Agent Of Gray Mold Disease Research Articles

Challenges and Opportunities Arising from Host-Botrytis cinerea Interactions to Outline Novel and Sustainable Control Strategies: The Key Role of RNA Interference.

The necrotrophic plant pathogenic fungus Botrytis cinerea (Pers., 1794), the causative agent of gray mold disease, causes significant losses in agricultural production. Control of this fungal pathogen is quite difficult due to its wide host range and environmental persistence. Currently, the management of the disease is still mainly based on chemicals, which can have harmful effects not only on the environment and on human health but also because they favor the development of strains resistant to fungicides. The flexibility and plasticity of B. cinerea in challenging plant defense mechanisms and its ability to evolve strategies to escape chemicals require the development of new control strategies for successful disease management. In this review, some aspects of the host-pathogen interactions from which novel and sustainable control strategies could be developed (e.g., signaling pathways, molecules involved in plant immune mechanisms, hormones, post-transcriptional gene silencing) were analyzed. New biotechnological tools based on the use of RNA interference (RNAi) are emerging in the crop protection scenario as versatile, sustainable, effective, and environmentally friendly alternatives to the use of chemicals. RNAi-based fungicides are expected to be approved soon, although they will face several challenges before reaching the market.

Plant Growth Promotion and Plant Disease Suppression Induced by Bacillus amyloliquefaciens Strain GD4a.

Botrytis cinerea, the causative agent of gray mold disease (GMD), invades plants to obtain nutrients and disseminates through airborne conidia in nature. Bacillus amyloliquefaciens strain GD4a, a beneficial bacterium isolated from switchgrass, shows great potential in managing GMD in plants. However, the precise mechanism by which GD4a confers benefits to plants remains elusive. In this study, an A. thaliana-B. cinerea-B. amyloliquefaciens multiple-scale interaction model was used to explore how beneficial bacteria play essential roles in plant growth promotion, plant pathogen suppression, and plant immunity boosting. Arabidopsis Col-0 wild-type plants served as the testing ground to assess GD4a's efficacy. Additionally, bacterial enzyme activity and targeted metabolite tests were conducted to validate GD4a's potential for enhancing plant growth and suppressing plant pathogens and diseases. GD4a was subjected to co-incubation with various bacterial, fungal, and oomycete pathogens to evaluate its antagonistic effectiveness in vitro. In vivo pathogen inoculation assays were also carried out to investigate GD4a's role in regulating host plant immunity. Bacterial extracellular exudate (BEE) was extracted, purified, and subjected to untargeted metabolomics analysis. Benzocaine (BEN) from the untargeted metabolomics analysis was selected for further study of its function and related mechanisms in enhancing plant immunity through plant mutant analysis and qRT-PCR analysis. Finally, a comprehensive model was formulated to summarize the potential benefits of applying GD4a in agricultural systems. Our study demonstrates the efficacy of GD4a, isolated from switchgrass, in enhancing plant growth, suppressing plant pathogens and diseases, and bolstering host plant immunity. Importantly, GD4a produces a functional bacterial extracellular exudate (BEE) that significantly disrupts the pathogenicity of B. cinerea by inhibiting fungal conidium germination and hypha formation. Additionally, our study identifies benzocaine (BEN) as a novel small molecule that triggers basal defense, ISR, and SAR responses in Arabidopsis plants. Bacillus amyloliquefaciens strain GD4a can effectively promote plant growth, suppress plant disease, and boost plant immunity through functional BEE production and diverse gene expression.

