New insights from short and long reads sequencing to explore cytochrome b variants in Plasmopara viticola populations collected from vineyards and related to resistance to complex III inhibitors.

Downy mildew is caused by Plasmopara viticola, an obligate oomycete plant pathogen, a devasting disease of grapevine. To protect plants from the disease, complex III inhibitors are among the fungicides widely used. They specifically target the mitochondrial cytochrome b (cytb) of the pathogen to block cellular respiration mechanisms. In the French vineyard, P. viticola has developed resistance against a first group of these fungicides, the Quinone outside Inhibitors (QoI), with a single amino acid substitution G143A in its cytb mitochondrial sequence. The use of QoI was limited and another type of fungicide, the Quinone inside Inhibitors, targeting the same gene and highly effective against oomycetes, was used instead. Recently however, less sensitive P. viticola populations were detected after treatments with some inhibitors, in particular ametoctradin and cyazofamid. By isolating single-sporangia P. viticola strains resistant to these fungicides, we characterized new variants in the cytb sequences associated with cyazofamid resistance: a point mutation (L201S) and more strikingly, two insertions (E203-DE-V204, E203-VE-V204). In parallel with the classical tools, pyrosequencing and qPCR, we then benchmarked short and long-reads NGS technologies (Ion Torrent, Illumina, Oxford Nanopore Technologies) to sequence the complete cytb with a view to detecting and assessing the proportion of resistant variants of P. viticola at the scale of a field population. Eighteen populations collected from French vineyard fields in 2020 were analysed: 12 showed a variable proportion of G143A, 11 of E203-DE-V204 and 7 populations of the S34L variant that confers resistance to ametoctradin. Interestingly, the long reads were able to identify variants, including SNPs, with confidence and to detect a small proportion of P. viticola with multiple variants along the same cytb sequence. Overall, NGS appears to be a promising method for assessing fungicide resistance of pathogens linked to cytb modifications at the field population level. This approach could rapidly become a robust decision support tool for resistance management in the future.

Durum Wheat Stress Tolerance Induced by Endophyte Pantoea agglomerans with Genes Contributing to Plant Functions and Secondary Metabolite Arsenal.

In the arid region Bou-Saâda at the South of Algeria, durum wheat Triticum durum L. cv Waha production is severely threatened by abiotic stresses, mainly drought and salinity. Plant growth-promoting rhizobacteria (PGPR) hold promising prospects towards sustainable and environmentally-friendly agriculture. Using habitat-adapted symbiosis strategy, the PGPR Pantoea agglomerans strain Pa was recovered from wheat roots sampled in Bou-Saâda, conferred alleviation of salt stress in durum wheat plants and allowed considerable growth in this unhostile environment. Strain Pa showed growth up to 35 °C temperature, 5-10 pH range, and up to 30% polyethylene glycol (PEG), as well as 1 M salt concentration tolerance. Pa strain displayed pertinent plant growth promotion (PGP) features (direct and indirect) such as hormone auxin biosynthesis, production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and ammonia and phosphate solubilization. PGPR features were stable over wide salt concentrations (0-400 mM). Pa strain was also able to survive in seeds, in the non-sterile and sterile wheat rhizosphere, and was shown to have an endophytic life style. Phylogenomic analysis of strain Pa indicated that Pantoea genus suffers taxonomic imprecision which blurs species delimitation and may have impacted their practical use as biofertilizers. When applied to plants, strain Pa promoted considerable growth of wheat seedlings, high chlorophyll content, lower accumulation of proline, and favored K+ accumulation in the inoculated plants when compared to Na+ in control non-inoculated plants. Metabolomic profiling of strain Pa under one strain many compounds (OSMAC) conditions revealed a wide diversity of secondary metabolites (SM) with interesting salt stress alleviation and PGP activities. All these findings strongly promote the implementation of Pantoea agglomerans strain Pa as an efficient biofertilizer in wheat plants culture in arid and salinity-impacted regions.

Emergence of boscalid-resistant strains of Erysiphe necator in French vineyards.

