Biological Control of Blast Disease of Rice (Oryza sativa L.) with Antagonistic Bacteria and Its Mediation by a Pseudomonas Antibiotic

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1 Biological Control of Blast Disease of Rice (Oryza sativa L.) with Antagonistic Bacteria Its Mediation by a Pseudomonas Antibiotic Samuel S. GNANAMANICKAM** Twng Wah MEW* Abstract Rice plant associated microorganisms both Pseudomonas spp. Bacillus spp. were isolated tested for antagonism against the rice blast fungus Pyricularia oryzae Cav. Field test of four selected strains in an upl rice farm in the Philippines afforded significant leaf blast reduction with rice var. UPLRi-5. Strain 7-14, identified as P. fluorescens was most effective. The crude extract prepared from this strain provided % inhibition of conidial germination at 1.0ppm. The antiblast extract also protected IR 50 rice seedlings from infection of P. oryzae. Evidence suggested that siderophore was unlikely to be involved in the mechanism of strain 7-14 in its antagonism against the rice blast fungus. In vitro test showed that inhibition of P. oryzae by strain 7-14 was not reversed by Fe amendments. Thus, the antiblast antibiotic, instead of siderophore production from Pseudomonas strain 7-14 protected rice seedlings from infection by P. oryzae. (Received October 23, 1991) Key words: rice, Pyricularia oryzae, biological control. INTRODUCTION Rice is the staple food for 2.7 billion people in Asia where 90% of the world's rice is grown eaten2). Blast caused by the fungus Pyricularia oryzae Cav. is a severe production constraint particularly in dryl unbunded upl rice cultures in tropical Asia where rice farmers generally have no access to fungicides often cannot afford purchased inputs. Because of the ability of the rice blast fungus to evolve new races, control of blast through breeeding for resistance has had only partial success in favorable environments. Therefore, we are searching for alternate disease management strategies that are nonpolluting inexpensive. Recent studies have shown the potential of root-colonizing microorganisms in inhibiting or displacing soilborne pathogens at the root-soil interface, thereby protect the root health of perennial annual plants10) such as cotton, potato, tobacco, flax, radish, cucumber, wheat, rice3,4,6-8,11-13,15). These microorganisms inhibit pathogens by producing antibiotics siderophores (compounds that chelate biologically available Fe), may produce substances that stimulate plant growth14,16,17). This paper describes new evidence of the potential of antagonistic bacteria to control rice blast, a destructive foliar disease, presents the basis for such antagonism in a fluorescent Pseudomonas strain. MATERIALS AND METHODS Isolation selection of bacterial strains. Initially we screened more than 400 bacterial

2 Ann. Phytopath. Soc. Japan 58 (3). July, strains in the laboratory. Of these, two fluorescent strains of Pseudomonas two strains of Bacillus causing maximum inhibition of P. oryzae were chosen for further evaluation in the field. Using stard procedures, mutant strains were generated by incorporating resistance to rifampicin (R) or rifampicin nalidixic acid (RN) (100ppm). A field experiment in a romized complete block design with four replications was conducted at an upl rice research site of the International Rice Research Institute (IRRI) at Cavinti, Philippines. Bacterial cells of test strains from 24-hr old cultures were scraped into a 1% carboxymethylcellulose buffer. Seed of rice variety UPLRi-5 (1.3kg/treatment) were coated with bacterial suspension incubated overnight in polyethylene bags at 25 Ž. Excess buffer-bacteria mixture was drained seeds were dried in sterile air for 12hr before sowing in 4 ~5m plots. At sowing, seeds had 109 colony-formingunits (cfu)/g. Seeds coated with the fungicide pyroquilon (Fongorene; CGA 49104) (8.0g/kg seed) untreated seeds were sown as checks. The crop also received three additional sprays (500ml/sq.m/ spray) with bacteria (108cfu/ml) or pyroquilon at 20, days after seeding. Bacterial multiplication was monitored from root shoot samples removed at 10-day intervals plated on nutrient broth-yeast extract agar (NBY) amended with R or RN. Studies on the mechanism of disease suppression. We next investigated the basis for biological suppression of P. oryzae by Pseudomonas strain 7-14 on the basis of its performance in the field test. Strain 7-14 was grown in 200-ml volumes of potato glucose broth placed in 500-ml Erlenmeyer flasks as shake culture for 5 days. Culture conditions for production of the antibiotics its isolation were those used by Gurusiddaiah et al.5) From a batch of 10 liter broth, an oily residue of mg/l was obtained after solvent extraction with CH2C12 concentration. The residue was further purified through Sephadex LH20 column chromatography twice. A partially purified substance obtained from selective elution of the column with methanol was chromatographed on t.l.c. (Merck Silica gel, F254 S 0.2mm, precoated on alumina) plates developed in chloroform: methylene chloride (9:1). The fluorescent antibiotics were further characterized by their Rf values, uv absorption biological activity. The two substances (AB1 AB2) were separately tested for inhibition of conidial germination in P. oryzae. Conidia were made up in sterile water at the concentration of 25,000 conidia/ml. Their germination was assessed in slide germination tests in the presence of 0 (check) to 100ppm concentration of the antibiotic after 1 to 6hr of incubation at 28 Ž. There were 6 replications for each concentration tested. Biological activity of the antibiotics were also assessed in three separate greenhouse tests. Seedlings of IR50 rice (3-4 leaf stage) were spray-inoculated with P. oryzae conidia (50,000/ml) suspended either in water or an aqueous solution (1.0ppm) of the two antibiotic substances together. Leaf blast lesions were counted 5 days after inoculation from 400 leaves. Selection of bacterial strains RESULTS AND DISCUSSION Among the four strains which were considered most efficient, the two fluorescent strains, were identified as of Pseudomonas fluorescens the nonfluorescent strains, as Bacillus spp. Species level identification of the Bacillus strains is incomplete. In plate tests, P. fluorescens strains Bacillus strains, caused maximum inhibition of the blast fungus, P. oryzae. The average diameter of the inhibition zones was 38.5mm for strain 7-14 (Fig. 1), 30.4mm for strain 4-15, 26.3mm for strain mm for strain The rifampicin (R) or rifampicin nalidixic acid (RN) resistant mutants of these strains retained their ability to inhibit P. oryzae. In our plate tests, inhibition of P. oryzae by strains was not reversed by amendments with FeCl3 (up to 10mm) in King's medium B or potato dextrose agar. Field experiments In the field experiment conducted at Cavinti, Philippines, strains of P. fluorescens afforded 59 47% leaf blast reduction in rice var. UPLRi-5. The Bacillus strains, gave 46

