-situ hybridisation[END_REF][START_REF] Palomo | Rapid identification of Clavibacter michiganensis subspecies sepedonicus based on the stable low molecular weight RNA (LMW RNA) profiles[END_REF]. Immunological techniques such as immunochromatographical lateral flow, Enzyme-Linked Immunosorbent Assay (ELISA) and immunofluorescence are based on the recognition of specific markers at the surface of pathogenic cells to detect and identify the pathogens [START_REF] Ronda | Evaluation of a PCR for detection of Ralstonia (Pseudomonas) solanacearum (race 3, biovar 2) and Clavibacter michiganensis subsp. sepedonicus and comparison with immuno-fluorescence microscopy, plating on semi-selective SMSA medium, pathogenicity test and fluorescent in-situ hybridisation[END_REF][START_REF] Merz | Improved immunological detection of Spongospora subterranea[END_REF][START_REF] Hughes | Molecular diagnostics and their importance for Rhizoctonia solani control in potatoes[END_REF]. Fungal pathogens display typical infrared spectra that differ from the spectra of substrate material such as potato; they can be early and rapidly detected by Fourier transform infrared (FT-IR) microscopically-based technique [START_REF] Erukhimovitch | Early and rapid detection of potato's fungal infection by Fourier transform infrared microscopy[END_REF]. Finally, monitoring of normal and disease-induced volatile profiles in stored potatoes or of the light reflected from plant in fields are valuable techniques to detect stress and thus pathogenic infections [START_REF] Ouellette | Volatile monitoring technique for disease detection in stored potatoes[END_REF][START_REF] Heath | Monitoring the presence and distribution of potato cyst nematodes: the potential use of light reflected from the crop[END_REF].

II. 2. Interactions between microorganisms, organisms and pathogens

Potato pathogens are not the only microorganisms living in the potato surroundings. A huge microbial biomass is associated and interacts with potatoes. About 10 7 bacteria colony forming units (CFU) per g of soil live in the potato rhizosphere and potato geocaulosphere which is the volume of soil surrounding the tubers [START_REF] Lazarovits | Edaphic soil levels of mineral nutrients, pH, organic matter, and cationic exchange capacity in the geocaulosphere associated with potato common scab[END_REF]. The structure of microbial and nematode communities in the geocaulosphere varies according to the plant age and other factors related to cultivar, nutritional status, biotic and abiotic stresses, etc. [START_REF] Al-Hazmi | Distribution, frequency and population density of nematodes associated with potato in Saudi Arabia[END_REF][START_REF] Krechel | Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood[END_REF][START_REF] Ferreira | Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles[END_REF][START_REF] Desgarennes | Nematode fauna associated with the rhizosphere of potato crop (Solanum tuberosum) grown in the region of Cofre de Perote, Veracruz, Mexico[END_REF][START_REF] Manici | Fungal community diversity and soil health in intensive potato cropping systems of the east Po valley, northern Italy[END_REF]. Scholte and Jacob, 1989;[START_REF] Krechel | Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood[END_REF][START_REF] Grosch | Effectiveness of 3 antagonistic bacterial isolates to control Rhizoctonia solani Kuhn on lettuce and potato[END_REF][START_REF] Back | Interactions between the potato cyst nematode Globodera rostochiensis and diseases caused by Rhizoctonia solani AG3 in potatoes under field conditions[END_REF][START_REF] Grosch | Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community[END_REF][START_REF] Santamarina | Influence of temperature and water activity on the antagonism of Trichoderma harzianum to Verticillium and Rhizoctonia[END_REF][START_REF] Mahmoud | Antagonistic activities of potato associated bacteria via their production of hydrolytic enzymes with special reference to pectinases[END_REF]Wilson et The microbial or faunal interactions in the geocaulosphere are involved in disease suppressiveness of the soil. Two classical types of suppressiveness of soil are known. General suppression is related to the global activity of the whole microbial biomass in the soil. In contrast, specific suppression is due to the specific activity of certain individuals or groups of microorganisms [START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF][START_REF] Weller | Microbial populations responsible for specific soil suppressiveness to plant pathogens[END_REF]. For instance, Serratia plymuthica, Pseudomonas spp., Bacillus spp., Streptomyces spp. and Trichoderma spp. [START_REF] Kumar | Studies on the antagonistic relationship of soybean spermosphere microflora with Rhizoctonia bataticola and Sclerotium rolfsii[END_REF][START_REF] Kamensky | Biocontrol of Botrytis cinerea and Sclerotinia sclerotiorum in the greenhouse by a Serratia plymuthica strain with multiple mechanisms of antifungal activity[END_REF][START_REF] Krechel | Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood[END_REF] are able to decrease the severity of several potato diseases (Table 1.6). They can be considered as biological control agents. Some biological control agents can act directly against fungal pathogens by enzymatic degradation of their cell walls [START_REF] Kamensky | Biocontrol of Botrytis cinerea and Sclerotinia sclerotiorum in the greenhouse by a Serratia plymuthica strain with multiple mechanisms of antifungal activity[END_REF][START_REF] Li | Broad spectrum anti-biotic activity and disease suppression by the potential biocontrol agent Burkholderia ambifaria BC-F[END_REF], by parasitismas it seems to be the case against numerous nematodes-( [START_REF] Nunez-Camargo | Fungi associated with Globodera rostochiensis cysts in Mexico[END_REF][START_REF] Papert | Physiological and DNA fingerprinting of the bacterial community of Meloidogyne fallax egg masses[END_REF], by antibiotics production [START_REF] Grosch | Effectiveness of 3 antagonistic bacterial isolates to control Rhizoctonia solani Kuhn on lettuce and potato[END_REF], by siderophore secretion that reduces the availability of iron needed by plant pathogens [START_REF] Bharadwaj | Arbuscular mycorrhizal fungal spore-associated bacteria affect mycorrhizal colonization, plant growth and potato pathogens[END_REF] or by interfering with communication between pathogens, i.e. by degrading molecules involved in the "quorum sensing" mechanisms of Pectobacterium spp. [START_REF] Dong | Insecticidal Bacillus thuringiensis silences Erwinia carotovora virulence by a new form of microbial antagonism, signal interference[END_REF]. Indirectly, biological control agents can lead to the plant strengthening and a better resistance to pathogen attacks by producing plant growth hormone or by inducing the production of plant defence molecules such as phytoalexins and PR proteins [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Larkin | Relative effects of biological amendments and crop rotations on soil microbial communities and soilborne diseases of potato[END_REF]. Mycorrhizal fungi also have a beneficial effect; inoculation with arbuscular mycorhizal fungus suppressed tuber dry rot and reduced stem canker and black scurf [START_REF] Bharadwaj | Arbuscular mycorrhizal fungal spore-associated bacteria affect mycorrhizal colonization, plant growth and potato pathogens[END_REF].

II. 3. Interactions between plants and pathogens

The major method to control potato diseases is to find resistant cultivars to a majority of pathogens especially since the use of chemicals is limited [START_REF] Inra | Réduire l'utilisation des pesticides et en limiter les impacts environnementaux: Ministère de l'agriculture et de la pêche[END_REF][START_REF] Paillotin | Paris: Ministère de l'agriculture et de la pêche[END_REF]. Different levels of resistance towards most of the soil-borne potato diseases have been observed among potato cultivars. Wild species of Solanum provide excellent sources of disease resistance genes that may be introgress into S. tuberosum genome by interspecific crossing [START_REF] Jansky | Multiple disease resistance in interspecific hybrids of potato[END_REF] (Table 1.7) and international structures such as the International Potato Center in Peru are aiming at preserving the genetic diversity of native potatoes. Varieties of potato which contain colour pigments are more and more utilized in current breeding programmes because cultivars producing anthocyanins can provide better resistance to soft rot or other diseases compared to white/yellow flesh cultivars [START_REF] Wegener | Soft-rot resistance of coloured potato cultivars (Solanum tuberosum L.): the role of anthocyanins[END_REF]. Cultivars resistant to several diseases were obtained, but simultaneous resistance to all pathogens is very difficult to achieve. Moreover, for some diseases, new genotypes of pathogen appear regularly and overcome plant defence turning the former resistant cultivars into susceptible ones. Hence the levels and durability of field resistance are often highly depending on numerous abiotic and biotic factors still not well known neither controlled. Pythium ultimum and Phytophthora erythroseptica [START_REF] Salas | Assessment of resistance of tubers of potato cultivars to Phytophthora erythroseptica and Pythium ultimum[END_REF][START_REF] Thompson | [END_REF] Solanum brevideus Pectobacterium spp. [START_REF] Ahn | Resistance to blackleg and tuber soft rot in interspecific somatic hybrids between S. brevidens and S. tuberosum ('Superior', 'Dejima', and dihaploid of 'Superior')[END_REF] Resistant potato cultivars resist to pathogenic attacks by plant defence reactions that generally lead to the production of suberin and anti-microbial agents, activation of defence genes and trigger hypersensitive cell death [START_REF] Levine | H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[END_REF] delaying the pathogen development in plant tissues until a wound periderm could form.

Susceptible cultivars produce non-uniform deposits of suberin making them less performing against pathogens (Finetti Sialer, 1990;[START_REF] Ray | Responses of potato tuber to infection by Fusarium sambucinum[END_REF].

The anti-microbial agents produced by potato can be glycoalkaloids (α-chaconin and α-solanine), phenolic compounds and phytoalexins, antimicrobial compounds produced by the plant after pathogen attacks (Okopnyi et al., 1983;Lyon, 1989;[START_REF] Ray | Responses of potato tuber to infection by Fusarium sambucinum[END_REF]Zagoskina et al., 2006;Baker et al., 2008;Lerat et al., 2009). Plants also produce inhibitors of virulence factors [START_REF] Kim | Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety "Golden Valley[END_REF]. An other plant defence reaction called systemic acquired resistance (SAR) spreads a signal through the surrounding cells. It allows plants to become highly resistant to subsequent infection by the original pathogen but also by a wide variety of other pathogens. For example, foliar SAR-inducing applications (benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester -BTH -and harpin) reduce the numbers of rootlesion nematodes (Pratylenchus spp.) and root knot nematodes, Meloidogyne chitwoodi by potato harvest [START_REF] Collins | Effect of foliar applied plant elicitors on microbial and nematode populations in the root zone of potato[END_REF].

III. Effects of cultural practices on the occurrence and development of soil-borne potato diseases

Each technical choice made by the farmers concerning the way of growing potatoes plays a predominant role on the quantitative and qualitative yield at the end of the cropping period. All cultural practices may impact disease development.

III. 1. Rotations

The most traditional way to control diseases is to use crop rotations that include a non host plant that can "sanitize" the soil [START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF]. Several studies show good results when potatoes are grown only one every three or four years (Table 1.8). The beneficial effect of crop rotation depends on the host range of the pathogen and its ability to survive in soil in the absence of its host plant thanks to dormant structures such as sclerotia or chlamydospores. Obviously, crop rotation must avoid including alternative hosts for the pathogen [START_REF] Peters | Influence of crop rotation and conservation tillage practices on the severity of soil-borne potato diseases in temperate humid agriculture[END_REF].

Susceptible weeds -such as hairy nightshade (Solanum sarrachoides) -have to be eliminated as they enable the pathogen to survive during the absence of the main host [START_REF] Boydston | Effect of hairy nightshade (Solanum sarrachoides) presence on potato nematodes, diseases, and insect pests[END_REF]. Crop rotation can also fail to control highly specialized pathogens, such as Globodera spp. or S. subterranea. These organisms are able to survive for long periods, either saprophytically or as dormant structures, in soil, and a very low inoculum density is sufficient to induce disease (Samaliev et al., 1998;[START_REF] Merz | Review: powdery scab of potato -increased knowledge of pathogen biology and disease epidemiology for effective disease management[END_REF]. Rotations with potatoes can include very diverse crops (Table 1.8). If some of those crops are beneficial effect, other might favour pathogen development and should not enter in the rotation, or at teast not as the crop preceding the potatoes.

III. 2. Fertilization and amendments

Supplying plants with micronutrients and macronutrients can be achieved with organic or inorganic fertilizers, either through soil application, foliar spray, or seed treatment [START_REF] Davis | Interactive effects of nitrogen and phosphorus on Verticillium wilt of Russet Burbank potato[END_REF][START_REF] Panique | Potassium rate and source effects on potato yield, quality, and disease interaction[END_REF][START_REF] Malakouti | The effect of micronutrients in ensuring efficient use of macronutrients[END_REF]. Adapted fertilization and amendment allow strong and healthy crops, which are less attractive to pathogens [START_REF] Khomyakov | The effects of potato saturation in rotations and of dosage rates of fertilizers on the development of diseases of tubers in storage. Trudy Vsesoyuznogo Nauchno-Issledovatel[END_REF]. Fertilization may also indirectly favour diseases by enhancing foliar development that maintain high level of humidity needed for example for the growth of Pectobacterium spp. (Rousselle et al., 1996).

Amendments contribute to control diseases by modifying soil properties, especially pH (see I.2.) and microbial activities. That could result in specific suppression caused by the stimulated specific antagonistic populations or in general suppression caused by increased microbial activities or both [START_REF] Lazarovits | Organic amendments as a disease control strategy for soilborne diseases of high-value agricultural crops[END_REF][START_REF] Steinberg | Soil suppressiveness to plant diseases[END_REF].

For some diseases, such as stem rot, organic fertilizers are more efficient than mineral ones in terms of disease suppression [START_REF] Amitava | Role of host nutrition and varieties on the development of stem rot of potato[END_REF]) (Table 1.8).

Among organic fertlizers composts are known to have the capacity to suppress diseases, depending on their degree of maturity (organic matter content, microbial activities). The causal agents of disease suppression brought by compost amendment of the soil are complexes of bacterial and fungal populations, which invade the pile during the curing stage, although some residual activity is probably related to fungistatic compounds occurring in the composts [START_REF] Raviv | The use of compost in growing media as suppressive agent against soilborne diseases[END_REF]. [START_REF] Khomyakov | The effects of potato saturation in rotations and of dosage rates of fertilizers on the development of diseases of tubers in storage. Trudy Vsesoyuznogo Nauchno-Issledovatel[END_REF]Polovny, 1995;[START_REF] Carnegie | The effect of date of haulm destruction and harvest on the development of dry rot caused by Fusarium solani var. coeruleum on potato tubers[END_REF][START_REF] Lui | Response surface models to predict potato tuber infection by Fusarium sambucinum from duration of wetness and temperature, and dry rot lesion expansion from storage time and temperature[END_REF][START_REF] Carter | Productivity parameters and soil health dynamics under longterm 2-year potato rotations in Atlantic Canada[END_REF][START_REF] Olsen | Efficacy of chlorine dioxide for disease control on stored potatoes[END_REF][START_REF] Peters | Influence of crop rotation and conservation tillage practices on the severity of soil-borne potato diseases in temperate humid agriculture[END_REF][START_REF] Cwalina-Ambroziak | Chemical control as a factor decreasing the infestation of potato tubers by pathogenic fungi[END_REF][START_REF] Raviv | The use of compost in growing media as suppressive agent against soilborne diseases[END_REF] Helminthosporium [START_REF] Lennard | Factors affecting the development of silver scurf (Helminthosporium solani) on potato-tubers[END_REF]Hide and Read, 1991;Firman and Allen, 1995;[START_REF] Carnegie | The effect of treating seed potato tubers with benzimidazole, imidazole and phenylpyrrole fungicides on the control of rot and skin blemish diseases[END_REF][START_REF] Carter | Productivity parameters and soil health dynamics under longterm 2-year potato rotations in Atlantic Canada[END_REF][START_REF] Olsen | Efficacy of chlorine dioxide for disease control on stored potatoes[END_REF][START_REF] Peters | Influence of crop rotation and conservation tillage practices on the severity of soil-borne potato diseases in temperate humid agriculture[END_REF] [START_REF] Meredith | Session 8C. Control of fungal diseases of potatoes[END_REF][START_REF] Fox | Some effects of cultural practices for the control of potato gangrene[END_REF]Croke and Logan & Copeland, 1979;Ostergaard and Henriksen, 1983;[START_REF] Bang | Cultivating measures for potatoes (Solanum tuberosum L.) and climatic factors affecting the gangrene pathogen Phoma foveata Foister. Vaxtskyddsrapporter[END_REF]Polovny, 1995;[START_REF] Carnegie | The effect of treating seed potato tubers with benzimidazole, imidazole and phenylpyrrole fungicides on the control of rot and skin blemish diseases[END_REF] Polyscytalum [START_REF] Johnston | Fungal pathogens and Pratylenchus penetrans associted with preceding crops of clovers, winterwheat, and annual ryegrass and their influence on succeeding potato crops on Prince Edward Island[END_REF]Firman and Allen, 1995;Hide and Read, 1995;[START_REF] Lakra | Effect of planting time and curing period on black scurf development and yield of potato[END_REF][START_REF] Klikocka | The influence of soil tillage systems and crop cultivation methods on potato tubers infection with Rhizoctonia solani Kuhn[END_REF][START_REF] Peters | Influence of crop rotation and conservation tillage practices on the severity of soil-borne potato diseases in temperate humid agriculture[END_REF][START_REF] Baljeet | Influence of depth of planting on development of black scurf of potato (Rhizoctonia solani)[END_REF][START_REF] Cwalina-Ambroziak | Chemical control as a factor decreasing the infestation of potato tubers by pathogenic fungi[END_REF][START_REF] Errampalli | Effect of a combination of chlorine dioxide and thiophanate-methyl pre-planting seed tuber treatment on the control of black scurf of potatoes[END_REF][START_REF] Nitzan | Ten Hoopen and Krauss[END_REF]Respiene and Minekiene, 2006;Zimmy et al., 2006;[START_REF] Henriksen | The effect of autumn ridging and inter-row subsoiling on potato tuber yield and quality on a sandy soil in Denmark[END_REF][START_REF] Raviv | The use of compost in growing media as suppressive agent against soilborne diseases[END_REF]Wilson et

Minimum tillage

Mancozeb, captan, metam sodium, 1,3-dichloropropene, chloropicrine [START_REF] Johnston | Fungal pathogens and Pratylenchus penetrans associted with preceding crops of clovers, winterwheat, and annual ryegrass and their influence on succeeding potato crops on Prince Edward Island[END_REF][START_REF] Davis | Effects of green manures on Verticillium wilt of potato[END_REF][START_REF] Soltani | Reduction of potato scab and verticillium wilt with ammonium lignosulfonate soil amendment in four Ontario potato fields[END_REF][START_REF] Taylor | Influence of tillage and method of metam sodium application on distribution and survival of Verticillium dahliae in the soil and the development of Verticillium wilt of potato[END_REF]Tsror et al., 2005;[START_REF] Omer | Detection, quantification, and vegetative compatibility of Verticillium dahliae in potato and mint production soils in the Columbia Basin of Oregon and Washington[END_REF] [START_REF] Bushkova | Some aspects of protection of potato and root cruciferous crops against bacterioses. Trudy Vsesoyuznogo Nauchno-Issledovatel[END_REF][START_REF] Khomyakov | The effects of potato saturation in rotations and of dosage rates of fertilizers on the development of diseases of tubers in storage. Trudy Vsesoyuznogo Nauchno-Issledovatel[END_REF][START_REF] Lewocz | Activity of some fungicides as limiting factors of black leg and soft rot of potato tubers[END_REF][START_REF] Saleh | Bacterial soft rot disease of tomato fruits in Florida, USA: Identification, response of some American and Egyptian cultivars of solanaceous plants and chemical control[END_REF]Karwasra and Parashar, 1988;Bartz, 1999;[START_REF] Olsen | Efficacy of chlorine dioxide for disease control on stored potatoes[END_REF][START_REF] Medina | Use of 1,4-naphthoquinones for control of Erwinia carotovora[END_REF][START_REF] Yaganza | Ultrastructural alterations of Erwinia carotovora subsp atroseptica caused by treatment with aluminum chloride and sodium metabisulfite[END_REF] No effect [START_REF] Meredith | Session 8C. Control of fungal diseases of potatoes[END_REF][START_REF] Volovik | Agrotechniques in the control of diseases[END_REF]Hide and Read, 1991;Conn and Lazarowits, 1999;[START_REF] Park | Chemical control of potato common scab (Streptomyces scabies)[END_REF][START_REF] Soltani | Reduction of potato scab and verticillium wilt with ammonium lignosulfonate soil amendment in four Ontario potato fields[END_REF][START_REF] Chaudhari | Management of common scab of potato[END_REF][START_REF] Mizuno | Judging conducive soils from clay mineralogical properties and soil chemical method to suppress potato common scab[END_REF][START_REF] Peters | Influence of crop rotation and conservation tillage practices on the severity of soil-borne potato diseases in temperate humid agriculture[END_REF]Scholte, 2005 [START_REF] Quiroz | [END_REF]Hague et al., 1982;[START_REF] Mulder | Effects of soil characteristics on the relationship between potato cyst nematodes and yield. II. Acidity (soil pH)[END_REF]Trifonova, 1997;De Ruijter and Haverkort, 1999;Molendijk, 1999;[START_REF] Minnis | Control of potato cyst nematodes and economic benefits of application of 1,3-dichloropropene and granular nematicides[END_REF] No effect Molendijk, 1999;[START_REF] Crow | Effects of potato-cotton cropping systems and nematicides on plant-parasitic nematodes and crop yields[END_REF][START_REF] Carter | Productivity parameters and soil health dynamics under longterm 2-year potato rotations in Atlantic Canada[END_REF]Coosemans, 2005;[START_REF] Hafez | Efficacy of fosthiazate for the control of Paratrichodorus spp. and Meloidogyne chitwoodi on potato[END_REF][START_REF] Charchar | Effects of fumigant and nonfumigant nematicides to control Meloidogyne spp. on potato and carrot[END_REF][START_REF] Ingham | Control of Meloidogyne chitwoodi in potato with shank-injected metam sodium and other nematicides[END_REF] Nacobbus aberrans False root-knot nematode

Planting in June or July

Abamectin and furateocarb, A.C. 92100, aldicarb, carbofuran, A.C. 64475 [START_REF] Quiroz | [END_REF][START_REF] Iriarte | Influence of time of sowing on potato yield and population density of Nacobbus aberrans[END_REF][START_REF] Main | Disinfection of potato seed infected with Nacobbus aberrans using mild nematicides[END_REF] Paratrichodorus and Trichodorus spp.

Stubby-root nematode (TRV vector)

With beet, oats, grasses Without sorghm-sundangrass or velvetbean , maize, wheat, cabbage, rape, barley Aldicarb (+ oxamyl), 1,3dichloropropene [START_REF] Barbez | The distribution of virus-vector nematodes in seed-potato fields in Flanders (Belgium) and its relation to some biotic and abiotic factors[END_REF][START_REF] Perez | Correlation between Paratrichodorus minor population levels and corky ringspot symptoms on potato[END_REF]Crow et al., 2001;[START_REF] Hafez | Efficacy of fosthiazate for the control of Paratrichodorus spp. and Meloidogyne chitwoodi on potato[END_REF] Pratylenchus spp.

Root-lesion nematode

With wheat, ryegrass, without red clover 1,3-dichloropropene, oxamyl, fosthiazate, cadusafos, carbofuran Organic [START_REF] Philis | Effect of cadusafos and carbofuran against Pratylenchus penetrans and some ectoparasitic nematodes infesting potato in Cyprus[END_REF][START_REF] Johnston | Fungal pathogens and Pratylenchus penetrans associted with preceding crops of clovers, winterwheat, and annual ryegrass and their influence on succeeding potato crops on Prince Edward Island[END_REF]Molendijk, 1999;[START_REF] Kimpinski | Differential effect of nematicide treatments on tuber yields in early-and late-maturing potato cultivars[END_REF][START_REF] Carter | Productivity parameters and soil health dynamics under longterm 2-year potato rotations in Atlantic Canada[END_REF]

III. 3. Tillage management

Potato cultivation traditionally involves intensive soil tillage throughout the cropping period. Mechanical tillage, ridging and harvesting entail intensive soil disturbance and modify the environmental conditions especially the microbial characteristics of soil, both on quantitative and qualitative aspects (FAO, 2008;[START_REF] Vian | Comparaison de différentes techniques de travail du sol en agriculture biologique: effet de la structure et de la localisation des résidus sur les microorganismes du sol et leurs activités de minéralisation du carbone et de l'azote[END_REF]. As an example, ploughing contributes to redistribute vertically the inoculum, which increases the probability of infection [START_REF] Taylor | Influence of tillage and method of metam sodium application on distribution and survival of Verticillium dahliae in the soil and the development of Verticillium wilt of potato[END_REF]. Over the last decades, there is tend to replace ploughing by techniques without soil inversion i.e. no tillage or superficial tillage, leading to efficient disease suppression [START_REF] Klikocka | The influence of soil tillage systems and crop cultivation methods on potato tubers infection with Rhizoctonia solani Kuhn[END_REF][START_REF] Peters | Influence of crop rotation and conservation tillage practices on the severity of soil-borne potato diseases in temperate humid agriculture[END_REF][START_REF] Vian | Comparaison de différentes techniques de travail du sol en agriculture biologique: effet de la structure et de la localisation des résidus sur les microorganismes du sol et leurs activités de minéralisation du carbone et de l'azote[END_REF] (Table 1.8). Indeed, rotation and conservation tillage practices can improve disease suppression by enhancing the antibiosis abilities of endophytic and root zone bacteria [START_REF] Peters | Developing diseasesuppressive soils through crop rotation and tillage management practices[END_REF]. Although minimum tillage is more beneficial for soil suppressiveness to disease, the plant growth and the macronutrient (N, P, K, Ca and Mg) contents in potato plant respond positively to a deeper soil caused by ploughing [START_REF] Boligowa | Yielding and quality of potato tubers depending on the kind of organic fertilisation and tillage method[END_REF][START_REF] Nunes | Potato plant growth and macronutrient uptake as affected by soil tillage and irrigation systems[END_REF].

III. 4. Planting, haulm destruction, lifting and harvesting

Planting, dehaulming, lifting and harvesting are decisive for disease expression (Table 1.8). For example, low planting density increases the yield per plant because the foliage has more space for growth. Also, sparse plants are less exposed to the attacks of disease than plants at high densities [START_REF] Milic | Influence of mineral nutrition and additional space upon yield of potato[END_REF].

Diseases can be reduced by adjusting planting, dehaulming and harvesting dates and cultivation of early-tuberizing cultivars combined with pre-harvesting desiccation of haulms and treatment of seed tubers with chemicals [START_REF] Sikka | Factors influencing quality of ware potatoes[END_REF].

Black scurf development on tubers has a positive correlation with the curing period (time between haulm destruction and harvest) because infection on tubers continues in the soil even after haulm destruction [START_REF] Lakra | Effect of planting time and curing period on black scurf development and yield of potato[END_REF].

III. 5. Pesticides

Pesticides are commonly used to control various pathogens altering potato tubers. They can be applied as soil fumigant (fumigants such as carbamates are not allowed in some European countries), sprayed or powdered directly on seed tubers after harvest or applied as granular (Hide et al., 1995;[START_REF] Tsror | Control of Verticiilium dahliae prior to potato production by soil fumigation with chloropicrin[END_REF][START_REF] Errampalli | Effect of a combination of chlorine dioxide and thiophanate-methyl pre-planting seed tuber treatment on the control of black scurf of potatoes[END_REF]. The chemicals have to be carefully chosen, since pathogens can adapt and become resistant (Table 1.8). Thiabendazole-resistance was detected in F.

avenaceum, F. culmorum, F. equiseti, and F. sporotrichioides (Fusarium dry rot) [START_REF] Ocamb | Benzimidazole resistance of Fusarium species recovered from potatoes with dry rot from storages located in the Columbia basin of Oregon and Washington[END_REF], in Polyscytalum pustulans (skin spot) [START_REF] Carnegie | Effects of fungicide and rate of application on the development of isolates of Polyscytalum pustulans resistant to thiabendazole and on the control of skin spot[END_REF] and in H. solani (silver scurf). Mefenoxam-resistance is known for Phytophthora erythroseptica (pink rot) populations [START_REF] Taylor | Biological significance of mefenoxam resistance in Phytophthora erythroseptica and its implications for the management of pink rot of potato[END_REF] and numerous treatments of carbendazim select resistant mutants of Sclerotium rolfsii (stem rot) [START_REF] Solunke | Survival ability of carbendazim resistant Sclerotium rolfsii in mixed population[END_REF]. Moreover, the use of numerous chemicals is nowadays regulated and many of them are no longer permitted in France.

