Botrytis cinerea is able to build-up resistance to pyrrolnitrin, an antibiotic produced by divers... more Botrytis cinerea is able to build-up resistance to pyrrolnitrin, an antibiotic produced by diverse biocontrol agents, possibly compromising the durability of this method of disease control. The development of two near-isogenic lines of B. cinerea differing in their level of resistance to pyrrolnitrin was compared in tomato plants and on PDA medium. In tomato plants, significant differences in the percentage of infected petioles 1 day after inoculation and in symptom progression on petioles and stems were observed between the resistant mutant and the sensitive wild-type parent, suggesting a difference in their level of aggressiveness. Cytohistological investigations revealed that conidia of both near-isogenic lines germinated 6 h after inoculation and mycelium developed within petiole tissues 12 h after inoculation. However, while the wild-type parent isolate spread throughout the petiole and rapidly invaded the stem tissues via the leaf-abscission zone 72 h after inoculation, the pyrrolnitrin-resistant mutant failed to extend beyond petiole tissues to invade the stem. Moreover, 72 h after inoculation, the mycelial development of the pyrrolnitrin-resistant mutant was accompanied by abnormal glycogen accumulation and chlamydospore-like cell formation. In contrast, wild-type parent mycelium was normally structured with intensive colonization of stem tissues. Additionally, on PDA medium the mycelium of the pyrrolnitrin-resistant mutant was less vigorous than the wild-type isolate. These results suggest that the acquisition of pyrrolnitrin-resistance in B. cinerea is accompanied by changes in mycelial structure and reduction in mycelial growth, leading to a noticeable loss of aggressiveness on tomato plants.
The objective of this work was to estimate the risk of a decrease in the efficacy of biocontrol a... more The objective of this work was to estimate the risk of a decrease in the efficacy of biocontrol as a result of selection pressure exerted by biocontrol agents on Botrytis cinerea, focusing on pyrrolnitrin, an antibiotic identified in diverse biocontrol agents having an effect on B. cinerea. To evaluate a possible decrease in sensitivity to pyrrolnitrin, 10 successive generations of five isolates of B. cinerea were produced in vitro in the presence of a sublethal dose (10 μg L−1) of the antibiotic. For one isolate, a significant reduction in the sensitivity to pyrrolnitrin at the fifth generation was observed with a resistance factor of c. 11. The production of 10 additional generations for four of these isolates, with increasing doses of pyrrolnitrin (100–4000 μg L−1), resulted in the development of variants of B. cinerea with high levels of resistance to the antibiotic (RF > 1000) and a reduced sensitivity in vitro to a pyrrolnitrin-producing bacterium. Reverse adaptation of resistant variants after 10 additional generations in the absence of selection pressure was not observed, suggesting stability of the resistance. Comparison of the pyrrolnitrin-resistant generations and their sensitive parental isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin resulted in a high fitness cost. Mycelial growth was reduced between 1·7 to 3·6 times and sporulation reduced 3·8 to 6·6 times that of sensitive parental isolates. Similarly, aggressiveness was 7 to 10 and 3 to 10 times lower for resistant isolates on tomato and apple, respectively. This study provides evidence that a fungal plant pathogen is able to gradually build up resistance to an antibiotic produced by a biocontrol agent.
Botrytis cinerea, Oidium neolycopersici and Bemisia tabaci are among the most important pests enc... more Botrytis cinerea, Oidium neolycopersici and Bemisia tabaci are among the most important pests encountered on tomato in greenhouses. In order to limit their development, biological methods of control have been elaborated. To protect pruning wounds against B. cinerea at crop level, the fungus Microdochium dimerum has shown a high efficacy. The formulated plant extract from the giant knotweed Reynoutria sachalinensis (Milsana) has shown a high efficacy to control powdery mildew of tomato. The myco-insecticide based on Lecanicillium muscarium (Mycotal) appears to be a promising biological control agent against whiteflies. The use of these products in a context of integrated protection of tomato requires that their efficacy is not altered when applied together. The objective of the present study was then to evaluate the compatibility of M. dimerum and plant extract with L. muscarium and vice versa. Compatibility tests were conducted on potted plants in controlled conditions. Mycotal had no significant effect on the susceptibility of tomato to either B. cinerea or O. neolycopersici, and neither M. dimerum nor Milsana had any effect on the susceptibility of tomato to infestation by B. tabaci. The efficacy of Mycotal was not altered by application of M. dimerum or Milsana, and the efficacy of M. dimerum and Milsana remained unaffected by application o f Mycotal. These results suggest that these biological products could be used together to intervene in tomato greenhouses.
