Necrotrophic Fungus Botrytis Cinerea Research Articles

Phenylalanine increases chrysanthemum flower immunity against Botrytis cinerea attack.

Flowers are the most vulnerable plant organ to infection by the necrotrophic fungus Botrytis cinerea. Here we show that pre-treatment of chrysanthemum (Chrysanthemum morifolium) flowers with phenylalanine (Phe) significantly reduces their susceptibility to B.cinerea. To comprehend how Phe treatment induces resistance, we monitored the dynamics of metabolites (by GC/LC-MS) and transcriptomes (by RNAseq) in flowers after Phe treatment and B.cinerea infection. Phe treatment resulted in accumulation of 3-phenyllactate and benzaldehyde, and in particular induced the expression of genes related to Ca2+ signaling and receptor kinases, implicating an induction of the defense response. Interestingly, the main effects of Phe treatment were observed in flowers exposed to B.cinerea infection, stabilizing the global fluctuations in the levels of metabolites and transcripts while reducing susceptibility to the fungus. We suggest that Phe-induced resistance is associated to cell priming, enabling rapid and targeted reprogramming of cellular defense responses to resist disease development. After Phe pre-treatment, the levels of the anti-fungal volatiles phenylacetaldehyde and eugenol were maintained and the level of coniferin, a plausible monolignol precursor in cell wall lignification, was strongly increased. In addition, Phe pre-treatment reduced ROS generation, prevented ethylene emission, and caused changes in the expression of a minor number of genes related to cell wall biogenesis, encoding the RLK THESEUS1, or involved in Ca2+ and hormonal signaling processes. Our findings point to Phe pre-treatment as a potential orchestrator of a broad-spectrum defense response which may not only provide an ecologically friendly pest control strategy but also offers a promising way of priming plants to induce defense responses against B.cinerea.

Transcription Factor PdeR Is Involved in Fungal Development, Metabolic Change, and Pathogenesis of Gray Mold Botrytis cinerea.

The necrotrophic fungus Botrytis cinerea releases extracellular enzymes that facilitate its penetration into a host. This study functionally characterized the gene pdeR of B. cinerea, which is predicted to encode a Zn(II)2Cys6 zinc finger transcription factor. To investigate the role of pdeR, deleted and complemented strains of pdeR in B. cinerea were generated, which were designated as ΔpdeR and PdeRc, respectively. The ΔpdeR strain exhibited impaired germination and growth compared to the wild-type and PdeRc strains, particularly when provided with maltose as the sole carbon source. When all of the strains were grown on a minimal medium containing polysaccharide as the sole carbon source, the ΔpdeR exclusively showed defects in polysaccharide hydrolysis with reduced gene expression encoding for amylase and cellulase. As far as the involvement of pdeR in carbon metabolism is concerned, metabolic changes were investigated in the ΔpdeR mutant. Comparisons of relative, normalized concentrations of each metabolite showed that the amounts of six metabolites including glucose and trehalose were significantly changed in the ΔpdeR strain. Based on pleiotropic changes derived from the deletion of pdeR, we hypothesized that pdeR has an important role in pathogenesis. When the ΔpdeR strain was inoculated onto pepper plant, the ΔpdeR strain did not cause expansion of the disease lesions from the infection sites, which grew on the surface without any penetration. Taken together, these results show that the deletion of pdeR affected the extracellular enzymatic activity, leading to changes in fungal development, metabolism, and virulence.

Characterization of the Role of a Non-GPCR Membrane-Bound CFEM Protein in the Pathogenicity and Germination of Botrytis cinerea.