III. 6. Organic farming versus conventional agriculture

Organic farming relies on agricultural techniques that exclude the use of chemical pesticides and recommend organic fertilization. As a result, the soil and tuber environment is quite different from the one caused by conventional practices and may induce disease suppression (Table 1.8). To reduce disease incidence or severity, the best adapted cultural system depends on the pathogen to control and varys strongly according to the soil type [START_REF] Messiha | Potato brown rot incidence and severity under different management and amendment regimes in different soil types[END_REF]. It has been reported

that farmers who switch from conventional to organic system faced critical pest or disease problems during a transition period of about 5 years but managed to control soil-borne diseases on the long-term [START_REF] Bruggen | Integrated approaches to root disease management in organic farming systems[END_REF]. However, organic farmers generally faced more sanitary problems than conventional farmers.

III. 7. Handling and storage

Inappropriate manipulation of tubers at harvest or during storage can provoke wounds that increase diseases such as black dot, Fusarium dry rots, silver scurf, gangrene, leak, pink rot, black leg and soft rot [START_REF] Meredith | Session 8C. Control of fungal diseases of potatoes[END_REF]Hide, 1994;[START_REF] Vanvuurde | Population dynamics of Erwinia carotovora subsp. atroseptica on the surface of intact and wounded seed potatoes during storage[END_REF][START_REF] Salas | The effect of wounding, temperature, and inoculum on the development of pink rot of potatoes caused by Phytophthora erythroseptica[END_REF][START_REF] Marcinkowska | Pathogenicity of some Phoma exigua var. exigua isolates[END_REF]Peters et al., 2008b) (Table 1.8). Significant measures of managing potato diseases include:

avoiding mechanical damage to potatoes during harvesting, shipping and sorting, curing the harmed parts thereby preventing infection and disease onset, avoiding manipulation of cold potato since potato tubers are more sensitive to injuries when cold, avoiding the exposure of table potato to light, and continuously providing stored potatoes with fresh air [START_REF] Milosevic | Some most important potato diseases and their control. Radovi Poljoprivrednog Fakulteta Univerziteta u Sarajevu[END_REF][START_REF] Scheid | What to do against storage diseases?[END_REF]. Most of the storage diseases decrease when the tubers are cured in dry conditions and stored at temperature below 4 or 5 °C, except gangrene (Table 1.8). However, tubers stored in a dry atmosphere show greater weight losses than tubers stored in a humid atmosphere, despite having less infection [START_REF] Lennard | Factors affecting the development of silver scurf (Helminthosporium solani) on potato-tubers[END_REF].

IV. Diseases management IV. 1. Risk assessment and decision support systems

Disease occurrence and development influenced by abiotic and biotic factors are difficult to predict. However, their prediction would be very useful to assess disease risk and consequently the potential yield loss and to choose the best disease control strategy. Current methods to evaluate yield losses are based on predictive models which commonly assign a value or score to each risk factor, such as cultivar resistance, inoculum density, cultural practices and environmental factors. The maximum score that can be assigned to each factor depends on the relative importance of the factor in determining the disease. For example, cultivar resistance is considered to be a major determinant of powdery scab severity, so this factor has a higher score than the zinc content of soil, which is thought to be less important [START_REF] Burgess | Development of an integrated control strategy for powdery scab of potatoes[END_REF]. Assessment of the risk for each factor and for each disease is performed by bioassays in fields or in growth chambers under controlled conditions. They are generally costly, laborious and time consuming.

Tolerant cultivars, that host pathogens without expressing symptoms, are a particular risk factor in potato production as they can maintain and increase the inoculum level in fields [START_REF] Merz | Review: powdery scab of potato -increased knowledge of pathogen biology and disease epidemiology for effective disease management[END_REF]. A tolerance threshold of the crop has to be determined. It takes into account the relationship between inoculum density and disease incidence or severity according to cultivar resistance (Table 1.4).

A score can also be attributed to each cultural practice in the equation of the model since they have various impacts on yield losses. For example, incidence and severity of Verticillium wilt decrease with long rotations [START_REF] Johnston | Fungal pathogens and Pratylenchus penetrans associted with preceding crops of clovers, winterwheat, and annual ryegrass and their influence on succeeding potato crops on Prince Edward Island[END_REF], but mint as a previous crop increases Verticillium wilt [START_REF] Omer | Detection, quantification, and vegetative compatibility of Verticillium dahliae in potato and mint production soils in the Columbia Basin of Oregon and Washington[END_REF]. Consequently, in the equation of the model, rotation length will be negatively correlated to yield losses whereas mint as previous crop will be positively correlated to yield losses due to Verticillium wilt.

On the same pattern, some predictable environmental factors such as nutrient content and soil pH can be scored. However, abiotic environmental factors are difficult to predict. For example, at planting time, rainfall and temperature conditions occurring at the critical growth phase of the disease are almost impossible to foresee.

As climatic conditions cannot be predicted at middle term, models of risk assessment are less reliable. However, no factor alone has a dramatic effect on the disease; and the beneficial reduction of a disease is usually achieved by the sum of optimized factors [START_REF] Harrison | Powdery scab disease of potato -A review[END_REF].

Mathematical modelling including all the data related to the environmental factors and to the results concerning plant resistance appeared to be helpful to evaluate risk, to overcome the scaling gap between bioassays in growth chamber and field application and to simulate scenario based on crop management [START_REF] Janvier | Soil health through soil disease suppression: Which strategy from descriptors to indicators?[END_REF].

Calculation of yield losses enables to identify a damage threshold and to determine the time at which disease control must be initiated. Indeed, yield loss threshold and economic threshold are different. Economic threshold is frequently higher than yield loss threshold; because up to a certain point, losing yield is less penalizing for farmers than spending money to avoid it. Calculation of economic thresholds beyond which control of diseases is profitable takes into account a damage function drift to potato yield, pathogen population density and crop selling prices. For example, application of control measures is found to be beneficial at an initial density of G.

rostochiensis higher than 8 eggs and larvae g -1 soil, while the damage threshold is at 2 eggs g -1 soil [START_REF] Samaliev | Relationship between initial population density of potato cyst nematode Globodera pallida Thorne and the yield of partially resistant potato varieties[END_REF]. Economic thresholds allow taking short-term strategic decisions such as choice of the cultivar, cultural practices, timing of crop establishment, seed treatment, planting density etc. and long-term strategic decisions such as define research priorities, design the breeding programs or develop integrated pest management strategies [START_REF] Savary | Quantification and modeling of crop losses: A review of purposes[END_REF]

(Figure 1.2).
Predicting models are used by farmers as decision support systems (DSS) and generally provide a theoretical yield to be obtained at the end of the cropping period, a monitoring of pest populations and comments and advices in order to increase the theoretical yield as much as possible (Been et al., 2005;Jorg et al., 2006). Some DSS are able to send real time alerts to farmers when several risk factors are combined and when control measures have to be taken immediately [START_REF] Dubois | Integrated control of potato late blight: MILPV, a new French decision support system[END_REF]. DSS are environmental and farmer friendly as they enable to increase economical yields by applying the right chemical doses at the right time and when disease pressure requires it, in order to reduce unnecessary environmental pollutions and treatment cost.

IV. 2. Control methods

Ways to control diseases are evolving since the use of chemicals is supposed to be reduced. In many cases, the most efficient long-term strategy is to use resistant cultivars when available. Otherwise, management strategies consist either in exclusion, avoiding contact between plant and pathogens, or by pest eradication, and leading to complete elimination or partial reduction of pathogen populations.

For the potato crop which is multiplied vegetatively, exclusion methods begin with the use of healthy tubers. Many soil-borne pathogens can be carried on by seed tubers and the use of certified seed potatoes is a major way to control or restrict the movement of pathogens of potato crops [START_REF] Andrade | Effect of using certified and uncertified potato seeds on the yield and sanitary conditions of a potato crop[END_REF]. Seed certification programs aim at warranting seed tuber quality to potato producers and favour the diffusion of genetic progress. The certified seed production process may be 8 to 10 years long. Strict rules established by the national regulation institutions (i.e. National

Potato Council in USA or GNIS -SOC (Groupement national interprofessionnel des semences -Service officiel de contrôle) in France) have to be respected and the seeds are regularly inspected for bacterial, viral, and fungal diseases, as well as varietal purity and identity. Each country is free to apply more or less severe rules.

Certification systems have been developed in most of the seed producing countries to cover the production of certified seed potatoes free from pathogens and pests [START_REF] Mcdonald | Disease control through crop certification: Herbaceous crops[END_REF][START_REF] Grousset | EPPO certification scheme for seed potatoes[END_REF][START_REF] Sahajdak | Potato health requirements arising from EU regulations and the Treaty of Accession[END_REF]). An international project of commercial and phytosanitary minimal guidelines (CEE-ONU S-1) is in progress. It is intended to serve as a minimal base consensus between the various standards established at "regional" levels (EU, NATTO, etc) (UNECE).

Eradication strategies aim at eliminating an established pathogen from plant propagation material or production sites. Eradication methods involve the use of pesticides, adapted cultural practices or biological control. Application of fungicides and nematicides are protecting strategies (see section III. 5. and table 1.8) whose application time and doses can be advised by DSS. However, pesticides are sometimes inefficient against pathogens, such as P. carotovorum [START_REF] Latour | Control of bacterial diseases of potato caused by Pectobacterium spp. (Erwinia carotovora)[END_REF], or their use is limited by environmental regulations. Consequently, alternative methods based on adapted cultural practices have to be recommended (see section III and tables 1.3 and 1.8). Some crops either susceptible or resistant may serve as baiting-crop, for example, resistant potato cultivars cropped just before the main potato crop decreased black scurf [START_REF] Scholte | Effect of potato used as a trap crop on potato cyst nematodes and other soil pathogens and on the growth of a subsequent main potato crop[END_REF]. Likewise, alfalfa can be used to avoid TRV transmitted by stubby root nematode, as this crop is a host for stubby-root nematode but immune to TRV [START_REF] Stevenson | Compendium of potato diseases[END_REF]. Cultivar precocity can be used to avoid some diseases. Since black dot and charcoal rot damages occur late in the growing season, early cultivars are generally recommended to control these diseases [START_REF] Stevenson | Compendium of potato diseases[END_REF]. When a disease is established in a production site, its spread must be avoided as much as possible. All diseased plants have to be eliminated or burned and tools should be properly disinfected before use in another field [START_REF] Salas | The effect of wounding, temperature, and inoculum on the development of pink rot of potatoes caused by Phytophthora erythroseptica[END_REF][START_REF] Latour | Control of bacterial diseases of potato caused by Pectobacterium spp. (Erwinia carotovora)[END_REF].

Natural interactions of plants and microorganisms with the pathogens are used as biological control to protect potato crops. There is a continuum from a conducive soil to a suppressive one [START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF] what means that in each soil, almost each pathogen can be potentially controlled by other microorganisms either by a specific antagonism or by competition with total microbial biomass (see section II. 2.

and table 1.6). Appropriate agricultural practices, thanks to the DSS should stimulate this potential to enhance or to maintain the soil suppressiveness to potato diseases.

Another approach consists in applying biocontrol agents. However, the choice of a biological control agent must take into account the potential risks to human health.

Even if Serratia grimesii and Burkholderia cepacia decrease dry rot and black scurf and stem canker respectively, they can cause human infections and are not recommended for biological control (Table 1.6) [START_REF] Grosch | Effectiveness of 3 antagonistic bacterial isolates to control Rhizoctonia solani Kuhn on lettuce and potato[END_REF][START_REF] Gould | Biocontrol of Fusarium sambucinum, dry rot of potato, by Serratia plymuthica 5-6[END_REF]. Moreover, indirect control such as strengthening of potato plants by mycorhization increases tuber yield and allow an integrated management of potato cyst nematode and root-knot nematode [START_REF] Sankaranarayanan | Influence of moisture and pH on the efficiency of VA-mycorrhiza, Glomus mosseae (Nicol and Gerd.) Gerd. and Trappe against Meloidogyne incognita (Kofoid and White) Chitw. on blackgram (Vigna mungo L.) Hepper[END_REF]Ryan et al., 2003). Biological control may also include the use of natural toxic compounds for pathogenic agents. Fumigation of essential oils is studied to control dry rot, gangrene, black scurf and stem canker [START_REF] Bang | Screening of natural plant volatiles to control the potato (Solanum tuberosum) pathogens Helminthosporium solani, Fusarium solani, Phoma foveata and Rhizoctonia solani[END_REF]. Fish emulsion and crushed crab shell are used against V. dahliae, V. albo-atrum and S. endobioticum respectively [START_REF] Hampson | Reduction of potato wart disease with crushed crabshell -suppression or eradication[END_REF][START_REF] Abbasi | Effect of fish emulsion used as a preplanting soil amendment on verticillium wilt, scab, and tuber yield of potato[END_REF]. Soil can be disinfected from pathogens by biofumigation or solar heating or both. For example, Brassica crops used in crop rotations and as green manures have been associated

with reductions in soil-borne pests and pathogens. These reductions have been attributed to the production of volatile sulfur compounds through the process of biofumigation, and to changes in soil microbial community structure [START_REF] Janvier | Soil health through soil disease suppression: Which strategy from descriptors to indicators?[END_REF]. Composting is also a sanitizing method which combines temperature, time and toxic compounds to control potato diseases. The composts the most frequently used on potato crop are organic wastes (sludge, manure, tea, etc.) that have undergone long, thermophilic, aerobic decomposition. The most effective compost composition and combinations of temperature and time have to be determined for each pathogen. As it decreases the pathogenic population or favours microbial enrichment of the soil, compost has generally a positive or no effect on disease suppression, and only rarely a disease stimulating effect (Termorshuizen et al., 2006). Sanitization is also performed on tubers before planting by hot water [START_REF] Janvier | Soil health through soil disease suppression: Which strategy from descriptors to indicators?[END_REF] or during storage with chemical treatments at high temperatures [START_REF] Secor | Susceptibility of Corynebacterium sepedonicum to disinfectants in vitro[END_REF]. However, heating may damage tubers resulting in fewer sprouts.

Biocontrol can also be performed by disrupting pathogens molecular pathways. P.

carotovorum quorum-sensing mechanism is controlled by a quorum-quenching strategy aiming at interrupting the quorum-sensing by using compounds or organisms able to cause interferences in the bacterial signal [START_REF] Latour | Control of bacterial diseases of potato caused by Pectobacterium spp. (Erwinia carotovora)[END_REF].

Finally, it is also possible to enhance plant defence reactions against soil-borne pathogens by foliar spraying with different inducers such as salicylic acid, dipotassium hydrogen phosphate and tri-potassium phosphate [START_REF] Mahmoud | Management of brown rot disease of potato[END_REF].

The different methods that were presented above are not items that have to be taken at random. Their combination generally gives better results than each of the method applied alone.

Decision support systems developed to predict yield losses allow choosing good control methods such as the use of healthy seeds, adapted pesticides, cultural practices and biological control agents for each potato diseases.

Conclusions

If a disease results from the interaction between the plant and a pathogen, its severity is influenced by soil abiotic and biotic factors affecting the plant, the pathogen, or both [START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF]. Biotic and abiotic factors are not independent, the abiotic factors modulating the biotic ones. They act both on the disease epidemiology, that means the environmental conditions which make the plant growing and the pathogen, present or latent on the crop, causing or not the disease. Moreover, some unfavourable factors for a given disease can be favourable to another. The multifaceted interactions between plants, pathogens and their environment make disease management complex since controlling every factor occurring in the disease development is quite impossible. Potato producers have to aim at limiting contact between plant and pathogens by using for example healthy seeds. Moreover, pathosystems are continuously changing since the pathogens genetically adapt to their hosts or to the environmental conditions implemented by human activities or not. In a system whose parameters vary continuously, the control strategies have to be adapted to each situation at every time.

This review aimed at being as exhaustive as possible about the factors impacting the Moreover, soil-borne diseases are difficult to study because soil is a complex environment in which numerous interactions occur and where detection of pathogens is not easily performed. However, researches on those diseases could be beneficial at long-term in case they would spread throughout the world. It would have been rather complex to consider air-borne diseases in addition to soil-borne diseases of potato. However, air-borne diseases such as late blight caused by Phytophthora infestans and early blight caused by Alternaria solani are responsible for huge economical losses and have to be considered with as much attention as soil-borne diseases. Finally, since few years, importance of potato tuber quality raised in developed countries where tubers are washed before selling. Indeed, washing tuber makes visible some superficial blemishes that were previously hidden by adhering soil. Consumer's habits changing, blemished tubers cannot be sold anymore and the losses take seriously damaging proportions for potato market.

The previous considerations acknowledge the fact that the plant disease problem can be reduced in short term thanks to solid knowledge in epidemiology and pathogens ecology, but in longer term control strategies must be adapted with the constant evolution of pathosystems.

Chapter 2

Review -Nomenclature and classification of potato tuber blemishes

Abstract

Since most of ware potato tubers are washed before selling, tubers blemishes are an important cause for downgrading and rejection of tuber lots. Tubers may be affected Potato tubers are subjected to a very large range of blemishes, which generally affect only upper layers of the tuber periderm and do not damage the inner part nor the nutritional quality of the potatoes. On one hand, typical blemishes are those that are largely studied and which have usually known causal origins, for example, common scab, netted scab, powdery scab, black scurf, black dot and silver scurf that are caused by thoroughly identified bacteria or fungi. Consequently the symptom terminology is consensual though with the exception for potato scabs (common, netted scab and russet scab). On the other hand, atypical blemishes, because still of unknown origin, might be frequently observed but they have been very rarely investigated (Perraton, personal data, 1996 and[START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF]. Since ware tubers are washed in order to remove adherent soil, there has been a constant feedback from growers and packers about a number of skin defects which do not appear to be associated with any of the typical blemishes, but which lead as likely to rejections or downgrading. These atypical blemishes are distributed on the tuber surface as more or less big patches of various aspects: shallow, irregular, and scaly, crackled or rough [START_REF] Jouan | Les principales causes d'altérations superficielles des tubercules[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Sexton | Some thoughts on elephant hide[END_REF]Selman et al., 2008).

Literature shows that terminologies concerning atypical blemishes as well as some typical blemishes like potato scabs differ according to the aspect of the blemish and the country where they are observed. It also appears that atypical blemishes are sometimes falsely attributed to pathogens causing other similar blemishes. Since several years, these issues are discussed among researchers and end-users; they highlight a lack of knowledge on the biology and symptomatology of those potato blemishes. Thus, the establishment of a consensual classification nomenclature of blemishes for all the scientific community is a prerequisite for further scientific investigation. At first, this paper briefly reviews the disorders leading to the alteration of the periderm. Then, based on the literature, as well as shared experience on assessment of tuber blemishes in collaborative research (Fiers et al., 2010) and extension projects [START_REF] Fnpppt | GNIS and ARVALIS-Institut du végétal. Guide pratique: Maladies, ravageurs et désordres de la pomme de terre[END_REF], it suggests a consensual nomenclature describing the main superficial blemishes, with special emphasis on the atypical blemishes of potato tubers.

Physiological disorders involved in the formation of atypical blemishes

The mechanisms underlying the appearance of the blemishes are not well known, but the most important lack of knowledge concerns the atypical blemishes.

The formation of atypical blemishes on the potato surface implies histological modifications and cellular disorders as a result of the plant response to an attack.

Atypical corky blemishes forming polygonal lesions (Figure 2.1.A) appear to be the result of one part of the tuber surface not growing as fast as the rest of the tuber [START_REF] Sexton | Some thoughts on elephant hide[END_REF]. These polygonal lesions may be due to localized stress that only affects one area of the tuber, while the rest of the tuber continues to grow and expand normally. They may occur when a stress uniformly slows down the growth of the whole tuber, but after the stress ends, the resumption of growth is not uniform across the tuber. If one area of the tuber surface fails to recover from the stress as fast as the rest of the tuber, it will grow slower than the surrounding tissues. If the tuber does not have a mechanism to respond to that stress, then polygonal lesions could develop within the tuber structure. Several authors suggest that polygonal lesions could be due to unfavourable environmental conditions, i.e high temperature, high organic matter content, high soil moisture or fertilization [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Turff | Fungus more complex than first thought[END_REF]Selman et al., 2008). The fungus R. solani was cited as well as responsible for the formation of polygonal lesions. The fungus growing on the tuber surface would delay the growth of the underlying tissues, which may result in deformed tubers. The pattern of the lesions on the skin would be related to the pattern of the branching hyphae of the fungus. Some of these hyphae may still be present on newly harvested tubers (Mulder et al., 2008).

Corky cracks and tuber cracks (Figures 2.1.G and 2.1.R) are also quoted as physiological reactions to a growth stress, either abiotic (fluctuation of soil moisture, herbicide) or biotic (disease, e.g., R. solani etc.). The main cause of tuber cracks is an irregular water uptake during the growing season. During a period of drought, the tuber stops growing. A water uptake induces a rapid rehydratation of the tuber. The internal turgor pressure becomes stronger than the peridermal resistance. The periderm breaks and heals progressively afterwards [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Fnpppt | GNIS and ARVALIS-Institut du végétal. Guide pratique: Maladies, ravageurs et désordres de la pomme de terre[END_REF].

Dry core blemish (Figure 2.1.S) is almost always allocated to R. solani, but it seems that the blemish only appears when the tuber is mechanically or naturally wounded.

Wireworms might be the main cause of natural wounding making fungal invasion easier [START_REF] Keiser | Influence of farming system, specific cultivation methods and site parameters on potato quality PhD thesis[END_REF].

It is likely that an unclear identification associated to a fuzzy terminology may explain the lack of publications about the mechanisms inducing corky spots and star-like

lesions (Figures 2.1.H to 2.1.J).
The majority of atypical blemishes affect the periderm integrity. Some of these blemishes, such as polygonal lesions, corky cracks or dry core are similar to the scars observed on tuber after they have been wounded. Tuber wound responses and wound-healing involve many biological processes, the most important of which being wound-induced suberization. The tissue distribution of suberized cell walls in plant suggests that suberization occurs wherever and whenever the plant needs to form a physical barrier [START_REF] Franke | Suberin -a biopolyester forming apoplastic plant interfaces[END_REF][START_REF] Pollard | Building lipid barriers: biosynthesis of cutin and suberin[END_REF]. As the wounded tuber tissue begins to heal, it forms a closing layer where the walls of existing parenchyma cells become suberized. In conjunction with the formation of the closing layer, a wound periderm forms under the closing layer [START_REF] Lulai | Skin-Set, Wound Healing, and Related Defects[END_REF]. The similarity between corky blemish and healed wound suggests that similar cellular mechanisms are induced by plants. Wounding, stress and/or invasion of plant tissues by pathogens elicit an oxidative burst of superoxide, hydrogen peroxide and hydroxyl radicals at the wound surface. These reactive oxygen forms play a role in protecting host tissue. Wounding activates superoxide radical formation that may have a role in the suberin synthesis [START_REF] Kumar | Strboh A homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers[END_REF]. In addition to this putative role i.e.

polymerization of phenolic monomers in lignin/suberin synthesis, the reactive oxygen forms serve as (a) signaling molecules for upregulating defence-related genes, (b) anti-microbial agents and catalysts for cross-linking cell wall proteins, and (c) molecules triggering hypersensitive cell death to contain infection [START_REF] Levine | H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[END_REF][START_REF] Simon-Plas | The plasma membrane oxidase NtrbohD is responsible for AOS production in elicited tobacco cells[END_REF][START_REF] Kumar | Strboh A homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers[END_REF].

Data about physiological processes resulting in atypical blemishes are rare however the understanding of these processes is needed to control tuber superficial alterations. Doubtless, the first step toward a full comprehension of these mechanisms is to remedy the confusing terminology of blemishes and to clearly identify, describe and name typical and atypical blemishes.

Critical analysis of current terminology for superficial blemishes on potato tubers

Morphological descriptions and pictures of blemishes have been published in several books and guides [START_REF] Radtke | Diseases and pests of the potato[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Fnpppt | GNIS and ARVALIS-Institut du végétal. Guide pratique: Maladies, ravageurs et désordres de la pomme de terre[END_REF]Mulder et al., 2008;[START_REF] Wale | Diseases, pests and disorders of potatoes[END_REF]. However depending on the authors, common names and descriptions of the blemishes differ. This induces ambiguities and misunderstandings between the different actors along the potato production chain whether they are scientists or extension workers (Table 2.1).

We firstly review typical blemishes, whose causes are known, and whose terminologies are straightforward. Albeit, they have been fully described in the literature, in practice they may be confused one with the others because of the similarity of symptoms. As an example, black dot caused by Colletotrichum coccodes 1993;[START_REF] Stevenson | Compendium of potato diseases[END_REF]. Inspection with a hand lens (10 x) will quickly differentiate the regularly spaced black dots from the bunched threads of silver scurf.

(
Though dealing with a pathosystem thoroughly studied [START_REF] Merz | Review: powdery scab of potato -increased knowledge of pathogen biology and disease epidemiology for effective disease management[END_REF] [START_REF] Goyer | Taxonomic studies on streptomycetes causing potato common scab: A review[END_REF][START_REF] Takeuchi | Phylogenetic analysis of Streptomyces spp. causing potato scab based on[END_REF][START_REF] Wanner | Aggressiveness of Streptomyces on Four Potato Cultivars and Implications for Common Scab Resistance Breeding[END_REF].

Russet scab (Figure 2.1.E) has only been described in the USA [START_REF] Bukhalid | Plant pathogenicity in the genus Streptomyces[END_REF],

in Japan [START_REF] Oniki | Causal agent of russet scab of potato[END_REF], in Canada [START_REF] Faucher | Characterization of Streptomyces causing russet scab in Quebec[END_REF], in India [START_REF] Khanna | Factors influencing the occurrence of russet scab on potato. Potato, global research & development[END_REF] and in Finlande [START_REF] Kreuze | Phylogenetic analysis of 16S rRNA genes and PCR analysis of the nec1 gene from Streptomyces spp. causing common scab, pitted scab, and netted scab in Finland[END_REF]. Russet scab and netted scab are visually very similar blemishes, which can be sometimes considered as one unique blemish [START_REF] Natsume | Phytotoxin produced by Streptomyces sp causing potato russet scab in Japan[END_REF]. However, netted scab tends to form a network at the tuber [START_REF] Stevenson | Compendium of potato diseases[END_REF]Mulder et al., 2008); S. reticuliscabiei and some isolates of S. europaeiscabiei cause netted scab (Bouchek-Mechiche et al., 2000a), and some isolates belonging to S. aureofaciens group cause russet scab [START_REF] Faucher | Characterization of Streptomyces causing russet scab in Quebec[END_REF][START_REF] Kreuze | Phylogenetic analysis of 16S rRNA genes and PCR analysis of the nec1 gene from Streptomyces spp. causing common scab, pitted scab, and netted scab in Finland[END_REF]. Likewise, tubers may present different types of superficial rugosity which are described by very diverse terms. A rough or scaled periderm of the tuber might be referred as a blemish called rough russeted skin or russeting (Figure 2.1.K). This blemish is a genetic varietal characteristic (eg. cultivar Russet Burbank), even though some authors suggest that physiological causes could also intervene [START_REF] Okazawa | On occurence of defected potato-tubers with rough russeted skin[END_REF][START_REF] Jong | Inheritance of russeting in cultivated diploid potatoes[END_REF]. Russeting differs from russet scab as it forms deeper and larger network on potato tubers. Another similar blemish induces a scuffed or feathered appearance of the tuber with a rubbed off periderm, called scuffing, skinning, excoriation or skin-set (Figure 2.1.L). This blemish is allocated to physiological or mechanical injury [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Lulai | Skin-Set, Wound Healing, and Related Defects[END_REF].

