Identification and Quantification of Glucosinolates and Phenolics in a Large Panel of Brassica napus Highlight Valuable Genetic Resources for Chemical Ecology and Breeding.

Glucosinolate (GLS) and phenolic contents in Brassicaceae contribute to biotic and abiotic stress responses. Breeding crop accessions harboring agroecologically relevant metabolic profiles require a characterization of the chemical diversity in Brassica germplasm. This work investigates the diversity of specialized metabolites in 281 accessions of B. napus. First, an LC-HRMS2-based approach allowed the annotation of 32 phenolics and 36 GLSs, revealing 13 branched and linear alkyl-GLSs and 4 isomers of hydroxyphenylalkyl-GLSs, many of which have been rarely reported in Brassica. Then, quantitative UPLC-UV-MS-based profiling was performed in leaves and roots for the whole panel. This revealed striking variations in the content of 1-methylpropyl-GLS (glucocochlearin) and a large variation of tetra- and penta-glucosyl kaempferol derivatives among accessions. It also highlighted two main chemotypes related to sinapoyl-O-hexoside and kaempferol-O-trihexoside contents. By offering an unprecedented overview of the phytochemical diversity in B. napus, this work provides a useful resource for chemical ecology and breeding.

Hatching of Globodera pallida Induced by Root Exudates Is Not Influenced by Soil Microbiota Composition.

Plant-parasitic nematodes are among the most harmful pests of cultivated crops causing important economic losses. The ban of chemical nematicides requires the development of alternative agroecological approaches to protect crops against nematodes. For cyst nematodes, egg hatching is stimulated by host plant root exudates. Inducing "suicide hatching" of nematode second-stage juveniles (J2), using root exudates in the absence of the host plant, may constitute an effective and innovative biocontrol method to control cyst nematodes. However, before considering the development of this approach, understanding the effect of soil biotic component on cyst nematode hatching by root exudates is a major issue. The effectiveness of this approach could be modulated by other soil organisms consuming root exudates for growth as soil microbiota, and this must be evaluated. To do that, four different native agricultural soils were selected based on their physicochemical properties and their microbiota composition were characterized by rDNA metabarcoding. To disentangle the effect of microbiota from that of soil on hatching, four recolonized artificial soils were obtained by inoculating a common sterile soil matrix with the microbiota proceeding from each agricultural soil. Each soil was then inoculated with cysts of the potato cyst nematode, Globodera pallida, and low or high doses of potato root exudates (PREs) were applied. After 40 days, viable J2 remaining in cysts were counted to determine the efficiency of root exudates to stimulate hatching in different soils. Results showed that (i) when physicochemical and microbiota compositions varied among native soils, the hatching rates remained very high albeit small differences were measured and no dose effect was detected and (ii) when only microbiota composition varied among recolonized soils, the hatching rates were also high at the highest dose of PREs, but a strong dose effect was highlighted. This study shows that abiotic and biotic factors may not compromise the development of methods based on suicide hatching of cyst nematodes, using root exudates, molecules inducing J2 hatch, or trap crops.

Soil microbiota influences clubroot disease by modulating Plasmodiophora brassicae and Brassica napus transcriptomes.

The contribution of surrounding plant microbiota to disease development has led to the 'pathobiome' concept, which represents the interaction between the pathogen, the host plant and the associated biotic microbial community, resulting or not in plant disease. The aim herein is to understand how the soil microbial environment may influence the functions of a pathogen and its pathogenesis, and the molecular response of the plant to the infection, with a dual-RNAseq transcriptomics approach. We address this question using Brassica napus and Plasmodiophora brassicae, the pathogen responsible for clubroot. A time-course experiment was conducted to study interactions between P. brassicae, two B. napus genotypes and three soils harbouring high, medium or low microbiota diversities and levels of richness. The soil microbial diversity levels had an impact on disease development (symptom levels and pathogen quantity). The P. brassicae and B. napus transcriptional patterns were modulated by these microbial diversities, these modulations being dependent on the host genotype plant and the kinetic time. The functional analysis of gene expressions allowed the identification of pathogen and plant host functions potentially involved in the change of plant disease level, such as pathogenicity-related genes (NUDIX effector) in P. brassicae and plant defence-related genes (glucosinolate metabolism) in B. napus.

pH effect on strain-specific transcriptomes of the take-all fungus.

