Physiol. 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). Figure 4. doi: 10.1126/science.1147113, Perez-Garcia, A., Romero, D., Fernandez-Ortuno, D., Lopez-Ruiz, F., De Vicente, A., and Tores, J. Biotechnol. In this study, we isolated a BCA against powdery mildew and identified it as a new member of B. amyloliquefaciens. Error bars indicate standard deviation among triplicates. Salicylic-acid induced resistance to Erwinia carotovora subsp carotovora in tobacco. Control 65, 1423. 3, 307319. 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. Error bars indicate standard deviation among triplicates. doi: 10.1016/j.biocontrol.2014.09.017, Palva, T. K., Hurtig, M., Saindrenan, P., and Palva, E. T. (1994). Appl. No use, distribution or reproduction is permitted which does not comply with these terms. doi: 10.1006/bcon.1996.0045, Vlot, A. C., Dempsey, D. A., and Klessig, D. F. (2009). Biochem. The PAL activities of either group reached the top level at 3rd day, while treatment group induced significantly stronger PAL activity (P < 0.05) than the control at the maximum inducing time point (Figure 3D). In order to further unravel the protective mechanisms of LJ02FB on disease control, the rhizosphere exudates of LJ02FB-treated cucumber were co-cultured with three common pathogen spores. Strain LJ02 produces H2S and can use citrate as sole carbon source. We inoculated both fractions onto cucumber cotyledons to treat plants and then extracted the SA samples from upper uninoculated true leaves and in roots after 0.5, 1, 3, 5, 7, and 11 days. Infect. Identification of a new spore wall protein from Encephalitozoon cuniculi. doi: 10.1016/J.Ab.2009.12.008, Wang, L. T., Lee, F. L., Tai, C. J., and Kasai, H. (2007). The final germination rate is about 10% for F. oxysporum (Figure 6A), less than 1% for B. cinerea (Figure 6B) and less than 2% for Alternaria spp. Therefore, BCA-induced production of SOD could provide an extra protection against pathogen infection of plants. A rapid assay for catechol oxidase and lactase using 2-nitro-5-thiobenzoic acid. Moreover, the quantity of rhizosphere fungi was determined after treated with LJ02FB in cucumber leaves. One strain was selected and named LJ02. (2015). was also made into fungi cake by a sterile puncher (0.5 cm in diameter) which was then inoculated onto the center of PDA plate. Yan, Z. F., Dolstra, O., Prins, T. W., Stam, P., and Visser, P. B. doi: 10.1111/j.1462-2920.2012.02860.x, Durrant, W. E., and Dong, X. Strain LJ02 colonies show light yellow on LB agar medium. (Figure 6E). As can be seen from Figure 7A, the accumulation of free SA in upper uninoculated cucumber leaves increased rapidly after 3-day treatment as compared to control. Rev. 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 of strain LJ02 and other Bacillus species from GenBank based on gyrB gene sequences. (2004). Wan, H. J., Zhao, Z. G., Qian, C. T., Sui, Y. H., Malik, A. Subsequently, the inhibition zone diameter was detected. (D) The quantity of pathogenic fungi in the rhizosphere of cucumber treated by different concentrations of LJ02FB in leaves. 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). Salicylic acid is an important plant-produced signaling molecule involved in SAR (Park et al., 2007). Thus, we established a method to detect the concentration of SA in LJ02FB-treated cucumber leaves (Figures 4A,B). 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. It is also positive for catalase, nitrate reduction, and VogesProskauer test and negative for oxidase, indole production and egg yolk reaction. This confirmed our speculation that LJ02 could stimulate the SA-mediated SAR response in cucumbers. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Interestingly, as an major defensive enzyme, SOD showed significantly elevated activity. Phytopathol. doi: 10.1111/j.1399-3054.1962.tb08052.x, Newman, M. A., Sundelin, T., Nielsen, J. T., and Erbs, G. (2013). Microbiol. (2006). Cell Fact. Antifungal effects of compost tea microorganisms on tomato pathogens. To further dissect the functions played by bacterium and its fermented substances of LJ02FB, we separated them by centrifugation and kept bacterial cells in 10 mM MgCl2 solution (LJ02BC) and filtered the supernatants to rule out the contamination of bacterial cells to harvest fermented substances (LJ02FS). 19, 711724. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Each pathogen fungus (F. oxysporum, B. cinerea, and Alternaria spp.) 10, 624634. 108, 16731678. 17, 155167. In addition, we also detected an increase of free SA (day 3 11) in cucumber roots with both LJ02BC- and LJ02FS-treatment (Figure 7B). Time course of changes in free SA activity in leaves (A) and in roots (B) of control and treatment group. doi: 10.1111/Mpp.12170, Feys, B. J., and Parker, J. E. (2000). In doing so, we could examine the effects of both fractions (LJ02BC and LJ02FS) on the production of SA in cucumber tissues with spatial and temporal scales. And it appeared that LJ02BC played a major role in this process, although the amount of free SA was lower as compared to that of leaves. Based on the long-range resistance responses, we have established a method that could easily detect the inhibitory effect induced by foliar BCAs. This is likely to be caused by the degradation of potential elicitors in fermentation broth. 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. Assessment of partial resistance to powdery mildew (Podosphaera pannosa) in a tetraploid rose population using a spore-suspension inoculation method. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Figure 1. Involvement of plant endogenous ABA in Bacillus megaterium PGPR activity in tomato plants. The content of free SA is known to increase in dozens of times when plant tissues are affected by incompatible pathogens and elicitors (Leon et al., 1995). Beijing: China Agriculture Press, 107108. doi: 10.4141/CJPS06035. Rev. 10, 153160. doi: 10.1128/Iai.74.1.239-247.2006. doi: 10.1016/j.phytochem.2009.06.009, Verberne, M. C., Verpoorte, R., Bol, J. F., Mercado-Blanco, J., and Linthorst, H. J. Relative expression of PR-1 gene in cucumber leaves after treated with LJ02FB (Treated) and PDB (Control). 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. 7, 356363. Our work demonstrated that the content of free SA in cucumber leaves was kept at a relatively higher level after spraying the LJ02FB. We found that PR-1 gene was stimulated after treated with LJ02FB in cucumber leaves at different time points. Flavonoid-peroxidase reaction as a detoxification mechanism of plant cells against H2O2. doi: 10.1016/0261-2194(95)00109-3, Robert-Seilaniantz, A., Grant, M., and Jones, J. D. (2011). Interestingly, LJ02FS could induce significantly higher amount of free SA at 5th day and declined to control level. 42, 185209. Different letters are significantly difference at P < 0.01 according to Duncan multiple range test. Glasshouse experiments on biocontrol of cucumber powdery mildew (Sphaerotheca fuliginea) by the Mycoparasites verticillium lecanii and Sporothrix rugulosa. Plant Microbe Interact. Induction of defense-related enzymes in soybean leaves by class IId bacteriocins (thuricin 17 and bacthuricin F4) purified from Bacillus strains. doi: 10.1016/j.micres.2011.02.004, Korkmaz, A., Uzunlu, M., and Demirkiran, A. R. (2007). doi: 10.1007/s10526-010-9324-8, Reuveni, M., Agapov, V., and Reuveni, R. (1996). Abscisic acid enhances resistance to Alternaria solani in tomato seedlings. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa. Nat. Annu. Perception of pathogenic or beneficial bacteria and their evasion of host immunity: pattern recognition receptors in the frontline. Although we did not detect a large amount of SA induced in roots, it is clearly that LJ02 could significant induce its production in a temporal manner. Ann. Overall, our work provides insights into SA-mediated defense response as a mode of action by B. amyloliquefaciens LJ02 against powdery mildew. In vitro and in vivo biocontrol of soil-borne phytopathogenic fungi by certain bioagents and their possible mode of action. Science 318, 113116. doi: 10.1038/nrmicro1129, Hunt, M. D., and Ryals, J. Microbiol. However, the excess production of H2O2 will lead to the production of POD that scavenge H2O2 (Yamasaki et al., 1997). Int. Different letters are significantly difference at P < 0.01 according to Duncan multiple range test. Therefore, we detected the diameter of inhibition zone against other pathogen fungi (F. oxysporum, B. cinerea, and Alternaria spp.) At present, gyrA, gyrB, rpoB, and rpoD gene sequences have been used for identifying the Bacillus sp. Physiol. Therefore, it is conceivably that similar substances will be produced in its close species, B. amyloliquefaciens and similar immune responses will be induced. Biol. We found extremely evident induced defensive response of root exudates against different pathogens including F. oxysporum, B. cinerea and Alternaria spp. To this end, the activity of systemic resistance-related enzymes was firstly examined. 58, 573578. Biocontrol 50, 165178. doi: 10.1111/j.1365-313X.2007.03369.x, Van der Ent, S., Van Wees, S. C. M., and Pieterse, C. M. J. Screening for the identification of potential biological control agents that induce systemic acquired resistance in sugar beet. LJ02 produces acid from xylose, sorbitol, L-arabinose, starch, inositol, sucrose, glucose, galactose, ribose, glycogen, and glycerol. It was shown that overexpression of PPO in transgenic tomato plants can lead to enhanced disease resistance (Li and Steffens, 2002). 26 Articles, This article is part of the Research Topic, Creative Commons Attribution License (CC BY). Science doi: 10.1126/science.aaa8764 [Epub ahead of print]. The main mechanisms of BCAs include the production of antibiotics, competition, plant growth promotion, and the induction of SAR and ISR (Ramarathnam et al., 2011). Rev. doi: 10.1016/j.ecoenv.2011.11.008. 10, 264272. Although it is similar to that described for disk diffusion method (On et al., 2015), our new method takes the advantage of the coordinate activation of anti-fungal substances by plant-BCA-pathogen interactions rather than traditional BCA-pathogen interactions. Ahmad, R., Kim, Y. H., Kim, M. D., Kwon, S. Y., Cho, K., Lee, H. S., et al. doi: 10.1016/j.biocontrol.2014.11.012, Farace, G., Fernandez, O., Jacquens, L., Coutte, F., Krier, F., Jacques, P., et al. In line with 7th day, the quantity of fungi at 14th day is still significantly less than that of control group (P < 0.01). All those results confirmed that LJ02FB treatment could indeed induce the production of SIR-related enzymes. 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). Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. Environ. Plant Signal. 53, 763775. Nat. 4:139. doi: 10.3389/fpls.2013.00139, On, A. N., Wong, F., Ko, Q., Tweddell, R. J., Antoun, H., and Avis, T. J.