Conidial Germination Rate Research Articles

Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana

ABSTRACT Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana’s pathogenic mechanisms.

Effect of temperature on in vitro germination and growth of Colletotrichum fioriniae, a new emerging pathogen of olive fruits.

Olive anthracnose induced by different Colletotrichum species causes dramatic losses of fruit yield and oil quality. The increasing incidence of Colletotrichum fioriniae (Colletotrichum acutatum species complex) as causal agent of olive anthracnose in Italy, is endorsing new studies on its biology, ecology, and environmental factors such as temperature. Five isolates from different sampling sites in Lazio region (Central Italy) were studied under controlled laboratory conditions aiming to better understand the differences of thermal development among the isolates and to lay the foundations of a future mathematical model able to describe the key aspects of the pathogen's life cycle. The mycelial growth rate and the conidial germination rate were assessed at seven different constant temperatures (5, 10, 15, 20, 25, 30, and 35°C) and fixed relative humidity (100% RH). The obtained dataset was analysed to estimate the parameters of mathematical functions that connect the mycelial growth rate and the spore germination with the environmental temperature. The parameters set provided as the result of this study constitute a key step forward in the biological knowledge of the species and the basis for future formulations of mathematical models that might be the core of decision support systems in an integrated pest management framework.

Roles of Three FgPel Genes in the Development and Pathogenicity Regulation of Fusarium graminearum.

Fusarium head blight (FHB) is a devastating fungal disease caused by Fusarium graminearum. Pectin lyase, a pectinase, acts on the α-1,4-glycosidic linkage of galacturonic acid primarily by β-elimination. In this study, three pectin lyase genes (FgPel1, 2, 3) in F. graminearum were selected, and deletion mutants (ΔFgPel1, 2, 3) were constructed by homologous recombination for functional characterization. The gene deletions affected the morphology and growth rate of F. graminearum on pectin medium at various concentrations, with the growth rate of ΔFgPel1 being more significant. The growth of ΔFgPel1 colonies slowed at pH 4, with optimal growth at pH 6.5, whereas ΔFgPel2 and ΔFgPel3 exhibited greater inhibition at pH 8. Colony morphology and diameter of the deletion mutants showed no significant differences compared to the wild-type strain PH-1, and there was no effect on conidial production or germination rate. Pathogenicity assays demonstrated that gene deletion significantly reduced the ability of F. graminearum to infest corn silks and wheat ears, and that ΔFgPel2 showed a more pronounced reduction in pathogenicity on wheat spikes. In summary, the pectin lyase genes (FgPel1, 2, 3) are involved in pectin utilization and are influenced by external pH conditions, which attenuate the pathogenicity of F. graminearum without affecting its vegetative growth or asexual spore formation. These findings elucidate the roles of these genes and provide a basis for controlling FHB.

MaAzaR, a Zn2Cys6/Fungus-Specific Transcriptional Factor, Is Involved in Stress Tolerance and Conidiation Pattern Shift in Metarhizium acridum.

Entomopathogenic fungi are valuable sources of biological pesticides, with conidial yield and quality being pivotal factors determining their broad applications. AzaR, a fungus-specific zinc-cluster transcription factor, is known to regulate the biosynthesis of polyketone secondary metabolites in Aspergillus niger; however, its role in pathogenic fungi remains unclear. This study investigated the role of MaAzaR in the growth, development, and environmental tolerance of Metarhizium acridum. MaAzaR deletion slowed down conidial germination rate, caused reduction in conidial yield, lowered fungal tolerance to UV radiation, did not affect fungal heat-shock tolerance, and increased fungal sensitivity to the cell-wall-destructive agent calcofluor white. Furthermore, MaAzaR deletion transformed microcycle conidiation to normal conidiation on the microcycle conidiation medium. Transcription profile analysis demonstrated that MaAzaR could regulate transformation of the conidiation pattern by controlling the expression of genes related to cell division, mycelium growth and development, and cell wall integrity. Thus, this study identified a new gene related to fungal conidiation and environmental tolerance, enriching our understanding of the molecular mechanism of microcycle conidiation and providing theoretical support and genetic resources for the development of high-yielding strains.

