New insights into mycotoxin risk management through fungal population genetics and genomics.

Mycotoxin contamination of food and feed is a major global concern. Chronic or acute dietary exposure to contaminated food and feed can negatively affect both human and animal health. Contamination occurs through plant infection by toxigenic fungi, primarily Aspergillus and Fusarium spp., either before or after harvest. Despite the application of various management strategies, controlling these pathogens remains a major challenge primarily because of their ability to adapt to environmental changes and selection pressures. Understanding the genetic structure of plant pathogen populations is pivotal for gaining new insights into their biology and epidemiology, as well as for understanding the mechanisms behind their adaptability. Such deeper understanding is crucial for developing effective and preemptive management strategies tailored to the evolving nature of pathogenic populations. This review focuses on the population-level variations within the two most economically significant toxigenic fungal genera according to space, host, and pathogenicity. Outcomes in terms of migration patterns, gene flow within populations, mating abilities, and the potential for host jumps are examined. We also discuss effective yet often underutilized applications of population genetics and genomics to address practical challenges in the epidemiology and disease control of toxigenic fungi.

First report of grapefruit rot caused by Colletotrichum gloeosporioides and C. karsti in France.

The grapefruit (Citrus paradisi) is a citrus hybrid tree (C. maxima & C. sinensis). Due to nutritional value and its bioactive compounds, the fruits are recognized as a functional food, valued as promoting health. French grapefruit production is low (7.5 Kt/year) but is confined to a restricted area in Corsica and benefits from a quality label, the economic impact of its cultivation being therefore locally significant. Since 2015 previously unreported symptoms have been repeatedly observed on grapefruits in more than half of the orchards in Corsica, with an incidence of 30% of fruits altered. Brown to black circular spots were observed on fruits and leaves, surrounded by chlorotic halos on the latter. On the mature fruit, lesions were round, 4 to 10 mm in diameter, brown and dry (e-Xtra 1). Although the lesions are superficial, the fruits cannot be marketed due to constraints linked to the quality label. 75 fungal isolates were obtained from symptomatic fruits or leaves collected in Corsica (in 2016, 2017, and 2021). Cultures obtained after 7 days on PDA at 25°C, were white to light grey in colour, forming concentric rings or dark spots on the agar surface. We did not observe any notable difference among the isolates except some evolved towards a more marked grey. Colonies tend to form a cottony aerial mycelium and orange conidial masses appear with age. The conidia were hyaline, aseptate, cylindrical with ends rounded, and measured 14.9 ± 0.95 µm length and 5.1 ± 0.45 µm width (n = 50). Cultural and morphological characteristics were similar to those described for C. gloeosporioides s. lat. or C. boninense s. lat. (Weir et al. 2012 ; Damm et al. 2012). Total genomic DNA was extracted from all isolates, and the ITS region of rDNA was amplified with ITS 5 & 4 primers, then sequenced (GenBank Accession Nos. OQ509805-808). For 90% of isolates GenBank BLASTn results were 100% identical to C. gloeosporioides isolates sequences, whereas for other isolates the resulting sequences were 100% identical to C. karsti or C. boninense isolates sequences. Four strains (three C. gloeosporioides with light colour differences, in order to see if there was diversity among isolates of C. gloeosporioides s. lato ; and one C. karsti) were further characterized by sequencing partial actin [ACT], calmodulin [CAL], chitin synthase [CHS-1], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ß-tubulin 2 [TUB2], for all strains ; glutamine synthetase [GS], Apn2-Mat1-2-1 intergenic spacer and partial mating type (Mat1-2) gene [ApMAT] for C. gloeosporioides s. lat., and HIS3 for C. boninense s. lat. (Weir et al. 2012 ; Silva et al, 2012) (GenBank Accession Nos. OQ509805-808 & OQ507698-724). Multilocus phylogenetic analyses carried out with the obtained and Genbank available sequences confirmed that 3 isolates (UBOCC-A-116036, -116038, & -116039) clustered within C. gloeosporioides s. s., while the other (UBOCC-A-116037) clustered within C. karsti (e-Xtra 2) 'Star ruby' grapefruits were surface sterilized then wound-inoculated with 20 µl of a conidial suspension (105 conidia ml-1) of UBOCC-A-116036 & 116037 isolates or 20 µl sterile water for control (ten fruits for each isolate or control). After 10 days incubation at 20°C, symptoms, identical to those initially observed, developed around the inoculation point, while controls inoculated with water remained symptomless. Fungal colonies re-isolated from the lesions were morphologically like the original isolates. Recently, various infections caused by some Colletotrichum sp. have strongly compromised citrus production in different Mediterranean countries: ie Italy (Aiello et al. 2015), Portugal (Ramos et al. 2016), Tunisia (Ben Hadj Daoud et al. 2019), Turkey (Uysal et al. 2022). In these studies, C. gloeosporioides s. s. and C. karsti were identified as the causal agents. These two species were the predominant Colletotrichum sp. associated with Citrus and allied genera in Europe (Guarnaccia et al. 2017). To our knowledge, our study is the first report of C. gloeosporioides and C. karsti causing anthracnose on grapefruit in France, which confirms the incidence of these two pathogens on the Mediterranean rim. Given the economic importance of citrus cultivation in the Mediterranean region, the presence of Colletotrichum spp. should deserves to be monitored, and a control strategy should be considered.

