biological fungicide for powdery mildew

2021/05/21

doi: 10.1016/j.biocontrol.2013.01.009, Arguelles-Arias, A., Ongena, M., Halimi, B., Lara, Y., Brans, A., Joris, B., et al. 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. Bergeys Manual of Systematic Bacteriology. Trends Immunol. doi: 10.1111/j.1364-3703.2008.00527.x, Pokora, W., Reszka, J., and Tukaj, Z. The number of CFU was recorded on each plate after 3 days.

Science doi: 10.1126/science.aaa8764 [Epub ahead of print]. 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. Evol. Altogether, the isolated BCA was referred to as B. amyloliquefaciens LJ02 based on its morphological, phylogenetic, physiological, and biochemical characterizations. Phylogenetic tree of strain LJ02 and other Bacillus species from GenBank based on gyrB gene sequences. 37, 6971. Li, L., and Steffens, J. C. (2002). This is likely to be caused by the degradation of potential elicitors in fermentation broth. doi: 10.1016/j.plaphy.2011.03.018, Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011). 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. Table 1. Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group. fungicide cure mildew powdery garden wasilla alaska natural adventures bush These authors have contributed equally to this work. Table 2. doi: 10.4161/psb.6.8.15780, Zhang, Z., Jiang, W., Jian, Q., Song, W., Zheng, Z., Wang, D., et al. Salicylic-acid induced resistance to Erwinia carotovora subsp carotovora in tobacco. Trends Genet. This further promoted us to note that LJ02 could induce a long-range defense response from leaves to roots in unknown mechanisms. (2004).

To further unravel the long-range effect of LJ02FB treatment, we tested the biocontrol activity of root exudates or secretions against common pathogenic fungi.

Specifically, strain LJ02 is aerobic and cannot grow under anaerobic conditions.

To this end, the activity of systemic resistance-related enzymes was firstly examined.

Flavonoid-peroxidase reaction as a detoxification mechanism of plant cells against H2O2.

Plant Pathol. (2015). 6, 11921194. Different letters are significantly difference at P < 0.01 according to Duncan multiple range test.

And it was shown that gyrB gene sequences provide higher resolution than 16S rRNA gene sequences (Wang et al., 2007). doi: 10.1016/j.biocontrol.2014.11.012, Farace, G., Fernandez, O., Jacquens, L., Coutte, F., Krier, F., Jacques, P., et al. 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. doi: 10.4141/CJPS06035. No use, distribution or reproduction is permitted which does not comply with these terms. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with Paenibacillus illinoisensis. 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. Int. doi: 10.1094/Mpmi-7-0356, Park, S. W., Kaimoyo, E., Kumar, D., Mosher, S., and Klessig, D. F. (2007). A one-way ANOVA was performed (*P < 0.05; **P < 0.01). 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). Time course of changes in free SA activity in leaves (A) and in roots (B) of control and treatment group. Most spores would germinate at 9 h in the preliminary period. Figure 1. Biotechnol. 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). Phytochemistry 70, 15811588. Plant Sci. 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. 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. Four pieces of MS solid medium cake were inoculated around the fungi cake at a distance of 2.5 cm (treatment group). Front. doi: 10.1631/jzus.B0820341, Mauch-Mani, B., and Slusarenko, A. J. 8, 63. doi: 10.1186/1475-2859-8-63, Bargabus, R. L., Zidack, N. K., Sherwood, J. E., and Jacobsen, B. J.

Environ. Microbiol. Moreover, we determined that TJ02FB-treated cucumber leaves secreted resistance-related substance into rhizosphere against a range of fungi pathogens, suggesting that LJ02 could elicit a long-range systemic resistance in cucumber against pathogens. 49, 693700. (2011). In vitro and in vivo biocontrol of soil-borne phytopathogenic fungi by certain bioagents and their possible mode of action. Acetyl salicylic acid alleviates chilling-induced damage in muskmelon seedlings. Plant Signal. (2015). Physiol. Res. Control 65, 1423. Sci. (C) spores with different treatments. (2009). 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. 19, 711724. In view of this long-term effect, we hypothesized that the systemic induced resistance (SIR) may take place when LJ02 was applied. The images were taken from a ultra-high resolution scanning electron microscope SU8010. Ann. Additionally, we also found that the activity of PPO was also stimulated during LJ02 treatment.

