Identification and Characterization of an Antifungal Gene Mt1 from Bacillus subtilis by Affecting Amino Acid Metabolism in Fusarium graminearum.

Fusarium head blight is a devastating disease that causes significant economic losses worldwide. Fusarium graminearum is a crucial pathogen that requires close attention when controlling wheat diseases. Here, we aimed to identify genes and proteins that could confer resistance to F. graminearum. By extensively screening recombinants, we identified an antifungal gene, Mt1 (240 bp), from Bacillus subtilis 330-2. We recombinantly expressed Mt1 in F. graminearum and observed a substantial reduction in the production of aerial mycelium, mycelial growth rate, biomass, and pathogenicity. However, recombinant mycelium and spore morphology remained unchanged. Transcriptome analysis of the recombinants revealed significant down-regulation of genes related to amino acid metabolism and degradation pathways. This finding indicated that Mt1 inhibited amino acid metabolism, leading to limited mycelial growth and, thus, reduced pathogenicity. Based on the results of recombinant phenotypes and transcriptome analysis, we hypothesize that the effect of Mt1 on F. graminearum could be related to the metabolism of branched-chain amino acids (BCAAs), the most affected metabolic pathway with significant down-regulation of several genes. Our findings provide new insights into antifungal gene research and offer promising targets for developing novel strategies to control Fusarium head blight in wheat.

Isolation and Optimization of a Broad-Spectrum Synthetic Antimicrobial Peptide, Ap920-WI, from Arthrobacter sp. H5 for the Biological Control of Plant Diseases.

Antimicrobial peptides (AMPs) are naturally occurring molecules found in various organisms that can help to defend against invading microorganisms and reduce the likelihood of drug resistance development. This study focused on the isolation of new AMPs from the genome library of a Gram-positive bacterium called Arthrobacter sp. H5. To achieve this, we used the Bacillus subtilis expression system and employed bioinformatics techniques to optimize and modify the peptides, resulting in the development of a new synthetic antimicrobial peptide (SAMP). Ap920 is expected to be a new antimicrobial peptide with a high positive charge (+12.5). Through optimization, a new synthetic antimicrobial peptide, Ap920-WI, containing only 15 amino acids, was created. Thereafter, the antimicrobial and antifungal activities of Ap920-WI were determined using minimum inhibitory concentration (MIC) and the concentration for 50% of maximal effect (EC50). The Ap920-WI peptide was observed to target the outer membrane of fungal hyphae, leading to inhibition of growth in Rhizoctonia Solani, Sclerotinia sclerotiorum, and Botrytis cinerea. In plants, Ap920-WI showed significant antifungal activity and inhibited the infestation of S. sclerotiorum on rape leaves. Importantly, Ap920-WI was found to be safe for mammalian cells since it did not show any hemolytic activity against sheep red blood cells. Overall, the study found that the new synthetic antimicrobial peptide Ap920-WI exhibits broad-spectrum activity against microorganisms and may offer a new solution for controlling plant diseases, as well as hold potential for drug development.

Isolation of Potato Endophytes and Screening of Chaetomium globosum Antimicrobial Genes.

Antimicrobial peptides (AMPs) have natural antibacterial activities that pathogens find difficult to overcome. As a result of this occurrence, AMPs can act as an important substitute against the microbial resistance. In this study, we used plate confrontation tests to screen out 20 potential endophytes from potato tubers. Among them, endophyte F5 was found to significantly inhibit the growth of five different pathogenic fungi. Following that, phylogenetic analysis revealed that the internal transcribed spacer (ITS) sequences were 99% identical to Chaetomium globosum corresponding sequences. Thereafter, the Bacillus subtilis expression system was used to create a C. globosum cDNA library in order to isolate the resistance genes. Using this approach, the resistance gene screening technology in the indicator bacteria built-in library was used to identify two antimicrobial peptides, CgR2150 and CgR3101, with broad-spectrum antibacterial activities. Furthermore, the results showed that CgR2150 and CgR3101 have excellent UV, thermal, and enzyme stabilities. Also, these two peptides can significantly inhibit the growth of various bacteria (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Clavibacter michiganensis, and Clavibacter fangii) and fungi (Fusarium graminearum, Rhizoctonia solani, and Botrytis cinerea). Scanning electron microscopy (SEM) observations revealed that CgR2150 and CgR3101 peptides act against bacteria by disrupting bacterial cell membranes. Moreover, hemolytic activity assay showed that neither of the two peptides exhibited significant hemolytic activity. To conclude, the antimicrobial peptides CgR2150 and CgR3101 are promising in the development of a new antibacterial agent and for application in plant production.

