Microsoft Word - 111 BIOTROPIA VOL. 13 NO. 2, 2006 : 111 - 121 ANTAGONISTIC BACTERIA AGAINST SCHIZOPHYLLUM COMMUNE FR. IN PENINSULAR MALAYSIA ANTARJO DIKIN', KAMARUZAMAN SIJAM', JUGAH KADIR' AND IDRIS ABU SEMANZ 'Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor D.E, Malaysia 2Plant Pathology and Weed Science Group, Biological Research Division Malaysian Palm Oil Board, 43000 Kajang, Selangor, D.E. Malaysia ABSTRACT Schizophyllum commune Fr., is one of the important fungi, causes brown germ and seed rot of oil palm. Biodiversity of antagonistic bacteria from oil palm plantations in Peninsular Malaysia is expected to support in development of biopesticide. Isolation with liquid assay and screening antagonistic bacteria using dual culture assay were carried out in the bioexploration. A total of 265 bacterial isolates from plant parts of oil palm screened 52 antagonistic bacterial isolates against 5. commune. Bacterial isolates were identified by using Biolog* Identification System i.e. Bacillus macroccanus, B. thermoglucosidasius, Burkholderia cepacia, B. gladioli, B. multivorans, B pyrrocinia, B. spinosa, Corynebacterium agropyri, C. misitidis, Enterobacter aerogenes, Microbacterium testaceum, Pseudomonas aeruginosa, P. citronellolis, Rhodococcus rhodochrous, Serratia ficaria, Serratia sp., S. marcescens, Staphylococcus sciuri, Sternotrophomonas maltophilia. Key words : Schizophyllum commune, biodiversity, antagonistic bacteria INTRODUCTION Schizophyllum commune Fr. causes brown germ and seed rot. Heavy infection decreased seed germination of oil palm about 60 percent (Dikin et al. 2003). Proper seed treatments are required for the control of S. commune in the oil palm seeds. Some synthetic fungicides were applied to reduce the loss of germination due to this pathogen, but the negative impact from toxic chemicals to the environment was difficult to avoid. The utilization of bacteria as biological control agents successfully controlled plant pathogen (Sharga and Lyon 1998; Bapat and Shah 2000). Many studies in exploration of beneficial organisms have been carried out such as Pseudomonas fluorescent for the control of Fusarium wilt of tomato (Dekkers et al. 1998). Streptomyces halstedii (K122) and S. coelicolor (K139) to inhibit the fungi belonging to Oomycetes, Zygomycetes, Deuteromycetes, Ascomycetes and Basidiomycetes (Frandberg and Schnurer 1998). Bacillus subtilis suppressed phytopathogenic microorganism (Phae et al. 1990). The isolation of antagonistic bacteria was early stage for development of biopesticide such as Pseudomonas fluorescent from rhizospheres (Dekkers et al. 1998), Bacillus licheniformis from leaves of citrus orchard at Letaba Estates, Corresponding address : antario_dikin@yahoo.com 111 BIOTROPIA VOL. 13 NO. 2,2006 Tzaneen (Jager and Kosten 1998), Streptomyces halstedii (K122) and S. coelicolor (K139) from cereal grains (Frandberg and Schnurer 1998), Bacillus subtilis from the composts (Phae et al. 1990), and Burkholderia cepacia from infected oil palm seeds (Dikin et al. 2003). The liquid assay technique was a simple method for isolation of bacteria. Fluorescent Pseudomonas from Pythium- diseased tulip roots was isolated by extraction of infected root in sterilized water (Weststeijn 1990). Malaysia is well known as a mega-biodiversity country with complex microbial association. The exploration of beneficial bacteria from oil palm plantations is expected to utilize the antagonistic bacteria from the same ecology of the oil palm pathogen itself. The purposes of the study were to isolate and to screen the antagonistic bacteria from different plant parts of oil palm for the control of Schizophyllum commune. MATERIALS AND METHODS Cultural Schizophyllum commune Fr. and plant part of oil palm The culture of S. commune was isolated from heavy infection of oil palm seeds. The fungus was confirmed based on their morphological characteristics and the pathogenic fungus of oil palm (Alexopoulus et al. 1996). The fungal isolate was sub-cultured onto PDA medium for further study. Randomized samples of plant parts such as fruits, seeds, rhizosphere, and plant debris were collected from oil palm fields in Selangor (UPM, Bangi, Kajang and Seri Kembangan), Guthrie, Layang-Layang, Johor in Peninsular region, Malaysia. Plant parts were used for isolation of potential bacteria. Isolation of Antagonistic Bacteria Plant parts of oil palm such as fruits, seeds, rhizospheres and plant debris were rinsed with tap water to remove the adhered soil on surface. Mesocarp of