The biocontrol bacterium Pseudomonas fluorescens Pf29Arp strain affects the pathogenesis-related gene expression of the take-all fungus Gaeumannomyces graminis var. tritici on wheat roots

The main effects of antagonistic rhizobacteria on plant pathogenic fungi are antibiosis, fungistasis or an indirect constraint through the induction of a plant defence response. To explore different biocontrol mechanisms, an in vitro confrontation assay was conducted with the rhizobacterium Pseudomonas fluorescens Pf29Arp as a biocontrol agent of the fungus Gaeumannomyces graminis var. tritici (Ggt) on wheat roots. In parallel with the assessment of disease extension, together with the bacterial and fungal root colonization rates, the transcript levels of candidate fungal pathogenicity and plant-induced genes were monitored during the 10-day infection process. The bacterial inoculation of wheat roots with the Pf29Arp strain reduced the development of Ggt-induced disease expressed as attack frequency and necrosis length. The growth rates of Ggt and Pf29Arp, monitored through quantitative polymerase chain reaction of DNA amounts with a part of the Ggt 18S rDNA gene and a specific Pf29Arp strain detection probe, respectively, increased throughout the interactions. Bacterial antagonism and colonization had no significant effect on root colonization by Ggt. The expression of fungal and plant genes was quantified in planta by quantitative reverse transcription-polymerase chain reaction during the interactions thanks to the design of specific primers and an innovative universal reference system. During the early stages of the tripartite interaction, several of the fungal genes assayed were down-regulated by Pf29Arp, including two laccases, a β-1,3-exoglucanase and a mitogen-activated protein kinase. The plant host glutathione-S-transferase gene was induced by Ggt alone and up-regulated by Pf29Arp bacteria in interaction with the pathogen. We conclude that Pf29Arp antagonism acts through the alteration of fungal pathogenesis and probably through the activation of host defences

Genetic evidence for differentiation of Gaeumannomyces graminis var. tritici into two major groups

International audienceThe aim of this study was to give a comparative overview of diversity in Gaeumannomyces graminis var. tritici (Ggt), the take-all plant pathogenic fungus. For this, 98 Ggt isolates of a worldwide collection were first compared with two sets of specific PCR tools that discriminate G1/G2 and A/B genetic groups. A correspondence was found between G1 and B on the one hand and between G2 and A on the other, and less frequent divergent genotypes were also found. For more insight into Ggt diversity, a deeper phylogenetic analysis was conducted with the DNA sequences of two regions: the coding region of the RAPD-PCR marker previously selected and used for genotyping G1/G2 isolates, corresponding to the gentisate 1,2-dioxygenase-like gene, and the rDNA region corresponding to a part of the 18S gene, 5.8S gene, ITS1 and ITS2 DNA sequences. The distribution of Ggt genotypes into two main groups was confirmed by this phylogenetic analysis. The results give some evidence of the congruence of the molecular markers used by different teams and describe for the first time at the level of a worldwide collection two major genetically different groups of Ggt. The use of a universal molecular descriptor of Ggt groups is useful for investigating the occurrence, distribution and changes in Ggt populations in relation to epidemic dynamics

Existence de deux groupes de génotypes dans les populations de Gaeumannomyces graminis var tritici, agent du piétin-échaudage du blé.

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Linear relationship between Gaeumannomyces graminis var. tritici (Ggt) genotypic frequencies and disease severity on wheat roots in the field

International audienceIn order to investigate potential links existing between Gaeumannomyces graminis var. tritici (Ggt) population structure and disease development during polyetic take-all epidemics in sequences of Ggt host cereals, seven epidemics in fields with different cropping histories were monitored during the seasons 2001/2002 (two fields), 2002/2003 (two fields) and 2003/2004 (three fields). Take-all incidence and severity were measured at stem elongation and Ggt populations were characterized. The 73 isolates collected in the two fields in 2001/2002 were distributed into two multilocus genotypes, G1 and G2 according to amplified fragment length polymorphism analysis. A monolocus molecular marker amplified by F-12 random amplification polymorphism DNA primer sizing between 1.9 and 2.0 kb that gave strictly the same distinction between the two multilocus genotypes was further applied to measure G1/G2 frequencies among Ggt populations in all fields (266 isolates). The ratios of G1 to G2 differed between fields with different cropping histories. A linear relationship between G2 frequency among Ggt populations and disease severity at stem elongation was measured during the three cropping seasons. When take-all decline was observed, G2 frequencies were low in first wheat crops, highest in short-term sequences and intermediate in longer sequences of consecutive crops of Ggt host cereals. This pattern could be the result of population selection by environmental conditions, in particular by microbial antagonism during the parasitic phase of the fungus. In order to better understand take-all epidemic dynamics, the distinction between these two genotypes could be a basis to develop models that link approaches of quantitative epidemiology and advances in population genetics of Gg

Méthode innovante de real-time PCR développée à partir du système KASPar : application au suivi simultané de deux génotypes d’un champignon phytopathogène sur racines de blé par quantification de SNP.