ABSTRACT Partial resistance to downy mildew (Pseudoperonospora cubensis) and complete resistance ... more ABSTRACT Partial resistance to downy mildew (Pseudoperonospora cubensis) and complete resistance to powdery mildew (Podosphaera xanthii races 1, 2, 3, and 5 and Golovinomyces cichoracearum race 1) were studied using a recombinant inbred line population between 'PI 124112' (resistant to both diseases) and 'Védrantais' (susceptible line). A genetic map of melon was constructed to tag these resistances with DNA markers. Natural and artificial inoculations of Pseudoperonospora cubensis were performed and replicated in several locations. One major quantitative trait loci (QTL), pcXII.1, was consistently detected among the locations and explained between 12 to 38% of the phenotypic variation for Pseudoperonospora cubensis resistance. Eight other Pseudoperonospora cubensis resistance QTL were identified. Artificial inoculations were performed with several strains of four races of Podosphaera xanthii and one race of G. cichoracearum. Two independent major genes, PmV.1 and PmXII.1, were identified and shown to be involved in the simple resistance to powdery mildew. Three digenic epistatic interactions involving four loci were detected for two races of Podosphaera xanthii and one race of G. cichoracearum. Co-localization between PmV.1, resistance genes, and resistance genes homologues was observed. Linkage between the major resistance QTL to Pseudoperonospora cubensis, pcXII.1, and one of the two resistance genes to powdery mildew, PmXII.1, was demonstrated.
... GISELE LAGUERRE,' MARC BARD IN,^ AND NOELLE AMARGER Institut national de la ... more ... GISELE LAGUERRE,' MARC BARD IN,^ AND NOELLE AMARGER Institut national de la recherche agronomique, Laboratoire de microbiologie des sols, 17, rue Sully, B V 1540, 21034 Dijon CPdex, France ... MSDJ MSDJ USDA USDA E. Martinez ORS MSDJ MSDJ USDA ...
The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol a... more The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient.
Epidemics of powdery mildew due to Leveillula taurica is an increasing problem in pepper producti... more Epidemics of powdery mildew due to Leveillula taurica is an increasing problem in pepper production areas, particularly in coastal regions or greenhouse cultivation. The highly resistant genitor 'H3' was submitted to genetic analysis and QTL mapping in order to promote the introgression of its oligogenic resistance into large and sweet-fruited cultivars. The doubled-haploid progeny from the cross 'H3' (resistant) by 'Vania' (susceptible) was tested for resistance under both natural field infection and artificial inoculation tests, and QTL detection was compared for those two methods. Seven genomic regions including additive QTLs and epistatic interactions were detected, explaining altogether the major part of genotypic variance. Two genomic regions were common to both the evaluation methods, whereas other QTLs were method-specific, reflecting the environment dependence of powdery mildew epidemics. Orthologies with tomato genomic regions carrying resistance genes to L. taurica and Oidium lycopersicum were revealed by comparative mapping with pepper. Tight linkages between the detected QTLs and virus resistance or fruit color traits in pepper were also shown, which adds to the agronomic importance of these regions in pepper breeding programs.
The behaviour of cucurbit powdery mildews (Podosphaera xanthii and Golovinomyces cichoracearum) a... more The behaviour of cucurbit powdery mildews (Podosphaera xanthii and Golovinomyces cichoracearum) and tomato powdery mildew (Oidium neolycopersici) infesting detached cotyledons of Lagenaria leucantha cv. ‘Minibottle’ was studied in order to develop an easy culture method for pure inoculum production. High spore production was found with a combination of mannitol (0.1 m), sucrose (0.02 m) and agar (8 g l−1) in the cotyledon survival medium. Sporulation on cotyledons and viability of conidia were affected by the age of culture for the three species of powdery mildew tested. The age of cotyledons had also an impact of the spore production. This method was used to produce large amounts of inoculum for P. xanthii, G. cichoracearum and O. neolycopersici and enable the development of other species of powdery mildew like Leveillula taurica. Freezing conidia in liquid nitrogen enabled the long-term conservation of P. xanthii without any loss of virulence. The same method was unsuccessful with G. cichoracearum, and L. taurica and partly successful with O. neolycopersici.
