Assessment of partial resistance to powdery mildew (Podosphaera pannosa) in a tetraploid rose population using a spore-suspension inoculation method. Control 65, 1423. 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). Physiol. 6:219. doi: 10.3389/fpls.2015.00219, Tsuda, K., Sato, M., Glazebrook, J., Cohen, J. D., and Katagiri, F. (2008). 10, 153160. All statistical analysis was performed with SPSS version 11.5 statistical software (SPSS, Chicago, IL, USA). doi: 10.1111/j.1365-313X.2007.03369.x, Van der Ent, S., Van Wees, S. C. M., and Pieterse, C. M. J. doi: 10.4265/bio.17.155, Alizadeh, H., Behboudi, K., Ahmadzadeh, M., Javan-Nikkhah, M., Zamioudis, C., Pieterse, C. M. J., et al. 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). HPLC chromatogram of SA standard (A) and sample (B), temporal changes of free SA (C) and conjugated SA (D) in cucumber leaves of control and treatment group. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa. 53, 763775. 57, 18461850. Additionally, we also found that the activity of PPO was also stimulated during LJ02 treatment. Spatial and temporal detection of SA with separated LJ02FB components (LJ02BC and LJ02FS). Harnessing useful rhizosphere microorganisms for pathogen and pest biocontrol, View all Error bars indicate standard deviation among triplicates. Eur. Overall, our work provides insights into SA-mediated defense response as a mode of action by B. amyloliquefaciens LJ02 against powdery mildew. doi: 10.1016/S0168-9525(00)02107-7, Garcia-Brugger, A., Lamotte, O., Vandelle, E., Bourque, S., Lecourieux, D., Poinssot, B., et al. Biol. Moreover, the quantity of rhizosphere fungi was determined after treated with LJ02FB in cucumber leaves. Therefore, it is likely that using gyrB as a phylogenetic marker is reliable in our study and could provide clues for bacillus determination. It is also positive for catalase, nitrate reduction, and VogesProskauer test and negative for oxidase, indole production and egg yolk reaction. 28, 27312739. SA as a key immune signal is involved in the regulation of downstream defense genes (Robert-Seilaniantz et al., 2011). (Figure 6E). 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. Cell Fact. Sci. J. Biotechnol. (D) The quantity of pathogenic fungi in the rhizosphere of cucumber treated by different concentrations of LJ02FB in leaves. doi: 10.1093/molbev/msr121, Tornero, P., Gadea, J., Conejero, V., and Vera, P. (1997). In BG part, the long-range resistance responses lead to the inhibition of several pathogens and their spore germination. Environ. Besides, the long-range elicitation of SA induction in roots may be involved in aforementioned long-range resistance. Both LJ02BC and LJ02FS could induce the production of free SA in upper leaves throughout all experiments. Time course of changes in SOD activity (A), PPO activity (B), POD activity (C), and PAL activity (D) in cucumber leaves of control and treatment group. Plant Physiol. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. 15, 4953. Yan, Z. F., Dolstra, O., Prins, T. W., Stam, P., and Visser, P. B. It was reported that foliar attack by the whitefly not only elicited AG resistance against a leaf pathogen, Xanthomonas axonopodis pv. Evol. Screening for the identification of potential biological control agents that induce systemic acquired resistance in sugar beet. 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. It is proved that H2O2 appears to be a key element involved in disease resistance to pathogens (Ahmad et al., 2010). This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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). Also the SA accumulation triggered by MAMPs is a major component of the MAMP-triggered signaling mechanism (Tsuda et al., 2008). doi: 10.1146/annurev-phyto-073009-114447, Romero, D., De Vicente, A., Zeriouh, H., Cazorla, F. M., Fernandez-Ortuno, D., Tores, J. Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with Paenibacillus illinoisensis. Hypothetical model of LJ02-mediated protection of cucumber plants. Effects of Pb on the oxidative stress and antioxidant response in a Pb bioaccumulator plant Vallisneria natans. We found that the accumulation of free SA on cucumber leaves increased rapidly after 5-day treatment as compared to control (Figure 4C). After treated for 48 h, cucumber seedlings were pulled from the MS solid medium. 10, 264272. For more details, please refer to main text. (2013). doi: 10.1146/annurev.phyto.050908.135202. Residues and dissipation kinetics of triazole fungicides difenoconazole and propiconazole in wheat and soil in Chinese fields. The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits. doi: 10.1094/MPMI-19-0711, Haas, D., and Defago, G. (2005). Int. 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. 99, 4656. 77, 486494. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Rev. (2003). 18, 779783. (2015). Xu, G. H., and Zheng, H. Y. vesicatoria, but also elevated resistance against the soil bacterium, Ralstonia solanacearum. Altogether, the isolated BCA was referred to as B. amyloliquefaciens LJ02 based on its morphological, phylogenetic, physiological, and biochemical characterizations. doi: 10.1111/j.1365-2745.2010.01756.x, Yi, H. S., Yang, J. W., Ghim, S. Y., and Ryu, C. M. (2011). 