The necrotrophic fungus Botrytis cinerea, is considered a major cause of postharvest losses in a wide range of crops. The common fungal extracellular membrane protein (CFEM), containing a conserved eight-cysteine pattern, was found exclusively in fungi. Previous studies in phytopathogenic fungi have demonstrated the role of membrane-bound and secreted CFEM-containing proteins in different aspects of fungal virulence. However, non-G protein-coupled receptor (non-GPCR) membrane CFEM proteins have not been studied yet in phytopathogenic fungi. In the present study, we have identified a non-GPCR membrane-bound CFEM-containing protein, Bcin07g03260, in the B. cinerea genome, and generated deletion mutants, ΔCFEM-Bcin07g03260, to study its potential role in physiology and virulence. Three independent ΔCFEM-Bcin07g03260 mutants showed significantly reduced progression of a necrotic lesion on tomato (Solanum lycopersicum) leaves. Further analysis of the mutants revealed significant reduction (approximately 20–30%) in conidial germination and consequent germ tube elongation compared with the WT. Our data complements a previous study of secreted ΔCFEM1 mutants of B. cinerea that showed reduced progression of necrotic lesions on leaves, without effect on germination. Considering various functions identified for CFEM proteins in fungal virulence, our work illustrates a potential new role for a non-GPCR membrane CFEM in pathogenic fungi to control virulence in the fungus B. cinerea.

Fungal-derived extracts induce resistance against Botrytis cinerea in Arabidopsis thaliana

Development of a sustainable agriculture is a current trend searching for alternative and more environmentally friendly strategies to control crop diseases. The necrotrophic fungus Botrytis cinerea, the causal agent of grey mould, affects crops pre- and post-harvest and cause huge economical losses. Microorganisms and microbially-derived elicitors are known to induce plant defences. Here, we study two fungal extracts derived from Colletotrichum acutatum (isolate M11). The crude extract, called CF (culture filtrate) and the semi-purified fraction ACF (axenic culture filtrate) consist in a blend of fungal-derived elicitors. Their activities were evaluated in the plant-pathogen system Arabidopsis thaliana-B. cinerea. Both CF and ACF induced local and systemic resistance, rendering A. thaliana resistant against grey mould disease. The extracts showed no antifungal activity since no inhibition of mycelium growth, changes in hyphal ultrastructure or damage in cell membrane permeability were observed. These findings would indicate that the protective effect was indirect and it was found that CF and ACF induced up-regulation of the defence-related genes PR1, WRKY70 and ERF6, as well as giving local callose deposition in treated leaves. Both fungal extracts have the potential to be used as a main component of a biologically based disease control product with activity against grey mould, with the advantage that the purification process of CF is simpler than for ACF.

SlREM1 Triggers Cell Death by Activating an Oxidative Burst and Other Regulators.

Programmed cell death (PCD), a highly regulated feature of the plant immune response, involves multiple molecular players. Remorins (REMs) are plant-specific proteins with varied biological functions, but their function in PCD and plant defense remains largely unknown. Here, we report a role for remorin in disease resistance, immune response, and PCD regulation. Overexpression of tomato (Solanum lycopersicum) REMORIN1 (SlREM1) increased susceptibility of tomato to the necrotrophic fungus Botrytis cinerea and heterologous expression of this gene triggered cell death in Nicotiana benthamiana leaves. Further investigation indicated that amino acids 173 to 187and phosphorylation of SlREM1 played key roles in SlREM1-triggered cell death. Intriguingly, multiple tomato REMs induced cell death in N benthamiana leaves. Yeast two-hybrid, split luciferase complementation, and coimmunoprecipitation assays all demonstrated that remorin proteins could form homo- and heterocomplexes. Using isobaric tags for relative and absolute quantitative proteomics, we identified that some proteins related to cell death regulation, as well as N benthamiana RESPIRATORY BURST OXIDASE HOMOLOG B (which is essential for reactive oxygen species production), were notably upregulated in SlREM1-expressing leaves. Heterologous expression of SlREM1 increased reactive oxygen species accumulation and triggered other cell death regulators. Our findings indicate that SlREM1 is a positive regulator of plant cell death and provide clues for understanding the PCD molecular regulatory network in plants.

Histochemical and Microscopic Studies Predict that Grapevine Genotype “Ju mei gui” is Highly Resistant against Botrytis cinerea