Prediction and Activity of a Cationic α-Helix Antimicrobial Peptide ZM-804 from Maize.

Antimicrobial peptides (AMPs) are small molecules consisting of less than fifty residues of amino acids. Plant AMPs establish the first barrier of defense in the innate immune system in response to invading pathogens. The purpose of this study was to isolate new AMPs from the Zea mays L. inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria. In silico, the Collection of Anti-Microbial Peptides (CAMPR3), a computational AMP prediction server, was used to screen a cDNA library for AMPs. A ZM-804 peptide, isolated from the Z. mays L. inbred line B73 cDNA library, was predicted as a new cationic AMP with high prediction values. ZM-804 was tested against eleven pathogens of Gram-negative and Gram-positive bacteria and exhibited high antimicrobial activities as determined by the minimal inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). A confocal laser scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes. SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato (Pst) DC3000 caused by ZM-804. In planta, ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by Pst DC3000. Moreover, four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response (HR) in tobacco leaves in response to low ZM-804 concentrations. ZM-804 exhibits low hemolytic activity against mouse red blood cells (RBCs) and is relatively safe for mammalian cells. In conclusion, the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control. Additionally, the ZM-804 peptide is considered a promising candidate for human and animal drug development.

Antimicrobial Peptides from Plants: A cDNA-Library Based Isolation, Purification, Characterization Approach and Elucidating Their Modes of Action.

Even in a natural ecosystem, plants are continuously threatened by various microbial diseases. To save themselves from these diverse infections, plants build a robust, multilayered immune system through their natural chemical compounds. Among the several crucial bioactive compounds possessed by plants' immune systems, antimicrobial peptides (AMPs) rank in the first tier. These AMPs are environmentally friendly, anti-pathogenic, and do not bring harm to humans. Antimicrobial peptides can be isolated in several ways, but recombinant protein production has become increasingly popular in recent years, with the Escherichia coli expression system being the most widely used. However, the efficacy of this expression system is compromised due to the difficulty of removing endotoxin from its system. Therefore, this review suggests a high-throughput cDNA library-based plant-derived AMP isolation technique using the Bacillus subtilis expression system. This method can be performed for large-scale screening of plant sources to classify unique or homologous AMPs for the agronomic and applied field of plant studies. Furthermore, this review also focuses on the efficacy of plant AMPs, which are dependent on their numerous modes of action and exceptional structural stability to function against a wide range of invaders. To conclude, the findings from this study will be useful in investigating how novel AMPs are distributed among plants and provide detailed guidelines for an effective screening strategy of AMPs.

Isolation of Antimicrobial Genes from Oryza rufipogon Griff by Using a Bacillus subtilis Expression System with Potential Antimicrobial Activities.

Antimicrobial genes are distributed in all forms of life and provide a primary defensive shield due to their unique broad-spectrum resistance activities. To better isolate these genes, we used the Bacillus subtilis expression system as the host cells to build Oryza rufipogon Griff cDNA libraries and screen potential candidate genes from the library at higher flux using built-in indicator bacteria. We observed that the antimicrobial peptides OrR214 and OrR935 have strong antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria, as well as several fungal pathogens. Owing to their high thermal and enzymatic stabilities, these two peptides can also be used as field biocontrol agents. Furthermore, we also found that the peptide OrR214 (MIC 7.7-10.7 µM) can strongly inhibit bacterial growth compared to polymyxin B (MIC 5-25 µM) and OrR935 (MIC 33-44 µM). The cell flow analysis, reactive oxygen burst, and electron microscopy (scanning and transmission electron microscopy) observations showed that the cell membranes were targeted by peptides OrR214 and OrR935, which revealed the mode of action of bacteriostasis. Moreover, the hemolytic activity, toxicity, and salt sensitivity experiments demonstrated that these two peptides might have the potential to be used for clinical applications. Overall, OrR214 and OrR935 antimicrobial peptides have a high-throughput bacteriostatic activity that acts as a new form of antimicrobial agent and can be used as a raw material in the field of drug development.