Sensitivity analysis and point mutations in BcSDHB confer cyclobutrifluram resistance in Botrytis cinerea from China

Botrytis cinerea is one of the most destructive pathogens worldwide. It can damage over 200 crops, resulting in significant yield and quality losses. Cyclobutrifluram, a new generation of succinate dehydrogenase inhibitors, exhibits excellent inhibitory activity against B. cinerea. However, the baseline sensitivity and resistance of B. cinerea to cyclobutrifluram remains poorly understood. This study was designed to monitor the sensitivity frequency distribution, assess the resistance risk, and clarify the resistance mechanism of B. cinerea to cyclobutrifluram. The baseline sensitivity of B. cinerea isolates to cyclobutrifluram was 0.89 μg/mL. Cyclobutrifluram-resistant B. cinerea populations are present in the field. Six resistant B. cinerea isolates investigated in this study possessed enhanced compound fitness index compared to the sensitive isolates according to mycelial growth, mycelial dry weight, conidiation, conidial germination rate, and pathogenicity. Cyclobutrifluram exhibited no cross-resistance with tebuconazole, fludioxonil, cyprodinil, or iprodione. Sequence alignment revealed that BcSDHB from cyclobutrifluram-resistant B. cinerea isolates had three single substitutions (P225F, N230I, or H272R). Molecular docking verified that these mutations in BcSDHB conferred cyclobutrifluram resistance in B. cinerea. In conclusion, the resistance risk of B. cinerea to cyclobutrifluram is high, and the point mutations in BcSDHB (P225F, N230I, or H272R) confer cyclobutrifluram resistance in B. cinerea. This study provided important insights into cyclobutrifluram resistance in B. cinerea and offered valuable information for monitoring and managing cyclobutrifluram resistance in the future.

VdPT1 Encoding a Neutral Trehalase of Verticillium dahliae Is Required for Growth and Virulence of the Pathogen.

Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene (VdPT1) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants (ΔVdPT1) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae. Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae. Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.

Comparative physiological and transcriptome analysis provide insights into the inhibitory effect of osthole on Penicillium choerospondiatis

Blue mold induced by Penicillium choerospondiatis is a primary cause of growth and postharvest losses in the fruit of Phyllanthus emblica. There is an urgent need to explore novel and safe fungicides to control this disease. Here, we demonstrated osthole, a natural coumarin compound isolated from Cnidium monnieri, exhibited a strong inhibitory effect on mycelia growth, conidial germination rate and germ tube length of P. choerospondiatis, and effectively suppressed the blue mold development in postharvest fruit of P. emblica. The median effective concentration of osthole was 9.86 mg/L. Osthole treatment resulted in cellular structural disruption, reactive oxygen species (ROS) accumulation, and induced autophagic vacuoles containing cytoplasmic components in fungal cells. Transcriptome analysis revealed that osthole treatment led to the differentially expressed genes mainly enriched in the cell wall synthesis, TCA cycle, glycolysis/ gluconeogenesis, oxidative phosphorylation. Moreover, osthole treatment led to increase genes expression involved in peroxisome, autophagy and endocytosis. Particularly, the autophagy pathway related genes (PcATG1, PcATG3, PcATG15, PcATG27, PcYPT7 and PcSEC18) were prominently up-regulated by osthole. Summarily, these results revealed the potential antifungal mechanism of osthole against P. choerospondiatis. Osthole has potentials to develop as a natural antifungal agent for controlling blue mold disease in postharvest fruits.

Xyloglucan-specific endo-β-1,4-glucanase (PdXEG1) gene is important for the growth, development and virulence of Penicillium digitatum