Influence of Maize Residues in Shaping Soil Microbiota and Fusarium spp. Communities.

Fusarium head blight (FHB) is a devastating fungal disease of small grain cereals including wheat. Causal fungal agents colonize various components of the field during their life cycle including previous crop residues, soil, and grains. Although soil and residues constitute the main inoculum source, these components have received much less attention than grains. This study aimed at disentangling the role of previous crop residues in shaping soil microbiota, including Fusarium spp. communities, in fields under wheat-maize rotation. Such knowledge may contribute to better understand the complex interactions between Fusarium spp. and soil microbiota. Dynamics of bacterial and fungal communities, with a special focus on Fusarium spp., were monitored in soils at 3 time points: during wheat cultivation (April 2015 and 2017) and after maize harvest (November 2016) and in maize residues taken from fields after harvest. Shifts in microbiota were also evaluated under mesocosm experiments using soils amended with maize residues. Fusarium graminearum and F. avenaceum were predominant on maize residues but did not remain in soils during wheat cultivation. Differences in soil bacterial diversity and compositions among years were much lower than variation between fields, suggesting that bacterial communities are field-specific and more conserved over time. In contrast, soil fungal diversity and compositions were more influenced by sampling time. Maize residues, left after harvest, led to a soil enrichment with several fungal genera, including Epicoccum, Fusarium, Vishniacozyma, Papiliotrema, Sarocladium, Xenobotryosphaeria, Ramularia, Cladosporium, Cryptococcus, and Bullera, but not with bacterial genera. Likewise, under mesocosm conditions, the addition of maize residues had a stronger influence on fungal communities than on bacterial communities. In particular, addition of maize significantly increased soil fungal richness, while bacteria were much less prone to changes. Based on co-occurrence networks, OTUs negatively correlated to Fusarium spp. were identified, such as those assigned to Epicoccum and Vishniacozyma. Altogether, our results allowed to gain a deeper insight into the complex microbiota interactions in soils, with bacteria and fungi responding differently to environmental disturbances.

Complete Genome Sequence of the Plant-Pathogenic Fungus Colletotrichum lupini.

Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phylogenetic Diversity and Effect of Temperature on Pathogenicity of Colletotrichum lupini.

Although lupin anthracnose caused by Colletotrichum lupini is a significant threat for spring and winter lupin crops, it has been poorly studied so far. This study aimed at characterizing the (i) phylogenetic, (ii) morphological, and (iii) physiological diversity of collected isolates from anthracnose-affected lupins. The genetic identification of representative isolates (n = 71) revealed that they were all C. lupini species, further confirming that lupin anthracnose is caused by this species. However, multilocus sequencing on these isolates and 16 additional reference strains of C. lupini revealed a separation into two distinct genetic groups, both of them characterized by a very low genetic diversity. The diversity of morphological characteristics of a selected subset of C. lupini isolates was further evaluated. To the best of our knowledge, microsclerotia production observed for some isolates has never been reported so far within the Colletotrichum acutatum species complex. Finally, the modeling of growth responses of a subset of C. lupini strains revealed the capacity of some strains to grow in vitro at 5°C. This ability was also evidenced in planta, because C. lupini DNA was detectable in plants from 14 days postinoculation at 5°C onward, whereas symptoms began to appear a week later, although at a very low level. Since lupin crops are planted during winter or early spring, growth studies in vitro and in planta demonstrated the capability of the species to grow at temperatures ranging from 5 to 30°C, with an optimum close to 25°C. In this study, C. lupini-specific primers were also designed for real-time quantitative PCR on fungal DNA and allowed the detection of C. lupini in asymptomatic field samples. These results open perspectives to detect earlier and limit the development of this pathogen in lupin crops.