3 382 日本植 物 病 理 学会 報 第58巻 第3号 平成4年7月 Fig. 1. Inhibition of Pyricularia oryzae in potato dextrose agar plate by fluorescent pseudomonad strain The plate on the right was inoculated in the center with a loopful of cells of 7-14 after the agar surfaces of both plates were spread with conidial suspension of P. oryzae. The plate on the left (check) did not receive bacterial inoculation. Fungal inhibition was observed after 4 days of incubation. Table 1. Effect Cavinti, a) Number of seed bacterization Philippines of blast lesions/cm2 b) Neck blast severity wet sprays with antagonistic bacteria on rice blast in cv. UPLRi-5 season leaf area. =n(1)+n(2)+n(3) n(9)/ Total number index n(1), n(2),etc. are number c) From in of tillerswith disease score 1 (resistant),2, or 9 (susceptible). 100 panicles per plot. 44% protection (Table 1). All these were significant reductions. Of all the treatments pyroquilon was most effective. Strain 7-14 was the most effective of all treatments for neck blast reduction (Table 1). There were also small increases in grain yield due to bacteria fongorene (pyroquilon) treatments. P. not cfu/g fluorescens detected tissue Pseudomonas strains in subsequent at 30 strains, had populations samples. days in spite of The 1.0 ~105cfu/g of their 0.5 ~105cfu/g Bacillus tissue lower population tissue strains, at 60 however were 110 effective up to 40 had days higher days after in reducing after seeding populations seeding. The leaf of were 0.9 ~106 fluorescent neck blast. Mechanism for biological diseases suppression Circumstantial evidence suggest that siderophore production, one of the important mechanisms known to mediate bacterial antagonism in fungal inhibition is unlikely to be involved in highly acidic soils9) such as the Cavinti soil, which has a ph of 4.0 Fe content that ranges from 3.7 to 4.6%1). Further, in our plate tests, inhibition of P. oryzase by strain 7-14 was not reversed by Fe amendments. Therefore, searching for antibiotics to explain the antiblast activity in strain 7-14 conducted.

4 Ann. Phytopath. Fig. 2. Inhibition of conidial germination Soc. Japan 58 (3). July, 1992 in Pyricularia oryzae by 383 antiblast substances produced by Pseudomonas strain Conidia were suspended either in water (left) or in an aqueous solution of AB1 AB2 at 1.0ppm concentration (right). Germination counts were made after 6 hours. Antiblast substances inhibited conidial germination from 70 to 100%. Fig. 3. Protection of IR50 rice seedlings from P. oryzae infection by treatment with Pseudomonas antibiotic. Leaves on the right are from seedlings that were spray-inoculated with conidia of P. oryzae suspended in water (50,000/ml). Leaves on the left showing fewer blast lesions are from seedlings that were spray-inoculated with the same level of conidia suspended in a 1.0ppm aqueous solution of antiblast substances (AB1 & AB2). Lesions were counted from 400 leaves per treatment on the 5th day after inoculation. In the three greenhouse tests, there was a mean 90% protection due to antibiotic treatment. A sample of 6.0g of crude extract made from 10 liter of culture fluids of strain 7-14 yielded 2.8mg of partially purified antibiotic at the end of two rounds of Sephadex LH 20 column chromatography. On t.l.c. this substance yielded a major UV-fluorescent (266nm) blue b (AB1) also a minor blue

