Biocontrol of the causal brown patch pathogen Rhizoctonia solani by Bacillus velezensis GH1-13 and development of a bacterial strain specific detection method

Abstract

Brown patch caused by the basidiomycete fungus Rhizoctonia solani is an economically important disease of cool-season turfgrasses. In order to manage the disease, different types of fungicides have been applied, but the negative impact of fungicides on the environment continues to rise. In this study, the beneficial bacteria Bacillus velezensis GH1-13 was characterized as a potential biocontrol agent to manage brown patch disease. The strain GH1-13 strongly inhibited the mycelial growth of turf pathogens including different anastomosis groups of R. solani causing brown patch and large patch. R. solani AG2-2(IIIB) hyphae were morphologically changed, and fungal cell death resulted from exposure to the strain GH1-13. In addition, the compatibility of fungicides with the bacterial strain, and the combined application of fungicide azoxystrobin and the strain in brown patch control on creeping bentgrass indicated that the strain could serve as a biocontrol agent. To develop strain-specific detection method, two unique genes from chromosome and plasmid of GH1-13 were found using pan-genome analysis of 364 Bacillus strains. The unique gene from chromosome was successfully detected using both SYBR Green and TaqMan qPCR methods in bacterial DNA or soil DNA samples. This study suggests that application of GH1-13 offers an environmentally friendly approach via reducing fungicide application rates. Furthermore, the developed pipeline of strain-specific detection method could be a useful tool for detecting and studying the dynamics of specific biocontrol agents.

Establishment and application of quantitative detection of Bacillus velezensis HMB26553, a biocontrol agent against cotton damping‐off caused by Rhizoctonia

“…Furthermore, Lee et al. conducted a pan‐genome analysis to identify the genomic features specific to B. velezensis strain GH1‐13 among 364 Bacillus strains, using orthologous protein coding sequences to identify both core conserved and strain‐specific genes 29 . Markers from various types of genes were known to be highly variable.…”

“…There were significant limitations that while a strain‐specific quantitative detection method based on the housekeeping gene bmmA had been previously established for B. velezensis B‐9987 and be used to design primers and probes, the method was only suitable for the quantitative detection of uncontaminated pure culture fermentation broth, as the gene was also present in certain strains of B. amyloliquefaciens , B. subtilis , and other species 32 . Similarly, pan‐genome analysis for B. velezensis GH1‐13 was conducted on 364 genome sequences of Bacillus strains specifically using orthologous protein coding sequences as strain‐specific genes, 29 but the database was limited and not compared with the entire NCBI database. However, our study had also some limitations as a small fraction of soil microorganisms were sequenced, with most still unknown.…”

“…NCBI stored over 516,545 prokaryotic and 28,211 eukaryotic genomes (https://www.ncbi.nlm.nih.gov/genome/browse#!/overview/, data collection date: March 18, 2023). Unique genes of a single strain could be identified by BLAST+ analysis against the publicly available genomic data, serving as markers for detecting a BCA in a strain‐specific manner 29 . Specific primers were designed to amplify the targeted gene and a TaqMan probe to increase sensitivity based on these unique genes 30 .…”

“…Unique genes of a single strain could be identified by BLAST+ analysis against the publicly available genomic data, serving as markers for detecting a BCA in a strain-specific manner. 29 Specific primers were designed to amplify the targeted gene and a TaqMan probe to increase sensitivity based on these unique genes. 30 Due to the precision and sensitivity of qPCR and TaqMan probe, this was a favorable method for high-throughput detection and quantification of BCA.…”

“…Similarly, Mendis et al used NCBI to perform a BLAST search and to align the whole genomes of alternate strains in B. firmus with the genome of B. firmus I-1582 to find unique regions. 27 Furthermore, Lee et al conducted a pan-genomeanalysis to identify the genomic features specific to B. velezensis strain GH1-13 among 364 Bacillus strains, using orthologous protein coding sequences to identify both core conserved and strain-specific genes 29. Markers from various types of genes were known to be highly variable.…”