Enhanced Nicotiana benthamiana immune responses caused by heterologous plant genes from Pinellia ternata.

BACKGROUND: Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F1 hybrid was proposed. RESULTS: By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F1 hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance. CONCLUSIONS: Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F1 hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

Pyramiding of nine transgenes in maize generates high-level resistance against necrotrophic maize pathogens.

Key message Nine transgenes from different categories, viz. plant defense response genes and anti-apoptosis genes, played combined roles in maize to inhibit the necrotrophic pathogens Rhizoctonia solani and Bipolaris maydis. Maize sheath blight and southern corn leaf blight are major global threats to maize production. The management of these necrotrophic pathogens has encountered limited success due to the characteristics of their lifestyle. Here, we presented a transgenic pyramiding breeding strategy to achieve nine different resistance genes integrated in one transgenic maize line to combat different aspects of necrotrophic pathogens. These nine genes, selected from two different categories, plant defense response genes (Chi, Glu, Ace-AMP1, Tlp, Rs-AFP2, ZmPROPEP1 and Pti4), and anti-apoptosis genes (Iap and p35), were successfully transferred into maize and further implicated in resistance against the necrotrophic pathogens Rhizoctonia solani and Bipolaris maydis. Furthermore, the transgenic maize line 910, with high expression levels of the nine integrated genes, was selected from 49 lines. Under greenhouse and field trial conditions, line 910 showed significant resistance against maize sheath blight and southern corn leaf blight diseases. Higher-level resistance was obtained after the pyramiding of more resistance transgenes from different categories that function via different mechanisms. The present study provides a successful strategy for the management of necrotrophic pathogens.

Complete genome sequence of a putative novel victorivirus from Ustilaginoidea virens.

Ustilaginoidea virens is the causal agent of a serious disease of rice. Here, we report the presence of five dsRNA bands ranging from about 1.2, 1.5, 1.7, and 1.8 to 5.6 kb in an isolate of this fungus from China and the complete sequence of the largest dsRNA segment, putatively representing the genome of a novel virus, designated as Ustilaginoidea virens RNA virus 1 (UvRV1), UvRV1, which has a genome length of 5567 bp and has two consecutive open reading frames (ORFs) with a five-nucleotide overlap. Phylogenetic analysis showed that UvRV1 belongs to the genus of Victorivirus in the family Totiviridae.

Genomic organization of a novel partitivirus from the phytopathogenic fungus Ustilaginoidea virens.

From the plant pathogen Ustilaginoidea virens, four double-stranded RNA (dsRNA) segments designated Uv-dsRNA1, -2, -3, and -4 were isolated, cloned, and sequenced. Uv-dsRNA1 (1775 bp) and -2 (1588 bp) potentially encode an RNA-dependent RNA polymerase (RdRp) and a viral coat protein (CP), respectively. Since the RdRp and CP sequences encoded by Uv-dsRNA1 and -2, respectively, are most closely related to, but clearly distinct from, those of viruses of the genus Partitivirus, they appear to be the two genome segments of a new partitivirus, for which the name Ustilaginoidea virens partitivirus 1 is proposed. In contrast, Uv-dsRNA3 (1352 bp) did not share significant sequence similarity with GenBank sequences, and the ORF of Uv-dsRNA4 (1119 bp) was only 32 % identical to a functionally unknown protein (GaRVMS2s3gp1) encoded by Gremmeniella abietina RNA virus MS2.

BsR1, a broad-spectrum antibacterial peptide with potential for plant protection.