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. The gyrB gene sequence of LJ02 was submitted to GenBank database and the accession number is KF501049. 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). Biol. Beijing: China Agriculture Press, 107108. Another interesting finding in our study is that the SA signaling pathways induced by beneficial bacterium such as LJ02 is likely to function from leaves (aboveground, AG) to roots (belowground, BG; Figure 7B). Thus, we established a method to detect the concentration of SA in LJ02FB-treated cucumber leaves (Figures 4A,B). Crit. (1995). B. Microbiol. It was also reported that enhanced resistance against phytopathogens by exogenous elicitor application is also associated with defense-related enzymes (Song et al., 2011). Salicylic acid is an important plant-produced signaling molecule involved in SAR (Park et al., 2007). Production of salicylic acid precursors is a major function of phenylalanine ammonia-lyase in the resistance of Arabidopsis to Peronospora parasitica. Sci. But the activity of PPO increased rapidly after 2 days and became significant different (P < 0.05) from the control at 3rd day. Acta Physiologiae Plantarum 25, 375384. (E) The comparison of the inhibition zone of cucumber rhizosphere MS solid medium against different pathogenic fungi (cm). These are conserved signatures of crucial microbial structures, such as cell wall components or motility organs (Boller and Felix, 2009) as well as cyclic lipopeptides found in B. subtilis (Farace et al., 2015). 15, 4953. Analytical Methods Manual of Soil Microorganisms (In Chinese).

Beneficial bacteria and pathogens are initially recognized as harmful invaders in order to limit their spreading (Trda et al., 2015). Control 80, 6369.

Error bars indicate standard deviation among triplicates. doi: 10.1016/J.Ab.2009.12.008, Wang, L. T., Lee, F. L., Tai, C. J., and Kasai, H. (2007). Besides, the long-range elicitation of SA induction in roots may be involved in aforementioned long-range resistance. doi: 10.1146/annurev-phyto-073009-114447, Romero, D., De Vicente, A., Zeriouh, H., Cazorla, F. M., Fernandez-Ortuno, D., Tores, J. 60, 379406. doi: 10.1146/annurev.arplant.57.032905.105346, Chen, X. H., Koumoutsi, A., Scholz, R., Schneider, K., Vater, J., Sussmuth, R., et al. Yan, Z. F., Dolstra, O., Prins, T. W., Stam, P., and Visser, P. B.

As is shown in Figure 3A, the treatment group had a significant increase in SOD production as compared to the control treatment. Error bars indicate standard deviation among triplicates. Plant Microbe Interact. 138, 520533. Microbiol. Plant Microbe Interact. Based on the long-range resistance responses, we have established a method that could easily detect the inhibitory effect induced by foliar BCAs. It was shown that overexpression of PPO in transgenic tomato plants can lead to enhanced disease resistance (Li and Steffens, 2002). 16, 177187. Anal. doi: 10.1099/ijs.0.64685-0, Wang, P., Zhang, S., Wang, C., and Lu, J. B. subtilis is known to produce cyclic lipopeptides such as surfactins, iturins and fengycins (Farace et al., 2015). Bacteriol. 10, 264272. doi: 10.1016/j.biocontrol.2014.09.017, Palva, T. K., Hurtig, M., Saindrenan, P., and Palva, E. T. (1994). Leon, J., Lawton, M. A., and Raskin, I. This confirmed our speculation that LJ02 could stimulate the SA-mediated SAR response in cucumbers. Overall, our work provides insights into SA-mediated defense response as a mode of action by B. amyloliquefaciens LJ02 against powdery mildew. J. Syst. Scanning microscope observation of LJ02 under two resolutions: 25,000 (A) and 5,000 (B). Therefore, we switched to the other method described by Yamamoto using gyrB as a marker to perform phylogenetic analysis (Yamamoto and Harayama, 1995). J.

MAMP (microbe-associated molecular pattern) triggered immunity in plants. doi: 10.1111/Mpp.12170, Feys, B. J., and Parker, J. E. (2000).

