Functional Differentiation of the Succinate Dehydrogenase Subunit SdhC Governs the Sensitivity to SDHI Fungicides, ROS Homeostasis, and Pathogenicity in Fusarium asiaticum.

Succinate dehydrogenase (SDH) is an integral component of the tricarboxylic acid cycle (TCA) and respiratory electron transport chain (ETC), targeted by succinate dehydrogenase inhibitors (SDHIs). Fusarium asiaticum is a prominent phytopathogen causing Fusarium head blight (FHB) on wheat. Here, we characterized the functions of the FaSdhA, FaSdhB, FaSdhC1, FaSdhC2, and FaSdhD subunits. Deletion of FaSdhA, FaSdhB, or FaSdhD resulted in significant growth defects in F. asiaticum. The FaSdhC1 or FaSdhC2 deletion mutants exhibited substantial reductions in fungal growth, conidiation, virulence, and reactive oxygen species (ROS). The FaSdhC1 expression was significantly induced by pydiflumetofen (PYD). The ΔFaSdhC1 mutant displayed hypersensitivity to SDHIs, whereas the ΔFaSdhC2 mutant exhibited resistance against most SDHIs. The transmembrane domains of FaSdhC1 are essential for regulating mycelial growth, virulence, and sensitivity to SDHIs. These findings provided valuable insights into how the two SdhC paralogues regulated the functional integrity of SDH, ROS homeostasis, and the sensitivity to SDHIs in phytopathogenic fungi.

Functional Plasticity, Redundancy, and Specificity of Lanosterol 14α-Demethylase in Regulating the Sensitivity to DMIs in Calonectria ilicicola.

Cytochrome P450 sterol 14α-demethylase (CYP51) is a key enzyme involved in the sterol biosynthesis pathway and serves as a target for sterol demethylation inhibitors (DMIs). In this study, the 3D structures of three CPY51 paralogues from Calonectria ilicicola (C. ilicicola) were first modeled by AlphaFold2, and molecular docking results showed that CiCYP51A, CiCYP51B, or CiCYP51C proteins individually possessed two active pockets that interacted with DMIs. Our results showed that the three paralogues play important roles in development, pathogenicity, and sensitivity to DMI fungicides. Specifically, CiCYP51A primarily contributed to cell wall integrity maintenance and tolerance to abiotic stresses, and CiCYP51B was implicated in sexual reproduction and virulence, while CiCYP51C exerted negative regulatory effects on sterol 14α-demethylase activity within the ergosterol biosynthetic pathway, revealing its genus-specific function in C. ilicicola. These findings provide valuable insights into developing rational strategies for controlling soybean red crown rot caused by C. ilicicola.

Resistance mechanism of Phomopsis longicolla to fludioxonil is associated with modifications in PlOS1, PlOS4 and PlOS5.

Phomopsis longicolla, a causal agent of soybean root rot, stem blight, seed decay, pod and stem canker, which seriously affects the yield and quality of soybean production worldwide. The phenylpyrrole fungicide fludioxonil exhibits a broad spectrum and high activity against phytopathogenic fungi. In this study, the baseline sensitivity of 100 P. longicolla isolates collected from the main soybean production areas of China to fludioxonil were determined. The result showed that the EC50 values of all the P. longicolla isolates ranged from 0.013 to 0.035 µg/ml. Furthermore, 12 fludioxonil-resistance (FluR) mutants of P. longicolla were generated from 6 fludioxonil-sensitive (FluS) isolates. and the resistance factors (RF) of 12 FluR mutants were >3500. Sequence alignment showed that multiple mutation types were found in PlOS1, PlOS4 or/and PlOS5 of FluR mutants. All the FluR mutants exhibited fitness penalty in mycelial growth, conidiation, virulence and osmo-adaptation. Under fludioxonil or NaCl treatment condition, the glycerol accumulation was significantly increased in FluS isolates, but was slightly increased in FluR mutants, and the phosphorylation level of most FluR mutants was significantly decreased when compared to the FluS isolates. Additionally, positive cross-resistance was observed between fludioxonil and procymidone but not fludioxonil and pydiflumetofen, pyraclostrobin or fluazinam. This is first reported that the baseline sensitivity of P. longicolla to fludioxonil, as well as the biological and molecular characterizations of P. longicolla FluR mutants to fludioxonil. These results can provide scientific directions for controlling soybean diseases caused by P. longicolla using fludioxonil.

