Defined microbial communities (also known as synthetic communities) are showing promising results for plant health but are lacking an efficient lab to field transition. This is the due to limited knowledge on how to efficiently modulate plant microbiota by considering complex environments and multi-kingdom interactions. In this study, we aimed to better understand the transmission and impact of multi-kingdom synthetic communities (SynComs) from the seed to seedling stage. We constructed 20 different SynComs using both a priori and random approaches, from pool of diverse strains including 24 bacteria, 11 yeasts and 10 filamentous fungi. SynComs were inoculated on Brassica napus seeds and we monitored both transmission and impact on the microbiota of 15-day-old seedlings grown in non-sterile soil. Optimization of the inoculation protocol showed that alginate coating improved bacterial, yeast and filamentous fungi concentrations by more than 2 log compared with other approaches. With this inoculation method, we observed contrasted seedling colonization profiles, with SynComs members representing between 1.1-45.7% of bacterial community and 3.2-36.6% of fungal community. Our multiple SynCom design revealed that strain selection is a more critical determinant of SynCom performance than assembly strategy. Even randomly assembled communities performed well, as long as they are drawn from a pool of ecologically relevant, well-adapted taxa. Based on these evidences, we identified key bacterial and fungal traits explaining efficient seedling colonization such as high abundance on inoculated seed and low in vitro lag-time. Despite low colonization levels, we observed that SynCom inoculation altered seeding bacterial community assembly in 14 SynComs. A total of 82 native bacterial ASVs were identified as responsive to SynCom inoculation, most likely originating from the soil. This shift indicates that SynComs influence community assembly by modulating the recruitment of environmental taxa, especially when SynCom strains were more integrated in multi-kingdom network structures. Finally, we identified four distinct SynComs profiles which either colonized strongly or not seedlings while shifting of not native microbiota. Altogether, these findings provide actionable directions for improving SynCom design, suggesting that leveraging ecological processes such as host adaptation, optimal inoculation density, and network integration could enhance both colonization efficiency and plant phenotypic outcomes.

Little is known about the effect of nitrogen nutrition on seedling susceptibility to seed-borne pathogens. We have previously shown that seedlings grown under high nitrate (5 mM) conditions are less susceptible than those grown under low nitrate (0.1 mM) and ammonium (5 mM) in the Arabidopsis-Alternaria brassicicola pathosystem. However, it is not known how seedling metabolism is modulated by nitrogen nutrition, nor what is its response to pathogen infection. Here, we addressed this question using the same pathosystem and nutritive conditions, examining germination kinetics, seedling development, but also shoot ion contents, metabolome and selected gene expression. Nitrogen nutrition clearly altered the seedling metabolome. A similar metabolomic profile was observed in inoculated seedlings grown at high nitrate levels and in not inoculated-seedlings. High nitrate levels also led to specific gene expression patterns (e.g. polyamine metabolism), while other genes responded to inoculation regardless of nitrogen supply conditions. Furthermore, the metabolites best correlated with high disease symptoms were coumarate, tyrosine, hemicellulose sugars and polyamines, and those associated with low symptoms were organic acids (tricarboxylic acid pathway, glycerate, shikimate), sugars derivatives and β-alanine. Overall, our results suggest that the beneficial effect of high nitrate nutrition on seedling susceptibility is likely due to nutritive and signaling mechanisms affecting developmental plant processes detrimental to the pathogen. In particular, it may be due to a constitutively high tryptophan metabolism, as well as down regulation of oxidative stress caused by polyamine catabolism.

The impact of the form of nitrogen (N) source (nitrate versus ammonium) on the susceptibility to Alternaria brassicicola, a necrotrophic fungus, has been examined in Arabidopsis thaliana at the rosette stage. Nitrate nutrition was found to increase fungal lesions considerably. There was a similar induction of defence gene expression following infection under both N nutritions, except for the phytoalexin deficient 3 gene, which was overexpressed with nitrate. Nitrate also led to a greater nitric oxide production occurring in planta during the saprophytic growth and lower nitrate reductase (NIA1) expression 7 days after inoculation. This suggests that nitrate reductase-dependent nitric oxide production had a dual role, whereby, despite its known role in the generic response to pathogens, it affected plant metabolism, and this facilitated fungal infection. In ammonium-grown plants, infection with A. brassicicola induced a stronger gene expression of ammonium transporters and significantly reduced the initially high ammonium content in the leaves. There was a significant interaction between N source and inoculation (presence versus absence of the fungus) on the total amino acid content, while N nutrition reconfigured the spectrum of major amino acids. Typically, a higher content of total amino acid, mainly due to a stronger increase in asparagine and glutamine, is observed under ammonium nutrition while, in nitrate-fed plants, glutamate was the only amino acid which content increased significantly after fungal inoculation. N nutrition thus appears to control fungal infection via a complex set of signalling and nutritional events, shedding light on how nitrate availability can modulate disease susceptibility.

