BIOTROPIA No. 7, 1994: 1-11 BIOLOGICAL CONTROL OF DAMPING-OFF FUNGI OF AGOHO (CASUARINA EQUISETIFOLIA L.) USING ANTAGONISTIC BACTERIA F.A. DELA PEÑA Mariano Marcos State University, Batac, Ilocos Norte Philippines 2906 A.K. RAYMUNDO, E.P. MILITANTE, M.U. GARCIA and L.U. DELA CRUZ University of the Philippines at Los Banos, College, Laguna Philippines 4031 ABSTRACT A series of laboratory and nursery experiments were conducted specifically to determine the efficacy of 85 strains of Bacillus species and 15 actinomycetes against six fungal pathogens isolated from damped-off agoho. These damping-off fungi were: Fusarium oxysporum Schet., Rhizoctonia solani Kuhn., Phytophthora parasitica Dastur, Pythium debaryanum Hesse, and two unidentified pathogens temporarily designated as Unk 1 and Unk 2. Preliminary test using the agar-plug technique revealed that 18 of the bacterial isolates could suppress two or more of the six damping-off fungi. Fusarium oxysporum was inhibited by 17 bacterial isolates, R. solani by 8 isolates, P. parasitica by 14 isolates and P. debaryanum by 15 isolates. The unidentified damping-off fungi Unk 1 and Unk 2 were inhibited by 13 and 9 isolates, respectively. Further screening using the agar-diffusion method disclosed that 10 isolates were effective antagonists with Bacillus subtilis (Code No. R060), Bacillus sp. (Code No. R071), and Streptomyces sp. (Code No. R086) as the consistent and most effective inhibitors. Application of the three most promising antagonistic bacteria as seed treatment s howed that they effectively inhibited the growth of the damping-off fungi in the laboratory as exhibited by an increase in percent germination. Bacillus subtilis however, was not able to antagonize the effect of P. debaryanum in this particular experiment. Seed germination and seedling survival were likewise improved with the application of the three most promising antagonistic bacteria as seed treatment. This was shown after three months under nursery conditions. There were possible mechanisms of control by the antagonistic bacteria against the damping-off fungi. The mycelium and spores of the pathogenic fungus may have been attacked and parasitized by the antagonist when they were simultaneously grown in culture media. There must have been a competitive interaction between the two microorganisms. Any essential requirement of microorganisms can serve as potentially possible basis for competition. Another was antibiosis which is an inhibitory effect exerted by an organism upon another organism through the production of antibiotic compounds. Moreover, several strains of bacteria are effective in lysing cell walls of pathogenic fungi under laboratory conditions. Lysis is often attributed to production of cell wall degrading enzymes like chitinase and gluconase t hat may hydrolyze major constituents of fungal cell walls. 1 BIOTROPIA No. 7, 1994 Furthermore, several strains of bacteria are effective in lysing cell walls of pathogenic fungi under laboratory conditions. Lysis is often attributed to production of cell wall degrading enzymes like chitinase and glucanase that may hydrolyze major constituents of fungal cell walls. As demonstrated by Kapoor and Kar (1979), hyphae showed lysis in addition to thickening of hyphae as well as formation of chlamydospores in abundance as an effect of Bacillus sp. on F. oxysporum f. sp. lycopersici. Inhibitory substance diffused into the media may initiate fungal cell wall lysis. Most bacteria are effective fast-growing colonizers which make such microorganisms efficient biological control agents of plant diseases. Seed treatment gave the antagonist the opportunity to be the first colonizer of the plant roots of the seedlings (Elad and Chet 1987) thus having an advantage for any competition for nutrients supplied by roots. Seed exudates probably occurred in the interaction between the antagonistic bacteria and damping-of f fungi in the root and is responsible at least to a degree for the observed biological control by introduced bacteria. Large populations of bacteria established