Biol. J. Syst. However, the different effects of LJ02BC and LJ02FS on the SA-mediated defense responses may attributed to certain unidentified MAMP factors that functions in association with bacterium itself. doi: 10.1007/s10526-010-9324-8, Reuveni, M., Agapov, V., and Reuveni, R. (1996). doi: 10.1016/j.it.2014.05.004, Keywords: powdery mildew, biocontrol agent, Bacillus amyloliquefaciens, salicylic acid, PR genes, systemic acquired resistance, Citation: Li Y, Gu Y, Li J, Xu M, Wei Q and Wang Y (2015) Biocontrol agent Bacillus amyloliquefaciens LJ02 induces systemic resistance against cucurbits powdery mildew. 114, 301308. SA as a key immune signal is involved in the regulation of downstream defense genes (Robert-Seilaniantz et al., 2011). Control 6, 353360. Therefore, it is likely that using gyrB as a phylogenetic marker is reliable in our study and could provide clues for bacillus determination. Saf. doi: 10.1111/j.1399-3054.1962.tb08052.x, Newman, M. A., Sundelin, T., Nielsen, J. T., and Erbs, G. (2013). Control 81, 101110. doi: 10.1111/j.1399-3054.2010.01348.x, PubMed Abstract | CrossRef Full Text | Google Scholar, Alamri, S., Hashem, M., and Mostafa, Y. S. (2012). Ann. 399, 257261. Biocontrol 56, 225235. doi: 10.1146/annurev-phyto-073009-114447, Romero, D., De Vicente, A., Zeriouh, H., Cazorla, F. M., Fernandez-Ortuno, D., Tores, J. PRR-mediated microbe sensing gives rise to a wide array of defense responses known as MAMP- or PAMP-triggered immunity (MTI/PTI; Zipfel, 2014). Evol. This is likely to be caused by the degradation of potential elicitors in fermentation broth. This phenomenon led us to conclude that LJ02 could induce a long-range of defense response by promoting the free SA production, which will then elicit the SA signaling pathways to enhance the immunity of cucumber plants systemically. And it was shown that gyrB gene sequences provide higher resolution than 16S rRNA gene sequences (Wang et al., 2007). Plant Microbe Interact. Environ. 77, 486494. doi: 10.1016/0261-2194(95)00109-3, Robert-Seilaniantz, A., Grant, M., and Jones, J. D. (2011). Int. Biological control of soil-borne pathogens by fluorescent pseudomonads. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Germination rate of F. oxysporum (A), B. cinerea (B), and Alternaria spp. 78, 2834. Interestingly, LJ02FS could induce significantly higher amount of free SA at 5th day and declined to control level. The rapid increase of POD activity was observed during the first 3 days after treatments and the difference between treatment and control became evident from 7th day (Figure 3C). Int. 57, 18461850. Microbiol. Perception of pathogenic or beneficial bacteria and their evasion of host immunity: pattern recognition receptors in the frontline. Plant Microbe Interact. It is responsible for inducing tolerance to a number of biotic and abiotic stresses, which is also a trigger for the production of SAR-related enzymes (Korkmaz et al., 2007). Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with Paenibacillus illinoisensis. Received: 03 April 2015; Accepted: 11 August 2015; Published: 28 August 2015. Error bars indicate standard deviation among triplicates. Infect. The number of CFU was recorded on each plate after 3 days. Plant Microbe Interact. Among PR genes, PR-1 expression is a paradigm for the co-regulation of PR genes and correlates well with the induction of SAR (Durrant and Dong, 2004). The tree was constructed by MEGA5.1 by the neighbor-joining method. Salicylic Acid, a multifaceted hormone to combat disease. Science 318, 113116. 28, 27312739. 