5 fluorescent b (AB2). The major principle (AB1) moved to Rf values of in CHCl3-acetone (4:1), in CHCl3-acetone (9:1) in CH2Cl2 (100%). Both AB1 AB2 had similar UV-spectra had a major absorption peak at 220nm minor absorption peak at 281nm. At 1.0ppm concentration, the antiblast antibiotics AB1 AB2 afforded % inhibition of conidial germination in P. oryzae (Fig. 2). They also protected IR50 rice seedlings from infection P. oryzae. In the three greenhouse tests made, there were an average of 23 blast lesions per leaf in the plants sprayed with conidia in water (check) while those seedlings sprayed with antibiotic-treated conidia had an average of 1.9 lesions per leaf (Fig. 3). The work on the isolation of antiblast substances, however incomplete as it may appear, suggest that these substances are quite different from the phenazine carboxylic acid characterized as the antibiotic produced by P. fluorescens strain 2-79 by Gurusiddaiah et al.5) shown to have an essential role in the biological control of Gaeumannomyces graminis var. graminis other root pathogens of wheat (16). It is also different from pyoluteorin pyrrolnitrin antibiotics (3, 5) by their spectral characteristics. Our results can be used to improve blast control with antagonistic bacteria. While no single cultivar may be suitable for all the different upl rice ecologies, biological control as a component of a disease management, may provide an additional option. However, cultivars can be improved on the basis of their responses to plant associated microorganisms through conventional breeding or genetic manipulation. Feasibility of these options needs further assessment in the different rice ecologies with a better comprehensive understing of the rice pathosystems the biological control agents. Literature 1. Anonymous (1987). Research Briefings: Report of the Research Briefing Panel on Biological Control in Managed Ecosystems. National Academy Press, Washington, D.C. 2. Anonymous (1989). Strategy Report-IRRI Toward 2000 Beyond. IRRI Los Banos, Laguna. 3. Champion, A.B., Barrett, E.L., Palleroni, N.J., Solderberg, K.L., Kunisawa, R., Contopulou R., Wilson, A.C. Doudoroff (1980). Evolution in Pseudomonas fluorescens. J. gen. Microbiol. 120: Garrote, B.P., Mercado, A., Jr. Garrity, D.P. (1986). Soil fertility management in acid upl environments. Phil. J. Crop Sci. 11: Gurusiddaiah, S., Weller, D.M., Sarkar, A. Cook, R.J. (1986). Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var. tritici Pythium spp. Antimicrob. Agents Chemother. 29: Howell, C.R. Stipanovic, R.D. (1979). Control of Rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens with an antibiotic produced by the bacterium. Phytopathology 69: Howell, C.R. Stipanovic, R.D. (1980). Suppression of Pythium ultimum-induced damping-off of cotton seedlings by Pseudomonas fluorescens its antibiotics, pyoluteorin. Phytopathology 70: Kloepper, J.W. (1983). Effect of seed piece inoculation with plant growth-promoting rhizobacteria on population of Erwinia carotovora on potato roots in daughter tubers. Phytopathology 73: Kloepper, J.W. Schroth, M.N. (1981). Relationship of in vitro antibiosis of plant growth-promoting rhizobacteria to plant growth the displacement of root microflora. Phytopaatology 71: Kloepper, J.W., Leong, J., Teintz, M. Schroth, M.N. (1980). Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature 286: Mew, T.W. Rosales, A.M. (1986). Bacterization of rice plants for control of sheath blight caused by Rhizoctonia solani. Phytopathology 76: Misaghi, I.J., Olsen, M.W., Cotty, P.J. Donndelinger, C.R. (1988). Fluorescent siderophore-mediated iron deprivation-a contingent biological control mechanism. Soil Biol. Biochem. 20: Rosales, A.M., Nuque, F.L. Mew, T.W. (1986). Biological control of bakanae disease of rice with antagonistic bacteria. Phil. Phytopathol. 22: Sakthivel, N. Gnanamanickam, S.S. (1987). Evaluation of Pseudomonas fluorescens for suppression of sheath rot disease for enhancement of grain yield in rice (Oryza sativa L.). Appl. Environ. Microbiol. 53: Sakthivel, N. Gnanamanickam, S.S. (1989). Incidence of different biovars of Pseudomonas fluorescens in flooded rice rhizosphere in India. Agric. Ecosystem Environ. 25: Sher, F.M. Baker, R. (1982). Effect of Pseudomonas putida a synthetic iron chelator on induction cited

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