Thirdly, we focus on atypical blemishes, whose causes are unknown, and whose terminologies unclear. They are often allocated to typical blemishes, although the symptoms are not exactly identical and the causes are not clearly demonstrated through the fulfilment of Koch's postulates. Atypical blemishes include several kinds of blemish which often have corky appearance (Figures 2.1.F to 2.1.J) and, to some degree, are visually similar to the ones due to known pathogens, namely Streptomyces spp. or R. solani. This is particularly true for netted scab-like blemishes (Figure 2.1.F and 2.1.H). The most common names encountered in North-American publications are elephant hide or alligator hide -for russet-skinned cultivars, or fishy skin, fish scale, and turtleback -for smooth-skinned cultivars [START_REF] Hart | Fish scale or elephant hide on potatoes[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Turff | Fungus more complex than first thought[END_REF]Selman et al., 2008) (Table 2.1). More descriptive names are used in Europe to describe these atypical corky blemishes, like cork, desquamation, scurfy appearance or scabby lesions [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Fnpppt | GNIS and ARVALIS-Institut du végétal. Guide pratique: Maladies, ravageurs et désordres de la pomme de terre[END_REF]. In some instances, these corky blemishes have sometimes been called rhizoscab because the occurrence of these atypical blemishes was suspected to be connected with R. solani [START_REF] Jouan | Les principales causes d'altérations superficielles des tubercules[END_REF]. Indeed, such symptoms are compared to the ones observed with tuber deformations and cracks (Figure 2.1.G) [START_REF] Stevenson | Compendium of potato diseases[END_REF]. However, the implication of R solani has so far never been clearly demonstrated [START_REF] Turff | Fungus more complex than first thought[END_REF][START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF].

In Europe, R. solani is also frequently associated with the occurrence of dry cores (Figure 2.1.S), which are restricted brown cavities up to several millimetres deep with a diameter of 3 to 6 millimetres [START_REF] Radtke | Diseases and pests of the potato[END_REF][START_REF] Keiser | Influence of farming system, specific cultivation methods and site parameters on potato quality PhD thesis[END_REF][START_REF] Wale | Diseases, pests and disorders of potatoes[END_REF]. Still this blemish is not mentioned in the American compendium of potato diseases ( 2001). In addition, several other atypical blemishes are often mentioned by farmers and extension workers dealing with thin-skinned potato cultivars grown under irrigation schemes; theses blemishes have the appearance of scurfy tissues around the lenticels (Figure 2.1.C). This blemish called enlarged lenticels is known to be due to excessive moisture or insufficient supply of oxygen. However, it is sometimes allocated to R. solani or Streptomyces spp. because enlarged lenticels may resemble scab lesions, although they are smaller and lighter in colour [START_REF] Stevenson | Compendium of potato diseases[END_REF].

Another interesting study case is the scab-like pale brown lesion with or without starshaped cracks (Figure 2.1.J) which is attributed to tobacco necrosis virus (TNV) (Mulder et al., 2008) whereas TNV tuber symptomatology has been illustrated by other authors [START_REF] Radtke | Diseases and pests of the potato[END_REF][START_REF] Jeffries | FAO/IPGRI technical guidelines for the safe movement of germplasm. No. 19: Potato[END_REF] Further more, in recently published potato disease guide (Mulder et al., 2008), atypical corky blemish called polygonal lesion (Figure 2.1.G) has been attributed as a consequence of Armillaria species infection : very few data are available about this pathosystem [START_REF] Jones | Amillaria dry rot of potato tubers in British Colombia[END_REF] and the fulfilment of Koch's postulate has probably never be attempted. By all means, this type of superficial blemish could be the result of any soil-borne microorganism, whether it is bacterial or fungal.

This survey about current terms used for designation of some typical and atypical blemishes highlights confusing situations and an obvious need of clarification. The following paragraph will endeavour to give a clearer and more structured nomenclature for classification of potato tuber blemishes.

Proposal of a nomenclature for potato blemishes

In order to resolve the nomenclatural problems about the common names of typical and atypical blemishes on potato tubers, we suggest the nomenclature hereafter, based on the visual aspect of blemishes rather than on their typical or atypical character. According to the naked-eye visual aspect, we can establish two categories of blemishes: the superficial blemishes and the raised ones (Table 2.3).

Pitted or raised blemishes

Dry core 1. Superficial blemishes alter only the external layers of the periderm and occasionally the external cortical cells but they never deeply damage the tuber flesh. They include:

-Discoloration such as black dot and silver scurf lesions, which form discoloured spots on the tuber surface (Figures 2.1.A and 2.1.B).

-Corky blemish such as o Enlarged lenticel, small, initially white turning brown, corky lesion surrounding the lenticels, that are clearly described (Figure 2 2. Pitted or raised blemish form recessing or projecting skin piece masses on the tuber (Table 2.3). They include:

-Pustules resulting from either o powdery scab, which forms pustules releasing a brownish and powdery mass consisting of cytosori at maturity (Figure 2 -Sclerotia or black scurf, typical potato blemish appearing as black superficial and irregularly shaped raised lumps, resting pellets of R. solani mycelia tightly attached to the periderm (Figure 2.1.Q).

-Tuber crack (Figure 2.1.R) being shallow to moderately deep splits of the tuber and for which this general term can be kept to describe all the kind of cracks without suggesting their origin or their cause.

-Dry core (Figure 2.1.S), a brown deep lesion for which terminology is commonly accepted.

The suggested nomenclature aims at harmonising the terms used to describe the different blemishes of potato tubers and could help avoiding several mistakes that can be made in the fields when blemished tubers are observed.

Conclusion

As the globalisation extends and because more and more multinational research projects are set up, there is a need for harmonising the terminology for scientific community and to make sure that we talk the same language about potato blemishes. Scientific papers often focus on a single disease with one or several symptoms, whereas pluridisciplinary approaches are les frequent.

Underlined is the problem of dealing with the potato overall pathosystems and more precisely the soil-borne pathosystems. By suggesting a detailed transversal view of potato blemish issue, this paper intends to make closer the points of view of scientific researchers and of more generalist persons dealing with potatoes such as growers, breeders, seed certification staff, etc. Reviewing the literature on this subject, it was obvious that a clarification of the terminology was necessary. This nomenclature is based on the study of the different terms used in the world to describe main potato blemishes. The terms the most widely employed and the richest in sense for the description of the blemishes were adopted and the terms which can bring confusions were eliminated.

Furthermore, this article shows the need of knowledge about the histological and physiological changes induced by abiotic and/or biotic stresses causing atypical blemishes. It appears as well that several hypotheses have been proposed to determine the causes of the atypical blemishes but reproducing potato-soil-borne pathosystems under controlled conditions appeared to be difficult task thus the Koch's postulates difficult to fulfill. The authors sincerely hope that this article will catch the attention of the potato community on this major economical problem of potato tuber blemishes and will facilitate future researches, the establishment of seed certification standards and the improvement of varietal assessment. Indeed, the determination of potato blemishes' causes often refers to known diseases while visual observations relate to symptoms of diverse origins. In the future, this new nomenclature will be optimized by being translated into several different languages, and will also be extended to other blemishes which are important for other potato areas, allowing the harmonization of potato blemish terminology in all the branches of the potato production systems. In a previous study (Fiers, 2010), most of tuber blemishes were classified according to the type of symptom: sclerotia, enlarged lenticels, skinning, russeting, common scab, netted scab and atypical corky blemishes. The latter including corky cracks, corky spots or "rhizoscab", star-like corky lesions and blemishes commonly called "elephant hide", described as irregular polygonal sunken corky lesions. These will hereafter be called polygonal lesions. Atypical blemishes frequently observed in ware potato production are the atypical corky blemishes, especially polygonal lesions and corky spots. Black scurf or sclerotia are known to be the long term survival form of R. solani [START_REF] Anderson | The genetics and pathology of Rhizoctonia solani[END_REF][START_REF] Bakali | Black scurf of potato[END_REF] and common and netted scab have been unequivocally demonstrated to be caused by Streptomyces spp. [START_REF] Lambert | Streptomyces scabies sp-nov, nom-rev[END_REF]. These typical blemishes (sclerotia, common scab and netted scab)

were integrated in this study as reference blemishes.

Production of all types of potato commodities aims at providing high quality tubers, either ware potatoes or seed tubers that need to meet the market demands related to the visual quality of the tubers. Atypical blemishes are then a predominant obstacle to the fulfilment of this quality requirement. Thus the determination of the causes of blemishes is needed. Assuming that atypical blemishes are of biological origin, two related hypotheses were considered: atypical blemishes are due to pathogenic microorganisms not yet identified or they are due to known pathogens producing atypical symptoms. Some atypical blemishes closely resemble netted scab caused by Streptomyces spp. but they are also occasionally attributed to R. solani Kühn (teleomorph: Thanatephorus cucumeris (Franck) Donk) [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF]. This is the reason why R.

solani and Streptomyces were investigated as well. The objectives of this study were (1) to isolate potential pathogens from atypical skin blemishes, and (2) test whether the isolated microorganisms are able to re-create the atypical blemishes on progeny tubers and so doing, verifying Koch's postulates. 3.1).

Materials and methods

Plant

Molecular identification of fungal isolates

For DNA extraction, all the collected fungal isolates were cultivated in tubes on PDA slants. Two ml of potato dextrose broth (PDB) was poured into PDA tubes and vortexed to disperse the spores, and the spores-PDB mix was poured into Roux flasks containing 100 ml of PDB. For non-sporulating fungi, 6 explants of PDA were directly placed into Roux flasks. Flasks were incubated at room temperature without shaking for 2 to 3 days. The mycelium was harvested by filtration, frozen at -80°C during 30 min, lyoph ilized and stored at -80°C.

The mycelium was ground in liquid nitrogen in a sterile mortar to obtain a mycelium powder. The DNA was extracted from 20 mg of mycelium powder using DNeasy plant mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer's protocol. The DNA quantity and quality were checked by electrophoresis on a 0.8 % agarose gel, revealed with ethidium bromide and visualized by UV trans-illumination. The DNA concentrations calculated with the image analysis software Bio-Profil Bio1D++ (Windows Application V11.9,

Copyright ©2004 Vilbert-Lourmat) were between 3.5 and 125 ng/µl.

For each fungal isolate, the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was amplified by PCR with the primers ITS1-F (CTTGGTCATTTAGAGGAAGTAA) and ITS4 (TCCTCCGCTTATTGATATGC) [START_REF] White | Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[END_REF][START_REF] Gardes | ITS primers with enhanced specificity for Basidiomycetes -Application to the identification of mycorrhizae and rusts[END_REF]. PCR amplifications were performed in a final volume of 50 µl by mixing 2 µl of DNA with 0.5 µM of each primer, 150 µM of dNTP, 6 U of Taq DNA polymerase (Q-Biogen, Evry, France)

and PCR reaction buffer. Amplification was conducted in a mastercycler (Eppendorf, Hambourg, Germany) with an initial denaturation of 3 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 50 °C, 1 min at 72°C, and a final extension of 10 min at 72°C. Aliquots of PCR produ cts were checked by electrophoresis on a 1 % agarose gel, revealed with ethidium bromide and visualized by UV trans-illumination.

The PCR products were sequenced by Beckman Coulters Genomics (Takeley, UK) using primers ITS1-F and ITS4. For each PCR product, sequences from both strands were assembled to produce a consensus sequence. Sequence identities were determined using BLAST analyses from the National Center for Biotechnology Information (NCBI) available on line.

Molecular identification of Streptomyces isolates

Streptomyces isolates stored at 4°C on PDA were cultivated in 2 5 ml of Luria Bertani (LB) media (10 g l -1 of bacto tryptone, 5 g l -1 of yeast extract, 10 g l -1 of NaCl; pH 7) for 6 days at 27°C. The DNA was ext racted using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's specifications.

The DNA quantity and quality were checked by electrophoresis as above and the DNA concentrations calculated as above were between 4 and 650 ng/µl.

For each Streptomyces isolate, the 16S rDNA was amplified by PCR with the primers 27F (AGAGTTTGATCCTGGCTCAG) [START_REF] Edwards | Isolation and direct complete nucleotide determination of entire genes -characterization of a gene coding for 16S-ribosomal RNA[END_REF] and 1392R

(ACGGGCGGTGTGTACA) [START_REF] Braker | Community structure of denitrifiers, Bacteria, and Archaea along redox gradients in pacific northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes[END_REF]. PCR reactions were performed in a final volume of 50 µl by mixing 10 µl of DNA with 0.2 µM of primer 27F, 0.2 µM of 1392R, 200 µM of dNTP, 12 U of Taq DNA polymerase (Q-Biogen) and PCR reaction buffer. Amplifications were conducted in a mastercycler (Eppendorf) with an initial denaturation of 3 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 57°C, 1 min at 72°C, and a final extension of 5 min at 72°C. Aliquots of PCR produc ts were checked by electrophoresis on a 1 % agarose gel as above.

The PCR products were sequenced using primers 27F and 1392R. For each PCR product, sequences from both strands were assembled to produce a consensus sequence. Sequences identities were determined using BLAST as above.

Pathogenicity tests

Strains representative of the most frequently isolated fungal and Streptomyces species were tested for their pathogenicity on potato tubers. In order to fulfil Koch's postulates for these strains, two different types of bioassays were set up: a pot-test with artificially infested soil and a test where potatoes were grown under hydroponic conditions.

Bioassays in soil

The For the Streptomyces inoculum, Streptomyces spp. were grown on 9 cm PDA plates. Prior to the inoculum setting, a specific substrate for the Streptomyces inoculum was prepared. One liter of vermiculite, mixed with 150 ml of deionized water was autoclaved for 1 h at 120°C, on two conse cutive days. 170 ml Say's media (sucrose 20 g l -1 ; asparagine 1.2 g l -1 ; K 2 HPO 4 0.6 g l -1 ; yeast extract 10 g l -1 ) was added to the vermiculite and autoclaved for 30 min at 115°C.

Streptomyces mat and spores were scraped from 7 to 10 day-old culture on oatmeal agar (oatmeal powder 50 g l -1 ; agar 23.5 g l -1 ) at 27 °C and ground in a sterile mortar with 8 ml sterile water. The resulting mixture was poured into the sterile vermiculite substratum and was incubated for 2 to 3 weeks at 27°C with daily shaking [START_REF] Wanner | Field isolates of Streptomyces differ in pathogenicity and virulence on radish[END_REF].

The potting mixture (one third of sand and two thirds of peat) used to grow the potatoes was steam-disinfected and stored at room temperature for 7 days to allow putative toxic compounds to be released. The fungal and Streptomyces inocula were mixed separately with about 6 l of disinfected potting mixture with a three-dimensional shaker (Turbula, System Schatz). The infested potting mixture was placed in 10 l plastic pots (25 cm diameter, 30 cm high). The pots infested with Streptomyces were prepared two weeks before planting, covered with a plastic cover and kept at room temperature for about 15 days to allow the establishment and multiplication of the bacteria.

One seed tuber was planted in each pot. Cultivar Charlotte was chosen for soil assays because it is one of the cultivars that is mostly cited in the sample collections (2006 and 2007) The intensity of sclerotia was scored according to the R. solani scale, from level 1 (1 % of the tuber surface covered by the blemish) to level 9 (>35 % of the tuber surface covered by the blemish). Other blemishes were scored according to the common and netted scab scale, from level 1 (4 % of the tuber surface covered by the blemish) to level 9 (> 60% of the tuber surface covered by the blemish). The intensity was calculated by averaging the intensity of the reproduced blemish for the 3 replicates (6 when the strain was tested twice (Table 3.2). Fungi and Streptomyces were isolated from the progeny tubers following the same protocol as above.

Co-inoculation tests under hydroponic conditions

In the second type of test, potatoes were grown under hydroponic conditions in order to follow the development of the blemishes in-situ. A total of 22 strains of fungi and Streptomyces spp. were tested in single and coinoculations, resulting in 49 different treatments (Table 3.3).

The experimental system (adapted from [START_REF] Gray | The growth of individual tubers[END_REF]) (Figure 3.2) consisted of a tray (40 cm in diameter and 10 cm in depth) with a 5 cm diameter hole in the centre, placed on a 5 l pot containing nutrient solution (N P K, 13 21 13 or N P K, 8 17 24, see below) (Algospeed Flo, Compo, France). A seed tuber was placed in the centre of the tray on a plastic screen of 1 cm mesh, maintaining the tuber in the tray, away from the water but allowing the roots to elongate into the nutrient solution. Certified seeds (Class A, 28-35) of the cultivar Bintje were used; this cultivar is known for its susceptibility to overall skin blemishes Two and a half months after plantation, when progeny tubers reached a sufficient size (i.e. > 2 cm long), they were inoculated with one or two strains.

Inoculations were performed using plugs of 10 day-old microbial cultures on PDA plates. Six visually healthy progeny tubers per plant were chosen. Three of them were wounded with a sterile toothpick and the three others were not. For each treatment, plugs of the inoculated strains were placed side by side on the tubers, on the wound for wounded tubers. For each treatment corresponding to single or double inoculations, three independent plants were inoculated. A control treatment corresponding to three non-inoculated plants was also conducted. Plants were grown until natural senescence and harvested individually.

After inoculation, newly formed tubers were observed weekly during 7 weeks.

The blemishes were scored when they appeared and all along their development.

At harvest, tubers from each plant were washed under running tap-water, airdried and placed in a paper bag. The number of tubers per plant was recorded and the tubers were scored individually for atypical corky blemishes with the scoring scale from 1 to 9.

Fungal and Streptomyces isolations on progeny tubers were done following the same protocol as above.

Results

Among the samples collected in 2006 and 2007, the blemishes the most frequently observed were polygonal lesions, representing 22 % of the total blemishes, followed by sclerotia (16 %), enlarged lenticels (15 %), corky spots (13 %), common scab (8 %), corky cracks (8 % ), star-like corky lesions (7 %), skinning (5 %), netted scab (4 %), and russeting (2 %). The atypical corky blemishes (polygonal lesions, corky spots, corky cracks and star-like corky lesions) all together represented 50 % of the observed blemishes.

Identification of fungi

Twenty six different fungal genera were found among the 349 isolates collected from blemishes and identified from their ITS sequence (Table 3.1).

The most represented genera were Fusarium (80 strains), Rhizoctonia (68 strains), Alternaria (46 strains), Penicillium (33 strains), and Clonostachys (27 strains). Most of the isolates were identified at the species level, however, some isolates were only identified at the genus level, because either their ITS sequence had the same percentage of similarity as two or more fungal species or the obtained ITS sequence was too short. Finally, at least 45 different fungal species were identified. In the case of R. solani isolates, the anastomosis groups (AG) were also determined based on ITS sequences (Table 3.1). Pathogenicity tests

1 F. culmorum 1 F. equiseti 1 1 1 F. graminearum 1 1 1 F. oxysporum 4 20 3 3 4 2 2 6 1 1 46 F. redolens 2 1 F. sambucinum 1 1 1 F. sambucinum or F. tumidum 1 F. solani 2 2 1 F. venenatum 1 2 1 1 Fusarium sp. 3 2 1 1 1 1 Gliomastix murmorum 1 Microdochium bolleyi 1 1 1 Microdochium sp. 1 Mortiella elongata 1 Mucor circenelloides 1 4 2 1 1 M. fragilis 1 M.

Bioassays in soil

The inoculated strains were re-isolated from the soil in 39% of the cases.

Fungi belonging to the Rhizoctonia solani species were more frequently reisolated than the other species.

The results of the two bioassays conducted in soil are presented in table 3.2.

Among the 62 fungal strains and the 8 Streptomyces strains inoculated, 30 fungi and 1 Streptomyces were re-isolated from the progeny tuber surfaces at the end of the tests. Several other fungi that were not initially inoculated in pots were also isolated such as Trichoderma spp., Mucor spp. and Colletotrichum spp. Among the inoculated fungi, R. solani was the species the most frequently re-isolated from tubers (16 strains out of 21 were recovered on the progeny tuber surface). Conversely, only one out of the 7 Alternaria strains tested and none of the 8 Clonostachys strains tested were recovered from the progeny tuber surface.

Non-inoculated control

Skinning, enlarged lenticels, polygonal lesions (a) The codification of the strains includes the year of isolation (two first digits), the geographical zone of isolation corresponding to the French departments (third and fourth digits) and the strain identification number (last digits and letters). Strains followed by an asterisk (*) were tested in the two different bioassays (2007; 2008). Strains followed by two asterisks (**) are reference strains, which were not sequenced in this study. (c) Intensities of blemishes were scored according to the official French scales of the tuber potato diseases (GNIS and FNPPPT). Sclerotia were scored from level 1 (1 % of the tuber surface covered by the blemish) to level 9 (>35 % of the tuber surface covered by the blemish) and the other blemishes were scored from level 1 (4 % of the tuber surface covered by the blemish) to level 9 (> 60% of the tuber surface is covered by the blemish).

(d) + means that the inoculated strain was isolated from the progeny tubers; -means that the inoculated strain was not isolated from the progeny tubers.

In all the treatments, all types of blemishes were observed with a variable intensity, but the original blemishes were rarely reproduced by the inoculated strains. There was no clear relationship between the type of blemish and the taxonomic status of the strains. However the strains of R. solani AG 3 were able to cause sclerotia (4 of the strains) as well as polygonal lesions (4 other strains)

and one strain generated corky crack, on the progeny tubers. One of 9 F.

oxysporum strains was able to cause polygonal lesions. One of the three strains of P. brevicompactum, two strains of R. solani, and the only strain of P. cucumerina caused enlarged lenticels. Among the strains of Streptomyces tested, none has produced the same blemish as the one from which it was isolated. Progeny tubers coming from the non-inoculated controls also showed blemishes: skinning (6 out of 6 tubers), enlarged lenticels (5 out of 6 tubers), and polygonal lesions (1 out of 6 tubers).

The intensity of reproduced blemishes varied from 1 to 6 with an average of 1.6, which means that in average, less than 6 % of tuber surfaces was covered by a given type of blemish. In general, blemishes intensities were low. Some tubers showed several different types of blemishes making it difficult to make a connection between the isolated strain and a specific blemish.

Co-inoculation tests under hydroponic conditions

Before inoculation, 43 % of the progeny tubers already displayed polygonal lesions with an average intensity of 3, meaning that 6 % of the tuber surface was covered by the blemish. The blemishes observed during the seven weeks following the inoculation, are listed in Table 3.3. Corky cracks, polygonal lesions, enlarged lenticels, skinning, and star-like corky blemishes were produced on progeny tubers grown under hydroponic conditions and inoculated with one or two selected strains. For each treatment, a variety of blemishes was observed without any relationship between the strain inoculated and blemishes.

In none of the treatments, the inoculated fungus or Streptomyces were reisolated from progeny tubers after harvest. However, numerous Mucor spp. and Penicillium spp. were isolated. The diversity of blemishes and of fungi isolated from the non-inoculated tubers was similar to the one from the inoculated tubers. Growing tubers under hydroponic conditions allowed the observation of formation and development of blemishes during tuber growth. It often appeared, on inoculated tubers as well as on non-inoculated tubers that wound and opened lenticel healing formed polygonal and star-like corky lesions. We also observed that polygonal lesions and corky cracks frequently resulted from the development of skinning and star-like corky lesions along with tuber growing period. Finally, crack formations were observed at the end of the growing period, when tubers tended to grow faster.

Discussion

The objective of this study was to investigate the microbial origin of potato skin tuber blemishes. We made a large collection of tubers showing different types of blemish, and isolated and identified microorganisms associated with these blemishes. At least 45 different species of fungi and 1 species of Streptomyces were isolated. Fusarium, Rhizoctonia, Alternaria, Penicillium and Clonostachys were the genera the most frequently isolated. Fusarium, Rhizoctonia and Alternaria are known to be present at the surface of potato tubers and in the rhizosphere of potato plants [START_REF] Cwalina-Ambroziak | Fungi colonizated of potato tubers (Solanum tuberosum L.) after harvest and after storage[END_REF][START_REF] Pieta | Antagonistic bacteria and fungi limiting potato infection by soil-borne pathogenic fungi[END_REF]. They are known to be responsible for dry rots, black scurf and stem canker and early blight, respectively, on potato crop [START_REF] Radtke | Diseases and pests of the potato[END_REF][START_REF] Stevenson | Compendium of potato diseases[END_REF]. Clonostachys, especially C. rosea (formerly Gliocladium roseum, [START_REF] Schroers | Classification of the mycoparasite Gliocladium roseum in Clonostachys as C. rosea, its relationship to Bionectria ochroleuca, and notes on other Gliocladium-like fungi[END_REF]) could be either a pathogenic agent causing dry rot [START_REF] Serfontein | A potato wilt disease in South Africa caused by Erwinia carotovora subspecies carotovora and Erwinia chrysanthemi[END_REF] or a biological agent controlling several potato soil-borne diseases [START_REF] Keinath | Potential of Gliocladium roseum for biocontrol of Verticillium dahliae[END_REF][START_REF] Davide | Evaluation of three nematophagous fungi and some nematicides for the control of potato cyst nematode Globodera rostochiensis[END_REF]. Penicillium species were isolated from potato tubers and identified as saprophytic strains (Cwalina-Ambroziak, 2002). To our knowledge it is the first time that P. brevicompactum, the most common Penicillium species found in our study, was isolated from potato tubers. Thus, tubers are an ecological niche favourable for the development of a diversity of fungi and bacteria, both pathogens and saprophytes.

Though quite diverse, the microflora present at the surface of tubers displaying blemishes might have been underestimated by the isolation technique. Extracting the DNA directly from the tuber blemishes could have revealed an even larger spectrum of microorganisms. However, such a direct approach was not adapted to our purpose since it would not have allowed us to isolate the microorganisms, characterize and test them individually in bioassays.

Only one sample of each blemish type, skinning and star-like corky lesion, was analysed for the isolation of Streptomyces. No Streptomyces were isolated from these blemishes. Further research needs to be done to know if Streptomyces does interfere with the formation of skinning and star-like corky lesions, or not.

A diversity of fungi was isolated in this study, direct observation using a microscope often allowed their identification at the genus level but further identification at the species level frequently required molecular tools. The ITS sequence and the 16S rDNA sequence are generally used as a species marker among fungi or Streptomyces, respectively. In our study, 75 % of the strains were identified at the species level by sequencing their ITS region or the 16S rDNA. Most of the remaining strains were not identified at the species level because of a too short sequence.

However, this does not stand for the strains belonging to the genus Alternaria.

Several of them could not be identified at the species level even with an almost complete ITS sequence because their ITS sequence matched two or more Alternaria species. In these cases, sequencing of the genes coding the elongation factor, the beta-tubulin, or the mitochondrial ribosomal large subunit (mtLSU) might have been more discriminative [START_REF] Peever | Molecular systematics of citrus-associated Alternaria species[END_REF]. On the other hand, the variability of ITS sequences is much higher in R. solani, allowing the differentiation of AG within the species [START_REF] Guillemaut | Typing of anastomosis groups of Rhizoctonia solani by restriction analysis of ribosomal DNA[END_REF]. In our study, three different AGs were identified.

The most important one was AG 3 -PT known to be the most frequent AG on potato [START_REF] Kuninaga | Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3[END_REF]. In addition, 6 strains of R. solani AG 2-1 and 4 strains of R.

solani AG 5 were also isolated. R. solani AG 3 -PT and AG 2-1 were mostly isolated from sclerotia whereas R. solani AG 5 was isolated from enlarged lenticels and russeting. Similar results were found in potato crops in France and Great-Britain [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Woodhall | Characterization of Rhizoctonia solani from potato in Great Britain[END_REF].

Concerning the identification of Streptomyces, identification at the species level has not been possible in all cases. A complementary identification method based on biochemical characteristics of streptomycetes could be used [START_REF] Bouchek-Mechiche | Streptomyces species isolated from potato scabs in France: numerical analysis of "Biotype-100" carbon source assimilation data[END_REF]. Otherwise, a specific PCR for each species of Streptomyces could allow a quicker and complementary detection. An amplification of the 16S rDNA, highly conserved, and amplification of the ribosomal intergenic spacer (RIS), more discriminative, could allow a better identification of Streptomyces species [START_REF] Lehtonen | Occurrence and survival of potato scab pathogens (Streptomyces species) on tuber lesions: quick diagnosis based on a PCR-based assay[END_REF][START_REF] Park | Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S-23S rDNA spacer region and denaturing gradient gel electrophoresis[END_REF].

The second part of the study was to assess the pathogenicity of the isolated strains.

Two different types of bioassays were set up. In the first one, the soil was artificially infested by the microbial strains in order to mimic field conditions. In the second one, potato plants were grown under hydroponic conditions and inoculated with one or two strains of potential pathogens, in order to follow the development of the blemishes.

