Regulation of nitrogen utilization and mycotoxin biosynthesis by the GATA transcription factor AaAreA in Alternaria alternata.

Alternaria alternata is a prevalent postharvest pathogen that generates diverse mycotoxins, notably alternariol (AOH) and alternariol monomethyl ether (AME), which are recurrent severe contaminants. Nitrogen sources modulate fungal growth, development, and secondary metabolism, including mycotoxin production. The GATA transcription factor AreA regulates nitrogen source utilization. However, little is known about its involvement in the regulation of nitrogen utilization in A. alternata. To examine the regulatory mechanism of AaAreA on AOH and AME biosynthesis in A. alternata, we analyzed the impact of diverse nitrogen sources on the fungal growth, conidiation and mycotoxin production. The use of a secondary nitrogen source (NaNO3) enhanced mycelial elongation and sporulation more than the use of a primary source (NH4Cl). NaNO3 favored greater mycotoxin accumulation than did NH4Cl. The regulatory roles of AaAreA were further clarified through gene knockout. The absence of AaAreA led to an overall reduction in growth in minimal media containing any nitrogen source except NH4Cl. AaAreA positively regulates mycotoxin biosynthesis when both NH4Cl and NaNO3 are used as nitrogen sources. Subcellular localization analysis revealed abundant nuclear transport when NaNO3 was the sole nitrogen source. The regulatory pathway of AaAreA was systematically revealed through comprehensive transcriptomic analyses. The deletion of AaAreA significantly impedes the transcription of mycotoxin biosynthetic genes, including aohR, pksI and omtI. The interaction between AaAreA and aohR, a pathway-specific transcription factor gene, demonstrated that AaAreA binds to the aohR promoter sequence (5'-GGCTATGGAAA-3'), activating its transcription. The expressed AohR regulates the expression of downstream synthase genes in the cluster, ultimately impacting mycotoxin production. This study provides valuable information to further understand how AreA regulates AOH and AME biosynthesis in A. alternata, thereby enabling the effective design of control measures for mycotoxin contamination.

Diversity study of Beauveria bassiana species for finding the most virulent strain to manage Bemisia tabaci in cotton.

Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: • 102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. • Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. • MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.

Use of Ozone as a Substrate Treatment for the Control of Trichoderma in the Production of Pleurotus ostreatus.

Pleurotus ostreatus is one of the most widely cultivated species in the world. It can be produced in many lignocellulosic substrates after carrying out a treatment to eliminate competing microorganisms. The most commonly used is pasteurization by steam or by immersion in hot water. The aim of this work is to evaluate if ozone can be employed as treatment for decontamination of the substrate used for the production of the edible mushroom P. ostreatus to control of green mold Trichoderma. Wheat straw was employed as a substrate. We used two different methodologies: bubbling ozone into a tank with water and the substrate, and injecting ozone into a closed tank with the substrate inside. Ten treatments were carried out including two treatments with inoculation by a spray of conidia of Trichoderma. The effect of ozone on the conidia was also evaluated. We found that the treatment of the substrate with ozone in immersed water resulted more effective (lower growth of Trichoderma) than injecting ozone into a closed tank. Anyway, we found that the contaminant fungi could grow on the substrate in both treatments with ozone. We observed that although ozone affected the conidia when it was bubbled into water, some of them still managed to survive and could germinate 72 h later. P. ostreatus could grow and produce fruiting bodies on a substrate that was previously treated with ozone and yields were not affected. Based on the results obtained, we conclude that ozone may not be an effective agent to control Trichoderma in highly contaminated substrates, at least in the experimental conditions that we used, for the production of P. ostreatus.

Complex nitrogen sources from agro-industrial byproducts: impact on production, multi-stress tolerance, virulence, and quality of Beauveria bassiana blastospores.

