Clonostachys rosea is a promising biocontrol fungus active against various plant fungal pathogens. In this study, the endochitinase-encoding gene Chi67-1, the expression of which is sharply upregulated in C. rosea 67-1 when induced by sclerotia, was transformed into the original isolate by protoplast transformation, and transformants were screened against Sclerotinia rot of soybean. The transformation efficiency was approximately 50 transformants per 1 × 107 protoplasts, and 68 stably heritable recombinants were assayed. The parasitic rates of 32.4% of the tested strains increased by more than 50% compared to 43.3% of the wild type strain in 16 h, and the Rc4-4 transformant showed a parasitic rate of 100% in 16 h. The control efficiencies of the selected efficient transformants to soybean Sclerotinia stem rot were evaluated in pots in the greenhouse, and the results revealed that Rc4-4 achieved the highest efficiency of 81.4%, which was 31.7% and 28.7% higher than the control achieved by the wide type and the pesticide carbendazim, respectively. Furthermore, the expression level of Chi67-1 was 107-fold higher in Rc4-4 than in the wild type, and accordingly, the chitinase activity of the recombinant increased by 140%. The results lay a foundation for the development of efficient genetically engineered strains of C. rosea.

Clonostachys rosea is a mycoparasite that has shown great potential in controlling various plant fungal pathogens. In order to find mycoparasitism-related genes in C. rosea, the transcriptome of the efficient isolate 67-1 in association with sclerotia of Sclerotinia sclerotiorum was sequenced and analysed. The results identified 26,351 unigenes with a mean length of 1,102 nucleotides, among which 18,525 were annotated in one or more databases of NR, KEGG, Swiss-Prot, GO and COG. Differentially expressed genes at 8 h, 24 h and 48 h after sclerotial induction were analysed and 6,890 unigenes were upregulated compared with the control without sclerotia. 713, 1,008 and 1,929 genes were specifically upregulated expressed, while 1,646, 283 and 529 genes were specifically downregulated, respectively. Gene ontology terms analysis indicated that these genes were mainly involved in metabolism of biological process, catalysis of molecular function and cellular component. The expression levels of 12 genes that were upregulated after encountering with S. sclerotiorum were monitored using real-time PCR. The results indicated that the quantitative detection was consistent with the transcriptome analysis. The study provides transcriptional gene expression information on C. rosea parasitizing S. sclerotiorum and forms the basis for further investigation of mycoparasitism-related genes of C. rosea.

Clonostachys rosea is an important mycoparasite, with great potential for controlling numerous plant fungal diseases. Understanding the mechanisms and modes of action will assist the development and application of this biocontrol fungus. In this study, the highly efficient C. rosea 67-1 strain was marked with the green fluorescent protein (GFP), and the transformant possessed the same biological characteristics as the wild-type strain. Fungal interactions with Botrytis cinerea during co-culture and encounter on tomato leaves were assessed by fluorescence confocal and electron microscopy. The results indicated that once the two fungi met, the hyphae of C. rosea grew alongside those of B. cinerea, then attached tightly to the host and developed special structures, via which the biocontrol fungus penetrated the host and absorbed nutrients, eventually disintegrating the cells of the pathogen. Mycoparasitism to B. cinerea was also observed on tomato leaves, suggesting that C. rosea can colonize on plants and act following the invasion of the pathogenic fungus.

Bacillus velezensis B006 is a biocontrol agent which functions through effective colonization and surfactin production. To reveal the surfactin-producing mechanism, gas chromatography–mass spectrometry based untargeted metabolomics was performed to compare the metabolite profiles of strain B006 grown in industrial media M3 and M4. Based on the statistical and pathway topology analyses, a total of 31 metabolites with a fold change of less than − 1.0 were screened as the significantly altered metabolites, which distributed in 15 metabolic pathways. Fourteen amino acids involving in the metabolisms of alanine/aspartate/glutamate, glycine/serine/threonine, arginine/proline, glutathione/cysteine/methionine and valine/leucine/isoleucine as well as succinic acid in TCA cycle were identified to be the hub metabolites. Aminoacyl-tRNA biosynthesis, glycerolipid metabolism, and pantothenate/CoA biosynthesis also contributed to surfactin production. To the best of our knowledge, this study is the first to investigate the metabolic pathways of B. velezensis on surfactin production, and will benefit the optimization of commercial fermentation for higher surfactin yield.

Fusarium oxysporum f. sp. cucumerinum (Foc) is the causal pathogen of cucumber Fusarium wilt resulting in losses to cucumber production. To investigate the effects of the selective pressures of host plants on the virulence of Foc, a low virulence isolate, foc‐3b, was successively inoculated on resistant and susceptible cucumber cultivars for five generations. The virulence of the original isolate diverged; virulence was significantly strengthened after serial passage on the resistant cultivar and weakened on the susceptible plants ( p ˂ .05). The expression of four virulence‐related genes of F. oxysporum , G‐protein α subunit gene fga1 , sucrose nonfermenting 1 gene snf1 , F‐box protein gene frp1 , and Class V chitin synthase gene chsV , was quantified using real‐time PCR . All genes were significantly upregulated after serial passage on the resistant cultivar, compared to the original strain, and the expression of snf1 was downregulated in strains re‐isolated from the susceptible plants ( p ˂ .05). A significant positive correlation was found between the expression levels of gene snf1 , frp1 , and chsV and disease severity of cucumber Fusarium wilt, suggesting these genes may impact virulence differentiation. This study will improve the management of cucumber Fusarium wilt and provide insight into the mechanisms underlying virulence of F. oxysporum .