Penicillium digitatum is a necrotrophic fungus responsible for the most severe postharvest rots of citrus fruit. However, the precise mechanisms underlying the interaction between P. digitatum and citrus fruit are still unknown. In this study, the xyloglucan-specific endo-β-1,4-glucanase gene in P. digitatum, designated as PdXEG1, was identified and characterized. Bioinformatics analysis showed that PdXEG1 is a member of the glycoside hydrolase 12 (GH12) family of the plant cell wall degrading enzymes (CWDEs) and is highly homologous with other XEG proteins. The molecular docking analysis revealed that the PdXEG1 protein can potentially recognize xyloglucan structures and exhibit hydrolytic activity. The knockout strain exhibited decreased growth rate, conidial yield, and conidial germination rate on the callus induction medium (CIM) (supplemented with citrus pomace). However, no discernible impact of PdXEG1 knockout was observed when the strain was cultured on the potato dextrose agar (PDA). The ΔPdXEG1 strain also displayed reduced xylanase and endoglucanase activities and attenuated in vivo pathogenicity and expansion ability. Correspondingly, PdXEG1 complementation restored these effects. Additionally, transcriptome analyses revealed that PdXEG1 knockdown markedly downregulated the expression of genes encoding various CWDEs, development-related regulators and transporter proteins, in addition to affecting the basal substance metabolism of carbohydrates, amino acids and fatty acids. In conclusion, these results suggested that the PdXEG1 gene is crucial for P. digitatum growth, spore production and pathogenicity during citrus fruit infection.

Calcium disodium edetate controls citrus green mold by chelating Mn2+ and targeting pyruvate synthesis in the pathogen

Edetate salts are a kind of food additive regarded as candidates for chemical fungicides because of their lower toxicity and higher safety for the environment and human health. The antifungal activities of edetate salts have been determined in some phytopathogens except Penicillium digitatum, which causes the most destructive fungal disease during the citrus postharvest period. In the present study, we investigated the molecular mechanisms of calcium disodium edetate (EDTA-Na2Ca) against P. digitatum in deepth. The results suggested that EDTA-Na2Ca inhibited the P. digitatum P44 strain in vitro at a minimum inhibitory/fungicidal concentration (MIC/MFC) of 50 mmol L−1. Meanwhile, EDTA-Na2Ca also inhibited P. digitatum in vivo by enhancing the host defense system. The P44 strain treated with EDTA-Na2Ca displayed a delayed conidial germination rate, expanded hyphal tips, thickened septa and cell walls, and destroyed mitochondria. Transcriptome sequencing and qRT-PCR analyses indicated that EDTA-Na2Ca suppressed the P44 strain primarily by disturbing the carbohydrate metabolism pathway, especially the synthesis of pyruvate in glycolysis. In addition, the adverse effects, including those on mycelial growth, pyruvate synthesis and superoxide dismutase activity, of EDTA-Na2Ca could be reversed by exogenous Mn2+, implying that Mn2+ chelation is an essential mode of action. In summary, the current work verifies that EDTA-Na2Ca is a potential antifungal drug that cures citrus green mold probably by chelating Mn2+ and targeting pyruvate synthesis in P. digitatum.

New Observation in Biocontrol of Penicillium caperatum against Fusarium oxysporum on Saposhnikovia divaricata and as a Plant Growth Promoter

Fusarium oxysporum, a common fungal pathogen that infects economic crops, causes Fusarium wilt disease to Saposhnikovia divaricata at an annual incidence rate of more than 15%. This study aimed to assess the potential of rhizospheric fungi as antifungal agents against Fusarium wilt of Saposhnikovia divaricata. In this study, 104 fungi were isolated from S. divaricata rhizospheric soil. Twelve rhizospheric strains that showed antagonistic activity against F. oxysporum, MR-16, MR-32, MR-38, etc., were screened out. Biocontrol activities of the twelve strains, especially MR-16, were subsequently characterized and evaluated. Strain MR-16 as potential stock for biocontrol had good antibiotic activity against F. oxysporum in vitro experiment. Based on the analysis of morphological properties and rDNA internal transcribed spacers (ITS), we identified an isolate MR-16 as Penicillium caperatum (GenBank No. OK287146.1), a new record of this species of China. The results of the in vitro antagonistic assay indicated that the conidial germination rate was significantly decreased, and the mycelia morphology of F. oxysporum induced change via the culture filtrate of P. caperatum MR-16, such as deformation and degradation. In an outdoor pot experiment, inoculation of S. divaricata plants with F. oxysporum created severe wilting symptoms; however, in inoculation trials, MR-16 effectively suppressed disease lesions, with a strong control efficacy of 60.76%. In addition, strain MR-16 could successfully colonize and form stable populations in the soil, and it showed a continuous positive growth-promoting effect on S. divaricata plants.

