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The PhoPR two-component system (TCS) is a signal transduction pathway to regulate the phosphate starvation response in Bacillus subtilis and regulated fengycin production in strain NCD-2 under low phosphate condition. The purpose of this study was to characterize the proteome level responses in the phoP-null mutant (MP) and the phoR-null mutant (MR), and to integrate the proteomics with the transcriptomic data obtained previously. The metabolic pathway for fengycin was predicted based on omics analysis as well as molecular genetics assay. Results showed the proteins and genes associated with biosynthesis of branched chain amino acids (BCAAs) were regulated by PhoPR TCS, and liquid chromatography mass spectrometry (LC-MS) analysis also confirmed that the production of BCAAs was down-regulated in the MP and MR mutants, when compared to wild-type strain NCD-2. Protein network analysis showed that the BCAA metabolism was linked to the biosynthesis of lipopeptides. The MP and MR strains decreased the fengycin production when cultured in modified Landy medium supplied with 0.42 mM phosphate, however, the fengycin production could be restored when the glutamic acid was replaced with BCAAs that were added to modified Landy medium. The lpdV gene, which is responsible for the BCAA degradation process, was deleted in strain NCD-2. Compared with the wild-type strain, the lpdV mutant produced significantly less fengycin in the medium supplied with BCAAs. Considered together, the results of this study indicate that the PhoPR TCS regulates fengycin production by affecting BCAA biosynthesis., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Background: Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore some secondary metabolite biosynthetic gene clusters and related antimicrobial compounds in strain NCD-2. An integrative approach combining genome mining and structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was adopted to interpret the chemical origins of metabolites with significant biological activities., Results: Genome mining revealed nine gene clusters encoding secondary metabolites with predicted functions, including fengycin, surfactin, bacillaene, subtilosin, bacillibactin, bacilysin and three unknown products. Fengycin, surfactin, bacillaene and bacillibactin were successfully detected from the fermentation broth of strain NCD-2 by UHPLC-QTOF-MS/MS. The biosynthetic gene clusters of bacillaene, subtilosin, bacillibactin, and bacilysin showed 100% amino acid sequence identities with those in B. velezensis strain FZB42, whereas the identities of the surfactin and fengycin gene clusters were only 83 and 92%, respectively. Further comparison revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. The biosynthetic enzyme-related gene srfAB for surfactin was divided into two parts. Bioinformatics analysis suggested that FenE in strain NCD-2 had a similar function to FenE and FenC in strain FZB42, and that FenA in strain NCD-2 had a similar function to FenA and FenD in strain FZB42. Five different kinds of fengycins, with 26 homologs, and surfactin, with 4 homologs, were detected from strain NCD-2. To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis., Conclusions: Our study revealed a number of gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, including a fengycin synthetic gene cluster that might be unique by using genome mining and UHPLC-QTOF-MS/MS. The production of fengycin, surfactin, bacillaene and bacillibactin might explain the biological activities of strain NCD-2.

The PhoRP two-component system (TCS), one of the most important signaling pathways in Bacillus subtilis, regulates cell physiological reactions mainly under phosphate starvation conditions. The mechanism by which PhoRP TCS regulates resistance towards antibiotics in B. subtilis strain NCD-2 was investigated in this study. Using phenotype microarray (PM) technology, the susceptibility of B. subtilis to 240 antimicrobial compounds was compared among the wild-type strain NCD-2, the phoR-null mutant (MR), and the phoP-null mutant (MP). Compared with the wild type, the MR mutant was more resistant to 13 antibiotics with different functions, and the MP mutant was more resistant to 14 antibiotics, of which 8 were 30S/50S ribosome-targeted. To investigate the molecular mechanisms involved in changing the level of antibiotic resistance, transcriptional analysis was performed to compare the differentially expressed genes among the wild-type strain and the MR and MP mutants. Compared with the wild-type strain, 294 genes were differentially expressed in the MR mutant, including 97 up-regulated genes and 197 down-regulated genes. Most of the differently expressed genes were associated with carbohydrate mechanism, amino acid mechanism, ABC-transporters and phosphotransferase systems. A total of 212 genes were differentially expressed in the MP mutant, including 10 up-regulated genes and 202 down-regulated genes, and most were associated with ribosome synthesis, amino acid metabolism, carbohydrate metabolism and ABC-transporters. The khtSTU operon (encoding the K + efflux pump) that was up-regulated in the MP mutant was deleted by in-frame deletion in the MP mutant. The phoP and khtSTU operon double mutant MPK showed decreased antibiotic resistance to doxycycline, chlortetracycline, spiramycin, puromycin, and paromomycin when compared with the MP mutant. Thus, the results indicated that the khtSTU operon was responsible for the PhoP-mediated multiple antibiotic resistance., (Copyright © 2020. Published by Elsevier GmbH.)

Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases. With the purpose of understanding the colonization characteristics of strain NCD-2, firstly, a constitutive expression promoter was cloned from strain NCD-2 and was used to construct GFP-labeled strain NCD-2. The GFP-labeled strain NCD-2 showed strong green fluorescence under planktonic cells and biofilm formation. The colonization characteristics of strain NCD-2 on different parts of cotton root were qualitatively observed by confocal laser scanning microscopy (CLSM). Results showed that strain NCD-2 mainly colonized on the zone of differentiation and elongation. Rhizosphere populations of B. subtilis strain NCD-2 on different cotton root were quantitatively evaluated by traditional plating count and quantitative PCR (qPCR) analysis in both autoclaved soil and non-autoclaved soil, respectively. Results showed that both traditional plating count and qPCR analysis showed similar trend for colonization characteristics of strain NCD-2. The greatest strain NCD-2 populations were in the root tip, at 9.19 × 10 7  CFU g -1 root and 6.75 × 10 7  CFU g -1 root as estimated by qPCR in non-autoclaved and autoclaved soil, respectively. This study provides a clearer understanding of the interactions between biocontrol agent and plant, as well as with the indigenous microorganisms in the soil.

Bacillus subtilis NCD-2 is an excellent biocontrol agent for tomato gray mold and cotton soil-borne diseases. The fengycin lipopeptides serve as a major role in its biocontrol ability. A previous study revealed that insertion of degQ with the mini-Tn10 transposon decreased the antifungal activity of strain NCD-2 against the growth of Botrytis cinerea. To clarify the regulation of degQ on the production of fengycin, we deleted degQ by in-frame mutagenesis. Compared with the wild-type strain NCD-2, the degQ-null mutant had decreased extracellular protease and cellulase activities as well as antifungal ability against the growth of B. cinerea in vitro. The lipopeptides from the degQ-null mutant also had significantly decreased antifungal activity against B. cinerea in vitro and in vivo. This result was confirmed by the decreased fengycin production in the degQ-null mutant that was detected by fast protein liquid chromatography analysis. Quantitative reverse transcription PCR further demonstrated that degQ positively regulated the expression of the fengycin synthetase gene. In addition, the degQ-null mutant also had a flatter colony phenotype and significantly decreased biofilm formation ability relative to the wild-type strain. All of those characteristics from degQ-null mutant could be restored to the strain NCD-2 wild-type level by complementation of intact degQ in the mutant. Therefore, DegQ may be an important regulator of fengycin production and biofilm formation in B. subtilis NCD-2., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Bacillus subtilis strain NCD-2 is strongly antagonistic toward phytopathogenic fungi, and functions as an excellent biocontrol agent for cotton soil-borne diseases. The aims of this study were to characterize the main active antifungal compound from strain NCD-2 and clarify its role in suppressing cotton damping-off disease. Strain NCD-2 and lipopeptide extract prepared from an NCD-2 culture strongly inhibited the growth of Rhizoctonia solani in vitro. The lipopeptides of strain NCD-2 were separated by fast protein liquid chromatography (FPLC) and the antifungal compound was identified as a cluster of fengycin homologs analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A fengycin-deficient mutant was obtained by in-frame deletion of the fengycin synthetase gene in B. subtilis NCD-2. Compared with the wild-type strain, this mutant showed decreased abilities to inhibit the growth of R. solani in vitro and to suppress cotton damping-off disease in vivo. Studies showed that the population of fengycin-deficient mutant was almost same as that of the wild-type NCD-2 strain in the cotton rhizosphere. However, the population of R. solani in the cotton rhizosphere colonized by the fengycin-deficient mutant was twice that in the cotton rhizosphere colonized by the NCD-2 wild-type strain. This study demonstrated that fengycin-type lipopeptides are the main antifungal active compounds produced by B. subtilis NCD-2. These compounds play a major role in restricting the population of R. solani in the cotton rhizosphere and in suppressing cotton damping-off disease., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Bacillus subtilis and its closely related species are indistinguishable from one another by morphological characteristics and 16S rDNA sequences. In this study, the partial phoR sequence was tested to determine the phylogenetic relationship of species in the B. subtilis group. Degenerate primers were developed according to the relatively conserved nucleotide sequences of phoR and the linked gene phoP in the B. subtilis group. The primers amplified a 1100 bp phoR fragment from strains representative of 6 species in the B. subtilis group. Based on the sequenced fragments, 26 type strains comprising these 6 species were clearly distinguished. At the intraspecies level, the phoR sequence similarities were 90%-100%, but at the interspecies level, the phoR sequence similarities were 32.8%-75%. Compared with the gyrB sequence, the phoR sequences showed a larger divergence especially at the interspecies levels. Therefore, the phoR sequence may be an efficient alternative marker for phylogenetic and taxonomic analysis of species in the B. subtilis group. Twenty-three Bacillus undomesticated isolates were tested for identification and phylogenetic analysis based on the phoR and gyrB sequences. The 23 isolates could be clearly delineated into 4 distinct groups, 10 as B. subtilis, 3 as B. mojavensis, 2 as B. atrophaeus, and 8 as B. amyloliquefaciens.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Bacillus subtilis NCD-2 (BS) decreased disease index of CVW, increased cotton yields. • BS did not decreased population of V. dahliae in rhizosphere. • BS increased bacterial and fungal diversities. • BS changed the microbial community structure. • Microbial communities structure was correlated with soil nutrients after BS applicaiton. Bacillus subtilis NCD-2 (BS) shows prominent biological control for cotton verticillium wilt (CVW). However, it is unknown whether the effects on the abundant and rare microbial communities in rhizosphere after application of BS. Therefore, we investigated the effects of BS treatment on the suppression of CVW and the microbial community structure in rhizosphere. BS treatment successfully suppressed CVW, achieving a control effect of 61.60%, and cotton yield increased by 6.07% compared with blank control. Moreover, application of BS resulted in 28.17%, 18.63%, and 5.08% increases in plant fresh weight, dry weight, and number of branches, respectively. However, the populations of Verticillium dahliae in rhizosphere did not decrease by BS treatment. Microbial diversity analysis showed that soil bacterial and fungal diversities were increased under BS treatment. Principal component analyses revealed that BS had significantly effect on the abundant and rare microbial communities. Higher abundances of Acidobacteria, Chloroflexi, and Planctomycetes were observed among abundant bacterial taxa in the BS treatment. Furthermore, the abundances of Arenimonas , Hydrogenophaga, Chaetomium , Aspergillus, Nectria, Psathyrella, Conocybe, and Chrysosporium were also significantly higher than those in blank control. Redundancy analysis indicated that the microbial communities structure in BS treatment were positively related to ammonium nitrogen (NH 4 +-N), inorganic phosphorus (IP), and pH, but negatively to nitrate nitrogen (NO 3 –-N), AK, and organic matter (OM). These research results may offer theoretical bases for comprehensive the mechanism of ecological prevention and control CVW by application of BS. [ABSTRACT FROM AUTHOR]

• Bacillus subtilis NCD-2 decreased incidence of Verticillium wilt, increased yield. • Broccoli residues reduced the DNA copy numbers of Verticillium dahliae in soil. • Broccoli residues decreased fungal community diversity. • Broccoli residues increased the abundance of beneficial microbes. • Broccoli residues return was effective method for controlling potato Verticillium wilt. Potato Verticillium wilt is an economically important disease that significantly decreases tuber yield. Biological control is an effective approach for controlling potato Verticillium wilt. The objective of this study was to evaluate the effects of treatment with Bacillus subtilis NCD-2 (BS), broccoli residues (BR), and combination among them (BB) on potato Verticillium wilt and the soil fungal community structure. Field experiments demonstrated that BS, BR and BB successfully suppressed potato Verticillium wilt, achieving control effects of 32.25%, 48.15%, and 37.85%, respectively. Compared to the untreated control, potato yield increased by 10.38%, 16.82% and 27.12% after the treatments of BS, BR and BB, respectively. Moreover, the BS treatment did not decrease the rhizosphere V. dahliae population, which was significantly decreased by BR (19.08%) and BB (30.46%). Illumina MiSeq analysis showed that soil fungal diversity in the rhizosphere was decreased under BS, BR and BB. Principal component and heatmap analyses showed that BS exerted a similar effect on the fungal community structure to the blank control. However, BR and BB greatly changed the fungal community structure. Moreover, the relative abundance of beneficial microbes such as Chaetomium and Mortierella was higher in the BS and BB soils than in the control. Redundancy analysis (RDA) showed that the fungal community structure under BR and BB was positively correlated with SOM, pH, AP and IP, while the opposite trend was observed under BS. The results suggested that BS seed treatment and BR return could be a useful strategy for potato production in sustainable agriculture; this research may provide a theoretical basis for understanding the mechanism of the ecological control of potato Verticillium wilt. [ABSTRACT FROM AUTHOR]