Recruiting grape‐isolated antagonistic yeasts for the sustainable bio‐management of Botrytis cinerea on grapes

AbstractBotrytis cinerea is the causative agent of grey mould disease in grapes, which was linked to significant postharvest losses. This study examined three grape‐isolated yeasts (Metschnikowia aff. fructicola, Metschnikowia pulcherrima, and Hansenispora uvarum) through in vitro and in vivo tests on detached grape berries against grey mould, as well as the elucidation of their possible mechanisms of action. The antifungal mechanism of action of yeasts was determined by the lytic enzyme activity, inhibition of spore germination, biofilm activity, iron depletion, diffusible metabolites, wound‐site colonisation, mycocin, and volatile organic compounds (VOCs) production. The highest in vitro efficacy (83.13%) was observed on M. aff. fructicola, followed by M. pulcherrima (82.10%) and H. uvarum (71.66%). Metschnikowia yeasts exhibited comparable enzyme activities, including protease, β‐1,3 glucanase, gelatinase chitinase, and cellulase, while H. uvarum had a poor enzymatic activity with chitinase and gelatinase. M. aff. fructicola showed relatively higher iron depletion activity than M. pulcherrima, while M. pulcherrima outperformed via diffusible metabolites. All yeast cultures significantly reduced spore germination by at least 86%. Overall, M. aff. fructicola exhibited the highest biocontrol activity with its iron depletion, inhibition of conidial germination, biofilm formation, VOCs, and well colonisation on grape berries. M. aff. fructicola 1‐UDM outperformed all other yeasts by significantly reducing disease incidence and lesion diameter values (93.4% and 94.3%, respectively). Remarkably, H. uvarum VOCs demonstrated potential as a biofumigant for suppressing grey mould. All yeasts are well adapted to their ecological niche to bio‐protect grapes from grey mould disease.

Understanding the Influence of Applying Two Culture Filtrates to Control Gray Mold Disease (Botrytis cinerea) in Tomato

Botrytis cinerea, a causal agent of gray mold disease, is one of the most destructive fungal pathogens that leads to substantial global economic crop losses, especially for tomato plants. The present study aims to investigate the inhibitory effect of two microbial culture filtrates (BCA filtrate alone and combined with salicylic acid) of Trichoderma longibrachiatum and Pseudomonas sp. against the phytopathogenic fungus B. cinerea on tomato plants. The biochemical modifications, gray mold disease incidence, and fruit quality parameters of the tomatoes were determined according to tested treatments. The results showed that both fungi and bacteria were able to solubilize phosphate and produce IAA and HCN. T. longibrachiatum could produce hydrolytic enzymes (chitinase, protease, and glucanase). Otherwise, Pseudomonas sp. showed the capacity to produce catalase and amylase enzymes. Both microbial culture filtrates inhibited the hyphae growth of B. cinerea. The biocontrol efficacy, in vitro, was significant: up to 50% in terms of the growth inhibition rate at a concentration of 40%. The tomato seedlings’ growth was promoted by the separate preventive treatments of each micro-organism culture filtrate. In addition, disease severity in the tomato seedlings and fruit was significantly reduced. Furthermore, the combined treatment of tomato fruit with culture filtrates and salicylic acid induced significant biochemical and physiological changes in fruit firmness, juice yield, total protein, and ROS enzyme activities. The culture filtrates of T. longibrachiatum and Pseudomonas sp. can be recommended as an effective microbial biofungicide to control gray mold disease under storage conditions.

Tobamovirus infection aggravates gray mold disease caused by Botrytis cinerea by manipulating the salicylic acid pathway in tomato.

Botrytis cinerea is the causative agent of gray mold disease, and infects more than 1400 plant species, including important crop plants. In tomato, B. cinerea causes severe damage in greenhouses and post-harvest storage and transport. Plant viruses of the Tobamovirus genus cause significant damage to various crop species. In recent years, the tobamovirus tomato brown rugose fruit virus (ToBRFV) has significantly affected the global tomato industry. Most studies of plant-microbe interactions focus on the interaction between the plant host and a single pathogen, however, in agricultural or natural environments, plants are routinely exposed to multiple pathogens. Here, we examined how preceding tobamovirus infection affects the response of tomato to subsequent infection by B. cinerea. We found that infection with the tobamoviruses tomato mosaic virus (ToMV) or ToBRFV resulted in increased susceptibility to B. cinerea. Analysis of the immune response of tobamovirus-infected plants revealed hyper-accumulation of endogenous salicylic acid (SA), upregulation of SA-responsive transcripts, and activation of SA-mediated immunity. Deficiency in SA biosynthesis decreased tobamovirus-mediated susceptibility to B. cinerea, while exogenous application of SA enhanced B. cinerea symptoms. These results suggest that tobamovirus-mediated accumulation of SA increases the plants' susceptibility to B. cinerea, and provide evidence for a new risk caused by tobamovirus infection in agriculture.

Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bio-inspired fungicide.

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.

Paraburkholderia phytofirmans PsJN delays Botrytis cinerea development on grapevine inflorescences.

Grapevine flowering is an important stage in the epidemiology of Botrytis cinerea, the causal agent of gray mold disease. To prevent infection and to minimize postharvest losses, the control of this necrotrophic fungus is mainly based on chemical fungicides application. However, there is a growing interest in other control alternatives. Among them, the use of beneficial microorganisms appears as an eco-friendly strategy. This study aims to investigate the effect of Paraburkholderia phytofirmans PsJN, root-inoculated or directly sprayed on fruiting cuttings inflorescences to control B. cinerea growth. For this purpose, quantification by real time PCR of Botrytis development, direct effect of PsJN on fungal spore germination and chemotaxis were assayed. Our results showed a significant protective effect of PsJN only by direct spraying on inflorescences. Moreover, we demonstrated an inhibition exerted by PsJN on Botrytis spore germination, effective when there was a direct contact between the two microorganisms. This study showed that PsJN is positively attracted by the pathogenic fungus B. cinerea and forms a biofilm around the fungal hyphae in liquid co-culture. Finally, microscopic observations on fruit cuttings revealed a co-localization of both beneficial and pathogenic microorganisms on grapevine receptacle and stigma that might be correlated with the protective effect induced by PsJN against B. cinerea via a direct antimicrobial effect. Taking together, our findings allowed us to propose PsJN as a biofungicide to control grapevine gray mold disease.

Metabolic and Transcriptomic Profiling of Lilium Leaves Infected With Botrytis elliptica Reveals Different Stages of Plant Defense Mechanisms.

Botrytis elliptica, the causal agent of gray mold disease, poses a major threat to commercial Lilium production, limiting its ornamental value and yield. The molecular and metabolic regulation mechanisms of Lilium's defense response to B. elliptica infection have not been completely elucidated. Here, we performed transcriptomic and metabolomic analyses of B. elliptica resistant Lilium oriental hybrid “Sorbonne” to understand the molecular basis of gray mold disease resistance in gray mold disease. A total of 115 differentially accumulated metabolites (DAMs) were detected by comparing the different temporal stages of pathogen infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed the differentially expressed genes (DEGs) and DAMs were enriched in the phenylpropanoid and flavonoid pathways at all stages of infection, demonstrating the prominence of these pathways in the defense response of “Sorbonne” to B. elliptica. Network analysis revealed high interconnectivity of the induced defense response. Furthermore, time-course analysis of the transcriptome and a weighted gene coexpression network analysis (WGCNA) led to the identification of a number of hub genes at different stages, revealing that jasmonic acid (JA), salicylic acid (SA), brassinolide (BR), and calcium ions (Ca2+) play a crucial role in the response of “Sorbonne” to fungal infection. Our work provides a comprehensive perspective on the defense response of Lilium to B. elliptica infection, along with a potential transcriptional regulatory network underlying the defense response, thereby offering gene candidates for resistance breeding and metabolic engineering of Lilium.

Clathrin Is Important for Virulence Factors Delivery in the Necrotrophic Fungus Botrytis cinerea.