The influence of temperature and vapour pressure deficit on conidia germination and germ tube production in an Australian Podosphaera xanthii isolate

Powdery mildew of cucurbits, caused by Podosphaera xanthii, is an economic constraint in cucurbit production worldwide. This study examined the influence of temperature and vapour pressure deficit (VPD) on the rate of conidial germination and the formation of germ tubes in an Australian P. xanthii isolate 12 – 48 h after inoculation. Two experiments were prepared by inoculating cucumber, cv. crystal salad, leaf-discs using a spore settling tower. The first experiment incubated inoculated cucumber leaf-discs at eight temperatures between 8 and 35 °C under saturated vapour pressure (SVP), the second compared 18 VPD conditions between 0.038 and 1.797 kPa, in six humidity chambers (33% – 99% relative humidity) and three temperatures (22 °C, 25 °C, 28 °C). Leaf-discs were cleared, stained and microscopically inspected for conidial germination and the number of germ tubes. The optimal temperature for germination was 28ºC at SVP, where more than 50% of conidia had germinated by 12 h, and 85% by 48 h. Fewer germinated conidia were recorded after 12 h at other temperature treatments between 17 °C and 31 °C. The germination percentage and germination rate were significantly lower when vapour pressure was between 0.13 and 2.5 kPa, with germination in response to VPD varying by approximately 10%, indicating difficulty associating conidial germination to VPD above 0.13 kPa. Germ tube production was highest between 25 ºC and 28 ºC at the lowest VPD treatment at near SVP, with more than 50% of the germinated conidia producing at least three germ tubes. Germination and formation of germ tubes significantly reduced when VPD increased. P. xanthii conidia were able to produce a primary germ tube under relatively dry conditions, such as 2.53 kPa, but these results show infection would be less likely and require longer incubation. This study provides the first crucial step in simulating powdery mildew infections on cucurbit plants and may lead to a model capable of providing risk forecasts or fungicide management decision support tools.

Synergistic Effects of Clonostachys rosea Isolates and Succinate Dehydrogenase Inhibitors Fungicides against Gray Mold on Tomato.

Gray mold caused by Botrytis cinerea is a devastating disease in tomatoes. Site-specific fungicide application is still key to disease management; however, chemical control has many drawbacks. Here, the combined application of a biological agent, Clonostachys rosea, with newly developed succinate dehydrogenase inhibitors (SDHI) fungicides showed stronger synergistic effects than the application of SDHI fungicides alone on tomato gray mold control. C. rosea 67-1 has been reported as an efficient biological control agent (BCA) for B. cinerea. Little information is currently available about the combination of C. rosea and fungicides in the control of gray mold. By testing the sensitivity to fungicides with different action mechanisms, C. rosea isolates showed high tolerance to SDHI fungicides (1000 μg mL-1) on PDA, and the conidial germination rate was almost not affected under 120 μg mL-1 of fluxapyroxad and fluopyram. In greenhouse experiments, the control effect of the combination of C. rosea and fluxapyroxad or fluopyram against tomato gray mold was significantly increased than the application of BCA or SDHI fungicides alone, and the combination allows a two-fold reduction of both the fungicide and BCA dose. Further, the biomass of B. cinerea and C. rosea on tomato plants was determined by qPCR. For B. cinerea, the trend of detection level for different treatments was consistent with that of the pot experiments, and the lowest biomass of B. cinerea was found when treated with C. rosea combined with fluxapyroxad and fluopyram, respectively. For C. rosea, qPCR assay confirmed its colonization on tomato plants when mixed with fluopyram and fluxapyroxad. These results indicated that combining C. rosea 67-1 with the SDHI fungicides could synergistically increase control efficacy against tomato gray mold.

VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae.