fruits, endosperm of seeds, and rhizosphere were sliced into 0.5-1.0 cm2 and then 50 g sliced plant parts were placed into 250 mL Erlemeyer flask added with 100 mL distilled water. Plant debris with 5. commune was collected under oil palm tree. Ten g of plant debris were cut off in size 1 cm and then transferred into a 250 mL Erlemeyer flask added with 100 mL sterilized water. Sample materials in flasks were placed on electric rotator at 100 rpm overnight at 26 ± 2°C. Fold serials (lO^-lO"4) dilutions of suspension were made, 0.5 mL suspension from each diluted suspension was streaked on King's B (KB) and Nutrient Agar (NA) agar media plates. Plates were incubated at 26-28°C for 48 hours. Bacterial colonies on plate were purified by streaking single bacterial colony onto NA medium plates. Each pure culture of bacteria was screened for the antagonistic bacteria based on dual culture (Dikin et al. 2002;Montealegree/a/. 2003). 112 Antagonistic bacteria - A. Dikin et al. Screening the antagonistic bacteria Screening of bacterial antagonist was carried out using dual culture assay. One 6-mm diameter of S. commune agar plug was placed at the centre of PDA medium in a Petri dish with 9 cm diameter. Bacterial isolate was streaked on PDA medium with a distance of 2.5 cm between S. commune agar plug and bacterial isolate. Plates were incubated for 7 days at 26 ± 2°C. The percentage of radial inhibition growth was measured with the formula: PIRG (%) = (1 - (fungal growth near to bacterial isolate /fungal growth other side at the same plate as control)) x 100%. Each treatment was replicated 3 times. Receded data were analyzed using SAS® Software. Treatment effect was tested by ANOVA and the means compared using Least Significant Different Test at 5% probability level (Okamoto et al. 1998; Anonymous 1999; Montealegreefa/. 2003). Identification of antagonistic bacteria Potential antagonistic bacterial isolates were identified by Biolog® identification system which followed the Biolog's procedures. Bacterial suspension was inoculated into GN or GP micro plates depending on gram reaction cluster, 145 uL per well using the 8-channeI repeating pipette. Microplate was covered with its lid and incubated at 28-30°C for 24 hours to allow the utilization of carbon sources. Reading result was directly done after inserting the incubated microplate into the Biolog's reader apparatus and its installed micro soft ware of Biolog® identification system for identifying bacteria up to the species level (Anonymous 2001). RESULTS AND DISCUSSION Isolation of antagonistic bacteria The number of bacterial isolates was extracted from samples of seeds, fruits, rhizospheres and plant debris of oil palm which grew on KB and NA media. A total of 265 bacterial isolates from plant parts of oil palm were found from different locations, Peninsular region, Malaysia. Separation of bacterial isolates was based on the morphological colony performance such as colony colour, elevation, the margin of colony and colony surface (Hayward 1983). Isolation of potential bacteria from plant parts of oil palm using liquid assay was effective and simple technique. The liquid assay and the agar plate media are commonly used for isolation of pathogenic bacteria from infected plant parts. Bacterial isolates from different plant parts of oil palm on NA and KB media grew well on the cultural plates. Dual culture assay screened 52 out of 265 bacterial isolates against S. commune. The number of antagonistic bacteria from each location isolated from different plant parts is presented in Table 1. 113   Antagonistic bacteria - A. Dikin et al. Based on Table 1, 96 bacterial isolates as the highest number were obtained from the rhizosphere followed by plant debris, 89 isolates. The average number of bacterial isolates from rhizosphere was 9.6 followed by 6.8 from plant debris, 6.1 from fruits, and 5.2 from seeds. The bacterial isolates obtained from plant debris were more diverse with the number of bacterial isolates higher than other plant parts such as rhizophere, fruit, and seed. Eighteen out of 89 antagonistic bacterial isolates were obtained from plant debris, followed by 15 out of 96 isolates from rhizosphere, 10 out of 43 isolates from fruit, and 9 out of 37 isolates from seed. The probability for isolation of antagonistic bacteria from each plant part was 20.2 percent, 15.6 percent, 23.2 percent, and 24.3 percent, respectively. More dominant bacteria in the rhizosphere and plant debris than seeds and fruits were due to the different