The soilborne fungus Gaeumannomyces tritici (G. tritici) causes the take-all disease on wheat roots. Ambient pH has been shown to be critical in different steps of G. tritici life cycle such as survival in bulk soil, saprophytic growth, and pathogenicity on plants. There are however intra-specific variations and we previously found two types of G. tritici strains that grow preferentially either at acidic pH or at neutral/alkaline pH; gene expression involved in pH-signal transduction pathway and pathogenesis was differentially regulated in two strains representative of these types. To go deeper in the description of the genetic pathways and the understanding of this adaptative mechanism, transcriptome sequencing was achieved on two strains (PG6 and PG38) which displayed opposite growth profiles in two pH conditions (acidic and neutral). PG6, growing better at acidic pH, overexpressed in this condition genes related to cell proliferation. In contrast, PG38, which grew better at neutral pH, overexpressed in this condition genes involved in fatty acids and amino acid metabolisms, and genes potentially related to pathogenesis. This strain also expressed stress resistance mechanisms at both pH, to assert a convenient growth under various ambient pH conditions. These differences in metabolic pathway expression between strains at different pH might buffer the effect of field or soil variation in wheat fields, and explain the success of the pathogen.

Long-lasting effects of antibiotics on bacterial communities of adult flies.

Insect symbionts benefit their host and their study requires large spectrum antibiotic use like tetracycline to weaken or suppress symbiotic communities. While antibiotics have a negative impact on insect fitness, little is known about antibiotic effects on insect microbial communities and how long they last. We characterized the bacterial communities of adult cabbage root fly Delia radicum in a Wolbachia-free population and evaluated the effect of tetracycline treatment on these communities over several generations. Three D. radicum generations were used: the first- and second-generation flies either ingested tetracycline or not, while the third-generation flies were untreated but differed with their parents and/or grandparents that had or had not been treated. Fly bacterial communities were sequenced using a 16S rRNA gene. Tetracycline decreased fly bacterial diversity and induced modifications in both bacterial abundance and relative frequencies, still visible on untreated offspring whose parents and/or grandparents had been treated, therefore demonstrating long-lasting transgenerational effects on animal microbiomes after antibiotic treatment. Flies with an antibiotic history shared bacterial genera, potentially tetracycline resistant and heritable. Next, the transmission should be investigated by comparing several insect development stages and plant compartments to assess vertical and horizontal transmissions of D. radicum bacterial communities.

Temporal dynamics of bacterial and fungal communities during the infection of Brassica rapa roots by the protist Plasmodiophora brassicae.

The temporal dynamics of rhizosphere and root microbiota composition was compared between healthy and infected Chinese cabbage plants by the pathogen Plasmodiophora brassicae. When inoculated with P. brassicae, disease was measured at five sampling dates from early root hair infection to late gall development. The first symptoms of clubroot disease appeared 14 days after inoculation (DAI) and increased drastically between 14 and 35 DAI. The structure of microbial communities associated to rhizosphere soil and root from healthy and inoculated plants was characterized through high-throughput DNA sequencing of bacterial (16S) and fungal (18S) molecular markers and compared at each sampling date. In healthy plants, Proteobacteria and Bacteroidetes bacterial phyla dominated the rhizosphere and root microbiota of Chinese cabbage. Rhizosphere bacterial communities contained higher abundances of Actinobacteria and Firmicutes compared to the roots. Moreover, a drastic shift of fungal communities of healthy plants occurred between the two last sampling dates, especially in plant roots, where most of Ascomycota fungi dominated until they were replaced by a fungus assigned to the Chytridiomycota phylum. Parasitic invasion by P. brassicae disrupted the rhizosphere and root-associated community assembly at a late step during the root secondary cortical infection stage of clubroot disease. At this stage, Flavisolibacter and Streptomyces in the rhizosphere, and Bacillus in the roots, were drastically less abundant upon parasite invasion. Rhizosphere of plants colonized by P. brassicae was significantly more invaded by the Chytridiomycota fungus, which could reflect a mutualistic relationship in this compartment between these two microorganisms.

