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Autophagy is the main intracellular degradation system by which cytoplasmic materials are transported to and degraded in the vacuole/lysosome of eukaryotic cells, and it also controls cellular differentiation and virulence in a variety of filamentous fungi. However, the contribution of the autophagic pathway to fungal development and pathogenicity in the important maize pathogen and mycotoxigenic fungus Fusarium verticillioides is still unknown. In this study, we characterized two autophagy-related proteins, FvAtg4 and FvAtg8. The F. verticillioides deletion mutants ΔFvAtg4 and ΔFvAtg8 were impaired in autophagosome formation, aerial hyphal formation, sexual growth, lipid turnover, pigmentation and fungal virulence. Interestingly, ΔFvAtg4 and ΔFvAtg8 were defective in fumonisin B1 (FB1) synthesis, which may have resulted from decreased intracellular levels of alanine in the mutants. Our results indicate that FvAtg4 and FvAtg8 contribute to F. verticillioides pathogenicity by regulating the autophagic pathway to control lipid turnover, fumonisin biosynthesis, and pigmentation during its infectious cycle.

Biofilms are architecturally complex communities of microbial cells held together by a self-produced extracellular matrix. Considerable research has focused on the environmental signals that trigger or inhibit biofilm formation by affecting cellular signalling pathways; however, response to soil cues in plant-associated Bacillus has remained largely unaddressed. Therefore, we aimed to investigate the effect of Zn(II) ions in biofilm formation of Bacillus amyloliquefaciens FZB42. We demonstrated that the biofilm formation of B. amyloliquefaciens FZB42 was abolished by Zn(II) at non-deleterious concentrations. Moreover, Zn(II) blocked matrix exopolysaccharide and TasA accumulations. Furthermore, the presence of Zn(II) suppressed expression of the response regulator Spo0F but not of sensor histidine kinases KinA-D. Suppression of phosphorelay by excess Zn interferes with sinI induction under biofilm-inducing conditions, leading to repression of transcription of operons epsA-O and tapA-sigW-tasA. Addition of Zn(II) decreased the intracellular Mn(II) level by competing for binding to the solute-binding protein MntA during Mn(II) uptake. These results suggest that the metal ion Zn(II) has a negative effect on biofilm formation in the plant growth promoting and biocontrol bacterium B. amyloliquefaciens FZB42.

Sclerotinia sclerotiorum is a devastating necrotrophic pathogen that infects multiple crops, and its control is an unremitting challenge. In this work, we attempted to gain insights into the pivotal role of lipopeptides (LPs) in the antifungal activity of Bacillus amyloliquefaciens EZ1509. In a comparative study involving five Bacillus strains, B. amyloliquefaciens EZ1509 harboring four LPs biosynthetic genes (viz. surfactin, iturin, fengycin, and bacilysin) exhibited promising antifungal activity against S. sclerotiorum in a dual-culture assay. Our data demonstrated a remarkable upsurge in LPs biosynthetic gene expression through quantitative reverse transcription PCR during in vitro interaction assay with S. sclerotiorum. Maximum upregulation in LPs biosynthetic genes was observed on the second and third days of in vitro interaction, with iturin and fengycin being the highly expressed genes. Subsequently, Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis confirmed the presence of LPs in the inhibition zone. Scanning electron microscope analysis showed disintegration, shrinkage, plasmolysis, and breakdown of fungal hyphae. During in planta evaluation, S. sclerotiorum previously challenged with EZ1509 showed significant suppression in pathogenicity on detached leaves of tobacco and rapeseed. The oxalic acid synthesis was also significantly reduced in S. sclerotiorum previously confronted with antagonistic bacterium. The expression of major virulence genes of S. sclerotiorum, including endopolygalacturonase-3, oxalic acid hydrolase, and endopolygalacturonase-6, was significantly downregulated during in vitro confrontation with EZ1509.

Fusarium graminearum and Fusarium verticillioides are fungal pathogens that cause diseases in cereal crops, such as Fusarium head blight (FHB), seedling blight, and stalk rot. They also produce a variety of mycotoxins that reduce crop yields and threaten human and animal health. Several strategies for controlling these diseases have been developed. However, due to a lack of resistant cultivars and the hazards of chemical fungicides, efforts are now focused on the biocontrol of plant diseases, which is a more sustainable and environmentally friendly approach. In the present study, the lipopeptide mycosubtilin purified from Bacillus subtilis ATCC6633 significantly suppressed the growth of F. graminearum PH-1 and F. verticillioides 7600 in vitro. Mycosubtilin caused the destruction and deformation of plasma membranes and cell walls in F. graminearum hyphae. Additionally, mycosubtilin inhibited conidial spore formation and germination of both fungi in a dose-dependent manner. In planta experiments demonstrated the ability of mycosubtilin to control the adverse effects caused by F. graminearum and F. verticillioides on wheat heads and maize kernels, respectively. Mycosubtilin significantly decreased the production of deoxynivalenol (DON) and B-series fumonisins (FB1, FB2 and FB3) in infected grains, with inhibition rates of 48.92, 48.48, 52.42, and 59.44%, respectively. The qRT-PCR analysis showed that mycosubtilin significantly downregulated genes involved in mycotoxin biosynthesis. In conclusion, mycosubtilin produced by B. subtilis ATCC6633 was shown to have potential as a biological agent to control plant diseases and Fusarium toxin contamination caused by F. graminearum and F. verticillioides.