The transmission of seed-borne pathogens by the germinating seed is responsible for major crop diseases. The immune responses of the seed facing biotic invaders are poorly documented so far. The Arabidopsis thaliana/Alternaria brassicicola patho-system was used to describe at the transcription level the responses of germinating seeds and young seedling stages to infection by the necrotrophic fungus. RNA-seq analyses of healthy versus inoculated seeds at 3 days after sowing (DAS), stage of radicle emergence, and at 6 and 10 DAS, two stages of seedling establishment, identified thousands of differentially expressed genes by Alternaria infection. Response to hypoxia, ethylene and indole pathways were found to be induced by Alternaria in the germinating seeds. However, surprisingly, the defense responses, namely the salicylic acid (SA) pathway, the response to reactive oxygen species (ROS), the endoplasmic reticulum-associated protein degradation (ERAD) and programmed cell death, were found to be strongly induced only during the latter post-germination stages. We propose that this non-canonical immune response in early germinating seeds compared to early seedling establishment was potentially due to the seed-to-seedling transition phase. Phenotypic analyses of about 14 mutants altered in the main defense pathways illustrated these specific defense responses. The unexpected germination deficiency and insensitivity to Alternaria in the glucosinolate deficient mutants allow hypothesis of a trade-off between seed germination, necrosis induction and Alternaria transmission to the seedling. The imbalance of the SA and jasmonic acid (JA) pathways to the detriment of the JA also illustrated a non-canonical immune response at the first stages of the seedling.

Glucosinolates and camalexin are secondary metabolites that, as phytoanticipins and phytoalexins, play a crucial role in plant defence. The present work proposes an improved analytical method for routine analysis and quantification of glucosinolates and camalexin in brassicaceous small-sized samples by using the very specific desulfation process of glucosinolates analysis and the specificity of fluorescence detection for camalexin analysis. The approach is based on a simultaneous ultrasound-assisted extraction followed by a purification on an anionexchange column. Final analyses are conducted by HPLC-UV-MS for desulfo-glucosinolates and HPLC coupled to a fluorescence detector (HPLC-FLD) for camalexin. The method is linear for glucosinolates (50-3500 mu M) and camalexin (0.025-5 mu g.mL-1) with an LOD/LOQ of 3.8/12.6 mu M and 0.014/0.046 mu g.mL-1 respectively. The method demonstrated adequate precision, accuracy and trueness on certified reference rapeseed. A practical application of our approach was conducted on different Brassicaceae genera (Barbarea vulgaris, Brassica nigra, Capsella bursa-pastoris, Cardamine hirsuta, Coincya monensis, Sinapis arvensis, and Sisymbrium officinale) and Arabidopsis thaliana genotypes (Columbia and Wassilewskija). Futhermore, different plant organs (seeds and leaves) were analysed, previously inoculated or not with the pathogenic fungus Alternaria brassicicola.

Background Seedling growth is an early phase of plant development highly susceptible to environmental factors such as soil nitrogen (N) availability or presence of seed-borne pathogens. Whereas N plays a central role in plant-pathogen interactions, its role has never been studied during this early phase for the interaction between Arabidopsis thaliana and Alternaria brassicicola , a seed-transmitted necrotrophic fungus. The aim of the present work was to develop an in vitro monitoring system allowing to study the impact of the fungus on A. thaliana seedling growth, while modulating N nutrition. Results The developed system consists of square plates placed vertically and filled with nutrient agar medium allowing modulation of N conditions. Seeds are inoculated after sowing by depositing a droplet of conidial suspension. A specific semi-automated image analysis pipeline based on the Ilastik software was developed to quantify the impact of the fungus on seedling aerial development, calculating an index accounting for every aspect of fungal impact, namely seedling death, necrosis and developmental delay. The system also permits to monitor root elongation. The interest of the system was then confirmed by characterising how N media composition [0.1 and 5 mM of nitrate (NO 3 − ), 5 mM of ammonium (NH 4 + )] affects the impact of the fungus on three A. thaliana ecotypes. Seedling development was strongly and negatively affected by the fungus. However, seedlings grown with 5 mM NO 3 − were less susceptible than those grown with NH 4 + or 0.1 mM NO 3 − , which differed from what was observed with adult plants (rosette stage). Conclusions The developed monitoring system allows accurate determination of seedling growth characteristics (both on aerial and root parts) and symptoms. Altogether, this system could be used to study the impact of plant nutrition on susceptibility of various genotypes to fungi at the seedling stage.