available nutrition and the requirement for bacterial growth. Dominance of bacteria in the rhizospheres and plant debris was due to complex interaction between microorganisms and plant parts. Plant debris such as decayed empty bunch, fronds, and rachis were good media for the fungal growth. Blotching symptom with water soak and brown colour in the fruiting bodies of S. commune was the indication of interaction between fungus and bacteria. Dual culture assay of S. commune against antagonistic bacteria is presented in Figure 1. Figure 1. a. Burkhoderia multivorans (bacterial code-50) inhibits the growth of S. commune on dual culture of PDA medium at 7-day after incubation at 26 ± 2°C b. Burkholderia cepacia inhibits the growth of S. commune on dual culture of PDA medium at 7-day after incubation at 26 ± 2°C Among 52 isolates from different plant parts and different sampling locations inhibited the mycelial growth of S. commune with various percentages of radial inhibition. Each bacterial isolate with radial growth inhibition of S. commune is presented in Table 2. The range of radial growth inhibition of antagonistic bacteria was 3.3 percent up to 95.2 percent from the bacterial code 29 and 10, respectively. In Table 2, there are 7 bacterial isolates with highest mean percentage of radial growth inhibition with the bacterial code 10, 8, 9, 14, 50, 7, and 2 with the percentage of inhibition of 95.2, 115 BIOTROPIA VOL. 13 NO. 2,2006 90.6, 83.2, 83.1, 83, 81.8, and 81.5, respectively. A number of antagonistic bacteria were isolated from plant parts with varied mean percentages of radial growth inhibition against S. commune. The bacterial isolates had high radial growth inhibition which were obtained from plant debris, rhizospheres, and fruit. Many authors have reported that certain antagonistic bacteria suppressed the growth of pathogenic fungus. In vitro study showed that Burkholderia cepacia from tomato's rhizospheres suppressed the growth of Fusarium oxysporum f.sp. lycopersicae stronger than S. commune. In contrast, B. cepacia from rhizhosperes of oil palm suppressed S. commune stronger than F. oxysporum f.sp. lycopersicae (Kamaruzaman and Dikin 2005). In this case , targeted potential antagonistic bacteria against S. commune should be isolated from the area of oil palm plantation. Identification of antagonistic bacteria Identification of antagonistic bacteria against S. commune based on Biolog® Identification system is presented in Table 3. 116 Antagonistic bacteria - A. Dikin et al. Table 3. Antagonistic bacteria based on Biolog* Identification System Plant Part Bacterial code Bacteria Rhizosphere 1 Burkholderia cepacia 6 Corynebacterium agropyri 7 Microbacterium testaceum 17 Pseudomonas citronellolis 20 Bacillus macroccanus 23 B. cepacia 24 Burkholderia spinosa 47 B. cepacia 49 Staphylococcus sciuri 50 Burkholderia multivorans 51 Burkholderia pyrrocinia 52 B. cepacia Fruit 3 B. cepacia 5 Serratia marcescens 9 C. agropyri 25 Pseudomonas aeruginosa 34 P. aeruginosa 38 P. aeruginosa 40 P. aeruginosa Seed 4 Serratia sp. 12 Bacillus thermoglucosidasius 18 B. pyrrocinia 22 B. cepacia 26 P. aeruginosa 37 Sternotrophomonas maltophilia Plant debris 2 S. mallophilia 8 Burkholderia gladioli 10 B. gladioli 11 B. cepacia 14 C. agropyri 16 Serratia ficaria 19 Enterobacter aerogenes 28 B. gladioli 30 P. aeruginosa 33 Corynebacterium masitidis 42 M. testaceum 43 Rhodococcus rhodochrous Table 3 presents the identified bacteria and non-identified bacteria by using Biolog Identification system. In the rhizosphere 12 identified species and 3 non-identified species were found. The identified species from rhizosphere were B. cepacia, C. agropyri, M. testaceum, P. citronellolis, B. macroccanus, B. spinosa, S. sciuri, B. multivorans, and B. pyrrocinia. Among 9 species, the dominant identified species in the rhizosphere was B. cepacia. 