Whole-Genome Sequence of the Orchid Anthracnose Pathogen Colletotrichum orchidophilum.

Colletotrichum orchidophilum is a plant-pathogenic fungus infecting a wide range of plant species belonging to the family Orchidaceae. In addition to its economic impact, C. orchidophilum has been used in recent years in evolutionary studies because it represents the closest related species to the C. acutatum species complex. Here, we present the first-draft whole-genome sequence of C. orchidophilum IMI 309357, providing a resource for future research on anthracnose of Orchidaceae and other hosts.

Gene family expansions and contractions are associated with host range in plant pathogens of the genus Colletotrichum.

BACKGROUND: Many species belonging to the genus Colletotrichum cause anthracnose disease on a wide range of plant species. In addition to their economic impact, the genus Colletotrichum is a useful model for the study of the evolution of host specificity, speciation and reproductive behaviors. Genome projects of Colletotrichum species have already opened a new era for studying the evolution of pathogenesis in fungi. RESULTS: We sequenced and annotated the genomes of four strains in the Colletotrichum acutatum species complex (CAsc), a clade of broad host range pathogens within the genus. The four CAsc proteomes and secretomes along with those representing an additional 13 species (six Colletotrichum spp. and seven other Sordariomycetes) were classified into protein families using a variety of tools. Hierarchical clustering of gene family and functional domain assignments, and phylogenetic analyses revealed lineage specific losses of carbohydrate-active enzymes (CAZymes) and proteases encoding genes in Colletotrichum species that have narrow host range as well as duplications of these families in the CAsc. We also found a lineage specific expansion of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein (NLPs) families within the CAsc. CONCLUSIONS: This study illustrates the plasticity of Colletotrichum genomes, and shows that major changes in host range are associated with relatively recent changes in gene content.

Shifts in Fusarium Communities and Mycotoxins in Maize Residues, Soils, and Wheat Grains throughout the Wheat Cycle: Implications for Fusarium Head Blight Epidemiology.

Fusarium Head Blight (FHB), predominantly caused by Fusarium species, is a devastating cereal disease worldwide. While considerable research has focused on Fusarium communities in grains, less attention has been given to residues and soil, the primary inoculum sources. Knowledge of Fusarium spp. diversity, dynamics, and mycotoxin accumulation in these substrates is crucial for assessing their contribution to wheat head infection and the complex interactions among Fusarium communities throughout the wheat cycle. We monitored six minimum-tillage wheat fields, with maize as the preceding crop, over two years. Soils, maize residues, and wheat grains were sampled at four stages. Fusarium composition was analyzed using a culture-dependent method, species-specific qPCR, and EF1α region metabarcoding sequencing, enabling species-level resolution. The Fusarium communities were primarily influenced by substrate type, accounting for 35.8% of variance, followed by sampling location (8.1%) and sampling stage (3.2%). Among the 32 identified species, F. poae and F. graminearum dominated grains, with mean relative abundances of 47% and 29%, respectively. Conversely, residues were mainly contaminated by F. graminearum, with a low presence of F. poae, as confirmed by species-specific qPCR. Notably, during periods of high FHB pressure, such as in 2021, F. graminearum was the dominant species in grains. However, in the following year, F. poae outcompeted F. graminearum, resulting in reduced disease pressure, consistent with the lower pathogenicity of F. poae. Source Tracker analysis indicated that residues were a more significant source of Fusarium contamination on wheat in 2021 compared to 2022, suggesting that F. graminearum in 2021 primarily originated from residues, whereas F. poae's sources of infection need further investigation. Additionally, multiple mycotoxins were detected and quantified in maize residues during the wheat cycle, raising the question of their ecological role and impact on the soil microbiota.

Genetic diversity and population structure of Botryosphaeria dothidea and Neofusicoccum parvum on English walnut (Juglans regia L.) in France.

