Mutation of Bacillus velezensis Using Corona Discharge

Bacillus velezensis is a kind of beneficial bacteria that is widely used in agriculture industry. Bacillus velezensis was irradiated with corona discharge generated by a needle-array high-voltage electrode. The results showed an improvement of activity of Bacillus velezensis by the corona discharge treatment was confirmed at an optimum input energy. Mutation of the Bacillus velezensis by the corona discharge treatment was also confirmed through an rRNA sequence alignment analysis. The enzyme activity of the mutated bacteria was greatly improved, which was a positive effect that can meet the production demand.

Effect of endophytic Bacillus and arbuscular mycorrhiza fungi (AMF) against Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici

Background Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (FOL) is a serious disease that causes significant economic losses in tomato production. Seventeen endophytic Bacillus isolates from tomato roots of Meghalaya were tested for antagonistic and plant growth promotion activities. Dominating arbuscular mycorrhiza fungi (AMF) spores were isolated from the rhizosphere soils of tomato grown in Meghalaya. The effect of different combinations of AMF and endophytic Bacillus on Fusarium wilt severity and growth of tomato plant under pot and field conditions was studied. Results The endophytic Bacillus isolates ERBS51 and ERBS10 showed a maximum inhibition against FOL, with 58.43 and 55.68%, respectively, in a dual culture experiment. ERBS51 and ERBS10 were identified as Bacillus velezensis and Bacillus sp., respectively, based on 16s rRNA sequencing. Both isolates were found positive for iturin A, surfactin, bacillomycin D, protease, cellulase, pectinase, alpha-amylase, siderophore, ammonia production and ZnCO3 solubilization. Funneliformis mosseae and Glomus fasciculatum were the dominating AMF species in tomato rhizosphere of Meghalaya. The result of pot and field experiments revealed that out of all the treatments, combination of Funneliformis mosseae + Glomus fasciculatum + Bacillus velezensis + Bacillus sp. was shown to be the best in reducing the severity of Fusarium wilt to 77.44 and 66.74%, respectively. F. mosseae + G. fasciculatum + B. velezensis + Bacillus sp. also recorded the highest in most growth attributes and yield. Conclusions Endophytic Bacillus (B. velezensis and Bacillus sp.) and AMF (F. mosseae and G. fasciculatum) were safe and effective biocontrol agents against Fusarium wilt of tomato.

Screening of Bacillus velezensis E2 and the Inhibitory Effect of Its Antifungal Substances on Aspergillus flavus

Aspergilus flavus is the main pathogenic fungus that causes food mold. Effective control of A. flavus contamination is essential to ensure food safety. The lipopeptides (LPs) produced by Bacillus strains have been shown to have an obvious antifungal effect on molds. In this study, an antagonist strain of Bacillus velezensis with obvious antifungal activity against A. flavus was isolated from the surface of healthy rice. Using HPLC-MS analysis, the main components of LPs produced by strain E2 were identified as fengycin and iturins. Further investigations showed that LPs could inhibit the spore germination, and even cause abnormal expansion of hyphae and cell rupture. Transcriptomic analyses showed that some genes, involved in ribosome biogenesis in eukaryotes (NOG1, KRE33) and aflatoxin biosynthesis (aflK, aflR, veA, omtA) pathways in A. flavus were significantly down-regulated by LPs. In conclusion, this study provides novel insights into the cellular and molecular antifungal mechanisms of LPs against grain A. flavus contamination.

Isolation and Characterization of Bacillus velezensis Strain P2-1 for Biocontrol of Apple Postharvest Decay Caused by Botryosphaeria dothidea

Botryosphaeria dothidea causes apple ring rot, which is among the most prevalent postharvest diseases of apples and causes significant economic loss during storage. In this study, we investigated the biocontrol activity and possible mechanism of Bacillus velezensis strain P2-1 isolated from apple branches against B. dothidea in postharvest apple fruit. The results showed strain P2-1, one of the 80 different endophytic bacterial strains from apple branches, exhibited strong inhibitory effects against B. dothidea growth and resulted in hyphal deformity. B. velezensis P2-1 treatment significantly reduced the ring rot caused by B. dothidea. Additionally, the supernatant of strain P2-1 exhibited antifungal activity against B. dothidea. Re-isolation assay indicated the capability of strain P2-1 to colonize and survive in apple fruit. PCR and qRT-PCR assays revealed that strain P2-1 harbored the gene clusters required for biosynthesis of antifungal lipopeptides and polyketides. Strain P2-1 treatment significantly enhanced the expression levels of pathogenesis-related genes (MdPR1 and MdPR5) but did not significantly affect apple fruit qualities (measured in fruit firmness, titratable acid, ascorbic acid, and soluble sugar). Thus, our results suggest that B. velezensis strain P2-1 is a biocontrol agent against B. dothidea-induced apple postharvest decay. It acts partially by inhibiting mycelial growth of B. dothidea, secreting antifungal substances, and inducing apple defense responses.

Structure of the plant growth-promoting factor YxaL from the rhizobacterium Bacillus velezensis and its application to protein engineering

The YxaL protein was isolated from the soil bacterium Bacillus velezensis and has been shown to promote the root growth of symbiotic plants. YxaL has further been suggested to act as an exogenous signaling protein to induce the growth and branching of plant roots. Amino acid sequence analysis predicted YxaL to exhibit an eight-bladed β-propeller fold stabilized by six tryptophan-docking motifs and two modified motifs. Protein engineering to improve its structural stability is needed to increase the utility of YxaL as a plant growth-promoting factor. Here, the crystal structure of YxaL from B. velezensis was determined at 1.8 Å resolution to explore its structural features for structure-based protein engineering. The structure showed the typical eight-bladed β-propeller fold with structural variations in the third and fourth blades, which may decrease the stability of the β-propeller fold. Engineered proteins targeting the modified motifs were subsequently created. Crystal structures of the engineered YxaL proteins showed that the typical tryptophan-docking interaction was restored in the third and fourth blades, with increased structural stability, resulting in improved root growth-promoting activity in Arabidopsis seeds. The work is an example of structure-based protein engineering to improve the structural stability of β-propellor fold proteins.