10, 153160. J. Zhejiang. Gram staining and microscopic observation showed that LJ02 is Gram-positive, endospore-forming and rod-shaped bacterium with a width of 0.70.9 m and a length of 1.83.0 m, indicative of a typical bacillus morphology (Figure 2). Phylogenetic tree was therefore constructed and demonstrated high homology to B. amyloliquefaciens (Figure 1). Leon, J., Lawton, M. A., and Raskin, I. MAMP (microbe-associated molecular pattern) triggered immunity in plants. Phylogenetic tree of strain LJ02 and other Bacillus species from GenBank based on gyrB gene sequences. doi: 10.1016/j.biocontrol.2003.11.005, Boller, T., and Felix, G. (2009). All experiments had at least five replicates. Four pieces of MS solid medium cake were inoculated around the fungi cake at a distance of 2.5 cm (treatment group). Thus, we established a method to detect the concentration of SA in LJ02FB-treated cucumber leaves (Figures 4A,B). As can be seen from our results (Figure 7) that MAMP associated with LJ02 is perhaps the underlying mechanism for the SA accumulation in cucumber tissues since we could detect the increase of SA production when treated with LJ02BC. Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Anal. (2006). To this end, we analyzed the level of transcription of PR-1 gene, a commonly used maker for SA-mediated expression (Tornero et al., 1997). BMC Plant Biol. Plant Sci. Controlling powdery mildew caused by Sphaerotheca fuliginea in cucumber by foliar sprays of phosphate and potassium salts. A cry for help from leaf to root. 22, Jinjing Road, Xiqing District, Tianjin 300384, China, wangyh@tjau.edu.cn; Qing Wei, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. At the first beginning, the identification of LJ02 as B. amyloliquefaciens is different from previous studies since we could not distinguish it from B. subtilis based on 16S rRNA comparison (data not shown). The images were taken from a ultra-high resolution scanning electron microscope SU8010. Microb. Plant Sci. 15, 583606. One strain was selected and named LJ02. 74, 239247. doi: 10.1146/annurev.phyto.050908.135202. Moreover, LJ02FS also has SA-inducing activity with relative lower level as compared to that of LJ02BC, indicating both fractions are important for SA-inducing activity. doi: 10.1016/j.micres.2011.02.004, Korkmaz, A., Uzunlu, M., and Demirkiran, A. R. (2007). Although 16S rRNA sequence has been commonly used as a simple method for the identification of microorganisms, it has limitations for constructing bacterial phylogenetic relationships due to the high percentage of sequence similarity between closely related species. 56, 976986. Scanning microscope observation of LJ02 under two resolutions: 25,000 (A) and 5,000 (B). doi: 10.1016/0003-2697(77)90262-7, Faheem, M., Raza, W., Zhong, W., Nan, Z., Shen, Q. R., and Xu, Y. C. (2015). (2015). Finally, we further dissect the roles played by LJ02BC and L02FS and found that LJ02 could really elicit SAR response in leaves as well as in roots. doi: 10.1016/j.biocontrol.2013.01.009, Arguelles-Arias, A., Ongena, M., Halimi, B., Lara, Y., Brans, A., Joris, B., et al. J. Ecol. It is also positive for catalase, nitrate reduction, and VogesProskauer test and negative for oxidase, indole production and egg yolk reaction. Most spores would germinate at 9 h in the preliminary period. doi: 10.1080/07352689609382371, Jung, W. J., Jin, Y. L., Kim, K. Y., Park, R. D., and Kim, T. H. (2005). A one-way ANOVA was performed (*P < 0.05; **P < 0.01). For more details, please refer to main text. Front. Physiol. Appl. (2015). Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Mol. Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine. doi: 10.1016/J.Ab.2009.12.008, Wang, L. T., Lee, F. L., Tai, C. J., and Kasai, H. (2007). (2004). And another interesting finding of this study is that AG whitefly feeding significantly increased the population density of beneficial BG microflora including actinomycetes and saprophytic fungi that may induce systemic resistance (Yang et al., 2011). Disease index and control efficacy of powdery mildew in cucumber. As is shown in Figure 3B, there was slight difference between the control and treatment group plants at the primary treatment period (<2 days). Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the hypersensitive response and development. Considerable decrease in spore germination and fungal growth were detected after treated with LJ02FB. 16, 449455. Rev. An 1.2 kb gyrB gene fragment was amplified from the strain LJ02 and sequenced. 7H2O: 0.5 g, agar: 15 g, 1% Rose Bangal: 0.33 mL, 1% streptomycin: 3 mL, distilled water: 1,000 mL; Xu and Zheng, 1986) and incubated at 25C. Acetyl salicylic acid alleviates chilling-induced damage in muskmelon seedlings. The SOD activity of treatment group increased gradually and peaked after 36 h. It is very interesting to notice that the SOD activity retained a higher level even after 11 days treatment, suggesting its major role in disease resistance under our conditions. 49, 693700. Subsequently, we found that when we separated the LJ02FB into LJ02BC and LJ02FS, LJ02BC played very important roles in inducing the production of free SA in both upper uninoculated leaves and in roots. Phytopathol. Besides, we found that the production of several resistance-related enzymes has been significantly stimulated upon LJ02 treatment, which was probably caused by the coordinate activation by enhanced SA levels in LJ02-treated plants. (1986). Different letters are significantly difference at P < 0.01 according to Duncan multiple range test. The data of disease index, CE, resistance enzyme activities, SA production, germination rate, and fungi pathogen counts in rhizospheres were analyzed by one-way ANOVA. MTI is a defense program with complex early signaling events including ion fluxes, mitogen-activated protein (MAP) kinase cascade activation and the production of reactive oxygen species (ROS; Garcia-Brugger et al., 2006). 16, 177187. Based on current data and those previous studies, we summarized our results and some hypotheses in Figure 8. Biochem. These authors have contributed equally to this work. A one-way ANOVA was performed (*P < 0.05; **P < 0.01). (2009). The inhibition against pathogen fungi of LJ02-induced secretions was detected as described previously with minor modifications (On et al., 2015). The physiological and biochemical properties were determined and shown in Table 2. Figure 6. Based on this findings, we further revealed that the rhizosphere MS solid medium of LJ02FB-treated cucumber could strongly inhibit the growth of F. oxysporum, B. cinerea, and Alternaria spp. (2009). The role of antibiosis and induced systemic resistance, mediated by strains of Pseudomonas chlororaphis, Bacillus cereus and B. amyloliquefaciens, in controlling blackleg disease of canola. It can be seen that upon inoculation of LJ02 and in AG part, the upper cucumber leaves synthesized elevated level of SA and at the same time, this could partially explain the inhibition of powdery mildews. The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits. 61, 11041109. Bacillus amyloliquefaciens sp nov, nom rev. In previous studies, only one-way signal transduction was investigated, such as AGBG, AGAG, and BGBG (Yi et al., 2011). 140, 2737. (2015). But the activity of PPO increased rapidly after 2 days and became significant different (P < 0.05) from the control at 3rd day. Mol. Rev. Very recently, it was discovered that SA could modulate colonization of the root microbiome by specific bacterial taxa, indicating that SA is likely to be important for the colonization or recruitment of beneficial microbes in rhizosphere (Lebeis et al., 2015). doi: 10.1007/s00425-002-0750-4, Li, S. M., Hua, G. G., Liu, H. X., and Guo, J. H. (2008). 19, 711724. A., and Chen, J. F. (2010). The gyrB gene sequence of LJ02 was aligned with published sequences of related taxa obtained from GenBank. The search for the BCA against powdery mildew has been a long standing practice for plant pathologists. Besides, the free SA was also increased in roots and it is possible that some beneficial bacteria could also be recruited to rhizospheres to protect plants from pathogenic invasions. Plant. 