Fungal Pathogen Botrytis Cinerea Research Articles

Characterization of two SGNH family cell death-inducing proteins from the horticulturally important fungal pathogen Botrytis cinerea based on the optimized prokaryotic expression system

Botrytis cinerea is one of the most destructive phytopathogenic fungi, causing significant losses to horticultural crops. As a necrotrophic fungus, B. cinerea obtains nutrients by killing host cells. Secreted cell death-inducing proteins (CDIPs) play a crucial role in necrotrophic infection; however, only a limited number have been reported. For high-throughput CDIP screening, we optimized the prokaryotic expression system and compared its efficiency with other commonly used protein expression systems. The optimized prokaryotic expression system showed superior effectiveness and efficiency and was selected for subsequent CDIP screening. The screening system verified fifty-five candidate proteins and identified two novel SGNH family CDIPs: BcRAE and BcFAT. BcRAE and BcFAT exhibited high expression levels throughout the infection process. Site-directed mutagenesis targeting conserved Ser residues abolished the cell death-inducing activity of both BcRAE and BcFAT. Moreover, the transient expression of BcRAE and BcFAT in plants enhanced plant resistance against B. cinerea without inducing cell death, independent of their enzymatic activities. Our results suggest a high-efficiency screening system for high-throughput CDIP screening and provide new targets for further study of B. cinerea-plant interactions.

Papiliotrema flavescens, a plant growth-promoting fungus, alters root system architecture and induces systemic resistance through its volatile organic compounds in Arabidopsis

The current trend in agricultural development is the establishment of sustainable agricultural systems. This involves utilizing and implementing eco-friendly biofertilizers and biocontrol agents as alternatives to conventional fertilizers and pesticides. A plant growth-promoting fungal strain, that could alter root system architecture and promote the growth of Arabidopsis seedlings in a non-contact manner by releasing volatile organic compounds (VOCs) was isolated in this study. 26S rDNA sequencing revealed that the strain was a yeast-like fungus, Papiliotrema flavescens. Analysis of plant growth-promoting traits revealed that the fungus could produce indole-3-acetic acid and ammonia and fix nitrogen. Transcriptome analysis in combination with inhibitor experiments revealed that P. flavescens VOCs triggered metabolic alterations, promoted auxin accumulation and distribution in the roots, and coordinated ethylene signaling, thus inhibiting primary root elongation and inducing lateral root formation in Arabidopsis. Additionally, transcriptome analysis and fungal infection experiments confirmed that pretreatment with P. flavescens stimulated the defense response of Arabidopsis to boost its resistance to the pathogenic fungus Botrytis cinerea. Solid-phase microextraction, which was followed by gas chromatography-mass spectrometry analysis, identified three VOCs (acetoin, naphthalene and indole) with significant plant growth-promoting attributes. Their roles were confirmed using further pharmacological experiments and upregulated expression of auxin- and ethylene-related genes. Our study serves as an essential reference for utilizing P. flavescens as a potential biological fertilizer and biocontrol agent.

Two transcription factors, RhERF005 and RhCCCH12, regulate rose resistance to Botrytis cinerea by modulating cytokinin levels.

Gray mold caused by the necrotrophic fungal pathogen Botrytis cinerea is one of the most destructive diseases in rose (Rosa spp.). Rose infection by B. cinerea leads to severe economic losses due to necrosis, tissue collapse, and rot. In rose, cytokinins (CKs) positively regulate a defense response to B. cinerea, but little is known about the underlying molecular mechanisms. Here, we characterized two ethylene/jasmonic acid-regulated transcription factors, RhEFR005 and RhCCCH12, that bind to the promoter region of PATHOGENESIS-RELATED 10.1 (RhPR10.1) and promote its transcription, leading to decreased susceptibility to B. cinerea. The RhEFR005/RhCCCH12-RhPR10.1 module regulated cytokinin content in rose, and the susceptibility of RhEFR005-, RhCCCH12-, and RhPR10.1-silenced rose petals can be rescued by exogenous CK. In summary, our results reveal that the RhERF005/RhCCCH12-RhPR10.1 module regulates the CK-induced defense response of rose to B. cinerea.

