Liqin Fan's research while affiliated with Zhoukou Normal University and other places

Objectives Microsclerotia (MS), anti-stress structures produced by many filamentous fungi, have been proven to be a great substitute for conidia in the production of insecticides within entomogenous fungi. NADPH oxidase (Nox) is a highly conserved ROS-response protein family that is widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether the formation of MS and pathogenicity in entomogenous fungi is regulated by the Nox inside. In this study, we reported the presence of NADPH oxidase homologs in a great potential biocontrol fungus, Metarhizium rileyi, and further showed multiple biological functions. Results Three Nox homologous genes in M. rileyi showed high expression throughout the entire process of MS formation. Targeted deletion of MrNoxA, MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, the anti-adversity assay showed that they are indispensable for growth, osmotic pressure and oxidative stress regulation in Metarhizium rileyi. Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria might be unaffected in mutants since there are no striking differences in virulence compared with WT by topical injections. Conclusion Our results revealed that NADPH oxidase plays important roles in growth regulation, MS formation and pathogenicity in M. rileyi, perhaps in the ROS response and hyphal polarity.

Insect pathogenic fungi, also known as entomopathogenic fungi, are one of the largest insect pathogenic microorganism communities, represented by Beauveria spp. and Metarhizium spp. Entomopathogenic fungi have been proved to be a great substitute for chemical pesticide in agriculture. In fact, a lot of functional genes were also already characterized in entomopathogenic fungi, but more depth of exploration is still needed to reveal their complicated pathogenic mechanism to insects. Metarhizium rileyi (Nomuraea rileyi) is a great potential biocontrol fungus that can parasitize more than 40 distinct species (mainly Lepidoptera: Noctuidae) to cause large-scale infectious diseases within insect population. In this study, a comparative analysis of transcriptome profile was performed with topical inoculation and hemolymph injection to character the infectious pattern of M. rileyi. Appressorium and multiple hydrolases are indispensable constituents to break the insect host primary cuticle defense in entomopathogenic fungi. Within our transcriptome data, numerous transcripts related to destruction of insect cuticle rather growth regulations were obtained. Most importantly, some unreported ribosomal protein genes and novel unannotated protein (hypothetical protein) genes were proved to participate in the course of pathogenic regulation. Our current data provide a higher efficiency gene library for virulence factors screen in M. rileyi, and this library may be also useful for furnishing valuable information on entomopathogenic fungal pathogenic mechanisms to host.

Microsclerotia (MS), the anti-stress structures produced by many filamentous fungi, have been proved to be a great substitute for conidia in production of insecticides in entomogenous fungi. NADPH oxidase (Nox) constitutes ROS-response protein family widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether Nox is required in the pathogenicity and formation of MS in insect pathogenic fungi. In this study, we reported the presence of NADPH oxidase homologs, which contain three different subunits (NoxAp/NoxBp catalytic subunit and NoxRp regulatory subunit), and further showed biological function in the entomopathogenic fungus, Metarhizium rileyi. Three Nox genes all showed high expression levels in the entire process of MS formation. Targeted deletion of MrNoxA , MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, our anti-adversity results showed that they are indispensable to regulate the growth, tolerate the osmotic pressure and oxidative stress in Metarhizium rileyi . Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria was impervious since there are no striking differences in virulence compared with WT and all mutants by topical injections. In general, our results revealed that NADPH oxidase may be involved in growth regulation, MS formation and pathogenicity by regulating the ROS metabolism and hyphal polarity.

Anthocyanins, an important class of pigmented and health-promoting plant secondary metabolites, are involved in protecting plants from stresses. However, the contribution of anthocyanins in plant resistance to biotic stresses is poorly understood compared with that in plant resistance to abiotic stresses. Here, we characterized the function of anthocyanins in protecting fruit from green mold, the major postharvest disease of citrus fruit. Compared with other oranges, ‘Tarocco’ orange, one of the most important blood oranges enriched in anthocyanins, showed reduced susceptibility to the necrotrophic fungus Penicillium digitatum (Pd) which causes citrus postharvest green mold. Pd infection induced the accumulation of anthocyanins in ‘Tarocco’ orange and exogenous treatment of anthocyanins significantly reduced the susceptibility of citrus fruit to Pd. The accumulation of reactive oxygen species (ROS), which was shown to contribute to plant infection of necrotrophic fungal pathogens, in ‘Tarocco’ orange was less than that in other oranges upon Pd infection. ROS content and associated gene expression in citrus fruit upon Pd infection were reduced after exogenous treatment of anthocyanins. Moreover, transcriptome analysis showed that a lot of genes probably participated in plant–pathogen interaction and anthocyanin biosynthesis were noticeably up-regulated in ‘Tarocco’ orange upon Pd infection. These findings highlight the contribution of anthocyanins to fruit disease resistance and provide significant insights into the control of citrus postharvest green mold.

Metarhizium rileyi, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field pests, especially noctuid pests. To explore the potential factors involved in the fungal pathogenicity, MrSte12, an important and conserved functional transcription factor in mitogen-activated protein kinase pathway was carried out by functional analysis. Homologous recombination was used to disrupt the MrSte12 gene in M. rileyi. The deletant fungal strain exhibited malformed hyphae and impaired conidiogenesis, and conidia could not be collected from △MrSte12 in vitro towards SMAY medium. Although conidia could be collected again supplemented with KCl within SMAY medium, the conidial germination, growth and stress tolerance were much weaker compared with that in WT. Additionally, △MrSte12 showed a dramatic reduction in virulence in intra-hemolymph injections and no pathogenicity in topical inoculations against noctuid pests, which is due to the failure of appressorium formation. Moreover, the content of chitin and β-1, 3-glucan in cell wall significantly reduced in mutant conidia. These results indicate that the MrSte12 gene markedly contributes to invasive growth and conidiation , as well as the major pathogenicity in M. rileyi.