The HOG-pathway related AaOS1 leads to dicarboximide-resistance in field strains of Alternaria alternata and contributes, together with the Aafhk1, to mycotoxin production and virulence.

BACKGROUND: Garlic leaf spot (GLS) caused by Alternaria alternata is one of the main diseases in the garlic production areas, and its management heavily relies on dicarboximide fungicides. However, the efficacy of dicarboximides against the GLS disease has decreased year on year. RESULTS: In the present study, 10 of 148 A. alternata strains separated from Jiangsu Province were moderately resistant (MR) to a dicarboximide fungicide procymidone (ProMR). Positive cross-resistance was observed between Pro and iprodione (Ipro) or fludioxonil (Fld), but not between Pro and fluazinam or azoxystrobin. Mutations at AaOS1, but not Aafhk1, were confirmed to confer the Pro resistance by constructing replacement mutants, whereas mutations at both AaOS1 and Aafhk1 decreased the gene expression level of AapksI, as well as the ability to produce mycotoxin AOH (polyketide-derived alternariol) and virulence. Additionally, more genes (AaOS1 and Aafhk1) harboring the mutations experienced a larger biological fitness penalty. CONCLUSION: To our knowledge, this is the first report on Pro resistance selected in garlic fields, and mutations at AaOS1 of A. alternata causing a decreased ability to produce the mycotoxin AOH. © 2024 Society of Chemical Industry.

The natural polycyclic tetramate macrolactam HSAF inhibit Fusarium graminearum through altering cell membrane integrity by targeting FgORP1.

Fusarium graminearum is a dominant phytopathogenic fungus causing Fusarium head blight (FHB) in cereal crops. Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam (PoTeM) isolated from Lysobacter enzymogenes that exhibits strong antifungal activity against F. graminearum. HSAF significantly reduces the DON production and virulence of F. graminearum. Importantly, HSAF exhibited no cross-resistance to carbendazim, phenamacril, tebuconazole and pydiflumetofen. However, the target protein of HSAF in F. graminearum is unclear. In this study, the oxysterol-binding protein FgORP1 was identified as the potential target of HSAF using surface plasmon resonance (SPR) combined with RNA-sequence (RNA-seq). The RNA-seq results showed cell membrane and ergosterol biosynthesis were significantly impacted by HSAF in F. graminearum. Molecular docking showed that HSAF binds with arginine 1205 and glutamic acid 1212, which are located in the oxysterol-binding domain of FgORP1. The two amino acids in FgORP1 are responsible for HSAF resistance in F. graminearum though site-directed mutagenesis. Furthermore, deletion of FgORP1 led to significantly decreased sensitivity to HSAF. Additionally, FgORP1 regulates the mycelial growth, conidiation, DON production, ergosterol biosynthesis and virulence in F. graminearum. Overall, our findings revealed the mode of action of HSAF against F. graminearum, indicating that HSAF is a promising fungicide for controlling FHB.

Antifungal Compound from the Predatory Bacterium Lysobacter enzymogenes Inhibits a Plant Pathogenic Fungus by Targeting the AAA ATPase VpVeb1.

Heat-stable antifungal factor (HSAF) isolated from Lysobacter enzymogenes is considered a potential biocontrol agent. However, the target of HSAF in phytopathogenic fungi remains unclear. In this study, we investigated the target of HSAF in Valsa pyri that causes fatal pear Valsa canker. Thirty-one HSAF-binding proteins were captured and identified by surface plasmon resonance (SPR) and high-performance liquid chromatography-mass spectrometry (LC-MS/MS), and 11 deletion mutants were obtained. Among these mutants, only ΔVpVEB1 showed decreased sensitivity to HSAF. Additionally, ΔVpVEB1 exhibited significantly reduced virulence in V. pyri. Molecular docking and SPR results revealed that HSAF bound to threonine 569 and glycine 570 of VpVeb1, which are crucial for AAA ATPase activity. Another study showed that HSAF could decrease the ATPase activity of VpVeb1, leading to the reduced virulence of V. pyri. Taken together, this study first identified the potential target of HSAF in fungi. These findings will help us better understand the model of action of HSAF to fungi.

Phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

Fusarium head blight caused by Fusarium asiaticum is an important cereal crop disease, and the trichothecene mycotoxins produced by F. asiaticum can contaminate wheat grain, which is very harmful to humans and animals. To effectively control FHB in large areas, the application of fungicides is the major strategy; however, the application of different types of fungicides has varying influences on the accumulation of trichothecene mycotoxins in F. asiaticum. In this study, phenamacril inhibited trichothecene mycotoxin accumulation in F. asiaticum; however, carbendazim (N-1H-benzimidazol-2-yl-carbamic acid, methyl ester) induced trichothecene mycotoxin accumulation. Additionally, phenamacril led to a lower level of reactive oxygen species (ROS) by inducing gene expression of the catalase and superoxide dismutase (SOD) pathways in F. asiaticum, whereas carbendazim stimulated ROS accumulation by inhibiting gene expression of the catalase and SOD pathways. Based on these results, we conclude that phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

FaSmi1 Is Essential for the Vegetative Development, Asexual Reproduction, DON Production and Virulence of Fusarium asiaticum.

Smi1 is a protein required for cell cycle progression, morphogenesis, stress response and life span of Saccharomyces cerevisiae. FaSmi1 was identified as a Smi1 homolog in a wheat scab pathogenic fungus Fusarium asiaticum strain 2021. The deletion of FaSmi1 leads to defects in mycelial growth, asexual reproduction, and virulence. The FaSmi1 deletion mutant also exhibited increased sensitivity to osmotic stresses generated by NaCl and KCl, but increased tolerance to oxidative stresses and cell wall integrity inhibitors. All of these defects were restored by genetic complementation of the mutant with the whole parental FaSmi1 gene. Interestingly, the antioxidant system-associated genes exhibit a lower expression level and the mycotoxins' DON content was decreased in the FaSmi1 deletion mutant compared with the parental strain 2021. These results indicate that FaSmi1 plays a critical role in the vegetative development, asexual reproduction, DON production and virulence of F. asiaticum.

Sterol 14α-Demethylase CaCYP51A and CaCYP51B are Functionally Redundant, but Differentially Regulated in Colletotrichum acutatum: Responsibility for DMI-Fungicide Resistance.

Colletotrichum acutatum, the main pathogen causing anthracnose on chili worldwide, is controlled by tebuconazole [a sterol C14-demethylation inhibitor (DMI) fungicide, abbreviated as Teb] with excellent efficacy. Our previous study exhibited that all C. acutatum isolates were sensitive to Teb while the Colletotrichum gloeosporioides population had developed resistance to Teb on the same fungicide-pressure selection. Therefore, the assessment of Teb-resistance in C. acutatum is impending. Twenty Teb-resistant (TebR) mutants obtained by fungicide domestication and ultraviolet (UV)-mutagenesis displayed similar fitness compared to parental isolates. Data in the current study exhibited that mutations at CaCYP51A and/or overexpression of CaCYP51s were responsible for Teb-resistance. Furthermore, the deletion mutants ΔCaCYP51A and ΔCaCYP51B played different roles in sensitivities to DMIs. Taken together, this study first reported that mutations at CaCYP51A and/or overexpression of CaCYP51s conferred resistance to Teb in C. acutatum, CaCYP51A and CaCYP51B are functionally redundant, but differentially regulated in DMI resistance.

Point Mutations in FgSdhC2 or in the 5' Untranslated Region of FgSdhC1 Confer Resistance to a Novel Succinate Dehydrogenase Inhibitor Flubeneteram in Fusarium graminearum.

Fusarium graminearum is one of the phytopathogenic fungi causing cereal fusarium head blight worldwide. Flubeneteram (Flu) is a novel succinate dehydrogenase inhibitor (SDHI) which exhibits strong fungicidal activity against F. graminearum. In this study, four Flu-resistant (FluR) mutants were generated by fungicide domestication from the wildtype strain PH-1. Sequencing alignment results of FgSdh from PH-1 and FluR mutants showed that all the mutations could be categorized into three resistant genotypes. Genotype I had an A-to-T mutation at the -57 bp of the 5' untranslated region (5'UTR) of FgSdhC1, while genotypes II and III carried nonsynonymous mutations conferring T77I or R86C in FgSdhC2, respectively. All the mutations conferring the Flu resistance and causing fitness penalty were validated. The genotype I mutant showed high Flu-resistance, while genotype II and III mutants exhibited low Flu resistance. Additionally, all the FluR genotypes showed distinct cross-resistance patterns among the five SDHIs.