Alternaria brassicicola causes black spot disease in Brassicaceae. During host infection, this necrotrophic fungus is exposed to various antimicrobial compounds, such as the phytoalexin brassinin which is produced by many cultivated Brassica species. To investigate the cellular mechanisms by which this compound causes toxicity and the corresponding fungal adaptive strategies, we first analyzed fungal transcriptional responses to short-term exposure to brassinin and then used additional functional approaches. This study supports the hypothesis that indolic phytoalexin primarily targets mitochondrial functions in fungal cells. Indeed, we notably observed that phytoalexin treatment of A. brassicicola disrupted the mitochondrial membrane potential and resulted in a significant and rapid decrease in the oxygen consumption rates. Secondary effects, such as Reactive oxygen species production, changes in lipid and endoplasmic reticulum homeostasis were then found to be induced. Consequently, the fungus has to adapt its metabolism to protect itself against the toxic effects of these molecules, especially via the activation of high osmolarity glycerol and cell wall integrity signaling pathways and by induction of the unfolded protein response.

Alternaria brassicicola is a necrotrophic fungus causing black spot disease and is an economically important seed-borne pathogen of cultivated brassicas. Seed transmission is a crucial component of its parasitic cycle as it promotes long-term survival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A. brassicicola pathosystem, showed that the level of susceptibility of the fungus to water stress strongly influenced its seed transmission ability. In this study, we gained further insights into the mechanisms involved in the seed infection process by analyzing the transcriptomic and metabolomic responses of germinated spores of A. brassicicola exposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteins that are assumed to be involved in cellular dehydration tolerance, was established in A. brassicicola based on the expression data and additional structural and biochemical criteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-like proteins showed that they were affected in their transmission to A. thaliana seeds, although their aggressiveness on host vegetative tissues remained intact.

ABSTRACT Alternaria brassicicola causes dark spot (or black spot) disease, which is one of the most common and destructive fungal diseases of Brassicaceae spp. worldwide. Here, we report the draft genome sequence of strain Abra43. The assembly comprises 29 scaffolds, with an N50 value of 2.1 Mb. The assembled genome was 31,036,461 bp in length, with a G+C content of 50.85%.

Seeds are involved in the transmission of microorganisms from one plant generation to another and consequently may act as the initial inoculum source for the plant microbiota. In this work, we assessed the structure and composition of the seed microbiota of radish (Raphanus sativus) across three successive plant generations. Structure of seed microbial communities were estimated on individual plants through amplification and sequencing of genes that are markers of taxonomic diversity for bacteria (gyrB) and fungi (ITS1). The relative contribution of dispersal and ecological drift in inter-individual fluctuations were estimated with a neutral community model. Seed microbial communities of radish display a low heritability across plant generations. Fluctuations in microbial community profiles were related to changes in community membership and composition across plant generations, but also to variation between individual plants. Ecological drift was an important driver of the structure of seed bacterial communities, while dispersal was involved in the assembly of the fungal fraction of the seed microbiota. These results provide a first glimpse of the governing processes driving the assembly of the seed microbiota.

Seeds are involved in the vertical transmission of microorganisms from one plant generation to another and consequently act as reservoirs for the plant microbiota. However, little is known about the structure of seed-associated microbial assemblages and the regulators of assemblage structure. In this work, we have assessed the response of seed-associated microbial assemblages of Raphanus sativus to invading phytopathogenic agents, the bacterial strain Xanthomonas campestris pv. campestris (Xcc) 8004 and the fungal strain Alternaria brassicicola Abra43. According to the indicators of bacterial (16S rRNA gene and gyrB sequences) and fungal (ITS1) diversity employed in this study, seed transmission of the bacterial strain Xcc 8004 did not change the overall composition of resident microbial assemblages. In contrast seed transmission of Abra43 strongly modified the richness and structure of fungal assemblages without affecting bacterial assemblages. The sensitivity of seed-associated fungal assemblage to Abra43 is mostly related to changes in relative abundance of closely related fungal species that belong to the Alternaria genus. Variation in stability of the seed microbiota in response to Xcc and Abra43 invasions could be explained by differences in seed transmission pathways employed by these micro-organisms, which ultimately results in divergence in spatio-temporal colonization of the seed habitat.