This study elaborates inter-kingdom signaling mechanisms, presenting a sustainable and eco-friendly approach to combat biotic as well as abiotic stress in wheat. Fusarium graminearum is a devastating pathogen causing head and seedling blight in wheat, leading to huge yield and economic losses. Psychrophilic Bacillus atrophaeus strain TS1 was found as a potential biocontrol agent for suppression of F. graminearum under low temperature by carrying out extensive biochemical and molecular studies in comparison with a temperate biocontrol model strain Bacillus amyloliquefaciens FZB42 at 15 and 25 °C. TS1 was able to produce hydrolytic extracellular enzymes as well as antimicrobial lipopeptides, i.e., surfactin, bacillomycin, and fengycin, efficiently at low temperatures. The Bacillus strain-induced oxidative cellular damage, ultrastructural deformities, and novel genetic dysregulations in the fungal pathogen as the bacterial treatment at low temperature were able to downregulate the expression of newly predicted novel fungal genes potentially belonging to necrosis inducing protein families (fgHCE and fgNPP1). The wheat pot experiments conducted at 15 and 25 °C revealed the potential of TS1 to elicit sudden induction of plant defense, namely, H2O2 and callose enhanced activity of plant defense-related enzymes and induced over-expression of defense-related genes which accumulatively lead to the suppression of F. graminearum and decreased diseased leaf area.

Fusarium graminearum is a destructive fungal pathogen that threatens the production and quality of wheat, and controlling this pathogen is a significant challenge. As the cost-effective homolog of melatonin, 5-methoxyindole showed strong activity against F. graminearum. In the present study, our results showed the strong adverse activity of 5-methoxyindole against F. graminearum by inhibiting its growth, formation, and conidia germination. In addition, 5-methoxyindole could induce malformation, reactive oxygen species (ROS) accumulation, and cell death in F. graminearum hyphae and conidia. In response to 5-methoxyindole, F. graminearum genes involved in scavenging reactive oxygen species were significantly downregulated. Overall, these findings reveal the mechanism of antifungal action of melatonin-homolog 5-methoxyindole. To the best of our knowledge, this is the first report that a novel melatonin homolog confers strong antifungal activity against F. graminearum, and 5-methoxyindole is a potential compound for protecting wheat plants from F. graminearum infection.

Bacillus strains are well studied for antagonistic effect against fungal pathogens, but the selection of potential antifungal strains is laborious and time-consuming process. Newly developed genetic markers and LC-MS based detection was undertaken simultaneously to detect eight antimicrobial compounds viz., surfactin, bacillomycin, iturin, fengycin/plipastatin, bacilysin, bacillaene, bacillibactin and plantazolicin in forty-seven Bacillus strains. Out of these strains, 19 were positive for the presence of marker genes encoding antimicrobial compounds. Bacillus strains FZB42, EZ1509, EZ1507, VM10, GBAC46, VM49 and LLCG43 possessed genes for maximum number of antimicrobial compounds. LC-MS analysis of antimicrobial compounds showed corresponding results except OKB105 and 168. Contrary to marker-based detection of genes, LC-MS analysis revealed that OKB105 can produce surfactin but unable to synthesize fengycin, while 168 was deficit in both compounds. To assay antifungal potential, 19 Bacillus strains and their methanolic extracts were tested in vitro to inhibit mycelial growth of S. sclerotiorum. Results revealed that EZ1509, VM10, GBAC46, VM49 and FZB42 showed highest inhibitory activity. A bioassay on detached rapeseed leaves demonstrated that strains VM10, EZ1509, FZB42 and GBAC46 were excellent in reducing lesion diameter, while, OKB105 and 168 were completely ineffective to control S. sclerotiorum. Interestingly, antifungal activity of Bacillus strains was positively co-related to the number of antimicrobial genes, indicating their role in antifungal activity of Bacillus strains. Our findings suggest that combining genetic markers and LC-MS analysis can rapidly screen Potential Bacillus strains with antifungal attributes and this screening method can serve as foundation for the development of new biopesticides.

The bacterium Pantoea ananatis is associated with devastating plant diseases that cause serious economic losses. Strain DZ-12 was previously isolated from maize brown rot leaves in Hebei Province, China and its genome sequencing revealed that it belongs to P. ananatis. It contains a large, endogenous plasmid, pDZ-12. Different studies have shown that virulence determinants are frequently carried on plasmids. To determine whether pDZ-12 from P. ananatis has any effect on pathogenicity, the plasmid was eliminated by substituting its native replication genes with temperature-sensitive replication genes. The resulting temperature-sensitive plasmid could be cured by growing cells at high temperature (37℃). Loss of pDZ-12 from P. ananatis DZ-12 led to a decreased disease severity in maize plants suggesting that the endogenous plasmid is important for pathogenesis. Loss of pDZ-12 also affected the ability of the bacterium to form biofilms. The study provides the first evidence that the endogenous plasmid of P. ananatis DZ-12 is important for pathogenesis in maize plants and carries genes involved in biofilm formation. This study also presents the first report on curing a plasmid from P. ananatis.

Background Microbial volatiles play an expedient role in the agricultural ecological system by enhancing plant growth and inducing systemic resistance against plant pathogens, without causing hazardous effects on the environment. To explore the effects of VOCs of Ralstonia solanacearum TBBS1 (Rs) on tobacco plant growth and on plant growth promoting efficiency of VOCs produced by Bacillus subtilis SYST2, experiments were conducted both in vitro and in planta. Results The VOCs produced by SYST2 significantly enhanced the plant growth and induced the systemic resistance (ISR) against wilt pathogen Rs in all experiments. The SYST2-VOCs significantly increased PPO and PAL activity and over-expressed the genes relating to expansin, wilt resistance, and plant defense while repressed the genes relating to ethylene production. More interestingly, VOCs produced by pathogen, Rs had no significant effect on plant growth; however, Rs-VOCs decreased the growth promoting potential of SYST2-VOCs when plants were exposed to VOCs produced by both SYST2 and Rs. The co-culture of SYST2 and Rs revealed that they inhibited the growth of each other; however, the inhibition of Rs by SYST2-VOCs appeared to be greater than that of SYST2 by Rs-VOCs. Conclusion Our findings provide new insights regarding the interaction among SYST2-VOCs, Rs-VOCs and plant, resulting in growth promotion and induced systemic resistance against the bacterial wilt pathogen Rs. This is the first report of the effect of VOCs produced by pathogenic microorganism on plant growth and on plant growth-promoting and systemic resistance-inducing potential of PGPR strain SYST2. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1083-6) contains supplementary material, which is available to authorized users.

