Lian-Ming Liang

Vesicular trafficking is fundamental in fungal cells; however, its regulatory contribution to pathogenicity remains poorly understood. Through large-scale screening in the nematophagous fungus Purpureocillium lavendulum, we identified PlGyp1, a Rab GTPase-activating protein, as a critical negative regulator of virulence. The ΔPlGyp1 mutant exhibited significantly enhanced nematicidal activity against Caenorhabditis elegans and Meloidogyne spp. Multiomics analyses revealed extensive transcriptional reprogramming, including the strong induction of nematicidal enzymes and secondary metabolite biosynthetic genes, accompanied by increased enzyme activities. Metabolomic profiling further identified robust overproduction of DOPA-aranotin, an epidithiodioxopiperazine derivative with potent nematicidal activity, driven by an upregulated aranotin-like biosynthetic gene cluster. Tomato plants treated with this mutant showed complete suppression of root gall formation caused by Meloidogyne incognita in pot experiments. These findings position PlGyp1 as a central hub linking vesicular trafficking, enzyme secretion, and secondary metabolism, providing a potential molecular target for genetically enhancing nematophagous fungi for sustainable biocontrol applications.

Nematode-trapping fungi, renowned for their specialized predatory structures that ensnare nematodes, offer a promising biological approach to managing plant-parasitic nematodes. However, the efficacy of these fungi is frequently hampered by biotic and abiotic factors within the soil, which can significantly impede fungal germination (fungistasis). To counteract these environmental challenges, certain nematode-trapping fungi have evolved to produce traps from their conidia, referred to as conidial traps. This adaptation allows them to bypass the inhibitory effects of their surroundings, enhancing their predatory capabilities. In this study, we explored how soil affects conidial trap formation in Drechslerella dactyloides . Our findings revealed that Acinetobacter spp. and Pantoea spp. present in soil extracts play pivotal roles in triggering the development of these traps. Using metagenomic sequencing, we mapped the shifts in bacterial communities and their relative abundances before and after incubation for up to 24 hours to optimize soil induction effects. This analysis highlighted the enrichment of specific functional genes in soil microbes and provided insights into the mechanisms driving conidial trap formation, based on changes in soil characteristics. Furthermore, through bacterial isolation procedures, we successfully cultured and characterized the bacteria responsible for this phenomenon, confirming their potent ability to stimulate the production of conidial traps in nematode-trapping fungi. This study not only underscores the critical role of bacterial diversity in modulating the life cycle transitions of nematode-trapping fungi but also sets the stage for the development of more effective and sustainable strategies to harness these fungi in the battle against pathogenic nematodes. IMPORTANCE: Predatory nematode-trapping fungi are important microbial antagonists of nematodes and can be developed into biocontrol agents. However, microbial biocontrol agents often suffer from inconsistent efficacy, primarily due to biotic and abiotic stresses in the rhizosphere soil. Drechslerella dactyloides , a nematode-trapping fungus, produces conidial traps in soil, serving as a survival strategy to overcome these stresses. In this study, we optimized soil suspensions to efficiently induce the formation of conidial traps. We found that bacteria in the soil directly trigger this formation. Metagenomic sequencing revealed bacterial enrichment during optimization, and we isolated and purified these bacteria with inducible activity. Our research deepens the understanding of this survival strategy of nematode-trapping fungi in nature, laying the foundation for enhancing the effectiveness of nematode biocontrol using this mechanism.

