2.MI664-Abdul Munif NEW Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.6.4.2ISSN 1978-3477, eISSN 2087-8575 Vol 6, No 4, December 2012, p 148-156 *Corresponding author; Phone: +62-251-8629364, E-mail: abdulmunif@ipb.ac.id Endophytic bacteria colonize healthy plant roots without causing apparent disease. More convey stress tolerance, induce systemic resistance, recently, endophytic bacteria have gained attention due to their interesting features related to plant growth and health stimulation. Studies on endophytic bacteria have been conducted. Some of the bacteria are known to increase nutrient availability, produce growth hormones, or deter plant pathogens (Hallmann et al. 1997; Buchenauer 1998). Several reports demonstrated that endophytic bacteria are plant-associated bacteria that colonize and persist in various healthy plants, such as fruits, vegetables, stems and roots (Mclnroy and Kloepper 1995; Sturz et al. 1997). Bacterial endophytes can be isolated from surface- sterilized plant tissue or extracted from internal plant tissue. Endophytic microorganisms enter plant tissue primarily through the root zone; however, aerial portions of plants, such as flowers, stems, and cotyledons, may also be used for entry (Quadt- Hallmann and Kloepper 1996). Specifically, the bacteria enter tissues via germinating radicles, Endophytic bacteria have gained attention due to their interesting features related to plant growth and health stimulation. The objective of this research was to determine the populations and spectrum of indigenous root endophytic bacteria from tomato (Lycopersicon esculentum) and the biocontrol activity of the bacteria for plant protection. The isolation procedure of these endophytic bacteria was done using surface-sterilization method using alcohol and sodium hypochlorite (NaOCl). General medium trypsic soy agar (TSA) was used as the growth medium for isolation. The total population density of endophytic bacteria recovered from tomato roots ranged -1 from 1.0 to 4.4 (in log scale) CFU g fresh root weight. A total of 564 strains of endophytic bacteria were isolated 10 from tomato plants grown in West Java, Indonesia. Endophytic bacterial strains were identified based on their fatty acid profile using FAME-GC-MIDI system. Fifty species and 32 genera of endophytic bacteria were found in association with tomato root. The most abundant endophytic bacterial genera were Bacillus spp and Pseudomonas spp. One hundred and eighty one bacterial strains were tested for their in vitro antagonism towards Rhizoctonia solani, Fusarium oxysporum f.sp. radicis-lycopersici, and F. oxysporum f.sp. lycopersici. Fourteen strains showed antagonism against R. solani, nine strains against F. oxysporum f.sp. radicis-lycopersici and seven strains against F. oxysporum f. sp. lycopersici. The close relationship between endophytic bacteria and their hosts make them ideal candidates for biological control and plant growth promotion. Key words : Bacillus spp., endophytic bacteria, Pseudomonas spp., tomato Bakteri endofit telah menarik perhatian karena potensinya untuk peningkatan pertumbuhan dan kesehatan tanaman. Tujuan penelitian ini adalah untuk mengetahui populasi dan spektrum bakteri endofit yang berasal dari akar tanaman tomat dan potensinya sebagai agen biokontrol untuk perlindungan tanaman. Prosedur isolasi bakteri endofit dilakukan dengan menggunakan metode sterilisasi permukaan dengan alkohol dan sodium hipoklorit (NaOCl), dan medium trypsic soy agar (TSA) sebagai media pertumbuhan untuk bakteri. Strain bakteri endofit diidentifikasi berdasarkan profil asam lemaknya dengan menggunakan FAME-GC-MIDI system. -1 Kepadatan total populasi bakteri endofit yang diisolasi dari akar tomat berkisar 1.0-4.4 (pada skala log ) CFU g 10 berat akar segar. Sebanyak 564 strain bakteri endofit berhasil diisolasi dari tanaman tomat dari daerah Jawa Barat, Indonesia. Hasil