Abiotic stress in plants pose a major threat to cereal crop production worldwide and cold stress is also notorious for causing a decrease in plant growth and yield in wheat. The present study was designed to alleviate cold stress on plants by inoculating psychrophilic PGPR bacteria belonging to Bacillus genera isolated from extreme rhizospheric environments of Qinghai-Tibetan plateau. The genetic screening of psychrophilic Bacillus spp. CJCL2, RJGP41 and temperate B. velezensis FZB42 revealed presence of genetic features corresponding to cold stress response, membrane transport, signal transduction and osmotic regulation. Subsequently, the time frame study for the expression of genes involved in these pathways was also significantly higher in psychrophilic strains as analyzed through qPCR analysis at 4 ℃. The inoculated cold tolerant Bacillus strains also aided in inducing stress response in wheat by regulating abscisic acid, lipid peroxidation and proline accumulation pathways in a beneficial manner. Moreover, during comparative analysis of growth promotion in wheat all three Bacillus strains showed significant results at 25 ℃. Whereas, psychrophilic Bacillus strains CJCL2 and RJGP41 were able to positively regulate the expression of phytohormones leading to significant improvement in plant growth under cold stress.

Nearly 1400 Bacillus strains growing in the plant rhizosphere were sampled from different sites on the Qinghai-Tibetan Plateau. Forty-five of the isolates, selected due to their biocontrol activity, were genome-sequenced and their taxonomic identification revealed that they were representatives of the Bacillus subtilis species complex (20) and the Bacillus cereus group (9). Majority of the remaining strains were found closely related to Bacillus pumilus, but their average nucleotide identity based on BLAST and electronic DNA/DNA hybridization values excluded closer taxonomic identification. A total of 45 different gene clusters involved in synthesis of secondary metabolites were detected by mining the genomes of the 45 selected strains. Except eight mesophilic strains, the 37 remaining strains were found either cold-adapted or psychrophilic, able to propagate at 10°C and below (Bacillus wiedmannii NMSL88 and Bacillus sp. RJGP41). Pot experiments performed at 10°C with winter wheat seedlings revealed that cold-adapted representatives of B. pumilus, B. safensis and B. atrophaeus promoted growth of the seedlings under cold conditions, suggesting that these bacilli isolated from a cold environment are promising candidates for developing of bioformulations useful for application in sustainable agriculture under environmental conditions unfavourable for the mesophilic bacteria presently in use.

Thirteen gene clusters involved in non-ribosomal and ribosomal synthesis of secondary metabolites with putative antimicrobial action have been identified within the genome of FZB42, the model for Gram-positive biocontrol strains. These gene clusters cover around ten percentage of the whole genome. Antimicrobial compounds not only suppress growth of plant pathogenic bacteria and fungi but could also stimulate induced systemic response (ISR) in plants. Recently, it has been found that besides secondary metabolites also a blend of volatile organic compounds (VOCs) is involved in the biocontrol effect exerted by FZB42 against plant pathogens suggesting complexity of biocontrol function. Cyclic lipopeptides and volatiles produced by plant-associated bacilli trigger pathways of induced systemic resistance (ISR), which protect plants against attacks of pathogenic microbes, viruses, and nematodes. Stimulation of ISR by bacterial metabolites is likely the main mechanism responsible for biocontrol action of FZB42.

Bacillus velezensis FZB42, the model strain for Gram-positive plant-growth-promoting and biocontrol rhizobacteria, has been isolated in 1998 and sequenced in 2007. In order to celebrate these anniversaries, we summarize here the recent knowledge about FZB42. In last 20 years, more than 140 articles devoted to FZB42 have been published. At first, research was mainly focused on antimicrobial compounds, apparently responsible for biocontrol effects against plant pathogens, recent research is increasingly directed to expression of genes involved in bacteria–plant interaction, regulatory small RNAs (sRNAs), and on modification of enzymes involved in synthesis of antimicrobial compounds by processes such as acetylation and malonylation. Till now, 13 gene clusters involved in non-ribosomal and ribosomal synthesis of secondary metabolites with putative antimicrobial action have been identified within the genome of FZB42. These gene clusters cover around 10% of the whole genome. Antimicrobial compounds suppress not only growth of plant pathogenic bacteria and fungi, but could also stimulate induced systemic resistance (ISR) in plants. It has been found that besides secondary metabolites also volatile organic compounds are involved in the biocontrol effect exerted by FZB42 under biotic (plant pathogens) and abiotic stress conditions. In order to facilitate easy access to the genomic data, we have established an integrating data bank ‘AmyloWiki’ containing accumulated information about the genes present in FZB42, available mutant strains, and other aspects of FZB42 research, which is structured similar as the famous SubtiWiki data bank.

Root colonization is important for the application of Plant Growth-Promoting Rhizobacteria (PGPR). Previously, chemotaxis was believed to be the major trait for colonization. However, because chemotaxis defective mutants are usually swarming impaired at the same time, in this study we clarified the roles played by swarming motility and chemotaxis in tomato root colonization by Bacillus subtilis SWR01. Tomato seeds were treated with B. subtilis SWR01 or mutants before being sown in Simons’ gnotobiotic system, and the colonization efficacy at root tips were observed after 2 weeks. Both microscopy and plate counts showed that the colonization efficacy of swarming defective and chemotaxis proficient mutants (ΔswrA, ΔminJ and ΔsrfAC) was about 5%–15% that of the parental strain SWR01, while the colonization efficacy of the swarming normal and chemotaxis impaired mutant ΔcheV was 78.9% that of the wild-type bacteria. These results demonstrate that while both chemotaxis and swarming motility are important in root colonization, and the role played by swarming is greater than that of chemotaxis. In addition, non-flagellated mutants (Δhag) showed 0.47% colonization efficacy relative to that of B. subtilis SWR01, suggesting more than 99% root colonization requires the presence of flagella. This study indicates new ways to enhance PGPR rhizoplane colonization, which is useful in agricultural applications.