The necrotrophic fungus Botrytis cinerea causes devastating pre- and post-harvest yield losses in grapevine (Vitis vinifera L.). Although B. cinerea has been well-studied in different plant species, there is limited information related to the resistance and susceptibility mechanisms of Vitis genotypes against B. cinerea infection. In the present study, leaves and berries of twenty four grape genotypes were evaluated against B. cinerea infection. According to the results, one genotype (Ju mei gui) was highly resistant (HR), one genotype (Kyoho) was resistant (R), eight genotypes were susceptible (S), and fourteen genotypes were highly susceptible (HS) against infection of B. cinerea in leaves. Whereas in the case of B. cinerea infection in grape berry, three genotypes were found to be highly resistant, three resistant, eleven genotypes susceptible, and seven were highly susceptible. To further explore the mechanism of disease resistance in grapevine, we evaluated “Ju mei gui” and “Summer black” in terms of B. cinerea progression, reactive oxygen species reactions, jasmonic acid contents, and the activities of antioxidant enzymes in leaf and fruit. We surmise that the resistance of “Ju mei gui” is due to seized fungal growth, minor reactive oxygen species (ROS) production, elevated antioxidant enzyme activity, and more jasmonic acid (JA) contents. This study provides insights into the resistance and susceptibility mechanism of Vitis genotypes against B. cinerea. This will help for the selection of appropriate germplasm to explore the molecular basis of disease resistance mechanisms in grapevine.

The BIR2/BIR3-Associated Phospholipase Dγ1 Negatively Regulates Plant Immunity.

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members, and for some of these, a specific function in resistance toward a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance toward the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.

Genomics and LC-MS Reveal Diverse Active Secondary Metabolites in Bacillus amyloliquefaciens WS-8.

Bacillus amyloliquefaciens is an important plant disease-preventing and growth-promoting microorganism. B. amyloliquefaciens WS-8 can stimulate plant growth and has strong antifungal properties. In this study, we sequenced the complete genome of B. amyloliquefaciens WS-8 by Pacific Biosciences RSII (PacBio) Single Molecule Real-Time (SMRT) sequencing. The genome consists of one chromosome (3,929,787 bp) and no additional plasmids. The main bacteriostatic substances were determined by genome, transcriptome, and mass spectrometry data. We thereby laid a theoretical foundation for the utilization of the strain. By genomic analysis, we identified 19 putative biosynthetic gene clusters for secondary metabolites, most of which are potentially involved in the biosynthesis of numerous bioactive metabolites, including difficidin, fengycin, and surfactin. Furthermore, a potential class II lanthipeptide biosynthetic gene cluster and genes that are involved in auxin biosynthesis were found. Through the analysis of transcriptome data, we found that the key bacteriostatic genes, as predicted in the genome, exhibited different levels of mRNA expression. Through metabolite isolation, purification, and exposure experiments, we found that a variety of metabolites of WS-8 exert an inhibitory effect on the necrotrophic fungus Botrytis cinerea, which causes gray mold; by mass spectrometry, we found that the main substances are mainly iturins and fengycins. Therefore, this strain has the potential to be utilized as an antifungal agent in agriculture.

Inactivation of UDP-Glucose Sterol Glucosyltransferases Enhances Arabidopsis Resistance to Botrytis cinerea.

Free and glycosylated sterols are both structural components of the plasma membrane that regulate their biophysical properties and consequently different plasma membrane-associated processes such as plant adaptation to stress or signaling. Several reports relate changes in glycosylated sterols levels with the plant response to abiotic stress, but the information about the role of these compounds in the response to biotic stress is scarce. In this work, we have studied the response to the necrotrophic fungus Botrytis cinerea in an Arabidopsis mutant that is severely impaired in steryl glycosides biosynthesis due to the inactivation of the two sterol glucosyltransferases (UGT80A2 and UGT80B1) reported in this plant. This mutant exhibits enhanced resistance against B. cinerea when compared to wild-type plants, which correlates with increased levels of jasmonic acid (JA) and up-regulation of two marker genes (PDF1.2 and PR4) of the ERF branch of the JA signaling pathway. Upon B. cinerea infection, the ugt80A2;B1 double mutant also accumulates higher levels of camalexin, the major Arabidopsis phytoalexin, than wild-type plants. Camalexin accumulation correlates with enhanced transcript levels of several cytochrome P450 camalexin biosynthetic genes, as well as of their transcriptional regulators WRKY33, ANAC042, and MYB51, suggesting that the Botrytis-induced accumulation of camalexin is coordinately regulated at the transcriptional level. After fungus infection, the expression of genes involved in the indole glucosinolate biosynthesis is also up-regulated at a higher degree in the ugt80A2;B1 mutant than in wild-type plants. Altogether, the results of this study show that glycosylated sterols play an important role in the regulation of Arabidopsis response to B. cinerea infection and suggest that this occurs through signaling pathways involving the canonical stress-hormone JA and the tryptophan-derived secondary metabolites camalexin and possibly also indole glucosinolates.