on planting material and roots become a partial sink for nutrients in the rhizophere, thus reducing the amount of carbon and nitrogen available to stimulate sporulation of fungal pathogen or for subsequent colonization of the root (Weller 1988). In addition, cell wall degrading enzymes are activated by nutrient deprivation imposed by microbial activity. Oospore germination by some damping-off fungi has already been shown to be affected by exogenous nutrients (Howell and Stipanovic 1980). Nutrients supplied by root exudates stimulate oospore germination in the rhizosphere. It therefore, appeared that bacteria may compete with germinating oospore for available carbon or nitrogen sources, and by eliminating these resources, the bacteria reduce the percentage of oospore germination. In addition, Elad and Chet (1987) stated that there was a significant positive correlation observed between inhibition of oospore germination and disease reduction by bacteria. CONCLUSION The promising antagonistic bacteria therefore, could reduce the incidence of damping-off disease of agoho. Its application as biological control agent could be a good substitute for soil fungicides. Although soil fumigation is very effective for the control of damping-off pathogens, it is very expensive, impractical and may create problems like environmental pollution and pathogen resistance. 10 Biological control of damping-off fungi of agoho - F.A. Dela Pena et al. REFERENCES DAYAN, M.P. and V.O. SINOHIN. 1989. Diagnosing Forest Tree Diseases (Fungal). Ecosystem Research and Development Bureau, College, Laguna. 29 p. DELA CRUZ, A. 1986. Microbial Antagonists of the Potato Soft-Rot Pathogen, Erwinia carotovora pv. carotovora (Jones) Bergey, Harrison, Breed, Hammer, and Hutton. (Unpublished MS Thesis, University of the Philippines at Los Banos, College, Laguna). DICKSON, J.G. 1956. Diseases of Field Crops. Me. Graw Hill Book, Co., New York, 517 p. ELA D, Y. and I. CHET. 1987. Possible role of competition for nutrients in biocontrol of Pythium damping-off by bacteria. Phytopathol. 1: 375-396. GARRET, S.D. 1965. Toward biological control of soil-borne plant pathogens: 4-16. In: K.F. Barker and W.C. Snyder (eds.), Ecology of Soil-Borne Plant Pathogens-, Prelude to Biological Control. University of California Press, California. HENIS, Y. and I. CHET. 1975. Microbiological control of plant pathogens. In: D. Perlman (ed.) Advances in Applied Microbiology. Vol. 19. Academic Press, New York. 332 p. HENIS, Y. and M. IMBAR. 1968. Effect of Bacillus subtilis on growth and sclerotium formation by Rhizoctonia solani. Phytopathol. New York. HOWELL, C.R. and R.D. STIPANOVIC. 1980. Suppression of Pythium ultimum induced damping-off of cotton seedlings by Pseudomonasfluorescens and antibiotic pyoluteorin. Phytopathol. 70: 712 - 715. JONES, R.K. 1985. Fungicides for bedding plants. Bedding Plants Inc. News 16(2): 3-4. KAPOOR, I.J. and B. KAR. 1989. Antagonism of Azotobacter and Bacillus to Fusarium oxysporum f. sp. lycopersici. Indian Phytopathol. 42(3): 400-404. LARSON, R. 1987. Growing concerns. Pest control: How much is enough. Marketletter 2: 5. NATIONAL RESEARCH COUNCIL. 1984. Casuarinas: Nitrogen Fixing Trees for Adverse Sites. National Academy Press, Washington D.C. 118 p. OLSEN, C.M. and K.F. BAKER. 1968. Selective heat treatment of soil and its effects on the inhibition of Rhizoctonia solani by Bacillus subtilis. Phytopathol. 58: 79-87. RAYMUNDO, A.K., E.T. SERRANO, G.D. REYES and T.O. ZULAYBAR. 1985. Isolation and identification of antibiotic-producing Bacillus from the soil. Phil. Agr. 68: 393-402. WELLER, D.M. 1988. Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Ann. Rev. Phythopathol. 26:379-407. 