Harpin HrpZ from the plant-pathogen Pseudomonas spp. elicits the hypersensitive response (HR), pathogen defense responses, and enhances growth in plants. To identify regions of HrpZ related to these bioactivities, we constructed 11 mutants of HrpZPsgS1, a 346-amino-acid harpin protein from P. savastanoi pv. glycinea S1. Results showed that proteins HrpZ74–204 and HrpZ1–194 could not induce macroscopic HR but could elicit microscopic HR in tobacco. The HR elicitation activity of mutant proteins with other C-terminal deletions in HrpZPsgS1, such as HrpZ1–102, HrpZ△195–238, HrpZ△241–248, HrpZ△254–298, and HrpZ△290–313, was reduced. The activity of the remaining mutants, other than HrpZ200–346, which lacks part of the N-terminus, was similar to wild-type. These results indicate that the C-terminus is indispensable for HR elicitation, and that parts of the N-terminus play a regulatory role. Also, mutants HrpZΔ89–124 and HrpZΔ254–298 enhanced growth in rice more than wild-type HrpZPsgS1. These mutants were also more effective at inducing resistance to Xanthomonas oryzae pv. oryzae in rice and to Tobacco Mosaic Virus (TMV) in tobacco. qRT-PCR assays showed that HrpZΔ89–124 and HrpZΔ254–298 induced higher levels of expression in genes related to HR, pathogen defense, and growth. Therefore, the modified proteins HrpZΔ89–124 and HrpZΔ254–298 may have potential for development as protein-type biocontrol agents.

Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up-regulated the expression of the plant defence gene PR-1a and the PR-1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA-dependent defence pathway is involved in cyclodipeptide-mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.

Fusarium graminearum (teleomorph: Ascomycota, Hypocreales, Gibberella , Gibberella zeae ) is a destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a significant challenge. In the present study, the commercially available strain Bacillus amyloliquefaciens ( Bacteria , Firmicutes , Bacillales , Bacillus ) FZB42 showed strong activity against F. graminearum . The lipopeptide bacillomycin D, produced by FZB42, was shown to contribute to the antifungal activity. Purified bacillomycin D showed strong activity against F. graminearum , and its 50% effective concentration was determined to be approximately 30 μg/ml. Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membranes and cell walls of F. graminearum hyphae and conidia. Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species and caused cell death in F. graminearum hyphae and conidia. F. graminearum secondary metabolism also responded to bacillomycin D challenge, by increasing the production of deoxynivalenol. Biological control experiments demonstrated that bacillomycin D exerted good control of F. graminearum on corn silks, wheat seedlings, and wheat heads. In response to bacillomycin D, F. graminearum genes involved in scavenging reactive oxygen species were downregulated, whereas genes involved in the synthesis of deoxynivalenol were upregulated. Phosphorylation of MGV1 and HOG1, the mitogen-activated protein kinases of F. graminearum , was increased in response to bacillomycin D. Taken together, these findings reveal the mechanism of the antifungal action of bacillomycin D. IMPORTANCE Biological control of plant disease caused by Fusarium graminearum is desirable. Bacillus amyloliquefaciens FZB42 is a representative of the biocontrol bacterial strains. In this work, the lipopeptide bacillomycin D, produced by FZB42, showed strong fungicidal activity against F. graminearum . Bacillomycin D caused morphological changes in the plasma membrane and cell wall of F. graminearum , induced accumulation of reactive oxygen species, and ultimately caused cell death in F. graminearum . Interestingly, when F. graminearum was challenged with bacillomycin D, the deoxynivalenol production, gene expression, mitogen-activated protein kinase phosphorylation, and pathogenicity of F. graminearum were significantly altered. These findings clarified the mechanisms of the activity of bacillomycin D against F. graminearum and highlighted the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against F. graminearum .

Harmful algal blooms, caused by massive and exceptional overgrowth of microalgae and cyanobacteria, are a serious environmental problem worldwide . In the present study, we looked for Bacillus strains with sufficiently strong anticyanobacterial activity to be used as biocontrol agents. Among 24 strains, Bacillus amyloliquefaciens FZB42 showed the strongest bactericidal activity against Microcystis aeruginosa , with a kill rate of 98.78%. The synthesis of the anticyanobacterial substance did not depend on Sfp, an enzyme that catalyzes a necessary processing step in the nonribosomal synthesis of lipopeptides and polyketides, but was associated with the aro gene cluster that is involved in the synthesis of the sfp -independent antibiotic bacilysin. Disruption of bacB , the gene in the cluster responsible for synthesizing bacilysin, or supplementation with the antagonist N -acetylglucosamine abolished the inhibitory effect, but this was restored when bacilysin synthesis was complemented. Bacilysin caused apparent changes in the algal cell wall and cell organelle membranes, and this resulted in cell lysis. Meanwhile, there was downregulated expression of glmS , psbA1 , mcyB , and ftsZ —genes involved in peptidoglycan synthesis, photosynthesis, microcystin synthesis, and cell division, respectively. In addition, bacilysin suppressed the growth of other harmful algal species. In summary, bacilysin produced by B. amyloliquefaciens FZB42 has anticyanobacterial activity and thus could be developed as a biocontrol agent to mitigate the effects of harmful algal blooms.