Fungi are the most prevalent plant pathogens, causing annually important damages. To infect and colonize their hosts, they secrete effectors including hydrolytic enzymes able to kill and macerate plant tissues. These secreted proteins are transported from the Endoplasmic Reticulum and the Golgi apparatus to the extracellular space through intracellular vesicles. In pathogenic fungi, intracellular vesicles were described but their biogenesis and their role in virulence remain unclear. In this study, we report the essential role of clathrin heavy chain (CHC) in the pathogenicity of Botrytis cinerea, the agent of gray mold disease. To investigate the importance of this protein involved in coat vesicles formation in eukaryotic cells, a T-DNA insertional mutant reduced in the expression of the CHC-encoding gene, and a mutant expressing a dominant-negative form of CHC were studied. Both mutants were strongly affected in pathogenicity. Characterization of the mutants revealed altered infection cushions and an important defect in protein secretion. This study demonstrates the essential role of clathrin in the infectious process of a plant pathogenic fungus and more particularly its role in virulence factors delivery.

Natural Compounds That Modulate the Development of the Fungus Botrytis cinerea and Protect Solanum lycopersicum.

Botrytis cinerea is the causal agent of gray mold disease and is responsible for the loss of millions of dollars in crops in worldwide. Currently, this pathogen exhibits increasing resistance to conventional fungicides; therefore, better control methods and novel compounds with a more specific mechanism of action but without biocidal effects, are required. In this work, several natural compounds to control B. cinerea were analyzed in vitro. Detected effects were dependent on the stage of fungus development, and 3-phenyl-1-propanol displayed the most potent inhibition of in vitro germination, germ tube development, and sporulation. However, it had lower protection of leaves and postharvest fruit in plant infection. Isoeugenol and 1-phenylethanol exhibited lower inhibition of in vitro germination and sporulation, but at the highest concentrations, they inhibited germ tube elongation. Although the lowest rates of foliage infection were recorded using isoeugenol and 3-phenyl-1-propanol, 1-phenylethanol significantly decreased the disease in postharvest tomato fruit, with an efficacy like Mancozeb, but at 18 times lower micromolar concentration. All compounds resulted in high cell viability after spores were removed from the treatment solution exhibited high cell viability, suggesting a non-biocidal effect. The diversity of in vitro and in-plant effects seems to indicate a different mechanism of action.

Effects of different levels of saline water on infection of tomato by Botrytis cinerea, the causal agent of gray mold

A greenhouse experiment was conducted to investigate the effect of different levels of NaCl salt on tomato upon B. cinerea infection the causal agent of gray mold disease. The disease assessment was recorded after inoculation by using the scale based on percentage leaf area affected, and the growth of the plants was recorded for each treatment. Three weeks after inoculation by conidial suspension, the estimated disease severity on plants of tomato was 35.18% compared to the control. The highest incidence disease increase of gray mold (39.21%) was obtained with using 300 mM of NaCl after inoculation with B. cinerea compared with the other concentrations and as well as distilled water. Under severe salt stress (150 and 300mM) increased susceptibility of gray mold disease severity were observed in plants inoculated with B. cinerea, while under mild salt stress (50mM of NaCl) this effect was reversed. The treatment of plant by B.cinerea has reduced the growth of the aerial part of tomato plants (39.06%) after three weeks inoculation compared to the control. Three levels of NaCl (50, 100 and 150mM) increased respectively the plant height from 12.73 to 29.84%, 0.28 to 27.16% for the fresh eight and 5.75 to 33.35% for dry weight compared to the plants inoculated and irrigated by distilled water. NaCl addition at 300mM on plants inoculated with B. cinerea decreased the height, fresh weight and dry weight at 0.99, 4.45 and 11.01% respectively.