Detection and Fitness of Dicarboximide-Resistant Isolates of Alternaria alternata from Dendrobium officinale, a Chinese Indigenous Medicinal Herb.

Black spot, caused by Alternaria alternata, poses a severe threat to the industry of Dendrobium officinale, a Chinese indigenous medicinal herb. Dicarboximide fungicides (DCFs) have been intensively used to control this disease for decades in China, and offer excellent efficacy. The resistance of phytopathogenic pathogens against DCFs are reportedly selected in fields; however, the DCF resistance of A. alternata from D. officinale is not well understood. The isolates of A. alternata with low procymidone resistance (ProLR) were detected in the commercial orchards of D. officinale in China in 2018 and biochemically characterized in this study. The result showed that the ProLR isolates were selected in the commercial orchards with a resistance frequency of 100%, and no significant difference in mycelial growth, sporulation, and virulence was observed among the ProLR and procymidone-sensitive (ProS) isolates. A positive cross-resistance pattern was exhibited between procymidone and iprodione. Results of amino acid sequence alignment of AaOS-1 from the tested isolates showed that all of the ProLR genotypes could be categorized into two groups, including group I (mutations at AaOs-1) and group II (no mutation). Under procymidone (5.0 µg/ml) treatment conditions, the AaOs-1 expression levels increased in the ProS isolates and ranged from approximately 2.94- to 3.69-fold higher than those under procymidone-free conditions, while the AaOs-1 expressions of the ProLR isolates were significantly lower than those in the ProS isolates under the same conditions. The data indicated that the mutations at AaOs-1 are involved in the DCF resistance of A. alternata selected in the D. officinale orchards.

Biological Characteristics and Molecular Mechanism of Procymidone Resistance in Stemphylium eturmiunum From Garlic.

Garlic leaf blight caused by Stemphylium eturmiunum was first reported in Jiangsu Province in China. The dicarboximide fungicide (DCF) procymidone is reported to possess broad-spectrum action in inhibiting filamentous fungi and is widely used to control leaf disease of various plants. Of 41 Stemphylium eturmiunum isolates collected in this study from commercial garlic farms in Pizhou and Dafeng counties of Jiangsu Province, eight isolates were resistant to procymidone. The following three phenotypes were categorized according to in vitro responses to DCFs: sensitive, low resistance to iprodione and procymidone, and high resistance to all iprodione and procymidone. The fitness of all resistant isolates was decreased in accordance with data on mycelial growth, conidiation, and virulence. After treatment with 10 µg/ml of procymidone for 4 h, mycelial intracellular glycerol concentrations of resistant isolates were significantly lower than those of sensitive isolates. Positive cross-resistance was observed between dicarboximides and phenylpyrroles, but there was no cross-resistance between dicarboximides and fluazinam or difenoconazole in the two resistant phenotypes. Nucleotide sequence alignment of two-component histidine kinase genes from sensitive and resistant isolates indicated that amino acid mutations were located at the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein and at the phosphatase domain of the N-terminal region and the response regulator domain of the C-terminal region. To our knowledge, this is the first report of DCF resistance in Stemphylium eturmiunum, and these findings will help establish a rational strategy to manage DCF-resistant populations of Stemphylium eturmiunum in the field.

Resistance risk assessment for a novel succinate dehydrogenase inhibitor pydiflumetofen in Fusarium asiaticum.

BACKGROUND: Fusarium asiaticum is one of predominant pathogens of Fusarium head blight (FHB) in China. Pydiflumetofen (Pyd) is a novel succinate dehydrogenase inhibitor (SDHI) which has been commercialized in China for the controlling of wheat FHB since 2019. In the current study, a risk assessment of the pydiflumetofen-resistance selected in Fusarium asiaticum was investigated. RESULTS: One PydMR mutant [resistance factor (RF) < 80] and four PydHR mutants (RF > 3000) were generated by fungicide-taming from 1000 mycelial discs of the wild-type strain 2021. Nucleotide sequences alignment results of FaSdh from the wild-type strain and resistant mutants showed that all the mutations were categorized into three genotypes, i.e. FaSdhBH248Y from PydMR mutant, both FaSdhC1 A64V and FaSdhC1 R67K from PydHR mutants. All the resistant mutants possessed no fitness penalty based on the data of mycelial linear growth, conidiation and virulence. In addition, the FaSdhC1 A64V mutants showed positive cross-resistance between pydiflumetofen and boscalid or thifluzamide, but no cross-resistance between pydiflumetofen and Y13149 or Y12196, while the FaSdhC1 R67K mutants exhibited positive cross-resistance between pydiflumetofen and boscalid, thifluzamide or Y12196, and no cross-resistance between pydiflumetofen and Y13149. Furthermore, positive cross-resistance between the five tested SDHIs was detected in the FaSdhBH248Y mutants. CONCLUSION: The results suggest a moderate to high resistance risk of F. asiaticum to pydiflumetofen, and provide essential data for monitoring the emergence of resistance and resistance management strategies for pydiflumetofen, which will be useful for scientific application of this fungicide in China.