Botryosphaeriaceae species are the major causal agents of walnut dieback worldwide, along with Diaporthe species. Botryosphaeria dothidea and Neofusicoccum parvum are the only two Botryosphaeriaceae species associated with this recently emergent disease in France, and little is known about their diversity, structure, origin and dispersion in French walnut orchards. A total of 381 isolates of both species were genetically typed using a sequence-based microsatellite genotyping (SSR-seq) method. This analysis revealed a low genetic diversity and a high clonality of these populations, in agreement with their clonal mode of reproduction. The genetic similarity among populations, regardless of the tissue type and the presence of symptoms, supports the hypothesis that these pathogens can move between fruits and twigs and display latent pathogen lifestyles. Contrasting genetic patterns between N. parvum populations from Californian and Spanish walnut orchards and the French ones suggested no conclusive evidence for pathogen transmission from infected materials. The high genetic similarity with French vineyards populations suggested instead putative transmission between these hosts, which was also observed with B. dothidea populations. Overall, this study provides critical insight into the epidemiology of two important pathogens involved in the emerging dieback of French walnut orchards, including their distribution, potential to mate, putative origin and disease pathways.

Genome evolution and transcriptome plasticity is associated with adaptation to monocot and dicot plants in Colletotrichum fungi.

BACKGROUND: Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes. RESULTS: Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation. CONCLUSIONS: These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.

Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding.

Walnut dieback can be caused by several fungal pathogenic species, which are associated with symptoms ranging from branch dieback to fruit necrosis and blight, challenging the one pathogen-one disease concept. Therefore, an accurate and extensive description of the walnut fungal pathobiome is crucial. To this end, DNA metabarcoding represents a powerful approach provided that bioinformatic pipelines are evaluated to avoid misinterpretation. In this context, this study aimed to determine (i) the performance of five primer pairs targeting the ITS region in amplifying genera of interest and estimating their relative abundance based on mock communities and (ii) the degree of taxonomic resolution using phylogenetic trees. Furthermore, our pipelines were also applied to DNA sequences from symptomatic walnut husks and twigs. Overall, our results showed that the ITS2 region was a better barcode than ITS1 and ITS, resulting in significantly higher sensitivity and/or similarity of composition values. The ITS3/ITS4_KYO1 primer set allowed to cover a wider range of fungal diversity, compared to the other primer sets also targeting the ITS2 region, namely, GTAA and GTAAm. Adding an extraction step to the ITS2 sequence influenced both positively and negatively the taxonomic resolution at the genus and species level, depending on the primer pair considered. Taken together, these results suggested that Kyo set without ITS2 extraction was the best pipeline to assess the broadest fungal diversity, with a more accurate taxonomic assignment, in walnut organs with dieback symptoms.

Pythium oligandrum: an example of opportunistic success.

Pythium oligandrum, a non-pathogenic soil-inhabiting oomycete, colonizes the root ecosystem of many crop species. Whereas most members in the genus Pythium are plant pathogens, P. oligandrum distinguishes itself from the pathogenic species by its ability to protect plants from biotic stresses in addition to promoting plant growth. The success of P. oligandrum at controlling soilborne pathogens is partly associated with direct antagonism mediated by mycoparasitism and antimicrobial compounds. Interestingly, P. oligandrum has evolved with specific mechanisms to attack its prey even when these belong to closely related species. Of particular relevance is the question of how P. oligandrum distinguishes between self- and non-self cell wall degradation during the mycoparasitic process of pathogenic oomycete species. The ability of P. oligandrum to enter and colonize the root system before rapidly degenerating is one of the most striking features that differentiate it from all other known biocontrol fungal agents. In spite of this atypical behaviour, P. oligandrum sensitizes the plant to defend itself through the production of at least two types of microbe-associated molecular patterns, including oligandrin and cell wall protein fractions, which appear to be closely involved in the early events preceding activation of the jasmonic acid- and ethylene-dependent signalling pathways and subsequent localized and systemic induced resistance. The aim of this review is to highlight the expanding knowledge of the mechanisms by which P. oligandrum provides beneficial effects to plants and to explore the potential use of this oomycete or its metabolites as new disease management strategies.

Water Microbiota in Greenhouses With Soilless Cultures of Tomato by Metabarcoding and Culture-Dependent Approaches.