IMPORTANCE: This study addresses the critical need for new antibacterial drugs in the face of bacterial multidrug resistance resulting from antibiotic overuse. It highlights the significance of antimicrobial peptides as essential components of innate immunity in animals and plants, which have been proven effective against multidrug-resistant bacteria and are difficult to develop resistance against. This study successfully synthesizes a broad-spectrum antibacterial peptide, BsR1, with strong inhibitory activities against various Gram-positive and Gram-negative bacteria. BsR1 demonstrates favorable stability and a mode of action that damages bacterial cell membranes, leading to cell death. It also exhibits biological safety and shows potential in enhancing disease resistance in rice. This research offers a novel approach and potential medication for antibacterial drug development, presenting a valuable tool in combating pathogenic microorganisms, particularly in plants.

Nonhost resistance of barley to different fungal pathogens is associated with largely distinct, quantitative transcriptional responses.

Nonhost resistance protects plants against attack by the vast majority of potential pathogens, including phytopathogenic fungi. Despite its high biological importance, the molecular architecture of nonhost resistance has remained largely unexplored. Here, we describe the transcriptional responses of one particular genotype of barley (Hordeum vulgare subsp. vulgare 'Ingrid') to three different pairs of adapted (host) and nonadapted (nonhost) isolates of fungal pathogens, which belong to the genera Blumeria (powdery mildew), Puccinia (rust), and Magnaporthe (blast). Nonhost resistance against each of these pathogens was associated with changes in transcript abundance of distinct sets of nonhost-specific genes, although general (not nonhost-associated) transcriptional responses to the different pathogens overlapped considerably. The powdery mildew- and blast-induced differences in transcript abundance between host and nonhost interactions were significantly correlated with differences between a near-isogenic pair of barley lines that carry either the Mlo wild-type allele or the mutated mlo5 allele, which mediates basal resistance to powdery mildew. Moreover, during the interactions of barley with the different host or nonhost pathogens, similar patterns of overrepresented and underrepresented functional categories of genes were found. The results suggest that nonhost resistance and basal host defense of barley are functionally related and that nonhost resistance to different fungal pathogens is associated with more robust regulation of complex but largely nonoverlapping sets of pathogen-responsive genes involved in similar metabolic or signaling pathways.

Corrigendum: Prediction and Characterization of Cationic Arginine-Rich Plant Antimicrobial Peptide SM-985 From Teosinte (Zea mays ssp. mexicana).

[This corrects the article DOI: 10.3389/fmicb.2020.01353.].

Maize endophytic microbial-communities revealed by removing PCR and 16S rRNA sequencing and their synthetic applications to suppress maize banded leaf and sheath blight.

Endophytic microbial-communities have specific beneficial functions and are considered key drivers for host plant health. The removing-PCR (R-PCR) is a simple culture-independent cost-effective method to identify endophytic microbial-communities. Microbial communities from maize plant grown in different soil types were identified and characterized via the R-PCR and 16S rRNA sequencing. Culture-dependent microbial community identified through 16S rRNA gene sequencing, further these bacterial communities screened for antagonistic assay against Rhizoctonia solani WH1, in vitro compatibility tests, plant-growth-promoting traits and BIOLOG identification. After that, synthetic-communities (SycomA and SycomB) were prepared by mixing different compatible bacterial-strains to use as an inoculant to suppress pathogens of maize. We identified 167 bacterial operational taxonomic units (OTUs) and unexpected 8 fungal OTUs through the R-PCR, whereas, 95 bacterial OTUs via 16S rRNA sequencing from maize leaves and roots. SycomA and SycomB treatments suppressed the disease level and promoted growth attributes more effectively as compare to the single bacterial-strain and control treatments. This study establishes an efficient approach to isolate, identify and characterize diverse endophytic microbial-community assembly in maize leaves and roots, to successfully apply particular microbes to improve crop growth in soils affected by soil-borne-pathogens.

Prediction and Characterization of Cationic Arginine-Rich Plant Antimicrobial Peptide SM-985 From Teosinte (Zea mays ssp. mexicana).