15, 848864. New York, Springer. 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). This is very intriguing since we used the fermentation broth of a beneficial bacterium rather than a pathogen to treat plant leaves. doi: 10.1111/j.1399-3054.1962.tb08052.x, Newman, M. A., Sundelin, T., Nielsen, J. T., and Erbs, G. (2013). However, we observed that LJ02BC could consistently induce twofold increase in free SA production, suggesting that microbe-associated molecular pattern (MAMP) may be responsible for SA stimulation (Newman et al., 2013). 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. Can. Figure 1. 15, 473497. Disease index and control efficacy of powdery mildew in cucumber. As is described previously, S. fuliginea is an obligate parasite which is hard to culture on nutrient medium (Perez-Garcia et al., 2009). Food Chem. doi: 10.1016/j.biocontrol.2013.01.009, Arguelles-Arias, A., Ongena, M., Halimi, B., Lara, Y., Brans, A., Joris, B., et al. It was shown that overexpression of PPO in transgenic tomato plants can lead to enhanced disease resistance (Li and Steffens, 2002). 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. 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. 3, 307319. Trends Genet. doi: 10.1111/j.1364-3703.2008.00527.x, Pokora, W., Reszka, J., and Tukaj, Z. A rapid assay for catechol oxidase and lactase using 2-nitro-5-thiobenzoic acid. 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. Phytopathol. The gyrB gene sequence of LJ02 was submitted to GenBank database and the accession number is KF501049. We proved that induced rhizosphere secretions in solid MS medium by treating plant leaves with BCAs was an effective method to examine the anti-fungal and long-range resistance activities of potential BCAs. LJ02 produces acid from xylose, sorbitol, L-arabinose, starch, inositol, sucrose, glucose, galactose, ribose, glycogen, and glycerol. doi: 10.1099/00207713-37-1-69, Ramarathnam, R., Fernando, W. G. D., and De Kievit, T. (2011). Analysis of defence enzymes induced by antagonistic bacterium Bacillus subtilis strain AR12 towards Ralstonia solanacearum in tomato. 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. Biochem. Relative expression of PR-1 gene in cucumber leaves after treated with LJ02FB (Treated) and PDB (Control). Biotechnol. Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29. It was also reported that enhanced resistance against phytopathogens by exogenous elicitor application is also associated with defense-related enzymes (Song et al., 2011). Crit. Scanning microscope observation of LJ02 under two resolutions: 25,000 (A) and 5,000 (B). Sci. As is shown in Figure 3B, there was slight difference between the control and treatment group plants at the primary treatment period (<2 days). Plants commonly use plasma-membrane localized pattern recognition receptors (PRRs) to perceive MAMPs/pathogen-associated molecular patterns (PAMPs; Newman et al., 2013). Analytical Methods Manual of Soil Microorganisms (In Chinese). and Pseudomonas sp. Nat. 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. 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). 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). Ecotoxicol. BMC Plant Biol. (2015). Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation. 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). (2011). J. Syst. Interestingly, LJ02FS could induce significantly higher amount of free SA at 5th day and declined to control level. 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. Germination rate of F. oxysporum (A), B. cinerea (B), and Alternaria spp. through plate cultivation test. Interestingly, as an major defensive enzyme, SOD showed significantly elevated activity. Briefly, 20 L of the LJ02FB was dropped onto the leaves including cotyledon under sterile condition at trefoil stage. Different letters are significantly difference at P < 0.01 according to Duncan multiple range test. (2015). Induced systemic resistance in cucumber and Arabidopsis thaliana by the combination of Trichoderma harzianum Tr6 and Pseudomonas sp Ps14. 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. Time course of changes in free SA activity in leaves (A) and in roots (B) of control and treatment group. We found that PR-1 gene was stimulated after treated with LJ02FB in cucumber leaves at different time points. (2006). Different letters are significantly difference at P < 0.01 according to Duncan multiple range test. Bergeys Manual of Systematic Bacteriology. One strain was selected and named LJ02. Int. Planta 215, 239247. (Figure 6C). (2004). Rev. Phytochemistry 70, 15811588. Control 6, 353360. Figure 6. Both group induced a rapid increase of PAL activity levels in cucumber leaves within 13 days. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. Wan, H. J., Zhao, Z. G., Qian, C. T., Sui, Y. H., Malik, A. doi: 10.1128/Iai.74.1.239-247.2006. Strain LJ02 produces H2S and can use citrate as sole carbon source. Interestingly, we further found a large body of evidences that LJ02 could induce SA-mediated SAR as one of its major mode of actions. Previous experiments had the same results after they inoculated the pathogens on various plants (Verberne et al., 2000). 