The carnivorous fungus Drechslerella dactyloides can form constricting rings through hyphal specialization to capture nematodes. The formation of constricting rings is a prerequisite for capturing nematodes and a characteristic of entering the carnivorous stage. Currently, there is limited research on the molecular mechanism of constricting ring formation. In this study, two D. dactyloides mutants unable to form constricting rings were obtained through UV irradiation mutagenesis, and their growth and development phenotypes were compared with the wild-type strain. Transcriptome comparisons revealed differences between the mutants and the wild-type strain in metabolic pathways related to cell wall structure, peroxisomes, lipid metabolism, and MAPK signal transduction, which we validated through qPCR. We further deleted one differentially expressed gene, DdSTE2, of the MAPK pathway and confirmed its role in conidiogenesis and trap formation in D. dactyloides. Together, our results indicate that the remodeling of cell wall structure, peroxisomes, lipid metabolism, and MAPK signal transduction pathways are involved in the formation and maturation of D. dactyloides constricting rings. We discuss the implications of these results for utilizing these fungi to control animal and plant parasitic nematodes.

Purpureocillium lavendulum is an important biocontrol agent against plant-parasitic nematodes, primarily infecting them with conidia. However, research on the regulatory genes and pathways involved in its conidiation is still limited. In this study, we employed Agrobacterium tumefaciens-mediated genetic transformation to generate 4,870 random T-DNA insertion mutants of P. lavendulum. Among these mutants, 131 strains exhibited abnormal conidiation, and further in-depth investigations were conducted on two strains (designated as #5-197 and #5-119) that showed significantly reduced conidiation. Through whole-genome re-sequencing and genome walking, we identified the T-DNA insertion sites in these strains and determined the corresponding genes affected by the insertions, namely Plhffp and Plpif1. Both genes were knocked out through homologous recombination, and phenotypic analysis revealed a significant difference in conidiation between the knockout strains and the wild-type strain (ku80). Upon complementation of the ΔPlpif1 strain with the corresponding wildtype allele, conidiation was restored to a level comparable to ku80, providing further evidence of the involvement of this gene in conidiation regulation in P. lavendulum. The knockout of Plhffp or Plpif1 reduced the antioxidant capacity of P. lavendulum, and the absence of Plhffp also resulted in decreased resistance to SDS, suggesting that this gene may be involved in the integrity of the cell wall. RT-qPCR showed that knockout of Plhffp or Plpif1 altered expression levels of several known genes associated with conidiation. Additionally, the analysis of nematode infection assays with Caenorhabditis elegans indicated that the knockout of Plhffp and Plpif1 indirectly reduced the pathogenicity of P. lavendulum towards the nematodes. The results demonstrate that Agrobacterium tumefaciens - mediated T-DNA insertion mutagenesis, gene knockout, and complementation can be highly effective for identifying functionally important genes in P. lavendulum.

Accurately recognizing pathogens by the host is vital for initiating appropriate immune response against infecting microorganisms. Caenorhabditis elegans has no known receptor to recognize pathogen-associated molecular pattern. However, recent studies showed that nematodes have a strong specificity for transcriptomes infected by different pathogens, indicating that they can identify different pathogenic microorganisms. However, the mechanism(s) for such specificity remains largely unknown. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum can infect the intestinal tract of the nematode C. elegans and the infection led to the accumulation of reactive oxygen species (ROS) in the infected intestinal tract, which suppressed fungal growth. Co-transcriptional analysis revealed that fungal genes related to anaerobic respiration and ethanol production were up-regulated during infection. Meanwhile, the ethanol dehydrogenase Sodh-1 in C. elegans was also up-regulated. Together, these results suggested that the infecting fungi encounter hypoxia stress in the nematode gut and that ethanol may play a role in the host–pathogen interaction. Ethanol production in vitro during fungal cultivation in hypoxia conditions was confirmed by gas chromatography-mass spectrometry. Direct treatment of C. elegans with ethanol elevated the sodh-1 expression and ROS accumulation while repressing a series of immunity genes that were also repressed during fungal infection. Mutation of sodh-1 in C. elegans blocked ROS accumulation and increased the nematode’s susceptibility to fungal infection. Our study revealed a new recognition and antifungal mechanism in C. elegans . The novel mechanism of ethanol-mediated interaction between the fungus and nematode provides new insights into fungal pathogenesis and for developing alternative biocontrol of pathogenic nematodes by nematophagous fungi. IMPORTANCE: Nematodes are among the most abundant animals on our planet. Many of them are parasites in animals and plants and cause human and animal health problems as well as agricultural losses. Studying the interaction of nematodes and their microbial pathogens is of great importance for the biocontrol of animal and plant parasitic nematodes. In this study, we found that the model nematode Caenorhabditis elegans can recognize its fungal pathogen, the nematophagous fungus Purpureocillium lavendulum , through fungal-produced ethanol. Then the nematode elevated the reactive oxygen species production in the gut to inhibit fungal growth in an ethanol dehydrogenase-dependent manner. With this mechanism, novel biocontrol strategies may be developed targeting the ethanol receptor or metabolic pathway of nematodes. Meanwhile, as a volatile organic compound, ethanol should be taken seriously as a vector molecule in the microbial–host interaction in nature.

