Bradysia difformis is one of the most damaging pests in mushroom production in China. In this study, eight Bacillus thuringiensis strains were analyzed for insecticidal activity in B. difformis. The strain JW-1 showed the highest insecticidal activity against B. difformis larvae, but did not inhibit the mycelial growth of Pleurotus ostreatus and P. geesteranus. The 16S rRNA gene (1397 bp) and cyt2 gene (792 bp) were obtained from strain JW-1. The phylogenetic tree based on 16S rRNA gene and Cyt2 toxin showed that strain JW-1 was a member of B. thuringiensis and Cyt2 toxin belonged to Cyt2Ba toxin cluster. The Cyt2Ba toxin from strain JW-1 was overexpressed in E. coli as a fusion protein and the fusion protein (70 kDa) was purified by Ni-IDA affinity chromatography. The purified Cyt2Ba fusion protein was toxic to B. difformis larvae (LC₅₀ was 2.25 ng/mL). The identification of Cyt2Ba from strain JW-1 and confirmation of the insecticidal activity of Cyt2Ba in B. difformis provided a new means of biological control of the important pest in mushroom 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 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.

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.

All living organisms, including animals, plants, fungi, and bacteria, use the olfactory system to recognize chemicals or pheromone from their environment. Insects detect a volatile substance using odorant receptors (ORs) or gustatory receptors (GRs) and ionotropic receptors (IRs). The gene families of the olfactory system in Acari are still not clear. In this study, we identified seven ORs, one GR, and five IRs from the transcriptome of the storage mite, Tyrophagus putrescentiae Schrank. No olfactory coreceptor was found in this transcriptome. Phylogenetic analysis of these gene families with other Arthropoda species revealed the conservation of carbon dioxide receptors in all tested flying insects and T. putrescentiae. Most of these ORs and GRs were unique to three mosquitoes (Anopheles gambiae Giles, Culex quinquefasciatus Say, and Aedes aegypti L.), Ixodes scapularis Say and Pediculus humanus L., indicating their involvement in specific aspects of both gustatory and olfactory perception. Some clades contained receptors obtained from all tested insect vector species, indicating a degree of conservation among some vector-dependent OR lineages. IRs family was a highly dynamic and independent original of the chemoreceptor genes subfamily. Our findings would make it possible for future research on the chemosensory recognition mechanism in Acari.