Biocontrol potential of Bacillus subtilis CTXW 7-6-2 against kiwifruit soft rot pathogens revealed by whole-genome sequencing and biochemical characterisation

Tingting Chen; Zhuzhu Zhang; Wenzhi Li; Jia Chen; Xuetang Chen; Bince Wang; Jiling Ma; Yunyun Dai; Haixia Ding; Weizhen Wang; Youhua Long

doi:10.3389/fmicb.2022.1069109

Biocontrol potential of Bacillus subtilis CTXW 7-6-2 against kiwifruit soft rot pathogens revealed by whole-genome sequencing and biochemical characterisation

Front Microbiol. 2022 Dec 1:13:1069109. doi: 10.3389/fmicb.2022.1069109. eCollection 2022.

Authors

Tingting Chen¹, Zhuzhu Zhang¹, Wenzhi Li¹, Jia Chen¹, Xuetang Chen¹, Bince Wang¹, Jiling Ma¹, Yunyun Dai¹, Haixia Ding^{1

2}, Weizhen Wang¹, Youhua Long^{1

3}

Affiliations

¹ Research Center for Engineering Technology of Kiwifruit, College of Agriculture, Institute of Crop Protection, Guizhou University, Guiyang, China.
² Department of Plant Pathology, Guizhou University, Guiyang, China.
³ Teaching Experimental Factory, Guizhou University, Guiyang, China.

Abstract

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.

Keywords: Bacillus subtilis; antifungal; biocontrol; kiwifruit soft rot; pulcherrimin.

Grants and funding

This research was supported by the National Natural Science Foundation of China (No. 32260707), Agricultural Research Projects of the Science and Technology Department of Guizhou Province (Nos. 2019 2403 and 2021 YIBAN237), the China Agriculture Research System of MOF (CARS-26), and the Talent Introducing Scientific Research Fund of Guizhou University (No. X2021029).