Streptomyces alfalfae XN-04 has been reported for the production of antifungal metabolites effectively to control Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum (Fov). In this study, we used integrated statistical experimental design methods to investigate the optimized liquid fermentation medium components of XN-04, which can significantly increase the antifungal activity and biomass of XN-04. Seven variables, including soluble starch, KNO₃, soybean cake powder, K₂HPO₄, MgSO₄·7H₂O, CaCO₃ and FeSO₄·7H₂O, were identified as the best ingredients based on one-factor-at-a-time (OFAT) method. The results of Plackett–Burman Design (PBD) showed that soluble starch, soybean cake powder and K₂HPO₄ were the most significant variables among the seven variables. The steepest climbing experiment and response surface methodology (RSM) were performed to determine the interactions among these three variables and fine-tune the concentrations. The optimal compositions of medium were as follows: soluble starch (26.26 g/L), KNO₃ (1.00 g/L), soybean cake powder (23.54 g/L), K₂HPO₄ (0.27 g/L), MgSO₄·7H₂O (0.50 g/L), CaCO₃ (1.00 g/L) and FeSO₄·7H₂O (0.10 g/L). A verification experiment was then carried out under the optimized conditions, and the results revealed the mycelial dry weight of S. alfalfae XN-04 reaching 6.61 g/L. Compared with the initial medium, a 7.47-fold increase in the biomass was achieved using the optimized medium. Moreover, the active ingredient was purified from the methanol extract of S. alfalfae XN-04 mycelium and then identified as roflamycoin (a polyene macrolide antibiotic). The results may provide new insights into the development of S. alfalfae XN-04 fermentation process and the control of the Fusarium wilt of cotton and other plant diseases.

Fusarium wilt of cotton, caused by the pathogenic fungal Fusarium oxysporum f. sp. vasinfectum (Fov), is a devastating disease of cotton, dramatically affecting cotton production and quality. With the increase of pathogen resistance, controlling Fusarium wilt disease has become a significant challenge. Biocontrol agents (BCAs) can be used as an additional solution to traditional crop breeding and chemical control. In this study, an actinomycete with high inhibitory activity against Fov was isolated from rhizosphere soil and identified as Streptomyces alfalfae based on phylogenetic analyses. Next, an integrative approach combining genome mining and metabolites detection was applied to decipher the significant biocontrol and plant growth-promoting properties of XN-04. Bioinformatic analysis and bioassays revealed that the antagonistic activity of XN-04 against Fov was associated with the production of various extracellular hydrolytic enzymes and diffusible antifungal metabolites. Genome analysis revealed that XN-04 harbors 34 secondary metabolite biosynthesis gene clusters. The ability of XN-04 to promote plant growth was correlated with an extensive set of genes involved in indoleacetic acid biosynthesis, 1-aminocyclopropane-1-carboxylic acid deaminase activity, phosphate solubilization, and iron metabolism. Colonization experiments indicated that EGFP-labeled XN-04 had accumulated on the maturation zones of cotton roots. These results suggest that S. alfalfae XN-04 could be a multifunctional BCA and biofertilizer used in agriculture.

Summary: Wheat scab, mainly caused by Fusarium graminearum , can decrease wheat yield and grain quality. Chemical pesticides are currently the main control method but have an inevitable negative consequence on the environment and in food safety. This research studies a promising substitute, Streptomyces pratensis S10, which was isolated from tomato leaf mould and shows a significant inhibition effect on F. graminearum based on antagonism assays. The biocontrol mechanism is studied by enhanced green fluorescent protein labelling, quantitative real‐time PCR, the Doskochilova 8 solvents system test and complete genome sequencing. Strain S10 can colonize in the wheat root, control wheat scab and decrease deoxynivalenol (DON) content. The control effects in vitro, planta and the plot experiments were 92.86%, 68.67% and 40.87% to 86.62%, respectively. S10 decreased DON content by inhibiting the mycelium growth and DON synthesis gene expression. The active substances of the S10 secondary metabolites had a high‐temperature resistance and 29 putative biosynthetic gene clusters in its genome. The S10 control mechanism is multivariate, which shows potential in controlling wheat scab.

In order to build integrated strains with superior growth-promoting and disease-suppression effects, the biological control efficacy of Fo47 solid agents combined with actinomycetes strains toward Fusarium oxysporum and Verticillium dahliae were investigated in experiments on watermelon, cotton and eggplant. Five actinomycetes strains were prepared by solid fermentation. The count of viable solid agents, initially with propagules at 10⁷–10¹¹ CFU/g, slowly decreased after being stored one year at room temperature. After being inoculated into sterile soil for 50 days, the viable count of strain Fo47 remained at a stable level. The suppressive effects of Fo47 combined with strain QLP12 on Fusarium wilt on watermelon and cotton, and Verticillium wilt on eggplant, reaching 58.47%, 50.73% and 58.82%, respectively. This was significantly better than the single strain Fo47 alone, and growth of these treated plants and the colonisation rate of Fo47 were increased substantially as well. These results indicate that solid integrated agents with a high viability count and superior stability in soil could increase disease suppression and promote plant growth by synergy with different strains. The increased suppression obtained by Fo47 combined with actinomycete strains was not due to a simple addition of different mechanisms of biocontrol agents. By being intelligently integrated, these combinations increase disease suppression and provide the best biocontrol effect.

Clubroot is a devastating disease threatening global cruciferous vegetable production caused by Plasmodiophora brassicae (Pb). We have evaluated the positive effects of the Alcaligenes faecalis Juj3 on cabbage growth promotion and Pb stress alleviation through pot and field experiments. The Juj3 strain was isolated from a healthy cabbage rhizosphere with growth-promoting characteristics and was identified as A. faecalis based on morphological traits and phylogeny. Seed germination assays revealed that Juj3 inoculation enhances cabbage bud shoot and root growth. In pot experiments, inoculation with Juj3 fermentation powder at cabbage sowing dates significantly improved the seedling biomass. Combining seed treatments with root irrigation after transplanting considerably reduced the clubroot disease index and resulted in appreciable biocontrol efficacy (83.7%). Gene expression analyses of cabbage after Juj3 inoculation showed that PR2 and EIN3 expression were significantly up-regulated. Physiologically, Juj3 inoculation enhanced cabbage chlorophyll content and root activity in a normal environment. Irrespective of whether plants were under normal environment or Pb stresses, Juj3 improved photosynthesis. Field trial analyses revealed that Juj3 exhibits satisfactory biocontrol efficacy in cabbage (51.4%) and Chinese cabbage (37.7%). Moreover, Juj3 could also enhance cabbage and Chinese cabbage biomass to improve the yield quality. These findings pave the way for future use of A. faecalis as biocontrol agents for clubroot and reveal the great potential of the rhizobacterium for plant growth-promoting applications in agriculture and horticulture.