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Biocontrol Mechanism of Bacillus subtilis C3 Against Bulb Rot Disease in Fritillaria taipaiensis P.Y.Li

ABSTRACT: Bulb rot disease has become one of the main diseases that seriously affects the yield and quality of Fritillaria taipaiensis P.Y.Li (F. taipaiensis). In this study, F. taipaiensis was used as the research object to explore the effect and mechanism of Bacillus subtilis C3 in preventing and curing bulb rot. Through isolation and verification of the pathogenic fungi, we determined for the first time that the pathogenic fungus that causes bulb rot in F. taipaiensis is Fusarium oxysporum. The results of the study showed that B. subtilis C3 inhibits the growth of pathogenic fungi, and the inhibition rate is as high as 60%. In the inhibition mechanism, strain C3 inhibits the conidiogenesis of pathogenic fungi and destroys the cell structure of its hyphae, causing protoplast exudation, chromatin concentration, DNA fragmentation, and ultimately cell death. Among the secondary metabolites of C3, antimicrobial proteins and main active components (paeonol, ethyl palmitate, and oxalic acid) inhibited the growth of F. oxysporum. The molecular weight of the antibacterial protein with the highest inhibition rate was approximately 50 kD. The results of a field experiment on the Taibai Mountain F. taipaiensis planting base showed that after the application of strain C3, the incidence of bulb rot in Fritillaria was reduced by 18.44%, and the ratio of bacteria to fungi in the soil increased to 8.21, which verified the control effect of C3 on Fritillaria bulb rot disease. This study provides a theoretical basis for the use of B. subtilis C3 to prevent and control bulb rot in Fritillaria.

SUBMITTER: Ku Y

PROVIDER: S-EPMC8515143 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The use of chemical fungicides against fungal pathogens adversely affects soil and plant health thereby resulting in overall environmental hazards. Therefore, biological source for obtaining antifungal agents is considered as an environment-friendly alternative for controlling fungal pathogens. RESULTS:In this study, seven endophytic bacteria were isolated from sugarcane leaves and screened for its antifungal activity against 10 fungal isolates belonging to the genera Alternaria, Cochliobolus, Curvularia, Fusarium, Neodeightonia, Phomopsis and Saccharicola isolated from diseased leaves of sugarcane. Among the seven bacterial isolates, SCB-1 showed potent antagonistic activity against the tested fungi. Based on the phenotypic data, Fatty Acid Methyl Esters (FAME) and 16S rRNA gene sequence analysis, the isolate SCB-1 was identified as Bacillus subtilis. The bacterial isolate was screened negative for chitinase production; however, chloroform and methanol extracts of the bacterial culture caused significant inhibition in the growth of the fungal isolates on semisolid media. Volatile component assay showed highest inhibitory activity against Saccharicola bicolor (SC1.4). A PCR based study detected the presence of the genes involved in biosynthesis of surfactin, bacillaene, difficidin, macrolactins and fengycin. Mass spectrometric analysis of the bacterial extract detected the presence of antifungal lipopeptide surfactin, but other metabolites were not detected. The biocontrol activity of the bacterial isolate was established when bacterial pretreated mung bean seeds were able to resist Fusarium infection, however, the untreated seeds failed to germinate. CONCLUSION:The antifungal potential of isolate Bacillus subtilis SCB-1 was established against taxonomically diverse fungal pathogens including the genera Saccharicola, Cochliobolus, Alternaria and Fusarium. The potent antifungal compound surfactin as well as volatiles produced by the bacterial isolate could be responsible for its bio-control activity against fungal infections.

Project description:Soft rot causes significant economic losses in the kiwifruit industry. This study isolated strain CTXW 7-6-2 from healthy kiwifruit tissue; this was a gram-positive bacterium that produced the red pigment pulcherrimin. The phylogenetic tree based on 16S ribosomal RNA, gyrA, rpoB, and purH gene sequences identified CTXW 7-6-2 as a strain of Bacillus subtilis. CTXW 7-6-2 inhibited hyphal growth of pathogenic fungi that cause kiwifruit soft rot, namely, Botryosphaeria dothidea, Phomopsis sp., and Alternaria alternata, by 81.76, 69.80, and 32.03%, respectively. CTXW 7-6-2 caused the hyphal surface to become swollen and deformed. Volatile compounds (VOC) produced by the strain inhibited the growth of A. alternata and Phomopsis sp. by 65.74 and 54.78%, respectively. Whole-genome sequencing revealed that CTXW 7-6-2 possessed a single circular chromosome of 4,221,676 bp that contained 4,428 protein-coding genes, with a guanine and cytosine (GC) content of 43.41%. Gene functions were annotated using the National Center for Biotechnology Information (NCBI) non-redundant protein, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of proteins, Gene Ontology, Pathogen-Host Interactions, Carbohydrate-Active enZYmes, and Rapid Annotations using Subsystem Technology databases, revealing non-ribosomal pathways associated with antifungal mechanisms, biofilm formation, chemotactic motility, VOC 3-hydroxy-2-butanone, cell wall-associated enzymes, and synthesis of various secondary metabolites. antiSMASH analysis predicted that CTXW 7-6-2 can produce the active substances bacillaene, bacillibactin, subtilosin A, bacilysin, and luminmide and has four gene clusters of unknown function. Quantitative real-time PCR (qRT-PCR) analysis verified that yvmC and cypX, key genes involved in the production of pulcherrimin, were highly expressed in CTXW 7-6-2. This study elucidates the mechanism by which B. subtilis strain CTXW 7-6-2 inhibits pathogenic fungi that cause kiwifruit soft rot, suggesting the benefit of further studying its antifungal active substances.

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Biocontrol Mechanism of Bacillus subtilis C3 Against Bulb Rot Disease in Fritillaria taipaiensis P.Y.Li

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