Although millet seeds used as inoculum were fully colonized, inoculated strains were not always detected in the soil at the end of the assay. This could be because the soil volume tested was too small, or because other species isolated from soil such as

Trichoderma or Mucor having a high growth rate, prevented the growth of the fungi of interest. Among the 72 fungal strains tested in soil, only 14 were able to reproduce the same blemishes they originated from; the other strains did not reproduce the blemish of origin. Among the 11 Streptomyces strains tested, none was able to reproduce the type of blemish it originated from. According to Koch's postulates, a

given strain can be considered as responsible for a symptom when it reproduces the original symptom and when the identical strain can be isolated at least once from the diseased progeny tubers [START_REF] Rapilly | Champignons des plantes: les premiers agents pathogènes reconnus dans l'histoire des sciences[END_REF]. The 14 potentially pathogenic strains belonged to the species F. oxysporum, P. brevicompactum, P. cucumerina, and R.

solani AG 3 -PT. Skinning, enlarged lenticels and polygonal lesions seemed to be the basal blemishes observed under the conditions of the assay rather than a result of the inoculum. Even though Fusarium spp. and more especially F. oxysporum were frequently isolated from blemishes and particularly associated with polygonal lesions, it seems that this species is well adapted to live on tuber surface as an opportunist.

Because, polygonal lesions were also observed on the non-inoculated controls, it is not possible, at this point, to infer the involvement of this species in the formation of polygonal lesions. Similarly, we cannot consider P. cucumerina, P. brevicompactum and R. solani AG 3 -PT as responsible for enlarged lenticels on potatoes. We know that a high level of humidity in the soil is favourable for the opening of lenticels as it was observed in the non-inoculated controls of the bioassays. Opened lenticels facilitate penetration of microorganisms. P. cucumerina, P. brevicompactum and R.

solani AG 3 -PT could be well adapted species to this specific niche and might be considered as opportunistic colonizers of the lenticels. Moreover, isolations made from soil from the bioassays showed that some species were better adapted to Chapter 3 -Diversity and pathogenicity of microorganisms from blemishes 123 survive in the soil than others (data not shown). R. solani did not spread homogenously into the soil since it was much more often recovered from the tuber surface than from the soil. Alternaria spp. and Clonostachys spp. were recovered neither from the soil nor from the tuber surface. While Clonostachys is known to be good saprophyte [START_REF] Serfontein | A potato wilt disease in South Africa caused by Erwinia carotovora subspecies carotovora and Erwinia chrysanthemi[END_REF], Alternaria and R. solani are not. R. solani behaves as a root inhabiting fungus rather than as a soil fungus [START_REF] Garrett | Pathogenic root-infecting fungi[END_REF].

The involvement of R. solani AG 3 -PT in the formation of polygonal lesions was observed in 4 independent replicates: this result confirms what is currently observed in open field conditions but needs further investigation under controlled conditions and artificial inoculation. Similarly, the contribution of R. solani AG 3 to the formation of corky crack was verified with only one strain. On the contrary, the pathogenicity of R. solani AG 3 -PT causing sclerotia was clearly demonstrated in four independent replicates. The involvement of R. solani AG 3 in the occurrence of sclerotia has already been demonstrated several times [START_REF] Anderson | The genetics and pathology of Rhizoctonia solani[END_REF][START_REF] Woodhall | Infection of potato by Rhizoctonia solani: effect of anastomosis group[END_REF].

Since the inoculation of single strains did not allow the reproduction of observed atypical blemishes, we assumed that a consortium of strains might be involved in the occurrence of blemishes. Co-inoculations, in the bioassay in hydroponic conditions, did not demonstrate clear fungal or Streptomyces contribution into the occurrence of skin blemishes. Blemishes like skinning or star-like corky lesions could be the early stage of other blemishes like polygonal lesions. We also found a connection between wound healing and the formation of polygonal lesions. Moreover, the appearance of severe polygonal lesions on the non-inoculated tubers under hydroponic conditions confirmed that this blemish could be skin response to other environmental factors. In the 60-70's and later in the 2000's, several publications suggested that polygonal lesions were due to an excess of humidity or organic matter [START_REF] Hart | Fish scale or elephant hide on potatoes[END_REF][START_REF] Sexton | Some thoughts on elephant hide[END_REF]. In our experimental set up, the high moisture content due to hydroponic conditions is probably one of the causes of some of the blemishes. Polygonal lesions could be the result of a physiological reaction of the plant to stressful abiotic factors.

Although, we noticed that cultivar Samba was particularly frequently associated with polygonal lesions, the genetic background of the potato germplasm was deliberately not taken into account in the expression of tuber blemishes because too little reliable data are available on the host-parasite relationships. However, the S. tuberosum -Streptomyces spp. complex is one of few that gather practical data at the cultivar level when assessed under controlled conditions and artificial inoculum [START_REF] Pasco | Resistance of potato genotypes to common and netted scab-causing species of Streptomyces[END_REF], although its expression can vary because it is highly dependant upon biotic and abiotic environmental factors.

This study showed the diversity of the microbial flora on the surface of potato tubers and demonstrated that, except for R. solani AG 3 -PT causing sclerotia, blemishes analyzed in this study could not be directly attributed to specific

Introduction

Potatoes (Solanum tuberosum L.) are cropped almost everywhere in the world and the production increases every year since several decades. In 2007, about 325 million tons were produced in the world. The quality of the tubers warranties good production sell. However, since fresh tubers are washed before selling in most of the developed countries, several kinds of blemishes became visible on the tuber surface.

Some of these blemishes (polygonal lesions, corky cracks, corky spots, star-like lesions with or without halo, rugosities, skinning) are caused by factors that are still not well known [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF]. In a previous study, it has been demonstrated that except for the fungus Rhizoctonia solani which causes sclerotia, the microorganisms present at the tuber surface were not directly implicated in the appearance of those blemishes. Conversely, a wide diversity of microorganisms is associated with potato surface (Fiers et al., 2010). Edaphic factors such as soil pH, organic matter rate, water supply, or the presence of particular nutrients could be ribosomal DNA (rDNA), respectively [START_REF] Liu | Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA[END_REF]. This method allows the characterization of the microbial community structures in the form of variable terminal restriction fragments (TRF). To go further and identify the microorganisms of the geocaulosphere potentially implicated in the occurrence of blemishes, the specific TRF of 56 microorganisms isolated from potatoes were determined using the same T-RFLP procedure.

Material and methods

Soil sampling

Soil sampling was done in 2008 in a potato field (cv. Juliette) located in Morbihan in Western France (48°05' N, 2°51' W) at t wo consecutive dates: T1 at the end of July, before haulm destruction and T2 at the end of August, after haulm destruction. The field was chosen because of recurrent problems of blemished tubers. A plot (15 m x 3 m) with 2 rows of potatoes (60 cm large) and 3 inter-rows (60 cm large) was delimited. At T1, 5 healthy plants and 5 plants whose progeny tubers were either healthy or blemished were chosen. At T2, 4 plants whose progeny tubers were either healthy or blemished and 4 blemished plants were chosen (Table 4.1).

There were no completely healthy plants in the plot at T2. Three tubers were collected from healthy plants, 3 healthy tubers and 3 blemished tubers were collected from partially-healthy and blemished plants, and 6 tubers were collected from blemished plants. About 100 g of soil from the geocaulosphere of the collected tubers were sampled; the soil samples were placed in a plastic bag with the corresponding tuber. At each sampling date, 5 samples of soil from the inter-rows were collected, pooled together and divided into 3 homogenised samples. A total of 99 soil samples were collected and brought back to the lab where they were immediately prepared for subsequent analyses. Each soil sample was stored in 10 tubes preserved at -20°C until the experiment proceeded. Potato tubers were observed and the superficial blemishes were scored according to chart approved by the French Ministry of Agriculture (GNIS and SOC, 2005). A picture of each tuber was taken. Bacterial and fungal community structures Nucleic acids were extracted from 1 g of soil of the 99 soil samples as described previously [START_REF] Edel-Hermann | Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils[END_REF]. Briefly, a physical disruption (bead-beater) and a chemical extractant (sodium dodecyl sulfate) at 70°C were used.

The crude nucleic acid extracts were purified twice using a polyvinylpolypyrrolidone spin column to remove coextracted humic acids and once using a Geneclean Turbot kit (Q-BIOgene, Illkirch, France). Purified DNA extracts were quantified by electrophoresis in agarose gels using dilutions of calf thymus DNA and stored at -20 °C.

The genetic structure of microbial communities in the different soil samples was investigated using T-RFLP of 16S and 18S ribosomal RNA (rRNA) genes for bacteria and fungi, respectively [START_REF] Edel-Hermann | Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils[END_REF][START_REF] Perez-Piqueres | Response of soil microbial communities to compost amendments[END_REF].

Bacterial 16S rDNA was amplified by PCR using the primer 27F (AGAGTTTGATCCTGGCTCAG) [START_REF] Edwards | Isolation and direct complete nucleotide determination of entire genes -characterization of a gene coding for 16S-ribosomal RNA[END_REF] labelled with the fluorescent dye D3 and the primer 1392R (ACGGGCGGTGTGTACA) [START_REF] Braker | Community structure of denitrifiers, Bacteria, and Archaea along redox gradients in pacific northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes[END_REF].

Fungal 18S rDNA was amplified by PCR using the primer nu-SSU-0817-5′

(TTAGCATGGAATAATRRAATAGGA) labelled with the fluorescent dye D3 and the primer nu-SSU-1536-3′ (ATTGCAATGCYCTATCCCCA) [START_REF] Borneman | PCR primers that amplify fungal rRNA genes from environmental samples[END_REF]. The labelled and unlabelled primers were synthesized by Sigma-Aldrich and Eurofins MWG Operon (Ebersberg, Germany), respectively. PCR amplification of 16S rDNA was carried out in 25 µL reactions including: Taq polymerase buffer, 200 µM dNTP, 0.2 µM of each primer, 2 U Taq polymerase, 20 ng DNA, and H 2 O. All PCR reagents were obtained from Q-BIOgene. Amplifications were conducted in a Mastercycler (Eppendorf, Hamburg, Germany) using the following reaction conditions: initial denaturation at 94°C for 3 min followed by 30 cycles of 1 min at 94°C, 1 min at 57°C and 1 min at 72°C and a final e longation for 10 min at 72°C.

PCR amplification of 18S rDNA was carried out using identical reagent conditions except the primers at a concentration of 0.25 µM each. The PCR conditions for 18S rDNA included an initial denaturation at 94°C for 3 min followed by 35 cycles of 1 min at 94°C, 1 min at 56°C and 1 min at 72°C and a fina l elongation for 10 min at 72°C.

Bacterial and fungal PCR reactions were checked by electrophoresis on a 1 % and 2 % agarose gel, respectively. PCR products were purified using the MinElute PCR purification kit (Qiagen, Courtaboeuf, France). Purified PCR products were quantified as above and 120 ng of purified PCR products were digested with 12 U of restriction enzyme in a final volume of 100 µl for 3 h at 37 °C . The restriction enzymes HaeIII Mean values for the relative intensity of peaks found in at least two of the three analyses were considered for further statistical analyses of microbial community structure. Moreover, the fragments between 60 to 640 bp corresponding to the size range of the standard were considered. The comparison of the TRF sizes between samples was automated by assigning them to discrete categories using the program

Lis with an interval of 1.25 bp [START_REF] Mougel | A mathematical method for determining genome divergence and species delineation using AFLP[END_REF]. The communities characterized by the sizes of the TRF and their intensity measured by the height of the peaks were compared by principal component analysis (PCA) using the ADE-4 software (Thioulouse et al., 1997). This ordination method summarizes multivariate data to a few variables or dimensions and provides an arrangement of the communities in a two-dimensional diagram based on their scores on the two first dimensions. The significance of the resulting structures was checked using Monte Carlo tests with 1 000 random permutations of the data.

Typing of microbial strains by T-RFLP

In order to identify microorganisms corresponding to characteristic TRF in the geocaulosphere, diverse fungi previously isolated from potato tubers were analysed using the same procedure of T-RFLP analysis as above. As Streptomyces bacteria are frequently associated to potatoes [START_REF] Bouchek-Mechiche | Streptomyces species isolated from potato scabs in France: numerical analysis of "Biotype-100" carbon source assimilation data[END_REF] France). Strains of Streptomyces stored at 4°C on potato dextrose agar (PDA) were cultivated in 25 mL of Luria Bertani media (10 g L -1 of bacto tryptone, 5 g L -1 of yeast extract, 10 g L -1 of NaCl; pH 7) for 6 days at 27°C. The DNA was ext racted using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's specifications. The DNA quantity and quality were checked by electrophoresis as above and the DNA concentrations calculated as above were between 6 and 650 ng µL -1 .

The fungal strains were cultivated in tubes on a PDA slope. Two mL of potato dextrose broth (PDB) were poured into each tube. Each tube was then vortexed to disperse the spores, and the mixture was poured into Roux flasks containing 100 mL of PDB. For fungi which do not sporulate (i.e. R. solani), 6 explants of PDA were directly placed into Roux flasks. Flasks were incubated at room temperature without shaking for 2 to 3 days. The mycelium was harvested by filtration, frozen at -80°C during 30 min, freeze dried and stored at -80°C. Th e mycelium was ground to powder with liquid nitrogen using cold sterile mortar and pestle. The DNA was extracted from 20 mg of mycelium powder using the DNeasy plant mini kit (Qiagen). The DNA quantity and quality were checked by electrophoresis on a 0.8 % agarose gel, revealed with ethydium bromide and visualized by UV transillumination. The DNA concentrations were between 3.5 and 125 ng µL -1 . The T-RFLP analysis of each strain was performed as above.

Results

Before haulm destruction (T1), 61 % of the tubers had blemishes. At this date, blemishes were light; they affected about 1 % of the tuber surface. The most frequently observed blemishes were superficial polygonal lesions (39 %), star-like lesions with (5 %) or without (9 %) halo and netted scab (7%). Other blemishes represented 1 %. After haulm destruction (T2), 86 % of the tubers were blemished, 52 % presented black scurf on at least 9 % of the tuber surface, 14 % were blemished by star-like lesions with halo and 13 % by star-like lesions without halo.

Seven percents of the tubers presented netted scab (Figure 4.1).

Before haulm destruction (T1) After haulm destruction (T2)

Polygonal lesion Black scurf

Light star-like lesion without halo Star-like lesion with halo Light netted scab T-RFLP analyses provided complex profiles for bacteria and fungi at each sampling time. The mean number of TRF per soil sample was 90 for the bacteria and 37 for the fungi. PCA showed that inter-row soils were always different from samples from geocaulospheres especially at T2 and they were more variable at T1 than at T2.

There were as much differences between tubers from the same plant as between tubers from different plants ( Analyses were performed on soil from the geocaulosphere of healthy plants (3 tubers, white squares), blemished plants (6 tubers, grey squares) or partially -healthy and blemished plants (3 healthy tubers, white circles and 3 blemished tubers, grey circles). Each plant was represented by a single number. Analyses of uncultivated soil were performed on three independent samples of inter-row soil as control (triangles). Each soil set is represented by its gravity centre. The branches show the divergence of the tubers from the same plant from the respective gravity centres.

A global analysis showed significant discrimination in bacterial community structures between the two periods of sampling, T1 and T2 (Figure 4.3a). However, the permutation test did not show significant difference between the community structures around blemished tubers from blemished plants at T2 and around blemished tubers from partially-blemished plants at T1. Among the peaks representing the bacterial TRF, five of them were high in intensity in all the treatments. One peak was intense only at T1, another was observed in all treatments except inter-row soils. Some peaks we only observed in inter-row soils.

The fungal global analysis revealed a marked discrimination between the two samplings T1 and T2 (Figure 4.3b). For all treatments, three fungal TRF were among the peaks of highest intensity. The TRF of ~ 583 bp was intense (5.5 % of the relative intensity in mean) in all the treatments except in inter-row soil at T2. In addition to the three main peaks, seven dominant peaks were observed in soils sampled at T1 and in soils sampled at T2, but they were different for the two sampling dates. One of the seven major TRF at T2, a peak around 565 bp, was particularly intense in fungal community structures around blemished tubers from blemished plants at T1 and T2.

Discussion

The main objective of the present study was to investigate whether the microbial community structure in the geocaulosphere of potato tubers depends on the occurrence of blemishes on the tubers. In addition, although the same TRF may correspond to different microorganisms, the analysis of a collection of strains with the same T-RFLP procedure can provide hypotheses concerning the microorganisms responsible for the variations observed in community structures. The occurrence of blemished tubers observed before and after haulm destruction increased by 41 % in 27 days and all the sclerotia appeared after haulm destruction.

The community structures of inter-row soils were different at T1 and T2. The slightest variability in inter-row soil sampled at T2 can be explained by climatic conditions or by a drought event at the end of the growing season. Inter-row soils were also generally distinct from the soil samples from geocaulosphere. This was confirmed by the fact that some TRF were specifically associated with inter-row soil samples.

Thus, non cultivated soils had different microbial community structures than soils where potatoes were grown. This implies that potato plants have an effect on the structure of the communities of their geocaulosphere, probably through phenological and physiological changes. The geocaulosphere is an evolving and alive environment that can be compared with the rhizosphere in which exudates are produced and have an effect on their environment.

Our study showed that the structures of microbial communities were homogeneous in the geocaulosphere of partially healthy and partially blemished plants. The microbial communities of healthy plants had different structures compared with the microbial communities of partially healthy and blemished plants, which were also different from the microbial community structures of blemished plants. Therefore, the microbial communities evolved according to the sanitary status of the plant.

The data showed significant differences between bacterial community structures before and after haulm destruction, especially among healthy tubers. In the case of healthy plants, the structure of the bacterial communities was different at T1 and T2, while in the case of blemished plants, the structure of the bacterial communities in the geocaulosphere of blemished tubers was similar at T1 and T2. The bacterial consortium was quite different for healthy tubers from healthy plant at T1 and for healthy tubers from partially-healthy plants at T2. Conversely, it was relatively homogenous and constant around blemished tubers from half blemished plant at T1

and blemished tubers from blemished plant at T2. The intermediate states, healthy tubers from partially-healthy plant at T1 and blemished tubers from partiallyblemished plant at T2, had in-between bacterial community structures. There was a progressive evolution from two different structures of the bacterial communities at T1 and T2 around healthy tubers toward a common structure around all blemished tubers. For fungi, the variations of community structures induced by time were higher than the variations induced by the health state of plants. The two distinct structures at T1 and T2 showed that there was a succession of the fungal communities in potato geocaulosphere along with time. Considering the analysis of the most intense peaks for each community, the differences of structures between T1 and T2 were much more marked for fungi than for bacteria. We observed that the main fungal TRF were very different at T1 and T2, whereas the most intense bacterial TRF were the same at T1 and T2. The distinction between T1 and T2 is not only due to the time delay between the two sampling dates. Although the weather report for 2008 showed that July and August had about the same climatic conditions in France, the physiological variations of plants and the impact of chemical products applied for haulm destruction on the microbial community structures should also be considered.

Data about the TRF lengths of reference strains could have allowed a more precise analysis of the complex soil profiles. Although some TRF provided information about the occurrence of putative fungal species in the geocaulosphere, the majority of the peaks in complex T-RFLP profiles could not have been associated with a particular species or genera. Although the T-RFLP analyses were performed on a representative sample of microorganisms living on the surface of the potato tubers (Fiers et al., 2010), only few microorganisms could be allocated to the main peaks observed for each profile. Moreover, our analysis could not determine the TRF lengths of some species because they were longer than 640 bp, the maximal detectable length with the size standard that was used. TRF data remain rare and databases providing information on the TRF lengths of various microorganism species could be an interesting tool allowing a more precise analysis of complex T-RFLP profiles. Several papers report works on databases development, but sources of error remain possible and existing databases are still poor concerning environmental microorganisms [START_REF] Matsumoto | Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota[END_REF][START_REF] Dickie | Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review[END_REF].

In our results, it appeared that all the determined TRF were not present in complex T-RFLP profiles. TRF corresponding to TRF of R. orzyae, V. dahliae, A. glauca and F.

griseum were not detected in any of the soil samples. Those fungi were absent from the geocaulosphere of the studied potatoes. The strains analyzed, isolated from blemished tubers were never detected in the geocaulosphere of healthy tubers. This

shows that those strains were specifically associated to the geocaulospheres of blemished tubers. It also happened that several genera had the same TRF length.

This was the case for the ~ 583 bp TRF which matched at least with four different genera: Alternaria, Ulocladium, Phoma and Epicoccum, and for the ~ 580 bp TRF which matched with Colletotrichum and Cylindrocarpon genera. Among the most intense peaks observed in soil community structures, some could correspond to some of the strains that were analysed. For example, the TRF of ~ 565 bp represented 5 % in average of the total intensity of each complex profile. This length corresponds to the mean TRF of R. solani. This fungal species or a fungus with the same TRF length was highly present in the geocaulosphere of blemished potato tubers after haulm destruction (T2). This coincided with the period of time that black scurf was first observed on blemished tubers in the experimental field. As we know that black scurf is caused by R. solani forming sclerotia, at the tuber surface (El [START_REF] Bakali | Black scurf of potato[END_REF], we can confirm that this particularly intense TRF of ~ 565 bp corresponded to R. solani. This is in agreement with the previous results

indicating that sclerotia are formed after haulm destruction [START_REF] Otrysko | Relations between the degree of dependence of daughter tubers on their mother plants and their infestation by Rhizoctonia solani[END_REF]. As just demonstrated, the fungal community structures of the geocaulosphere of potato tuber evolve by succession of different fungal community structures in time.

The apparition of a R. solani population around blemished tubers after haulm destruction is the only case for which a causality link between the presence of this fungus and the occurrence of specific blemishes can be proposed (Fiers et al., 2010).

For the other microorganisms, it is so far difficult to conclude whether the modification of the structure of their communities has provided conditions for, or was a consequence of the occurrence of blemishes, but this study clearly demonstrates a direct relationship between these simultaneous events.

Introduction

The fungus Rhizoctonia solani (teleomoph Thanatephorus cucumeris, Kühn, 1858) is a widely spread plant pathogen which cause severe damages on numerous plant species. R. solani includes many related but genetically different subspecific groups. The hyphae of the closely related strains can fuse and, hence, form an anastomosis group (AG), whereas the distantly related strains are unable to anastomose [START_REF] Parmeter | Rhizoctonia solani : biology and pathology[END_REF][START_REF] Kuninaga | DNA base sequence homology in Rhizoctonia solani Kuhn IV. Genetic[END_REF][START_REF] Carling | Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani[END_REF].

The known strains of R. solani can be classified into at least 13 AGs, but the classification is not strictly fixed as some 'bridging strains' are able to anastomose with strains of at least two AGs [START_REF] Parmeter | Rhizoctonia solani : biology and pathology[END_REF][START_REF] Carling | Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani[END_REF][START_REF] Sharon | Classification of Rhizoctonia spp. using rDNA-ITS sequence analysis supports the genetic basis of the classical anastomosis grouping[END_REF]. Each AG is either host specific or with a wide host range [START_REF] Ogoshi | Ecology and Pathogenicity of Anastomosis and Intraspecific Groups of Rhizoctonia-Solani Kuhn[END_REF][START_REF] Carling | Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani[END_REF]. For example, AG 2 is associated with diverse host plants but AG 8 is more specifically associated with cereals. AG 3 is divided into two genetically different subgroups: AG 3 PT which is associated with potatoes, and AG 3 TB, associated with tobacco [START_REF] Kuninaga | Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3[END_REF][START_REF] Woodhall | Infection of potato by Rhizoctonia solani: effect of anastomosis group[END_REF]. R. solani AG 3 PT reduces tuber quality by producing sclerotia (black scurf) on progeny potato tubers. The pathogen can also infect underground organs (stems, stolons and roots) which affect crop yield (tuber size and number) (El [START_REF] Bakali | Black scurf of potato[END_REF]. Beside those typical symptoms, R. solani is associated with several types of blemishes on potato tubers (Fiers et al., 2010). Other AGs of R. solani are sometimes considered as potential pathogens of potato in France and in the United Kingdom, although the Koch's postulates have still not been assessed [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF]Woodhall et al., 2008).

The AG differentiation is traditionally carried out by pairing unknown isolates with reference strains and by identification of the hyphal anastomosis reaction [START_REF] Carling | Grouping in Rhizoctonia solani by hyphal anastomosis reaction[END_REF][START_REF] Guillemaut | Typing of anastomosis groups of Rhizoctonia solani by restriction analysis of ribosomal DNA[END_REF]. However, this method is time consuming, needs experienced eyes and does not show the diversity among AGs. Sequencing of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) is now a more convenient and rapid method to differentiate R. solani AG. However this region is less variable for detection of differences between isolates of the same AG [START_REF] Kuninaga | Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3[END_REF][START_REF] Woodhall | Characterization of Rhizoctonia solani from potato in Great Britain[END_REF]Lehtonen et al., 2008b). The gene tef-1α encoding the translation elongation factor 1α and more variable than the ITS region has already proved its usefulness to reveal genetic variability within fungal genera, including Fusarium [START_REF] Geiser | FUSARIUM-ID v. 1.0: A DNA sequence database for identifying Fusarium[END_REF] and Trichoderma (Anees et al., 2010b). This genetic marker could be useful to reflect polymorphism both between and within AG of R. solani. In addition to targeted DNA markers, strategies based on amplified fragment length polymorphism (AFLP) allow a large number of DNA fragments to be screened in order to assess intraspecific variability to differentiate closely related isolates and reveal clonal lineages [START_REF] Mcdonald | The population genetics of fungi: Tools and techniques[END_REF].

The aim of this study was to characterize the genetic diversity of isolates of R. solani collected from the different potato-growing areas in France. The genetic variability between and within AG was evaluated using ITS and tef-1α sequencing, together with AFLP analysis.

Material and methods

A collection of 73 isolates of R. solani was collected from potato tubers of several varieties and from 9 different French departments producing potato in 2006 and 2007 (TABLE 18). The potato tubers were affected by various superficial blemishes (Fiers et al., 2010). In addition, 31 isolates of R. solani from other countries were analyzed: 6 from Finland, 1 from Germany, 1 from Japan, 4 from Morocco, 1 from the Netherlands, 2 from Poland, 1 from Spain, 12 from Switzerland, and 3 from the United Kingdom (Table 5.1).

DNA of all the fungal isolates was extracted from freeze dried powder of mycelium using the DNeasy plant mini kit according to the protocol previously described (Fiers et al., 2010). For each fungal isolate, the ITS region of the rDNA and part of the tef-1α gene were amplified by PCR. ITS PCR was performed with the primers ITS1-F (CTTGGTCATTTAGAGGAAGTAA) and ITS4 (TCCTCCGCTTATTGATATGC) [START_REF] White | Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[END_REF][START_REF] Gardes | ITS primers with enhanced specificity for Basidiomycetes -Application to the identification of mycorrhizae and rusts[END_REF] ITS and tef-1α PCR products were sequenced by Beckman Coulters Genomics (Takeley, UK) using primers ITS1-F and ITS4 and EF1-645F and EF1-1190R, respectively. For each PCR product, sequences from both strands were assembled to produce a consensus sequence using the software SeqMan (DNASTAR Lasergene, GATC Biotech SARL, Marseille, France). The identities of the sequences were determined using BLAST analyses from the National Center for Biotechnology Information (NCBI) available on line. For each DNA region, sequences were aligned using ClustalX (Thompson et al., 1997). The multiple sequence alignments were carried out with PHYLO-WIN [START_REF] Galtier | SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny[END_REF] using the Kimura's two parameters distance model [START_REF] Kimura | A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF] and the neighbor-joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The topology of the resulting tree was tested by bootstrapping with 1000 resamplings of the data. Phylogenetic trees were drawn using the NJPLOT program [START_REF] Perrière | www-Query: an on line retrieval system for biological sequence banks[END_REF].

The digestion and ligation of 125 ng of extracted DNA were carried out according to the manufacturer's specifications of the AFLP Core Reagent kit (Invitrogen) [START_REF] Vos | AFLP -A new technique for DNA-fingerprinting[END_REF]. The ligation products were pre-amplified by PCR. PCR amplifications were performed in a final volume of 25.5 µL by mixing 2.5 µL of DNA with 0.3 µM of each primer E-0 (GACTGCGTACCAATTC) and M-0 (GATGAGTCCTGAGTAA), 212 µM of dNTP, 2.5 U of Taq DNA polymerase (Q-Biogen, Evry, France) and PCR reaction buffer. Amplification was conducted in a thermal cycler GeneAmp PCR system 9600 (Perkin Elmer Applied Biosystems, Foster City, Calif.) with 20 cycles of 30 s at 94 C, performed on a 41 cm polyacrylamide gel (Li-Cor, Germany) with 6.5 % acrylamide KB (Li-Cor), during 10h30 at 1 500 V for a resolution of the fragments from 50 to 700 bp. A size standard (50-700 bp sizing standard, Li-Cor) and a strain of reference for which the profile is known, were added on each gel in at least five wells. A picture of each gel was taken. For each strain, the analysis was performed in duplicates from independent DNA extracts.