Resistance to Seven Site-Specific Fungicides in Botrytis cinerea from Greenhouse-Grown Ornamentals.

The fungal pathogen Botrytis cinerea is a notorious problem on many floriculture greenhouse hosts including petunia, geranium, and poinsettia; these key crops contribute to the $6.43 billion U.S. ornamental industry. While growers use cultural strategies to reduce relative humidity and free moisture to limit Botrytis blight, fungicides remain a primary component of control programs. Isolates (n = 386) of B. cinerea sampled from symptomatic petunia, geranium, and poinsettia in Michigan greenhouses from 2018 to 2021 were screened for resistance to eight fungicides belonging to seven Fungicide Resistance Action Committee (FRAC) groups. Single-spored isolates were subjected to a germination-based assay using previously defined discriminatory doses of each fungicide. Resistance was detected to thiophanate-methyl (FRAC 1; 94%), pyraclostrobin (FRAC 11; 80%), boscalid (FRAC 7; 67%), iprodione (FRAC 2; 65%), fenhexamid (FRAC 17; 38%), cyprodinil (FRAC 9; 38%), fludioxonil (FRAC 12; 21%), and fluopyram (FRAC 7; 13%). Most isolates (63.5%) were resistant to at least four FRAC groups, with 8.7% of all isolates demonstrating resistance to all seven FRAC groups tested. Resistance frequencies for each fungicide were similar among crops, production regions, and growing cycles but varied significantly for each greenhouse. Phenotypic diversity was high, as indicated by the 48 different fungicide resistance profiles observed. High frequencies of resistance to multiple fungicides in B. cinerea populations from floriculture hosts highlight the importance of sustainable and alternative disease management practices for greenhouse growers.

Codon Optimization Enables the Geneticin Resistance Gene to Be Applied Efficiently to the Genetic Manipulation of the Plant Pathogenic Fungus Botrytis cinerea.

Botrytis cinerea can infect almost all of the important horticultural crops and cause severe economic losses globally every year. Modifying candidate genes and studying the phenotypic changes are among the most effective ways to unravel the pathogenic mechanism of this crop killer. However, few effective positive selection markers are used for B. cinerea genetic transformation, which limits multiple modifications to the genome, especially genes involving redundant functions. Here, we optimized a geneticin resistance gene, BcNPTII, based on the codon usage preference of B. cinerea. We found that BcNPTII can greatly increase the transformation efficiency of B. cinerea under G418 selection, with approximately 30 times higher efficiency than that of NPTII, which is applied efficiently to transform Magnaporthe oryzae. Using the gene replacement method, we successfully knocked out the second gene BOT2, with BcNPTII as the selection marker, from the mutant ΔoahA, in which OAHA was first replaced by the hygromycin resistance gene HPH in a field strain. We obtained the double knockout mutant ΔoahA Δbot2. Our data show that the codon-optimized BcNPTII is an efficient positive selection marker for B. cinerea transformation and can be used for various genetic manipulations in B. cinerea, including field wild-type strains.

Construction of a Mycoviral Infectious Clone for Reverse Genetics in Botrytis cinerea.

The most important advances in our understanding of the viral life cycle, such as genome replication, packaging, transmission, and host interactions, have been made via the development of viral infectious full-length clones. Here, we describe the detailed protocols for the construction of an infectious clone derived from Botrytis virus F (BVF), a mycoflexivirus infecting the plant pathogenic fungus Botrytis cinerea, the determination of the complete sequence of the cloned mycovirus, the preparation of fungal protoplasts, and the transfection of protoplasts using transcripts derived from the BVF infectious clone.