Computational analysis of the Plasmodiophora brassicae genome: mitochondrial sequence description and metabolic pathway database design.

Plasmodiophora brassicae is an obligate biotrophic pathogenic protist responsible for clubroot, a root gall disease of Brassicaceae species. In addition to the reference genome of the P. brassicae European e3 isolate and the draft genomes of Canadian or Chinese isolates, we present the genome of eH, a second European isolate. Refinement of the annotation of the eH genome led to the identification of the mitochondrial genome sequence, which was found to be bigger than that of Spongospora subterranea, another plant parasitic Plasmodiophorid phylogenetically related to P. brassicae. New pathways were also predicted, such as those for the synthesis of spermidine, a polyamine up-regulated in clubbed regions of roots. A P. brassicae pathway genome database was created to facilitate the functional study of metabolic pathways in transcriptomics approaches. These available tools can help in our understanding of the regulation of P. brassicae metabolism during infection and in response to diverse constraints.

Can soil microbial diversity influence plant metabolites and life history traits of a rhizophagous insect? A demonstration in oilseed rape.

Interactions between plants and phytophagous insects play an important part in shaping the biochemical composition of plants. Reciprocally plant metabolites can influence major life history traits in these insects and largely contribute to their fitness. Plant rhizospheric microorganisms are an important biotic factor modulating plant metabolites and adaptation to stress. While plant-insects or plant-microorganisms interactions and their consequences on the plant metabolite signature are well-documented, the impact of soil microbial communities on plant defenses against phytophagous insects remains poorly known. In this study, we used oilseed rape (Brassica napus) and the cabbage root fly (Delia radicum) as biological models to tackle this question. Even though D. radicum is a belowground herbivore as a larva, its adult life history traits depend on aboveground signals. We therefore tested whether soil microbial diversity influenced emergence rate and fitness but also fly oviposition behavior, and tried to link possible effects to modifications in leaf and root metabolites. Through a removal-recolonization experiment, 3 soil microbial modalities ("high," "medium," "low") were established and assessed through amplicon sequencing of 16S and 18S ribosomal RNA genes. The "medium" modality in the rhizosphere significantly improved insect development traits. Plant-microorganism interactions were marginally associated to modulations of root metabolites profiles, which could partly explain these results. We highlighted the potential role of plant-microbial interaction in plant defenses against Delia radicum. Rhizospheric microbial communities must be taken into account when analyzing plant defenses against herbivores, being either below or aboveground.

Strain-specific variation in a soilborne phytopathogenic fungus for the expression of genes involved in pH signal transduction pathway, pathogenesis and saprophytic survival in response to environmental pH changes.