Bacillus amyloliquefaciens FZB42 is a plant growth-promoting rhizobacterium that induces resistance to a broad spectrum of pathogens. This study analyzed the mechanism by which FZB42 restricts leaf disease caused by Phytophthora nicotianae in Nicotiana benthamiana. The oomycete foliar pathogen P. nicotianae is able to reopen stomata which had been closed by the plant innate immune response to initiate penetration and infection. Here, we showed that root colonization by B. amyloliquefaciens FZB42 restricted pathogen-mediated stomatal reopening in N. benthamiana. Abscisic acid (ABA) and salicylic acid (SA)-regulated pathways mediated FZB42-induced stomatal closure after pathogen infection. Moreover, the defense-related genes PR-1a, LOX, and ERF1, involved in the SA and jasmonic acid (JA)/ethylene (ET) signaling pathways, respectively, were overexpressed, and levels of the hormones SA, JA, and ET increased in the leaves of B. amyloliquefaciens FZB42-treated wild type plants. Disruption of one of these three pathways in N. benthamiana plants increased susceptibility to the pathogen. These suggest that SA- and JA/ET-dependent signaling pathways were important in plant defenses against the pathogen. Our data thus explain a biocontrol mechanism of soil rhizobacteria in a plant.

Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides.

Rhizobacterial volatile organic compounds (VOCs) play an important role in the suppression of soil-borne phytopathogens. In this study, the VOCs produced by a soil-isolate, Bacillus subtilis FA26, were evaluated in vitro for their antibacterial activity against Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. The VOCs emitted by FA26 inhibited the growth of Cms significantly compared with the control. Scanning and transmission electron microscopy analyses revealed distorted colony morphology and a wide range of abnormalities in Cms cells exposed to the VOCs of FA26. Varying the inoculation strategy and inoculum size showed that the production and activity of the antibacterial VOCs of FA26 were dependent on the culture conditions. Headspace solid-phase microextraction/gas chromatography–mass spectrometry analyses revealed that FA26 produced 11 VOCs. Four VOCs (benzaldehyde, nonanal, benzothiazole and acetophenone) were associated with the antibacterial activity against Cms. The results suggested that the VOCs produced by FA26 could control the causal agent of bacterial ring rot of potato. This information will increase our understanding of the microbial interactions mediated by VOCs in nature and aid the development of safer strategies for controlling plant disease.

Fusarium verticillioides (teleomorph, Gibberella moniliformis) is an important plant pathogen that causes seedling blight, stalk rot, and ear rot in maize (Zea mays). During infection, F. verticillioides produce fumonsins B1 (FB1) that pose a serious threat to human and animal health. Recent studies showed that Set1, a methyltransferase of H3K4, was responsible for toxin biosynthesis in filamentous fungi. However, to date, the regulation of FvSet1 on FB1 biosynthesis remains unclear. In the current study, we identified only one Set1 ortholog in F. verticillioides (FvSet1) and found that the deletion of FvSET1 led to various defects in fungal growth and pathogenicity. More interestingly, the FvSET1 deletion mutant (ΔFvSet1) showed a significant defect in FB1 biosynthesis and lower expression levels of FUM genes. FvSet1 was also found to play an important role in the responses of F. verticillioides to multiple environmental stresses via regulating the phosphorylation of FvMgv1 and FvHog1. Taken together, these results indicate that FvSet1 plays essential roles in the regulation of FB1 biosynthesis, fungal growth and virulence, as well as various stress responses in F. verticillioides.

Volatile organic compounds (VOCs) produced by various bacteria have significant potential to enhance plant growth and to control phytopathogens. Six of the most effective antagonistic Bacillus spp. were used in this study against Ralstonia solanacearum (Rsc) TBBS1, the causal agent of bacterial wilt disease in tobacco. Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22 had the strongest inhibitory effect against Rsc. Thirteen VOCs produced by FZB42 and 10 by LSSC22 were identified using gas chromatography-mass spectrometry analysis. Benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene significantly inhibited the colony size, cell viability, and motility of pathogens and negatively influenced chemotaxis. Transmission and scanning electron microscopy revealed severe morphological and ultra-structural changes in cells of Rsc. Furthermore, VOCs altered the transcriptional expression level of PhcA (a global virulence regulator), type III secretion system (T3SS), type IV secretion system (T4SS), extracellular polysaccharides and chemotaxis-related genes, which are major contributors to pathogenicity, resulting in decreased wilt disease. The VOCs significantly up-regulated the expression of genes related to wilt resistance and pathogen defense. Over-expression of EDS1 and NPR1 suggest the involvement of SA pathway in induction of systemic resistance. Our findings provide new insights regarding the potential of antibacterial VOCs as a biocontrol tool against bacterial wilt diseases.

Bacterial volatiles play a significant role in promoting plant growth by regulating the synthesis or metabolism of phytohormones. In vitro and growth chamber experiments were conducted to investigate the effect of volatile organic compounds (VOCs) produced by the plant growth promoting rhizobacterium Bacillus subtilis strain SYST2 on hormone regulation and growth promotion in tomato plants. We observed a significant increase in plant biomass under both experimental conditions; we observed an increase in photosynthesis and in the endogenous contents of gibberellin, auxin, and cytokinin, while a decrease in ethylene levels was noted. VOCs emitted by SYST2 were identified through gas chromatography-mass spectrometry analysis. Of 11 VOCs tested in glass jars containing plants in test tubes, only two, albuterol and 1,3-propanediole, were found to promote plant growth. Furthermore, tomato plants showed differential expression of genes involved in auxin (SlIAA1. SlIAA3), gibberellin (GA20ox-1), cytokinin (SlCKX1), expansin (Exp2, Exp9. Exp 18), and ethylene (ACO1) biosynthesis or metabolism in roots and leaves in response to B. subtilis SYST2 VOCs. Our findings suggest that SYST2-derived VOCs promote plant growth by triggering growth hormone activity, and provide new insights into the mechanism of plant growth promotion by bacterial VOCs.