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Simultaneous monitoring of two fungal genotypes on plant roots by single nucleotide polymorphism quantification with an innovative KASPar quantitative PCR

International audienceAn innovative quantitative PCR-based method derived from the Kompetitive Allele Specific PCR Assay Reagent (KASPar) system was developed to quantify the genomic DNA from two coexisting genotypes on the same tissues of a host-plant. For this purpose, the classical end-point KASPar method was evolved to a real-time method thanks to the addition of an adapted measurement step after each PCR cycle. It was applied to the quantification of the two genotypes G1 and G2 of the Gaeumannomyces graminis var. tritici (Ggt) soilborne fungus, pathogenic on wheat roots. Specific primers targeting a single nucleotide polymorphism from the ITS sequence were used allowing simultaneous quantification of both genotypes in the same reaction. The assays were applied to quantify fungal DNA of each genotype, aside or mixed together, after DNA extraction from fungal pure cultures and from single or co-inoculated roots in artificial medium or in soil. The detection and quantification lower limits for the two genotypes were 1.25 pg and 5 pg for DNA from fungal pure cultures, and 1.8 pg and 7 pg for DNA from fungal inoculated roots. The advantages of this cost-effective method are the high levels of specificity, sensitivity and reproducibility. Moreover, the accuracy of the method is independent of the copy numbers of the target sequences. The method is the first one to adapt the non-quantitative genotyping KASPar system to a quantitative application of two known genotypes of a species simultaneously and is suitable for simultaneous genotype-specific quantification of any other organisms (fungi, bacteria, plants)

Sensitivity to pH and ability to modify ambient pH of the take-all fungus Gaeumannomyces graminis var. tritici

International audiencepH is one of the major ambient factors affecting life history traits of soilborne phytopathogenic fungi. The diversity of phenotypic responses to pH changes has not been extensively explored within fungal populations. To investigate this question, the ability of 82 strains of a worldwide collection of the take-all agent Gaeumannomyces graminis var. tritici (Ggt) to grow in controlled pH conditions, reflecting their pH sensitivity, was measured. Of these 82 strains, 37 belonged to the G1 type and 45 to the G2 type, the two main genetic groups identified in Ggt populations. The experiments were conducted in Petri dishes on Fahraeus solid media buffered at pH 46, 60 or 70 with citrate-disodium phosphate solutions. The 82 strains exhibited a wide range of hyphal growth rates at the three pH levels. Ten statistically different pH profiles were described. The G2 strains grew significantly better than the G1 on the slightly acidic (pH 60) and the neutral (pH 70) buffered media. The ability of three strains to change ambient pH was also measured on unbuffered Fahraeus solid media initially adjusted to pH 56 or 80. All three strains were able to alkalinize the acidic medium. However, important variations between strains in the intensity, range and persistence of this alkalinization were measured. These results provide the first evidence of intraspecific variability in pH sensitivity within soilborne fungal species

Asymmetric outcome of community coalescence of seed and soil microbiota during early seedling growth

Seed microbial community constitutes a primary inoculum for plant microbiota assembly. Still, the persistence of seed microbiota when seeds encounter soil during plant emergence and early growth is barely documented. Here, we characterized the interchange event or coalescence of seed and soil microbiota and how it structured seedling bacterial and fungal communities. We performed eight contrasted coalescence events to identify drivers influencing seedling microbiota assembly: four seed lots of two Brassica napus genotypes were sown in two soils of contrasted diversity. We found that seedling root and stem microbiota were influenced by soil diversity but not by initial seed microbiota composition. A strong selection on the two-source communities occurred during microbiota assembly, with only 8-32% of soil taxa and 0.8-1.4% of seed-borne taxa colonizing seedlings. The recruitment of seedling microbiota came mainly from soil (35-72% of diversity) and not from seeds (0.3-15%). The outcome of seed and soil microbiota coalescence is therefore strongly asymmetrical with a dominance of soil taxa. Interestingly, seedling microbiota was primarily composed of initially rare taxa (from seed, soil or unknown origin) and sub-dominant soil taxa. Our results suggest that plant microbiome engineering success based on native seed or soil microbiota will rely on rare and sub-dominant taxa in source communities

Transmission of Seed and Soil Microbiota to Seedling

International audienceThe seed microbial community constitutes an initial inoculum for plant microbiota assembly. Still, the persistence of seed microbiota when seeds encounter soil during plant emergence and early growth is barely documented. We characterized the encounter event of seed and soil microbiota and how it structured seedling bacterial and fungal communities by using amplicon sequencing. We performed eight contrasting encounter events to identify drivers influencing seedling microbiota assembly. To do so, four contrasting seed lots of two Brassica napus genotypes were sown in two soils whose microbial diversity levels were manipulated by serial dilution and recolonization. Seedling root and stem microbiota were influenced by soil but not by initial seed microbiota composition or by plant genotype. A strong selection on the seed and soil communities occurred during microbiota assembly, with only 8% to 32% of soil taxa and 0.8% to 1.4% of seed-borne taxa colonizing seedlings. The recruitment of seedling microbiota came mainly from soil (35% to 72% of diversity) and not from seeds (0.3% to 15%). Soil microbiota transmission success was higher for the bacterial community than for the fungal community. Interestingly, seedling microbiota was primarily composed of initially rare taxa (from seed, soil, or unknown origin) and intermediate-abundance soil taxa

Influence d’une bactérie antagoniste sur l’expression in planta de gènes du pouvoir pathogène de Gaeumannomyces graminis var. tritici.

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