We investigated the impact of various complex organic nitrogen sources on the submerged liquid fermentation of Beauveria bassiana, a versatile entomopathogenic fungus known for producing hydrophilic yeast-like single cells called blastospores. Specifically, we examined yeast extract, autolyzed yeast, inactive yeast, cottonseed flour, corn bran, and corn gluten meal as nitrogen compounds with different carbon-to-nitrogen (C:N) ratios. Our comprehensive analysis encompassed blastospore production, tolerance to abiotic stresses, shelf stability after drying, and virulence against mealworm larvae, crucial attributes for developing effective blastospore-based biopesticides. Notably, cottonseed flour emerged as the optimal nitrogen source, yielding up to 2.5 × 109 blastospores/mL within 3 days in a bioreactor. These blastospores exhibited the highest tolerance to heat stress and UV-B radiation exposure. The endogenous C:N ratio in blastospore composition was also impacted by nitrogen sources. Bioassays with mealworm larvae demonstrated that blastospores from cottonseed flour were the most virulent, achieving faster lethality (lower LT50) and requiring a lower inoculum (LC50). Importantly, blastospores produced with cottonseed flour displayed extended viability during storage, surpassing the retention of viability compared to those from autolyzed yeast over 180 days at 4°C. Despite differences in storage viability, both nitrogen sources conferred similar long-term blastospore bioactivity against mealworms. In summary, this research advances our understanding of the crucial impact of complex organic nitrogen selection on the phenotypic traits of blastospores in association with their intracellular C:N ratio, contributing to the production of ecologically fit, shelf-stable, and virulent propagules for effective pest biocontrol programs. IMPORTANCE: Biological control through entomopathogenic fungi provides essential ecological services in the integrated management of agricultural pests. In the context of submerged liquid fermentation, the nutritional composition significantly influences the ecological fitness, virulence and quality of these fungi. This study specifically explores the impact of various complex organic nitrogen sources derived from agro-industrial byproducts on the submerged liquid fermentation of Beauveria bassiana, a versatile entomopathogenic fungus known for producing hydrophilic yeast-like blastospores. Notably, manipulating the nitrogen source during submerged cultivation can influence the quality, fitness, and performance of blastospores. This research identifies cottonseed flour as the optimal low-cost nitrogen source, contributing to increased production yields, enhanced multi-stress tolerance, heightened virulence with extended shelf life and long-term bioactivity. These findings deepen our understanding of the critical role of nitrogen compound selection in liquid media formulation, facilitating the production of ecologically fit and virulent blastospores for more effective pest biocontrol programs.

FgPfn participates in vegetative growth, sexual reproduction, pathogenicity, and fungicides sensitivity via affecting both microtubules and actin in the filamentous fungus Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum species complexes (FGSG), is an epidemic disease in wheat and poses a serious threat to wheat production and security worldwide. Profilins are a class of actin-binding proteins that participate in actin depolymerization. However, the roles of profilins in plant fungal pathogens remain largely unexplored. Here, we identified FgPfn, a homolog to profilins in F. graminearum, and the deletion of FgPfn resulted in severe defects in mycelial growth, conidia production, and pathogenicity, accompanied by marked disruptions in toxisomes formation and deoxynivalenol (DON) transport, while sexual development was aborted. Additionally, FgPfn interacted with Fgα1 and Fgß2, the significant components of microtubules. The organization of microtubules in the ΔFgPfn was strongly inhibited under the treatment of 0.4 µg/mL carbendazim, a well-known group of tubulin interferers, resulting in increased sensitivity to carbendazim. Moreover, FgPfn interacted with both myosin-5 (FgMyo5) and actin (FgAct), the targets of the fungicide phenamacril, and these interactions were reduced after phenamacril treatment. The deletion of FgPfn disrupted the normal organization of FgMyo5 and FgAct cytoskeleton, weakened the interaction between FgMyo5 and FgAct, and resulting in increased sensitivity to phenamacril. The core region of the interaction between FgPfn and FgAct was investigated, revealing that the integrity of both proteins was necessary for their interaction. Furthermore, mutations in R72, R77, R86, G91, I101, A112, G113, and D124 caused the non-interaction between FgPfn and FgAct. The R86K, I101E, and D124E mutants in FgPfn resulted in severe defects in actin organization, development, and pathogenicity. Taken together, this study revealed the role of FgPfn-dependent cytoskeleton in development, DON production and transport, fungicides sensitivity in F. graminearum.