Effectiveness of Four Synthetic Fungicides in the Control of Post-Harvest Gray Mold of Strawberry and Analyses of Residues on Fruit

Fungicides are usually applied on strawberries to manage gray mold, induced by the fungal pathogen Botrytis cinerea. In this study, four reduced-risk fungicides (formulations of pyrimethanil, PYR, 175 mL/hL; boscalid, BOS, 80 g/hL; combination fludioxonil, FLU, +cyprodinil, CYP, 110 g/hL) were applied before harvest for the management of post-harvest diseases of strawberries. The resulting fungicide residues on the strawberry fruit were also quantified. Strawberry fruits were harvested at 0, 4, 8, and 12 days following treatment (dft) and kept at 20 ± 1 °C for 4 days or cold-stored for 7 days at 0.5 ± 1 °C, followed by a 4-day shelf life at 20 ± 1 °C. All fungicides significantly reduced gray mold, according to the McKinney Index. At 0 dft and 4 days of shelf life, the FLU + CYP completely prevented post-harvest strawberry gray mold, while PYR and BOS reduced the disease by 88% and 42%, respectively, in comparison to the untreated control. For the duration of experiment, fungicide residues were always below the maximum residue levels, and FLU was the most degraded, thanks to the enzymatic pool of the strawberries. Monitoring fungicide residues in strawberries is essential to provide the consumer information on the safety of this widely consumed fruit. The present study points out the safety of strawberry fruits for consumers, even if the treatment strategy implies the use of fungicide mixtures before the consumption, with fungicide levels always being below the MRL.

Exogenous melatonin orchestrates multiple defense responses against Botrytis cinerea in tomato leaves

Infection of tomato leaves and fruits by necrotrophic fungal pathogen Botrytis cinerea causes considerable economic loss. Melatonin has multiple physiological functions and is considered a potential biocontrol agent for its positive effects on plant innate immunity. In tomato, melatonin reduces Botrytis-caused post-harvest fruit decay by activating jasmonic acid (JA) signaling. In this study, we analyzed the effects of melatonin on resistance to Botrytis and associated defense mechanisms in tomato leaves. Treatment of detached tomato leaves with melatonin at a concentration as low as 100 µM induced resistance to Botrytis based on reduced lesion size and fungal growth. Melatonin-induced resistance to gray mold was associated with activation of a complex set of defense mechanisms in tomato leaves. First, melatonin suppressed Botrytis-induced cell death and accumulation of reactive oxygen species (ROS), which was correlated with increased activities of ROS-scavenging enzymes and enhanced expression of their corresponding genes. Second, melatonin treatment induced callose synthase genes and promoted rapid and strong callose deposition in tomato leaves. Melatonin also induced other anti-microbial genes encoding chitinases, β-1,3-glucanases, phenylalanine ammonialyases and polyphenol oxidases. Third, melatonin increased Botrytis-induced expression of genes encoding SlWRKY33, SlMYC2 and SlERF transcription factors, which activate defense-related genes expression through JA and ethylene (ET) signaling pathways. By contrast, melatonin suppressed salicylic acid (SA) signaling based on reduced expression of the SlPR1 marker gene, which is known to antagonize defense against Botrytis. These results collectively indicate that exogenous melatonin orchestrates multiple signaling pathways to activate a complex set of defense mechanisms against Botrytis in tomato leaves.

A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference.

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection.

Interaction with the entomopathogenic fungus Beauveria bassiana influences tomato phenome and promotes resistance to Botrytis cinerea infection.

Plants are central to complex networks of multitrophic interactions. Increasing evidence suggests that beneficial microorganisms (BMs) may be used as plant biostimulants and pest biocontrol agents. We investigated whether tomato (Solanum lycopersicum) plants are thoroughly colonized by the endophytic and entomopathogenic fungus Beauveria bassiana, and how such colonization affects physiological parameters and the phenotype of plants grown under unstressed conditions or exposed to the pathogenic fungus Botrytis cinerea. As a positive control, a strain of the well-known biocontrol agent and growth inducer Trichoderma afroharzianum was used. As multitrophic interactions are often driven by (or have consequences on) volatile organic compounds (VOCs) released by plants constitutively or after induction by abiotic or biotic stresses, VOC emissions were also studied. Both B. bassiana and T. afroharzianum induced a significant but transient (one to two-day-long) reduction of stomatal conductance, which may indicate rapid activation of defensive (rejection) responses, but also limited photosynthesis. At later stages, our results demonstrated a successful and complete plant colonization by B. bassiana, which induced higher photosynthesis and lower respiration rates, improved growth of roots, stems, leaves, earlier flowering, higher number of fruits and yield in tomato plants. Beauveria bassiana also helped tomato plants fight B. cinerea, whose symptoms in leaves were almost entirely relieved with respect to control plants. Less VOCs were emitted when plants were colonized by B. bassiana or infected by B. cinerea, alone or in combination, suggesting no activation of VOC-dependent defensive mechanisms in response to both fungi.