Water supply, in hydroponic greenhouses, can originate from groundwater, surface water or rainwater stored in open tanks. To limit contamination of water supply, several methods have been used including active and passive methods such as slow filtration techniques which consist in passing the nutrient solutions slowly through filters. The purpose of this study was to describe the microbiota associated with water sampled before entering greenhouses and in recirculating nutrient solutions, either before or after running through a biofiltration system. Metabarcoding analysis revealed that water ecosystems were unique niches for diverse bacterial and fungal communities. Microbial composition varied greatly across storage conditions (groundwater vs. rainwater) and among greenhouses, suggesting that water microbiota is site- and storage-condition-specific. Nonetheless, we found that microbiota structure in open-stored water (either coming from ground or rain) shared a higher degree of similarity than with water directly pumped out of the ground. Open-stored waters were characterized by predominant taxa, notably those involved in aerobic chemoheterotrophy, such as the Sphingomonadaceae and Hyphomicrobiaceae families. Water directly collected from the ground showed the lowest levels of fungal and bacterial richness while also characterized by a significantly higher level of bacterial equitability and an enrichment in taxa involved in N-cycling. Slow filtration allowed reducing cultivable bacterial loads as well Pythium spp. and Fusarium oxysporum propagules, based on culture-dependent results, without compromising microbiota richness and diversity. Although compositional structure was similar following biofiltration, significant differences in bacterial (but not fungal) taxa abundance were reported, with primarily an enrichment of Chelativorans, Mycobacterium, and Gemmata as well as a depletion of Rhodobacter, Aminobacter, and Ellin329. The exact mechanisms by which such taxa would be favored at the expense of other remained unknown. Besides the accurate description of microbiota found in water at both taxonomical and predicted functional levels, our study allowed comparing the water microbiota between various storage system and following biofiltration. Although preliminary, our results provide a first insight into the potential microbial diversity, which can increase ecosystem functionality.

Deciphering the Infectious Process of Colletotrichum lupini in Lupin through Transcriptomic and Proteomic Analysis.

The fungal phytopathogen Colletotrichum lupini is responsible for lupin anthracnose, resulting in significant yield losses worldwide. The molecular mechanisms underlying this infectious process are yet to be elucidated. This study proposes to evaluate C. lupini gene expression and protein synthesis during lupin infection, using, respectively, an RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach. Patterns of differentially-expressed genes in planta were evaluated from 24 to 84 hours post-inoculation, and compared to in vitro cultures. A total of 897 differentially-expressed genes were identified from C. lupini during interaction with white lupin, of which 520 genes were predicted to have a putative function, including carbohydrate active enzyme, effector, protease or transporter-encoding genes, commonly described as pathogenicity factors for other Colletotrichum species during plant infection, and 377 hypothetical proteins. Simultaneously, a total of 304 proteins produced during the interaction were identified and quantified by mass spectrometry. Taken together, the results highlight that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes with unknown or poorly-described functions were found to be specifically associated with the early or late stages of infection, suggesting that they may be of importance for pathogenicity. This study, conducted for the first time on a species belonging to the Colletotrichum acutatum species complex, presents an opportunity to deepen functional analyses of the genes involved in the pathogenicity of Colletotrichum spp. during the onset of plant infection.

Influence of Pythium oligandrum biocontrol on fungal and oomycete population dynamics in the rhizosphere.

Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.

Combined Metabarcoding and Co-occurrence Network Analysis to Profile the Bacterial, Fungal and Fusarium Communities and Their Interactions in Maize Stalks.

Fusarium Head Blight (FHB) is one of the most devastating diseases of cereals worldwide, threatening both crop production by affecting cereal grain development, and human and animal health by contaminating grains with mycotoxins. Despite that maize residues constitute the primary source of inoculum for Fusarium pathogenic species, the structure and diversity of Fusarium spp. and microbial communities in maize residues have received much less attention than in grains. In this study, a metabarcoding approach was used to study the bacterial, fungal and Fusarium communities encountered in maize stalks collected from 8 fields in Brittany, France, after maize harvest during fall 2015. Some predominant genera found in maize residues were cereal or maize pathogens, such as the fungal Fusarium, Acremonium, and Phoma genera, and the bacterial Pseudomonas and Erwinia genera. Furthermore, a high predominance of genera with previously reported biocontrol activity was found, including the bacterial Sphingomonas, Pedobacter, Flavobacterium, Pseudomonas, and Janthinobacterium genera; and the fungal Epicoccum, Articulospora, Exophiala, and Sarocladium genera. Among Fusarium spp., F. graminearum and F. avenaceum were dominant. We also found that the maize cultivar and previous crop could influence the structure of microbial communities. Using SparCC co-occurrence network analysis, significant negative correlations were obtained between Fusarium spp. responsible for FHB (including F. graminearum and F. avenaceum) and bacterial OTUs classified as Sphingomonas and fungal OTUs classified as Sarocladium and Epicoccum. Considering that isolates belonging to these taxa have already been associated with antagonist effect against different Fusarium spp. and/or other pathogenic microorganisms and due to their predominance and negative associations with Fusarium spp., they may be good candidates as biocontrol agents. Combining the use of Fusarium-specific primers with universal primers for bacteria and fungi allowed us to study the microbial communities, but also to track correlations between Fusarium spp. and other bacterial and fungal genera, using co-occurrence network analysis. Such approach could be a useful tool as part of a screening strategy for novel antagonist candidates against toxigenic Fusarium spp., allowing the selection of taxa of interest.