Exogenous Indole-3-Acetic Acid Suppresses Rice Infection of Magnaporthe oryzae by Affecting Plant Resistance and Fungal Growth.

Auxin is an important phytohormone that regulates diverse biologic processes, including plant growth and immunity. Indole-3-acetic acid (IAA), known as one of the main forms of auxin, is able to activate plant immunity. However, it is unknown whether IAA enhances plant resistance and/or suppresses the growth of the fungal pathogen Magnaporthe oryzae. Here, we found that IAA could induce expression levels of pathogenesis-related genes to enhance disease resistance and could control the development of blast disease through inhibiting M. oryzae infection. Exogenous IAA suppressed mycelial growth and delayed spore germination by inhibiting fungal endogenous IAA biosynthesis and impairing redox homeostasis, respectively. When applied to a field test, two IAA analogues, 1-naphthaleneacetic acid and 2,4-dichlorophenoxy acetic acid, can effectively control rice blast disease. Our study advances the understanding of IAA in controlling rice blast disease through suppressing pathogen growth and enhancing plant resistance.

The Role of Cytokinins and Abscisic Acid in the Growth, Development and Virulence of the Pathogenic Fungus Stagonospora nodorum (Berk.).

Cytokinins (CKs) and abscisic acid (ABA) play an important role in the life of both plants and pathogenic fungi. However, the role of CKs and ABA in the regulation of fungal growth, development and virulence has not been sufficiently studied. We compared the ability of two virulent isolates (SnB and Sn9MN-3A) and one avirulent isolate (Sn4VD) of the pathogenic fungus Stagonospora nodorum Berk. to synthesize three groups of hormones (CKs, ABA and auxins) and studied the effect of exogenous ABA and zeatin on the growth, sporulation and gene expression of necrotrophic effectors (NEs) and transcription factors (TFs) in them. Various isolates of S. nodorum synthesized different amounts of CKs, ABA and indoleacetic acid. Using exogenous ABA and zeatin, we proved that the effect of these hormones on the growth and sporulation of S. nodorum isolates can be opposite, depends on both the genotype of the isolate and on the concentration of the hormone and is carried out through the regulation of carbohydrate metabolism. ABA and zeatin regulated the expression of fungal TF and NE genes, but correlation analysis of these parameters showed that this effect depended on the genotype of the isolate. This study will contribute to our understanding of the role of the hormones ABA and CKs in the biology of the fungal pathogen S. nodorum.

Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.

The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.

Microplastic influences the ménage à trois among the plant, a fungal pathogen, and a plant growth-promoting fungal species.

Microplastics (MP) can influence a plethora of fungal species within the rhizosphere. Nevertheless, there are few studies on the direct impacts of MPs on soil fungi and their intricate interplay with plants. Here, we investigated the impact of polyethylene microspheres (PEMS) on the ecological interactions between Fusarium solani, a plant pathogenic fungus, and Trichoderma viride, a fungal plant growth promotor, within the rhizosphere of Solanum lycopersicum (tomato). Spores of F. solani and T. viride were pre-incubated with PEMS at two concentrations, 100 and 1000 mg L-1. Mycelium growth, sporulation, spore germination, and elongation were evaluated. Tomato seeds were exposed to fungal spore suspensions treated with PEMS, and plant development was subsequently assessed after 4 days. The results showed that PEMS significantly enhanced the sporulation (106.0 % and 70.1 %) but compromised the spore germination (up to 27.3 % and 32.2 %) and radial growth (up to -5.2% and -21.7 %) of F. solani and T. viride, respectively. Furthermore, the 100 and 1000 mg L-1 concentrations of PEMS significantly (p<0.05) enhanced the mycelium density of T. viride (9.74 % and 22.30 %, respectively), and impaired the germ-tube elongation of F. solani after 4 h (16.16 % and 11.85 %, respectively) and 8 h (4 % and 17.10 %, respectively). In addition, PEMS amplified the pathogenicity of F. solani and boosted the bio-enhancement effect of T. viride on tomato root growth. Further, PEMS enhanced the bio-fungicidal effect of T. viride toward F. solani (p<0.05). In summary, PEMS had varying effects on F. solani and T. viride, impacting their interactions and influencing their relationship with tomato plants. It intensified the beneficial effects of T. viride and increased the aggressiveness of F. solani. This study highlights concerns regarding the effects of MPs on fungal interactions in the rhizosphere, which are essential for crop soil colonization and resource utilization.