UNLABELLED Both Bacillus subtilis and harpins stimulate plant growth and defence against various plant pathogens. In this study, B. subtilis 168 and two derivatives, surfactin producer OKB105 and combined surfactin and HpaGX ooc producer OKBHF, were applied to tomato plants to investigate the mechanisms underlying this effect. To evaluate colonization ability, strains were labelled with green fluorescent protein (GFP). Although biofilm distribution of the three strains was similar on root surfaces, Colonization populations of the two surfactin producers were approximately 2- to 3-fold higher than that of strain 168, and this was accompanied by significantly increased tomato growth. These results suggest that efficient colonization, possibly facilitated by surfactin production, enhanced the efficiency of plant growth promotion by B. subtilis. All three B. subtilis treatments caused plants to have less severe disease symptoms after inoculation with Ralstonia solanacearum, with plants treated with OKBHF being the most resistant, suggesting that hpaGX ooc improves biocontrol efficiency of B. subtilis. Analysis of defence-related genes showed a synergistic effect of HpaGX ooc on B. subtilis enhancement of the expression of the pathogenesis-related genes PR1b1 and PR-P2. In contrast, expression of the defence-related genes PINI and PINII was suppressed. SIGNIFICANCE AND IMPACT OF THE STUDY Bacillus subtilis and harpins are biological control agents with respective advantages. In this study, combinations of the both were applied to tomato in the form of hpaGX ooc -expressing B. subtilis, showed much better effects on resistance to wilt disease, and equivalent effects on plant growth promotion compared with the progenitor strain have a great potential in agricultural use.

Parasitic nematodes of plants are important plant pathogens that represent a significant financial burden on agriculture. This study evaluated the efficacy of Bacillus spp. as nematode biocontrol agents and identified Bacillus genes associated with nematicidal activity. Culture by products of Bacillus subtilis strains OKB105 and 69 and Bacillus amyloliquefaciens strains FZB42 and B3 were used to treat Aphelenchoides besseyi, Ditylenchus destructor, Bursaphelenchus xylophilus and Meloidogyne javanica, respectively. The highest mortality rates were observed at 12 h when combinations of either A. besseyi/B3, D. destructor/OKB105, B. xylophilus/69 or M. javanica/OKB105 resulted in 10.6%, 27.6%, 35.6% and 100% mortality rates, respectively. Supernatant analysis demonstrated that the nematicidal active ingredients of strain OKB105, with a molecular weight of

In previous studies, Bacillus amyloliquefaciens C06 has been proven to be effective in controlling brown rot of stone fruit caused by Monilinia fructicola. When tested in vitro, cell-free filtrate of B. amyloliquefaciens C06 significantly inhibited mycelial growth and conidial germination of the fungal pathogen. This study aimed to determine the role of the antifungal compound(s) in the cell-free filtrate of B. amyloliquefaciens C06 by an approach combining a DNA-based suppression subtractive hybridization (SSH) method with MALDI-TOF-MS analysis. It was demonstrated that B. amyloliquefaciens C06 harbored two genes, bmyC and fenD, involved in biosynthesis of bacillomycin D and fengycin, two lipopeptides belonging to the iturin and fengycin family, respectively. To determine the roles of bacillomycin D and fengycin of B. amyloliquefaciens C06 in suppressing M. fructicola, the mutants of B. amyloliquefaciens C06 deficient in producing bacillomy- cin D, fengycin or both were constructed, and evaluated in vitro together with the wild-type B. amyloliquefaciens C06. The results indicated that bacillomycin D and fengycin jointly contributed to the inhibition of conidial germination of M. fructicola, and fengycin played a major role in suppressing mycelial growth of the fungal pathogen.

Bacillussubtilis is well known for its biocontrol activity against several plant pathogens and for its role in promoting plant growth. HpaGXooc, from rice pathogenic bacterium Xanthomonasoryzae pv. oryzicola, is a member of the harpin group of proteins. It is known to elicit hypersensitive cell death in non-host plants, thereby inducing disease and insect resistance in the plants and enhancing plant growth. In our previous experiment, we constructed a genetically engineered strain—B. subtilis OKBHF—through the introduction of the gene encoding HpaGXooc into B. subtilis OKB105 in order to combine the effects of HpaGXooc and wild-type PGPR in improving the plant growth rate and for biological control. In this study, we evaluated the use of treating the tomato plant with B. subtilis OKBHF. The results of greenhouse experiments demonstrated that OKBHF treatment had a significant effect on increasing the height, fresh weight, and flower and fruit number and obviously lowered the disease severity of Cucumber mosaic virus (CMV) infection at 28 days postinoculation (dpi). Subsequent reverse transcription-polymerase chain reaction analysis revealed the molecular mechanisms of HpaGXooc and B. subtilis in the tomato plant, suggesting their synergistic roles in inducing enhanced expression of three expansin genes LeEXP2, LeEXP5, and LeEXP18, which regulate plant cell growth, and two defense-related genes Pti4 and Pti6, which activate the expression of a wide array of PR genes and one defense gene, PR-1a.

Bacillus spp., as a type of plant growth-promoting rhizobacteria (PGPR), were studied with regards promoting plant growth and inducing plant systemic resistance. The results of greenhouse experiments with tobacco plants demonstrated that treatment with the Bacillus spp. significantly enhanced the plant height and fresh weight, while clearly lowering the disease severity rating of the tobacco mosaic virus (TMV) at 28 days post-inoculation (dpi). The TMV accumulation in the young non-inoculated leaves was remarkably lower for all the plants treated with the Bacillus spp. An RTPCR analysis of the signaling regulatory genes Coi1 and NPR1, and defense genes PR-1a and PR-1b, in the tobacco treated with the Bacillus spp. revealed an association with enhancing the systemic resistance of tobacco to TMV. A further analysis of two expansin genes that regulate plant cell growth, NtEXP2 and NtEXP6, also verified a concomitant growth promotion in the roots and leaves of the tobacco responding to Bacillus spp.