RNA sequencing-based transcriptome analysis of mature strawberry fruit infected by necrotrophic fungal pathogen Botrytis cinerea

Botrytis cinerea, the causal agent of gray mold disease, is regarded as the most impactful postharvest fungal pathogen, as it causes serious losses in fresh fruits, ornamentals, and vegetables worldwide. To understand the molecular defense mechanisms of the affected plants, we analyzed transcriptome using RNA sequencing (RNA-seq) of mature strawberry fruit that were infected by B. cinerea at 12, 24, and 48 h. Overall, 18 cDNA libraries were produced generating 923,482,642,150 base pair reads. A total of 1459 differentially expressed genes (DEGs) were identified (≥2.0-fold change and FDR ≤ 0.05), among which only 26 were down-regulated. The majority of changes in gene expression occurred 24 h after inoculation, before significant lesion development, suggesting that this is an important stage during B. cinerea infection. Further analysis revealed that the DEGs were predominately involved in pathogen recognition (pattern recognition receptors), signaling transduction (MAPK cascades, CDPKs), defense response (transcription factors, secondary metabolism biosynthesis, pathogenis-related), as well as different sorts of transporter encoding genes. These data provided a global insight into mature strawberry fruit-B. cinerea interaction and might be used for fruit postharvest pathogens and diseases management.

Different levels of resistance to cyprodinil and iprodione and lack of fludioxonil resistance in Botrytis cinerea isolates collected from pistachio, grape, and pomegranate fields in California

Botrytis cinerea is the causal agent of gray mold disease and can infect fruits of important crops like grape, pistachio, and pomegranate, causing significant yield losses worldwide. In this study, mycelial growth assays in vitro were used to evaluate the sensitivities of 160 single-spore isolates of Botrytis cinerea collected from commercial grape (n = 58), pistachio (n = 74), and pomegranate (n = 28) fields in California to cyprodinil, fludioxonil, and iprodione. Based on the effective concentration at which mycelial growth was inhibited by 50% (EC50 values), 100, 83, and 38% of the isolates from the 3 hosts were sensitive (EC50 < 1 μg/mL) to fludioxonil, cyprodinil, and iprodione, respectively. Thirteen, 3, and 1% of the isolates showed low (CyLR), weak (CyWR), or high (CyHR) resistance to cyprodinil, respectively; Sixty-two percent of the isolates tested showed low resistance (1 < EC50 < 10 μg/mL) toward iprodione (IpLR) and were detected in all three hosts. Absence of cross-resistance was not observed between cyprodinil, iprodione, and fludioxonil. Sequence analysis of the two-component histidine kinase target gene (Bos1) revealed that some of the IpLR phenotype isolates were associated with either I365N/S, Q369P, and/or N373S mutations. Laboratory studies showed that IpLR, CyLR, and CyWR phenotypes were not associated with fitness penalties for mycelial growth rate on non-amended media and pathogenicity on detached grape berries. Furthermore, preventative applications of commercial formulations of iprodione (Rovral) and cyprodinil (Vangard) were effective against gray mold caused by Ip- and Cy-sensitive isolates, but failed to control disease caused by IpLR and, CyLR and CyWR isolates, respectively. The generated information will serve as a basis for implementing fungicide use recommendations against bunch rot of grapes, blossom and shoot blight of pistachio, and gray mold of pomegranate.

Synergistic effect of the combined bio-fungicides ε-poly-l-lysine and chitooligosaccharide in controlling grey mould (Botrytis cinerea) in tomatoes

The antifungal properties and the induction of resistance by ε-poly-l-lysine (ε-PL) and chitooligosaccharide (COS) were examined to find an alternative to synthetic fungicides currently used in the control of the devastating fungal pathogen Botrytis cinerea, the causal agent of grey mould disease of tomatoes. As presented herein, this combined treatment (200 mg/L ε-PL + 400 mg/L COS) was found to have optimal in vitro antifungal activities, achieving an inhibition rate of 90.22%. In vivo assays with these combined bio-fungicides, under greenhouse conditions using susceptible tomato plants, demonstrated good protection against severe grey mould. In field tests, the combined bio-fungicides had a control effect of up to 66.67% against tomato grey mould. To elucidate the mechanisms of the combined bio-fungicide-induced resistance in the tomato, plants were subjected to three treatments: 1) inoculation with B. cinerea after spraying with 200 mg/L ε-PL alone, 2) inoculation with the combined bio-fungicides, and 3) inoculation with 400 mg/L COS alone. Compared to the control (sterile water), increases in salicylic acid (SA) and jasmonic acid (JA) levels and increased phenylalanine ammonia lyase (PAL), peroxidase (POD), and superoxide dismutase (SOD) activities were observed. Catalase (CAT) activity and abscisic acid (ABA) and gibberellin (GA) levels decreased, particularly in the combined bio-fungicide-treated plants. Altogether, these findings reveal that the combined bio-fungicides (200 mg/L ε-PL + 400 mg/L COS) should be an excellent biocontrol agent candidate that combines direct antifungal activity against B. cinerea with plant resistance.