Formulation of essential oils-loaded solid lipid nanoparticles-based chitosan/PVA hydrogels to control the growth of Botrytis cinerea and Penicillium expansum.

Botrytis cinerea and Penicillium expansum are phytopathogenic fungi that produce the deterioration of fruits. Thus, essential oil (EO) has emerged as a sustainable strategy to minimize the use of synthetic fungicides, but their volatility and scarce solubility restrict their application. This study proposes the EO of Oreganum vulgare and Thymus vulgaris-loaded solid lipid nanoparticles (SLN) based chitosan/PVA hydrogels to reduce the infestation of fungi phytopathogen. EO of O. vulgare and T. vulgaris-loaded SLN had a good homogeneity (0.21-0.35) and stability (-28.8 to -33.0 mV) with a mean size of 180.4-188.4 nm. The optimization of EO-loaded SLN showed that the encapsulation of 800 and 1200 µL L-1 of EO of O vulgare and T. vulgaris had the best particle size. EO-loaded SLN significantly reduced the mycelial growth and spore germination of both fungi pathogen. EO-loaded SLN into hydrogels showed appropriate physicochemical characteristics to apply under environmental conditions. Furthermore, rheological analyses evidenced that hydrogels had solid-like characteristics and elastic behavior. EO-loaded SLN-based hydrogels inhibited the spore germination in B. cinerea (80.9 %) and P. expansum (55.7 %). These results show that SLN and hydrogels are eco-friendly strategies for applying EO with antifungal activity.

FpPEX5 and FpPEX7 are involved in the growth, reproduction, DON toxin production, and pathogenicity in Fusarium pseudograminearum.

Fusarium crown rot, caused by Fusarium pseudograminearum, is a devastating disease affecting the yield and quality of cereal crops. Peroxisomes are single-membrane organelles that play a critical role in various biological processes in eukaryotic cells. To functionally characterise peroxisome biosynthetic receptor proteins FpPEX5 and FpPEX7 in F. pseudograminearum, we constructed deletion mutants, ΔFpPEX5 and ΔFpPEX7, and complementary strains, ΔFpPEX5-C and ΔFpPEX7-C, and analysed the functions of FpPEX5 and FpPEX7 proteins using various phenotypic observations. The deletion of FpPEX5 and FpPEX7 resulted in a significant deficiency in mycelial growth and conidiation and blocked the peroxisomal targeting signal 1 and peroxisomal targeting signal 2 pathways, which are involved in peroxisomal matrix protein transport, increasing the accumulation of lipid droplets and reactive oxygen species. The deletion of FpPEX5 and FpPEX7 may reduce the formation of toxigenic bodies and decrease the pathogenicity of F. pseudograminearum. These results indicate that FpPEX5 and FpPEX7 play vital roles in the growth, asexual reproduction, virulence, and fatty acid utilisation of F. pseudograminearum. This study provides a theoretical basis for controlling stem rot in wheat.

RNA-dependent RNA polymerases regulate ascospore discharge through the exonic-sRNA-mediated RNAi pathway.