11 BIOTROPIA No. 7, 1994 Pre-emptive colonization was also a possible mechanism of control. Seed treatment gave the antagonist the opportunity to be the first colonizers of the plant roots and moved along the roots of the seedlings thus having an advantage for any competition of nutrients. Most bacteria are effective fast-growing colonizers which make such microorganisms efficient biological control agents of plant diseases. Key Words: Biological control. Fungal diseases, Casuarina equisetifolia, Antagonistic bacteria INTRODUCTION Agoho (Casuarina equisetifolia L.) is a good fuel wood species in scores of tropical countries like the Philippines. It has particularly been valuable for stabilizing sand dunes as it withstands salt sprays, thrives/grows in Infertile soil and is drought-tolerant (National Research Council 1984). These indispensable characteristics make it highly recommended for reforestation and afforestation purposes. Damping-off disease however, is the major threat in the production of sufficient and quality planting stocks. Damping-off is a term applied to any disease that results in the decay of seeds in the soil before seedling emergence, or rapid rotting usually at the soil line of recently emerged seedlings (Dayan and Sinohin 1989). It is caused by a number of soil-inhabiting fungi that are facultative parasites and not specialized as to host. The ubiguity of damping-off fungi necessitates effective control measures because there are times when seedlings in the entire seedbed are wiped out resulting to shortage of supply for field planting. Chemical control is usually not feasible due to the expensive price of fungicides. Moreover, they can cause environmental pollution and may even induce pathogen resistance (Jones 1985; Larson 1987). Biological control perhaps offers one of the best alternatives. As defined by Garret (1965), biological control is any condition or practice under which survival or activity of a pathogen is reduced through the agency of any organism (except man himself) with the result that there is a reduction in the incidence of the disease caused by the pathogen. One of the ways that biological control may operate to suppress pathogen is introduction of biocontrol agents or antagonists. It is for this purpose that a study on the biological control of damping-off fungi of agoho using antagonistic bacteria was conducted. Specifically, the study aimed to determine the efficacy of promising antagonistic bacteria in the laboratory and under nursery conditions. 2 Biological control of damping-off fungi of agoho - F.A. Dela Pena et al. MATERIALS AND METHODS The antagonistic activity of 100 bacterial isolates consisting of 85 strains of Bacillus species and 15 actinomycetes was determined against six fungal pathogens isolated from damped-off agoho. The damping-off fungi were: Fusarium oxysporum Schet., Rhizoctonia solani Kuhn., Phytophthora parasitica Dastur, Pythium debaryanutn Hesse, and two unidentified pathogens temporarily designated as Unk 1 and Unk 2. Preliminary screening using the agar-plug technique involved simultaneous inoculation of the pathogen (damping-off fungus) and the antagonist (bacterial isolate). Bacterial isolates showing good antagonistic activity were then subjected to the agar-diffusion method and further tested in the laboratory and under nursery conditions as seed treatment. Agar-Plug Technique The cell suspension of Bacillus species was prepared by growing the organism in tryptone glucose yeast extract agar (TGYA) in a flat bottle for 24 -48 hr under room temperature. After incubation, the growth was flooded with sterile distilled water and then aseptically dislodged from the agar. The resulting suspension was adjusted to 0.30 optical density (OD) at 540 nm using a Spectronic 20 to have the desired population of 10 9 cells/ml (Dela cruz 1986). The cell suspension of actinomycete was prepared by growing the organism in yeast-malt extract broth (YMB) for 48 - 72 hr on a shaker at room temperature. The broth culture was then pooled to make the necessary amount needed and homogenized using an osterizer for 5 min. A 10% suspension was made by diluting 100 ml of the prepared actinomycete suspension into 900 ml sterile distilled water (Dela Cruz 1986). Agar plugs were prepared by initially growing the damping-off fungus on potato dextrose agar (PDA) under room temperature for seven days or until sufficient growth was