Background: Image analysis is increasingly used in plant phenotyping. Among the various imaging techniques that can be used in plant phenotyping, chlorophyll fluorescence imaging allows imaging of the impact of biotic or abiotic stresses on leaves. Numerous chlorophyll fluorescence parameters may be measured or calculated, but only a few can produce a contrast in a given condition. Therefore, automated procedures that help screening chlorophyll fluorescence image datasets are needed, especially in the perspective of high-throughput plant phenotyping.Results: We developed an automatic procedure aiming at facilitating the identification of chlorophyll fluorescence parameters impacted on leaves by a stress. First, for each chlorophyll fluorescence parameter, the procedure provides an overview of the data by automatically creating contact sheets of images and/or histograms. Such contact sheets enable a fast comparison of the impact on leaves of various treatments, or of the contrast dynamics during the experiments. Second, based on the global intensity of each chlorophyll fluorescence parameter, the procedure automatically produces radial plots and box plots allowing the user to identify chlorophyll fluorescence parameters that discriminate between treatments. Moreover, basic statistical analysis is automatically generated. Third, for each chlorophyll fluorescence parameter the procedure automatically performs a clustering analysis based on the histograms. This analysis clusters images of plants according to their health status. We applied this procedure to monitor the impact of the inoculation of the root parasitic plant Phelipanche ramosa on Arabidopsis thaliana ecotypes Col-0 and Ler.Conclusions: Using this automatic procedure, we identified eight chlorophyll fluorescence parameters discriminating between the two ecotypes of A. thaliana, and five impacted by the infection of Arabidopsis thaliana by P. ramosa. More generally, this procedure may help to identify chlorophyll fluorescence parameters impacted by various types of stresses. We implemented this procedure at http://www.phenoplant.org freely accessible to users of the plant phenotyping community.

During their life cycle, fungi face adverse environmental conditions associated with alterations in water status. Phytopathogenic fungi are faced with this type of stress during the infection process, especially when they colonize seeds. Although these organisms are particularly effective to adapt to these water potential decreases, these coping mechanisms are so far very poorly described, particularly in filamentous fungi. Alternaria brassicicola is a seed-borne fungal pathogen responsible for the black spot disease on Brassicaceae plants. Alteration of Brassicaceae seed quality is one of the most damaging effects of the black spot. Beyond contribution to pathogen dissemination, the presence of the fungus on the seeds compromises seedling germination and survival. To better understand the determinism of fungus transmission to seeds, we previously established a reliable Arabidopsis-based pathosystem allowing investigations of A. brassicicola transmission to seeds. In particular, we showed that two mutants strain ∆abhog and ∆abnik exhibiting higher susceptibility to osmotic and water stress were highly impaired in seed transmission ability. Transcriptomic analyzes, carried out under different experimental in vitro conditions inducing these types of stress (addition of sorbitol or Poly Ethylene Glycol (PEG) or by incubation under low relative humidity (1% RH)), allowed us to identify additional mechanisms potentially involved in the fungal adaptive responses. In particular, these analyzes revealed a pool of over-expressed genes encoding putative proteins which share physiochemical features typical of hydrophilin-like proteins. We initiated studies of some of these hydrophilins by generating respective Knock-Out mutants. Functional studies has been carried out to determine whether these mutants were impaired in their adaptive response to water stress and other types of stress (such as oxidative stress) and whether hydrophilins are involved in pathogenicity. Additional transcriptomic assays conducted on ∆abhog and ∆abnik strain growth under sorbitol exposure revealed that numerous hydrophilins are regulated by these two genes.

Glucosinolates are brassicaceous secondary metabolites that have long been considered as chemical shields against pathogen invasion. Isothiocyanates (ITCs), are glucosinolate-breakdown products that have negative effects on the growth of various fungal species. We explored the mechanism by which ITCs could cause fungal cell death using Alternaria brassicicola, a specialist Brassica pathogens, as model organism. Exposure of the fungus to ICTs led to a decreased oxygen consumption rate, intracellular accumulation of reactive oxygen species (ROS) and mitochondrial-membrane depolarization. We also found that two major regulators of the response to oxidative stress, i.e., the MAP kinase AbHog1 and the transcription factor AbAP1, were activated in the presence of ICTs. Once activated by ICT-derived ROS, AbAP1 was found to promote the expression of different oxidative-response genes. This response might play a significant role in the protection of the fungus against ICTs as mutants deficient in AbHog1 or AbAP1 were found to be hypersensitive to these metabolites. Moreover, the loss of these genes was accompanied by a significant decrease in aggressiveness on Brassica. We suggest that the robust protection response against ICT-derived oxidative stress might be a key adaptation mechanism for successful infection of host plants by Brassicaceae-specialist necrotrophs like A. brassicicola.

This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).