Isolation, Identification, and Antibacterial Mechanisms of Bacillus amyloliquefaciens QSB-6 and Its Effect on Plant Roots

Abstract

Apple replant disease (ARD) is a common problem in major apple planting areas, and biological factors play a leading role in its etiology. Here, we isolated the bacterial strain QSB-6 from the rhizosphere soil of healthy apple trees in a replanted orchard using the serial dilution method. Strain QSB-6 was provisionally identified as Bacillus amyloliquefaciens based on its morphology, physiological and biochemical characteristics, carbon source utilization, and chemical sensitivity. Maximum likelihood analysis based on four gene sequences [16S ribosomal RNA gene (16S rDNA), DNA gyrase subunit A (gyrA), DNA gyrase subunit B (gyrB), and RNA polymerase subunit B (rpoB)] from QSB-6 and other strains indicated that it had 100% homology with B. amyloliquefaciens, thereby confirming its identification. Flat standoff tests showed that strain QSB-6 had a strong inhibitory effect on Fusarium proliferatum, Fusarium solani, Fusarium verticillioides, Fusarium oxysporum, Alternaria alternata, Aspergillus flavus, Phoma sp., Valsa mali, Rhizoctonia solani, Penicillium brasilianum, and Albifimbria verrucaria, and it had broad-spectrum antibacterial characteristics. Extracellular metabolites from strain QSB-6 showed a strong inhibitory effect on Fusarium hyphal growth and spore germination, causing irregular swelling, atrophy, rupture, and cytoplasmic leakage of fungal hyphae. Analysis of its metabolites showed that 1,2-benzenedicarboxylic acid and benzeneacetic acid, 3- hydroxy-, methyl ester had good inhibitory effects on Fusarium, and increased the length of primary roots and the number of lateral roots of Arabidopsis thaliana plantlet. Pot experiments demonstrated that a QSB-6 bacterial fertilizer treatment (T2) significantly improved the growth of Malus hupehensis Rehd. seedlings. It increased root length, surface area, tips, and forks, respiration rate, protective enzyme activities, and the number of soil bacteria while reducing the number of soil fungi. Fermentation broth from strain QSB-6 effectively prevented root damage from Fusarium. terminal restriction fragment length polymorphism (T-RFLP) and quantitative PCR (qPCR) assays showed that the T2 treatment significantly reduced the abundance of Fusarium in the soil and altered the soil fungal community structure. In summary, B. amyloliquefaciens QSB-6 has a good inhibitory effect on Fusarium in the soil and can significantly promote plant root growth. It has great potential as a biological control agent against ARD.

Biocontrol Potential of The Phloridin-Degrading Bacillus Licheniformis XNRB-3 Against Apple Replant Disease

“…Bacterial suspensions were made by suspending cells cultured on NA plates in 10 mM MgSO 4 . The density of the bacterial suspensions was adjusted to 1×10 8 CFU ml −1 , The spores of Fusarium obtained refers to the method of Duan et al (2021). Each suspension (about 50 µL) were then placed on a microscope slide, and kept at 20 ℃ on moistened, sterilized lter paper placed in Petri dishes for 24 h. The germination percentages of conidia (200 conidia for each treatment) were measured using the Nikon BX-51 uorescence microscope.…”

“…The selected isolates were identi ed through 16S ribosomal RNA gene (16S rDNA), DNA gyrase subunit A (gyrA), DNA gyrase subunit B (gyrB), and RNA polymerase subunit B (rpoB) (Yamamoto and Harayama, 1995;Chun and Bae, 2000;Zalila-Kolsi et al, 2016;Somerville et al, 2020). PCR ampli cation was performed in an Applied Biosystems 2720 Thermal Cycler (Applied Biosystems Inc., USA) refer to the method of Duan et al (2021). The primers and annealing temperatures are presented in Table S2.…”

“…The supernatant (extracellular medium) was passed through a 0.22-µm Nylon66 microporous membrane to obtain CFCF. Effect of cell-free culture ltrate on Fusarium hyphae refers to the method of Duan et al (2021). The samples were sent to Keshang Biotech Co., Ltd. for scanning electron microscope observation.…”

“…The peak area normalization method was used to calculate the relative content of each component. The chromatographic and mass spectrometry conditions according to the method of Duan et al (2021).…”

“…The differences between samples were calculated by Bray-curtis, and analysis of similarity (ANOSIM) was conducted to identify the signi cant differences between the fungal communities(Liu et al, 2019a;Xu et al, 2020). Richness index (SR) and evenness index (E) were calculated by Bio-dap software were calculated by Bio-dap software(Duan et al, 2021).…”