Far-red radiation increases dry mass partitioning to fruits but reduces Botrytis cinerea resistance in tomato

The addition of far-red (FR, 700–800 nm) radiation to standard growth light triggers a set of photomorphogenic responses collectively termed shade avoidance syndrome. Recent research showed that additional FR increased fruit yield in greenhouse tomato production. However, the mechanism behind this increase is not clear; nor is it known whether there is a trade-off between growth and defense against plant diseases in tomato under additional FR. The aims of this study were 1) to quantify the effect of additional FR on tomato fruit growth, 2) to explain this effect based on underlying growth components and 3) to examine the FR effect on resistance against the necrotrophic fungus Botrytis cinerea. Tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown for four months with 30 or 50 μmol m−2 s−1 of FR added to 150 μmol m−2 s−1 red + blue or white background LED lighting. Growth and development parameters were recorded, and a growth component analysis was conducted. Bioassays for resistance against B. cinerea were conducted on leaf samples collected from each light treatment. Additional FR increased total fruit dry mass per plant by 26–45%. FR affected multiple growth components, among which the fraction of dry mass partitioned to fruits was the most prominent with a 15–35% increase. Truss appearance rate was increased 11–14% by FR while instantaneous net photosynthesis rate was not affected. FR also resulted in more severe disease symptoms upon infection with B. cinerea. In conclusion, additional FR increases tomato fruit production mainly by increasing dry mass partitioning to fruits, rather than improving photosynthesis or increasing total plant biomass. However, FR also reduces resistance of tomato leaves against B. cinerea.

The molecular dialogue between grapevine inflorescence/berry and Botrytis cinerea during initial, quiescent and egression infection stages

Grape quality and yield are affected by bunch rot disease, caused by the necrotrophic fungus Botrytis cinerea. Primary infection often occurs at blooming, although the fungus remains quiescent until maturity and egresses at ripening, causing bunch rot. The molecular dialogue between B. cinerea and the grapevine inflorescence/berry from bloom until maturity is not completely elucidated, although its understanding is vital to implement proper management. In this study, a molecular characterization of the B. cinerea-flower/berry interaction was achieved using transcriptomic and metabolic analysis of the host and the pathogen. Open flowers from fruiting cuttings of 'Pinot Noir' were infected with green fluorescent protein (GFP)-labelled B. cinerea, and samples were collected at 24 and 96 h post-inoculation (hpi) and at 4 and 12 weeks post-inoculation (wpi). Our results indicated that penetration of the flower epidermis by B. cinerea at 24 hpi induced genes encoding virulence factors, representing the effort of the pathogen to invade the host. On the other hand, grapevine flowers responded rapidly, involving genes associated with the accumulation of pathogenesis-related (PR) proteins, stilbenoids, reactive oxygen species and cell-wall reinforcement. At 96 hpi, the transcriptional reaction appeared largely diminished in both the host and the pathogen. Afterwards, infected berries continued their developmental program without any visible symptoms. The interaction between the fungus and the hard, green berries was transcriptionally active. At 12 wpi, the egressed B. cinerea expressed almost all virulence- and growth-related genes to enable the pathogen to colonize the berries. In response to egression, ripe berries reprogramed different defence responses, though they were ineffective.

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from Serratia marcescens.

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.

Overexpression of the VvSWEET4 Transporter in Grapevine Hairy Roots Increases Sugar Transport and Contents and Enhances Resistance to Pythium irregulare, a Soilborne Pathogen.