Plant mRNAs move into a fungal pathogen via extracellular vesicles to reduce infection

Cross-kingdom small RNA trafficking between hosts and microbes modulates gene expression in the interacting partners during infection. However, whether other RNAs are also transferred is unclear. Here, we discover that host plant Arabidopsis thaliana delivers mRNAs via extracellular vesicles (EVs) into the fungal pathogen Botrytis cinerea. A fluorescent RNA aptamer reporter Broccoli system reveals host mRNAs in EVs and recipient fungal cells. Using translating ribosome affinity purification profiling and polysome analysis, we observe that delivered host mRNAs are translated in fungal cells. Ectopic expression of two transferred host mRNAs in B.cinerea shows that their proteins are detrimental to infection. Arabidopsis knockout mutants of the genes corresponding to these transferred mRNAs are more susceptible. Thus, plants have a strategy to reduce infection by transporting mRNAs into fungal cells. mRNAs transferred from plants to pathogenic fungi are translated to compromise infection, providing knowledge that helps combat crop diseases.

Determination of the Mycovirome of a Necrotrophic Fungus.

Next-generation sequencing (NGS) of total RNA has allowed the detection of novel viruses infecting different hosts, such as fungi, increasing our knowledge on virus horizontal transfer events among different hosts, virus diversity, and virus evolution. Here, we describe the detailed protocols for the isolation of the plant pathogenic fungus Botrytis cinerea, from grapevine plants showing symptoms of the mold gray disease, the culture and maintenance of the isolated B. cinerea strains, the extraction of total RNA from B. cinerea strains for NGS, the bioinformatics pipeline designed and followed to detect mycoviruses in the sequenced samples, and the validation of the in silico detected mycoviruses by different approaches.

The Raf-like MAPKKKs STY8, STY17, and STY46 negatively regulate Botrytis cinerea resistance by limiting MKK7 protein accumulation in Arabidopsis.

Plant diseases, which seriously damage crop production, are in most cases caused by fungal pathogens. In this study, we found that the Raf-like MAPKKKs STY8 (SERINE/THREONINE/TYROSINE KINASE 8), STY17, and STY46 negatively regulate resistance to the fungal pathogen Botrytis cinerea through jasmonate response in Arabidopsis. Moreover, STY8/STY17/STY46 homologs negatively contribute to chitin signaling. We further identified MKK7 as the MAPKK component interacting with STY8/STY17/STY46 homologs. MKK7 positively contributes to resistance to B. cinerea and chitin signaling. Furthermore, we found that STY8/STY17/STY46 homologs negatively affect the accumulation of MKK7, in accordance with the opposite roles of MKK7 and STY8/STY17/STY46 homologs in defense against B. cinerea. These results provide new insights into the mechanisms precisely regulating plant immunity via Raf-like MAPKKKs.

GREEN BIOCHEMICAL PROTECTION OF POSTHARVEST TABLE GRAPES AGAINST GRAY MOLD (BOTRYTIS CINEREA) USING 7S PROTEINS

The 7S globulins (seed storage glycoprotein) isolated from soybean and chickpea seeds have the antifungal potential against the pathogenic fungus (Botrytis cinerea) causing gray mold in grapes assessing table grapes in vitro or postharvest by comparing with synthetic fungicide, Switch 62.5 WG. Conventional microbiological procedures estimated the in-vitro antifungal potential of the 7S globulins, such as linear growth curves and scanning electron microscopy (SEM). Soybean-7S significantly inhibited the in-vitro growth of Botrytis cinerea by about 64.44%, 66.64%, and 76.67% when applied at 50, 100, and 200 μg/mL, respectively, followed by chickpea 7S with growth reduction of 52.22%, 54.44%, and 66.67%, respectively. The synthetic fungicide (Switch 62.5 WG) induced higher growth inhibition extents (83.33% and 86.66%) when applied at 50 and 200 μg/mL, respectively. The 7S-exposed B. cinerea displayed swollen hyphae compared with the control under scanning electron microscope examination. The 7S derived from soybean and chickpea inhibited gray mold development in table grapes when applied at 200 and 400 μg/mL for 30 days after infection with B. cinerea. The maintained disease severity was also minimal (40% and 25% for soybean-7S and chickpea-7S, respectively). An increased level of treatment (400 μg/mL) highly reduced the disease severity to only 7.5% after 30 days of storage at cold conditions for both proteins. The 7S globulin from legume seeds can be an alternative to synthetic fungicides for controlling B. cinerea as a postharvest treatment. Developing these legume proteins as natural fungicides could also progress for the safe control of various plant pathogens, causing drastic crop losses.

