Exploring Synergistic Effects of Levan and Levan-Metabolizing Bacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat.

Boosting plant immunity by priming agents can lower agrochemical dependency in plant production. Levan and levan-derived oligosaccharides (LOS) act as priming agents against biotic stress in several crops. Additionally, beneficial microbes can promote plant growth and protect against fungal diseases. This study assessed possible synergistic effects caused by levan, LOS and five levan- and LOS-metabolizing Bacillaceae (Bacillus and Priestia) strains in tomato and wheat. Leaf and seed defense priming assays were conducted in non-soil (semi-sterile substrate) and soil-based systems, focusing on tomato-Botrytis cinerea and wheat-Magnaporthe oryzae Triticum (MoT) pathosystems. In the non-soil system, seed defense priming with levan, the strains (especially Bacillus velezensis GA1), or their combination significantly promoted tomato growth and protection against B. cinerea. While no growth stimulatory effects were observed for wheat, disease protective effects were also observed in the wheat-MoT pathosystem. When grown in soil and subjected to leaf defense priming, tomato plants co-applied with levan and the bacterial strains showed increased resistance to B. cinerea compared with plants treated with levan or single strains, and these effects were synergistic in some cases. For seed defense priming in soil, more synergistic effects on disease tolerance were observed in a non-fertilized soil as compared to a fertilized soil, suggesting that potential prebiotic effects of levan are more prominent in poor soils. The potential of using combinations of Bacilliaceae and levan in sustainable agriculture is discussed.

Characterization of a Bacillus subtilis S-16 Thiazole-Synthesis-Related Gene thiS Knockout and Antimicrobial Activity Analysis.

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.

Arabinose Plays an Important Role in Regulating the Growth and Sporulation of Bacillus subtilis NCD-2.

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.

First Report of Verticillium Wilt of Watermelon Caused by Verticillium dahliae in China.

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.

Genome mining and UHPLC-QTOF-MS/MS to identify the potential antimicrobial compounds and determine the specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2.

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.

Qualitative and Quantitative Analyses of the Colonization Characteristics of Bacillus subtilis Strain NCD-2 on Cotton Root.

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.

Identification of a New Genotype of Fusarium oxysporum f. sp. vasinfectum on Cotton in China.

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, ß-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.

Complete genome sequence of Bacillus velezensis B31, a potential biocontrol agent with ability of tolerance to fusaric acid and antagonism against Fusarium oxysporum.

Bacillus velezensis B31 is tolerant to fusaric acid, exhibits antagonism against Fusarium oxysporum, and has an excellent control effect on tomato fusarium wilt. Here, we present the complete genome sequence of B31, which contains 4,056,755 bp DNA with a G + C ratio of 46.39%. The genome has 3,838 protein-coding genes.

Improving crop health by synthetic microbial communities: Progress and prospects.

Crop health directly affects yields and food security. At present, agrochemicals such as fertilizers and pesticides are mainly used in agricultural production to promote crop health. However, long-term excessive utilization of agrochemicals will damage the ecological environment of farmlands and increase the safety risk of agricultural products. It is urgent to explore efficient and environment-friendly agricultural products. Rhizosphere microbiome are considered as the second genome of plants, which are closely related to crop health. Understanding the key functional microbes, microbe-microbe interactions, and plant-microbe interactions are fundamental for exploring the potential of beneficial microbes in promoting crop health. However, due to the heterogeneity and complexity of the natural environment, stimulating the function of indigenous microorganisms remains uncertain. Synthetic microbial community (SynCom) is an artificial combination of two or more different strain isolates of microorganisms, with different taxonomic, genetic, or functional characteristic. Because of the advantages of maintaining species diversity and community stability, SynCom has been widely applied in the fields of human health, environmental governance and industrial production, and may also have great potential in promoting crop health. We summarized the concept and research status of SynCom, expounded the principles and methods of constructing SynCom, and analyzed the research on the promotion of crop health by exploring the mechanism of plant-microbe interactions, promoting plant growth and development, and improving stress resistance. Finally, we envisaged the future prospects to guide the using SynCom to improve crop health.

