BABA-induced pathogen resistance: a multi-omics analysis of the tomato response reveals a hyper-receptive status involving ethylene.

Prior exposure to microbial-associated molecular patterns or specific chemical compounds can promote plants into a primed state with stronger defence responses. ß-aminobutyric acid (BABA) is an endogenous stress metabolite that induces resistance protecting various plants towards diverse stresses. In this study, by integrating BABA-induced changes in selected metabolites with transcriptome and proteome data, we generated a global map of the molecular processes operating in BABA-induced resistance (BABA-IR) in tomato. BABA significantly restricts the growth of the pathogens Oidium neolycopersici and Phytophthora parasitica but not Botrytis cinerea. A cluster analysis of the upregulated processes showed that BABA acts mainly as a stress factor in tomato. The main factor distinguishing BABA-IR from other stress conditions was the extensive induction of signaling and perception machinery playing a key role in effective resistance against pathogens. Interestingly, the signalling processes and immune response activated during BABA-IR in tomato differed from those in Arabidopsis with substantial enrichment of genes associated with jasmonic acid (JA) and ethylene (ET) signalling and no change in Asp levels. Our results revealed key differences between the effect of BABA on tomato and other model plants studied until now. Surprisingly, salicylic acid (SA) is not involved in BABA downstream signalization whereas ET and JA play a crucial role.

Population Genomics Reveals Molecular Determinants of Specialization to Tomato in the Polyphagous Fungal Pathogen Botrytis cinerea in France.

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help to prevent the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the gray mold pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host of origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and nonsynonymous sites, and analyses of presence and absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.

Striking Similarities Between Botrytis cinerea From Non-agricultural and From Agricultural Habitats.

Investigations into life history of microorganisms that cause plant diseases have been limited mostly to contexts where they are in interaction with plants, and with cropped or otherwise managed vegetation. Therefore, knowledge about the diversity of plant pathogens, about potential reservoirs of inoculum and about the processes that contribute to their survival and adaptation is limited to these contexts. The agro-centric perspective of plant pathogen life histories is incoherent with respect to the capacity of many of them to persist as saprophytes on various substrates. In this context we have investigated the ubiquity of the broad host range necrotrophic fungus Botrytis cinerea, outside of agricultural settings and have determined if the populations in these natural habitats can be distinguished phenotypically and phylogenetically from populations isolated from diseased crops. Over a period of 5 years, we isolated B. cinerea from 235 samples of various substrates collected in France including rainfall, snowpack, river, and lake water, epilithic biofilms in mountain streams, leaf litter and plant debris, rock surfaces, bird feathers and healthy wild plants from outside of agricultural fields. All substrates except rock surfaces harbored B. cinerea leading us to establish a collection of purified strains that were compared to B. cinerea from diseased tomato, grapes and various other crops in France. Phylogenetic comparisons of 321 strains from crop plants and 100 strains from environmental substrates based on sequences of 9 microsatellite markers revealed that strains from crops and the environment could not be distinguished. Furthermore, the genetic diversity of strains outside of agriculture was just as broad as within agriculture. In tests to determine the aggressiveness of strains on tomato stems, the mean disease severity caused by strains from environmental substrates was statistically identical to the severity of disease caused by strains from tomato, but was significantly greater than the severity caused by strains from grape or other crops. Our results suggest that highly diverse populations of this plant pathogen persist outside of agriculture in association with substrates other than plants and that this part of their life history is compatible with its capacity to maintain its potential as plant pathogen.

Tomato root microbiota and Phytophthora parasitica-associated disease.

BACKGROUND: Interactions between pathogenic oomycetes and microbiota residing on the surface of the host plant root are unknown, despite being critical to inoculum constitution. The nature of these interactions was explored for the polyphagous and telluric species Phytophthora parasitica. RESULTS: Composition of the rhizospheric microbiota of Solanum lycopersicum was characterized using deep re-sequencing of 16S rRNA gene to analyze tomato roots either free of or partly covered with P. parasitica biofilm. Colonization of the host root surface by the oomycete was associated with a shift in microbial community involving a Bacteroidetes/Proteobacteria transition and Flavobacteriaceae as the most abundant family. Identification of members of the P. parasitica-associated microbiota interfering with biology and oomycete infection was carried out by screening for bacteria able to (i) grow on a P. parasitica extract-based medium (ii), exhibit in vitro probiotic or antibiotic activity towards the oomycete (iii), have an impact on the oomycete infection cycle in a tripartite interaction S. lycopersicum-P. parasitica-bacteria. One Pseudomonas phylotype was found to exacerbate disease symptoms in tomato plants. The lack of significant gene expression response of P. parasitica effectors to Pseudomonas suggested that the increase in plant susceptibility was not associated with an increase in virulence. Our results reveal that Pseudomonas spp. establishes commensal interactions with the oomycete. Bacteria preferentially colonize the surface of the biofilm rather than the roots, so that they can infect plant cells without any apparent infection of P. parasitica. CONCLUSIONS: The presence of the pathogenic oomycete P. parasitica in the tomato rhizosphere leads to a shift in the rhizospheric microbiota composition. It contributes to the habitat extension of Pseudomonas species mediated through a physical association between the oomycete and the bacteria.