Recent Advances in Mechanisms Underlying Defense Responses of Horticultural Crops to Botrytis cinerea

Horticultural crops are a crucial component of agriculture worldwide and have great economic value. The notorious plant fungal pathogen Botrytis cinerea can cause gray mold disease in over 200 horticultural crops, leading to severe economic losses. Investigating the mechanisms underlying plant defense responses to pathogens is crucial for developing new strategies for effectively controlling plant diseases, and much progress has occurred in the mechanisms underlying defense responses of horticultural crops to B. cinerea mainly due to the completion of genome sequencing and the establishment of efficient tools for functional genomics. In this review, recent progress in mechanisms underlying defense responses and natural products that can enhance the resistance of horticultural crops to B. cinerea are summarized, and future research directions are also discussed.

The role of WRKY transcription factors, FaWRKY29 and FaWRKY64, for regulating Botrytis fruit rot resistance in strawberry (Fragaria × ananassa Duch.)

BackgroundThe cultivated strawberry (Fragaria × ananassa Duch.) is one of the most economically important horticultural crops worldwide. Botrytis fruit rot (BFR) caused by the necrotrophic fungal pathogen Botrytis cinerea is the most devasting disease of cultivated strawberries. Most commercially grown strawberry varieties are susceptible to BFR, and controlling BFR relies on repeated applications of various fungicides. Despite extensive efforts, breeding for BFR resistance has been unsuccessful, primarily due to lack of information regarding the mechanisms of disease resistance and genetic resources available in strawberry.ResultsUsing a reverse genetics approach, we identified candidate genes associated with BFR resistance and screened Arabidopsis mutants using strawberry isolates of B. cinerea. Among the five Arabidopsis T-DNA knockout lines tested, the mutant line with AtWRKY53 showed the greatest reduction in disease symptoms of BFR against the pathogen. Two genes, FaWRKY29 and FaWRKY64, were identified as orthologs in the latest octoploid strawberry genome, ‘Florida Brilliance’. We performed RNAi-mediated transient assay and found that the disease frequencies were significantly decreased in both FaWRKY29- and FaWRKY64-RNAi fruits of the strawberry cultivar, ‘Florida Brilliance’. Furthermore, our transcriptomic data analysis revealed significant regulation of genes associated with ABA and JA signaling, plant cell wall composition, and ROS in FaWRKY29 or FaWRKY64 knockdown strawberry fruits in response to the pathogen.ConclusionOur study uncovered the foundational role of WRKY transcription factor genes, FaWRKY29 and FaWRKY64, in conferring resistance against B. cinerea. The discovery of susceptibility genes involved in BFR presents significant potential for developing resistance breeding strategies in cultivated strawberries, potentially leveraging CRISPR-based gene editing techniques.

Spermine deficiency shifts the balance between jasmonic acid and salicylic acid-mediated defence responses in Arabidopsis.