In order to investigate the effect of LJ02FB on this process, the expression of PR-1 in cucumber leaves was studied after treated with LJ02FB. (Figure 6E).

Plants commonly use plasma-membrane localized pattern recognition receptors (PRRs) to perceive MAMPs/pathogen-associated molecular patterns (PAMPs; Newman et al., 2013). Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa.

Mol. Figure 6. (2006).

Identification of a new spore wall protein from Encephalitozoon cuniculi. Eur. doi: 10.1080/07352689609382371, Jung, W. J., Jin, Y. L., Kim, K. Y., Park, R. D., and Kim, T. H. (2005). 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. All those results confirmed that LJ02FB treatment could indeed induce the production of SIR-related enzymes. 4:139. doi: 10.3389/fpls.2013.00139, On, A. N., Wong, F., Ko, Q., Tweddell, R. J., Antoun, H., and Avis, T. J. Figure 7. Therefore, we detected the diameter of inhibition zone against other pathogen fungi (F. oxysporum, B. cinerea, and Alternaria spp.) Univ. 42, 185209.

Interestingly, we further found a large body of evidences that LJ02 could induce SA-mediated SAR as one of its major mode of actions. Control 6, 353360. (2011). This is very intriguing since we used the fermentation broth of a beneficial bacterium rather than a pathogen to treat plant leaves. Error bars indicate standard deviation among triplicates. doi: 10.1016/j.phytochem.2009.06.009, Verberne, M. C., Verpoorte, R., Bol, J. F., Mercado-Blanco, J., and Linthorst, H. J. Plant Pathol. Interplay of signaling pathways in plant disease resistance. doi: 10.1111/j.1399-3054.1962.tb08052.x, Newman, M. A., Sundelin, T., Nielsen, J. T., and Erbs, G. (2013). Physiological and biochemical characteristics of strain LJ02. 108, 16731678. Xu, Y. J., Takvorian, P., Cali, A., Wang, F., Zhang, H., Orr, G., et al.

A one-way ANOVA was performed (*P < 0.05; **P < 0.01). 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. Each pathogen fungus (F. oxysporum, B. cinerea, and Alternaria spp.) Environ. The LJ02FS is also functional to stimulate the SA production both in upper leaves and roots but not as potent as that of LJ02BC. 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. 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). Assessment of partial resistance to powdery mildew (Podosphaera pannosa) in a tetraploid rose population using a spore-suspension inoculation method. We found extremely evident induced defensive response of root exudates against different pathogens including F. oxysporum, B. cinerea and Alternaria spp. During initial screening of LJ02, greenhouse trials indicated that LJ02 is effective in controlling cucurbits powdery mildew over 20 days.

Different letters are significantly difference at P < 0.01 according to Duncan multiple range test. Rev. Cell Fact. 6:883. doi: 10.3389/fmicb.2015.00883.

(2015). This work was supported in part by National Natural Science Foundation of China (31171892; 31300066) and Extension of Agricultural Science and Technology Project funded by Tianjin Agricultural Commission (201304160).

56, 976986. 53, 763775. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).

Biol. (2006). It was reported that foliar attack by the whitefly not only elicited AG resistance against a leaf pathogen, Xanthomonas axonopodis pv. Analysis of defence enzymes induced by antagonistic bacterium Bacillus subtilis strain AR12 towards Ralstonia solanacearum in tomato. doi: 10.1093/molbev/msr121, Tornero, P., Gadea, J., Conejero, V., and Vera, P. (1997). Annu. This is possibly caused by the degradation of functional elicitors in LJ02FS. 99, 4656. Interestingly, LJ02FS could induce significantly higher amount of free SA at 5th day and declined to control level. Abscisic acid enhances resistance to Alternaria solani in tomato seedlings. The tree was constructed by MEGA5.1 by the neighbor-joining method.

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). A rapid assay for catechol oxidase and lactase using 2-nitro-5-thiobenzoic acid. (2013). Science 318, 113116.

PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. J. Biotechnol.