1
Li-Cor gel pictures were analyzed with ONE-Dscan software (Scanalytics BD Biosciences-Bioimaging, version 2.05), measuring the band sizes of the 115 most intense bands between 100 and 500 bp for each profile. Categories grouping fragments, whose weight differed by less than 1.5 bp, were created with LisAFLP program [START_REF] Mougel | A mathematical method for determining genome divergence and species delineation using AFLP[END_REF]. The LecPCR application (ADE-4 v. 2001Thioulouse et al., 1997) was then applied to the data to transform the fragment weights matrix into a binary matrix. The binary matrix indicated the absence (0) or presence (1) of each fragment for each profile. Finally, only fragments present in the two repeats performed for each strain were included in the analysis. Genetic relationships between each profile was estimated using the Nei and Li similarity index [START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF] and bootstrap values corresponding to the appearance frequency of branches in 1000 data permutations was calculated with Treecon software (Vandepeer and Dewachter, 1994 version 1.3b). The similarity matrix was represented by a dendrogram with the UPGMA (unweighted pair grouping method with arithmetic mean) algorithm [START_REF] Sneath | Numerical taxonomy. The principles and practice of numerical classification (A Series of books in biology)[END_REF].

Results

PCR amplification of the ITS region with the primers ITS1-F and ITS 4 gave a single product of approximately 700 bp for each isolate. The ITS1, 5.8S and the ITS2 regions were sequenced for 100 isolates (Table 5.1). For all isolates, BLAST analyses allowed to determine the corresponding AG on the basis of 99 to 100 % sequence similarity with corresponding sequences. Among the 73 French isolates, 60 were identified as R. solani AG 3 PT, the remaining were AG 2-1 (8 isolates) and AG 5 (5 isolates). A total of 64 polymorphic sites was identify in ITS1 but only 24 polymorphic sites in ITS2. Among the large number of AG 3 PT isolates analyzed, 10 and 3 variable sites were observed in ITS1 and ITS2, respectively (Figure 5.1).

Heterogeneity within isolates was observed for 48 out of the 100 isolates. This heterogeneity was observed as two overlapping peaks at the same position in the electrophoregram, indicating that multiple ITS types might exist within isolates.

Among the AG 3 PT sequences, this heterogeneity within isolates was observed at all the 13 variable sites identified within the AG. Conversely, only 2 and 4 sites were identified to be polymorphic within isolates among AG 2-1 and AG 5 sequences; however the number of sequences was also lower with 11 and 6 isolates from AG 2-1 and AG 5, respectively.

All the sequences were assigned to a type number from m1 to m10 for the 10 monomorphic sequences observed and p1 to p29 for the 29 sequences with polymorphic sites, coded as m and p followed by a number designate the different monomorphic and polymorphic sequence types, respectively (Figure 5.1).

Comparisons of all sequences allowed identifying 39 ITS types. Four types were identified within AG 2-1 (type m1, m2, p1, p2), 33 within AG 3 PT (type m3 -m9 and type p3 -p28) and 2 within AG 5 (type m10 and p29) (Table 5.1).

The 52 sequences without any polymorphic site were compared in a phylogenetic tree (Figure 5.2). The three AG were genetically different as they appeared on three distinct branches of the tree with significant bootstrap values above 99 %. Isolates belonging to AG 5 were more similar to isolates from AG 2-1 than to isolates from AG 3 PT (Figure 5. The PCR amplification of the tef-1α gene with the primers EF1-645F and EF1-1190R gave a single product of approximately 400 bp. The tef-1α gene was sequenced for 86 isolates (Table 5.1). In BLAST analyses, all sequences matched a sequence of the same AG of R. solani than the AG identified through ITS sequence, with a similarity of 93 to 100 %. Among the 86 sequenced isolates 13 belonged to AG 2-1, 68 to AG 3 PT and 5 to AG 5.

A total of 60 variable sites were identified among tef-1α sequences (Figure 5.3).

Among the 68 sequences of AG 3 PT, 20 variable sites were depicted. Among AG 2-1 and AG 5 isolates, 42 and 8 variable sites were observed, respectively. Like in the ITS sequences, some heterogeneity within isolates was observed for 64 isolates out of the 86, such as polymorphic sites, which revealed polymorphism within the tef-1α gene in the same individual. This heterogeneity was observed at 17, 14 and 5 variable sites observed within AG 3 PT, AG 2-1 and AG 5 sequences, respectively (Figure 5.3). According to the variations in the tef-1α sequences, 37 different elongation factor types were identified: 7 within AG 2-1 (types m1 -m3 and types p1-p4), 28 within AG 3 PT (type m4 -m7 and type p5 -p28) and 2 within AG 5 (type p29 and p30) (Table 5.1).

A phylogenetic tree was built up on the basis of the 23 tef-1α sequences without any Combined data from ITS sequences and tef-1α sequences revealed a total of 59 twolocus sequence types among the 82 isolates for which both loci were analyzed (Table 5.1). Among the 59 types, only 10 two-locus sequence types were monomorphic for both loci. Eighty-nine isolates were analyzed by the AFLP method. The number of bands analyzed (from 100 to 500 bp) was 60 -102 per profile, with an average of 78 bands, for a total of 254 useful markers. The phylogenetic tree of AFLP profiles showed three groups supported by significant bootstrap values above 96 %, corresponding to the three AGs (Figure 5.5). Regarding AFLP profiles, the AG 5 isolates were more related to the AG 3 PT isolates than to the AG 2-1 isolates, as it was shown on the tree of tef-1α sequences. A significant diversity was observed within each AG with so many AFLP profiles as isolates, but no particular clusters could be identified within AGs. Among AG 3 PT isolates, the French isolates were spread all over the tree. At a smaller scale, isolates originating from the same French Department (Finistère) were also found all along the tree, showing no direct relation of the diversity observed with the geographic origin.

Discussion

In this study, 104 isolates of R. solani, all sampled from blemished potato tubers, were characterized by sequencing of the ITS region and part of the tef-1α gene and by AFLP fingerprinting. The 73 French isolates were representative of all area producing potatoes in France; they were compared with 31 isolates originating from other countries. AG 3 PT was found to be predominant, including 82 % of the French isolates. The remaining isolates belonged to AG 2-1 and AG 5. R. solani isolates belonging to AG 3 are frequently isolated from potato tubers and are known to be pathogens for this crop [START_REF] Kuninaga | Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3[END_REF], but AG 2-1 and AG 5 are more rarely isolated from blemished tubers and their pathogenicity has still not been demonstrated [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Woodhall | Infection of potato by Rhizoctonia solani: effect of anastomosis group[END_REF]Fiers et al., 2010).

Several distinct types of the ITS region and of the tef-1α gene were identified among the R. solani isolates and surprisingly, in the majority of isolates the coexistence of multiple types was observed. Polymorphism in the ITS region and tef-1α gene within isolates can be due to the existence of different ribosomal DNA units within the same nucleus or different sequences in different nuclei. This hypothesis could be confirmed after cloning [START_REF] Justesen | Genetic diversity in potato field populations of Thanatephorus cucumeris AG-3, revealed by ITS polymorphism and RAPD markers[END_REF]. This heterogeneity results from mutations that would be fixed. Several ITS sequence types within the same individual were also identified for other fungi such as Sclerotium spp. [START_REF] Almeida | Genotypic diversity among Brazilian isolates of Sclerotium rolfsii[END_REF], Ascochyta spp. [START_REF] Fatehi | Detection of multiple rRNA-ITS regions in isolates of Ascochyta[END_REF] and Botrytis spp. [START_REF] Yohalem | Taxonomic and nomenclatural clarification of the onion neck rotting Botrytis species[END_REF]. Among R. solani populations, ITS polymorphism was studied within AG 2-1 and AG 3 [START_REF] Justesen | Genetic diversity in potato field populations of Thanatephorus cucumeris AG-3, revealed by ITS polymorphism and RAPD markers[END_REF][START_REF] Pannecoucque | Detection of rDNA ITS polymorphism in Rhizoctonia solani AG 2-1 isolates[END_REF]. The same 10 within-isolates polymorphic sites previously identified by [START_REF] Justesen | Genetic diversity in potato field populations of Thanatephorus cucumeris AG-3, revealed by ITS polymorphism and RAPD markers[END_REF] among ITS1 sequences of R. solani AG 3 were also detected in our study in addition to others sites. The finding of two different ITS types or elongation factor types within the same isolate and the possible association with two different nuclear types is consistent with the heterokaryotic nature of R. solani which has been confirmed by other DNA marker methods that revealed heterozygosity in isolates of R. solani AG 3 [START_REF] Ceresini | Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci[END_REF].

Both loci, ITS region and tef-1α gene, showed a considerable level of variability among R. solani isolates. Concerning the ITS region, we found that ITS1 sequences were much more variable than ITS2 sequences, with 24 % and 11 % of variable sites in ITS1 and ITS2, respectively. Our results are in agreement with the ITS variability recently described by [START_REF] Nilsson | Intraspecific ITS Variability in the Kingdom Fungi as Expressed in the International Sequence Databases and Its Implications for Molecular Species Identification[END_REF] among R. solani. The second locus used, tef-1α gene, was found even more polymorphic, with 27 % of variable sites among all three detected AGs of R. solani, and 9 % of variable sites among AG 3 PT isolates.

However, all this variability could not be illustrated in trees, since dimorphic sites were excluded from the phylogenetic analysis.

The different sequence types obtained for one DNA locus did not match exactly the sequence types obtained for the other locus. However, in both ITS and tef-1α trees, the AG 3 PT branch that diverged from other AG 3 PT sequences corresponded to the same isolates MIAE00354 and MIAE00355.

The analysis of ITS sequences is a wide spread method for specific identifications of fungi and our results indicated that the gene tef-1α showed a greater diversity among AGs of R. solani than the ITS region. This shows the complementarity of sequences from two or more loci in multilocus sequence typing (MLST) approaches. Such MLST strategy becomes widely used to resolve phylogenetic relationships among fungi such as Fusarium [START_REF] Nitschke | Differentiation of Eleven Fusarium spp. Isolated from Sugar Beet, Using Restriction Fragment Analysis of a Polymerase Chain Reaction-Amplified Translation Elongation Factor 1 alpha Gene Fragment[END_REF][START_REF] O'donnell | A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex[END_REF] or Trichoderma [START_REF] Kullnig-Gradinger | Phylogeny and evolution of the genus Trichoderma: a multigene approach[END_REF]Anees et al., 2010b). These recent studies have shown the interest of including tef-1α gene in such studies. However, the use of this gene to resolve genetic diversity within R. solani has not been previously published and only 33 sequences of tef-1α gene of R. solani were available in Genbank before this study. We used primers EF1-645F and EF1-1190R originally described for Alternaria spp. [START_REF] Andersen | A polyphasic approach to the taxonomy of the Alternaria infectoria speciesgroup[END_REF]. We adapted PCR conditions to amplify part of the tef-1α gene from R. solani. Multigene approaches may be useful for more precise molecular identifications of species or AGs, when a unique locus such as ITS does not always provide clear information (Anees et al., 2010b).

ITS and tef-1α types did not show any relationship with the geographical origin of the isolates or the cultivar of origin. Moreover, AFLP data did not show particular structure among the isolates belonging to the same AG. Our findings support the hypothesis that R. solani populations are constantly always evolving following different genetic events. The absence of sporulation of R. solani may limit the dissemination of clonal isolates and may prevent the structuring of the populations.

On the other hand, the diversity of the R. solani populations could be enriched by the spread of the fungal genes taking place within the field and at larger extent, between French departments and between several countries separated by several hundred kilometers. As tubers are exchanged on the international market, seed born inoculum could be the predominant cause of the long distance dispersal of the fungus. Indeed, potato tubers are vegetatively multiplied what increases the risk of fungal transmission through infected seed. Therefore, these newly introduced genomes increase the diversity of the population by bringing in and exchanging genes with the endemic isolates.

Despite the large diversity at the intraspecific level within R. solani, no evident relationship between cultivars and associated populations of R. solani could have been depicted. Since there is no difference of pathogenicity between isolates of R. solani (Fiers et al., 2010), it seems that the severity of the disease depends mainly on environmental conditions and perhaps on the behavior of the cultivar. This aspect of the plant -pathogen interaction was not previously studied in depth.

Our study is the first evidence of genetic variability among R. solani associated with blemished potato tubers in France. However, the genetic diversity of R. solani populations isolated from blemished tubers is neither dependant on the geographical origin of the isolates or on the host cultivar. The lack of population structure suggests a constant evolution within R. solani. Such evolution should be promoted by frequent genetic events, genetic mixing and anthropogenic activities. Further researches are needed to determine the phenotypical characteristics of the isolates in order to set up adapted control strategies against R. solani.

Introduction

Potato tuber quality is a predominant aspect of the potato production but can be altered by numerous damages essentially due to pathogenic attacks. Indeed, about 40 different diseases caused by fungi, bacteria, nematodes or viruses may damage potato crop all around the world. Quality of potato tubers may also be changed by environmental conditions due to natural phenomena -high soil temperature is responsible for heat necrosis in vascular ring tissues [START_REF] Stevenson | Compendium of potato diseases[END_REF] -or anthropogenic activities -mechanic harvesting and grading induce damages on tubers [START_REF] Peters | Damage of potato tubers, a review[END_REF]. Knowledge about the causes of the different kinds of tuber quality problems is generally proportional to the economical importance of the damages.

Potato blemishes are superficial alterations of the tubers that alter only the upper layers of the periderm (Fiers et al., submitted). Very few studies are available on the subject because blemishes became only recently an important economical issue [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Woodhall | Infection of potato by Rhizoctonia solani: effect of anastomosis group[END_REF]. A few years ago potatoes were not washed before being sold so, the superficial blemishes were hidden by the soil adhering to the tubers and thus, they were not considered as a problem. In the 80's, the habits of consumers began to change and tubers were washed to present "clean" products on the store shelves. Since then, blemished tubers were downgraded and the economical losses became more and more important. Although blemishes were observed since a long time [START_REF] Peters | Elephant hide of potato[END_REF][START_REF] Hart | Fish scale or elephant hide on potatoes[END_REF], researches to determine their causes only began in the 2000's. Several hypotheses were proposed about the origin of the blemishes. The soil-borne fungus Rhizoctonia solani was supposed to be predominantly involved in the occurrence of some superficial blemishes but not all of them [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Woodhall | Infection of potato by Rhizoctonia solani: effect of anastomosis group[END_REF]. Environmental stresses such as high soil temperature, proximity of decaying organic matter, excessive moisture etc.

were also assumed to be responsible for the visual alterations of the tubers [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Sexton | Some thoughts on elephant hide[END_REF]Selman et al., 2008) but a clear demonstration of the respective involvement of biotic and non biotic factors is still lacking. In the previous chapters, we tested the hypothesis of a pathogenic origin and we demonstrated that indeed, R. solani causes black scurf or sclerotia attachment to the tubers but none of the 283 other soil-borne microorganisms associated to the various blemishes found on potato tubers were responsible for the appearance of those blemishes (Fiers et al., 2010).

The second hypothesis about non biotic factors will be addressed in this part through a specific focus on the environmental conditions or cultural practices that can cause the occurrence of potato blemishes. Testing individually every factor of climate, soil characteristics and crop management plan would have been very tedious and not really representative of the field conditions. Thus, in order to be as close as possible to the real crop conditions, we decided to realize a survey with farmers to determine favourable and unfavourable environmental parameters to the development of tuber blemishes. Moreover, we performed statistical analyses to depict any putative relationship between the potato cultivar and the occurrence of potato blemishes. Indeed, it has been shown that the same biotic or/and abiotic factor can induce blemishes of various aspects and that potato cultivars differ in their susceptibility to the blemishes [START_REF] Lutomirska | The influence of cultivar and meteorological factors during vegetation season on black scurf of potato tubers[END_REF][START_REF] Wanner | Aggressiveness of Streptomyces on Four Potato Cultivars and Implications for Common Scab Resistance Breeding[END_REF].

Material and methods

The survey was conducted in the main French potato production basins in the North and the West of France with 45 potato producers chosen according to the amount of blemished tubers they harvested in 2007. A semi structured questionnaire was send to the farmers. They were asked to give data on their cultural practices and the soil characteristics of the field. The survey takes into account soil characteristics such as soil depth, pH value, cation exchange capacity (CEC) value, organic matter rate, texture, and structure (Figure 6.1). Soil structure was considered as "good" when the soil was aerated, fragmented with a good filtering coefficient. A "bad" soil structure was characterized by a compact and asphyxiated soil. Cultural practices were evaluated by comparing delays between the dates of planting and dehaulming and between the date of dehaulming and harvesting, rotation length, preceding crops, irrigation and drainage during the potato crop. Meteorological data were also collected. The amounts of water received by the crop, i.e. precipitations + irrigation, and the average temperature during all the growing season were taken into account.

For each parameter, answers were distributed in 2 to 4 categories that were related to the occurrence of blemishes with a Chi square (Khi 2 ) test (α = 0.05).

In addition, blemished potato tubers were collected in 2006 and 2007 in the same French areas and in Switzerland, Italy (Sicilia), and Morocco. All the surveyed producers provided samples collected in 2007. Each sample was collected from a single field. In 2006 and 2007, a collection of 92 and 122 samples of tubers presenting blemishes from 29 and 45 different cultivars respectively was set up. A picture of each tuber was taken; blemishes were classified in 4 classes (sclerotia, atypical corky blemishes, potato scabs and enlarged lenticels) and they were scored according to the French notation scale edited by the French official service of control and certification (SOC) (GNIS and SOC, 2005). For each year, a test of conformity with the binomial law was performed (α = 0.05) in order to test the conformity of the occurrence of the blemishes with the theoretical probability. A significant high frequency of occurrence of a given blemish on a cultivar would reveal a specific sensibility of the cultivar to that blemish. The dependence between the lines and the columns of the contingency tables was tested with a Chi square test. The relationships between the potato cultivars and the blemishes were represented by factorial correspondence analysis (FCA). The statistical tests were performed with the XLStat Pro 7.5 software (Addinsoft, version 7.5). More than 53 % of the farmers answered the survey. The average rate of answers to every question was 67.7 %. Despite, the good answering rate of the survey, the total number of fulfilled questionnaires was too weak to reach a pertinent statistical signification. Nevertheless, interesting tendencies could be shown.

Results

Among

Average surveyed fields had a soil depth between 60 and 90 cm, a pH value between 6 and 7, the CEC value was under 15 meq / 100 g of soil and the organic matter rate was between 2 and 4 %. Soils were generally loamy, with a good structure. Delay between planting and dehaulming was commonly longer than 100 days and delay between dehaulming and harvesting was shorter than 40 days. There were as many rotations longer than 5 years than rotations shorter than 5 years and cereals were the most frequent crops included in the rotation. In general, no intercrop was used and there were neither drainage nor irrigation (Figure 6.1).

Results showed significant effects of pH and delay between dehaulming and harvesting (α = 0.05). A pH value between 6 and 7 increased the occurrence of atypical corky blemishes and a period longer than 40 days between dehaulming and harvesting favoured the development of potato scabs symptoms (Figure 6.1). Results nearby the significant threshold indicated that a rotation length between 3 and 5 years increased the risk of sclerotia occurrence whereas a rotation longer than 5 years decreased the risk. About 70 % of the farmers grew cereals before potatoes in the rotation without any positive or negative effect on the occurrence of the blemishes. Climatic conditions, especially pluviometry below 350 mm had also an impact on blemishes by enhancing the occurrence of sclerotia all along the growing season.

Concerning the cultivars-blemishes relationships, data of 2006 and 2007 were not treated together, because the two sampling years have had very different climatic conditions in France. In 2006, the temperatures were exceptionally high (+ 1 or + 2°C higher than seasonal averages) and the pluviometry was normal. In 2007, the cropping season was fresh and very humid. The majority of the sample comes from the North West of France where the precipitations in 2007 were 2 to 3 times higher than the seasonal average (Météo France). The Chi-square test showed that the lines and the columns were significantly independent (α = 0.05) for the two years.

The FCA represented 88.59 % of the total variability in 2006 (F1: 50.10 % and F2: 38.49 %, Figure 6.2) and 87.14 % of the variability in 2007 (F1: 49.97 % and F2: 37.17 %, Figure 6.3).

In 2006, the FCA showed that the cultivars Adriana, Chérie and Désirée were distinct from the other cultivars. Moreover, the cultivar Chérie was significantly associated with potato scabs (coefficient = 0.001). The tests of conformity with the binomial law indicated that Amandine and Anoe were displaying significantly more enlarged lenticels than the other cultivars, with coefficient values of 0.020 and 0.031 respectively (α = 0.05). The FCA showed also that cultivars Spunta was significantly more susceptible to atypical corky blemishes than the other cultivars with a coefficient of 0.039. Other cultivars seem not to be particularly susceptible to a specific blemish (Figure 6.2).

Discussion

In Brittany, our major sampling place, the soils are a bit acidic with an average of 6.3 compared with the range of values (from 5 to > 8) displayed by agricultural soils in France [START_REF] Arrouays | RMQS -Outil cartographique de la Base de données de analyses de terre[END_REF]. Almost 30 % of the surveyed fields belonged to the category of pH between 6 and 7, and those pH values seemed to be favourable to the occurrence of atypical corky blemishes. Nevertheless, pH is a complex factor that affects several other parameters. It determines the availability of soil nutrients and elements contents that can impact the potato sanitary status [START_REF] Stengel | [END_REF]. Indeed a deficiency or an excess of one of the macronutrients (nitrogen, phosphorus, potassium) or micronutrients (iron, manganese, aluminium, etc.) is a source of stress for the plant and it can result into blemish occurrence on tubers (Zorn, 1995;[START_REF] Sarkar | In vitro characterization of manganese toxicity in relation to phosphorus nutrition in potato (Solarium tuberosum L.)[END_REF]). The element rates of each surveyed soils should be taken into account to determine the real role played by pH in the appearance of blemishes.

On the other hand, we showed that a period of time between dehaulming and harvesting longer than 40 days was harmful for potato as the severity of potato scabs developing on tubers increased. This confirms the results obtained previously indicating that the quality of ware potatoes can be controlled by adjusting planting and harvesting dates [START_REF] Sikka | Factors influencing quality of ware potatoes[END_REF]. Minimum disease severity is generally recorded when tubers are directly harvested along with foliage. However, with delay in date of haulm cut, there is gradual and significant increase in tuber yield. As long as tubers are attached to the green plants, the photosynthates keep on accumulating in tubers, i.e. the biological sink. Once the green top is removed the tubers in soil attached to stolons and roots, are likely to be more liable to various infections including that of Streptomyces spp. and R. solani [START_REF] Sangeeta | Effect of haulm cutting and harvesting time on Rhizoctonia solani of potato[END_REF].

Time interval between dehaulming and harvesting has to be long enough to allow for the convenient growth of the tubers, but also short enough to avoid the formation of potato scab. The dehaulming -harvesting delay has to be calculated according to the pressure exerted by Streptomyces spp. inoculum on the crop. Concerning the rotation length, it is known since a long time that long rotations allow the decrease or the eradication of pathogen inoculums [START_REF] Peters | Developing diseasesuppressive soils through crop rotation and tillage management practices[END_REF]. The longer the time between two potato crops, the less the concentration of R. solani inoculum, the less severe black scurf will be. Rotation length is an important parameter to take into account as well as the rotation composition. Cereals in the rotation did not increase nor decrease the appearance of the blemishes. Consequently this crop can be integrated into the rotation without risk.

Our results did not show any effects of high soil temperature, heterogeneous distribution of the organic matter in soil and excessive moisture on the occurrence of blemishes, as it has been previously suggested [START_REF] Stevenson | Compendium of potato diseases[END_REF][START_REF] Sexton | Some thoughts on elephant hide[END_REF]Selman et al., 2008). However, we showed that excessive moisture decreased the occurrence of sclerotia. In literature, papers on this subject are rather contradictory. Some authors claimed that black scurf is favoured by high soil moisture [START_REF] Lakra | Relationship between irrigation levels and black scurf development in potato crop[END_REF][START_REF] Bakali | Black scurf of potato[END_REF], others showed that irrigation decreased black scurf (Hide et al., 1994), or that there was no influence of rainfall on sclerotia occurrence [START_REF] Lutomirska | The influence of cultivar and meteorological factors during vegetation season on black scurf of potato tubers[END_REF]. Nevertheless, rainfall and irrigation are not systematically correlated with high soil moisture content. Indeed, well drained soils avoid overcoming the water holding capacity of the fields even after strong rains or too important irrigation.

Since, the number of answered questionnaires was low, this study can not provide irrefutable information or concrete advises on favourable or unfavourable environmental traits and cultural practices for the management of healthy potato crop. Nevertheless, our results are good indicators to highlight the points that need to be watched in order to avoid blemish occurrence. Some factors are favourable to some blemishes and unfavourable to others. Therefore, a large scale survey should be undergone among the producers with some more specific and precise questions issuing from the present survey such as soil pH, soil texture, organic matter (soil content and dispersion in the soil), delay around haulm destruction, rotation length and previous crops, moisture (irrigation, pluviometry and drainage) and soil temperatures.

The choice of the potato cultivar is a major cultural practice that allows adapting the crop to the market expectations, to the environmental conditions and to the sanitary risks. This work aimed at established relationships if any between potato cultivars and superficial blemishes. We could have studied the relationships between the cultivars and the geographical origins of the samples or between the blemishes and the geographical origins, but it would have been biased since some cultivars are more frequently cropped in some areas because of the climate and the way the potatoes will be used (seeds, ware, or processing).

Despite the different climatic conditions in the two years, the 2 AFC showed that the susceptibility of the cultivars to the blemishes was rather constant in 2006 and 2007.

This confirms previous results claiming that the relative susceptibility of a cultivar to a given blemish remains approximately the same whatever the environmental conditions. For example, a susceptible cultivar will always have more severe blemishes or symptoms than a resistant cultivar whatever the climatic conditions (INRA and Agrocampus, 2006;2007;2008). Some cultivars were represented by only one sample whereas other cultivars were represented by at most 28 samples. Indeed, some cultivars are more frequently used than others. The results concerning cultivars represented by one or two samples can not be considered as representative of the cultivar's behaviour.

Our results also showed that the cultivars with a common genetic background are liable to the same blemishes, what is in agreement with previous studies [START_REF] Bozec | Evaluation au champ de la résistance de la pomme de terre aux gales communes[END_REF].

New potato cultivar assessment takes into account several crop and tuber characteristics, especially disease resistance of the cultivars. However the aggressiveness of each pathogen is expressed according to the response of the cultivar and the environmental conditions. Resistance assessments frequently reveal that common scab is the only tested disease among the superficial blemishes affecting the potato tuber. Indeed, Solanum tuberosum -Streptomyces spp.

relationship is one of the few potato parasitic complexes that gather available data at the cultivar level, although the behaviour of the potato phenotypes against Streptomyces spp. still remains environment-dependant [START_REF] Pasco | Resistance of potato genotypes to common and netted scab-causing species of Streptomyces[END_REF][START_REF] Anonymous | Actualité semences pommes de terre[END_REF]. The description and the awareness about atypical superficial blemishes vary according to countries and remain frequently unclear. That is one of the reasons why official assessments for registration of new potato cultivars do not integrate cultivar behaviour to atypical blemishes into the evaluation criteria yet, even if some official assessments were recently validated in France about pitted vs. netted scab [START_REF] Pasco | Resistance of potato genotypes to common and netted scab-causing species of Streptomyces[END_REF] and in Switzerland, about tuber deformations due to R.

solani [START_REF] Reust | Rapport: essais principaux nouvelles variétés de pommes de terre 2007: Agroscope ACW Changin-Wädenswil -Agroscope ART Reckenholz-Tänikon[END_REF].