The complete mitochondrial genome of Drechslerella dactyloides was characterized in this study. This mitogenome is a closed circular molecule of 246860 bp in length with a GC content of 26.16%, including 87 predicted protein-coding genes, 29 transfer RNA genes, and two rRNA gens. Phylogenetic analyses based on concatenated amino acid sequences at 14 conserved mitochondrial protein-coding genes showed that D. dactyloides was closely related to Dactylellina haptotyla.

Plant-parasitic nematodes cause severe economic losses to agriculture. As important biocontrol agents, nematophagous fungi evolved the ability to obtain nitrogen sources from nematodes. However, the impact of nitrogen sources on the growth and development of these fungi is largely unknown. In this study, we aimed to better understand how nitrogen sources could influence vegetative growth and conidiation through epigenetic regulation in the nematophagous fungus, Purpureocillium lavendulum. Through nutrition screening, we found a phenomenon of the fungus, limited colony extension with a large amount of conidia production when cultured on PDA media, can be altered by adding ammonia nitrate. Characterized by site-directed mutagenesis, the histone H3K14 acetylation was found to be involved in the alternation. Furthermore, the acetyltransferase PlGCN5 was responsible for H3K14 acetylation. Knockout of Plgcn5 severely diminished conidiation in P. lavendulum. Chip-seq showed that H3K14ac distributed in conidiation regulating genes, and genes in the MAPK pathway which may be the downstream targets in the regulation. These findings suggest that histone modification and nitrogen sources coordinated lifestyle regulation in P. lavendulum, providing new insight into the mechanism of growth regulation by nutritional signals for the carnivorous fungus.

A novel actinobacterium, designated KC 17012T, was isolated from lead zinc tailings collected from Lanping, Yunnan, PR China. Comparative 16S rRNA gene sequencing showed that KC 17012T belonged to the genus Streptomyces and was most closely related to the type strains of Streptomyces neyagawaensis (98.34%), Streptomyces panaciradicis (98.34%) and Streptomyces heilongjiangensis (98.27%). Phylogenetic tree analysis revealed strain KC 17012T formed a distinct clade. The genome size was 8.64 Mbp with a DNA G+C content of 70.8%. Digital DNA–DNA hybridization and average nucleotide identity values between the genome sequence of strain KC 17012T and those of S. neyagawaensis JCM 4796T (25.3 and 81.5 %) and S. panaciradicis NBRC 109811T (30.1 and 85.7 %) were below the thresholds of 70 and 96% for prokaryotic conspecific assignation. The strain formed long straight aerial hyphae which generated regular short rod spores with spiny surfaces. Growth occurred at 10–45 °C, pH 6–8 and with 0–9 % NaCl (w/v). Strain KC 17012T contained ll-diaminopimelic acid and the major whole-cell hydrolysates included glucose, mannose and ribose. The menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The major cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified lipid and one unidentified phospholipid. On the basis of the results of a polyphasic taxonomic study, it is concluded that KC 17012T represents a novel species of the genus Streptomyces , for which the name Streptomyces plumbidurans sp. nov., is proposed. The type strain is KC 17012T (CGMCC 4.7704T=JCM 35204T).