Biochem. In addition, we also detected an increase of free SA (day 3 11) in cucumber roots with both LJ02BC- and LJ02FS-treatment (Figure 7B). The MS solid medium containing rhizosphere secretions was made into cake by a sterile puncher (0.5 cm in diameter). Can. 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. After treated for 48 h, cucumber seedlings were pulled from the MS solid medium. PRR-mediated microbe sensing gives rise to a wide array of defense responses known as MAMP- or PAMP-triggered immunity (MTI/PTI; Zipfel, 2014). Effects of Pb on the oxidative stress and antioxidant response in a Pb bioaccumulator plant Vallisneria natans.

doi: 10.1094/MPMI.1997.10.5.624, Trda, L., Boutrot, F., Claverie, J., Brule, D., Dorey, S., and Poinssot, B. (2015). In addition, we also observed an increase of conjugated SA (day 3 5) with a subsequent decline in production from LJ02FB-treated leaves (Figure 4D).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Control 30, 342350. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. 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. Plant pattern-recognition receptors. 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). Microbiol. 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. All experiments had at least five replicates. Mol. Plant. 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).

The gyrB gene sequence of LJ02 was aligned with published sequences of related taxa obtained from GenBank. doi: 10.1111/j.1365-313X.2007.03369.x, Van der Ent, S., Van Wees, S. C. M., and Pieterse, C. M. J. Induced systemic resistance in cucumber and Arabidopsis thaliana by the combination of Trichoderma harzianum Tr6 and Pseudomonas sp Ps14. 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. Annu. 87, 581585. Residues and dissipation kinetics of triazole fungicides difenoconazole and propiconazole in wheat and soil in Chinese fields. We found that PR-1 gene was stimulated after treated with LJ02FB in cucumber leaves at different time points. 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). 47, 177206. Microbiol. doi: 10.1146/annurev.phyto.050908.135202. (2012).

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.

6:219. doi: 10.3389/fpls.2015.00219, Tsuda, K., Sato, M., Glazebrook, J., Cohen, J. D., and Katagiri, F. (2008). Figure 5. Both LJ02BC and LJ02FS could induce the production of free SA in upper leaves throughout all experiments.

Plant.

Error bars indicate standard deviation among triplicates. Biochem. Nat.

Mol.

Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. Jasmonate signaling in plant interactions with resistance-inducing beneficial microbes.

Figure 2.

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. In BG part, the long-range resistance responses lead to the inhibition of several pathogens and their spore germination. 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. Hypothetical model of LJ02-mediated protection of cucumber plants. Front.

A. Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29. Saf. Activities of superoxide dismutase (SOD) isoforms during growth of Scenedesmus (Chlorophyta) species and strains grown in batch-cultures. 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. SA as a key immune signal is involved in the regulation of downstream defense genes (Robert-Seilaniantz et al., 2011). Infect. 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). Controlling powdery mildew caused by Sphaerotheca fuliginea in cucumber by foliar sprays of phosphate and potassium salts. 28, 27312739. To be specific, when treated with LJ02FB, cucumber seedlings produced significantly high amount of SOD, POD, PPO, and PAL. Biol. 57, 18461850. 61, 11041109. doi: 10.1105/tpc.8.2.203, Murashige, T., and Skoog, F. (1962). 15, 848864. Actually, it was reported that PAL and PPO are the main SAR-related enzymes in plants (Jung et al., 2011). Systemic acquired resistance signal transduction. (2006). PAL was also enhanced during LJ02 treatment. 15, 583606. As is shown in Figure 6D, after treated with LJ02FB in cucumber leaves, the number of rhizosphere fungi declined significantly. doi: 10.1007/BF03175560, Li, J., Yan, Q., Zhao, L. H., Zhang, S. M., Wang, Y. X., and Zhao, X. Y.

Rev. The search for the BCA against powdery mildew has been a long standing practice for plant pathologists.

Plant Physiol. One-hundred-fifteen isolations were collected to test their antagonistic activity according to their inhibitory activity. 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.

Figure 8. Microbiol. 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). (2004).

All features are highly identical to those of B. amyloliquefaciens described in Manual of Systematic Bacteriology (Vos et al., 2009). Received: 03 April 2015; Accepted: 11 August 2015; Published: 28 August 2015. The main function of SOD is eliminating the cellular superoxide radical (O2), and the increase of SOD activity can lead to the accumulation of H2O2.