Plipastatin A1 produced by a marine sediment-derived Bacillus amyloliquefaciens SH-B74 contributes to the control of gray mold disease in tomato.

Certain Bacillus species have the capacity to produce cyclic lipopeptides and these lipopeptides are promising determinants contributing to the biocontrol of plant diseases. In the current study, a cyclic lipopeptide plipastatin A1 was isolated from the fermentation broth of a marine sediment-derived Bacillus amyloliquefaciens SH-B74 by the combination of solid-phase extraction and reversed-phase high-performance liquid chromatography, and its structure was identified by tandem mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and gas chromatography-mass spectrometry together with nuclear magnetic resonance analysis. Moreover, data from activity evaluation revealed that plipastatin A1 has excellent in vitro activity on the suppression of the conidia germination of B. cinerea, the causal agent of gray mold disease in tomato. Furthermore, plipastatin A1 can successfully decrease the incidence of gray mold disease on tomato leaves at 50µM concentration. This study indicates that B. amyloliquefaciens SH-B74 appears to be a potentially sustainable pesticide to control gray mold disease in tomato plants, and its cyclic lipopeptide plipastatin A1 plays an important role in the in vitro and in planta biocontrol of B. cinerea.

Small RNAs from Bemisia tabaci Are Transferred to Solanum lycopersicum Phloem during Feeding.

The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

Antifungal Activity of Pseudomonas frederiksbergensis CMAA 1323 Isolated from the Antarctic Hair Grass Deschampsia antarctica

Aims: Epiphytic bacteria, isolated from Deschampsia antarctica, were screened for their potential to inhibit the plant pathogen Botrytis cinerea, the causal agent of gray mold disease of strawberry pseudofruits. This phytopathogenic fungus is more active and the disease is more serious in temperate climate where the temperatures are lower. Methodology: The approach involved the isolation and characterization of bacteria from the phyllosphere of D. antarctica and the evaluation of their antifungal activity. Results: A total of 56 bacterial strains were isolated and only one of them, identified by the sequencing of 16S rDNA gene, as Pseudomonas frederiksbergensis CMAA 1323, inhibited the mycelial growth and the conidial germination of the fungus. Its ethyl acetate extract also inhibited Original Research Article Melo et al.; BMRJ, 14(3): 1-11, 2016; Article no.BMRJ.25314 2 the mycelial growth. Analysis of the bioactive fraction by using liquid chromatography coupled to a mass spectrometry (LC-MS) revealed the presence of cyclo(Pro-Val), a compound belonging to the class of diketopiperazines (DKPs). Conclusion: These findings support the potential use of the psychrophilic bacterium P. frederiksbergensis as promising antifungal agent, and highlight the need for more studies with this bacterium in the biological control of plant pathogenic fungi.

DHN melanin biosynthesis in the plant pathogenic fungus Botrytis cinerea is based on two developmentally regulated key enzyme (PKS)-encoding genes.