Antimicrobial peptides (AMPs) are effective against different plant pathogens and newly considered as part of plant defense systems. From prokaryotes to eukaryotes, AMPs can exist in all forms of life. SM-985 is a cationic AMP (CAMP) isolated from the cDNA library of Mexican teosinte (Zea mays ssp. mexicana). A computational prediction server running with different algorithms was used to screen the teosinte cDNA library for AMPs, and the SM-985 peptide was predicted as an AMP with high probability prediction values. SM-985 is an arginine-rich peptide and composed of 21 amino acids (MW: 2671.06 Da). The physicochemical properties of SM-985 are very promising as an AMP, including the net charge (+8), hydrophobicity ratio of 23%, Boman index of 5.19 kcal/mol, and isoelectric point of 12.95. The SM-985 peptide has amphipathic α-helix conformations. The antimicrobial activity of SM-985 was confirmed against six bacterial plant pathogens, and the MIC of SM-985 against Gram-positive indicators was 8 µM, while the MIC of SM-985 against Gram-negative indicators was 4 µM. The SM-985 interacting with the bacterial membrane and this interaction were examined by treatment of the bacterial indicators with FITC-SM-985 peptide, which showed a high binding affinity of SM-985 to the bacterial membrane (whether Gram-positive or Gram-negative). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the treated bacteria with SM-985 demonstrated cell membrane damage and cell lysis. In vivo antimicrobial activity was examined, and SM-985 prevented leaf spot disease infection caused by Pst DC3000 on Solanum lycopersicum. Moreover, SM-985 showed sensitivity to calcium chloride salt, which is a common feature of CAMPs.

Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system.

Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. We chose Bacillus subtilis as a target pathogen indicator and transferred the Aegilops tauschii Cosson cDNA library into B. subtilis cells. Expression of the candidate antimicrobial gene can inhibit B. subtilis cell growth. Using this strategy, we screened six genes that have an internal effect on the indicator bacteria. Then, the secreted proteins were extracted and tested; two genes, AtR100 and AtR472, were found to have strong external antimicrobial activities with broad-spectrum resistance against Xanthomonas oryzae pv. oryzicola, Clavibacter fangii, and Botrytis cinerea. Additionally, thermal stability tests indicated that the antimicrobial activities of both proteins were thermostable. Furthermore, these two proteins exhibited no significant hemolytic activities. To test the feasibility of application at the industrial level, liquid fermentation and spray drying of these two proteins were conducted. Powder dilutions were shown to have significant inhibitory effects on B. cinerea. Fluorescence microscopy and flow cytometry results showed that the purified protein impaired and targeted the cell membranes. This study revealed that these two antimicrobial peptides could potentially be used for replacing antibiotics, which would provide the chance to reduce the emergence of drug resistance.

A quinone-dependent dehydrogenase and two NADPH-dependent aldo/keto reductases detoxify deoxynivalenol in wheat via epimerization in a Devosia strain.

The Fusarium mycotoxin deoxynivalenol (DON) is typically controlled by fungicides. Here, we report DON detoxification using enzymes from the highly active Devosia strain D6-9 which degraded DON at 2.5 µg/min/108 cells. Strain D6-9 catabolized DON to 3-keto-DON and 3-epi-DON, completely removing DON in wheat. Genome analysis of three Devosia strains (D6-9, D17, and D13584), with strain D6-9 transcriptomes, identified three genes responsible for DON epimerization. One gene encodes a quinone-dependent DON dehydrogenase QDDH which oxidized DON into 3-keto-DON. Two genes encode the NADPH-dependent aldo/keto reductases AKR13B2 and AKR6D1 that convert 3-keto-DON into 3-epi-DON. Recombinant proteins expressed in Escherichia coli efficiently degraded DON in wheat grains. Molecular docking and site-directed mutagenesis revealed that residues S497, E499, and E535 function in QDDH's DON-oxidizing activity. These results advance potential microbial and enzymatic elimination of DON in agricultural samples and lend insight into the underlying mechanisms and molecular evolution of DON detoxification.

UPLC-QTOF-MS metabolomics analysis revealed the contributions of metabolites to the pathogenesis of Rhizoctonia solani strain AG-1-IA.