Bacterial blight and bacterial leaf streak are serious, economically damaging, diseases of rice caused by the bacteria Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. Bacillus amyloliquefaciens FZB42 was shown to possess biocontrol activity against these Xanthomonas strains by producing the antibiotic compounds difficidin and bacilysin. Analyses using fluorescence, scanning electron and transmission electron microscopy revealed difficidin and bacilysin caused changes in the cell wall and structure of Xanthomonas. Biological control experiments on rice plants demonstrated the ability of difficidin and bacilysin to suppress disease. Difficidin and bacilysin caused downregulated expression of genes involved in Xanthomonas virulence, cell division and protein and cell wall synthesis. Taken together, our results highlight the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against bacterial diseases of rice and the utility of difficidin and bacilysin as antimicrobial compounds.

According to the change of environment, soil-dwelling Bacillus species differentiate into distinct subpopulations, such as spores and competent cells. Rap-Phr systems have been found to be involved in this differentiation circuit by interacting with major regulatory proteins, such as Spo0A, ComA, and DegU. In this study, we report that the plasmid-born RapQ-PhrQ system found in Bacillus amyloliquefaciens B3 affects three regulatory pathways in the heterologous host Bacillus subtilis. Expression of rapQ in B. subtilis OKB105 strongly suppressed its sporulation efficiency, transformation efficiency, and surfactin production. Co-expression of phrQ or addition of synthesized PhrQ pentapeptide in vitro could compensate for the suppressive effects caused by rapQ. We also found that expression of rapQ decreased the transcriptional level of the sporulation-related gene spoIIE and surfactin synthesis-related gene srfA; meanwhile, the transcriptional levels of these genes could be rescued by co-expression of phrQ and in vitro addition of PhrQ pentapeptide. Electrophoretic mobility shift (EMSA) result also showed that RapQ could bind to ComA without interacting with ComA binding to DNA, and PhrQ pentapeptide antagonized RapQ activity in vitro. These results indicate that this new plasmid-born RapQ-PhrQ system controls sporulation, competent cell formation, and surfactin production in B. subtilis OKB105.

Background Plant growth-promoting rhizobacteria (PGPR) are soil beneficial microorganisms that colonize plant roots for nutritional purposes and accordingly benefit plants by increasing plant growth or reducing disease. However, the mechanisms and pathways involved in the interactions between PGPR and plants remain unclear. In order to better understand these complex plant-PGPR interactions, changes in the transcriptome of the typical PGPR Bacillus subtilis in response to rice seedlings were analyzed. Results Microarray technology was used to study the global transcriptionl response of B. subtilis OKB105 to rice seedlings after an interaction period of 2 h. A total of 176 genes representing 3.8% of the B. subtilis strain OKB105 transcriptome showed significantly altered expression levels in response to rice seedlings. Among these, 52 were upregulated, the majority of which are involved in metabolism and transport of nutrients, and stress responses, including araA, ywkA, yfls, mtlA, ydgG et al. The 124 genes that were downregulated included cheV, fliL, spmA and tua, and these are involved in chemotaxis, motility, sporulation and teichuronic acid biosynthesis, respectively. Conclusions We present a transcriptome analysis of the bacteria Bacillus subtilis OKB105 in response to rice seedings. Many of the 176 differentially expressed genes are likely to be involved in the interaction between Gram-positive bacteria and plants.

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.

Bacillus amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters P repB and P spac from plasmids pMK3 and pLOSS, respectively. These strains contained two antibiotic resistance genes, and markerless strains were constructed by deleting the chloramphenicol resistance cassette and promoter region bordered by two lox sites (lox71 and lox66) using Cre recombinase expressed from the temperature-sensitive vector pLOSS-cre. The vector-encoded spectinomycin resistance gene was removed by high temperature (50 °C) treatment. RT-PCR and qRT-PCR results indicated that P repB and especially P spac significantly increased expression of the bac operon, and FZBREP and FZBSPA strains produced up to 170.4 and 315.6 % more bacilysin than wild type, respectively. Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). Both the size and degree of ring rot-associated necrotic tubers were decreased compared with the wild-type strain, which confirmed the protective effects and biocontrol potential of these genetically engineered strains.

The genome of rhizobacterium Bacillus amyloliquefaciens subsp. plantarum strain NAU-B3 is 4,196,170 bp in size and harbors 4,001 genes. Nine giant gene clusters are dedicated to the nonribosomal synthesis of antimicrobial lipopeptides and polyketides. Remarkably, NAU_B3 contains a large inversion within the central portion of the genome.

Lectins are proteins of non-immune origin that specifically interact with carbohydrates, known to play important roles in the defense system of plants. In this study, in order to study the function of a new soybean lectin (SBL), the corresponding encoding gene lec-s was introduced into tobacco plants via Agrobacterium-mediated transformation. Southern blot analyses had revealed that the lec-s gene was stable integrated into the chromosome of the tobacco. The results of the reverse transcription polymerase chain reaction (RT-PCR) also indicated that the lec-s gene in the transgenic tobacco plants could be expressed under the control of the constitutive CaMV35S promoter. Evaluation agronomic of the performance had showed that the transgenic plants could resist to the infection of Phytophthora nicotianae. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed SBL significantly (P.0.05) reduced the weight gain of larvae of the beet armyworm (Spodoptera exigua). Further on, the lectins retarded the development of the larvae and their metamorphosis. These findings suggest that soybean lectins have potential as a protective agent against pathogens and insect pests through a transgenic approach.