Sugar transport and partitioning play key roles in the regulation of plant development and responses to biotic and abiotic factors. During plant/pathogen interactions, there is a competition for sugar that is controlled by membrane transporters and their regulation is decisive for the outcome of the interaction. SWEET sugar transporters are the targets of extracellular pathogens, which modify their expression to acquire the sugars necessary to their growth (Chen et al., 2010). The regulation of carbon allocation and sugar partitioning in the interaction between grapevine (Vitis vinifera) and its pathogens is poorly understood. We previously characterized the SWEET family in V. vinifera and showed that SWEET4 could be involved in resistance to the necrotrophic fungus Botrytis cinerea in Arabidopsis (Chong et al., 2014). To study the role of VvSWEET4 in grapevine, we produced V. vinifera cv. Syrah hairy roots overexpressing VvSWEET4 under the control of the CaMV 35S promoter (VvSWEET4OX). High levels of VvSWEET4 expression in hairy roots resulted in enhanced growth on media containing glucose or sucrose and increased contents in glucose and fructose. Sugar uptake assays further showed an improved glucose absorption in VvSWEET4 overexpressors. In parallel, we observed that VvSWEET4 expression was significantly induced after infection of wild type grapevine hairy roots with Pythium irregulare, a soilborne necrotrophic pathogen. Importantly, grapevine hairy roots overexpressing VvSWEET4 exhibited an improved resistance level to P. irregulare infection. This resistance phenotype was associated with higher glucose pools in roots after infection, higher constitutive expression of several genes involved in flavonoid biosynthesis, and higher flavanol contents. We propose that high sugar levels in VvSWEET4OX hairy roots provides a better support to the increased energy demand during pathogen infection. In addition, high sugar levels promote biosynthesis of flavonoids with antifungal properties. Overall, this work highlights the key role of sugar transport mediated by SWEET transporters for secondary metabolism regulation and pathogen resistance in grapevine.

The Arabidopsis Pleiotropic Drug Resistance Transporters PEN3 and PDR12 Mediate Camalexin Secretion for Resistance to Botrytis cinerea.

Plant defense often depends on the synthesis and targeted delivery of antimicrobial metabolites at pathogen contact sites. The pleiotropic drug resistance (PDR) transporter PENETRATION3 (PEN3)/PDR8 in Arabidopsis (Arabidopsis thaliana) has been implicated in resistance to a variety of fungal pathogens. However, the antimicrobial metabolite(s) transported by PEN3 for extracellular defense remains unidentified. Here, we report that PEN3 functions redundantly with another PDR transporter (PDR12) to mediate the secretion of camalexin, the major phytoalexin in Arabidopsis. Consistent with this, the pen3 pdr12 double mutants exhibit dramatically enhanced susceptibility to the necrotrophic fungus Botrytis cinerea as well as severe hypersensitivity to exogenous camalexin. PEN3 and PDR12 are transcriptionally activated upon B. cinerea infection, and their expression is regulated by the mitogen-activated protein kinase 3 (MPK3) and MPK6, and their downstream WRKY33 transcription factor. Further genetic analysis indicated that PEN3 and PDR12 contribute to B. cinerea resistance through exporting not only camalexin but also other unidentified metabolite(s) derived from Trp metabolism, suggesting that PEN3 and PDR12 have multiple functions in Arabidopsis immunity via transport of distinct Trp metabolic products.

Specific molecular interactions between Vitis vinifera and Botrytis cinerea are required for noble rot development in grape berries

Under peculiar climatic conditions, the beneficial form of the necrotrophic fungus Botrytis cinerea can develop on grape berries as a latent infection, known as noble rot, which induces positive biochemical and metabolic changes in the berries, including an increase in the sugar content and the production of aromatic compounds that improve wine quality. The infected berries undergo rapid withering, which is required to produce famous sweet white wines such as Sauternes and Tokaj. To gain insight into the molecular interactions between grapevine berries (Vitis vinifera) and B. cinerea during the establishment of noble rot, we prepared a large-scale transcriptomics dataset representing noble rot development and carried out a comparative meta-analysis with gray mold infection and natural post-harvest withering. In particular, we artificially induced berry botrytization of two grape varieties (Garganega and Möller-Thurgau) and sampled them at different stages of noble rot for comparative whole-transcriptome analysis, highlighting important common transcriptional reprogramming in both varieties reflecting an accelerated withering process. Simultaneously, we analyzed the modulation of B. cinerea genes and compared the expression profile during noble rot development with the previously reported gray mold infection profile, revealing the onset of an infection process by the fungus in its beneficial form associated with reduced virulence. This, together with the restrained plant defense response observed in botrytized berries, may favour the development of noble rot instead of gray mold. Finally, the comprehensive meta-analysis of gene expression during noble rot infection, gray mold and post-harvest withering led to the identification of key genes specifically modulated during noble rot infection.

