A method to determine antifungal activity in seed exudates by nephelometry.

BACKGROUND: One of the levers towards alternative solutions to pesticides is to improve seed defenses against pathogens, but a better understanding is needed on the type and regulation of existing pathways during germination. Dormant seeds are able to defend themselves against microorganisms during cycles of rehydration and dehydration in the soil. During imbibition, seeds leak copious amounts of exudates. Here, we developed a nephelometry method to assay antimicrobial activity (AA) in tomato seed exudates as a proxy to assess level of defenses. RESULTS: A protocol is described to determine the level of AA against the nonhost filamentous fungus Alternaria brassicicola in the exudates of tomato seeds and seedlings. The fungal and exudate concentrations can be adjusted to modulate the assay sensitivity, thereby providing a large window of AA detection. We established that AA in dormant seeds depends on the genotype. It ranged from very strong AA to complete absence of AA, even after prolonged imbibition. AA depends also on the stages of germination and seedling emergence. Exudates from germinated seeds and seedlings showed very strong AA, while those from dormant seeds exhibited less activity for the same imbibition time. The exudate AA did not impact the growth of a pathogenic fungus host of tomato, Alternaria alternata, illustrating the adaptation of this fungus to its host. CONCLUSIONS: We demonstrate that our nephelometry method is a simple yet powerful bioassay to quantify AA in seed exudates. Different developmental stages from dormant seed to seedlings show different levels of AA in the exudate that vary between genotypes, highlighting a genetic diversity x developmental stage interaction in defense. These findings will be important to identify molecules in the exudates conferring antifungal properties and obtain a better understanding of the regulatory and biosynthetic pathways through the lifecycle of seeds, from dormant seeds until seedling emergence.

Natural Products Targeting the Fungal Unfolded Protein Response as an Alternative Crop Protection Strategy.

Discovering new solutions for crop protection is a major challenge for the next decades as a result of the ecotoxicological impact of classical fungicides, the emergence of fungicide resistances, and the consequence of climate change on pathogen distribution. Previous work on fungal mutants deficient in the unfolded protein response (UPR) supported that targeting this pathway is a promising plant disease control strategy. In particular, we showed that the UPR is involved in fungal virulence by altering cell protection against host defense compounds, such as phytoalexins and phytoanticipins. In this study, we evaluated natural products targeting fungal IRE1 protein (UPR effector) and consequently increasing fungal susceptibility to plant defenses. Developing an in vitro cell-based screening assay allowed for the identification of seven potential IRE1 inhibitors with a focus on polyhydroxylated prenylated xanthones. Inhibition of hac1 mRNA splicing, which is mediated by IRE1, was then validated for the most active compound, namely, γ-mangostin 3. To study the mode of interaction between the binding site of IRE1 and active xanthones, molecular docking was also undertaken, revealing similar and novel interactions between the known inhibitor and the binding site. Eventually, active xanthones applied at subtoxic doses induced a significant reduction in necrosis size for leaves of Brassica oleracea inoculated with Alternaria brassicicola and Botrytis cinerea.

Pentadecaibins I-V: 15-Residue Peptaibols Produced by a Marine-Derived Trichoderma sp. of the Harzianum Clade.

In the course of investigations on peptaibol chemodiversity from marine-derived Trichoderma spp., five new 15-residue peptaibols named pentadecaibins I-V (1-5) were isolated from the solid culture of the strain Trichoderma sp. MMS1255 belonging to the T. harzianum species complex. Phylogenetic analyses allowed precise positioning of the strain close to T. lentiforme lineage inside the Harzianum clade. Peptaibol sequences were elucidated on the basis of their MS/MS fragmentation and extensive 2D NMR experiments. Amino acid configurations were determined by Marfey's analyses. The pentadecaibins are based on the sequences Ac-Aib1-Gly2-Ala3-Leu4-Aib/Iva5-Gln6-Aib/Iva7-Val/Leu8-Aib9-Ala10-Aib11-Aib12-Aib13-Gln14-Pheol15. Characteristic of the pentadecaibin sequences is the lack of the Aib-Pro motif commonly present in peptaibols produced by Trichoderma spp. Genome sequencing of Trichoderma sp. MMS1255 allowed the detection of a 15-module NRPS-encoding gene closely associated with pentadecaibin biosynthesis. Pentadecaibins were assessed for their potential antiproliferative and antimicrobial activities.

Responses to Hydric Stress in the Seed-Borne Necrotrophic Fungus Alternaria brassicicola.

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.

A flavoprotein supports cell wall properties in the necrotrophic fungus Alternaria brassicicola.

BACKGROUND: Flavin-dependent monooxygenases are involved in key biological processes as they catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. Flavoprotein monooxygenases are frequently encountered in micro-organisms, most of which require further functional and biocatalytic assessment. Here we investigated the function of the AbMak1 gene, which encodes a group A flavin monooxygenase in the plant pathogenic fungus Alternaria brassicicola, by generating a deficient mutant and examining its phenotype. RESULTS: Functional analysis indicates that the AbMak1 protein is involved in cell wall biogenesis and influences the melanization process. We documented a significant decrease in melanin content in the Δabmak1 strain compared to the wild-type and complemented strains. We investigated the cell wall morphology and physical properties in the wild-type and transformants using electron and atomic force microscopy. These approaches confirmed the aberrant morphology of the conidial wall structure in the Δabmak1 strain which had an impact on hydrophilic adhesion and conidial surface stiffness. However, there was no significant impairment in growth, conidia formation, pathogenicity or susceptibility to various environmental stresses in the Δabmak1 strain. CONCLUSION: This study sheds new light on the function of a fungal flavin-dependent monooxygenase, which plays an important role in melanization.

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.