Ascospores, forcibly released into the air from perithecia, are the primary inoculum for Fusarium head blight. In Fusarium graminearum, the biological functions of four RNA-dependent RNA polymerases (RdRPs) (Fgrdrp1-4) have been reported, but their regulatory mechanisms are poorly understood and the function of Fgrdrp5 is still unknown. In this study, we found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays an important role in ascospore discharge, and they all participate in the generation of turgor pressure in a polyol-dependent manner. Moreover, these three genes all affect the maturation of ascospores. Deep sequencing and co-analysis of small RNA and mRNA certified that Fgrdrp1, Fgrdrp2, and Fgrdrp5 partly share their functions in the biogenesis and accumulation of exonic small interference RNA (ex-siRNA), and these three RdRPs negatively regulate the expression levels of ex-siRNA corresponding genes, including certain genes associated with ascospore development or discharge. Furthermore, the differentially expressed genes of deletion mutants, those involved in lipid and sugar metabolism or transport as well as sexual development-related transcription factors, may also contribute to the defects in ascospore maturation or ascospore discharge. In conclusion, our study suggested that the components of the dicer-dependent ex-siRNA-mediated RNA interference pathway include at least Fgrdrp1, Fgrdrp2, and Fgrdrp5. IMPORTANCE: We found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays important roles in ascospore maturation and ascospore discharge of Fusarium graminearum. These three RNA-dependent RNA polymerases participate in the biogenesis and accumulation of exonic small interference RNA and then regulate ascospore discharge.

Does sorghum phenolic extract have antifungal effect?

This study aimed to evaluate the antifungal effect of SC319 sorghum phenolic extract (SPE) on the Aspergillus, Fusarium, Penicillium, Stenocarpella, Colletotrichum, and Macrophomina genera. SPE was extracted by 20% ethanol and used in four assays: (1) against Fusarium verticillioides in solid (PDA) and liquid (PD) potato dextrose media; (2) Minimum Inhibitory Concentration (MIC) assay with 16 fungi isolates; (3) Conidial Germination Rate (CGR) with 14 fungi isolates and (4) Growth Curve (GC) with 11 fungi isolates. There was no reduction in the mycelial growth (colony diameter and dry weight) and in the number of Fusarium verticillioides spores in assay 1 (PDA and PD). The colony's dry weight was almost six times higher in the presence than in the absence of SPE. All SPE samples presented MIC (assay 1) above the maximum concentration tested (5000 µg.mL-1) for the 16 isolates. Also, there was no inhibitory effect of SPE on conidia germination rate (CGR). Oppositely, in GC assay, the control had a higher CFU count than the samples with SPE in 24 h. This result suggests that SPE can delay the fungal growth in the first hours of incubation, which is an important finding that may help reduce the severity of fungal diseases in plants. However, further studies are needed to confirm these results, including sorghum genotypes with different profiles of phenolic compounds. Although the SC319 SPE was not effective as an antifungal agent, it may have potential as a growth promoter of beneficial fungi in the food and pharmaceutical industries.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Leaf litter from Cynanchum auriculatum Royle ex Wight leads to root rot outbreaks by Fusarium solani, hindering continuous cropping.

Cynanchum auriculatum Royle ex Wight (CA) is experiencing challenges with continuous cropping obstacle (CCO) due to soil-borne fungal pathogens. The leaf litter from CA is regularly incorporated into the soil after root harvesting, but the impact of this practice on pathogen outbreaks remains uncertain. In this study, a fungal strain D1, identified as Fusarium solani, was isolated and confirmed as a potential factor in CCO. Both leave extract (LE) and root extract (RE) were found to inhibit seed germination and the activities of plant defense-related enzymes. The combinations of extracts and D1 exacerbated these negative effects. Beyond promoting the proliferation of D1 in soil, the extracts also enhanced the hypha weight, spore number, and spore germination rate of D1. Compared to RE, LE exhibited a greater degree of promotion in the activities of pathogenesis-related enzymes in D1. Additionally, caffeic acid and ferulic acid were identified as potential active compounds. LE, particularly in combination with D1, induced a shift in the composition of fungal communities rather than bacterial communities. These findings indicate that the water extract of leaf litter stimulated the growth and proliferation of fungal strain D1, thereby augmenting its pathogenicity toward CA and ultimately contributing to the CCO process.