Understanding the causes of population subdivision is of fundamental importance, as studying barr... more Understanding the causes of population subdivision is of fundamental importance, as studying barriers to gene flow between populations may reveal key aspects of the process of adaptive divergence and, for pathogens, may help forecasting disease emergence and implementing sound management strategies. Here, we investigated population subdivision in the multihost fungus Botrytis cinerea based on comprehensive multiyear sampling on different hosts in three French regions. Analyses revealed a weak association between population structure and geography, but a clear differentiation according to the host plant of origin. This was consistent with adaptation to hosts, but the distribution of inferred genetic clusters and the frequency of admixed individuals indicated a lack of strict host specificity. Differentiation between individuals collected in the greenhouse (on Solanum) and outdoor (on Vitis and Rubus) was stronger than that observed between individuals from the two outdoor hosts, probably reflecting an additional isolating effect associated with the cropping system. Three genetic clusters coexisted on Vitis but did not persist over time. Linkage disequilibrium analysis indicated that outdoor populations were regularly recombining, whereas clonality was predominant in the greenhouse. Our findings open up new perspectives for disease control by managing plant debris in outdoor conditions and reinforcing prophylactic measures indoor.
ABSTRACT Gray mold, caused by Botrytis cinerea, is a common threat for greenhouse production of t... more ABSTRACT Gray mold, caused by Botrytis cinerea, is a common threat for greenhouse production of tomatoes. Control of this disease can be difficult even with chemical treatments, and alternative methods are needed. Nitrogen (N) fertilization is known to modify the impact of pathogens on plants. However, there is scarce knowledge about the effect of fertilization on the efficacy of biocontrol. Here, we studied the effect of N fertilization on biocontrol agents Trichoderma atroviride and Microdochium dimerum that protect tomato against B. cinerea. Plants were grown for 2 months in a greenhouse with a soil-less drip irrigation system. Differential N fertilization (five concentrations of nitrate) was applied for the last 4 weeks prior to leaf pruning, biocontrol agent application, and B. cinerea inoculation. Results show that increasing N fertilization up to 10 mmol/L reduced disease by half for controls. High N fertilization also increased biocontrol, with a protection index rising from nearly 0 to up to 100 % depending on the biocontrol agent and the pressure of the pathogen. Indeed, high N fertilization delayed stem symptoms and slowed lesion expansion. To our knowledge, this is the first report of an effect of N fertilization on the efficacy of biocontrol against an airborne disease. Therefore, adapting N fertilization is a promising technique to protect greenhouse tomato.
Clarifying the role of precipitation in microbial dissemination is essential for elucidating the ... more Clarifying the role of precipitation in microbial dissemination is essential for elucidating the processes involved in disease emergence and spread. The ecology of Pseudomonas syringae and its presence throughout the water cycle makes it an excellent model to address this issue. In this study, 90 samples of freshly fallen rain and snow collected from 2005-2011 in France were analyzed for microbiological composition. The conditions favorable for dissemination of P. syringae by this precipitation were investigated by (i) estimating the physical properties and backward trajectories of the air masses associated with each precipitation event and by (ii) characterizing precipitation chemistry, and genetic and phenotypic structures of populations. A parallel study with the fungus Botrytis cinerea was also performed for comparison. Results showed that (i) the relationship of P. syringae to precipitation as a dissemination vector is not the same for snowfall and rainfall, whereas it is the same for B. cinerea and (ii) the occurrence of P. syringae in precipitation can be linked to electrical conductivity and pH of water, the trajectory of the air mass associated with the precipitation and certain physical conditions of the air mass (i.e. temperature, solar radiation exposure, distance traveled), whereas these predictions are different for B. cinerea. These results are pertinent to understanding microbial survival, emission sources and atmospheric processes and how they influence microbial dissemination.
Although Botrytis cinerea is known for its ability to produce high amounts of spores on diseased ... more Although Botrytis cinerea is known for its ability to produce high amounts of spores on diseased plants, enabling it to complete rapidly numerous developmental cycles in favorable environments, population genetics studies of this fungus indicate enormous diversity and limited clonal spread. Here, we report an exception to this situation in the settings of commercial tomato greenhouses. The genotypic characterization of 712 isolates collected from the air and from diseased plants, following the development of gray mold epidemics in four greenhouses in southern France, revealed the presence of a few predominant genotypes in a background of highly diverse populations. The comparison of genotypic profiles for isolates collected in the air or on the plants was compatible with the hypothesis of an entry in the greenhouse of substantial amounts of inoculum from the outside environment but it also highlighted the importance of secondary inoculum produced within the crop. The overall results of this work suggest that sporulation could be an important target for disease management strategies in the greenhouse.
Strains of Sphaerotheca fuliginea, one of the causal agents of powdery mildew of cucurbits, were ... more Strains of Sphaerotheca fuliginea, one of the causal agents of powdery mildew of cucurbits, were examined for differences in virulence, mating type and DNA polymorphism. The 28 strains were chosen to be diverse according to host and geographic origin. Characterization of virulence phenotypes was based on the expression of symptoms on 4 species of cucurbits and 6 cultivars of melon. Two pathotypes, capable of attacking either cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ and squash cv. ‘Diamant’ or cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ were observed. Tests on melon cultivars revealed 3 races. In tests of sexual compatibility with reference strains, heterothallism was observed for all isolates. Frequency of the two mating types differed significantly in the population. DNA polymorphism was determined both by restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers (ITS) and 5.8S DNA amplified by the polymerase chain reaction and by random amplified polymorphic DNA (RAPD). For any one of the 11 restriction enzymes tested all strains presented an identical pattern of ITS RFLP. RAPD analysis, using 22 primers which provided reproducible patterns, revealed a relatively low degree of polymorphism. Furthermore, cluster analysis based on RAPD data (152 markers) did not separate groups within the species S. fuliginea. No association could be found between virulence, mating type, geographical and host origin and RAPD patterns. The lack of association between phenotypic and molecular markers and the close fit to linkage equilibrium for the characters examined suggest that recombination may play a role in populations of S. fuliginea.
Botrytis cinerea is able to build-up resistance to pyrrolnitrin, an antibiotic produced by divers... more Botrytis cinerea is able to build-up resistance to pyrrolnitrin, an antibiotic produced by diverse biocontrol agents, possibly compromising the durability of this method of disease control. The development of two near-isogenic lines of B. cinerea differing in their level of resistance to pyrrolnitrin was compared in tomato plants and on PDA medium. In tomato plants, significant differences in the percentage of infected petioles 1 day after inoculation and in symptom progression on petioles and stems were observed between the resistant mutant and the sensitive wild-type parent, suggesting a difference in their level of aggressiveness. Cytohistological investigations revealed that conidia of both near-isogenic lines germinated 6 h after inoculation and mycelium developed within petiole tissues 12 h after inoculation. However, while the wild-type parent isolate spread throughout the petiole and rapidly invaded the stem tissues via the leaf-abscission zone 72 h after inoculation, the pyrrolnitrin-resistant mutant failed to extend beyond petiole tissues to invade the stem. Moreover, 72 h after inoculation, the mycelial development of the pyrrolnitrin-resistant mutant was accompanied by abnormal glycogen accumulation and chlamydospore-like cell formation. In contrast, wild-type parent mycelium was normally structured with intensive colonization of stem tissues. Additionally, on PDA medium the mycelium of the pyrrolnitrin-resistant mutant was less vigorous than the wild-type isolate. These results suggest that the acquisition of pyrrolnitrin-resistance in B. cinerea is accompanied by changes in mycelial structure and reduction in mycelial growth, leading to a noticeable loss of aggressiveness on tomato plants.