Depression of Fungal Polygalacturonase Activity in Solanum lycopersicum Contributes to Antagonistic Yeast-Mediated Fruit Immunity to Botrytis.

The acquisition of susceptibility to necrotrophy over the course of ripening is one of the critical factors limiting shelf life. In this study, phytopathology and molecular biology were employed to explore the roles of pectinase in fruit susceptibility and ripening. Solanum lycopersicum fruit softened dramatically from entirely green to 50% red, which was accompanied by a continuously high expressed SlPG2 gene. The necrotrophic fungus Botrytis cinerea further activated the expression of SlPGs and SlPMEs to accelerate cell wall disassembly, while most of the polygalacturonase inhibitor proteins encoding genes expression were postponed in ripe fruit following the pathogen attack. Pectin induced the antagonistic yeast to secrete pectinolytic enzymes to increase fruit resistance against gray mold. The activities of pathogenic pectinase of B. cinerea were correspondingly depressed in the pectin-inducible yeast enzyme elicited ripe fruit. These data suggest that pectinase is a molecular target for regulation of disease resistance during fruit ripening.

The Importance of Selecting Appropriate Rotation and Tank-Mix Partners for Novel SDHIs to Enhance Botrytis Fruit Rot Control in Strawberry.

Botrytis fruit rot (BFR), caused by the necrotrophic fungus Botrytis cinerea, is the most important disease of strawberry and is mainly controlled by applications of fungicides from multiple chemical groups. To develop more effective and sustainable BFR management programs, field trials were conducted to evaluate the efficacy of fluopyram and penthiopyrad, two newly registered succinate dehydrogenase inhibitors (SDHIs), rotated or tank mixed with the multisites thiram and captan or the single-sites fludioxonil and fenhexamid. The treatments were applied at two different strawberry fields during the 2013-14 and 2014-15 seasons. Overall, tank mixtures of fluopyram and penthiopyrad increased yield and reduced BFR better than rotations with the same fungicides. The multisite thiram tank mixed with fluopyram reduced BFR incidence by 63 to 86% versus 56 to 84% when the two fungicides were rotated. Thiram tank mixed with penthiopyrad reduced BFR incidence by 55 to 72% versus 42 to 66% when rotated. Captan rotated or tank mixed with fluopyram had a positive effect on yield and BFR incidence, whereas the combination of captan with penthiopyrad had negative impacts. Similarly, the single-site fenhexamid had significant positive impacts when rotated or tank mixed with fluopyram but resulted in poor BFR control when combined with penthiopyrad. The rotation of fludioxonil with both SDHIs had a significant positive effect, although its combination with fluopyram was more effective. The multirotation consisting of both SDHIs and different multi- and single-site fungicides did not provide a greater efficacy than the dual rotation or tank-mixture programs. Our findings suggest more scrutiny is needed when recommending tank-mixture or rotation partners for new fungicides to ensure compatibility and enhanced BFR management. Future recommendations should emphasize the importance of such selections at an early stage for delaying fungicide resistance development and extending the lifespan of at-risk fungicides.

Microbial Antagonism Toward Botrytis Bunch Rot of Grapes in Multiple Field Tests Using One Bacillus ginsengihumi Strain and Formulated Biological Control Products.