Mutations at sterol 14α-demethylases (CYP51A&B) confer the DMI resistance in Colletotrichum gloeosporioides from grape.

BACKGROUND: Grape anthracnose caused by the ascomycete fungus Colletotrichum gloeosporioides has been widely controlled by demethylation inhibitors (DMIs) for decades in China. The resistance status and mechanism of C. gloeosporioides against DMIs is not well understood. RESULTS: All difenoconazole-resistant (DfnR ) isolates from vineyards exhibited decreased fitness. Positive cross-resistance was detected between DMI triazoles. Sequence alignment results from the DfnR and DfnS isolates revealed that multiple mutations are distributed at CgCYP51A, concomitant with mutations at CgCYP51B. The half maximal effective concentration (EC50 ) values of single deleted and complemented mutants of CgCYP51A and CgCYP51B showed that ΔCgCYP51A became more sensitive to difenoconazole, but not ΔCgCYP51B. Furthermore, all single complemented mutants had a stronger biological fitness than the progenitor strain. All the defectives of ΔCgCYP51A and ΔCgCYP51B could be restored by complementation of the whole corresponding gene from the resistant strains. Relative gene expression of CgCYP51A and CgCYP51B in most of the mutants was greatly upregulated relative to the progenitor isolate when treated with difenoconazole at the same concentration. Moreover, the extension of five amino acids (GNETI) caused by mutation at the stop codon of CgCYP51A, concurrent with other seven amino acid substitutions and the synonymous mutation P10P (CCG → CCT), significantly enhanced DMI resistance. CONCLUSION: The DMI resistance of C. gloeosporioides selected in vineyards is conferred by mutations at CgCYP51s, and validated by a genetics method. The roles of CgCYP51A and CgCYP51B overlap, and are counter-balanced, but cannot be replaced reciprocally. © 2020 Society of Chemical Industry.

Mutations and Overexpression of CYP51 Associated with DMI-Resistance in Colletotrichum gloeosporioides from Chili.

Chili anthracnose caused by Colletotrichum spp. is an annual production concern for growers in China. Sterol C14-demethylation inhibitors (DMIs, such as tebuconazole) have been widely used to control this disease for more than three decades. In the current study, of 48 isolates collected from commercial chili farms in Jiangsu Province of China during 2018 and 2019, 8 single-spore isolates were identified as Colletotrichum gloeosporioides and the rest were identified as C. acutatum. To determine whether the DMI resistance of isolates develops in the field, mycelial growth of the 48 isolates was measured in culture medium with and without tebuconazole. In all, 6 of the 8 C. gloeosporioides isolates were resistant to tebuconazole, but all 40 of the C. acutatum isolates were sensitive to tebuconazole. The fitness cost of resistance was low based on a comparison of fitness parameters between the sensitive and resistant isolates of C. gloeosporioides. Positive cross-resistance was observed between tebuconazole and difenconazole or propiconazole, but not prochloraz. Alignment results of the CgCYP51 amino acid sequences from the sensitive and resistant isolates indicated that mutations can be divided into three genotypes. Genotype I possessed four substitutions (V18F, L58V, S175P, and P341A) at the CgCYP51A gene but no substitutions at CgCYP51B, while genotype II had five substitutions (L58V, S175P, A340S, T379A, and N476T) at CgCYP51A, concomitant with three substitutions (D121N, T132A, and F391Y) at CgCYP51B. In addition, genotype III contained two substitutions (L58V and S175P) at CgCYP51A, concomitant with one substitution (T262A) at CgCYP51B. Molecular docking models illustrated that the affinity of tebuconazole to the binding site of the CgCYP51 protein from the resistant isolates was decreased when compared with binding site affinity of the sensitive isolates. Our findings provide not only novel insights into understanding the resistance mechanism to DMIs, but also some important references for resistance management of C. gloeosporioides on chili.