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

A highly sensitive quantitative PCR (qPCR) method was developed for detection and quantification of Bacillus velezensis HMB26553 in cotton rhizosphere. The study aimed to develop a quantitative detection method for the strain HMB26553, and explore the relationship between its colonization of the cotton rhizosphere and its control effect. The whole genome sequence of strain HMB26553 was obtained by genome sequencing and a unique specific sequence pB-gene0026 on plasmid plaBV2 was identified by using high-throughput alignment against NCBI. Plasmid plaBV2 could be stably genetically inherited. Based on this sequence, specific primers for amplifying 106 bp and a minor groove binder (MGB) TaqMan probe for enhancing sensitivity were designed. The copy number of plaBV2 in strain HMB26553, which was 2, was confirmed by internal reference primers and the MGB TaqMan probe based on housekeeping gene gyrB. The established detection technique based on these primers and probes had high specificity and sensitivity compared to traditional plate counting method, with a detection limit of 1.5 copy genome. Using this method, the study discovered a likely correlation between the quantity of colonization in cotton rhizosphere and efficacy against cotton damping-off caused by Rhizoctonia after seed soaking and irrigation with strain HMB26553. Thus, this method provides scientific support for the rational application of strain HMB26553 in the future.

Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1.

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15-C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.

Screening Biocontrol Agents for Cash Crop Fusarium Wilt Based on Fusaric Acid Tolerance and Antagonistic Activity against Fusarium oxysporum.

Fusarium wilt, caused by Fusarium oxysporum, is one of the most notorious diseases of cash crops. The use of microbial fungicides is an effective measure for controlling Fusarium wilt, and the genus Bacillus is an important resource for the development of microbial fungicides. Fusaric acid (FA) produced by F. oxysporum can inhibit the growth of Bacillus, thus affecting the control efficacy of microbial fungicides. Therefore, screening FA-tolerant biocontrol Bacillus may help to improve the biocontrol effect on Fusarium wilt. In this study, a method for screening biocontrol agents against Fusarium wilt was established based on tolerance to FA and antagonism against F. oxysporum. Three promising biocontrol bacteria, named B31, F68, and 30833, were obtained to successfully control tomato, watermelon, and cucumber Fusarium wilt. Strains B31, F68, and 30833 were identified as B. velezensis by phylogenetic analysis of the 16S rDNA, gyrB, rpoB, and rpoC gene sequences. Coculture assays revealed that strains B31, F68, and 30833 showed increased tolerance to F. oxysporum and its metabolites compared with B. velezensis strain FZB42. Further experiments confirmed that 10 µg/mL FA completely inhibited the growth of strain FZB42, while strains B31, F68, and 30833 maintained normal growth at 20 µg/mL FA and partial growth at 40 µg/mL FA. Compared with strain FZB42, strains B31, F68, and 30833 exhibited significantly greater tolerance to FA.

Colonization Ability of Bacillus subtilis NCD-2 in Different Crops and Its Effect on Rhizosphere Microorganisms.

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.

Effect of incorporation of broccoli residues into soil on occurrence of verticillium wilt of spring-sowing-cotton and on rhizosphere microbial communities structure and function.

Cotton verticillium wilt (CVW) represented a typical plant soil-borne disease and resulted in widespread economic losses in cotton production. However, the effect of broccoli residues (BR) on verticillium wilt of spring-sowing-cotton was not clear. We investigated the effects of BR on CVW, microbial communities structure and function in rhizosphere of two cotton cultivars with different CVW resistance using amplicon sequencing methods. Results showed that control effects of BR on CVW of susceptible cultivar (cv. EJ-1) and resistant cultivar (cv. J863) were 58.49% and 85.96%, and the populations of V. dahliae decreased by 14.31% and 34.19%, respectively. The bacterial diversity indices significantly increased in BR treatment, while fungal diversity indices significantly decreased. In terms of microbial community composition, the abilities to recruit bacteria and fungi were enhanced in BR treatment, including RB41, Gemmatimonas, Pontibacter, Streptomyces, Blastococcus, Massilia, Bacillus, and Gibberella, Plectosphaerella, Neocosmospora, Aspergillus and Preussia. However, the relative abundances of Sphingomonas, Nocardioides, Haliangium, Lysobacter, Penicillium, Mortierella and Chaetomidium were opposite tendency between cultivars in BR treatment. According to PICRUSt analysis, functional profiles prediction showed that significant shifts in metabolic functions impacting KEGG pathways of BR treatment were related to metabolism and biosynthesis. FUNGuild analysis indicated that BR treatment altered the relative abundances of fungal trophic modes. The results of this study demonstrated that BR treatment decreased the populations of V. dahliae in soil, increased bacterial diversity, decreased fungal diversity, changed the microbial community structure and function, and increased the abundances of beneficial microorganisms.

Functional Analyses of the Bacillus velezensis HMB26553 Genome Provide Evidence That Its Genes Are Potentially Related to the Promotion of Plant Growth and Prevention of Cotton Rhizoctonia Damping-Off.

Bacillus spp. is one kind of the important representative biocontrol agents against plant diseases and promoting plant growth. In this study, the whole genomic sequence of bacterial strain HMB26553 was obtained. A phylogenetic tree based on the genome and ANI (average nucleotide identity), as well as dDDH (digital DNA-DNA hybridization), was constructed, and strain HMB26553 was identified as Bacillus velezensis. Fourteen biosynthetic gene clusters responsible for secondary metabolite were predicted via anti-SMASH, and six secondary metabolites were identified by UHPLC-QTOF-MS/MS (ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight tandem mass spectrometry). When the phytopathogen Rhizoctonia solani was treated with B. velezensis HMB26553, the mycelial structure changed, ROS (reactive oxygen species) accumulated, and the mitochondrial membrane potential decreased. Characteristics of strain HMB26553 were predicted and confirmed by genomic information and experiments, such as producing IAA, siderophore, extracellular enzymes and biofilm, as well as moving and promoting cotton growth. All these results suggested the mechanisms by which B. velezensis HMB26553 inhibits pathogen growth and promotes cotton growth, which likely provided the potential biocontrol agent to control cotton Rhizoctonia damping-off.

Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain.

IMPORTANCE: DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.

phoR sequences as a phylogenetic marker to differentiate the species in the Bacillus subtilis group.

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.

iTRAQ-based proteomic analysis of Bacillus subtilis strain NCD-2 regulated by PhoPR two-component system: A comparative analysis with transcriptomics revealed the regulation for fengycin production by branched chain amino acids.

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.

Insights into complex infection by two Pectobacterium species causing potato blackleg and soft rot.

Pectobacterium spp. are causative agents of blackleg and soft rot of potato. However, little is known about the relationship between the pathogenicity of mixed infections of different Pectobacterium spp. at different temperatures. In this study, two pectinolytic strains of Pectobacterium spp. were isolated from the same potato plant with typical symptoms of blackleg and identified as P. brasiliense and P. carotovorum by multilocus sequence analysis (MLSA), whole-genome phylogenetic tree construction, average nucleotide identity (ANI) analysis and digital DNA-DNA hybridization (dDDH). Plant cell wall degrading enzyme, including pectinases, cellulases and proteases, as the most important virulence factors, as well as pathogenicity toward potato tuber, were compared between the strains P. brasiliense BL-2 and P. carotovorum BL-4 at 28 â„ƒ. The results showed that P. carotovorum had higher cell wall-degrading enzyme activities and brought more severe disease symptoms to potato tubers than P. brasiliense. Moreover, the pathogenicity of P. carotovorum and P. brasiliense increased with increasing temperature (20, 25, 28, 32 â„ƒ). The pathogenicity was more severe when P. carotovorum strain BL-4 was co-inoculated with P. brasiliense strain BL-2, especially when the former exhibited an advantage in bacterial number at the initial time. The results of this study provide new insight for understanding the pathogenicity caused by mixed infections with different species of Pectobacterium spp., and they may provide some guidance for controlling potato blackleg and soft rot.

Unlocking the bacterial contact-dependent antibacterial activity to engineer a biocontrol alliance of two species from natural incompatibility to artificial compatibility.

Plant growth-promoting rhizobacteria (PGPR) contain various biocontrol bacteria with broad-spectrum antimicrobial activity, and their single species has been extensively applied to control crop diseases. The development of complex biocontrol community by mixing two or more PGPR members together is a promising strategy to enlarge the efficacy and scope of biocontrol. However, an effective method to assess the natural compatibility of PGPR members has not yet been established to date. Here, we developed such a tool by using the bacterial contact-dependent antibacterial activity (CDAA) as a probe. We showed that the CDAA events are common in two-species interactions in the four selected representative PGPRs, represented by the incompatible interaction of Lysobacter enzymogenes strain OH11 (OH11) and Lysobacter antibioticus strain OH13 (OH13). We further showed that the CDAA between OH11 and OH13 is jointly controlled by a contact-dependent killing device, called the type IV secretion system (T4SS). By deleting the respective T4SS synthesis genes, the T4SS in both strains was co-inactivated and this step unlocked  their natural CDAA, resulting in an engineered, compatible mutant alliance that co-displayed antibacterial and antifungal activity. Therefore, this study reveals that releasing bacterial CDAA is effective to rationally engineer the biocontrol community.