Botrytis bunch rot (BBR), caused by the necrotrophic fungus Botrytis cinerea, is a major disease of wine and table grapes worldwide. Due to negative effects of pesticides on the environment and human health, alternative control strategies against BBR, such as biological control agents (BCAs), are required to produce high-quality grapes and wines with high standards of food safety. However, few biological control products against BBR are available, and their efficacy is sometimes variable. This study aimed to evaluate and compare (1) the efficacy of new bacterial BCA strains developed at INRA Bordeaux and (2) the BBR reductions achieved by commercial biocontrol products that are already registered or close to being registered. During three consecutive seasons, 10 field experiments were established in six different experimental vineyards in southwestern France. Spray applications were performed at key phenological stages (five or six during the season), or at high BBR-risk periods late in the season according to a Disease Risk Index model. At harvest, BBR incidence and severity (% of symptomatic berries per bunch) were visually determined. The experiments included four bacterial strains at an early experimental stage, particularly Bacillus ginsengihumi (S38). Nine commercial BCA products were also tested, including Bacillus subtilis, Bacillus amyloliquefaciens, Aureobasidium pullulans, Ulocladium oudemansii, and Candida sake. Among the four experimental bacterial strains, only B. ginsengihumi S38 significantly controlled the BBR, presenting reductions in the average severity ranging from 35 to 60%, compared to untreated control, throughout the three seasons. Several commercial BCAs achieved significant reductions in BBR severity ranging from 21 to 58%, although not in every trial. The treatments that achieved higher reductions in severity rates were based on C. sake (45%), B. subtilis (54%), and B. amyloliquefaciens (58%). The efficacy of those BCAs was consistent throughout the studied seasons. The results confirmed the suitability of several biological control products under the conditions in vineyards in southwestern France, while also highlighting the good performance of the novel experimental BCA B. ginsengihumi S38 strain, which achieved similar control rates to the products registered for commercial use. The major factors involved in the variability in the results are also discussed.

Molecular and Functional Characterization of Elicitor PeBC1 Extracted from Botrytis cinerea Involved in the Induction of Resistance against Green Peach Aphid (Myzus persicae) in Common Beans (Phaseolus vulgaris L.).

Elicitors are biofactors that induce resistance in plants against different insect pests. This in vitro study evaluated the impact of a novel elicitor protein PeBC1, extracted from a necrotrophic fungus Botrytis cinerea, on the development and fecundity parameters of green peach aphid (Myzus persicae) on common beans (Phaseolus vulgaris L.). Three different concentrations of PeBC1 elicitor (i.e., 33.56, 25.43, 19.33 µg mL−1) were applied at three different temperature regimes (i.e., 18, 21, and 25 °C). Elicitor treatments were applied topically on the bean plants at 3-leaf stage and newly emerged (0–6 h old) apterous adult aphids were exposed to these treated leaves. In addition to the biological parameters of aphids, the relative expression levels of key genes associated with jasmonic acid (JA) and salicylic acid (SA) plant defense pathways were also determined through RT-qPCR. Results of bioassays revealed that the application of PeBC1 elicitor protein exhibited pronounced and significant (p < 0.05) sub-lethal effects on green peach aphids. The fecundity was reduced and the nymphal development time was prolonged by different concentrations of PeBC1 elicitor and temperature regimes. Gene expression studies showed that the exogenous application of PeBC1 induced a significant upregulation of the expression levels of JA and SA pathway-associated genes in bean plants. As compared to control, elicitor-treated plants exhibited an induced resistance against aphids. Our findings suggest the potential use of PeBC1 elicitor protein in future bio-intensive management strategies against sap-sucking insect pests such as green peach aphids.

Heterologous Expression of the Grapevine JAZ7 Gene in Arabidopsis Confers Enhanced Resistance to Powdery Mildew but Not to Botrytis cinerea.

Jasmonate ZIM-domain (JAZ) family proteins comprise a class of transcriptional repressors that silence jasmonate-inducible genes. Although a considerable amount of research has been carried out on this gene family, there is still very little information available on the role of specific JAZ gene members in multiple pathogen resistance, especially in non-model species. In this study, we investigated the potential resistance function of the VqJAZ7 gene from a disease-resistant wild grapevine, Vitis quinquangularis cv. “Shang-24”, through heterologous expression in Arabidopsis thaliana. VqJAZ7-expressing transgenic Arabidopsis were challenged with three pathogens: the biotrophic fungus Golovinomyces cichoracearum, necrotrophic fungus Botrytis cinerea, and semi-biotrophic bacteria Pseudomonas syringae pv. tomato DC3000. We found that plants expressing VqJAZ7 showed greatly reduced disease symptoms for G. cichoracearum, but not for B. cinerea or P. syringae. In response to G cichoracearum infection, VqJAZ7-expressing transgenic lines exhibited markedly higher levels of cell death, superoxide anions (O2¯, and H2O2 accumulation, relative to nontransgenic control plants. Moreover, we also tested the relative expression of defense-related genes to comprehend the possible induced pathways. Taken together, our results suggest that VqJAZ7 in grapevine participates in molecular pathways of resistance to G. cichoracearum, but not to B. cinerea or P. syringae.