• Novel qPCR diagnostics for Bacillus subtilis NCD-2 and Pseudomonas protegens FD6. • Inoculant qPCR detection limits independent of soil type and wheat root tissue. • Soil and wheat rhizosphere colonization by strain FD6 greater than strain NCD-2. • Differential in-vitro antibiosis of wheat root pathogens by strains NCD-2 and FD6. • Inoculant colonization suppressed Rhizoctonia solani and increased wheat growth. Bacillus subtilis strain NCD-2 and Pseudomonas protegens strain FD6 produce a range of antimicrobial metabolites known to be effective against soil-borne plant pathogens. Quantitative qPCR assays were designed from sequence characterized amplified fragment length polymorphisms (AFLP) diagnostic for each inoculant genotype. The qPCR minimum detection thresholds (MDT) for NCD-2 and FD6 ranged between 5.2 × 103 (log 3.71) and 1.5 × 106 (log 6.18) genome (cell) equivalents g−1 of soil or wheat root tissue and were independent of soil type. Wheat rhizosphere populations of strains FD6 and NCD-2 significantly declined during the first 14 days postinoculation and then remained stable for the next 14 days. Colonization of wheat roots, rhizosphere soil and bulk (non-rhizosphere) soil by FD6 was significantly greater than that of NCD-2. Strains FD6 and NCD-2 significantly decreased in vitro growth of wheat root pathogenic Rhizoctonia solani AG-8, Pythium irregulare and Gaeumannomyces graminis var. tritici , fungal antibiosis by FD6 greater than NCD-2. Wheat rhizosphere colonization by NCD-2 and FD6 significantly decreased abundance of R. solani AG-8 in the rhizosphere by 3- and 13-fold respectively, pathogen suppression increasing (21%–29%) foliar biomass of wheat seedlings to levels not significantly different to the non-diseased control. The qPCR assays developed in this study were used to monitor soil and rhizosphere populations of P. protegens FD6 and B. subtilis NCD-2 and their suppressive efficacies against R. solani AG-8 in wheat. These assays can be applied to monitor the dynamics of these inoculants in suppressing Rhizoctonia root rot and other crop diseases. [ABSTRACT FROM AUTHOR]

• B. subtilis strain NCD-2 is a biocontrol agent for cotton verticillium wilt. • Fengycin was main antifungal compound against V. dahliae. • Surfactin mainly affected the root colonization of strain NCD-2. • Both fengycin and surfactin contribute to the biocontrol efficiency. Bacillus subtilis strain NCD-2 is a promising biocontrol agent for soil borne plant diseases. A previous study indicated that strain NCD-2 could produce surfactin and fengycin simultaneously. In this study, the exact roles of surfactin and fengycin in strain NCD-2 against cotton verticillium wilt were determined by knockout of the surfactin synthetase gene srfAA and the fengycin synthetase gene fenC. Compared to the wild-type strain NCD-2, the fengycin-deficient mutant ΔFenC decreased the antifungal ability toward Verticillium dahliae , but the surfactin-deficient mutant ΔSrfAA mainly decreased the swarming and biofilm formation abilities of strain NCD-2. Comparatively, the fengycin and surfactin double mutant ΔFenC/SrfAA decreased the antifungal activity and swarming and biofilm formation abilities to more significant levels. The colonization abilities of the wild type strain NCD-2 and its derivative mutants were compared in the cotton rhizosphere. Compared to the wild-type strain NCD-2, ΔSrfAA and ΔFenC/SrfAA exhibited the poorest colonization ability. RT-qPCR results confirmed that knockout of the srfAA and fenC genes decreased the expression of biofilm formation-related genes. The biocontrol abilities of the strains against cotton verticillium wilt were evaluated in a greenhouse. All mutants decreased the biocontrol abilities against cotton verticillium wilt, while ΔFenC/SrfAA showed the poorest biocontrol ability, followed by ΔFenC. Taken together, fengycin was the main antifungal active compound, and surfactin affected root colonization. Both the antifungal ability and root colonization of strain NCD-2 played important roles in suppressing cotton verticillium wilt. [ABSTRACT FROM AUTHOR]