identifikasi menunjukkan 50 spesies dan 32 genera bakteri endofit ditemukan berasosiasi dengan akar tomat. Genus bakteri endofit paling banyak ditemukan adalah Bacillus spp. dan Pseudomonas spp. Sebanyak 181 isolat bakteri endofit diuji antagonismenya secara in vitro terhadap cendawan patogen. Sebanyak 14 isolat bakteri menunjukkan reaksi antagonisme terhadap Rhizoctonia solani, 9 strain terhadap Fusarium oxysporum f.sp. radicis-lycopersici, dan 7 strain terhadap F. oxysporum f. sp. lycopersici. Hubungan yang sangat dekat antara bakteri endofit dan tanaman inangnya menjadikan bakteri endofit sangat berpotensi sebagai agen biokontrol dan pemacu pertumbuhan tanaman. Kata kunci: Bacillus spp., bakteri endofit, Pseudomonas spp., tomat . Isolation of Endophytic Bacteria from Tomato and Their Biocontrol Activities against Fungal Diseases 1 2 2 ABDUL MUNIF *, JOHANNES HALLMANN , AND RICHARD A SIKORA 1 Department of Plant Protection, Institut Pertanian Bogor (IPB), Kampus IPB Darmaga, Bogor 16680,Indonesia; 2 Institut fur Pflanzenkrankheiten, University of Bonn, Nussallee 9, D 53119 Bonn, Germany secondary roots, stomata, or as a result of foliar damage. Endophytes inside a plant may either become localized at the point of entry or spread throughout the plant (Quadt-Hallmann et al. 1997). Variations in the populations of endophytic bacteria are attributed to plant source, plant age, tissue type, time of sampling, and environment. Generally, bacterial populations are larger in roots and lower in the stems and leaves (Hallmann et al. 1997). Endophytic bacteria may be considered a new source of biocontrol agents (Backman and Sikora 2008). Some reports have demonstrated that bacterial endophytes are able to improve plant growth and reduce disease symptoms caused by plant pathogens such as Fusarium oxysporum subsp. vasinfectum on cotton, Verticillium albo-atrum, and Rhizoctonia solani on potato (Hallmann et al. 1997), Clavibacter michiganensis subsp. sepedonicum on potato (Van Buren et al. 1993), and plant-parasitic nematodes (Munif et al. 2000; Vertrivelkalai et al. 2010). When compared with rhizobacteria, endophytic bacteria have distinct advantages: 1) they colonize an ecological niche similar to that of vascular plant pathogens, i.e. vascular wilt pathogens and sedentary plant parasitic nematodes; 2) they face less competition with other microorganisms; 3) they have access to sufficient supply of nutrients; 4) they are less exposed to rhizosphere's abiotic and antibiotic stress factors; 5) their bioactive metabolites are better translocated within the host plant (Hallmann et al. 1997; Buchenauer 1998). The exploitation of the beneficial properties of endophytic bacteria requires a basic knowledge of the endophytic bacteria, such as distribution within the plant tissue, diversity, population dynamics, and characterization. The objective of this research was to determine the variations and types of indigenous endophytic bacteria from tomato plants grown in West Java, Indonesia, and its biological control potential against fungal disease. MATERIALS AND METHODS Isolation of Endophytic Bacteria. A total of 20 tomato plants were uprooted at random from tomato fields in the districts Bogor, Sukabumi, and Bandung, West Java, Indonesia. The tomato plants were approximately two months old. The tomato roots were transported to the laboratory for immediate processing. Tomato roots were washed under running tap water to remove adherent soil particles and then blotted dry on tissue paper. To simplify root processing, the root system was divided into three parts: top, middle, and bottom. The top section was discarded, whereas the middle and bottom parts were examined. The root was weighed and then surface sterilized with 3% and 6% sodium hypochlorite containing 0.01% Tween 20 for 3 min, followed by four rinses in sterile 0.01 M potassium phosphate buffer (PB) at pH 7.0 (80 g NaCl, 2 g KCl, 11.5 g Na HPO , 2 g KH PO ). To confirm 2 4 2 4 complete surface-sterilization (sterility check), the surface sterilized roots were imprinted on tryptic soy agar (TSA). If bacterial growth occurred within 48 h, samples were discarded. The tomato roots were then macerated with a sterile mortar and pestle in three times PB (w/v). The macerate was decanted into sterile conical flasks and shaken for 30 sec. A dilution series was made and 100 µL of each dilution was plated onto 1/10 strength TSA with a Drigalski spatula (plate spreader). Petri plates were incubated at 24 °C for 2-3 d and colony forming units (CFU) were calculated. Three replicates were made per dilution. On each Petri plate a zone containing approximately 30 bacterial colonies was marked. All bacterial colonies from this zone were transferred and purified on full strength TSA. The bacterial colonies were stored in tryptic soy broth (TSB) plus 20% glycerol at -20 °C. Identification of Endophytic Bacteria. All bacterial strains were identified based on their fatty acid profile using fatty acid methyl ester (FAME) analysis by gas-chromatography and the software Microbial Identification System (MISI Microbial ID, Newark, Delware, USA). The extraction procedure for whole-cell fatty acid from endophytic bacteria was done following the method described by Sasser (1990). Harvesting. Bacteria from stock cultures (-80 °C) were pre-cultivated for 24 h on tryptic soy broth agar (TSBA) and then streaked on TSBA for identification. Each strain was streaked with a loop onto TSBA in four zones with increasing dilution and incubated at 28 °C for 24 h. The bacteria from zone 2 and 3 were harvested. A loopful of bacteria (about 50 mg) was used to inoculate medium tube. Saponification. Lipids were saponified by adding 1.0 mL of 15% NaOH in 50% methanol Reagent 1 (sodium hydroxide/certified ACS 45 g, methanol /reagent grade 150 mL, and deionized distilled water 150 mL) into each sample tube and the tube was sealed with a teflon-lined cap, then mixed for 5 sec. The tube was heated in a waterbath at 100 °C for 30 min, and the content was mixed again. Methylation. The samples were methylated by Volume 6, 2012 Microbiol Indones 149 adding 2 mL of 6 N HCl in 50% methanol (Reagent 2: 6.0 N hydrochloride acid 325 mL, methanol (reagent grade) 275 mL), mixed for 5 sec, placed in a waterbath 80 °C for 10 min, and then rapidly cooled in water. Extraction. The FAMEs were extracted from this solution by adding 1 mL of methyl tert-butyl ester (MTBE) hexane mix (50:50, v/v) (Reagent 3: hexane (HPLC grade) 200 mL, methyl tert butyl ether (MTBE) 200 mL), rotated for 10 min. The top, organic phase was transferred with a Pasteur pipette to test tubes. Washing. The organic phase was then washed using 3.0 ml of diluted NaOH (Reagent 4: sodium hydroxide 10.8 g, deionized distilled water 900 mL). The organic phase was then transferred to 2 mL vials for subsequent analysis by gas chromatography using an HP 5890 (Hewlett Packard, MIS, ID, Newak, Delware, USA). In vitro Antagonism of Endophytic Bacteria towards Plant Pathogenic Fungi. A total of 181 bacterial endophytes were tested for antibiosis against fungal pathogens R. solani, F. oxysporum subsp. radicis-lycopersici, and F. oxysporum subsp. lycopersici. Isolates R. solani and F. oxysporum subsp. lycopersici were obtained from the fungal collection of the Institut fuer Pflanzenkrankheiten, University of Bonn, while isolate F. oxysporum subsp. radicis- lycopercisi was obtained from the fungal collection of the Humboldt University of Berlin. The fungi were grown on potato dextrose agar (PDA) at pH = 6.4 and stored in potato dextrose broth plus 20% glycerol at -80 °C. In vitro antibiosis was tested on PDA using the dual-culture technique. Fungal plugs (d = 10 mm) of 6 d old cultures were placed at the center of each Petri dish and the