Metarhizium anisopliae, an entomopathogenic fungus, has been widely used for the control of agricultural and forestry pests. However, sporulation degeneration occurs frequently during the process of successive culture, and we currently lack a clear understanding of the underlying mechanisms. In this study, the metabolic profiles of M. anisopliae were comparatively analyzed based on the metabolomics approach of gas chromatography–mass spectrometry (GC–MS). A total of 74 metabolites were detected in both normal and degenerate strains, with 40 differential metabolites contributing significantly to the model. Principal component analysis (PCA) and potential structure discriminant analysis (PLS-DA) showed a clear distinction between the sporulation of normal strains and degenerate strains. Specifically, 23 metabolites were down-regulated and 17 metabolites were up-regulated in degenerate strains compared to normal strains. The KEGG enrichment analysis identified 47 significant pathways. Among them, the alanine, aspartate and glutamate metabolic pathways and the glycine, serine and threonine metabolism had the most significant effects on sporulation, which revealed that significant changes occur in the metabolic phenotypes of strains during sporulation and degeneration processes. Furthermore, our subsequent experiments have substantiated that the addition of amino acids could improve M. anisopliae’s spore production. Our study shows that metabolites, especially amino acids, which are significantly up-regulated or down-regulated during the sporulation and degeneration of M. anisopliae, may be involved in the sporulation process of M. anisopliae, and amino acid metabolism (especially glutamate, aspartate, serine, glycine, arginine and leucine) may be an important part of the sporulation mechanism of M. anisopliae. This study provides a foundation and technical support for rejuvenation and production improvement strategies for M. anisopliae.

The Pseudomonas strain T1-3-2^T isolated from the cone of Cunninghamia laceolata exhibited growth-promoting and antifungal activity. Strain T1-3-2^T was characterized by a polyphasic taxonomy and complete genome sequencing analysis to explore its taxonomic position and biocontrol potentials fully. The results revealed that strain T1-3-2^T shares a high degree of similarity with Pseudomonas eucalypticola and is distinct from any known Pseudomonas species. The G + C content was 61.65%, and the difference was greater than 1 compared to “P. eucalypticola”. Additionally, values of the average nucleotide identity blast (ANIb), average nucleotide identity MUMmer (ANIm), and DNA-DNA hybridization (DDH) between T1-3-2^T and its closest known related species, “P. eucalypticola”, were below the thresholds necessary for species delineation. Furthermore, the T1-3-2^T strain exhibited the distinctions with the multiple polar flagella and the specific quinone system with MK8 compared with that of “P. eucalypticola”. Collectively, these findings affirm the designation of strain T1-3-2^T as a new Pseudomonas species, proposed to be named Pseudomonas silvicola, with T1-3-2^T as the type strain. Genomic analyses revealed strain T1-3-2^T contains three circular DNA contigs, including a 7,613,303 bp chromosome and two plasmids (952,764 bp and 84,880 bp). Bioinformatics analyses further offered potential insight into the molecular mechanisms whereby this strain can promote plant growth and control disease, revealing encoded genes related to antibiotic and secondary metabolite production, the uptake and biosynthesis of siderophores, and pyoverdine biosynthesis. These genomic data offer a valuable foundation for future efforts to apply the T1-3-2^T strain in research contexts.