The ergosterol biosynthesis pathway plays an important role in model pathogenic bacteria Saccharomyces cerevisiae, but little is known about the biosynthesis of ergosterol in the pathogenic fungus Verticillium dahliae. In this study, we identified the VdERG2 gene encoding sterol C-8 isomerase from V. dahliae and investigated its function in virulence by generating gene deletion mutants (ΔVdERG2) and complemented mutants (C-ΔVdERG2). Knockout of VdERG2 reduced ergosterol content. The conidial germination rate and conidial yield of ΔVdERG2 significantly decreased and abnormal conidia were produced. In spite of VdERG2 did not affect the utilization of carbon sources by V. dahliae, but the melanin production of ΔVdERG2 was decreased in cellulose and pectin were used as the sole carbon sources. Furthermore, the ΔVdERG2 mutants produced less microsclerotia and melanin with a significant decrease in the expression of microsclerotia and melanin-related genes VaflM, Vayg1, VDH1, VdLAC, VdSCD and VT4HR. In addition, mutants ΔVdERG2 were very sensitive to congo red (CR), sodium dodecyl sulfate (SDS) and hydrogen peroxide (H2O2) stresses, indicating that VdERG2 was involved in the cell wall and oxidative stress response. The absence of VdERG2 weakened the penetration ability of mycelium on cellophane and affected the growth of mycelium. Although ΔVdERG2 could infect cotton, its pathogenicity was significantly impaired. These phenotypic defects in ΔVdERG2 could be complemented by the reintroduction of a full-length VdERG2 gene. In summary, as a single conservative secretory protein, VdERG2 played a crucial role in ergosterol biosynthesis, nutritional differentiation and virulence in V. dahliae.

Effect of Rhizospheric Fungus on Biological Control of Root Rot (Fusarium equiseti) Disease of Saposhnikovia divaricata

Saposhnikovia divaricata is a high-demand medicinal plant containing various bioactive metabolites (e.g., chromone). However, root rot disease leads to a dramatic reduction in the yield and quality of S. divaricata. The use of rhizospheric microorganisms is one of the best strategies for biological control. In this study, a total of 104 fungi isolated from the rhizospheric soil of S. divaricata plants were examined for their different antifungal properties. Subsequently, strain MR-57 was selected as a potential stock for biocontrol due to its broad-spectrum antagonistic activity against pathogens, including F. equiseti. Based on the analysis of morphological properties and rDNA internal transcribed spacers (ITSs), strain MR-57 was identified as Acrophialophora jodhpurensis (GenBank No. OK287150.1), a newly recorded species for China. In an in vitro antifungal assay, the culture filtrate of strain MR-57 significantly reduced the conidial germination rate and induced alterations in the mycelia morphology of F. equiseti, such as deformation and degradation. To assess the antifungal efficacy of MR-57 against root rot disease and the properties promoting the growth of S. divaricata, pot experiments were performed under natural outdoor conditions. The results indicated that co-inoculation with MR-57 delayed the occurrence of S. divaricata root rot and showed a control efficacy of 65.41% (p < 0.05) based on the measurement of suppressed disease lesions. Additionally, MR-57 successfully colonized and formed a stable population in the soil in which S. divaricata was grown, and it exhibited a consistently positive effect on the promotion of the growth of S. divaricata plants. In short, Acr. jodhpurensis MR-57 could be considered for the development of a potential biocontrol agent for the management of S. divaricata root rot caused by F. equiseti.

Investigation on tolerance of Metarhizium flavoviride Ma130821 to environmental stress factors and responses on biological control of larvae of Potosia brevitarsis Lewis

AbstractEntomopathogenic fungi of Metarhizium spp., are one of the candidates for eco‐friendly pest control instead of chemical control agents. But the development involved in producing a wide range of mycoinsecticides and application of biotechnology widely is restricted, usually due to the defect of slow action and sensitivity to environmental stresses. This study evaluates the tolerance of conidia of Metarhizium flavoviride Ma130821 to high temperature exposure, humidity level, ultraviolet radiation and the pathogenicity against larvae of Potosia brevitarsis Lewis under environmental stresses. Results showed that M. flavoviride Ma130821 had the higher tolerance ability to 40°C with continuous exposure for 24 h, the conidial germination rate was 54.00%, radial growth was 1.59 mm/day and sporulation was 9.37 × 106 conidia/cm2, the ability to desiccation with humidity of 53% (85.33%, 1.94 mm/d, 2.13 × 107 conidia/cm2), the ability of uvioresistant effect with 900 s radiation (98.11%, 0.91 mm/d, 8.87 × 106 conidia/cm2). The data of bioassay at artificial adverse condition had been fitted to the time–concentration–mortality (TCM) model. The cumulative mortality of P. brevitarsis larvae was 76.67% with conidial concentrations of 3.41 × 108 conidia/mL. The low lethal concentration (LC50) in 7 days after inoculation (DAI) and short lethal time (LT50) was 6.70 × 109 conidia/mL and 8.02 days, respectively. The mortality also reached to 73.33% at natural condition, with conidial concentration of 3.75 × 108 conidia/mL, but the LT50 delayed to 14.08 days. Our results demonstrate that entomopathogenic fungi M. flavoviride Ma130821, with significantly and superior antistress capacity and high virulence, could be considered a promising biocontrol agent against white grubs.