UNLABELLED: Reports suggest that Bacillus spp. can be used to increase plant growth and resistance to disease, but the molecular mechanisms underlying the interaction between Bacillus spp. and plant is not completely understood. In the present study, to clarify these underlying mechanisms, the interaction between Bacillus cereus and rice was investigated using two-dimensional gel electrophoresis. Through comparative analysis, a total of 31 differentially expressed proteins were obtained upon B. cereus NMSL88 treatment, including 22 proteins that were up-regulated and nine that were down-regulated. These data indicated that certain proteins involved in plant growth and development were up-regulated, such as xyloglucan endotransglycosylase. Interestingly, proteins involved in defence were also up-regulated, including peroxidases, glutathione S-transferases and kinases. Thus, proteins associated with disease resistance characteristics were induced in the plants after exposure to B. cereus NMSL88. In addition, several proteins involved in protein and lipid metabolism showed significant changes in expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report to reveal the molecular mechanisms involved in rice seedlings in response to inoculation with Bacillus cereus at the level of proteome. The results demonstrated that B. cereus NMSL88 can up-regulate the expression of proteins related to plant growth and defence, and lead to enhanced plant growth and disease resistance.

Bacillus amyloliquefaciens C06, an effective biological agent in controlling brown rot of stone fruit caused by Monilinia fructicola, was also found to produce extra-cellular mucilage and form mucoid colonies on semi-solid surfaces. This study aimed to characterize the extra-cellular mucilage produced by B. amyloliquefaciens C06 using transposon mutagenesis and biochemical and physical analyses. The mucilage production in B. amyloliquefaciens C06 was demonstrated to be associated with ywsC gene expression and characterized to be of high molecular weight, consisted of only glutamic acid and linked with non-peptide bonds, thus identified as gamma-polyglutamic acid (gamma-PGA). Compared with wild type B. amyloliquefaciens C06, its mutants deficient in producing gamma-PGA, e.g. M106 and C06DeltaywsC showed less efficiency in biofilm formation, surface adhesion and swarming ability. It was also demonstrated that gamma-PGA was not essential for C06 to form colony on semi-solid surfaces, but was able to improve its colony structure. In vivo evaluation showed that disruption of gamma-PGA production in C06DeltaywsC impaired its efficiency of colonizing apple surfaces.

Bacillus amyloliquefaciens C06, a potential agent in biological preservation of post-harvest fruit, was found to secrete extra-cellular γ-polyglutamic acid (γ-PGA) in liquid culture. In this work, M306, a transposon mutant of B. amyloliquefaciens C06, defective in forming structured colony and displaying enhanced ability of producing γ-PGA, was obtained. Inverse PCR and quantitative reverse transcription PCR (qRT-PCR) analysis demonstrated that the defective phenotype in M306 was associated with an ORF showing high similarity to RBAM_034550 from B. amyloliquefaciens FZB42. In this paper, the ORF was designated pbrA, standing for γ-PGA production and biofilm formation regulatory factor. qRT-PCR analysis also indicated that pbrA down-regulated mRNA expression of epsD and yqxM, the crucial genes involved in biofilm formation, but affected little on expression of ywtB, the gene directing γ-PGA synthesis. Evaluations in γ-PGA productivity of wild-type C06 and its mutants C06ΔepsA and C06ΔtasA, respectively, deficient in producing exopolysaccharides (EPS) and TasA, revealed that γ-PGA overproduction in M306 was probably due to the redistributed metabolic flux caused by defective production of EPS.

The early stage of plant-rhizobacteria interaction, affected by plant root exudates and plant-rhizobacteria surface contact, is considered to be critical for plant growth-promoting rhizobacteria colonizing plant roots and initiating the beneficial effects on plant growth. However, little is known about the mechanisms of plant-rhizobacteria surface contact involved in early stage of plant-rhizobacteria interaction. In order to reveal the molecular mechanisms of the surface contact, a rhizobacterium Bacillus amyloliquefaciens B55 was interacted with plant roots of rice R109 and used to perform a cDNA-based suppression-subtractive hybridization. Seven differentially expressed DNA fragments were identified. Except for the two fragments showing no matches to any known sequences in the Genbank, the other five fragments were found to have high homologies with the genes encoding 2-oxoglutarate dehydrogenase E1 component OdhA, aspartate ammonia-lyase AnsB, and hypothetical protein proposed to be involved in surface adhesion, acetolactate decarboxylase AlsD, and DNA mismatch repair protein MutL, respectively. The induced RNA expression levels of two putative genes ansB and odhA and an unmatched DNA fragment BD33 were verified by RT-PCR analysis.

HpaGXooc, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. To express and secret the HpaGXooc protein in Bacillus subtilis, we constructed a recombinant expression vector pM43HF with stronger promoter P43 and signal peptide element nprB. The SDS-PAGE and Western blot analysis demonstrated the expression of the protein HpaGXooc in B. subtilis. The ELISA analysis determined the optimum condition for HpaGXooc expression in B. subtilis WBHF. The biological function analysis indicated that the protein HpaGXooc from B. subtilis WBHF elicits hypersensitive response (HR) and enhances the growth of tobacco. The results of RT-PCR analysis revealed that HpaGXooc induces expression of the pathogenesis-related genes PR-1a and PR-1b in plant defense response.

Summary Engineering durable nonspecific resistance to phytopathogens is one of the ultimate goals of plant breeding. However, most attempts to reach this goal fail as a result of rapid changes in pathogen populations and the sheer diversity of pathogen infection mechanisms. In this study, we show that the expression of a harpin-encoding gene (hrf1), derived from Xanthomonas oryzae pv. oryzae, confers nonspecific resistance in rice to the blast fungus Magnaporthe grisea. Transgenic plants and their T1–T7 progenies were highly resistant to all major M. grisea races in rice-growing areas along the Yangtze River, China. The expression of defence-related genes was activated in resistant transgenic plants, and the formation of melanized appressoria, which is essential for foliar infection, was inhibited on plant leaves. These results suggest that harpins may offer new opportunities for generating broad-spectrum disease resistance in other crops.