A novel metabarcoding approach to investigate Fusarium species composition in soil and plant samples.

The genus Fusarium contains more than 300 species, most of which are plant pathogens. Appropriate molecular tools for accurately and rapidly describing temporal and spatial shifts in Fusarium communities would be useful for the development of control strategies. Here, we present a new Fusarium-specific primer pair targeting the translation elongation factor 1-α (EF1α) gene with amplicons of ~430 bp, suitable for MiSeq metabarcoding sequencing. Mock Fusarium communities were used to evaluate its resolution and to optimize read filtering and downstream analyses. The use of the DADA2 pipeline coupled with operational taxonomic unit (OTU) picking at 98% similarity cut-off significantly increased the accuracy of read filtering. Building a phylogenetic tree using a manually curated database as a reference allowed taxonomic assignment at the species or species-complex level. This methodology was tested on soil and maize residue samples collected from crop fields. Up to 18 Fusarium OTUs, belonging to 17 species and 8 species complexes, were obtained, with F. oxysporum being the most abundant species in soil samples, while F. graminearum and F. avenaceum were the most abundant in maize residues. We demonstrated the high performance of this workflow which could be further used for profiling Fusarium species composition and dynamics during the cultivation cycle.

Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster.

Lupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01-0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting. PCR-based detection systems exhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific C. lupini primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of C. lupini, 23 different Colletotrichum species, and 21 different organisms isolated from seeds of Lupinus albus cv. Multitalia, L. luteus cv. Mister, and L. angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/10,000 infected seed.

Radical scavenging, antioxidant and antimicrobial activities of halophytic species.

For the first time, both antioxidant and antimicrobial activities are simultaneously reported in halophytic plants, particularly on polar fractions. Chloroformic and methanolic extracts of the halophytes Eryngium maritimum L., Crithmum maritimum L. and Cakile maritima Scop. were tested for their antimicrobial activities against 12 bacterial and yeast strains. In addition, radical scavenging and antioxidant activities were assessed, as well as total phenol contents. Only one bacterial strain (Listeria monocytogenes) was not inhibited by plants extracts, and apolar (chloroformic) fractions were generally more active than polar (methanolic) ones. Eryngium maritimum presented the weakest radical scavenging activity (ABTS IC(50)=0.28 mg ml(-1)), as well as the lowest total phenol content (16.4 mg GAE g(-1) DW). However, the three halophytic species had relatively strong total antioxidant activities (from 32.7 to 48.6 mg ascorbate equivalents g (-1) DW). Consequences on the potential use of these plants in food or cosmetic industry are discussed.

Combined Metabarcoding and Multi-locus approach for Genetic characterization of Colletotrichum species associated with common walnut (Juglans regia) anthracnose in France.

Juglans regia (walnut) is a species belonging to the family Juglandaceae. Broadly spread in diverse temperate and subtropical regions, walnut is primarily cultivated for its nuts. In France, Colletotrichum sp. on walnut was detected for the first time in 2007; in 2011 the disease led to 50-70% losses in nut production. A combined approach of metabarcoding analysis and multi-locus genetic characterization of isolated strains has been used for taxonomic designation and to study the genetic variability of this pathogen in France. Evidence indicates that four Colletotrichum species are associated with walnut in France: 3 belong to the C. acutatum species complex and 1 to the C. gloeosporioides species complex. Results also show that C. godetiae is the most abundant species followed by C. fioriniae; while C. nymphaeae and another Colletotrichum sp. belonging to the C. gloeosporioides complex are found rarely. Representative isolates of detected species were also used to confirm pathogenicity on walnut fruits. The results show a high variability of lesion's dimensions among isolates tested. This study highlights the genetic and pathogenic heterogeneity of Colletotrichum species associated with walnut anthracnose in France providing useful information for targeted treatments or selection of resistant cultivars, in order to better control the disease.