117 BIOTROPIA VOL. 13 NO. 2,2006 Seven identified species and 3 non-identified species of antagonistic bacteria were found in fruits. The identified species from fruits were B. cepacia, S. marcescens, C. agropyri, and P. aeruginosa. Among the 4 species, the dominant identified species in the fruit was P. aeruginosa. P. aeruginosa was isolated from the rhizospheres and plant debris. This bacteria was recognized as the supplier of mineral which was required for metabolism process of plant from the access of bacterial metabolites (Hofte et al. 1993; Abdullah et al. 2003). There were complex microorganisms around rhizospheres to compete with each other for survival which showed the synergism and antagonism interaction. Infected plant debris with S. commune around the rhizosphere was to bait the potential antagonistic bacteria. Six identified species and 3 non-identified species of antagonistic bacteria in seeds were found. The identified species from fruits were Serratia sp., B. thermoglucosidasius, B. pyrrocinia, B. cepacia, P. aeruginosa, and S. maltophilia. In the plant debris 12 identified species and 6 non-identified were found. The identified species were S. maltophilia, B. gladioli, B. cepacia, C. agropyri, S. ficaria, E. aeogenes, P. aeruginosa, C. masitidis, M. testaceum, and R. rhodochrous. The dominant species from plant debris was B. gladioli. B. cepacia and B. gladioli were dominantly found in the rhizospheres and plant debris, respectively. The presence of B. cepacia in the rhizospheres of oil palm was the same evident with the presence of B. cepacia in the rhizospheres of banana to protect plant infection caused by Fusarium oxysporum f. sp cubense. B. cepacia colonizes the surface of hyphae and fungal macrospores (Pan et al. 1997). B. gladioli was found in the rhizospheres and potential antagonistic bacteria against S. commune, however the implication for biological control was less recognized. The identified species of antagonistic bacteria from different plant parts were Bacillus macmccanus, B. thermoglucosidasius, Burkholderia cepacia, B. gladioli, B. multivorans, B pyrrocinia, B. spinosa, Corynebacterium agropyri, C. misitidis, Enterobacter aerogenes, Microbacterium testaceum, Pseudomonas aeruginosa, P. citronellolis, Rhodococcus rhodochrous, Serratia ficaria, Serratia sp., S. marcescens, Staphylococcus sciuri, and Sternotrophomonas maltophilia. Out of these species were new recorded species of antagonistic bacteria i.e. B. thermoglucosidasius, B. multivorans, B. spinosa, C. agropyri, C. misitidis, Enterobacter aerogenes, P. citronellolis, Rhodococcus rhodochrous, Serratia ficaria, and Staphylococcus sciuri. However, B. cepacia, B pyrrocinia P. aeruginosa, S. marcescens and S. maltophilia were reported as biocontrol agents (Burkhead et al. 1994; Kobayashi et al. 1995; Suparman et al. 2002; Szczech and Shoda 2004). Avirulent isolate of B. gladioli strain 1064A is used for suppressing the incidence of bacterial seedling blight of rice caused by B. plantarii (Miyagawa 2000). P. aeruginosa is grouped as fluorescent Pseudomonads based on the production of a fluorescens pigment on KB medium (Sand et al. 1980). The bacterium produces siderophores as plant growth promoter (Hofte et al. 1993) and broad spectrum antagonistic bacteria against pathogenic fungi (Haas et al. 1991). 118 Antagonistic bacteria - A. Dikin et at. Several species of fluorescent Pseudomonads were known to be antagonistic bacteria and used as biological control agents. P. aeruginosa 7NSSK2 was able to suppress Pythium splendens, the causal pre and post-emergence damping-off and root rot of many crops such as tomato (Tambong et al. 1998). P. aeruginosa and S. marcescens were isolated from plant part of oil palm, these isolates were confirmed as biocontrol agent for suppressing Sclerotium rolfsii and Rhizoctonia solani (Ordentliche/a/. 1987). CONCLUSIONS A number of bacterial isolates from plant part such as seeds, fruits, rhizospheres, and plant debris under oil palm trees were potential antagonistic bacteria against S. commune. A total of 52 out of 265 bacterial isolates were identified as the antagonistic bacteria against S. commune. The identified antagonistic bacteria using Biolog® Identification System were as follows : Agrobacterium agropyri, Bacillus macroccanus, B. thermoglucosidasius, Burkholderia cepacia, B. gladioli, B. multivorans, B pyrrocinia, B. spinosa, Corynebacterium agropyri, C. misitidis, Enterobacter aerogenes, Microbacterium testaceum, Pseudomonas aeruginosa, P. citronellolis, Rhodococcus rhodochrous, Serratia ficaria, Serratia sp., S. marcescens, Staphylococcus sciuri, and Sternotrophomonas maltophilia. Some of bacterial isolates were recognized as biocontrol agents of plant pathogenic fungi and the rest of isolates have yet to be studied for their status. All of these species are required for further studies on the production of their secondary metabolites which might be potential substances to inhibit the growth of S. commune. ACKNOWLEGDMENT The study is partially supported by IRPA project, Malaysian Government (Vote No. 54400) as part of the Ph.D. thesis of the first author. 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