endophytic bacteria were streaked in two lines approximately 2 cm from the fungus. The bacterial strains were precultured on tryptic soy agar (TSA) for 2 d at 24 °C. Plates containing fungus alone served as controls. Petri dishes were maintained at 25 °C until radial growth in the control reached the border of the plate. Antibiosis were scored as ' + ' in the presence of an inhibition zone or as ' - ' in the absence. The experiment was replicated twice. RESULTS Population of Endophytic Bacteria. The population densities of indigenous endophytic bacteria in tomato roots between the plants varied. The lowest -1 endophytic population was 1.8 (in log scale) CFU g 10 fresh root after surface sterilization with 3% NaOCl -1 and 1.0 (in log scale) CFU g fresh root after surface 10 sterilization with 6% NaOCl. The highest endophytic -1 populations were 4.4 (in log scale) CFU g fresh root 10 after surface sterilization with 3% NaOCl and 3.0 (in -1 log scale) CFU g fresh root surface sterilization with 10 6% NaOCl (Table 1). Spectrum of Endophytic Bacteria from Tomato Roots. A broad range of bacterial genera and species were recovered from tomato roots in West Java. In total, 564 isolates of bacteria were identified by FAME-GC and MIDI system. Fatty acid profile is likely to become more widely used and will lead to easier identification through comparison to the literature as well as reference strains (Sasser 1990). Based on the similarity index (SI), isolates were identified at the species level for SI > 0.4, at the genus level for SI 0.2-0.4, and remained unidentified for SI < 0.2. Fifty one bacterial species comprising 32 genera were found in tomato roots from West Java, Indonesia. The most abundant endophytic bacterial species were B. megaterium (14.31%) followed by P. putida (12.44%) and Serratia marcescens (10.85%) (Table 2). In vitro Antagonism of Endophytic Bacteria towards Fungal Pathogens. A total of 181 strains of endophytic bacteria were studied for their in vitro antagonistic activity towards R. solani, F. oxysporum subsp. radicis-lycopersici, and F. oxysporum subsp. lycopersici on PDA. Fourteen strains (7.7 %) showed antagonism against R. solani, nine strains (5.0 %) against F. oxysporum subsp. radicis-lycpersici, and seven strains (3.9 %) against F. oxysporum subsp. lycopersici (Table 3). The level of antagonism expressed as size of the inhibition zone varied from weak (<1 cm) to medium (1 to 2 cm) and strong (>2cm) (Rocha et al. 2009). Isolates of endophytic bacteria with pronounced antagonism towards R. solani included strains of Bacillus megaterium, Burkholderia cepacia, Comamonas oxydovorans, Enterobacter intermedius, Pseudomonas chlororaphis, P. mendocina, P. putida, P. stutzeri, P. savastanoi, P. syringae, and S. marcescens. Whereas, bacterial species with antagonism towards F. oxysporum subsp. radicis-lycopersici and F. oxysporum subsp. lycopersici included Bacillus megaterium, Burkholderia cepacia, P. chlororaphis, P. putida, and S. marcescens. In this dual culture assay, of 181 endophytic bacteria strains could inhibit R. solani, F. oxysporum subsp. radicis-lycopersici, and F. oxysporum subsp. lycopersici in varying degrees (Fig 1). The inhibitory rates of the total bacterial strains were less than 10% (Table 3). 150 MUNIF ET AL. Microbiol Indones X-100 for 90 min and with 75% ethanol for 5 min, followed by 0.5% NaOCl in 0.05 Triton X-100 for 60 min. Sturz et al. (1997) isolated endophytic bacteria from red clover and potato. The root tissue of red clover and potato tuber were rinsed with 95% ethanol, then surface sterilized with 5% NaOCl for 3 min, followed by 2% solution of hydrogen peroxidase for 3 min, and then three washes with sterile distilled water. Well defined isolation procedures for recovering DISCUSSIONS Sodium hypochlorite is commonly used as a surface disinfectant for recovering bacterial endophytes, mainly