FgLEU1 Is Involved in Leucine Biosynthesis, Sexual Reproduction, and Full Virulence in Fusarium graminearum.

Fusarium head blight (FHB) caused by Fusarium graminearum is a significant disease among cereal crops. In F. graminearum, biosynthesis of leucine, which is a branched chain amino acid, is achieved by converting α-isopropylmalate to β-isopropylmalate catalyzed by isopropylmalate isomerase encoded by LEU1. Considering the potential for targeting this pathway by fungicides, we characterized the gene FgLEU1 (FGSG-09589) in the Fusarium graminearum genome using bioinformatics methods. For functional characterization, we constructed a deletion mutant of FgLEU1 (ΔLEU1) through homologous recombination. Compared with the wild-type strain PH-1, ΔLEU1 showed slower colony growth and fewer aerial mycelia. Leucine addition was needed to ensure proper mutant growth. Further, ΔLEU1 showed decreased conidial production and germination rates, and could not produce ascospores. Moreover, ΔLEU1 showed complete loss of pathogenicity and reduced ability to produce deoxynivalenol (DON) and aurofusarin. Upstream and downstream genes of FgLEU1 were significantly upregulated in ΔLEU1. Contrary to previous reports, the deletion mutant was more resistant to osmotic stress and cell wall-damaging agents than the wild-type. Taken together, FgLEU1 plays a crucial role in leucine synthesis, aerial mycelial growth, sexual and asexual reproduction, pathogenicity, virulence, and pigmentation in Fusarium graminearum, indicating its potential as a target for novel antifungal agents.

Resistance of peanut to web blotch caused by Phoma arachidicola is related to papillae formation and the hypersensitive response

AbstractPeanut (Arachis hypogaea) is an important economic crop that is susceptible to various foliar diseases. Web blotch, which is caused by Phoma arachidicola, is one of the most serious peanut foliar diseases because it affects the leaves and results in substantial yield losses. In this study, the P. arachidicola infection process and the response of peanut plants to infection were revealed through a cytological study of resistant and susceptible peanut varieties. Conidial germination rates, appressorium formation and resistance responses, including papillae formation and the hypersensitive response (HR), were analysed at different infection stages. Notably, the basal infection response of susceptible varieties resulted in limited papillae formation and a single HR, which was followed by programmed cell death. The papillae formation frequency and HR frequency were significantly greater in the resistant varieties than in the susceptible varieties at 84 h postinoculation. The primary difference between the two resistant varieties was the superior HR of Zheng8903 compared with that of Yuhua15. The complex amphidiploid peanut genome may explain the complexity and diversity of the resistance responses. The interactions of functional orthologs should be clarified and perhaps modulated in future investigations. The results of this study revealed the differences in the resistance responses among peanut germplasm and may be relevant for the breeding of new peanut varieties highly resistant to P. arachidicola.

Development of Cordyceps javanica BE01 with enhanced virulence against Hyphantria cunea using polyethylene glycol-mediated protoplast transformation.