Polyamines are small aliphatic polycations present in all living organisms. In plants, the most abundant polyamines are putrescine (Put), spermidine (Spd) and spermine (Spm). Polyamine levels change in response to different pathogens, including Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the regulation of polyamine metabolism and their specific contributions to defence are not fully understood. Here we report that stimulation of Put biosynthesis by Pst DC3000 is dependent on coronatine (COR) perception and jasmonic acid (JA) signalling, independently of salicylic acid (SA). Conversely, lack of Spm in spermine synthase (spms) mutant stimulated galactolipids and JA biosynthesis, and JA signalling under basal conditions and during Pst DC3000 infection, whereas compromised SA-pathway activation and defence outputs through SA-JA antagonism. The dampening of SA responses correlated with COR and Pst DC3000-inducible deregulation of ANAC019 expression and its key SA-metabolism gene targets. Spm deficiency also led to enhanced disease resistance to the necrotrophic fungal pathogen Botrytis cinerea and stimulated endoplasmic reticulum (ER) stress signalling in response to Pst DC3000. Overall, our findings provide evidence for the integration of polyamine metabolism in JA- and SA-mediated defence responses, as well as the participation of Spm in buffering ER stress during defence.

A novel biocontrol agent Bacillus velezensis K01 for management of gray mold caused by Botrytis cinerea

Gray mold is a destructive plant disease caused by a fungal pathogen Botrytis cinerea. The use of plant growth promoting rhizobacteria (PGPR) has proven to be a promising method to control this disease. Bacillus velezensis K01 was isolated from the rhizosphere of planting tomatoes. Strain K01 has a range of roles, including the ability to solubilize phytate phosphorus, stimulate resistant response, and produce indoleacetic acid (IAA), protease, cellulase, and antimicrobial substances. Strain K01 was found to inhibit 12 phytopathogenic fungi and 5 phytopathogenic bacteria. Specially, strain K01 demonstrated a biocontrol efficiency of over 78% against gray mold caused by B. cinerea on the leaves and fruits of tomato and pepper. Additionally, K01 was found to promote the growth of maize seedlings. Further genomic analysis revealed that K01 belongs to B. velezensis, which is consistent with phylogenetic analysis, average nucleotide polymorphism (ANI), and digital DNA–DNA hybridization (dDDH). The genome of strain K01 had a size of 3,927,799 bp and deduced 3866 predicted genes, with an average guanine-cytosine (GC) content of 46.5%. Based on the analyses of genomic secondary metabolites, over 18.4% of the genome was annotated to 12 gene clusters related to antimicrobial metabolite synthesis. Additionally, genome annotation and comparative genomics identified several genes associated with plant growth promotion and environmental adaption. These findings suggest that B. velezensis K01 has the potential to serve as a new biocontrol agent for management of gray mold on tomato and pepper.

Small Cationic Cysteine-Rich Defensin-Derived Antifungal Peptide Controls White Mold in Soybean.

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action possess potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. GMA4CG_V6 exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro with an MIC value of 24 µM. At this concentration, internalization of this peptide into fungal cells occurred prior to discernible membrane permeabilization. GMA4CG_V6 markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Its spray application on soybean plants provided robust control of this disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has potential for development as a bio-fungicide for white mold control in soybean.

Different Size Formulations of Fluopyram: Preparation, Antifungal Activity, and Accumulation in the Fungal Pathogen Botrytis cinerea.

Nanotechnology is revolutionizing the efficient production and sustainable development of modern agriculture. Understanding the pesticide activity of both nano- and conventional methods is useful for developing new pesticide formulations. In this study, three solid fluopyram formulations with varying particle sizes were developed, and the mechanisms underlying the difference in the antifungal activity among these formulations were investigated. Wet media milling combined with freeze drying was used to prepare fluopyram nanoparticles (FLU-NS) and a micron-sized solid formulation (FLU-MS), and a jet grinding mill was employed to fabricate fluopyram wettable powder (FLU-WP). The mean particle sizes of FLU-NS, FLU-MS, and FLU-WP were 366.8 nm, 2.99 μm, and 10.16 μm, respectively. Notably, FLU-NS displayed a toxicity index against Botrytis cinerea (gray mold) that was approximately double those of FLU-MS and FLU-WP. Similar trends were noticed in the antifungal tests on Alternaria solani. The uptake of FLU-NS by B. cinerea was approximately twice that of FLU-MS and FLU-WP, indicating that fluopyram nanoparticles are more easily taken up by the pathogen (B. cinerea), and display better bioactivity than the larger fluopyram particles. Therefore, the nanosizing of pesticides appears to be a viable strategy to enhance efficiency without increasing the amount of pesticide used.