because it is non-toxic and easy to handle. The concentration of NaOCl varied between 0.5 and 5% and the incubation time between 3 and 90 min. For example, Misaghi and Donndelinger (1990) treated cotton plants with 1.0% NaOCl in 0.05 Triton Volume 6, 2012 Microbiol Indones 151 Table 1 Population density of endophytic bacteria isolated from tomato from West Java, Indonesia, following surface- sterilization with sodium hypochlorite (NaOCl) Sample Origin of samples 3 % NaOCl 6 % NaOCl Sterility a) check Log CFU/g a) fresh root Bacterial c) isolates Sterility a) check Log CFU Bacterial T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 Bogor Bogor Bogor Bogor Bogor Bogor Bogor Bogor Sukabumi Sukabumi Sukabumi Sukabumi Sukabumi Sukabumi Bandung Bandung Bandung Bandung Bandung Bandung + + - - - - + - - - - - + - + + - - + + - - 3.3 3.4 2.8 3.6 - 3.8 3.8 3.5 4 4 - 4 - - 3.6 2.9 - - - - 29 30 35 31 - 30 30 19 32 38 - 32 - - 24 21 - - - - - - - + - - - - - - + - - + - - - - 1.6 (d) n.d 1 1.7 1.5 - 2.2 1.9 1.2 2.2 1.0 2.9 - 2.3 n.d - n.d n.d 3.0 2.5 10 - 6 30 30 - 11 12 16 12 18 30 - 30 - - - - - 8 Total 12 351 17 213 564 b) fresh root c) isolates a) - : Samples free from surface contamination, + : Samples with surface contamination b) Means of the population density of two replications c) - : Bacterial isolates were not taken, root samples were contaminated d) n.d : Non detectable; population density below detection limit disinfectants. The total number of plants passing the sterility check was higher for 6% NaOCl (85%) than for 3% NaOCl (60%). However, at 6% NaOCl the total population density was lower than that of 3% NaOCl (Table 1). The relatively lower number of bacterial endophytes at 6% NaOCl was due to the fact that surface sterilization was too strong and probably killed some endophytic bacteria within the internal root tissue. Hallmann et al. (1997) reported that slight variations in the concentration and incubation time of the surface disinfestation process greatly affects the number and type of bacteria isolated. Population densities of bacterial endophytes have been shown to be greatest in plant roots. In this experiment, the population densities of endophytic bacteria isolated from tomato roots were in the range -1 from 1.0 to 3.0 (in log scale) CFU g fresh weight 10 endophytic bacteria are a prerequisite for research with endophytes. Due to their secluded niche inside plant tissues, endophytic bacteria are most commonly isolated from macerated plant tissue following surface sterilization (Hallmann et al. 1997). Disinfectants generally used for isolation of endophytic bacteria include sodium hypochlorite (NaOCl), ethanol, hydrogen peroxide (H O ), mercury chloride (HgCl) or 2 2 a combination of two or more of these disinfectants followed by several washes in sterile distilled water or buffer solutions (He et al. 2009). To reduce surface tension of the solvent and therefore allow the disinfectant to penetrate into micro niches on the root surface, Tween 20 or Tween 80 can be added (Yasuda et al. 2009). In this study, 3% and 6% NaOCl supplemented with 0.01% Tween 20 were used as surface 152 MUNIF ET AL. Microbiol Indones Table 2 Species diversity of endophytic bacteria recovered from tomato roots in West Java, Indonesia using FAME-GC (in percent of 564 isolates) Species Percent of total isolates 1. Acidovorax avinae 2. Agrobacterium radiobacter 3. Alcaligenes xylosoxydans 4. Alteromonas haloplanktis 5. Arthrobacter citreus 6. Aureobacterium barkeri 7. Bacillus cereus 8. Bacillus circulans 9. Bacillus mycoides 10. Bacillus megaterium 11. Bacillus pumilus 12. Bacillus sphaericus 13. Bacillus thuringiensis 14. Burkholderia cepacia 15. Burkholderia gladioli 16. Burkholderia pickettii 17. Cellulomonas cellulans 18. Chryseobacterium balustinum 19. Chryseobacterium meningosepticum 20. Chryseobacterium indologens 21. Curtobacterium flaccumfaciens 