The soilborne fungus Gaeumannomyces graminis var. tritici (Ggt) causes take-all, a wheat root disease. In an original strain-specific way, a previous study indicates that inside the Ggt species, some strains grow preferentially at acidic pH and other strains at neutral/alkaline pH. The most important mechanism for a fungal response to the environmental pH is the Pal pathway which integrates the products of the six pal genes and the transcription factor PacC. To evaluate whether the Ggt strain-specific growth in function of the ambient pH is mediated via the Pal pathway, a transcriptional study of the genes encoding this pathway was carried out. This study provided the first evidence that the pH signalling pathway similar to those described in other fungi operated in Ggt. The pacC gene was induced at neutral pH whatever the strain. In an original way, the expression of Ggt genes coding for the different Pal proteins depended on the strain and on the ambient pH. In the strain growing better at acidic pH, few pal genes were pH-regulated, and some were overexpressed at neutral pH when regulated. In the strain growing better at neutral pH, underexpression of most of the pal genes at neutral pH occurred. The strains displayed higher gene expression in the ambient pH that unfavoured their growth as if it was a compensation system. All pH taken together, a globally weaker Pal transcript level occurred in the strains that were less sensitive to acidic pH, and on the contrary, the strain growing better on neutral pH showed higher Pal mRNA levels. The expression of genes involved in pathogenesis and saprophytic growth was also regulated by the ambient pH and the strain: each gene displayed a specific pH-regulation that was similar between strains. But all pH taken together, the global transcript levels of four out of six genes were higher in the strain growing better on neutral pH. Altogether, for the first time, the results show that inside a species, conditions affecting environmental pH modulate the expression of genes in an original strain-specific way.

Genomic analysis of the biocontrol strain Pseudomonas fluorescens Pf29Arp with evidence of T3SS and T6SS gene expression on plant roots.

Several bacterial strains of the Pseudomonas genus provide plant growth stimulation, plant protection against pests or bioremediation. Among these bacteria, P. fluorescens Pf29Arp reduces the severity of take-all, a disease caused by the pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) on wheat roots. In this study, we obtained a draft genome of Pf29Arp and subsequent comparative genomic analyses have revealed that this bacterial strain is closely related to strains of the 'P. brassicacearum-like' subgroup including P. brassicacearum ssp. brassicacearum NFM421 and P. fluorescens F113. Despite an overall chromosomal organization similar to these strains, a number of features including antibiotic synthesis gene clusters from secondary metabolism are not found in the Pf29Arp genome. But Pf29Arp possesses different protein secretion systems including type III (T3SS) and type VI (T6SS) secretion systems. Pf29Arp is the first Pseudomonas sp. strain described with four T6SS clusters (cluster I, II, III and IV). In addition, some protein-coding genes involved in the assembly of these secretion systems are basally expressed during Pf29Arp colonization of healthy wheat roots and display different expression patterns on necrotized roots caused by Ggt. These data suggest a role of T3SS and T6SS in the Pf29Arp adaptation to different root environments.

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 ß-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.

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

In 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 Ggt.

Changes in population structure of the soilborne fungus Gaeumannomyces graminis var. tritici during continuous wheat cropping.

A method was developed to assess the genetic structure of Gaeumannomyces graminis var. tritici (Ggt) populations and test the hypothesis of an association between disease level in the field with changes in pathogen populations. A long-term wheat monoculture experiment, established since 1994, generated different take-all epidemics with varying the number of wheat crop successions in the 1999-2000 cropping season. Genetic polymorphism in Ggt populations was investigated over natural, local epidemics. Four populations of 30 isolates were isolated from necrotic wheat roots in a first, third, fourth, and sixth wheat crop in the same year. Each Ggt isolate was characterized with RAPD (Random Amplification Polymorphism DNA) markers and AFLP (Amplified Fragment Length Polymorphism) fingerprinting. Seventeen multilocus genotypes based on the combination of RAPD and AFLP markers were identified among all these populations. The 120 isolates were divided into two main groups, G1 and G2, according to bootstrap values higher than 86%, except for an unique isolate from the third wheat crop. Within each group, populations ranged between 93 and 100% similarity. Both groups included isolates collected from the first, third, fourth or sixth wheat crop. However, G1 group profiles dominated amongst isolates sampled in the first and the sixth wheat crops, whereas G2 group profiles largely dominated amongst isolates collected from the third and fourth wheat crops. Aggressiveness of group G2 (38%) was significantly greater than that of G1 (29.5%). These results suggest that changes in Ggt population structure occur during continuous wheat cropping. The distinction of two Ggt groups provides a simple basis for further spatio-temporal analysis of Ggt population during polyetic take-all decline.