To explore the pathogenesis of Rhizoctonia solani and its phytotoxin phenylacetic acid (PAA) on maize leaves and sheaths, treated leaf and sheath tissues were analyzed and interpreted by ultra-performance liquid chromatography-mass spectrometry combined with chemometrics. The PAA treatment had similar effects to those of R. solani on maize leaves regarding the metabolism of traumatin, phytosphingosine, vitexin 2'' O-beta-D-glucoside, rutin and DIBOA-glucoside, which were up-regulated, while the synthesis of OPC-8:0 and 12-OPDA, precursors for the synthesis of jasmonic acid, a plant defense signaling molecule, was down-regulated under both treatments. However, there were also discrepancies in the influences exhibited by R. solani and PAA as the metabolic concentration of zeaxanthin diglucoside in the R. solani infected leaf group decreased. Conversely, in the PAA-treated leaf group, the synthesis of zeaxanthin diglucoside was enhanced. Moreover, although the synthesis of 12 metabolites were suppressed in both the R. solani- and PAA-treated leaf tissues, the inhibitory effect of R. solani was stronger than that of PAA. An increased expression of quercitrin and quercetin 3-O-glucoside was observed in maize sheaths treated by R. solani, while their concentrations were not changed significantly in the PAA-treated sheaths. Furthermore, a significant decrease in the concentration of L-Glutamate, which plays important roles in plant resistance to necrotrophic pathogens, only occurred in the R. solani-treated sheath tissues. The differentiated metabolite levels may be the partial reason of why maize sheaths were more susceptible to R. solani than leaves and may explain the underlying mechanisms of R. solani pathogenesis.

Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system.

Antimicrobial genes are found in all classes of life. To efficiently isolate these genes, we used Bacillus subtilis and Escherichia coli as target indicator bacteria and transformed them with cDNA libraries. Among thousands of expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external effect from antimicrobial proteins working from outside of the cells) and the high throughput ability of screening. We found that B. subtilis is more efficient and reliable than E. coli. Using the B. subtilis expression system, we identified 19 novel, broad-spectrum antimicrobial genes. Proteins expressed by these genes were extracted and tested, exhibiting strong external antibacterial, antifungal and nematicidal activities. Furthermore, these newly isolated proteins could control plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for clinical medicine, as they exhibited no haemolytic activity. Based on our findings, we speculated that plant, animal and human pathogenic bacteria, fungi or even cancer cells might be taken as the indicator target cells for screening specific resistance genes.

Isolated Bacillus subtilis strain 330-2 and its antagonistic genes identified by the removing PCR.

Plant growth-promoting bacteria (PGPB) may trigger tolerance against biotic/abiotic stresses and growth enhancement in plants. In this study, an endophytic bacterial strain from rapeseed was isolated to assess its role in enhancing plant growth and tolerance to abiotic stresses, as well as banded leaf and sheath blight disease in maize. Based on 16S rDNA and BIOLOG test analysis, the 330-2 strain was identified as Bacillus subtilis. The strain produced indole-3-acetic acid, siderophores, lytic enzymes and solubilized different sources of organic/inorganic phosphates and zinc. Furthermore, the strain strongly suppressed the in vitro growth of Rhizoctonia solani AG1-IA, Botrytis cinerea, Fusarium oxysporum, Alternaria alternata, Cochliobolus heterostrophus, and Nigrospora oryzae. The strain also significantly increased the seedling growth (ranging 14-37%) of rice and maize. Removing PCR analysis indicated that 114 genes were differentially expressed, among which 10%, 32% and 10% were involved in antibiotic production (e.g., srfAA, bae, fen, mln, and dfnI), metabolism (e.g., gltA, pabA, and ggt) and transportation of nutrients (e.g., fhu, glpT, and gltT), respectively. In summary, these results clearly indicate the effectiveness and mechanisms of B. subtilis strain 330-2 in enhancing plant growth, as well as tolerance to biotic/abiotic stresses, which suggests that the strain has great potential for commercialization as a vital biological control agent.