In the previous chapters, we concluded that no causality links could be established between the microorganisms associated to blemishes and the occurrence of the blemishes. In this chapter, we are inclined to think that some non biotic factors including climate and agricultural practices are involved in the appearance of the blemishes and also that susceptible cultivars could exacerbate this appearance.

Therefore, it should be possible in the future to overcome the problem by innovative management of the production although it is still very difficult to adopt a course of action avoiding all the problems.

General discussion

In the global current context of massive demographic increase, potatoes are desirable to feed the global populations and to contribute to their economical and social development. Obviously, the issues and constrains of potato production are not the same according to the country. Developing countries aim at supplying enough staple potato production to meet increasing food demands, whereas developed countries have to provide good quality products matching with the consumers' expectations. Among other factors, the economical development of fresh potato producers in developed countries depends on the visual quality of the tubers. Indeed, the losses due to the downgrading or rejecting of blemished tubers hamper the proper evolution of the fresh potato industry. Consumers are more and more demanding concerning food quality. The perception of fresh fruits and vegetables has changed since populations became more urban and are less familiar with the production systems. Consumers who were more familiar with the geographical origin of fruit and vegetable production mentioned non-sensory attributes -mode of production, variety -more often than those with less contact, who, in contrary mentioned sensory attributes -aspect, odour, touch - [START_REF] Peneau | Freshness of fruits and vegetables: concept and perception Thesis[END_REF]. Concerning fresh potatoes, blemished tubers are not well perceived by the consumers, even if they do not affect the nutritional quality of the product. In the long term, efforts should be made to make the consumers aware of the quality of fresh potatoes despite some visual blemishes, but at the present time solutions to avoid the occurrence of blemishes are needed. The determination of their causes is a crucial point that has to blemishes that can be observed on tuber surface (chapter 2). Some atypical potato blemishes, such as polygonal lesions, were observed since a long time and were named "elephant hide", especially in USA. The first paper we found in the literature about this issue dates from 1966 [START_REF] Peters | Elephant hide of potato[END_REF]. Other authors approached briefly the subject in the 70's [START_REF] Hart | Fish scale or elephant hide on potatoes[END_REF]. They mentioned some observations and suggested few probable causes but no actual study was carried out. In the 2000's, few papers dealing with blemishes were recorded. Two of them studied more in depth the implication of Rhizoctonia solani in the occurrence of the atypical blemishes [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Woodhall | Infection of potato by Rhizoctonia solani: effect of anastomosis group[END_REF]. Nevertheless, the overview of those rare previous studies showed the heterogeneity and confusion in the different names used for the blemishes. Seeing the importance of the problem for the potato community, it was necessary to examine the matter in more detail, with experts from the seed, ware and processed potato sector, researchers and breeders, and the first thing to do was to harmonise the nomenclature and the classification of Those variations allowed collecting very diverse types of blemishes during the two years.

Two causes were possible to explain the appearance of the blemishes. Either they were due to one or several pathogens or they resulted from a reaction of the plant to unfavourable environmental conditions [START_REF] Agrios | Plant pathology[END_REF]. Our first goal was to make an exhaustive isolation and identification of the microorganisms living at the surface of the tubers (chapter 3). However, the scope of the study constrained us to target some microorganisms. A large diversity of fungi was identified but the method we used allowed us isolating only cultivable fungi. Concerning bacteria, we targeted

Streptomyces, known to be implicated in several potato diseases especially netted scab that causes blemishes, very similar to the atypical blemishes. The pathogenicity of the Streptomyces isolates and the fungi the most frequently isolated from the surface of the blemished tubers was tested in bioassays with infested soil. This classical technique aimed at favouring the development of a specific microorganism in the soil and at fulfilling the Koch's postulates. Koch's postulates fulfilment is the only known method to prove rigorously the pathogenicity of a microorganism [START_REF] Rapilly | Champignons des plantes: les premiers agents pathogènes reconnus dans l'histoire des sciences[END_REF]. The fulfilment of the four postulates was not easy, especially as the bioassays were conducted in a non sterile environment. Aerial microorganisms could develop in the soil and establish interactions with the tested microorganism or the tuber. Some microbial interactions were tested by inoculating tubers with combinations of two microorganisms. In all those tests, blemishes were produced showing that we succeeded in creating favourable conditions to blemish formation, but apart from R.

solani, there was no relationship between an inoculated microorganism and the occurrence of the blemishes. We enlarged the domain of investigations from the surface of the tuber to the surrounding geocaulosphere and from the artificial conditions of the greenhouse to the natural conditions of a field in which superficial blemishes were previously regularly observed (chapter 4). This approach based on the analysis of the microbial structure in the soil close to the altered and the non altered potato was used to overcome the practical limitation of the approach based on isolation. Such an approach was already successfully used to identify the fungal populations of Trichoderma spp. involved in the dynamics of infectious patches caused by R. solani in sugar beet crop (Anees et al., 2010a). Unfortunately, no noticeable peak (TRF) which could have indicated the relative abundance of a microbial group to identify in the geocaulosphere of blemished tubers compared to the one of healthy tubers was recorded, apart, once again, the TRF corresponding to the R. solani group. Although modifications of the structures of the fungal and bacterial communities were observed at the different sampling dates and in the vicinity of blemished tubers, we could not conclude that these modifications were responsible for the appearance of the blemishes or if, at the contrary, the blemished potato had specific exudates that could change the population ratios among the microbial communities. Anyway, this non-a priori approach gave the same results as the Pasteurian one based on the fulfilment of Koch postulates. No causality between the presence of microorganisms and the appearance of atypical blemishes was found apart in the case of R. solani. We concluded that R. solani was actually responsible for sclerotia but the other blemishes were not of pathogenic origin.

We therefore focused on this pathogen to know more about its diversity, although it is not really involved in atypical blemishes but it is in quite typical and already described blemishes called sclerotia. R. solani is a very common soil-borne pathogen with a large diversity of host plants and causing various diseases under diverse environmental conditions. Julius Kühn observed this fungus for the first time on potato in 1858. R. solani can cause seed decay, damping off, root rot or stem canker on bean, cotton, cabbage, sugar beet, alfalfa, tomato, potato etc [START_REF] Parmeter | Rhizoctonia solani : biology and pathology[END_REF]. In fact, there is a relative host specificity of the different anastomosis groups (AG) of R. solani. On potato, we isolated especially AG 3, AG 2-1 and AG 5. AG 3 and more precisely the subgroup associated with potato, AG 3 PT is known to cause black scurf and stem canker on potato tubers [START_REF] Kuninaga | Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3[END_REF]. AG 2-1 and AG 5 were isolated at lesser extent than AG 3 but they are supposed to be implicated in the appearance of atypical blemishes. Nevertheless, Koch's postulates were not fulfilled for these two AGs neither in chapter 3 nor in other studies [START_REF] Campion | Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France[END_REF][START_REF] Woodhall | Infection of potato by Rhizoctonia solani: effect of anastomosis group[END_REF]. It is very difficult to eradicate R. solani once the pathogen is in the field. The fungus may cause up to 30 % of losses in the potato production [START_REF] Banville | Yield Losses and Damage to Potato Plants Caused by Rhizoctonia-Solani Kuhn[END_REF]. Moreover, chemical treatments against plant diseases are more and more limited because of the current regulations for the protection of the environment all over the world. Many chemicals previously used are now forbidden.

New control techniques are studied; they include unfavourable cultural practices for the pathogen (chapter 6), sustainable chemical treatments and biological control [START_REF] Wilson | Biological and chemical control and their combined use to control different stages of the Rhizoctonia disease complex on potato through the growing season[END_REF]. Chapter 6 of this thesis mentioned that abundant irrigation and rotation longer than 5 years could decrease the soil inoculum of R. solani. On the other hand, microbial cocktails, growth hormones and several other stimulators of plant defence response are tested [START_REF] Mahmoud | Management of brown rot disease of potato[END_REF]. Those new control methods are currently tested by Végénov-Bretagne Biotechnologie Végétale (BBV) to assess their efficacy against potato blemishes. The application of such control methods implies a deep knowledge of the epidemiology and the ecology of the pathogen.

Even if R. solani has been studied for more than 150 years, uncertainties remain concerning its ecology, especially classification and taxonomy. R. solani is a multinucleate fungus. This can introduce many variations in its genome. In chapter 5, the study of the phylogenetic relationships between the French strains of R. solani compared with strains from other countries showed that there was no geographical structure of the R. solani population and no relationship between the host cultivars and the isolates of R. solani. This suggests that frequent genetic events and genetic mixing take place at a large scale all over the world, without any structure of the population could have been drawn, probably because of the limited dissemination of R. solani. The spread of genomes probably occurs because of the global exchange market of seed tubers inducing numerous international transportations of potatoes potentially contaminated by mycelium or sclerotia of R. solani [START_REF] Justesen | Genetic diversity in potato field populations of Thanatephorus cucumeris AG-3, revealed by ITS polymorphism and RAPD markers[END_REF].

The second possible cause of the blemishes was related to stressful environmental conditions. Potato tubers are subterranean stems. They develop in a complex environment which is the soil and which harbours both biotic and abiotic components. The survey we did was quite limited and we discussed, in chapter 6, that this was a restraint preventing us from general and definitive conclusion.

Anyway, we saw that the physico-chemical traits of the soil such as pH and water availability interfere with the plant health status and impact the occurrence of atypical corky blemishes and sclerotia. This leads us to the conclusion that this hypothesis was more likely to bring us with the identification of the causes of the appearance of blemishes than the microbial one, although they are not exclusive mutually. However it is clear and we mentioned it, that a large scale survey should be conducted to provide us with more precise information about the list and the relative importance of the abiotic factors which seemed to be involved in the appearance of blemishes.

Large scale means both geographic areas, climatic conditions and agricultural practices but also a large number of parameters surrounding the plant itself including aerial part, geocaulosphere and rhizosphere of the potato. Biotic factors should also be included as parameters to measure as they interact with abiotic factors and thus might have an indirect role on the occurrence or development of the atypical blemishes. The rhizosphere is a favourable area for microbial development. Carbon, nitrogen, phosphorus, potassium and other micronutriments are released by the plants in the rhizosphere and they are a source of energy for the microorganisms. In return, microorganisms contribute to the plant nutrition by altering minerals and through symbiotic associations such as mycorhization or formation of nodosities [START_REF] Stengel | [END_REF]. Exudates released by the plants modify the microenvironment of the rhizosphere. The soil pH and the chemical balances change increasing the availability of nutrients for the plant. Moreover, those major modifications change the structure of the microbial communities in the rhizosphere. As evocated in chapter 4, the structures of the microbial communities of the potato geocaulosphere evolve in time and according to the phenology of the plant. Similar mechanisms could take place in rhizosphere and geocaulosphere. Indeed, we showed that the microbial communities and especially the bacterial communities were different in the geocaulosphere of healthy plants according the period of time, but they evolve toward an identical structure in the geocaulosphere of blemished tubers. This showed that there are many mutual interactions between plants and soil-borne microorganisms in which the human can interfere in order to improve the agronomic systems.

We showed also in our study that the cultivar has to be chosen according the future use of the progeny tubers and also according to the sanitary risks incurred in a given field: amount of pathogenic inoculum, climatic conditions, soil characteristics, etc.

Choice of unadapted cultivar will increase the sanitary risks. If the weather conditions are also unfavourable, it could be hard to control the risk even with good cultural practices and adapted control methods. Most of European potato cultivars belong to Solanum tuberosum species but about 10 other Solanum species are cultivated in South America and 200 wild species are registered (FAO, 2008). Disease and insect resistance of natural populations, or traditional cultivars developed by Andean farmers, is an important legacy. Genetic selection of potatoes for disease resistance did probably not begin before the end of the late blight epidemic in Ireland between 1845 and 1849. Since then, continuous progresses are made to breed new cultivars resistant to the new diseases and with good intrinsic qualities. Therefore, the susceptibility of the potato plant, i.e. the cultivar, has to be included in the set of biotic and abiotic data to be considered to evaluate the risk of blemish appearance. Some efforts were already done to propose models aiming at evaluating the risk of production losses [START_REF] Savary | Quantification and modeling of crop losses: A review of purposes[END_REF]. Similar efforts should be done using statistical analysis to identify indicators and bioindicators of the beneficial or deleterious environment of the potato during crop production but more precisely during the period during which blemishes appear [START_REF] Janvier | Soil health through soil disease suppression: Which strategy from descriptors to indicators?[END_REF]. Such approach should stimulate the build up of decision support systems to prevent blemishes.

This thesis tackled topical issues at several scales. The project was initially consecutive to repeated requests of technical staff in the fresh-washed potato production to limit the annual non negligible amount of fresh potatoes that were down-graded due to poor visual quality. Because the tuber is also the plant for planting, ie the seed, then a strong demand for high visual quality seeds has challenged seed potato growers to produce very high quality seeds; with the hope that this measure would solve the tuber blemish issue in the following growing season. Seed potato producers in Brittany, together with fresh potato growers have then launched this project in order to find out solutions while respecting the current environmental regulations. This problem concerns also the global agriculture which needs to produce abundant and qualitative food products and has to protect the planet at the same time. Those preoccupations meet the first and the seventh millennium development goals of the United Nations Organisation: End poverty and hunger and ensure environmental sustainability (ONU, 2000). Measures introduced to reach those goals depend largely on our capacity to manage an alternative agriculture with beneficial socioeconomic and environmental impacts. This is the aim of agroecology. Agroecology provides the scientific basis to address the production by a biodiverse agroecosystem able to sponsor its own functioning [START_REF] Altieri | Agroecology: The science of natural resource management for poor farmers in marginal environments[END_REF].

Agroecology initially dealt primarily with crop production and protection aspects, in recent decades new dimensions such as environmental, social, economic, ethical and development issues are becoming relevant [START_REF] Wezel | Agroecology as a science, a movement and a practice. A review[END_REF]. Thus, by studying the causes of the occurrence of potato tuber blemishes at a microscopic scale, this project dealt with quite larger scales that aim at developing global agriculture, improving the socio-economical conditions for farmers and finding sustainable techniques for crop management.

Future prospects

Based on the conclusions we drawn back from our study, and also, as an obvious corollary, on the analysis of the literature we did, I would suggest first a short term practical perspective about a common terminology we should have about potato blemishes.

But I think also that the approaches we used, although they provided a lot of information and partial responses to the original question, were not totally satisfying to the growers as we did not give a clear and definitive answer towards the causality of blemishes, we just provided with promising tracks. That is why I would propose some middle term perspectives about the i) plant-pathogen interactions and plant defence reactions, ii) the definition of indicators of the soil health to built up decision support systems for the growers and iii) an integrated pest management study directly related to the previous proposed perspectives.

Practical perspectives

New nomenclature and classification were suggested in order to harmonize further works on potato blemishes. It has been written in English for a scientific journal which will be available for a limited number of potato scientists and researchers. The value of such nomenclature is to be spread at all levels in the potato industry and among extension staff. Translations in several languages of this nomenclature and publication in technical reviews could allow farmers, technicians as well as engineers or researchers to exchange about blemishes with the same words and the same meanings. This nomenclature could be used in certification shemes to allow the identification and the precise description of tuber blemishes that are still rarely taken into account in the criteria of certification of the tubers.

Research perspectives i) Plant-pathogen interactions and plant defence reactions

In addition to the microbial mechanisms and the environmental conditions we evoked during this study and that may lead to the reduction of the pathogenic attacks, plants interact with their environment, especially the soil environment through the rhizosphere activity. In the case of potato, the soil surrounding tuber is called geocaulosphere and can be compared to the rhizosphere. The impact of the geocaulosphere on the soil charcteristics and the microbial communities is not well known. We suggest that plant rhizosphere and potato geocaulosphere could be compared according to the release of exudates and their impact on the surrounding microbial communities. Regarding the lack of knowledge about this question it would be interesting to study the similarities and differences between rhizosphere and geocaulosphere in order to better understand the interaction between plants, especially potato, soil and microorganisms. Plants are able to defend themselves from pathogenic or environmental stresses by setting up several different mechanisms. The first defence system is mechanical; it includes physical barriers avoiding the entrance and spread of the pathogen in the plant. The second mechanism induces biochemical reactions that produce substances either toxic for the pathogens or inhibiting their growth and that modify the plant physiology. The physiological mechanisms of the blemish formation have never been studied but it is known that after an attack, plant cells produce suberin that forms a cork layer on the cell walls [START_REF] Pollard | Building lipid barriers: biosynthesis of cutin and suberin[END_REF]. The accumulation of suberin on the upper layers of the tuber periderm could lead to the formation of superficial corky structures such as those observed on blemished tubers. Even in the same host, defence reactions are different depending on the age of the plant, the cultivar or the weather conditions [START_REF] Agrios | Plant pathology[END_REF]. The physiological mechanisms of the plant leading to blemish formation are not well understood. They could be related to a hypersensitive response of the plant and all the physiological changes it induces. Are the plant defence process the same in response to a pathogenic or an environmental stress?

What are the differences? Are some beneficial microorganisms able to potentialize the plant defence reactions? What are the genes implicated? What are the physiological functioning leading to the formation of the blemishes themselves?

Those are some questions that need to be answered in the next years in order to understand the mechanisms underlying the tuber blemish formation. Concerning R.

solani, few studies report that this fungus produces phytotoxins leading to plant infections [START_REF] Gvozdeva | Interaction of proteinases secreted by the fungal plant pathogen Rhizoctonia solani with natural proteinase inhibitors produced by plants[END_REF][START_REF] Brooks | Sensitivity to a phytotoxin from Rhizodonia solani correlates with sheath blight susceptibility in rice[END_REF] but plant -pathogen interactions need to be clarified.

ii) Definition of indicators of the soil health to built up decision aid supports for the growers Soil health and soil quality are closed concepts that allow soil functions to be related to specific purpose i.e. to maintain an appropriate productivity and good quality of the products while simultaneously reducing the effects on the environment and contributing to human health. However, the former takes into account more clearly plant health and therefore the phytosanitary aspect of the soil than soil quality [START_REF] Janvier | Soil health through soil disease suppression: Which strategy from descriptors to indicators?[END_REF]. Soil health assessment includes the quantification of indicators of soil quality. Such indicators may be derived from specific soil parameters obtained from different disciplines of soil science, but descriptive indicators which are inherently qualitative can also be used in assessing soil health and soil quality [START_REF] Schjonning | Soil quality managementconcepts and terms[END_REF]. Soil quality indicators condense an enormous complexity in the soil and therefore could be used as management tools to positively influence soil health. Nevertheless, they have to be chosen to be usefully used and not too complex to handle by policy makers or land managers [START_REF] Doran | Soil health and sustainability: managing the biotic component of soil quality[END_REF]. In our study, we showed the potential implication of soil characteristics and cultural practices in the occurrence of the blemishes thanks to the statistical analyses of a survey conducted with some farmers from the West of France. But we concluded that more indicators would be useful to identify the beneficial and deleterious components preventing from or leading to blemishes so that agricultural practices could promote the beneficial components. Therefore, we propose to conduct a survey at a larger scale than the one we previously used to collect a larger amount of both specific and descriptive parameters. Such a survey could be conducted among the population of French producers of seed potato whose number is estimated around 915 people (FNPPPT and GNIS, 2007). A representative sample of this population should be determined to provide information on both geographic areas, climatic conditions and agricultural practices but also a large number of parameters surrounding the plant iii) Integrated pest management to control soil-borne pathogens of potatoes

The soil microflora is composed of very diverse species that can be pathogenic or non pathogenic for the plant. The severity of a disease is regulated by the non pathogenic flora. Their ways of action are either active or passive. The antagonistic microorganisms reduce actively the pathogenic populations by parasitism or by antibiosis, whereas other species can compete passively with the pathogens and deprive them from essential nutrients or ecological niche [START_REF] Alabouvette | Soil suppressiveness to diseases induced by soilborne plant pathogens[END_REF][START_REF] Stengel | [END_REF]. New environmental friendly control methods are inspired by those natural mechanisms. They consist in inoculating antagonistic populations in diseased fields or in favouring the development and the activity of the beneficial populations by sustainable cultural practices. The occurrence of sclerotia due to R.

solani could be controlled by field inoculation of efficient antagonists such as Trichoderma spp. [START_REF] Wilson | Biological and chemical control and their combined use to control different stages of the Rhizoctonia disease complex on potato through the growing season[END_REF]. Cultural practices showing a reduction of the occurrence of some tuber blemishes in chapter 6 could also be a part of the sustainable management systems leading to the production of unblemished potatoes. Storage conditions were briefly evocated in this work. Indeed, important damages may occur if tubers are stored in bad conditions [START_REF] Cwalina-Ambroziak | Fungi colonizated of potato tubers (Solanum tuberosum L.) after harvest and after storage[END_REF]. There was no study on the development of atypical blemishes during storage but generally fast drying of tubers after harvesting permits to avoid the development of several diseases [START_REF] Scheid | What to do against storage diseases?[END_REF]. Unfortunately, cultural management practices alone are not currently fully efficient to control diseases, causing the potato industry to become over-reliant on the use of agrochemicals for effective management.

Current research efforts are directed at the identification and incorporation of genetic resistance into cultivars with acceptable agronomical characteristics to provide more effective disease management [START_REF] Gudmestad | Management of soilborne diseases of potato[END_REF].

The appropriate analyses of the databases built up during the previous proposal of perspective supported by the built up of mathematical models would be in perfect adequacy with the philosophy developed within the ENDURE network. Development of integrated pest management could be a sustainable solution to produce potatoes that will respond to the preoccupations of all the potato industry that aim at producing a fresh mass consumer product with high qualitative and quantitative yields, good presentation and environment friendly modes of production (Figure 7.1).

Supervision of master students

Figure

  Figure 0.1 Diagram of the thesis outline Figure 1.1 Symptoms caused by some potato soil-borne diseases Figure 1.2 Input and output parameters of yield loss calculation models Figure 2.1 Classification of potato blemishes Figure 3.1 Pictures of atypical corky blemishes Figure 3.2 Scheme of the experimental set up of the assay under hydroponic conditions Figure 4.1 Pictures of some blemishes observed before and after haulm destruction on potato tubers.

Figure 4. 2

 2 Figure 4.2Principal component analysis of bacterial 16S and fungal 18S terminal restriction fragment length polymorphism data sets before and after haulm destruction of the potato plants.

Figure 4. 3

 3 Figure 4.3Principal component analysis of 16S and 18S terminal restriction fragment length polymorphism data sets before and after haulm destruction of the potato plants.

Figure 5. 1

 1 Figure 5.1 Sequence alignment of part of the ITS1 and ITS2 regions among isolates of Rhizoctonia solani collected from potatoes.

Figure 5. 2

 2 Figure 5.2Neighbour-joining tree of 52 monomorphic ITS sequences of Rhizoctonia solani isolated from potato tubers.

Figure 5. 3

 3 Figure 5.3Sequence alignment of part of the tef-1α gene among isolates of Rhizoctonia solani collected from potatoes.

Figure 5. 4

 4 Figure 5.4Neighbour-joining tree of 23 tef-1α sequences of Rhizoctonia solani isolated from potato tubers from France and other countries.

Figure 5. 5

 5 Figure 5.5Phylogenetic relationships among 89 strains of Rhizoctonia solani inferred from AFLP data using a UPGMA analysis of Nei -Li distances.

Figure 6. 1

 1 Figure 6.1Percentage of answers for each category of surveyed parameters.

Figure

  Figure 6.2 Correspondence analyses of blemishes x potato cultivars in 2006 Figure 6.3 Correspondence analyses of blemishes x potato cultivars in 2007 Figure 7.1 Diagram of the research perspectives issuing from the thesis

  have launched a 3-year Program of Collaborative Research (PRC) with the financial support of the Regional Council of Brittany and in collaboration with the laboratory of Soil and Environmental Microbiology (MSE) at INRA (National Institute of Agronomical Research). The locally based industrial company, Végénov-Bretagne Biotechnologie Végétale (BBV), has also contributed to the project. The objectives of this project are -To propose clear and consensual terminology and classification of the different blemishes that can be observed on the tuber surface.

Figure 0 . 1

 01 Figure 0.1 Diagram of the thesis outline

Figure 1 . 1

 11 Figure 1.1 Symptoms caused by some potato soil-borne diseases, a. Black leg (Pectobacterium spp.), b. Root-knot nematode (Meloidogyne incognita), c. Black dot (Colletotrichum coccodes), d. Potato virus Y (PVY).

Figure 1 . 2

 12 Figure 1.2 Input and output parameters of yield loss calculation models

  occurrence and development of the soil-borne potato diseases. To our knowledge, such a work putting in relation numerous potato diseases and comparing their development conditions, the ecology of the causal pathogens and their abiotic and biotic interactions responds to a clear demand from both scientists, extension services, breeders, and farmers. Studies dealing with potato diseases frequently consider only one or few diseases at the same time. Thus, this review constitutes by itself a decision support system since the optimal factors limiting disease development are listed. Nevertheless, the data collected here deal more with diseases known in developed counties and those which cause severe economical losses. Knowledge about minor diseases such as Phoma leaf spot, Rosellinia black rot and Thecaphora smut are extremely rare, probably because these diseases occur in very isolated areas. Phoma leaf spot was recorded only in Bolivia and Peru, Rosellinia black rot was described in South America and Africa and Thecaphora smut in South America and Mexico.

  by a diversity of superficial blemishes among which typical and atypical blemishes can be distinguished. Typical blemishes are those for which the causal agents are known. They are more studied than atypical blemishes for which the causal agents are still unknown, sometimes putative and not yet scientifically identified.Researchers, growers and technical staff dealing with such blemishes all over the world use different terminologies to describe each of the blemishes. Besides major tuber diseases like silver scurf, black dot and powdery scab, for which common names of symptoms are usually well defined and consensual, there is a wide range of atypical blemishes and some typical ones for which the terminology is more disparate and may induce confusion and misunderstandings. Physiological mechanisms involved in the appearance of atypical blemishes are reviewed; this highlights the scarcity of available data on the subject and the need for carrying out complementary studies based on a consensual nomenclature. After a critical worldwide bibliographical review of the most commonly used terminology of tuber blemishes, we put forward a proposal for a new nomenclature harmonizing common names for each kind of blemish.IntroductionSince several decades potato (Solanum tuberosum L.) crop area increases especially in developing countries. Some 325 millions tons of tubers are produced annually in the world(FAO, 2008). In western Europe, most fresh potato lots are washed before selling (Commission of the European Communities, 2007): this radical procedure has positively prevented a too harsh decline of the consumption of table potatoes but has had an adverse effect of highlighting tuber skin defects. As a consequence, visual quality of tubers has become a very important cause of rejection or downgrading of potatoes leading to potential important economical losses for farmers.

  Figure 2.1.A) and silver scurf caused by Helminthosporium solani (Figure 2.1.B) may be misinterpreted by inexperienced eye because of the similarity of the discoloured patches they form on tuber surface. Black dot causes diffuse grey-brown spots with black microsclerotia, whereas silver scurf blemishes are clear silvery spots with very fine black punctuations and induce a separation of the periderm (El Imane-Collet,

  .1.C). o Netted scab (produced by Streptomyces spp.) which forms typical, very superficial and regular polygonal corky lesions (Figure 2.1.D) o Russet scab (produced by strains belonging to S. aureofasciens group), which is very similar to netted scab, with different conditions of development (Figure 2.1.E) o Atypical corky blemish (unknown origin) that can appear under different forms, we suggest to distinguish 4 categories: Elephant hide, turtleback or scabby lesion (Figure 2.1.F), characterized by the polygonal framework it forms on the tuber periderm. We suggest using the term polygonal lesion to describe this blemish. Corky crack (Figure 2.1.G) refers to corky lesions present on split tubers; Corky spot, formerly named rhizoscab (Figure 2.1.H), is similar to polygonal lesions, but forms little corky patch instead of large corky plates. The term rhizoscab should not be employed henceforth because it suggests that R. solani might be responsible for this blemish, even though the Koch's postulates have so far never been fulfilled; Star-like corky lesion with or without halo (Figures 2.1.I and 2.1.J); -Rugosity including o russeting, which refers to a rough or scaled periderm of the tuber (Figure 2.1.K), o skinning, which describes the blemish quite similar to russeting but forming a detached periderm (Figure 2.1.L).