Botrytis cinerea is the causal agent of gray mold disease in various plant species and produces grayish macroconidia and/or black sclerotia at the end of the infection cycle. It has been suggested that the pigmentation is due to the accumulation of 1,8-dihydroxynaphthalene (DHN) melanin. To unravel its basis and regulation, the putative melanogenic and regulatory genes were identified and functionally characterized. Unlike other DHN melanin-producing fungi, B. cinerea and other Leotiomycetes contain two key enzyme (PKS)-encoding enzymes. Bcpks12 and bcpks13 are developmentally regulated and are required for melanogenesis in sclerotia and conidia respectively. BcYGH1 converts the BcPKS13 product and contributes thereby to conidial melanogenesis. In contrast, enzymes acting downstream in conversion of the PKS products (BcBRN2, BcSCD1 and BcBRN1) are required for both, sclerotial and conidial melanogenesis, suggesting that DHN melanogenesis in B. cinerea follows a non-linear pathway that is rather unusual for secondary metabolic pathways. Regulation of the melanogenic genes involves three pathway-specific transcription factors (TFs) that are clustered with bcpks12 or bcpks13 and other developmental regulators such as light-responsive TFs. Melanogenic genes are dispensable in vegetative mycelia for proper growth and virulence. However, DHN melanin is considered to contribute to the longevity of the reproduction structures.

Rapid Assessment of Gray Mold (Botrytis cinerea) Infection in Grapes with a Biosensor System

Botrytis cinerea is the causative agent of gray mold disease, which causes considerable economic losses to winemakers. The extent of gray mold infection of winegrapes is commonly visually estimated, a method that is prone to assessor bias. Here, we used rapid and simple enzyme-based screening consisting of carbon-electrode, screen-printed amperometric biosensors to estimate gluconic acid and glycerol concentration in winegrapes infected with different degrees of B. cinerea. The lower limits of quantification of the screen-printed amperometric biosensors were 3 mg/L for gluconic acid (corresponding to an infection rate of less than 1%) and 35 mg/L for glycerol; the response times with a flow rate of 0.5 mL/min were in a range of 0.5 to 2 min in the linear ranges of the two assays. This study demonstrates the efficacy of amperometric biosensors for rapid analysis of gluconic acid and glycerol in grapes. The measurements confirmed that concentrations of both compounds are highly correlated with the rate of B. cinerea infection (R2 = 0.98). Thus, the biosensor developed to measure gluconic acid in grapes (or must) was more precise and gave a faster response than methods that currently exist for determining the rate of B. cinerea infection of grape berries.

Fenhexamid Resistance in Botrytis cinerea from Strawberry Fields in the Carolinas Is Associated with Four Target Gene Mutations.

Botrytis cinerea, the causal agent of gray mold disease, is one of the most important pathogens of strawberry. Its control in commercial strawberry fields is largely dependent on the application of fungicides during bloom and fruit maturation. The hydroxyanilide fenhexamid is one of the most frequently used fungicides in the southeast of the United States for gray mold control. It inhibits the 3-ketoreductase (Erg27) of the ergosterol biosynthesis pathway and, due to this site-specific mode of action, is at risk for resistance development. Single-spore isolates were collected from 11 commercial strawberry fields in North and South Carolina and subjected to a conidial germination assay that distinguished sensitive from resistant phenotypes. Of the 214 isolates collected, 16.8% were resistant to fenhexamid. Resistance was found in three of four locations from North Carolina and in four of seven locations from South Carolina, indicating that resistance was widespread. Mutations in Erg27 (T63I, F412S, F412C, and F412I) were associated with resistance, with F412S the predominant and most widespread mutation. In this study, mutations T63I and F412C in field isolates of B. cinerea are described for the first time. Detached fruit studies showed that field rates of Elevate 50 WDG (fenhexamid) controlled sensitive but not resistant isolates carrying any of the four mutations. Resistant isolates produced the same lesion size and number of sporulating lesions on fruit sprayed with Elevate 50 WDG as on untreated controls, showing the fungicide's loss of efficacy against those isolates. A rapid polymerase chain reaction method was developed to quickly and reliably distinguish isolates sensitive or resistant to fenhexamid in the Carolinas and to determine the mutation associated with resistance. The presence of fenhexamid-resistant strains in B. cinerea from strawberry fields in the Carolinas must be considered in future resistance management practices for sustained gray mold control.