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[Aims] Verticillium wilt (VW) of cotton was effectively controlled by application of broccoli residues (BR) to soil. Information regarding the variation in bacterial communities in rhizosphere of cotton cultivars with different VW resistance levels under BR treatment is still lacking and finally to provide guidance for screening effective biocontrol bacteria. [Methods] Real-time fluorescence quantitative PCR was used to determine the population of Verticillium dahliae, the effects of BR on the bacterial community structure in rhizosphere were determined by high-throughput sequencing technology. [Results] Results showed that control effects for susceptible cultivar (cv. EJ-1) and resistant cultivar (cv. J863) on VW after BR treatment were 51.76% and 86.15%, the population of V. dahliae decreased by 18.88% and 30.27%, respectively. High-throughput sequencing showed that ACE and Chao1 indices were increased by application of BR. Actinobacteria, Proteobacteria, Bacteroidetes, Gemmatimonadetes, Acidobacteria, and Firmicutes were the most dominant phyla, and relative abundances of these bacterial taxa significantly differed between cultivars. Additionally, Bacillus stably increased in rhizosphere following BR treatment. Redundancy analysis (RDA) showed that relative abundances of Bacillus, Lysobacter, Streptomyces, Rubrobacter, Gemmatimonas, Bryobacter and Nocardioides were correlated with occurrence of VW. Field experiments demonstrated that dressing cotton seeds with Bacillus subtilis NCD-2 could successfully reduce occurrence of VW, and control effects for EJ-1 and J863 were 35.26% and 31.02%, respectively. [Conclusions] The application of BR changed the bacterial community structure in cotton rhizosphere, decreased the population of V. dahliae in soil, and increased the abundance of beneficial microorganisms, thus significantly reducing the occurrence of VW.

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 population of V. 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 NH4+-N, IP, and pH, but negatively to NO3--N, AK, and OM. These research results may offer theoretical bases for comprehensive the mechanism of ecological prevention and control CVW by application of BS.

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 of the secondary metabolite biosynthetic gene clusters and related bioactive compounds in strain NCD-2. An integrative approach, which combined genome mining with structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was conducted to interpret the chemical origins of the significant biological activities in strain NCD-2. Results Genome mining revealed that strain NCD-2 contained nine gene clusters having predicted functions involving secondary metabolites with bioactive abilities. They encoded six known products 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. Bacillaene, subtilosin, bacillibactin, and bacilysin related biosynthetic gene clusters showed 100% amino acid sequence similarity with B. velezensis strain FZB42，however, the biosynthetic gene clusters for surfactin and fengycin showed 83% and 92%, respectively. Further comparison of gene clusters encoding fengycin and surfactin revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. Moreover, biosynthetic enzyme-related gene srfAB for surfactin had divided into two parts. Bioinformatics analysis predicted that FenE function in strain NCD-2 was same to that of FenE and FenC in strain FZB42, and FenA function in strain NCD-2 was same to that of FenA and FenD in strain FZB42. Five kinds of fengycin, 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 and unique gene cluster related to fengycin synthesis. Conclusions It was found that there were many gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, and the fengycin synthetic gene cluster 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.

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 × 107 CFU g−1 root and 6.75 × 107 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 strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator PhoP and its sensor kinase PhoR compose a two-component system in B. subtilis. In this study, the phoR- and phoP-knockout mutants were constructed by in-frame deletion and the role of PhoR/PhoP on the production of fengycin was determined. Inactivation of phoR or phoP in B. subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type. The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate, and the lipopeptides from phoR-null mutant or phoP-null mutant almost lost the inhibition ability against B. cinerea growth compared to the lipopeptides from strain NCD-2 wild type. Fast protein liquid chromatography (FPLC) analysis of the lipopeptides showed that inactivation of phoR or phoP genes reduced the production of fengycin by strain NCD-2. The fengycin production abilities were compared for bacteria under low-phosphate medium (LPM) and high-phosphate medium (HPM), respectively. Results indicated that the regulation of fengycin production by the PhoR/PhoP two-component system occurred in LPM but not in HPM. Reverse transcriptional-PCR confirmed that the fengycin synthetase gene fenC was positively regulated by phoP when cultured in LPM. All of these characteristics could be partially restored by complementation of intact phoR or phoP gene in the mutant. These data indicated that the PhoR/PhoP two-component system greatly regulated fengycin production and antifungal ability in B. subtilis NCD-2 mainly under low-phosphate conditions.

Watermelon (Citrullus lanatus T.) is one of the most important economic crops in China. Soil-borne diseases are becoming more and more serious with longer growing seasons and continuous cropping of watermelon in greenhouses. In May 2020, symptoms were observed on plants in greenhouses located at Xingtai, Hebei province of China and included wilted leaves, chlorosis and plant death. Among the 26 greenhouses examined, symptomatic plants were observed in 17 greenhouses. The incidences of infected plants ranged from 1% to 35%, and caused an average 10% yield loss. Symptoms began on lower part of the plants and progressed upward to the vines and leaves. At the early stage of infection, the edge of watermelon leaves changed from green to yellow, and became soft. As the disease progressed, infected leaves wilted and desicated. The vascular tissue of the stem exhibited a uniform brown discoloration that often extended throughout the vine. To identify the causal agent, small pieces approximate 3.0×3.0 mm size of infected stem tissues were collected and sterilized with 0.5% sodium hypochlorite solution for 1 min, rinsed three times with sterile water and transferred onto potato dextrose agar (PDA) medium amended with 100 μg·mL-1 of chloramphenicol. The plates were incubated at 25°C for 3 days in the dark and fungal isolates were purified using the single-spore isolation method. A total of 22 fungal isolates with identical colony morphology were collected from diseased plants. The color of the fungal colonies on PDA medium was creamy-white with an abundance of mycelia that darken after 5 days growth due to the formation of microsclerotia. Fungal colonies consisted of fine, hyaline hyphae with verticillate conidiophores producing hyaline, ellipsoidal to oval conidia with an average size of 5.12×3.41 μm (n=50). The morphological characters of the fungal isolates were identical to those of Verticillium dahliae Kleb. described by Hawksworth and Talboys (Hawksworth, D. and Talboys, P, 1970). Pathogenicity tests were performed by soaking 30 watermelon seedlings with wounded root tips in the fungal conidial suspension (1x107 conidium/mL) for 30 min (Ma, et al, 2004). The same number of non-inoculated watermelon seedlings was used as a control. All plants were kept in a greenhouse at 25°C and 90%-95% relative humidity. Seven days post-inoculation (dpi), leaves of treated plants began to show symptoms of wilt. At 10-dpi, lower leaves wilted and dry and by 15-dpi, whole plants were dead. Pathogenicity tests were repeated three times with consistent results. The pathogen was re-isolated from the diseased plants and displayed identical morphological characteristics to the original isolates. To further identity the pathogens, the ribosomal DNA Internal Transcribed Spacer (rDNA-ITS) region was amplified by PCR (White et al., 1990; Liu et al., 1999; Bellemain et al.. 2010). The amplicon was sequenced and showed 99%-100% identity to the ITS region of the V. dahliae reference strains deposited in the NCBI database (MK093977.1, MK287620.1, MT348570.1 and LC549667.1, respectively). Based on morphological and ITS sequence information, the fungal pathogen was identified as V. dahliae. V. dahliae is an economically important pathogen with a wide host range worldwide. The discovery of Verticillium wilt on watermelons indicates that there might be a risk of Verticillium wilt when watermelons are planted in subsequent crops of the host plants of the disease, such as cotton or eggplant. To our knowledge, this is the first report of V. dahliae causing Verticillium wilt of watermelon in China. Financed: the Special Fund for Agro-scientific Research in the Public Interest, China (201503109) References: Hawksworth, D. and Talboys, P. 1970. Description of Pathogenic Fungi and Bacteria, CMI, Surrey. Ma, P., et al. 2004. A New Inoculation Method for Verticillium Wilt on Cotton and Its Application in Evaluating Pathogenesis and Host Resistance. Acta Phytopathologica Sinica, 34(6): 536-541. White, T. J., et al. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. PCR protocols: a guide to methods and applications, 18(1), 315-322. Bellemain, E., et al. 2010. ITS as an Environmental DNA Barcode for Fungi: an in Silico Approach Reveals Potential PCR Biases. BMC microbiology, 10(1), 1-9. Liu, Y. J., et al. 1999. Phylogenetic Relationships Among Ascomycetes: Evidence from an RNA Polymerse II SubunitMol. Biol. Evol. 16:1799-1808.

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.

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.

Genetic composition of Fusarium oxysporum f. sp. vasinfectum strains, including race 3, 7, and 8, Australian genotype strain, and 80 strains collected from China, were studied using amplified fragment length polymorphism (AFLP). Based on AFLP analysis, these strains were separated into four groups. Race 3, strain CN3, was the only strain in group A. Race 8, strain CN8, was the only strain in group B. Race 7, strain CN7, was grouped with 75 strains from China in group C. The Australian genotype strain ATCC96291 was grouped with five strains from China in group D. Evolution of the five native strains in group D was studied using multigene genealogies. Phylogenetic tree analysis revealed that the five strains of group D had a closer genetic relationship to the Australian genotype strain than the other races based on the combined elongation factor, β-tubulin, and phosphate permase gene sequence data. Group D was further tested for pathogenicity and virulence on four cotton cultivars from Upland (Gossypium hirsutum) and Sea Island (G. barbadense) cotton. All five strains caused typical Fusarium wilt symptoms on all four cotton cultivars but virulence were relatively low compared with race 3, race 7, and race 8.

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.

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.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most important diseases of Brassicaceae family. Bacillus spp. that produce lipopeptide antibiotics are often used as biocontrol agents to suppress soilborne diseases. B. subtilis NCD-2 isolated from the cotton rhizosphere has been found to inhibit pathogenic fungi through the production of fengycin-type cyclopeptides. In this study, the biocontrol efficacy of B. subtilis NCD-2 against P. brassicae and the role of fengycin in disease suppression were evaluated on Chinese cabbage in glasshouse. B. subtilis NCD-2 reduced clubroot disease incidence and severity up to 47.7% and 51.6%, respectively, while the fengycin defective mutant ΔfenC and a PhoP/PhoR two-component regulatory system mutant, ΔphoP, were unable to prevent this disease. In vitro, P. brassicae resting spore cells were lysed when treated with standard fengycin and crude fengycin extracts of the wild type B. subtilis NCD-2, but not of its mutant ΔfenC. Moreover, the population dynamics of B. subtilis and P. brassicae was determined using quantitative real-time PCR (qPCR). The results were surprising that the wild type B. subtilis NCD-2 and its mutant ΔfenC colonized at similar rates in the rhizosphere, while levels of P. brassicae DNA in the rhizosphere and root tissue were reduced following B. subtilis NCD-2 treatment, but there was no effect of the mutant ΔfenC. Thus, the production of fengycins in B. subtilis NCD-2 may play a promising role in the suppression of P. brassicae in the rhizosphere and reduction of incidence of clubroot disease in Chinese cabbage.

The Bacillus subtilis strain NCD-2 is an important biocontrol agent against cotton verticillium wilt and cotton sore shin in the field, which are caused by Verticillium dahliae Kleb and Rhizoctonia solani Kuhn, respectively. A mutant of strain NCD-2, designated M216, with decreased antagonism to V. dahliae and R. solani, was selected by mini-Tn10 mutagenesis and in vitro virulence screening. The inserted gene in the mutant was cloned and identified as the phoR gene, which encodes a sensor kinase in the PhoP/PhoR two-component system. Compared to the wild-type strain, the APase activities of the mutant was decreased significantly when cultured in low phosphate medium, but no obvious difference was observed when cultured in high phosphate medium. The mutant also grew more slowly on organic phosphate agar and lost its phosphatidylcholine-solubilizing ability. The suppression of cotton seedling damping-off in vivo and colonization of the rhizosphere of cotton also decreased in the mutant strain when compared with the wild type strain. All of these characteristics could be partially restored by complementation of the phoR gene in the M216 mutant.

Bacillus subtilis BAB-1, isolated from cotton rhizosphere soil, is an excellent biocontrol agent for tomato gray mold. The genome of B. subtilis strain BAB-1 was fully sequenced and annotated, genes encoding the antifungal active compound were identified, and multiple sets of regulatory systems were found in the genome.