Involvement of the two L-lactate dehydrogenase in development and pathogenicity in Fusarium graminearum.

Lactate dehydrogenase (LDH) widely exists in organisms, which catalyzes the interconversion of pyruvate into lactate with concomitant interconversion of NADH and NAD+. In this study, two L-type lactate dehydrogenase genes FgLDHL1 and FgLDHL2 were characterized in an ascomycete fungus Fusarium graminearum, a causal agent of wheat head blight. Both the single-gene deletion mutants of FgLDHL1 or FgLDHL2 exhibited phenotypic defects in vegetative growth, sporulation, spore germination, L-lactate biosynthesis and activity. Additionally, the two L-lactate dehydrogenases were involved in the utilization of carbon sources and maintenance of redox homeostasis during spore germination. Pathogenicity assays showed that ΔFgLDHL1 exhibits reduced virulence on wheat spikelets and on corn stigmas, suggesting that it was indirectly correlated with a reduced level of deoxynivalenol accumulation. These results indicate that FgLDHL1 and FgLDHL2 play multiple roles in the developmental processes and pathogenesis in F. graminearum, and help understand the functional diversity of D-/L-lactate dehydrogenase in phytopathogenic fungi.

Involvement of a dihydrodipicolinate synthase gene (FaDHDPS1) in fungal development, pathogenesis and stress responses in Fusarium asiaticum.

BACKGROUND: Dihydrodipicolinate synthase (DHDPS) is an allosteric enzyme, which catalyzes the first unique step of lysine biosynthesis in prokaryotes, higher plants and some fungi. To date, the biological roles of DHDPS in filamentous fungi are poorly understood. RESULTS: In this study, on the basis of comparative genome resequencing, a DHDPS gene was found to be specific in Fusarium asiaticum, named FaDHDPS1, which showed high amino acid identity to that of entomopathogenic fungus. Subcellular localization of the FaDHDPS1-GFP fusion protein was mainly concentrated in the cytoplasm of conidia and dispersed in the cytoplasm during conidial germination. To reveal the biological functions, both deletion and complementation mutants of FaDHDPS1 were generated. The results showed that the FaDHDPS1 deletion mutant was defective in conidiation, virulence and DON biosynthesis. In addition, deletion of FaDHDPS1 resulted in tolerance to sodium pyruvate, lysine, low temperature and Congo red. CONCLUSION: Results of this study indicate that FaDHDPS1 plays an important role in the regulation of vegetative differentiation, pathogenesis and adaption to multiple stresses in F. asiaticum.

Resistance risk assessment of Fusarium oxysporum f. sp. melonis against phenamacril, a myosin inhibitor.

Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (FOM) is one of the most notorious seed-borne diseases worldwide. Phenamacril is a cyanoacrylate fungicide with novel chemical structure and strong inhibitive activity against FOM. To evaluate the risk of FOM developing phenamacril resistance, five phenamacril-resistant mutants with >800µgml-1 minimum inhibitory concentration were obtained by repeated exposure to the fungicide in the laboratory. Compared with the parental isolate, four of the five phenamacril-resistant mutants showed enhanced biological fitness in sporulation and virulence, but not in sensitivity to various stresses (oxidative and osmotic pressure, cell membrane and wall inhibitor). No positive cross-resistance was observed among phenamacril and the other five fungicides, including azoxystrobin, carbendazim, boscalid, fluazinam and tebuconazole. Sequencing alignment results of the myosin 5 from the five resistant mutants and the parental strain indicated that the three resistant mutants fo-2, fo-3 and fo-4 had a single point mutation (S175L), which may confer the resistance of FOM against phenamacril. Interestingly, the resistant mutant fo-4 harbored not only one mutation (S175L) at myosin 5, but also the other mutation (A52G) at ß2-tublin. Our data supported that resistance risk of Fusarium oxysporum f. sp. melonis against phenamacril was between the moderate to high level.

The Autophagy Gene BcATG8 Regulates the Vegetative Differentiation and Pathogenicity of Botrytis cinerea.

Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8, a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (ΔBcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the ΔBcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinereaIMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea.