Bacillus subtilis strain NCD-2 is a promising biocontrol agent for soil-borne plant diseases and shows potential for promoting the growth of some crops. The purposes of this study were to analyze the colonization ability of strain NCD-2 in different crops and reveal the plant growth promotion mechanism of strain NCD-2 by rhizosphere microbiome analysis. qRT-PCR was used to determine the populations of strain NCD-2, and microbial communities’ structures were analyzed through amplicon sequencing after application of strain NCD-2. Results demonstrated that strain NCD-2 had a good growth promotion effect on tomato, eggplant and pepper, and it was the most abundant in eggplant rhizosphere soil. There were significantly differences in the types of beneficial microorganisms recruited for different crops after application of strain NCD-2. PICRUSt analysis showed that the relative abundances of functional genes for amino acid transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, and defense mechanisms were enriched in the rhizospheres of pepper and eggplant more than in the rhizospheres of cotton, tomato and maize after application of strain NCD-2. In summary, the colonization ability of strain NCD-2 for five plants was different. There were differences in microbial communities’ structure in rhizosphere of different plants after application of strain NCD-2. Based on the results obtained in this study, it was concluded that the growth promoting ability of strain NCD-2 were correlated with its colonization quantity and the microbial species it recruited.

Bacillus subtilis S-16 isolated from sunflower-rhizosphere soil is an effective biocontrol agent for preventing soilborne diseases in plants. Previous research revealed that the volatile organic compounds (VOCs) produced by the S-16 strain have strong inhibitory effects on Sclerotinia sclerotiorum. The identification of the VOCs of S-16 using gas chromatography-tandem mass spectrometry (GC-MS/MS) revealed 35 compounds. Technical-grade formulations of four of these compounds were chosen for further study: 2-pentadecanone, 6,10,14-trimethyl-2-octanone, 2-methyl benzothiazole (2-MBTH), and heptadecane. The major constituent, 2-MBTH, plays an important role in the antifungal activity of the VOCs of S-16 against the growth of Sclerotinia sclerotiorum. The purpose of this study was to determine the impact of the thiS gene’s deletion on the 2-MBTH production and to conduct an antimicrobial activity analysis of the Bacillus subtilis S-16. The thiazole-biosynthesis gene was deleted via homologous recombination, after which the contents of 2-MBTH in the wild-type and mutant S-16 strains were analyzed using GC-MS. The antifungal effects of the VOCs were determined using a dual-culture technique. The morphological characteristics of the Sclerotinia sclerotiorum mycelia were examined via scanning-electron microscopy (SEM). Additionally, the lesion areas on the sunflower leaves with and without treatment with the VOCs from the wild-type and mutant strains were measured to explore the effects of the VOCs on the virulence of the Sclerotinia sclerotiorum. Moreover, the effects of the VOCs on the sclerotial production were assessed. We showed that the mutant strain produced less 2-MBTH. The ability of the VOCs produced by the mutant strain to inhibit the growth of the mycelia was also reduced. The SEM observation showed that the VOCs released by the mutant strain also caused more flaccid and gapped hyphae in the Sclerotinia sclerotiorum. The Sclerotinia sclerotiorum treated by the VOCs produced by the mutant strains caused more damage to the leaves than that treated by the VOCs produced by the wild type and the mutant-strain-produced VOCs inhibited sclerotia formation less. The production of 2-MBTH and its antimicrobial activities were adversely affected to varying degrees by the deletion of thiS.

A microbial fungicide developed from Bacillus subtilis NCD-2 has been registered for suppressing verticillium wilt in crops in China. Spores are the main ingredient of this fungicide and play a crucial role in suppressing plant disease. Therefore, increasing the number of spores of strain NCD-2 during fermentation is important for reducing the cost of the fungicide. In this study, five kinds of carbon sources were found to promote the metabolism of strain NCD-2 revealed via Biolog Phenotype MicroArray (PM) technology. L-arabinose showed the strongest ability to promote the growth and sporulation of strain NCD-2. L-arabinose increased the bacterial concentration and the sporulation efficiency of strain NCD-2 by 2.04 times and 1.99 times compared with D-glucose, respectively. Moreover, L-arabinose significantly decreased the autolysis of strain NCD-2. Genes associated with arabinose metabolism, sporulation, spore resistance to heat, and spore coat formation were significantly up-regulated, and genes associated with sporulation-delaying protein were significantly down-regulated under L-arabinose treatment. The deletion of msmX, which is involved in arabinose transport in the Bacillus genus, decreased growth and sporulation by 53.71% and 86.46% compared with wild-type strain NCD-2, respectively. Complementing the mutant strain by importing an intact msmX gene restored the strain’s growth and sporulation.

Bacillus subtilis NCD-2 showed a promising biocontrol effect against plant soil-borne diseases, and was developed as a commercial microbial fungicide against cotton verticillium wilt in China. Spores were main ingredient of the fungicide and played the crucial role in biological control of plant diseases. Therefore, the key to reducing the cost of fungicide was to find ways to increase the number of spores of strain NCD-2 during fermentation. In this study, 755 substances were evaluated by Phenotype Microarray technology, and 5 kinds of carbon sources and 1 kind of nitrogen source were found to promote the metabolism of strain NCD-2. Among carbon sources L-arabinose showed strongest ability to promote the bacteria growth and sporulation of strain NCD-2. When L-arabinose was used as a single carbon source, it could increase the bacteria concentration and the sporulation efficiency of strain NCD-2 by 2.04 times and 1.99 times, respectively, compared with D-glucose. Moreover, L-arabinose could significantly decrease the autolysis of strain NCD-2 revealed by microscopy observation. The mechanism for promoting the sporulation by L-arabinose was analysed by RNA-seq sequencing. Results showed that total of 790, 923 and 1270 genes were up-regulated and 639, 850 and 1001 genes were down-regulated under L-arabinose supplies when cultured at 8 h, 12 h and 16 h, respectively. Fourteen differentially expressed genes associated with arabinose transportation and sporulation were selected for qRT-PCR analysis, and the result showed basically consistent expression trend with transcriptome. Notably, genes associated with arabinose metabolism, sporulation, spore resistance to heat and spore coat formation were significantly up-regulated, and genes associated with sporulation-delaying protein were significantly down-regulated under L-arabinose supplies. msmX gene which was involved in arabinose transport in Bacillus genus was deleted, and the mutant decreased the growth and sporulation by 53.71% and 86.46%, respectively, when compared to strain NCD-2 wild type. Complementary of the mutant by importing intact msmX gene could restore the growth and sporulation of the mutant strain. In conclusion, arabinose played an important role in regulating the growth and sporulation of strain NCD-2.

The SUN family protein MoSun4 in Magnaporthe oryzae has been previously implicated in mitophagy and has potential as a target for reducing rice blast, but its role as a secreted protein remains poorly understood. In this study, signal peptide prediction and yeast secretion assays confirmed MoSun4 signal peptide function, and co-localization revealed the localization of the extra-invasive hyphal membrane (EIHM)-associated apoplastic compartment or matrix, establishing MoSun4 as a secreted protein. In addition, deletion of the MoSUN4 gene reduced the hyphal growth, conidiation and virulence of M. oryzae. We further showed that MoSun4 is involved in regulating the expression of virulence-related genes, including multiple genes involved in cell wall degradation (eglC, eglD), secondary metabolism (gliK), and melanin biosynthesis (SDH1, BUF1, Cmr1, ALB1). Collectively, our study reveals that MoSun4 is a secreted protein that contributes to pathogenicity and is associated with altered expression of virulence-related genes, providing new insights into the functions of SUN family proteins.