obtained. A sterile 1-cm-diameter cork borer was used to obtain the agar plugs from these culture plates. The agar plugs inoculated on agar plates were initially prepared by plating 1 ml of bacterial cell suspension on TGYA for Bacillus species and yeast-malt extract agar (YMA) for actinomycetes. Inoculation of the agar plugs was done in an upside-down position to provide a direct contact between the pathogen and the antagonist. The assay plates were incubated at room temperature for 3 to 5 days, after which growth of the pathogen was observed. This experiment utilized a Completely Randomized Design (CRD) with three replications. 3 BIOTROPIA No. 7, 1994 Agar-Diffusion Method Cultures (24 to 48-hr-old) of the potential antagonistic bacteria were used to inoculate tryptone glucose yeast extract broth (TGYB) for Bacillus species and YMB for actinomycetes. These were then incubated at room temperature on a shaker. After 48 hr (for Bacillus spp.) and 72 hr (for actinomycetes) incubation, the broth cultures were centrifuged at 14 000 rpm, 0°C, for 15 min. The supernatant was used for the microbial assay. One loopful of growth from 48-hr-old culture of the damping-off fungus was suspended in 2 ml sterile distilled water, mixed and the suspension inoculated into 100 ml PDA top agar. This was used to seed the PDA plates and then allowed to solidify (Raymundo et al. 1985). Following the solidification of the top agar, four cup cylinders were positioned on the plate and these were then filled with 0.1 ml of appropriate supernatant from each potential antagonist. After incubation for 48 hr or until sufficient growth was obtained, the assay plates were examined and the diameter of zones of inhibition was measured. This experiment utilized a CRD with three replications and three subreplications. Significant values were further subjected to Duncan's Multiple Range Test (DMRT). Antagonistic bacteria showing potential to control the damping-off fungi in the agar-diffusion method were further screened as seed treatment in the laboratory and under nursery conditions. Laboratory Experiment Newly prepared cell suspensions of the antagonistic bacteria were used to coat the agoho seeds by soaking for at least 15 min. before sowing onto sterile germination plates lined with tissue paper. The germination plates were initially inoculated with 5 ml fungal cell suspension prepared by suspending a loopful of growth of a 48-hr-old culture onto 100 ml sterile distilled water. Twenty agoho seeds treated with the antagonistic bacteria were sown in each inoculated germination plate. Untreated seeds sown in an uninoculated germination plate served as the control. All seeds sown were disease-free and previously surface-sterilized. The plates were incubated at room temperature for 15 days. Watering was regulated using sterile distilled water. Seed germination and the occurrence of damping-off disease were regularly assessed. The experiment utilized a CRD with three replications. 4 Biological control of damping-off fungi of agoho - F.A. Dda Pena et al. Nursery Experiment The promising antagonistic bacteria were further screened as seed treatment under nursery conditions. A newly prepared bacterial cell suspension corresponding to each treatment was used to coat the agoho seeds. This was done by soaking for at least 15 min. before sowing into a pot with naturally infested soil. Untreated seeds sown in naturally infested soil served as the control. Another treatment made use of untreated seeds sown in a soil medium subjected to sterilization in an autoclave for 1 hr at 15 psi for three consecutive days. There were 20 disease-free and previously surface-sterilized seeds sown in every pot. Seed germination and the occurrence of damping-off disease were assessed. The percentage of survival of the seedlings was determined after three months when the study was terminated. The experiment utilized a CRD with five replications. Significant values were further subjected to DMRT. RESULTS AND DISCUSSION In vitro assay of the antagonistic activity of the 100 bacterial