The grapevine powdery mildew Erysiphe necator (E. necator) is an obligate pathogen. Powdery mildew-diseased vines show an important reduction in plant size, winter hardiness and grape yield. Even a low-level infection with powdery mildew was shown to taint wine and ultimately reduce wine quality. For many years, succinate dehydrogenase inhibitor (SDHI) fungicides, mainly the new generation active ingredients (AIs) boscalid, penthiopyrad and fluopyram, have been widely used to control powdery mildew in grapevines. The repeated use of fungicides (mainly boscalid) has resulted in the emergence of resistant microorganisms such as Botrytis cinerea (B. cinerea). However, boscalid resistance was never observed in E. necator. In this study, a large-scale survey of French grapevine field populations of E. necator revealed many field populations with low sensitivity to boscalid. Single spore strains originating from collected resistant populations showed Half maximal effective concentration (EC50) values greater than 100 mg L-1, and strains originating from boscalid sensitive populations showed EC50 values lower than 1 mg L-1. The complete nucleotide sequences of the EnSdhB succinate dehydrogenase of sensitive and resistant single spore strains revealed that H242R and H242Y substitutions in the EnSdhB succinate dehydrogenase subunit conferred E. necator resistance to boscalid. No cross-resistance of E. necator strains bearing H242R and H242Y substitutions in EnSdhB succinate dehydrogenase to fluxapyroxad and fluopyram was noticed. Therefore, our results highlight the emergence of resistance to boscalid activity in French vineyards and warrant the need of the implementation of risk assessment strategies to maintain effective grapevine protection against powdery mildew.

Exposure of marine mussels to diclofenac: modulation of prostaglandin biosynthesis.

Human pharmaceuticals, such as nonsteroidal anti-inflammatory drugs (NSAIDs), are an emerging threat to marine organisms. NSAIDs act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid into prostaglandins. One experiment was carried out whereby marine mussels were exposed for 72 h to 1 and 100 µg/L diclofenac (DCF). A specific and sensitive method using liquid chromatography high-resolution tandem mass spectrometry was developed to quantify DCF in mussel tissues. The developed method could also clearly identify and quantify COX products, i.e., prostaglandin levels, and be used to assess their modulation following DCF exposure. Prostaglandin-D2 (PGD2) was always found below the detection limit (20 µg/kg dry weight (dw)). Basal prostaglandin-E2 (PGE2) concentrations ranged from below the detection limit to 202 µg/kg dw. Exposure of 100 µg/L resulted in a significant reduction in PGE2 levels, whereas a downward trend was observed at 1 µg/L exposure. No difference was observed for prostaglandin-F2α (PGF2α) levels between controls and exposed organisms.

Comparative Genomics of Bacillus amyloliquefaciens Strains Reveals a Core Genome with Traits for Habitat Adaptation and a Secondary Metabolites Rich Accessory Genome.

The Gram positive, non-pathogenic endospore-forming soil inhabiting prokaryote Bacillus amyloliquefaciens is a plant growth-promoting rhizobacterium. Bacillus amyloliquefaciens processes wide biocontrol abilities and numerous strains have been reported to suppress diverse bacterial, fungal and fungal-like pathogens. Knowledge about strain level biocontrol abilities is warranted to translate this knowledge into developing more efficient biocontrol agents and bio-fertilizers. Ever-expanding genome studies of B. amyloliquefaciens are showing tremendous increase in strain-specific new secondary metabolite clusters which play key roles in the suppression of pathogens and plant growth promotion. In this report, we have used genome mining of all sequenced B. amyloliquefaciens genomes to highlight species boundaries, the diverse strategies used by different strains to promote plant growth and the diversity of their secondary metabolites. Genome composition of the targeted strains suggest regions of genomic plasticity that shape the structure and function of these genomes and govern strain adaptation to different niches. Our results indicated that B. amyloliquefaciens: (i) suffer taxonomic imprecision that blurs the debate over inter-strain genome diversity and dynamics, (ii) have diverse strategies to promote plant growth and development, (iii) have an unlocked, yet to be delimited impressive arsenal of secondary metabolites and products, (iv) have large number of so-called orphan gene clusters, i.e., biosynthetic clusters for which the corresponding metabolites are yet unknown, and (v) have a dynamic pan genome with a secondary metabolite rich accessory genome.

Proteomic analysis of proteins secreted by Botrytis cinerea in response to heavy metal toxicity.

Although essential in many cellular processes, metals become toxic when they are present in excess and constitute a global environmental hazard. To overcome this stress, fungi have evolved several mechanisms at both intracellular and extracellular levels. In particular, fungi are well known for their ability to secrete a large panel of proteins. However, their role in the adaptation of fungi to metal toxicity has not yet been investigated. To address this question, here, the fungus Botrytis cinerea was challenged to copper, zinc, nickel or cadmium stress and secreted proteins were collected and separated by 2D-PAGE. One hundred and sixteen spots whose volume varied under at least one tested condition were observed on 2D gels. Densitometric analyses revealed that the secretome signature in response to cadmium was significantly different from those obtained with the other metals. Fifty-five of these 116 spots were associated with unique proteins and functional classification revealed that the production of oxidoreductases and cell-wall degrading enzymes was modified in response to metals. Promoter analysis disclosed that PacC/Rim101 sites were statistically over-represented in the upstream sequences of the 31 genes corresponding to the varying unique spots suggesting a possible link between pH regulation and metal response in B. cinerea.