167, 1419. In view of this long-term effect, we hypothesized that the systemic induced resistance (SIR) may take place when LJ02 was applied. It is proved that H2O2 appears to be a key element involved in disease resistance to pathogens (Ahmad et al., 2010). Yamasaki, H., Sakihama, Y., and Ikehara, N. (1997). doi: 10.1146/annurev.phyto.42.040803.140421, Esterbauer, H., Schwarzl, E., and Hayn, M. (1977). Front. Physiol. Control 65, 1423. Immun. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. Recently, the bidirectional signal exchanges between AGBG was identified as another type of induced resistance response. Analytical Methods Manual of Soil Microorganisms (In Chinese). Plant. PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29. Phytochemistry 70, 15811588. Eur. Typically, SAR can perceive the invasion of pathogens by initiate defensive responses via the synthesis of SA and the coordinate activation of a large set of PR proteins and resistance-related enzymes (Feys and Parker, 2000). Duncans multiple-range test was applied when one-way ANOVA revealed significant differences (P < 0.05). doi: 10.1016/j.phytochem.2009.06.009, Verberne, M. C., Verpoorte, R., Bol, J. F., Mercado-Blanco, J., and Linthorst, H. J. Specifically, strain LJ02 is aerobic and cannot grow under anaerobic conditions. Rev. It was reported that purified surfactin, mycosubtilin, and plipastatin could be perceived by grapevine plant cells to stimulate grapevine innate immune responses including SA-mediated pathways (Farace et al., 2015). doi: 10.1111/j.1365-2745.2010.01756.x, Yi, H. S., Yang, J. W., Ghim, S. Y., and Ryu, C. M. (2011). Furthermore, we have proved that LJ02-induced long-range resistance is an appropriate indicator to examine the biocontrol ability, especially the SAR-inducing activity, of different BCAs using solid rhizosphere medium. 18, 779783. Nat. Plant J. Residues and dissipation kinetics of triazole fungicides difenoconazole and propiconazole in wheat and soil in Chinese fields. It was reported that foliar attack by the whitefly not only elicited AG resistance against a leaf pathogen, Xanthomonas axonopodis pv. B. 3, 1st Beichen West Road, Chaoyang District, Beijing 100101, China, qingwei2013@im.ac.cn. Altogether, the isolated BCA was referred to as B. amyloliquefaciens LJ02 based on its morphological, phylogenetic, physiological, and biochemical characterizations. Annu. Environ. Identification of a new spore wall protein from Encephalitozoon cuniculi. Strain LJ02 produces H2S and can use citrate as sole carbon source. Each pathogen fungus (F. oxysporum, B. cinerea, and Alternaria spp.) doi: 10.1111/Mpp.12170, Feys, B. J., and Parker, J. E. (2000). doi: 10.1146/annurev.arplant.57.032905.105346, Chen, X. H., Koumoutsi, A., Scholz, R., Schneider, K., Vater, J., Sussmuth, R., et al. Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection against various abiotic stresses. Plant Signal. Phytopathol. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. All those results showed that LJ02FB could elicit the defense responses from plant leaves to rhizosphere, suggesting a long-range systemic resistance is induced under our test conditions. Furthermore, the expression of PR-1 gene was detected by qRT-PCR. This is possibly caused by the degradation of functional elicitors in LJ02FS. Activities of superoxide dismutase (SOD) isoforms during growth of Scenedesmus (Chlorophyta) species and strains grown in batch-cultures. Error bars indicate standard deviation among triplicates. Error bars indicate standard deviation among triplicates. Error bars indicate standard deviation among triplicates. (C) spores with different treatments. 