isolates using the agar-plug technique revealed that only 18 inhibited two or more of the damping-off fungi of agoho (Table 1). Fusarium oxysporum was inhibited by 17 bacterial isolates, R. solani by 8 isolates, P. parasitica by 14 isolates, and P. debaryanum by 15 isolates. The unidentified damping-off fungi Unk 1 and Unk 2 were inhibited by 13 and 9 isolates, respectively. Of those that showed inhibitory effects, 15 belonged to the genus Bacillus while three were actinomycetes. Further screening using the agar-diffusion method disclosed that 10 were promising antagonists with B. subiilis (Code No. R060), Bacillus sp. (Code No. R071), and Streptomyces sp. (Code No. R086) as the consistent and most effective inhibitors (Table 2). Some of the strains listed for inhibitory effects gave values below that of the reference antibiotic mycostatin (200 ug/ml) while others did not produce any inhibition zone, thus there was no antimicrobial activity observed. Effect of the three promising antagonistic bacteria on percent germination of agoho seeds inoculated with the damping-off fungi in the laboratory is shown in Table 3. The control which was not inoculated with any of the six pathogens gave the highest seed germination of 81.67%. It was however, surpassed though not significantly different by seed treatment with Bacillus sp. (Code No. R071) against P. parasitica resulting to 83.33%. Inoculation with any of the damping-off fungi and with no seed treatment with any antagonist gave the lowest percent germination. Bacillussubtilis (Code No. R060) however, was not able to antagonize 5 BIOTROPIA No. 7, 1994 the effect of P. debaryanum in this particular experiment. Application or treatment with B. subtilis may not have been of optimum concentration to affect an increase in percent germination of seeds inoculated with P. debaryanum. This could also be due to the fact that this pathogenic fungus is considered very virulent (Dickson 1956). Under nursery conditions, the antagonists suppressed the growth of the damping-off fungi (Table 4 and Fig. 1). Seed treatments gave percent germination values of 74, 69 and 78 for B. subtilis, Bacillus sp., and Streptomyces sp., respectively. Seed coating with Streptomyces sp. gave a comparable value with untreated seeds sown in sterilized soil (83 %). Table 1. Inhibitory effects of the bacterial isolates on growth of the damping-off fungi of agoho based on agar-plug technique a Code b Classification c Inhibitory effect d __________ Number F. oxysporum R. solani P. parasitica P. debaryanum Unk 1 Unk 2 R013 B + - + + - + ROM B - + - - + + R059 B + + + + - - R060 B + + + + + + R063 B + + + + - + R070 B + + - + + - R071 B + + + + + + R072 B + - + + + - R073 B + - + + - - R074 B + - + + + - R075 B + - + - + - R076 B + - + + - + R078 B + - - + - - R079 B + - - + - - R080 B + - + + + - R086 A + + + + + + R095 A + + + + + + R099 A + - + - + + Total number of Active isolates = 18 17 8 14 15 13 9 a Results based on three replications b Treatments with no antimicrobial activity against any of the six damping-off fungi are not listed c Either belonging to the genus Bacillus (B) or an actinomycete (A) d Positive sign ( + ) means with inhibitory effect: negative sign (-) means no inhibitory effect 6 Biological control of damping-off fungi of agoho - F.A. Dela Pena et al. Table 2. In vitro assay of the antagonistic activity of the different bacterial isolates against the damping-off fungi of agoho using agar-diffusion method Inhibition zone b (mean diameter in mm) F. oxysporum R. solani P. parasitica P. debaryanum Unk 1 Unk 2 Control 0 f 0 d 0 d 0 e 0 d 0 e R013 0 f 0 d 0 d 0 e 11.97c 0 e R060 21.65c 29.63a 27.05a 9.31b 19.21a 17.85c R071 29.22b 19.09b 10.06a 11.16a 14.30b 34.67a R072 0 f 0 d 9.89c 0 e 0 d 0 e R074 0 f 10.71c 0 d 0 e 0 d 11.37d R07S 9.73e 11.80c 0 d 0 e 11.20c 10.21d R078 9.62e 0 d 0 d 0 e 0 d 0 a R079 0 f 12.58c 0 d 0 e 0 d 0 e R086 31.42a 27.69a 18.43b 0 e 14.56b 25.26b R095 9.75e 11.96c 9.24c 9.00c 0 d 11.04d m200 d 14.05d 12.64c 9.43c 8.67d 11.84c 11.96d a Treatments with no