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. Immun. Figure 8. 15, 583606. (2009). Microbiol. The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Li, L., and Steffens, J. C. (2002). Plant Microbe Interact. Table 2. 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). 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. Biochem. doi: 10.1094/MPMI.1997.10.5.624, Trda, L., Boutrot, F., Claverie, J., Brule, D., Dorey, S., and Poinssot, B. Phylogenetic tree of strain LJ02 and other Bacillus species from GenBank based on gyrB gene sequences. Thus, we come to the conclusion that functional SA production was enhanced in cucumber leaves to stimulate SA-mediated defense response, such as the expression of resistance-related enzymes. Received: 03 April 2015; Accepted: 11 August 2015; Published: 28 August 2015. doi: 10.1007/s11738-003-0019-y, Porcel, R., Zamarreno, A. M., Garcia-Mina, J. M., and Aroca, R. (2014). Front. 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). (2004). One-hundred-fifteen isolations were collected to test their antagonistic activity according to their inhibitory activity. 140, 2737. Jasmonate signaling in plant interactions with resistance-inducing beneficial microbes. In line with 7th day, the quantity of fungi at 14th day is still significantly less than that of control group (P < 0.01). Plant J. 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. 78, 2834. Annu. Control 80, 6369. Furthermore, the expression of PR-1 gene was detected by qRT-PCR. 6, 11921194. doi: 10.1016/0003-2697(77)90262-7, Faheem, M., Raza, W., Zhong, W., Nan, Z., Shen, Q. R., and Xu, Y. C. (2015). except 16S rRNA gene sequence (Yamamoto and Harayama, 1995). These authors have contributed equally to this work. Production of salicylic acid precursors is a major function of phenylalanine ammonia-lyase in the resistance of Arabidopsis to Peronospora parasitica. Evol. doi: 10.1016/j.jbiotec.2008.10.011, Chen, Y., Yan, F., Chai, Y., Liu, H., Kolter, R., Losick, R., et al. doi: 10.1111/j.1462-2920.2012.02860.x, Durrant, W. E., and Dong, X. Besides, the examination of rhizosphere secretions in solid MS medium was proved to be an effective method that identifies potential BCAs with resistance-induction and anti-fungal activities. Mol. 108, 16731678. Systemic acquired resistance signal transduction. 138, 520533. (C) spores with different treatments. In view of this long-term effect, we hypothesized that the systemic induced resistance (SIR) may take place when LJ02 was applied. 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). The cake made from sterile MS solid medium without root secretions was used as a control. Phylogenetic analysis of LJ02 was determined using gyrB gene as a reference (Yamamoto and Harayama, 1995). doi: 10.1016/j.biocontrol.2003.11.005, Boller, T., and Felix, G. (2009). doi: 10.1146/annurev.arplant.57.032905.105346, Chen, X. H., Koumoutsi, A., Scholz, R., Schneider, K., Vater, J., Sussmuth, R., et al. In previous studies, only one-way signal transduction was investigated, such as AGBG, AGAG, and BGBG (Yi et al., 2011). Front. Rev. 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. Annu. Leon, J., Lawton, M. A., and Raskin, I. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. Salicylic acid is an important plant-produced signaling molecule involved in SAR (Park et al., 2007). Front. Microb. 61, 11041109. Those results led us to conclude that SAR is indeed induced by LJ02 and MAMP is likely to be responsible for the triggering of SA-dependent defense such as SA synthesis and the subsequent pathogen inhibition (Porcel et al., 2014). 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). During initial screening of LJ02, greenhouse trials indicated that LJ02 is effective in controlling cucurbits powdery mildew over 20 days. Most spores would germinate at 9 h in the preliminary period. 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. (2006). 16, 449455. 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. doi: 10.1006/bcon.1996.0045, Vlot, A. C., Dempsey, D. A., and Klessig, D. F. (2009). 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. (A) A cucumber seedling under normal condition; (B) LJ02 inoculation elicits SA-dependent signaling pathway in both directions: AGAG and AGBG. Strain LJ02 colonies show light yellow on LB agar medium. The MS solid medium containing rhizosphere secretions was made into cake by a sterile puncher (0.5 cm in diameter). J. Ecol. Biol. Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Plant Sci. Vos, P., Garrity, G., Jones, D., Krieg, N. R., Ludwig, W., Rainey, F. A., et al. Nat. 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. (2009). doi: 10.1038/77347, Verhaar, M. A., Hijwegen, T., and Zadoks, J. C. (1996). At present, gyrA, gyrB, rpoB, and rpoD gene sequences have been used for identifying the Bacillus sp. A cry for help from leaf to root. doi: 10.1126/science.1147113, Perez-Garcia, A., Romero, D., Fernandez-Ortuno, D., Lopez-Ruiz, F., De Vicente, A., and Tores, J.