99, 4656. Evaluation of biological control agents for managing cucurbit powdery mildew on greenhouse-grown melon. Assessment of partial resistance to powdery mildew (Podosphaera pannosa) in a tetraploid rose population using a spore-suspension inoculation method. Planta 215, 239247. (2011). It was shown to catalyze the deamination of L-phenylalanine to produce cinnamic acid, a substrate feeding into several biosynthetic routes to various classes of phenylpropanoid-derived secondary plant products, including SA (Mauch-Mani and Slusarenko, 1996). No use, distribution or reproduction is permitted which does not comply with these terms. 4:139. doi: 10.3389/fpls.2013.00139, On, A. N., Wong, F., Ko, Q., Tweddell, R. J., Antoun, H., and Avis, T. J. (E) The comparison of the inhibition zone of cucumber rhizosphere MS solid medium against different pathogenic fungi (cm). Microbiol. 37, 6971. 168, 396403. Rev. doi: 10.1126/science.1147113, Perez-Garcia, A., Romero, D., Fernandez-Ortuno, D., Lopez-Ruiz, F., De Vicente, A., and Tores, J. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. To further unravel the long-range effect of LJ02FB treatment, we tested the biocontrol activity of root exudates or secretions against common pathogenic fungi. A. Rev. Spatial and temporal detection of SA with separated LJ02FB components (LJ02BC and LJ02FS). Hypothetical model of LJ02-mediated protection of cucumber plants. doi: 10.1099/00207713-37-1-69, Ramarathnam, R., Fernando, W. G. D., and De Kievit, T. (2011). 6:219. doi: 10.3389/fpls.2015.00219, Tsuda, K., Sato, M., Glazebrook, J., Cohen, J. D., and Katagiri, F. (2008). 3, 307319. Greenhouse trials showed the CE on cucurbits powdery mildew of 70, 90, and 85% after treated with 1% LJ02FB for 7, 14, and 21 days, respectively (Table 1). Abscisic acid enhances resistance to Alternaria solani in tomato seedlings. A one-way ANOVA was performed (*P < 0.05; **P < 0.01). Xu, Y. J., Takvorian, P., Cali, A., Wang, F., Zhang, H., Orr, G., et al. 8, 63. doi: 10.1186/1475-2859-8-63, Bargabus, R. L., Zidack, N. K., Sherwood, J. E., and Jacobsen, B. J. Subsequently, the inhibition zone diameter was detected. The production of free SA and the expression of SA-dependent PR-1 gene in cucumber leaves were also enhanced markedly after treating with LJ02FB. 7, 356363. Acta Physiologiae Plantarum 25, 375384. As is shown in Figure 5, at 2nd day and 3rd day after treated with LJ02FB, the expression of PR-1 gene in treatment group was significantly higher than that of control group, indicating that SA-mediated defense gene expression is induced in LJ02FB-treated cucumber leaves. Induction of defense-related enzymes in soybean leaves by class IId bacteriocins (thuricin 17 and bacthuricin F4) purified from Bacillus strains. J. As is shown in Figure 3A, the treatment group had a significant increase in SOD production as compared to the control treatment. Wan, H. J., Zhao, Z. G., Qian, C. T., Sui, Y. H., Malik, A. Figure 7. Nat. PPOs function as catalyzing the oxygen-dependent oxidation of phenols to quinones and are assumed to be involved in plant defense against pests and pathogens (Li and Steffens, 2002). Plant Physiol. (Figure 6C). Harnessing useful rhizosphere microorganisms for pathogen and pest biocontrol, View all Biochem. The MS solid medium containing rhizosphere secretions was made into cake by a sterile puncher (0.5 cm in diameter). 15, 473497. Table 1. Therefore, we intended to identify the LJ02 based on its phylogeny, morphology, physiological, and biochemical properties. Early signaling events induced by elicitors of plant defenses. Relative expression of PR-1 gene in cucumber leaves after treated with LJ02FB (Treated) and PDB (Control). Bacteriol. (2000). Microbiol. (2011). Generally, it was believed that beneficial bacteria can make the plant more tolerant to pathogens by stimulating ISR, which was shown to protect above-ground plant tissues and acts through roots to leaves (Van der Ent et al., 2009). Microbiol. Moreover, the quantity of rhizosphere fungi was determined after treated with LJ02FB in cucumber leaves. Ecotoxicol. Salicylic-acid induced resistance to Erwinia carotovora subsp carotovora in tobacco.
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