  .1.M) or o common scab. We suggest that the international scientific community should henceforth use the term common scab to describe pitted, raised or shallow lesions caused by Streptomyces bacteria. The general term common scab used in Europe, and referring either to netted scab or (raised) common scab, should be replaced by the term potato scabs (Table 2.2). Three different kinds of common scab blemishes can be distinguished: pitted scab (Figure 2.1.N) is commonly used to describe those deep, sometimes star-shaped lesions and the term should be conserved; raised scab (Figure 2.1.O) instead of erumpent scab, which is less frequently used; shallow scab (Figure 2.1.P) instead of superficial scab which is a too general term.

Figure 2 . 1

 21 Figure 2.1 Classification of potato blemishes

Figure 3 . 1

 31 Figure 3.1 Pictures of atypical corky blemishes (a) Irregular polygonal sunken corky blemishes or polygonal lesions; (b) Corky crack; (c) Corky spot; (d) Star-like corky lesions without halo; (e) Star-like corky lesions with halo

  bioassays were conducted in 2007 and 2008 in a greenhouse from April to September. Healthy potato tubers were grown in pots containing soil artificially infested with fungal or Streptomyces isolates. Thirty-three strains were tested in 2007 and 48 strains in 2008. Nine strains were tested twice (Table 3.2). In addition, four reference strains were tested: Fusarium sambucinum (TFSa, isolated in France 2007), F. solani var. coeruleum (T FSC1, isolated in France 2006) R. solani AG 3 (i4 / 9729-1, isolated in France 1999), and R. solani AG 2-1 (0799-001 2N WA, isolated in Morocco 2007).Fungal inoculum was prepared on autoclaved millet seeds. Forty grams of seeds were mixed in jars with 32 ml of sterile deionised water and autoclaved for 45 min at 110°C on two consecutive days and for 20 min at 120°C the third day. The jars were stored at room temperature during 4 days before inoculation to allow the release of putative toxic compounds (mainly NH 3 ). Six plugs of 12 day-old fungal cultures on PDA were introduced and mixed with the millet seeds. The cultures were incubated at room temperature for 3 weeks with regular shaking.

  and because it has a medium to high overall susceptibility to atypical superficial blemishes(FNPPPT and GNIS, 2007, Chatot, personnal data). Commercial certified seeds (Class A; 25-32) were used and visually examined for absence of any skin blemish. Plants were grown in a glasshouse at room temperature with minimal temperature of 10°C and maximal temperature of 25°C with a 16 h day len gth, for approximately 4 months, until natural maturity; additional light (200 W/m 2 ) was provided when needed. Plants were regularly watered, but soil moisture content was not monitored. Fertilizing watering was carried out every week with a fertilizer solution (N P K, 20 20 20). Plants were harvested in September. A non inoculated control and three replicate pots per treatment were set up.At harvest, one gram of soil from each pot was spread on PDA in a Petri dish to check the survival and viability of the inoculated fungi and bacteria during the assay. After 4 days, the presence of the inoculated microorganisms was checked under a microscope.Progeny tubers from the same plant were washed under running tap-water, airdried, weighed and stored in a paper bag. The number of tubers per plant was recorded and the tubers larger than 3 cm long were scored individually according to the different classes of blemishes. The scoring scale edited by the French official service of control and certification (SOC)(GNIS and SOC, 2005) was adapted to black scurf, netted scab and common scab scales, each with 10 different levels of disease severity. Black scurf scale was: 0 = no lesion, 1 = 1 % of area covered by lesions; 3 = 4% of area covered by lesions; 5 = 9 % of area covered by lesions; 7 = 14 % of area covered by lesions; 9 = 35 % of area covered by lesions. Netted and common scab scales scored tuber as following: 0 = no lesion, 1 = 4 % of area covered by lesions; 3 = 6% of area covered by lesions; 5 = 30 % of area covered by lesions; 7 = 45 % of area covered by lesions; 9 = 60 % of area covered by lesions

Figure 3 . 2

 32 Figure 3.2 Scheme of the experimental set up of the assay under hydroponic conditions

  (b) Collection MIAE, Microorganisms of Interest for Agriculture and Environment (INRA Dijon, France).

  microorganisms. Evidences indicate that stress factors induce a differential response of the plant and the diversity of the microorganisms associated with the potato could be a natural situation. Cultivar susceptibility is also probably a key to blemish development and thus control. Complementary studies about the environmental conditions responsible for development of potato blemishes and the assessment of susceptible and resistant cultivars are needed.AbstractAppearance of atypical blemishes on potato tubers is unexplained. The involvement of soil borne microorganisms has so far never been demonstrated except for Rhizoctonia solani causing black scurf. To test if the microbial communities of the geocaulosphere of tubers are associated to the occurrence of blemishes, the bacterial and fungal community structures of the soil surrounding healthy and blemished tubers were analysed by terminal restriction fragment length polymorphism (T-RFLP) before and after haulm destruction.The same T-RFLP procedure was used to characterize microorganisms previously isolated from blemished potatoes. Bacterial communities around healthy tubers evolved between the two sampling dates; they changed toward a single structure around blemished tubers. Fungal community structures around healthy and blemished tubers were different one from the others and at the two sampling dates what reveals the succession of different populations along with the sanitary status of the potato and with the season. However, although differential evolutions of the structures of microbial communities were shown to be related to the blemish occurrences, no causality link was depicted apart from the one supposed to be R. solani population that significantly increased around blemished tubers after haulm destruction.

  involved. In addition to abiotic factors, the microflora harboured by the soil surrounding the tuber (geocaulosphere) might influence the potato development and the occurrence of blemishes. The term 'geocaulosphere' built up with the same etymological bases as the term 'rhizosphere' refers to the soil of the nearby environment of tubers, under the influence of the subterranean stems.Microorganisms in the geocaulosphere could have an indirect impact on tubers and create favourable (i.e. stimulation of defence mechanisms) or unfavourable conditions (i.e. modification of soil pH or soil moisture) which might affect potato development and blemish occurrence. To assess this hypothesis, the bacterial and fungal community structures of the geocaulosphere of healthy, blemished and partially-healthy and blemished plants were compared at different periods of time by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S and 18S

(

  Promega) and MspI (Q-BIOgene) were used to digest 16S and 18S PCR products, respectively.Fluorescently-labelled terminal restriction fragments (TRF) were separated and detected using a capillary electrophoresis sequencer CEQ TM 2000XL (Beckman Coulter, Fullerton, CA, USA). Analyses were run with the Frag-4-30s-70min method, including a denaturation of 2 min at 90°C, an injec tion at 2000 V during 30 s and a separation at 4 800 V during 70 min. The sizes of the TRF were determined by comparison with Size Standard-600 (Beckman Coulter). Fungal and bacterial community structures were characterised by the size and fluorescence intensity of the TRF. For each PCR product, the T-RFLP analysis was performed in triplicate.

Figure 4 . 1

 41 Figure 4.1 Pictures of some blemishes observed before (T1) and after (T2) haulm destruction on potato tubers.

Figure 4 . 2

 42 Figure 4.2 Principal component analysis of bacterial 16S (a and c) and fungal 18S (b and d) terminal restriction fragment length polymorphism data sets before, T1 (a and b) and after, T2 (c and d) haulm destruction of the potato plants.Analyses were performed on soil from the geocaulosphere of healthy plants (3 tubers, white squares), blemished plants (6 tubers, grey squares) or partially -healthy and blemished plants (3 healthy tubers, white circles and 3 blemished tubers, grey circles). Each plant was represented by a single number. Analyses of uncultivated soil were performed on three independent samples of inter-row soil as control (triangles). Each soil set is represented by its gravity centre. The branches show the divergence of the tubers from the same plant from the respective gravity centres.

Figure 4 . 3

 43 Figure 4.3 Principal component analysis of 16S (a) and 18S (b) terminal restriction fragment length polymorphism data sets before (T1) and after (T2) haulm destruction of the potato plants.Analyses were performed on soil from the geocaulosphere of healthy plants (3 tubers, white squares), blemished plants (6 tubers, grey squares) or partially -healthy partially -blemished plants (3 healthy tubers, white circles and 3 blemished tubers, grey circles). Analyses of uncultivated soil were performed on three independent samples of inter-row soil as control (triangles). Each soil set is represented by its gravity centre. The branches show the divergence of the tubers from the same plant from the respective gravity centres.

  Likewise, TRF of ~ 581 bp could correspond to Trichoderma sp. This TRF represented 10.7 % of the total intensity of the T-RFLP profile of inter-row soil at T2.Another peak representing 10 % of the total intensity of the profile of blemished tubers at T2 was 583 bp long. This TRF could be associated to Alternaria, Ulocladium, Phoma or Epicoccum. Trichoderma spp., Alternaria spp., Ulocladium spp., Phoma spp. and Epicoccum spp. are not pathogenic for potatoes; however, they are good saprophytes and can easily develop in soil. Alternaria spp. were particularly associated with blemished tubers(Fiers et al., 2010), probably because it takes advantage of the niche created by the blemish formation. The bacterial TRF of ~ 222 bp was detected in some inter-row soil samples. This TRF length can be associated to the TRF of Streptomyces, a potentially pathogenic bacterium of potato causing netted scab, observed on some of the analyzed tubers.

  in a final volume of 50 µL by mixing 2 µl of DNA with 0.5 µM of each primer, 150 µM of dNTP, 6 U of Taq DNA polymerase (Q-Biogen, Evry, France) and PCR reaction buffer. PCR amplifications of tef-1α were performed with primers EF1-645F (TCG TCG TYA TCG GMC ACG TCG A) and EF1-1190R (TAC CAG TGA TCA TGT TCT TGA TGA) (Andersen et al., 2009) in a final volume of 25 µL by mixing 1 µL DNA with 0.068 µM of each primer, 1.5 mM MgCl 2 , 150 µM dNTP, 1 U Taq DNA polymerase (Q-Biogen, Evry, France) and PCR reaction buffer. Amplifications were conducted in a mastercycler (Eppendorf, Hambourg, Germany). ITS amplification consisted in an initial denaturation of 3 min at 94 C, followed by 35 cycles of 1 min at 94 C, 1 min at 50 C, 1 min at 72 C, and a final extension of 10 min at 72 C. Amplification of tef-1α was carried out with an initial denaturation of 5 min at 94 C, followed by 40 cycles of 30 s at 94 C, 30 s at 52 C, and 80 s at 72 C and a final extension of 7 min at 72 C. Aliquots of PCR products were checked by electrophoresis on a 1 % agarose gel, revealed with ethydium bromide and visualized by UV trans-illumination.

  min at 56 C, and 1 min at 72 C. A 1:50 dilution was performed with 1.5 µL of preamplified product diluted in 73.5 µL TE buffer. Selective amplification was performed in a final volume of 20 µL by mixing 5 µL of DNA with 0.4 µM of E-AA 5′-IRD800 fluorescent primer (GACTGCGTACCAATTCAA) (MWG), 0.30 µM of M-C primer (GATGAGTCCTGAGTAAC), 202.5 µM of dNTP, 0.5 U of Taq DNA polymerase (Q-Biogen) and PCR reaction buffer. PCR was conducted in a thermal cycler GeneAmp PCR system 9600 with a first cycle at 94 C during 30 s, 65 C during 30 s, and 72 C during 1 min. The 12 following cycles were performed by reducing the annealing temperature of 0.7 C at each cycle. Then, 23 cycles were performed at 94 C during 30 s, 56 C during 30 s, and 72 C during 1 min. Aliquots of PCR products were checked by electrophoresis on a 1 % agarose gel, revealed with ethydium bromide and visualized by UV trans-illumination. Separation of the amplified fragments was

  2). The tree of ITS sequences showed 7 different ITS types among AG 3 PT. Type m3 comprised one isolate (MIAE00267) from France(Côtes d'Armor) and isolated from cultivar Charlotte. Type m5 included 12 isolates from France (Finistère, Eure-et-Loir, Pas-de-Calais). They were isolated from cultivar Spunta, Samba, Pamela, Chérie, Amandine, Hermes and Juliette. ITS type m4 comprised 11 isolates from France (Finistère, Morbihan, Côtes d'Armor), Switzerland and United Kingdom. They were isolated from cultivars Charlotte, Juliette, Atlas, Gourmandine, and Bellini. Type m7 included 4 isolates all from France (Finistère) isolated from cultivar Samba or a hybrid. The ITS types m6, m8 and m9 were grouped in the same branch, distantly related from the other types in AG 3 PT. Type m6 comprised 5 isolates fromFrance (Finistère) and United Kingdom isolated from cultivars Charlotte and Juliette. Type m8 comprised 2 isolates from France (Finistère) isolated from cultivars Spunta and Bintje. Type m9 comprised 3 isolates from Poland and France (Finistère) isolated from Urgenta and unknown cultivars.Two different ITS types were observed among AG 2-1. Type m2 included 7 isolates from France (Somme) and United Kingdom. They were isolated from a hybrid cultivar and an unknown cultivar. Type m1 included 2 isolates from Finland and France (Côtes d'Armor) and they were isolated from an unknown cultivar and cultivar Juliette. Finally, monomorphic isolates from AG 5 were grouped in only one ITS type (m10). They were 5 isolates fromFrance (Eure-et-Loir, Lot-et-Garonne) and Finland isolated from cultivars Chérie, Samba, a hybrid and an unknown cultivar. .|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AG 2-1 m1 CCCATTCATTTGGGCATGTGCACACCTTTCTCTTTCATCCCACACACACCTGTGAACCTGTGAGACAGATGGGGAATTTACTTGTTGCTTTTTGGAATAC m2 .T....T.....................C...........................................................C........... AG 3 m3 T.T...T....A...............CC............................T..........T...........T.....AT......T....A m4 T.T...T....A...............CC............................T..........T...........T.....AT......T....A m5 T.T...T....A...............CC............................T..........T...........T.....AT......T....A m6 T.T...T....A...............C.............................T..........T...........T.....AT.A....T....A m7 T.T...T....A...............CC............................T..........T...........T.....AT......T....A m8 T.T...T....A...............C.............................T..........T...........T.....AT.A....T....A m9 T.T...T....A...............C.............................T..........T...........T.....AT.A....T....A AG 5 m10 .T...GA....................A..........-...............C..T......G...............T.GA...T.....AAG.ACA AG 2-1 p1 ......T....A................C.............T......................T.................................. p2 .T....T.....................C...........................................................C........... AG 3 p3 T.T...T....A...............CY............................T..........T...........T.....AT.W....T....A p4 T.T...T....A...............C.............................T..........T...........T.....AT.A....T....A p5 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p6 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p7 T.T...T....A...............CY............................T..........T...........T.....AT.W....T....A p8 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p9 T.T...T....A...............CY............................T..........T...........T.....AT.A....T....A p10 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p11 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p12 T.T...T....A...............CC............................T..........T...........T.....AT.W....T....A p13 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p14 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p15 T.T...T....A...............C.............................T..........T...........T.....AT.A....T....A p16 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p17 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p18 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p19 T.T...T....A...............CY............................T..........T...........T.....AT.A....T....A p20 T.T...T....A...............CC............................T..........T...........T.....AT.W....T....A p21 T.T...T....A...............CY............................T..........T...........T.....AT.W....T....A p22 T.T...T....A...............C.............................T..........T...........T.....AT.A....T....A p23 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p24 T.T...T....A...............CC............................T..........T...........T.....AT.W....T....A p25 T.T...T....A...............CC............................T..........T...........T.....AT......T....A p26 T.T...T....A...............CY............................T..........T...........T.....AT.W....T....A p27 T.T...T....A...............CY............................T..........T...........T.....AT.W....T....A p28 T.T...T....A...............CY............................T..........T...........T.....AT.W....T....A AG 5 p29 .T...GA....................A..........-...............C..T......G...............T.GA...T.....AAG..|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AG 2-1 m1 AAAGGCAATAGGTTATTGGACCCT-CTGTCTACTCAATTAATATAAACTCAATTTATTTTAAAACGAATGTAATGGATGTAACACATCTC m2 ........................-.............................A.........T......................... AG 3 m3 ..T.AT....AA.C....A.....T............C.C.......A...............TG-........................ m4 ..T.AT....A..C....A.....T............C.C.......A...............TG-........................ m5 ..T.AT....AA.C....A.....T............C.C.......A...............TG-........................ m6 ..TAAT....A..C.C..A.....T............C.C.......................TG-........................ m7 ..T.AT....A..C....A.....T............C.C.......A.........CA....TG-........................ m8 ..TAAT....A..C.C..A.....T............C.C.......................TG-........................ m9 ..TAAT....A..C.C..A.....T............C.C.....C.................TG-........................ AG 5 m10 .TT..TGC.G.--------.....-............-.-...................AG...T..................G.....A AG 2-1 p1 ........................-.......................C..............CT......................... p2 ........................-.......................Y.....A.........T......................... AG 3 p3 ..TRAT....A..C.Y..A.....T............C.C.......M...............TG-........................ p4 ..TAAT....A..C.C..A.....T............C.C.......................TG-........................ p5 ..T.AT....AR.C....A.....T............C.C.......A...............TG-........................ p6 ..T.AT....A..C....A.....T............C.C.......A.........WY....TG-........................ p7 ..TRAT....A..C....A.....T............C.C.......M...............TG-........................ p8 ..T.AT....AA.C....A.....T............C.C.......A...............TG-........................ p9 ..TRAT....A..C.Y..A.....T............C.C.......M...............TG-........................ p10 ..T.AT....AA.C....A.....T............C.C.......A...............TG-........................ p11 ..T.AT....AA.C....A.....T............C.C.....M.A...............TG-........................ p12 ..T.AT....AA.C....A.....T............C.C.....M.A...............TG-........................ p13 ..T.AT....AR.C....A.....T............C.C.......A...............TG-........................ p14 ..T.AT....AR.C....A.....T............C.C.......A.........YW....TG-........................ p15 ..TAAT....A..C.C..A.....T............C.C.....M.................TG-........................ p16 ..T.AT....A..C....A.....T............C.C.......A.........YW....TG-........................ p17 ..T.AT....AA.C....A.....T............C.C.......A.........YW....TG-........................ p18 ..T.AT....AA.C....A.....T............C.C.......A.........YW....TG-........................ p19 ..TRAT....AR.C.Y..A.....T............C.C.......M...............TG-........................ p20 ..T.AT....A..C....A.....T............C.C.......A........W......TG-........................ p21 ..TRAT....A..C.Y..A.....T............C.C.....M.M...............TG-........................ p22 ..TRAT....A..C.C..A.....T............C.C.......M...............TG-........................ p23 ..T.AT....AR.C....A.....T............C.C.......A.........YW....TG-........................ p24 ..T.AT....A..C....A.....T............C.C.......A...............TG-........................ p25 ..T.AT....A..C....A.....T............C.C.....M.A...............TG-........................ p26 ..TRAT....A..C.Y..A.....T............C.C.......M...............TG-........................ p27 ..TRAT....A..C.Y..A.....T............C.C.......M.........YW....TG-........................ p28 ..TRAT....AR.C.Y..A.....T............C.C.....C.M...............TG-........................ AG 5 p29 .TT..TGC.G.--------.....-............-..CATA..CYCA.T..AT..AG...TG-.................G.....A .|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AG 2-1 m1 TTCTTCAAAGTAAATCTTTTG-TTAACTCAATTGGTTTCACTTTGGTATTGGAGGTCTTTTGCAGCTTCACACCTGCTCCTCTTTGTTCATTAGCTGGAT m2 .....................-....T....C.......................................................G............ AG 3 m3 A........A.C........TG...........A....G.TC.............................................GT........... m4 A........A.C........TG...........A....G.T..............................................GT........... m5 A........A.C........TG...........A....G.T.........................................

  ......A.C........TG...........A....G.TY.............................................GT........... p11 A........A.C........TG...........A....G.T..............................................GT........... p12 A........A.C........TG...........A....G.T..............................................GT........... p13 A........A.C........TG...........A....G.T..............................................GT........... p14 A........A.C........TG...........A....G.TY.............................................GT........... p15 A........A.C........TG...........A....G.T..............................................GT........... p16 A........A.C........TG...........A....G.T..............................................GT........... p17 A........A.C........TG...........A....G.TY.............................................GT........... p18 A........A.C........TG...........A....G.T..............................................GT........... p19 A........A.C........TG...........A....G.T.................A............................GT........... p20 A........A.C........TG...........A....G.T..............................................GT........... p21 A........A.C........TG...........A....G.T..............................................GT........... p22 A........A.C........TG...........A....G.T.................A............................GT........... p23 A........A.C........TG...........A....G.T..............................................GT........... p24 A........A.C........TG...........A....G.T.................W............................GT........... p25 A........A.C........TG...........A....G.T..............................................GT........... p26 A........A.C........TG...........A....G.T.................A............................GT........... p27 A........A.C........TG...........A....G.T.................W............................GT........... p28 A........A.C........TG...........A....G.T..............................................GT........... AG 5 p29 A...................-G...............CY.....................-.......................Y.............|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|... AG 2-1 m1 CTCAGTGTTATGCTTGGTTCCACTCGGCGTGATAAATTATCTATCGCTGAGGACACTGTAAAAGGTGGCCAAGGTAAATGCAGATGAACCGCTTCTAA m2 .................................................................................................. AG 3 m3 .........................A.....................................A.................................. m4 .........................A.....................................A.................................. m5 .........................A.....................................A.................................. m6 .........................A.....................................A.................................. m7 .........................A.....................................A.................................. m8 .........................A.....................................A.................................. m9 .........................A.....................................A.................................. AG 5 m10 .........................A.........G.........................C............G..................

Figure 5 . 1

 51 Figure 5.1 Sequence alignment of part of the ITS1 (a) and ITS2 (b) regions among isolates of Rhizoctonia solani collected from potatoes. Y indicates C and T, W indicates A and T, M indicates A and C and R indicates A and G. Polymorphic bases indicated in grey are common to those found by Justesen et al (2003) in the ITS1 region.

Figure 5 . 2

 52 Figure 5.2 Neighbour-joining tree (Kimura two-parameter distance) of 52 monomorphic ITS sequences of Rhizoctonia solani isolated from potato tubers. Bootstrap values (≥ 60 %) are shown near the equivalent branches.

  polymorphic site, together with one sequence of AG 5 (MIAE00279) including only one polymorphic site. Indeed all the sequences of R. solani AG 5 analyzed comprised at least one polymorphic site (Figure5.4). The phylogenetic tree showed 4 different elongation factor types within AG 3 PT. Type m6 comprised one isolate (MIAE00351) from Japan; type m5 comprised 7 isolates from France (Finistère), Switzerland and Finland isolated from cultivar Spunta, Samba, Naviga and Bellini. Type m4 comprised 9 isolates from different department of France (Côtes d'Armor, Finistère and Loiret) isolated from cultivars Atlas, Bintje, Charlotte, Juliette, Spunta and Urgenta. Finally type m7 included 2 isolates from Poland isolated from unknown cultivars. All the strains belonging to the type m7 in tef-1α analysis corresponded to type m9 in ITS analysis; both being distantly related to other AG 3 PT sequences (Figures 5.2 and 5.4). Among AG 2-1 sequences, three elongation factor types m1, m2 and m3 were identified.

Figure 5 . 3

 53 Figure 5.3 Sequence alignment of part of the tef-1α gene among isolates of Rhizoctonia solani collected from potatoes. Y indicates C and T, W indicates A and T, M indicates A and C and R indicates A and G.

Figure 5 . 4

 54 Figure 5.4 Neighbour-joining tree (Kimura two-parameter distance) of 23 tef-1α sequences of Rhizoctonia solani isolated from potato tubers from France and other countries. Strain MIAE00279 belonging to AG 5 includes one polymorphic site. Bootstrap values (≥ 60 %) are shown near the equivalent branches

Figure 5 . 5

 55 Figure 5.5 Phylogenetic relationships among 89 strains of Rhizoctonia solani inferred from AFLP data using a UPGMA analysis of Nei -Li distances. Bootstrap values (≥ 50%) are shown above the equivalent branches

  the collected tubers, 38 % presented atypical corky blemishes in 2006 and 40 % in 2007, 34 % of the tubers had sclerotia both in 2006 and 2007, 15 % had potato scabs symptoms in 2006 and 10 % in 2007, whereas 13 % presented enlarged lenticels in 2006 and 16 % in 2007.

Figure 6 . 2 Figure 6 . 3

 6263 Figure 6.2 Correspondence analyses of blemishes x potato cultivars in 2006 Numbers between brackets represent the number of scored samples per related cultivar.

  be clarified in order to optimize its control. A global review of the knowledge about soil-borne potato diseases was proposed in chapter 1 on this thesis. Data about the conditions of appearance and development of diseases are essential to understand the mechanisms underlying host -pathogen interactions and to set up efficient methods to control diseases. The first chapter of this work can be considered as a decision support system to avoid damages on potato crop since it resumes all the favourable and unfavourable conditions affecting the occurrence and the development of the soil-borne diseases of potato. Nevertheless, very little information were collected in the literature on the blemishes despite the evident need of knowledge on this subject. Consequently, the demand of extension people and farmers for further researches aiming at solving this problem was foreseeable. Ce qui se conçoit bien s'énonce clairement et les mots pour le dire arrivent aisement -What is well conceived is expressed clearly, and the words to say it come easily-wrote Nicolas Boileau-Despréaux, a famous French poet and writer in 1674 (Boileau-Despréaux 1674). This sentence sums up the first objective of this work that was to propose clear and consensual terminology and classification of the different

  the blemishes in order to avoid misunderstandings and favour collaborative researches between different laboratories. Several choices aiming at keeping the consensual names and changing those which could introduce confusion had to be made. They imply that the potato community changes its denomination habits and adopts the new nomenclature in order to work on common and clear foundations at a global scale.The second objective of this study was to identify the origin of the blemishes altering potato surfaces. Tubers were collected for two consecutive years, 2006 and 2007. The climatic conditions were very different during the two growing periods, spring and summer were hot and dry in 2006 and they were cold and wet in 2007.

  itself including aerial part, geocaulosphere and rhizosphere of the potato. It should provide with detailed information about the list and the relative importance of the abiotic factors which seemed to be involved in the appearance of blemishes. Biotic factors should also be included as parameters to measure as they interact with abiotic factors and thus might have an indirect role on the occurrence or development of the atypical blemishes. Appropriate multivariate statistical analyses combined with mathematical model based on the data set will identify the most appropriate indicators to be handled by the growers, i.e. a decision support system to manage the soil health to prevent blemishes and to conduct healthy potato crops.

Figure 7 . 1 Fiers

 71 Figure 7.1 Diagram of the research perspectives issuing from the thesis

Table 0 .1

 0 Potato production by area in 2007

	Table 1.1	Potato soil-borne pathogens
	Table 1.2	Favourable climatic conditions for potato soil-borne
		diseases development
	Table 1.3	Favourable edaphic conditions for potato soil-borne
		diseases development
	Table 1.4	Inoculum sources and correlation between inoculum
		density and soil-borne potato diseases severity
	Table 1.5	Genetic variability, strategies of conservation and attack of
		the pathogens and detection methods
	Table 1.6	Detrimental and beneficial associations of microorganisms
		with potato soil-borne pathogens
	Table 1.7	Resistance of wild potato cultivars toward pathogens
	Table 1.8	Cultural practices favourable to reduce disease
		development
	Table 2.1	Description of potato tubers main blemishes
	Table 2.2	Nomenclature of potato scabs in the world and suggestion
		for a consensus
	Table 2.3	Suggestion of a nomenclature for the superficial blemishes
		of potato tubers
	Table 3.1	Fungal and Streptomyces species isolated from the
		different blemishes
	Table 3.2	Strains tested in the two bioassays conducted in infected
		soil
	Table 3.3	Strains tested in the bioassay conducted with single and
		co-inoculations under hydroponic conditions
	Table 4.1	Collected soil samples, corresponding number of plants
		and graphic representations
	Table 4.2	Terminal restriction fragment (TRF) lengths of fungal
		strains.
	Table 5.1	Isolates of Rhizoctonia solani analyzed in this study

Table 0

 0

	.1 Potato production by area in 2007
	Harvested surface (ha)	Quantity (tonnes)	Yield (tonnes/ha)

Africa

Potatoes are grown in more than 100 countries, mainly in Asia (135 million tons) and Europe (130 million tons) (Table

Table 1 .1

 1 Potato soil-borne pathogens.

		Main symptoms	Patho
	TUBERS	OTHER PARTS	genici a ty test	Distribution	Ref

Table 1 .2

 1 Favourable climatic conditions for potato soil-borne diseases development

	Optimal level of humidity	Optimal light duration

Table 1 .3

 1 Favourable edaphic conditions for potato soil-bornediseases development

			Optimal soil texture			Optimal
	Pathogen	Disease	Mainly sandy or light soils	Mainly clay soils or heavy	Optimal soil pH	Optimal soil nutrient concentrations	content organic matter	References
	FUNGI & OOMYCETES
	Colletotrichum coccodes	Black dot	X		6 -7	Low nitrogen level	Kang et al., 2003; Nitzan and Tsror, 2003;
							Tsror, 2004
	Fusarium spp.	Fusarium dry	X		F. solani	High Fe level	variable
		rots			> 5.3	Low Ca, borax and P
					F. roseum	levels
					: No effect

Table 1 .4

 1 Inoculum sources and correlation between inoculum density and soil-borne potato diseases severity

				Correlation between inoculum
	Pathogen	Disease	Inoculum source	density and disease severity	Ref
				(max or min thresholds)
	FUNGI & OOMYCETES
	Colletotrichum coccodes	Black dot	Soil > Seed tuber	Disease severity remains constant	Lees, 2003; Nitzan et al., 2008
				above a threshold of soil-borne
				inoculum
				(0.5 to 1.7 g of inoculum per litre of
				soil)
	Fusarium spp.	Fusarium dry rots	Soil, seed tuber	Positive correlation (> 10 4 conidia ּml -1 for F. sulphureum
				> 105 conidia ּml

-1 for F. coeruleum)

Table 1 .5

 1 Genetic variability, strategies of conservation and attack of the pathogens and detection methods.

	Pathogen	Disease	Genetic variability a	Conservation and overwintering	ways Main penetration	Detection methods b	References
	FUNGI & OOMYCETES
	Colletotrichum coccodes	Black dot	6 or 7 VCG pathogenic for	At least 8 years at 10 cm	Mechanical	Q and RT-PCR , Fourier transform
			potato	depth in the soil as sclerotia		infrared (FT-IR)

2 species: G. pallida and G. rostochiensis Until 8 years in the soil as cysts

							de Boer,
							2009
	Streptomyces spp.	Common and	Common scab: S. scabies, S.	Conidia	Enzymatic	Conventional and RT-PCR, RFLP, rRNA	Rudkiewicz and Sikorski, 1984; Bouchek-
		netted scab	europaeiscabiei, S.			sequence analysis, carbon source	Mechiche et al., 2000; Flores-Gonzalez et al.,
			stelliscabiei, S. acidiscabiei, S.			utilisation, repetitive BOX profiles	2008; Loria et al., 2008; Mulder et al.,2008; Zhao
			turguidiscabiei and maybe				et al, 2008
			some others.
			Netted scab: S. reticuliscabiei
			and some isolates of S.
			europaeiscabiei
	NEMATODES
	Belonolaimus longicaudatus Sting nematode			Mechanical	Centrifugal-flotation method,	Crow et al., 2000
						morphological detection
	Ditylenchus destructor	Potato rot		About 4 months in favourable	Mechanical	Extraction in water, morphological	Shojaei et al., 2006; EPPO, 2008; Ilyashenka and
		nematode		conditions		identification, PCR-RFLP	Ivaniuk, 2008
	Globodera pallida,	Potato cyst
	Globodera rostochiensis	nematode

2009 Meloidogyne spp. Root-knot nematode At least 7 species: M

	. hapla,
	M. chitwoodi, M. fallax
	(Mediterranean soils), M.
	arenaria, M. incognita, M.
	javanica and M. mayaguensis
	(Mediterranean and tropical
	soils)

Table 1 .6

 1 Detrimental and beneficial associations of microorganisms with potato soil-

borne pathogens Pathogen Disease Organisms enhancing diseases Organisms reducing diseases References FUNGI & OOMYCETES

	Colletotrichum coccodes Black dot	V. dahliae, S. subterranea		Tsror 2004; Merz and Falloon, 2009
	Fusarium spp.	Fusarium dry rots	P. atrosepticum, Meloidogyne spp.	Serratia plymuthica, D. destructor	Munzert et al, 1977; Jensen, 1978; Gould et al., 2008;
			D. destructor, S. subterranea		Merz and Falloon, 2009
	Helminthosporium solani Silver scurf		Acremonium strictum, Pseudomonas putida,	Elson et al., 1997; Rivera-Varas et al., 2007
				Nocardia globerula, Xanthomonas campestris
	Macrophomina	Charcoal rot		Trichoderma harzianum, Bacillus subtilis, P.	Kumar and Khare, 1990; Gupta et al., 1999
	phaseolina			aeruginosa
	Phoma andigena var.	Phoma leaf spot
	andina
	Phoma spp.	Gangrene
	Phytophthora	Pink rot	S. subterranea	Enterobacter sp., E. cloacae, Pseudomonas	Merz and Falloon, 2009; Schisler et al., 2009
	erythroseptica			sp., P. fluorescens
	Polyscytalum pustulans Skin spot
	Pythium ultimum var.	Leak		Pseudomonas fluorescens, Burkholderia	Li et al., 2002; Bardin et al., 2004
	ultimum			ambifaria
	Rhizoctonia solani AG3 Black scurf / Stem	G. rostochiensis, Meloidogyne spp. + V.	Paenibacillus polymyxa, Bacillus licheniformis,
		canker	dahliae, Pratylenchus neglectus + V.	P. fluorescens, Chryseobacterium gleum,
			dahliae	Lysobacter enzymogenes, Streptomyces spp.,
				Verticillium biguttatum + Gliocladium roseum +
				Azotobacter chroococcum, Trichoderma spp.,
				non pathogenic Rhizoctonia spp.

Table 1 .7

 1 Resistance of wild potato cultivars toward pathogens

	Cultivar	Resistance	References
	Solanum vernei	Spongospora subterranea	Merz and Falloon, 2009
	Solanum acaule	Clavibacter michiganensis var.	Laurila et al., 2003
		sepedonicus
	Solanum commersonii	Ralstonia solanacearum	Kim-Lee et al., 2005
	Solanum bulbocastanum	Meloidogyne chitwoodi	Nitzan et al., 2009
	Snowder (Solanum
	etuberosum x Solanum
	berthaultii)

Table 1 .8 Cultural practices favourable to reduce disease development Pathogen Disease Favourable rotation Favourable fertilisation & amendments Tillage Favourable planting, lifting, and harvesting methods Pesticides Cultural systems Storage Ref

 1

	FUNGI & OOMYCETES
	Colletotrichum coccodes	Black dot	Long rotations		Mouldboard	Avoid water stress,	Increased by oxamyl		Dry curing	Hide and Read, 1991; Andrivon et al.,
			(>5years)		ploughing at	Early harvesting	Decreased by imazalil,		and/or	1997; Denner et al., 2000; Esfahani and
			With wheat, red		30 cm	Short interval	tolchlofos-methyl, mancozeb,		temperatures	Bak, 2004; Glais-Varlet et al., 2004;
			clover, alfalfa, rye,			between haulm	thiabendazole, fenpiclonil and		below 5°C	Cwalina-Ambroziak and Czajka, 2006;
			maize, orchard			destruction and	propiconazole			Nitzan et al., 2006
			grass, fallow, barley			harvesting
			Without yellow
			mustard, soybean,
			spring canola
	Fusarium spp.	Fusarium dry	No monoculture,	Composted	Minimum	Early harvesting	Chlorine dioxide, fenpiclonil and	Organic	Dry curing
		rots	minimum 3 years of	manure	tillage	Short interval	a mixture of thiabendazole and		and/or low
			rotation with red			between haulm	imazalil, mancozeb		temperatures
			clover			destruction and			below 4°C
						harvesting, wound
						healing

  ;Respiene and Minekiene, 2006

	Ralstonia solanacearum	Brown rot	Without solanaceous	Calcium	4 deep	Tri-potassium phosphate,	Depends on	Kishore et al., 1996; Mahmoud, 2007;
			plants.	superphospha	ploughings	bleaching powder	the soil type	Messiha et al., 2007
			With barley and flax	te	after harvest
	Streptomyces scabiei, S.	Common and	With lupin, soybean,	Ammonium	Subsoiling	Increased by oxamyl, 3% boric
	acidiscabiei, S.	netted scab	winter rye or	lignosulfate,		acid, streptomycin, streptomycin
	europeiscabiei		serradilla	potassium,		sulfate, daminozide, DL-
			Without: sugar beet,	phosphate,		ethionine
			carrots, pasture	compost,
				swine manure

  Streptomyces (Figures 2.1.N, 2.1.O, 2.1.P) simply because they produce visually similar puffy pustules[START_REF] Jouan | Les principales causes d'altérations superficielles des tubercules[END_REF].Secondly, typical blemishes, whose causes are known, but whose terminologies are unclear are considered. The confusion may come from the different common names used for the same typical blemish or from the same name used for different blemishes. Diseases caused by Streptomyces spp. are the most striking example. In Europe, and especially in France, the term common scab refers to at least two different diseases caused by Streptomyces spp. (Table2.2): i) raised scab, a raised blemish sometimes looking like craters on tuber surface (Figure2.1.O), and ii) netted scab, a symptom forming corky network (Figure2.1.D)(Bouchek-Mechiche et al., 2000b). Netted scab was only described in European countries. In North America or

, one may still mistakes between some types of powdery scab lesions caused by Spongospora subterranea (Figure 2.1.M) and some common scab symptoms caused by some strains of Asia, the term common scab describes one disease including several types of lesions. They are round to star-shaped, clearly defined corky lesions called pitted scab (Figure 2.1.N), raised or erumpent scab (Figure 2.1.O) and shallow scab (Figure 2.1.P)

Table 2 .1

 2 Description of potato tubers main blemishes

	Common name(s)	Description of tuber blemishes	Cited causes	Possible	References
	found in the				confusion
	literature
	Black dot		Diffuse brown spots with black punctuations	Colletotrichum coccodes	Silver scurf	Jouan, 1997; Stevenson
			called sclerotia on the upper surface			et al., 2001; FNPPPT et
							al., 2008; Wale et al.,
							2008
	Silver scurf		Clear silvery patches with very fine black	Helminthosporium solani	Black dot	Stevenson et al., 2001;
			punctuations and separation of the skin			FNPPPT et al., 2008;
			layers				Selman et al., 2008
	Powdery scab	Whitish pustules releasing a brownish and	Spongospora subterranea f.	pitted and raised	Jouan, 1997; Christ, 2001;
			powdery mass consisting of cytosori at	sp. subterranea	scab	FNPPPT et al., 2008;
			maturity				Mulder et al., 2008; Merz
							& Falloon, 2009
		Pitted scab		Roughly, star-shaped, shallow,		Powdery scab,
				sometimes crater-like lesions		tobacco necrosis
						virus
	Common scab	Raised scab / Erumpent scab Shallow scab /	Round to star-shaped, clearly defined lesions	Raised lesions Superficial lesions	Streptomyces scabiei, S. europaeiscabiei, S. stelliscabiei, S. acidiscabiei, S. turgidiscabiei S. reticuliscabiei	Russeting, polygonal lesions, shallow scab, netted scab, tobacco necrosis virus Russeting, polygonal lesions,	Faucher et al., 1993; Bouchek-Mechiche et al., 2000a; Bouchek-Mechiche et al., 2000b; Stevenson et al., 2001; Mulder et al., 2008
		Superficial				raised scab, netted
		scab				scab, tobacco
						necrosis virus
	Netted scab		Superficial net-like structures limited to	Streptomyces reticuliscabiei,
			typical polygonal scab plates often	S. europaeiscabiei
			associated with necrosis of all underground
			parts of the potato plant, including roots

Table 2 .2

 2 Nomenclature of potato scabs in the world and suggestion for a consensus

	Former nomenclatures Europe America and Asia		New nomenclature
				Pitted scab		Pitted scab
		Raised	Common	Raised scab		Common	Raised scab
	Common scabs	scab Netted scab	scab Not described Shallow scab	Potato scabs	scab Netted scab Shallow scab
	Russet scab (in Finland)	Russet scab		Russet scab

Table 2 .3

 2 Suggestion of a nomenclature for the superficial blemishes of potato tubers

	Name(s) used in literature

Table 3 .1

 3 Fungal and Streptomyces species isolated from the different blemishes

	Fungal or Streptomyces species Sclerotia	Polygonal lesions	Corky cracks	Corky spots	Star-like corky lesions	Enlarged lenticels	Skinning Russeting	Common scab	Netted scab	Other	Total
	Absidia glauca							1
	Alternaria arborescens		1
	A. citri					1
	A. longissima		1		1						1
	Alternaria sp.	3	17		4	4	4	3	1	3		2	41
	Bjerkandera adusta		1				1	1
	Bjerkandera sp.		3					1
	Ceratobasidium sp.				1
	Cercophora grandiuscula						1
	Cladosporium cladosporioides					1		1
	Cladosporium sp.	1	1			1	1			1
	Clonostachys rosea	2	12			1	6	1		5		27
	Colletotrichum coccodes	1	2	1			1			2
	Colletotrichum sp.		2
	Cylindrocarpon olidum		1
	Epicoccum nigrum					1				4	1	1
	Fusarium avenaceum					1

Table 3 .2

 3 Strains tested in the two bioassays conducted in infected soil

	Bioassays results
	Observed blemishes

Table 3 .3

 3 Strains tested in the bioassay conducted with single and co-inoculations under hydroponic conditions

	Observed blemishes in the bioassay	(a)
	Double inoculations	(b)
		Streptomyces spp.

Table 4 .1

 4 Collected soil samples, corresponding number of plants and graphic representations.

			Sanitary status of			Symbols for
		N° of plant	potato plants	progeny tubers	Number of samples	graphic representation (PCA)
	T1	C	Inter-row soil
		1	Half-healthy half-blemished	Healthy
		1		Blemished
		2	Half-healthy half-blemished	Healthy
		2		Blemished
		3	Half-healthy half-blemished	Healthy
		3		Blemished
		4	Half-healthy half-blemished	Healthy
		4		Blemished
		5	Half-healthy half-blemished	Healthy
		5		Blemished
		6	Healthy	Healthy
		7	Healthy	Healthy
		8	Healthy	Healthy
		9	Healthy	Healthy
		10	Healthy	Healthy
	T2	C	Inter-row soil
		11	Half-healthy half-blemished	Healthy
		11		Blemished
		12	Half-healthy half-blemished	Healthy
		12		Blemished
		13	Half-healthy half-blemished	Healthy
		13		Blemished
		14	Half-healthy half-blemished	Healthy
		14		Blemished
		15	Blemished	Blemished
		16	Blemished	Blemished
		17	Blemished	Blemished
		18	Blemished	Blemished

Table 4 .2

 4 Terminal restriction fragment (TRF) lengths of fungal strains.

	TRF length (bp) a	Species b
	155.14 ± 0.04	Rhizopus orzyae
	306.81 ± 0.04	Mortiella elongata
	524.76 ± 0.32	Verticillium dahliae
	556.94 ± 0.27	Absidia glauca
	558.36 ± 0.18	Fusidium griseum
	558.56 ± 0.20	Penicillium spp. (9)
	561.34 ± 0.37	Macrophomina phaseolina
	564.59 ± 0.08	Bjerkandera adusta
	565.19 ± 0.31	Rhizoctonia solani (3)
	580.06 ± 0.37	Colletotrichum sp.
	580.10 ± 0.19	Cylindrocarpon olidum
	580.86 ± 0.10	Trichoderma spp. (T. velutinum; Trichoderma spp.)
	580.94 ± 0.25	Cladosporium cladosporioides
	583.04 ± 0.14	Alternaria spp. (4)
	583.13 ± 0.28	Ulocladium capsicum
	583.22 ± 0.21	Phoma exigua
	583.40 ± 0.12	Epicoccum nigrum
	> 640	Cercophora grandiuscula, Clonostachys rosea, Fusarium

spp., Gliomastix murmorum, Microdochium bolleyi, Mucor spp., Neonectria radicicola, Plectosphaerella cucumerina, Stereum rugosum, Trichocladium asperum, Trichoderma tomentosum a values are mean of three replicates TRF > 640 bp are out of the range of detection b Numbers in brackets indicate the number of strains analyzed when it was more than 1.

Table 5 .1

 5 Isolates of Rhizoctonia solani analyzed in this study

	Anastomosis	Strains	MIAE	Geographical origin		Host of origin	ITS	tef-1α	Two-
	group		accession number a			(potato cultivar)	sequence type b	sequence type b	locus type
	2-1	0722-092 2 B PDA	MIAE00269	France	Côtes d'Armor	Juliette	m1	m1	1
	2-1	0629-011 1 WA	MIAE00231	France	Finistère	Rosanna	p1	m2	2
	2-1	0680-006 2 Aβ WA	MIAE00208	France	Somme	Hybride	m2	p1	3
	2-1	0680-006 1 PDA	MIAE00262	France	Somme	Hybride	m2	p1	3
	2-1	0680-006 1 WA	MIAE00263	France	Somme	Hybride	m2	p1	3
	2-1	0680-006 2 A PDA	MIAE00264	France	Somme	Hybride	m2	p1	3
	2-1	0680-006 2 Aα WA	MIAE00265	France	Somme	Hybride	m2	p1	3
	2-1	0680-006 2 Bβ WA	MIAE00266	France	Somme	Hybride	m2	p1	3
	2-1	173-4	MIAE00348	Finland		Unknown	Nd	m1
	2-1	R114	MIAE00349	Finland		Unknown	Nd	p2
	2-1	R25	MIAE00357	Finland		Unknown	m1	p3	4
	2-1	0799-001 2 N WA	MIAE00189	Morocco		Unknown	p2	p4	5
	2-1	Y25	MIAE00350	United Kingdom		Unknown	m2	m3	6
	3	0602-001 1 B PDA	MIAE00072	France	Aisne	Unknown	p3	p5	7
	3	0722-001 1 B PDA	MIAE00267	France	Côtes d'Armor	Charlotte	m3	m4	8
	3	0722-001 2 A PDA	MIAE00268	France	Côtes d'Armor	Charlotte	m4	p6	9
	3	0628-006 1 A WA	MIAE00150	France	Eure-et-Loir	Amandine	m5	p7
	3	0628-006 3 A WA	MIAE00152	France	Eure-et-Loir	Amandine	p4	Nd
	3	0628-006 3 A PDA	MIAE00219	France	Eure-et-Loir	Amandine	p4	Nd
	3	0728-012 PDA	MIAE00270	France	Eure-et-Loir	Amandine	p5	p8
	3	0728-091 A PDA	MIAE00271	France	Eure-et-Loir	Ditta	p3	Nd
	3	0629-049 2 WA	MIAE00006	France	Finistère	Juliette	p6	p9
	3	0629-033 2 B WA	MIAE00082	France	Finistère	Juliette	m6	m4
	3	0629-036 1 A WA	MIAE00083	France	Finistère	Juliette	m4	p10
	3	0629-038 3 A WA	MIAE00087	France	Finistère	Samba	m7	p9
	3	0629-039 2 A WA	MIAE00090	France	Finistère	Amandine	p7	Nd
	3	0629-040 2 A WA	MIAE00092	France	Finistère	Samba	p8	p9
	3	0629-040 2 A PDA	MIAE00093	France	Finistère	Samba	p8	p9
	3	0629-004 1A WA	MIAE00164	France	Finistère	Spunta	m8	p11
	3	0629-014 3 WA	MIAE00165	France	Finistère	Chérie	p9	p9
	3	0629-023 1 A PDA	MIAE00170	France	Finistère	Charlotte	m6	p12
	3	0629-030 2 PDA	MIAE00176	France	Finistère	Samba	m5	Nd
	3	0629-055 3 WA	MIAE00179	France	Finistère	Pamela	p10	p11
	3	0629-059 3 PDA	MIAE00185	France	Finistère	Hybride	m7	p13
	3	0629-004 1 B WA	MIAE00222	France	Finistère	Spunta	p4	m5
	3	0629-004 3 WA	MIAE00224	France	Finistère	Spunta	p9	m5
	3	0629-004 4 WA	MIAE00225	France	Finistère	Spunta	p10	p14
	3	0629-005 1 A PDA	MIAE00226	France	Finistère	Spunta	m5	m4
	3	0629-005 1 WA	MIAE00227	France	Finistère	Spunta	m5	m4
	3	0629-005 3 B PDA	MIAE00228	France	Finistère	Spunta	p11	p15
	3	0629-005 3 WA	MIAE00229	France	Finistère	Spunta	p12	p15
	3	0629-014 1 WA	MIAE00232	France	Finistère	Chérie	m5	p9
	3	0629-014 2 WA	MIAE00233	France	Finistère	Chérie	p5	p9
	3	0629-017 4 WA	MIAE00235	France	Finistère	Charlotte	p8	p16
	3	0629-021 1 A WA	MIAE00236	France	Finistère	Atlas	m4	m4
	3	0629-023 1 B WA	MIAE00237	France	Finistère	Charlotte	m6	m4
	3	0629-023 2 B WA	MIAE00238	France	Finistère	Charlotte	m6	m4
	3	0629-030 2 A WA	MIAE00239	France	Finistère	Samba	m5	p9
	3	0629-038 1 A PDA	MIAE00240	France	Finistère	Samba	p4	m5
	3	0629-038 1 WA	MIAE00241	France	Finistère	Samba	p4	m5
	3	0629-038 3 A PDA	MIAE00242	France	Finistère	Samba	m7	p17
	3	0629-038 3 B WA	MIAE00243	France	Finistère	Samba	m7	p9
	3	0629-049 1 A PDA	MIAE00248	France	Finistère	Juliette	p13	p18
	3	0629-049 1 Bα PDA	MIAE00249	France	Finistère	Juliette	m5	p19
	3	0629-051 1 A WA	MIAE00250	France	Finistère	Marine	p8	p20
	3	0629-055 2 PDA	MIAE00251	France	Finistère	Pamela	m5	p14

  .....GT........... m6 A........A.C........TG...........A....G.T.................A............................GT........... m7 A........A.C........TG...........A....G.T..............................................GT........... m8 A........A.C........TG...........A....G.T..............................................GT........... m9 A........A.C........TG...........A....G.T..............................................GT........... AG 5 m10 A...................-G...............CT.....................-.......................C............... AG 2-1 p1 .......Y............-G....T.............................T..............................G............ p2 ....................-G....T....C.......................................................G............ AG 3 p3 A........A.C........TG...........A....G.T.................W............................GT........... p4 A........A.C........TG...........A....G.T.................W............................GT........... p5 A........A.S........TG...........A....G.T..............................................GT........... p6 A........A.C........TG...........A....G.T..............................................GT........... p7 A........A.C........TG...........A....G.T.................W............................GT........... p8 A........A.S........TG...........A....G.T..............................................GT........... p9 A........A.C........TG...........A....G.T.................A............................GT........... p10 A..

  ..... AG 2-1 p1 .................................................................................................. p2 .................................................................................................. AG 3 p3 .........................A.....................................A.................................. p4 .........................A.....................................A.................................. p5 .........................A.....................................A.................................. p6 .........................A.....................................A.................................. p7 .........................A.....................................A.................................. p8 .........................A.....................................A.................................. p9 .........................A.....................................A..................................

  |....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AG 2-1 m1 CATTGAAAAGTTCGAGAAGGAAGCTGCTGAGCTCGGCAAGGGCTCCTTCAAGTATGGTGCGTACGCATATACTACTCGATCGA--CCGCCACTGATTAGT m2 ...C..........................................................G.............T.G....CCGT--........... m3 ...C................................A.........................G...............G....--TT.TT.......... AG 3 PT m4 G.........................................T...................G......C...G....C....GTTT........C.... m5 G.........................................T...................G......C...G....CC...GTTT..T.....C.... m6 G.........................................T...................G......C...G....C....GTTT..T.....C.... m7 G.........................................T...................G..........G....C....GT.T..T.....C.... AG 2-1 p1 ...C................................A.........................G...............G....--TT.TT......Y...

	10	20	30	40	50	60	70	80	90	100
	....

Russeting, polygonal lesions, russet scab[START_REF] Bukhalid | Plant pathogenicity in the genus Streptomyces[END_REF][START_REF] Bouchek-Mechiche | Streptomyces species isolated from potato scabs in France: numerical analysis of "Biotype-100" carbon source assimilation data[END_REF] Bouchek-Mechiche et al., 2000a; Scholte, 2005;[START_REF] Fnpppt | GNIS and ARVALIS-Institut du végétal. Guide pratique: Maladies, ravageurs et désordres de la pomme de terre[END_REF] Mulder et al., 2008

Zorn W. Potassium deficiency in potatoes.Kartoffelbau. 1995, 46 (2): 50-51.

Remerciements

Acknowledgements

Marie Fiers was financially supported by a PhD funding from the National Association of Technical Research (ANRT) (CIFRE n°1085/2006.

This work was part of a Program of Collaborative Research (PRC) between Bretagne

Plants and Germicopa, subsidized by the Regional Council of Brittany.

Acknowledgements

The authors wish to thank all technical partners, namely Pom' Alliance and Germicopa Seed Production, for supplying tuber samples, Bretagne Plants for supplying strains T FSa and T FSC1 and INRA, Station de Pathologie Végétale, Le Rheu (France) for supplying the strains 9529b and i4 (9729-1). We also thank H. Friberg for comments on the manuscript. Marie Fiers was financially supported by a PhD funding from the National Association of Technical Research (ANRT) (CIFRE n°1085/2006). This work was part of a wider project: Program of Collaborative Research (PRC) between Bretagne Biotechnologie Végétale (BBV), Bretagne Plants and Germicopa, funded by the Region Brittany.

Acknowledgments

The authors thank Jόzefa Kapsa, Brice Dupuis, Jean-Marie Torche, James Woodhall, Jari Valkonen, and Shiro Kuninaga for providing R. solani isolates.

Marie Fiers was financially supported by a PhD funding from the National Association of Technical Research (ANRT) (CIFRE n°1085/2006.

This work was part of a Program of Collaborative Research (PRC) between Bretagne

Plants and Germicopa, subsidized by the Regional Council of Brittany.

Je veux remercier tout d

Earthworms and nematodes favour pathogens mobility by transporting them through the soil [START_REF] Jensen | Interrelations of nematodes and other organisms in disease complexes. International Potato Center. Report of the 2nd planning conference on the developments in the control of nematode pests of potatoes[END_REF] (Table 1.6). Nematodes enhance potato diseases because they act as vectors of the pathogens. They also enhance the diseases either by facilitating the development of other pathogens -acting as mechanical wound agents and providers of necrotic tissues for pathogen penetration or nutrition -or by benefiting of their attacks as opportunistic microorganisms [START_REF] Jensen | Interrelations of nematodes and other organisms in disease complexes. International Potato Center. Report of the 2nd planning conference on the developments in the control of nematode pests of potatoes[END_REF].

Aknowledgements

The authors wish to thank Jean-Michel Gravoueille and Bernard Quéré for supplying photographs.

Submitted in FEMS Microbiology ecology

Aknowledgement

The authors wish to thank Jean-Philippe Guillermic for providing his potato field.

Marie Fiers was financially supported by a PhD funding from the National Association of Technical Research (ANRT) (CIFRE n°1085/2006.

This work was part of a Program of Collaborative Research (PRC) between Bretagne

Plants and Germicopa, subsidized by the Regional Council of Brittany.

Chapter 5

Genetic diversity of Rhizoctonia solani associated with potato tubers in France

Chapter 5

Genetic diversity of Rhizoctonia solani associated with potato tubers in France

Marie Fiers, Véronique Edel-Hermann, Cécile Héraud, Nadine Gautheron, Catherine

Chatot, Yves Le Hingrat, Karima Bouchek-Mechiche and Christian Steinberg

Submitted in Mycologia

Abstract

The soil borne fungus Rhizoctonia solani is a pathogen for many plants and causes severe damages in crops all around the world. Strains of R. solani from the anastomosis group (AG) 3 attack potatoes that lead to great yield losses and to the downgrading of the production. The study of the genetic diversity of the strains of R. Our results showed that superficial blemish occurrences depended on abiotic factors and that some potato varieties are particularly susceptible to some blemishes. Abiotic factors may also impact biotic ones which enhances blemish formation. Integrated management of soil characteristics, cultural practices and the choice of the varieties are of major importance to control blemishes on the surface of potato tubers and to warranty the good cosmetic quality of the production.