Cordyceps javanica has promising application prospects as an entomopathogenic fungus with a wide range of hosts. To enhance the virulence of C. javanica, a polyethylene glycol (PEG)-mediated protoplast genetic transformation system was constructed. Strains overexpressing the subtilisin-like protease genes CJPRB and CJPRB1 and the tripeptidyl peptidase gene CJCLN2-1 were constructed with this system, and the effects of these strains on Hyphantria cunea were tested. The aminoglycoside G418 was used at 800 μg ml−1 to screen the transformants. C. javanica hyphae were degraded with an enzyme mixture to obtain protoplasts at 1.31 × 107 protoplasts ml−1. The transformation of 2 μg of DNA into 1,000 protoplasts was achieved with 20% PEG2000, and after 6 h of recovery, the transformation efficiency was 12.33 ± 1.42 transformants μg−1 plasmid. The LT50 values of CJPRB, CJPRB1, and CJCLN2-1-overexpressing C. javanica strains were 1.32-fold, 2.21-fold, and 2.14-fold higher than that of the wild-type (WT) strain, respectively. The three overexpression strains showed no significant differences from the WT strain in terms of colony growth, conidial yield, and conidial germination rate. However, the infection rate of the CJPRB1 strain was faster than that of the WT strain, with infection occurring within 4–5 days. The CJCLN2-1 strain had a significantly higher mortality rate than the WT strain within 4–10 days after infection. A C. javanica genetic transformation system was successfully constructed for the first time, and an overexpression strain exhibited enhanced virulence to H. cunea compared with the WT strain.

2,3-Butanedione suppresses gray mold of postharvest fruit by activating the autophagy of Botrytis cinerea

The volatile organic compounds produced by plants and microorganisms have been widely used for postharvest disease control due to their high antifungal capacity. In this research, a volatile product of citrate metabolism, 2,3-butanedione, was assayed against gray mold caused by Botrytis cinerea on postharvest fruit. 2,3-Butanedione treatment exhibited a distinct inhibitory effect on colony growth, conidial germination rate and germ tube length of B. cinerea in vitro and efficiently mitigated the disease prevalence of inoculated fruit under both artificial and natural inoculation conditions. Transcriptome analysis displayed that 2,3-butanedione treatment led to down-regulated expression of genes associated with arginine biosynthesis, ribosome biogenesis and several pathogenicity-related genes, including Bcpg1–3, BcNEP1, Bcboa2 and Bmp1. Furthermore, treatment led to increased genes expression related to proteolysis, peroxisome and autophagy. Among them, the autophagy pathway was most prominent, and key genes in this pathway (BcATGs and BcVPS45) were activated by 2,3-butanedione treatment. Moreover, numerous autophagic vacuoles containing cytoplasmic components were observed in the 2,3-butanedione-treated fungal cells. These results suggest that 2,3-butanedione inhibits gray mold on postharvest fruit by not only interfering the gene expression of fungal growth and pathogenicity in B. cinerea, but also inducing autophagic activity. Collectively, these results provide a theoretical basis for elucidating the underlying antifungal mechanism of 2,3-butanedione against B. cinerea.

Antifungal Potential of Bacillus velezensis CE 100 for the Control of Different Colletotrichum Species through Isolation of Active Dipeptide, Cyclo-(D-phenylalanyl-D-prolyl).

Colletotrichum species are important fungal pathogens causing anthracnose of tropical and subtropical fruit and vegetable crops. Dual culture assay indicated that Bacillus velezensis CE 100 was a strong antagonist against C. acutatum, C. coccodes, C. dematium, and C. gloeosporioides. The volatile organic compounds produced by B. velezensis CE 100 affected mycelial growth of Colletotrichum species tested in our study and caused twisted hyphal structures of all these fungal species. Chloroform crude compounds of B. velezensis CE 100 inhibited four Colletotrichum species in a concentration-dependent manner and induced severe damage in hyphal morphology of these fungal pathogens, including swelling, bulging, and multiple branching. Moreover, the active cyclic dipeptide, cyclo-(D-phenylalanyl-D-prolyl), was isolated from chloroform crude extract and identified by nuclear magnetic resonance (NMR) and mass spectrometry. The inhibitory effect of cyclo-(D-phenylalanyl-D-prolyl) on conidial germination of C. gloeosporioides occurred in a concentration-dependent manner. The conidial germination rate was completely inhibited by a concentration of 3 mg/mL of cyclo-(D-phenylalanyl-D-prolyl). Scanning electron micrographs revealed that the exposure to cyclic dipeptide resulted in seriously deformed hyphae and conidia with shriveled surfaces in dipeptide-treated C. gloeosporioides. Therefore, active dipeptide-producing B. velezensis CE 100 is a promising biocontrol agent for Colletotrichum species causing anthracnose.