Agrifood systems and the microbial safety of fresh produce: Trade-offs in the wake of increased sustainability.

Fresh produce has been a growing cause of food borne outbreaks world-wide prompting the need for safer production practices. Yet fresh produce agrifood systems are diverse and under constraints for more sustainability. We analyze how measures taken to guarantee safety interact with other objectives for sustainability, in light of the diversity of fresh produce agrifood systems. The review is based on the publications at the interface between fresh produce safety and sustainability, with sustainability defined by low environmental impacts, food and nutrition security and healthy life. The paths for more sustainable fresh produce are diverse. They include an increased use of ecosystem services to e.g. favor predators of pests, or to reduce impact of floods, to reduce soil erosion, or to purify run-off waters. In contrast, they also include production systems isolated from the environment. From a socio-economical view, sustainability may imply maintaining small tenures with a higher risk of pathogen contamination. We analyzed the consequences for produce safety by focusing on risks of contamination by water, soil, environment and live stocks. Climate change may increase the constraints and recent knowledge on interactions between produce and human pathogens may bring new solutions. Existing technologies may suffice to resolve some conflicts between ensuring safety of fresh produce and moving towards more sustainability. However, socio-economic constraints of some agri-food systems may prevent their implementation. In addition, current strategies to preserve produce safety are not adapted to systems relying on ecological principles and knowledge is lacking to develop the new risk management approaches that would be needed.

Is the efficacy of biological control against plant diseases likely to be more durable than that of chemical pesticides?

The durability of a control method for plant protection is defined as the persistence of its efficacy in space and time. It depends on (i) the selection pressure exerted by it on populations of plant pathogens and (ii) on the capacity of these pathogens to adapt to the control method. Erosion of effectiveness of conventional plant protection methods has been widely studied in the past. For example, apparition of resistance to chemical pesticides in plant pathogens or pests has been extensively documented. The durability of biological control has often been assumed to be higher than that of chemical control. Results concerning pest management in agricultural systems have shown that this assumption may not always be justified. Resistance of various pests to one or several toxins of Bacillus thuringiensis and apparition of resistance of the codling moth Cydia pomonella to the C. pomonella granulovirus have, for example, been described. In contrast with the situation for pests, the durability of biological control of plant diseases has hardly been studied and no scientific reports proving the loss of efficiency of biological control agents against plant pathogens in practice has been published so far. Knowledge concerning the possible erosion of effectiveness of biological control is essential to ensure a durable efficacy of biological control agents on target plant pathogens. This knowledge will result in identifying risk factors that can foster the selection of strains of plant pathogens resistant to biological control agents. It will also result in identifying types of biological control agents with lower risk of efficacy loss, i.e., modes of action of biological control agents that does not favor the selection of resistant isolates in natural populations of plant pathogens. An analysis of the scientific literature was then conducted to assess the potential for plant pathogens to become resistant to biological control agents.

Resistance to bio-insecticides or how to enhance their sustainability: a review.

After more than 70 years of chemical pesticide use, modern agriculture is increasingly using biological control products. Resistances to conventional insecticides are wide spread, while those to bio-insecticides have raised less attention, and resistance management is frequently neglected. However, a good knowledge of the limitations of a new technique often provides greater sustainability. In this review, we compile cases of resistance to widely used bio-insecticides and describe the associated resistance mechanisms. This overview shows that all widely used bio-insecticides ultimately select resistant individuals. For example, at least 27 species of insects have been described as resistant to Bacillus thuringiensis toxins. The resistance mechanisms are at least as diverse as those that are involved in resistance to chemical insecticides, some of them being common to bio-insecticides and chemical insecticides. This analysis highlights the specific properties of bio-insecticides that the scientific community should use to provide a better sustainability of these products.

Population structure and temporal maintenance of the multihost fungal pathogen Botrytis cinerea: causes and implications for disease management.

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.

Features of air masses associated with the deposition of Pseudomonas syringae and Botrytis cinerea by rain and snowfall.

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.

Remarkable predominance of a small number of genotypes in greenhouse populations of Botrytis cinerea.

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.

Functional and structural comparison of pyrrolnitrin- and iprodione-induced modifications in the class III histidine-kinase Bos1 of Botrytis cinerea.

Dicarboximides and phenylpyrroles are commonly used fungicides against plant pathogenic ascomycetes. Although their effect on fungal osmosensing systems has been shown in many studies, their modes-of-action still remain unclear. Laboratory- or field-mutants of fungi resistant to either or both fungicide categories generally harbour point mutations in the sensor histidine kinase of the osmotic signal transduction cascade.In the present study we compared the mechanisms of resistance to the dicarboximide iprodione and to pyrrolnitrin, a structural analogue of phenylpyrrole fungicides, in Botrytis cinerea. Pyrrolnitrin-induced mutants and iprodione-induced mutants of B. cinerea were produced in vitro. For the pyrrolnitrin-induced mutants, a high level of resistance to pyrrolnitrin was associated with a high level of resistance to iprodione. For the iprodione-induced mutants, the high level of resistance to iprodione generated variable levels of resistance to pyrrolnitrin and phenylpyrroles. All selected mutants showed hypersensitivity to high osmolarity and regardless of their resistance levels to phenylpyrroles, they showed strongly reduced fitness parameters (sporulation, mycelial growth, aggressiveness on plants) compared to the parental phenotypes. Most of the mutants presented modifications in the osmosensing class III histidine kinase affecting the HAMP domains. Site directed mutagenesis of the bos1 gene was applied to validate eight of the identified mutations. Structure modelling of the HAMP domains revealed that the replacements of hydrophobic residues within the HAMP domains generally affected their helical structure, probably abolishing signal transduction. Comparing mutant phenotypes to the HAMP structures, our study suggests that mutations perturbing helical structures of HAMP2-4 abolish signal-transduction leading to loss-of-function phenotype. The mutation of residues E529, M427, and T581, without consequences on HAMP structure, highlighted their involvement in signal transduction. E529 and M427 seem to be principally involved in osmotic signal transduction.

Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic fungi.

Understanding the mechanisms responsible for divergence and specialization of pathogens on different hosts is of fundamental importance, especially in the context of the emergence of new diseases via host shifts. Temporal isolation has been reported in a few plants and parasites, but is probably one of the least studied speciation processes. We studied whether temporal isolation could be responsible for the maintenance of genetic differentiation among sympatric populations of Ampelomyces, widespread intracellular mycoparasites of powdery mildew fungi, themselves plant pathogens. The timing of transmission of Ampelomyces depends on the life cycles of the powdery mildew species they parasitize. Internal transcribed spacer sequences and microsatellite markers showed that Ampelomyces populations found in apple powdery mildew (Podosphaera leucotricha) were genetically highly differentiated from other Ampelomyces populations sampled from several other powdery mildew species across Europe, infecting plant hosts other than apple. While P. leucotricha starts its life cycle early in spring, and the main apple powdery mildew epidemics occur before summer, the fungal hosts of the other Ampelomyces cause epidemics mainly in summer and autumn. When two powdery mildew species were experimentally exposed to Ampelomyces strains naturally occurring in P. leucotricha in spring, and to strains naturally present in other mycohost species in autumn, cross-infections always occurred. Thus, the host-related genetic differentiation in Ampelomyces cannot be explained by narrow physiological specialization, because Ampelomyces were able to infect powdery mildew species they were unlikely to have encountered in nature, but instead appears to result from temporal isolation.

Peach Rusty Spot Is Caused by the Apple Powdery Mildew Fungus, Podosphaera leucotricha.

Peach rusty spot, an economically important disease of peach (Prunus persica var. persica), appears as necrotic spots on fruit. The etiology of the disease is still not well understood, although it has long been suspected that the causal agent is the apple powdery mildew pathogen, Podosphaera leucotricha. This work confirmed this hypothesis based on cross-inoculation experiments and analysis of rDNA internal transcribed spacer sequences polymerase chain reaction amplified from rusty spot and peach powdery mildew lesions. Cross-inoculations of apple and peach leaves with P. leucotricha and P. pannosa, the causal agent of peach powdery mildew, showed that (i) young peach fruit, up to 5 cm in diameter, developed symptoms typical of rusty spot following inoculation with P. leucotricha; (ii) leaves of 'Jonagold' apple seedlings developed powdery mildew infections when inoculated by touching young rusty spot lesions to their surfaces; (iii) P. leucotricha sporulated on young peach fruit up to 5 cm in diameter; and (iv) peach leaves and young shoots were not susceptible to P. leucotricha, whereas P. pannosa infected all the green parts of peach. A field experiment revealed that there was only a 2- to 3-week period of time during early peach fruit development when the epidermis was susceptible to P. leucotricha. An outcome of this study is that now a clear distinction can be made between the symptoms caused by P. pannosa and P. leucotricha on peach.

Microsatellite stability in the plant pathogen Botrytis cinerea after exposure to different selective pressures.

The stability of microsatellite markers was investigated in the spore-producing fungus Botrytis cinerea exposed to four growth conditions. This knowledge is essential in order to differentiate mutations from genetic exchanges or recombination in population genetics studies. It is also important when using strains from collections that need to be regularly propagated on medium. Successive spore generations of four isolates of the fungus were realised in plates on different agar media: a nutrient-rich medium, a nutrient-poor medium, a medium supplemented with the antibiotic pyrrolnitrin and a medium supplemented with the fungicide iprodione. The stability of nine microsatellite markers was studied by comparing the molecular pattern obtained between the wild type parent strains and the final generations obtained. The results showed that, despite the phenotypic changes observed in some generations, no changes were observed in the allele size at nine microsatellite loci whatever the selective pressure endured by the fungus. This is the first study that reveals long-term stability of microsatellite markers of a spore-producing fungus exposed to different stresses.

Inoculum production and long-term conservation methods for cucurbits and tomato powdery mildews.

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 gl(-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.

Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999.

Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.