Con7 is a key transcription regulator for conidiogenesis in the plant pathogenic fungus Fusarium graminearum.

The mycelium of the plant pathogenic fungus Fusarium graminearum exhibits distinct structures for vegetative growth, asexual sporulation, sexual development, virulence, and chlamydospore formation. These structures are vital for the survival and pathogenicity of the fungus, necessitating precise regulation based on environmental cues. Initially identified in Magnaporthe oryzae, the transcription factor Con7p regulates conidiation and infection-related morphogenesis, but not vegetative growth. We characterized the Con7p ortholog FgCon7, and deletion of FgCON7 resulted in severe defects in conidium production, virulence, sexual development, and vegetative growth. The mycelia of the deletion mutant transformed into chlamydospore-like structures with high chitin level accumulation. Notably, boosting FgABAA expression partially alleviated developmental issues in the FgCON7 deletion mutant. Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, the chitin synthase gene Fg6550 (FGSG_06550) showed significant upregulation in the FgCON7 deletion mutant, and altering FgCON7 expression affected cell wall integrity. Further research will focus on understanding the behavior of the chitin synthase gene and its regulation by FgCon7 in F. graminearum. This study contributes significantly to our understanding of the genetic pathways that regulate hyphal differentiation and conidiation in this plant pathogenic fungus. IMPORTANCE: The ascomycete fungus Fusarium graminearum is the primary cause of head blight disease in wheat and barley, as well as ear and stalk rot in maize. Given the importance of conidia and ascospores in the disease cycle of F. graminearum, precise spatiotemporal regulation of these biological processes is crucial. In this study, we characterized the Magnaporthe oryzae Con7p ortholog and discovered that FgCon7 significantly influences various crucial aspects of fungal development and pathogenicity. Notably, overexpression of FgABAA partially restored developmental defects in the FgCON7 deletion mutant. ChIP-qPCR analysis confirmed a direct genetic link between FgABAA and FgCON7. Furthermore, our research revealed a clear correlation between FgCon7 and chitin accumulation and the expression of chitin synthase genes. These findings offer valuable insights into the genetic mechanisms regulating conidiation and the significance of mycelial differentiation in this plant pathogenic fungus.

The CsPbs2-interacting protein oxalate decarboxylase CsOxdC3 modulates morphosporogenesis, virulence, and fungicide resistance in Colletotrichum siamense.

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides.

Synthesis, Characterization, and Antifungal Activity of Benzimidazole-Grafted Chitosan against Aspergillus flavus.

Aspergillus flavus contamination in agriculture and food industries poses threats to human health, leading to a requirement of a safe and effective method to control fungal contamination. Chitosan-based nitrogen-containing derivatives have attracted much attention due to their safety and enhanced antimicrobial applications. Herein, a new benzimidazole-grafted chitosan (BAC) was synthesized by linking the chitosan (CS) with a simple benzimidazole compound, 2-benzimidazolepropionic acid (BA). The characterization of BAC was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (1H and 13C NMR). Then, the efficiency of BAC against A. flavus ACCC 32656 was investigated in terms of spore germination, mycelial growth, and aflatoxin production. BAC showed a much better antifungal effect than CS and BA. The minimum inhibitory concentration (MIC) value was 1.25 mg/mL for BAC, while the highest solubility of CS (16.0 mg/mL) or BA (4.0 mg/mL) could not completely inhibit the growth of A. flavus. Furthermore, results showed that BAC inhibited spore germination and elongation by affecting ergosterol biosynthesis and the cell membrane integrity, leading to the permeabilization of the plasma membrane and leakage of intracellular content. The production of aflatoxin was also inhibited when treated with BAC. These findings indicate that benzimidazole-derived natural CS has the potential to be used as an ideal antifungal agent for food preservation.

Nitric oxide-mediated regulation of Aspergillus flavus asexual development by targeting TCA cycle and mitochondrial function.

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

The Gene vepN Regulated by Global Regulatory Factor veA That Affects Aflatoxin Production, Morphological Development and Pathogenicity in Aspergillus flavus.

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus.