Verticillium wilt is a severe disease caused by Verticillium dahliae, which afflicts many crops, particularly cotton in the Xinjiang province of China. Chemical fungicides are harmful to the environment, and biological control agents against the fungal pathogens of plants provide an alternative to chemicals. Biological control agents include antagonistic bacteria or metabolic products. We investigated the inhibitory effects of two plant-growth-promoting rhizobacterial (PGPR) strains, namely Bacillus tequilensis C-9 and Sphingobacterium A1, against V. dahliae in vitro and in the field. We used the mixed fermentation of two bacteria to inhibit V. dahliae. Antimicrobial assays were performed and showed that spore production and germination, and the virulence protein of V. dahliae were reduced after treatment with cell-free mixed fermentation broth. In field studies, there was improvement in the disease severity, along with declined incidence and index after the treatment in the bud and bell stages. To explore the molecular mechanisms of this inhibition, we studied six metabolism-related genes by real-time quantitative polymerase chain reaction (qRT-PCR). The results showed that in response to mixed fermentation broth, some genes related to growth and development were significantly upregulated, explaining the structural changes observed in V. dahliae. Some genes that regulate oxidative stress were also dramatically induced, indicating that mixed bacterial fermentation had a significant impact on V. dahliae. These results support the idea that the cell-free mixed fermentation broth of Bacillus tequilensis C-9 and Sphingobacterium A1, when applied for biocontrols, can inhibit V. dahliae in vitro and in vivo.Abbreviations: ANOVA: analysis of variance; BE: biocontrol efficacy (%); CFU: colony-forming unit; DI: disease incidence rates (DI %); DS: disease status; PDA: potato dextrose agar; PDB: potato dextrose broth (liquid medium); PGPR: plant-growth-promoting rhizobacteria ARTICLE HISTORY

Background: Verticillium dahliae, the causal agent of Verticillium wilt, is notoriously invasive in many crops and has been involved in numerous epidemics worldwide. Bacillus species, as representatives of biocontrol bacteria, produce a variety of lipopeptides (LPs), which are useful as biofungicides to many pathogenic fungi, including Verticillium dahliae. This study will explore the mechanism of resistance of V. dahliae to Bacillus and biocontrol bacteria.Results: By using in vitro confrontation bioassays, we found that under the stress induced by Bacillus, the spore vitality of V. dahliae with larger colonies was higher, and more abundant microsclerotia were formed. Then, according to the RNA-Seq analysis, the target of rapamycin (TOR) and mitophagy pathways were enriched among the significantly upregulated 542 genes observed in two co-culture groups with different colony sizes. In addition, in the group of V. dahliae with large colonies, the pathways related to cell wall synthesis, microsclerotia formation and the clearance of reactive oxygen species were regulated, and the expression of genes was up-regulated.Conclusion: This study found that the larger colonies of V. dahliae were more resistant to the antagonistic actions of Bacillus and the likelihood of the formation of homeostasis. Therefore, the prevention of Verticillium wilt by Bacillus is more effective than the treatment of an active fungal infection. These transcriptomic insights provide direction for the use of fungicides in the prevention and treatment of diseases such as Verticillium wilt.

Verticillium dahliae causes disease symptoms in its host plants; however, due to its rapid variability, V. dahliae is difficult to control. To analyze the reason for this pathogenic differentiation, 22 V. dahliae strains with different virulence were isolated from a cotton farm. The genetic diversity of cotton varieties make cotton cultivars have different Verticillium wilt resistance, so the Xinluzao 7 (susceptible to V. dahliae), Zhongmian 35 (tolerant), and Xinluzao 33 (resistant) were used to investigate the pathogenicity of the strains in a green house. Vegetative compatibility groups (VCGs) assays, Internal Transcribed Spacer (ITS) PCR, and pathogenicity analysis showed that SHZ-4, SHZ-5, and SHZ-9 had close kinship and significantly different pathogenicity. Transcriptome sequencing of the three strains identified 19 of 146 unigenes in SHZ-4_vs_ SHZ-5, SHZ-5_vs_ SHZ-9, and SHZ-4_vs_ SHZ-9. In these unigenes, three proteinase and four polysaccharide degrading hydrolases were found to be associated with the pathogenicity. However, due to a number of differentially expressed genes in the transport, these unigenes not only played a role in nutrition absorption but might also contribute to the resistance of sugar-induced hyperosmosis. Moreover, the tolerance ability was positively related to the pathogenicity of V. dahliae. This resistance to sugar-induced hyperosmosis might help V. dahliae to access the nutrition of the host. The pathogenicity of V. dahliae correlated with the resistance of sugar-induced-hyperosmosis, which provides clues for the cultivation of V. dahliae resistant varieties.