The objective of this work was to estimate the risk of a decrease in the efficacy of biocontrol a... more The objective of this work was to estimate the risk of a decrease in the efficacy of biocontrol as a result of selection pressure exerted by biocontrol agents on Botrytis cinerea, focusing on pyrrolnitrin, an antibiotic identified in diverse biocontrol agents having an effect on B. cinerea. To evaluate a possible decrease in sensitivity to pyrrolnitrin, 10 successive generations of five isolates of B. cinerea were produced in vitro in the presence of a sublethal dose (10 μg L−1) of the antibiotic. For one isolate, a significant reduction in the sensitivity to pyrrolnitrin at the fifth generation was observed with a resistance factor of c. 11. The production of 10 additional generations for four of these isolates, with increasing doses of pyrrolnitrin (100–4000 μg L−1), resulted in the development of variants of B. cinerea with high levels of resistance to the antibiotic (RF > 1000) and a reduced sensitivity in vitro to a pyrrolnitrin-producing bacterium. Reverse adaptation of resistant variants after 10 additional generations in the absence of selection pressure was not observed, suggesting stability of the resistance. Comparison of the pyrrolnitrin-resistant generations and their sensitive parental isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin resulted in a high fitness cost. Mycelial growth was reduced between 1·7 to 3·6 times and sporulation reduced 3·8 to 6·6 times that of sensitive parental isolates. Similarly, aggressiveness was 7 to 10 and 3 to 10 times lower for resistant isolates on tomato and apple, respectively. This study provides evidence that a fungal plant pathogen is able to gradually build up resistance to an antibiotic produced by a biocontrol agent.
Botrytis cinerea, Oidium neolycopersici and Bemisia tabaci are among the most important pests enc... more Botrytis cinerea, Oidium neolycopersici and Bemisia tabaci are among the most important pests encountered on tomato in greenhouses. In order to limit their development, biological methods of control have been elaborated. To protect pruning wounds against B. cinerea at crop level, the fungus Microdochium dimerum has shown a high efficacy. The formulated plant extract from the giant knotweed Reynoutria sachalinensis (Milsana) has shown a high efficacy to control powdery mildew of tomato. The myco-insecticide based on Lecanicillium muscarium (Mycotal) appears to be a promising biological control agent against whiteflies. The use of these products in a context of integrated protection of tomato requires that their efficacy is not altered when applied together. The objective of the present study was then to evaluate the compatibility of M. dimerum and plant extract with L. muscarium and vice versa. Compatibility tests were conducted on potted plants in controlled conditions. Mycotal had no significant effect on the susceptibility of tomato to either B. cinerea or O. neolycopersici, and neither M. dimerum nor Milsana had any effect on the susceptibility of tomato to infestation by B. tabaci. The efficacy of Mycotal was not altered by application of M. dimerum or Milsana, and the efficacy of M. dimerum and Milsana remained unaffected by application o f Mycotal. These results suggest that these biological products could be used together to intervene in tomato greenhouses.
ABSTRACT Partial resistance to downy mildew (Pseudoperonospora cubensis) and complete resistance ... more ABSTRACT Partial resistance to downy mildew (Pseudoperonospora cubensis) and complete resistance to powdery mildew (Podosphaera xanthii races 1, 2, 3, and 5 and Golovinomyces cichoracearum race 1) were studied using a recombinant inbred line population between 'PI 124112' (resistant to both diseases) and 'Védrantais' (susceptible line). A genetic map of melon was constructed to tag these resistances with DNA markers. Natural and artificial inoculations of Pseudoperonospora cubensis were performed and replicated in several locations. One major quantitative trait loci (QTL), pcXII.1, was consistently detected among the locations and explained between 12 to 38% of the phenotypic variation for Pseudoperonospora cubensis resistance. Eight other Pseudoperonospora cubensis resistance QTL were identified. Artificial inoculations were performed with several strains of four races of Podosphaera xanthii and one race of G. cichoracearum. Two independent major genes, PmV.1 and PmXII.1, were identified and shown to be involved in the simple resistance to powdery mildew. Three digenic epistatic interactions involving four loci were detected for two races of Podosphaera xanthii and one race of G. cichoracearum. Co-localization between PmV.1, resistance genes, and resistance genes homologues was observed. Linkage between the major resistance QTL to Pseudoperonospora cubensis, pcXII.1, and one of the two resistance genes to powdery mildew, PmXII.1, was demonstrated.
... GISELE LAGUERRE,' MARC BARD IN,^ AND NOELLE AMARGER Institut national de la ... more ... GISELE LAGUERRE,' MARC BARD IN,^ AND NOELLE AMARGER Institut national de la recherche agronomique, Laboratoire de microbiologie des sols, 17, rue Sully, B V 1540, 21034 Dijon CPdex, France ... MSDJ MSDJ USDA USDA E. Martinez ORS MSDJ MSDJ USDA ...
The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol a... more The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient.
Epidemics of powdery mildew due to Leveillula taurica is an increasing problem in pepper producti... more Epidemics of powdery mildew due to Leveillula taurica is an increasing problem in pepper production areas, particularly in coastal regions or greenhouse cultivation. The highly resistant genitor 'H3' was submitted to genetic analysis and QTL mapping in order to promote the introgression of its oligogenic resistance into large and sweet-fruited cultivars. The doubled-haploid progeny from the cross 'H3' (resistant) by 'Vania' (susceptible) was tested for resistance under both natural field infection and artificial inoculation tests, and QTL detection was compared for those two methods. Seven genomic regions including additive QTLs and epistatic interactions were detected, explaining altogether the major part of genotypic variance. Two genomic regions were common to both the evaluation methods, whereas other QTLs were method-specific, reflecting the environment dependence of powdery mildew epidemics. Orthologies with tomato genomic regions carrying resistance genes to L. taurica and Oidium lycopersicum were revealed by comparative mapping with pepper. Tight linkages between the detected QTLs and virus resistance or fruit color traits in pepper were also shown, which adds to the agronomic importance of these regions in pepper breeding programs.
The behaviour of cucurbit powdery mildews (Podosphaera xanthii and Golovinomyces cichoracearum) a... more The behaviour of cucurbit powdery mildews (Podosphaera xanthii and Golovinomyces cichoracearum) and tomato powdery mildew (Oidium neolycopersici) infesting detached cotyledons of Lagenaria leucantha cv. ‘Minibottle’ was studied in order to develop an easy culture method for pure inoculum production. High spore production was found with a combination of mannitol (0.1 m), sucrose (0.02 m) and agar (8 g l−1) in the cotyledon survival medium. Sporulation on cotyledons and viability of conidia were affected by the age of culture for the three species of powdery mildew tested. The age of cotyledons had also an impact of the spore production. This method was used to produce large amounts of inoculum for P. xanthii, G. cichoracearum and O. neolycopersici and enable the development of other species of powdery mildew like Leveillula taurica. Freezing conidia in liquid nitrogen enabled the long-term conservation of P. xanthii without any loss of virulence. The same method was unsuccessful with G. cichoracearum, and L. taurica and partly successful with O. neolycopersici.
Understanding the causes of population subdivision is of fundamental importance, as studying barr... more Understanding the causes of population subdivision is of fundamental importance, as studying barriers to gene flow between populations may reveal key aspects of the process of adaptive divergence and, for pathogens, may help forecasting disease emergence and implementing sound management strategies. Here, we investigated population subdivision in the multihost fungus Botrytis cinerea based on comprehensive multiyear sampling on different hosts in three French regions. Analyses revealed a weak association between population structure and geography, but a clear differentiation according to the host plant of origin. This was consistent with adaptation to hosts, but the distribution of inferred genetic clusters and the frequency of admixed individuals indicated a lack of strict host specificity. Differentiation between individuals collected in the greenhouse (on Solanum) and outdoor (on Vitis and Rubus) was stronger than that observed between individuals from the two outdoor hosts, probably reflecting an additional isolating effect associated with the cropping system. Three genetic clusters coexisted on Vitis but did not persist over time. Linkage disequilibrium analysis indicated that outdoor populations were regularly recombining, whereas clonality was predominant in the greenhouse. Our findings open up new perspectives for disease control by managing plant debris in outdoor conditions and reinforcing prophylactic measures indoor.
ABSTRACT Gray mold, caused by Botrytis cinerea, is a common threat for greenhouse production of t... more ABSTRACT Gray mold, caused by Botrytis cinerea, is a common threat for greenhouse production of tomatoes. Control of this disease can be difficult even with chemical treatments, and alternative methods are needed. Nitrogen (N) fertilization is known to modify the impact of pathogens on plants. However, there is scarce knowledge about the effect of fertilization on the efficacy of biocontrol. Here, we studied the effect of N fertilization on biocontrol agents Trichoderma atroviride and Microdochium dimerum that protect tomato against B. cinerea. Plants were grown for 2 months in a greenhouse with a soil-less drip irrigation system. Differential N fertilization (five concentrations of nitrate) was applied for the last 4 weeks prior to leaf pruning, biocontrol agent application, and B. cinerea inoculation. Results show that increasing N fertilization up to 10 mmol/L reduced disease by half for controls. High N fertilization also increased biocontrol, with a protection index rising from nearly 0 to up to 100 % depending on the biocontrol agent and the pressure of the pathogen. Indeed, high N fertilization delayed stem symptoms and slowed lesion expansion. To our knowledge, this is the first report of an effect of N fertilization on the efficacy of biocontrol against an airborne disease. Therefore, adapting N fertilization is a promising technique to protect greenhouse tomato.
Clarifying the role of precipitation in microbial dissemination is essential for elucidating the ... more Clarifying the role of precipitation in microbial dissemination is essential for elucidating the processes involved in disease emergence and spread. The ecology of Pseudomonas syringae and its presence throughout the water cycle makes it an excellent model to address this issue. In this study, 90 samples of freshly fallen rain and snow collected from 2005-2011 in France were analyzed for microbiological composition. The conditions favorable for dissemination of P. syringae by this precipitation were investigated by (i) estimating the physical properties and backward trajectories of the air masses associated with each precipitation event and by (ii) characterizing precipitation chemistry, and genetic and phenotypic structures of populations. A parallel study with the fungus Botrytis cinerea was also performed for comparison. Results showed that (i) the relationship of P. syringae to precipitation as a dissemination vector is not the same for snowfall and rainfall, whereas it is the same for B. cinerea and (ii) the occurrence of P. syringae in precipitation can be linked to electrical conductivity and pH of water, the trajectory of the air mass associated with the precipitation and certain physical conditions of the air mass (i.e. temperature, solar radiation exposure, distance traveled), whereas these predictions are different for B. cinerea. These results are pertinent to understanding microbial survival, emission sources and atmospheric processes and how they influence microbial dissemination.
Although Botrytis cinerea is known for its ability to produce high amounts of spores on diseased ... more Although Botrytis cinerea is known for its ability to produce high amounts of spores on diseased plants, enabling it to complete rapidly numerous developmental cycles in favorable environments, population genetics studies of this fungus indicate enormous diversity and limited clonal spread. Here, we report an exception to this situation in the settings of commercial tomato greenhouses. The genotypic characterization of 712 isolates collected from the air and from diseased plants, following the development of gray mold epidemics in four greenhouses in southern France, revealed the presence of a few predominant genotypes in a background of highly diverse populations. The comparison of genotypic profiles for isolates collected in the air or on the plants was compatible with the hypothesis of an entry in the greenhouse of substantial amounts of inoculum from the outside environment but it also highlighted the importance of secondary inoculum produced within the crop. The overall results of this work suggest that sporulation could be an important target for disease management strategies in the greenhouse.
Strains of Sphaerotheca fuliginea, one of the causal agents of powdery mildew of cucurbits, were ... more Strains of Sphaerotheca fuliginea, one of the causal agents of powdery mildew of cucurbits, were examined for differences in virulence, mating type and DNA polymorphism. The 28 strains were chosen to be diverse according to host and geographic origin. Characterization of virulence phenotypes was based on the expression of symptoms on 4 species of cucurbits and 6 cultivars of melon. Two pathotypes, capable of attacking either cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ and squash cv. ‘Diamant’ or cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ were observed. Tests on melon cultivars revealed 3 races. In tests of sexual compatibility with reference strains, heterothallism was observed for all isolates. Frequency of the two mating types differed significantly in the population. DNA polymorphism was determined both by restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers (ITS) and 5.8S DNA amplified by the polymerase chain reaction and by random amplified polymorphic DNA (RAPD). For any one of the 11 restriction enzymes tested all strains presented an identical pattern of ITS RFLP. RAPD analysis, using 22 primers which provided reproducible patterns, revealed a relatively low degree of polymorphism. Furthermore, cluster analysis based on RAPD data (152 markers) did not separate groups within the species S. fuliginea. No association could be found between virulence, mating type, geographical and host origin and RAPD patterns. The lack of association between phenotypic and molecular markers and the close fit to linkage equilibrium for the characters examined suggest that recombination may play a role in populations of S. fuliginea.
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