antimicrobial activity against any of the six damping-off fungi are not listed b Results based on three replications with three subreplications c Means with the same letter within the column are not significantly different at 1 % level using DMRT d Reference antibiotic mycostatin (200 ug/ml) Table 3. Effects of the promising antagonistic bacteria on percent germination of agoho inoculated with damping-off fungi under laboratory conditions Treatments Percent germination (means) F. oxysporum R. solani P. parasitica P. debaryanum Unk 1 Unk 2 Control 91.67a 87.67a 81.67a 81.67a 81.67a 81.67a Pathogen alone 18.33d 1.67b 38.33c 0 d 0 e 26.67d Pathogen + R060 65.00b 68.33a 66.67b 0 d 71.67b 58.33b Pathogen + R071 50.00c 66.67a 83.33a 63.33b 60.00c 41.67c Pathogen + R086 75.00ab 76.67a 61.67b 30.33c 51.67d 71.67ab Means with the same letter within the column are not significantly different at 1 % level using DMRT Furthermore, Table 4 and Fig. 1 show that percent survival of seedlings was increased by seed treatment with values of 94, 85, and 95 for B. subtilis, Bacillus sp. and Streptomyces sp., respectively. These were significantly different with the control (62%). Seed coating with B. subtilis and Streptomyces sp. gave comparable values with untreated seeds sown in sterilized soil (99%). 7 Treatments a BIOTROPIA No. 7, 1994 Figure 1. Effects of the three promising antagonistic bacteria on percent germination and survival (after three months) of agoho under nursery conditions. Bars with the same fill marked with different letter(s) are not significantly different at the 5% level using DMRT 8 Biological control of damping-off fungi of agoho - F.A. Dda Pena et al. Table 4. Effects of the three promising antagonistic bacteria on percent germination and survival (after three months) of agoho under nursery conditions Treatments Germination Survival A 57d 62c B 83a 99a C 74bc 94a D 69c 85b E 78ab 95a Means followed by a common letter are not significantly different at 5% level by DMRT. Legend: A - Untreated seeds sown in naturally infested soil B - Untreated seeds sown in sterilized soil C - B. subtilis treated seeds sown in naturally infested soil D - Bacillus sp. treated seeds sown in naturally infested soil E - Streptomyces sp. treated seeds sown in naturally infested soil Results of the assay methods revealed that a considerable number of Bacillus sp. and actinomycetes could inhibit the growth of damping-off fungi in culture. The mycelium and spores of the pathogenic fungi may have been attacked and parasitized by the antagonists when they were simultaneously grown in culture media utilizing the agar-plug technique. There must have been a competitive interaction between the two microorganisms as supported by studies of Henis and Chet (1975). Any essential requirement of microorganisms can serve as potentially possible basis for competition. Competition for nutrients between the antagonist and germinating spores of the pathogenic fungus could have taken place. Olsen and Baker (1968) added that B. subtilis which developed along the mycelial wall of R. solani apparently obtained nutrients either from wall components or contents leaking from senescent fungal cells. In the agar-diffusion method, antibiosis which is an inhibitory effect exerted by an organism upon another organism through the production of antibiotic compounds may have likely occurred. The supernatant used in the assay demonstrated the possible role of antibiotics in biological control. Moreover, most strains of B. subtilis or any other species of the genus Bacillus and actinomycetes produce a wide variety of antibiotics which inhibit filamentous fungi. Bacillus subtilis in particular, produces a subtilinlike antibiotic (Henis and Chet 1975) in addition to bulbiformin, mycosubtilin and bacillomycin (Henis and Inbar 1968) which have antifungal activity. Some of the antibiotics produced by such strains may be extracellular, diffusable in solid agar, and could inhibit the growth of fungal pathogens. 9 1.pdf 10.pdf 11.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf