Cyclic dimeric guanosine monophosphate (c-di-GMP) is synthesized by diguanylate cyclase (DGC) with the GGDEF domain. As a ubiquitous bacterial second messenger, it regulates diverse life-activity phenotypes in some bacteria. Although 38 genes encoding GGDEF-domain-containing proteins have been identified in the genome of the Pseudomonas glycinae strain MS82, whether c-di-GMP functions as a facilitator or repressor of life-activity phenotypes is poorly understood. In this study, one of the 38 genes containing a GGDEF domain in MS82, PafS was investigated to explore its regulatory function in bacterial life activities. The PafS-deletion mutant ΔPafS and reversion mutant PafS-comp were constructed by the method of biparental conjugation and homologous recombination. The life activities of the mutants, such as antifungal activity, biofilm formation ability, polysaccharide content, and motor behavior, were explored. The results showed that all life-activity phenotypes were significantly reduced after knocking out PafS, whereas all were significantly restored to a similar level to that of MS82 after the complementation of PafS. These results suggested that PafS plays an important role in the regulation of a range of cellular activities by c-di-GMP in P. glycinae MS82.

Cordycepin is a key active component of Cordyceps militaris, but the molecular mechanism underlying temperature-regulated biosynthesis remains unclear. In this study, Cordyceps militaris strain KN-1 was used as experimental material, with low-temperature (15 °C), control (20 °C), and high-temperature (25 °C) treatments applied during the fruiting body stage. Transcriptomics, untargeted metabolomics, weighted gene co-expression network analysis (WGCNA), and Reverse Transcription quantitative PCR (RT-qPCR) validation were integrated to elucidate the molecular mechanism of temperature-mediated cordycepin biosynthesis. The results showed that 25 °C increased fruiting body cordycepin content by 84%, while 15 °C reduced it. Transcriptomic analysis identified differentially expressed genes (DEGs) enriched in transmembrane transport and fatty acid metabolism, and untargeted metabolomics revealed differential metabolites (DAMs) enriched in lipids and organic acids, indicating that temperature primarily affects Cordyceps militaris membrane function. WGCNA showed that the MEblue module was positively correlated with cordycepin (r = 0.93), with Major Facilitator Superfamily (MFS) members accounting for the highest proportion (47.1%) that may affect cordycepin transmembrane transport. Multi-omics analysis indicated that high temperature promotes cordycepin accumulation through the synergistic regulation of multiple pathways: upregulating genes in the pentose phosphate pathway, purine metabolism, and cordycepin biosynthetic gene cluster (Cns1–Cns3), increasing protective agent pentostatin content, downregulating cordycepin-degrading genes, and enhancing cordycepin transmembrane transport. This study clarifies the molecular mechanism of temperature-mediated cordycepin accumulation, providing a theoretical basis for improving cordycepin production via temperature regulation, optimizing Cordyceps militaris strain quality, and facilitating efficient industrial production.

Volvariella volvacea, the Chinese mushroom, is a high-temperature grass-rot fungus with great production potential, yet its low yield limits industrial development. Exogenous sodium acetate (NaAc) has been shown to increase yield by promoting indole-3-acetic acid (IAA) synthesis during the primordium stage, but the underlying mechanism remains unclear. In this study, the aldehyde dehydrogenase gene VvALDH, highly expressed at the primordium stage, was cloned and functionally characterized. VvALDH encodes a 1509 bp cDNA with a conserved aldehyde dehydrogenase domain. Using Agrobacterium-mediated transformation, overexpression lines showed a 4.76-fold increase in VvALDH expression, accompanied by higher biomass (38%), yield (83%), and IAA content (34%), while RNAi lines showed opposite trends. These results demonstrate that VvALDH promotes IAA biosynthesis, enhances primordium differentiation, and increases yield. Further analysis revealed its involvement in multiple IAA biosynthetic pathways, including indolepyruvate, tryptamine, and tryptophan side-chain oxidase pathways. This work clarifies the molecular basis of NaAc-mediated yield improvement and provides a theoretical foundation for genetic and cultivation strategies in V. volvacea.

Cyclic diguanylate (c-di-GMP) affects bacterial physiological and biochemical functions like biofilm, motility, virulence, and bacterial secretion systems. GGDEF/EAL-domain proteins, participating in c-di-GMP synthesis and degradation, are widely present in Pseudomonas, with various structures and functions. Pseudomonas glycinae MS82 is a rhizosphere bacterium that protects mushroom against the pathogenic fungi. Although 14 genes encoding GGDEF/EAL-domain proteins have been identified in the genome of MS82, c-di-GMP regulation is poorly understood as a facilitator or repressor of physiological phenotypes. Here, PafQ and PafR, encoding the proteins with the tandem GGDEF/EAL domain, were functionally analyzed and found to regulate antifungal activity. Individual deletion mutants of PafQ and PafR were constructed in P. glycinae MS82 through biparental conjugation and homologous recombination. Subsequently, antifungal activity, biofilm formation, motility, and expression of the genes related to antifungal substance synthesis were examined and contrasted with those of wild-type P. glycinae MS82. Most phenotypes of physiological activities were significantly reduced after knocking out PafQ or PafR. In other members of the genus Pseudomonas, homologous genes of PafQ and PafR possess different functions in c-di-GMP regulation. In P. glycinae, the positive regulation of PafQ and PafR on fungistatic substance synthesis, biofilm formation, and motility is crucial in the biocontrol of mushroom diseases.

Pleurotus ostreatus cultivation is often affected by pest infestations, which contaminate the bag by eating nutrients and mycelium. This contamination eventually leads to a decline in the quality and yield of edible mushrooms and affects farmers’ income. Therefore, pesticides are commonly used for pest control. To examine the impact of insecticides on the growth of P. ostreatus, this study quantified the activities of antioxidant enzymes, including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and phenylalanine deaminase (PAL), in the mushroom under different insecticide treatments. Additionally, transcriptome sequencing was performed to investigate the underlying regulatory mechanisms. The findings indicated that dinotefuran, diflubenzuron, chlorantraniliprole, and beta-cypermethrin treatments resulted in a significant reduction in catalase and peroxidase activities in P. ostreatus. Conversely, the application of beta-cypermethrin and chlorantraniliprole significantly enhanced PAL and SOD activities in the mycelium. PAL activity was significantly increased in all the mixed substrates, whereas only spray treatments with diflubenzuron resulted in a significant increase in PAL activity. SOD activity in the substrates was reduced by diflubenzuron in the mixed treatment and chlorantraniliprole in the spray treatment. In contrast, all other treatments resulted in a significant increase in SOD activity in the substrates. Transcriptome sequencing revealed that differential genes were predominantly enriched in valine, leucine, and isoleucine degradation, fatty acid degradation, tyrosine metabolism, ascorbate and aldarate metabolism, and histidine metabolism, among others. These biological processes are hypothesized to be involved in the growth regulatory effects of insecticides on the mycelium and ascospores of P. ostreatus. The reliability of the transcriptomic data was also validated through quantitative real-time polymerase chain reaction.

Bradysia difformis is a notorious pest in mushroom production in China. Biological control using Bacillus thuringiensis (Bt) offers an environmentally friendly and effective strategy against this pest. Here, we show that the complete genome of strain JW-1 consists of one circular chromosome and seven circular plasmids. JW-1-Plasmid 4 comprises 127,921 bp with a GC content of 33.9%, and is predicted to contain 131 genes, including six insecticidal genes: cry4Aa, cry4Ba, cry10Ab, cry11Aa, cyt1Aa, and cyt2Ba. A 3542-bp fragment containing the cry4Aa gene was amplified from this strain. Phylogenetic analysis based on Cry4 toxin sequences showed that JW-1 Cry4 toxin belongs to the Cry4Aa toxin cluster. A Cry4Aa fusion protein was subsequently expressed in E. coli and purified using Ni-IDA affinity chromatography. A larval feeding assay showed that purified Cry4Aa was toxic to B. difformis larvae, with an LC₅₀ of 2.71 ng/mL. These results confirmed the identity and bioactivity of Cry4Aa from strain JW-1, offering a promising biological control agent against this major pest.