22. Comamonas ocidovorans 23. Enterobacter tylorae 24. Erwinia amylovora 25. Gluconobacter oxidans 26. Kluyvera cryocrescens 0.46 3.46 1.61 0.23 0.46 1.61 1.61 1.15 0.46 14.31 0.23 2.54 1.85 2.07 1.15 0.46 0.69 1.15 0.23 0.69 0.23 0.23 1.15 0.23 0.69 2.31 27. Lactobacillus fermentum 28. Macrococcus lutens 29. Methylobacterium mesophilicum 30. Ochrobactrum anthropi 31. Paenibacillus pabuli 32. Paracoccus denitrificans 33. Peinococcus erythromixa 34. Phyllobacterium myrcinacearum 35. Phyllobacterium rubiacearum 36. Pseudomonas aeruginosa 37. Pseudomonas chlororaphis 38. Pseudomonas corugata 39. Pseudomonas fluorescens 40. Pseudomonas flekteus 41. Pseudomonas mendocina 42. Pseudomonas putida 43. Pseudomonas savastanoi 44. Rathayibacter rathayi 45. Spingobacterium multirorum 46. Staphylococcus sciari 47. Stenotrophomonas maltophilia 48. Serratia marcescens 49. Weeksella verosa 50. Xanthobacter agilis 51. Xanthomonas campestris 52. Unidentified 1.84 0.69 0.69 2.76 0.23 0.23 0.23 0.23 0.23 0.46 2.76 0.23 3.70 0.69 2.54 12.44 0.23 0.23 0.23 0.23 1.15 10.85 0.23 1.85 0.23 13.86 Percent of total isolates SpeciesNo No indigenous bacterial endophytes found for roots and stems from cotton and sweet corn ranging between 4.0 -1 to 6.0 (in log scale) CFU g fresh weight. Similar 10 results for the population densities of bacterial (when surface sterilized with 6% NaOCl) and from 3.0 -1 to 4.4 (in log scale) CFU g fresh weight (when 10 surface sterilized with 3% NaOCl). McInroy and Kloepper (1995) reported common population sizes of Volume 6, 2012 Microbiol Indones 153 Species Number of strains tested Number of strains with towards antibiosis activity Rs 1) Forl 2) Fol 3) Agroba cterium radiobacter 4 0 4) 0 0 Aureobacterium esteroaromaticum 2 0 0 0 Bacillus cereus 3 0 0 0 Bacillus circulans 2 0 0 0 Bacillus megaterium 28 3 3 3 Bacillus sphaericus 3 0 0 0 Bacillus thuringiensis 3 0 0 0 Burkholderia cepacia 3 2 1 0 Comamonas oxidovorans 3 1 0 0 Comamonas testoteroni 2 0 0 0 Chryseobacterium meningsepticum 2 0 0 0 Chryseobacterium indologens 4 0 0 0 Curtobacterium flaccumfaciens 2 0 0 0 Chryseobacterium balustinum 6 0 0 0 Enterobacter tylorae 2 0 0 0 Gluconobacter oxidans 3 0 0 0 Kluyvera cryocrescens 7 0 0 0 Micrococcus varians 2 0 0 0 Ochrobactrum anthropi 4 0 0 0 Paracoccus denitrificans 2 0 0 0 Phyllobacterium myrcinacearum 1 0 0 0 Pseudomonas aeruginosa 3 0 0 0 Pseudomonas chlororaphis 9 1 1 1 Pseudomonas corrugata 2 0 0 0 Pseudomonas fluorescens 8 0 0 0 Pseudomonas mendocina 3 1 1 0 Pseudomonas putida 27 3 1 2 Pseudomonas syringae 2 1 1 0 Pseudomonas stutzeri 3 1 1 0 Serratia marcescens 14 2 1 1 Xanthobacter agilis 4 0 0 0 Unidentified 8 0 0 0 Total 181 14 11 7 Table 3 In vitro antagonism of endophytic bacteria from tomato roots towards Rhizoctonia solani, Fusarium oxysporum subsp. radicis-lycopersici, and F. oxysporum subsp. lycopersici five days after inoculation on PDA media 1) Rhizoctonia solani 2) Fusarium oxysporum f.sp. radicis0lycopersici 3) Fusarium oxysporum f.sp. lycopersici 4) 0: No antibiosis effect on PDA medium endophytes were obtained from different plant roots including alfalfa, citrus twigs, sugar beet, cotton, grapevine, pine seedlings, red clover and potato of 2.0 -1 to 6.0 (in log scale) CFU g fresh root (Misaghi and 10 Donndelinger 1990; Bell et al. 1995; Hallmann et al. 1997; Sturz et al. 1997). Population densities of bacterial endophytes differ depending on the types of plant tissue studied and seem to be highest in the root and lower in the stem and decreases acropetally. The root is thought to be the preferred site of bacterial entrance into the plant, which would explain the high bacterial numbers in the root at early growth stages. The root system seems to provide a more buffered habitat with regards to water availability and temperature changes ( et al. 2004; He et al. 2009; Sarr et al. 2010). The present experiments showed that tomato plants were found to host a diverse spectrum of endophytic bacteria. The spectrum of endophytic bacteria isolated was similar to those found for other locations and/or other host plants. McInroy and Sessitsch 154 MUNIF ET AL. Microbiol Indones Kloepper (1995) reported 46 bacterial species in 31 genera in roots and stems of sweet corn and 45 species in 32 genera in roots and stems of cotton. In grapevine 24 bacterial species in 13 genera were found (Bell et al. 1995), in clover roots 31 species in 14 genera (Sturz et al. 1997), in cotton roots 43 bacterial species of 28 genera and in potato tubers 24 bacterial species of 13 genera (Hallmann et al. 1997). It should be mentioned that, these are only the culturable bacteria. Almost nothing is known about the non-culturable bacteria inside root tissue. The diversity of endophytic bacteria reported in this study has many similarities with the endophytic spectrum detected in other crops. Some bacterial genera, namely, Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Chryseobacterium, Enterobacter, Pseudomonas, and Stenotrophomonas have been commonly recovered from roots of cucumber, sugar beet, corn, and lemon (McInroy and Kloepper 1995; Mahaffee and Kloepper 1997; Hallmann et al. 1997). Factors like crop rotation, organic matter, temperature, C A D B Fig 1 In vitro antagonisms of 3 endophytic bacteria isolated from tomato root towards Rhizoctonia solani. The figures show growth of R. solani on PDA alone (A), and in the presence of endophytic bacteria showing various degrees of inhibition: strong inhibition by Pseudomonas spp. MT004 (B), moderate inhibition by P. putida MT019 (C), P. savastanoi f.sp. fraxinus (D). Images were taken 5 d after fungal inoculation. rainfall, soil physical, and chemical properties affect the bacterial spectrum and these factors were the source of endophytic colonizers in these tests (Mahaffee and Kloepper 1997; Sessitsch et al. 2004). In the present study, in vitro antibiosis was tested on potato dextrose agar (PDA). The choice of the growth medium can affect the degree of inhibition as demonstrated in these experiments (Yang et al. 2011). Strains of endophytic bacteria were screened for in vitro inhibition of R. solani, F. oxysporum f.sp. radicis- lycopersici, and F. oxysporum subsp. lycopersici on PDA. Testing in vitro antagonism of bacteria against fungal pathogens provides a rapid method to pre-select biological control candidates based on antibiosis. Results showed that some bacteria inhibited the growth in vitro of R. solani (8.3%), F. oxysporum subsp. radicis-lycopersici (6.6%), and F. oxysporum subsp. lycopersici (3.9%). Yang et al. (2011) reported that the inhibitory rates of endophytic bacteria in dual test assay against Botrytis cinerea were varied and less than 40%. This result might be affected by medium used for antibiosis in vitro assay. Fuchs and Defago (1990) studied the in vitro antagonism of bacteria towards fungal disease Phomopsis sclerotioides on PDA, Malt Agar (MA), and King's B Medium Agar (KBA). Some bacteria, especially Pseudomonas fluorescent, inhibited fungal pathogens on KBA, but not on PDA or MA and vice versa, whereas other bacteria showed antibiosis on all three media. In conclusion, endophytic bacteria can be isolated with surface-sterilized method. More than fifty one species of endophytic bacteria were recovered from tomato roots and three most abundant species, B. megaterium, P. putida, and S. marcescens, were frequently found in association with tomato roots from West Java, Indonesia and showed antibiosis activity against fungal diseases. The close relationship between endophytic bacteria and their hosts make them ideal candidates for biological control and plant growth promotion. REFERENCES Backman PA, Sikora RA. 2008. Endophytes: An emerging tool for biological control. Biol Control. 46(6):1-3. doi:10.1016/j.biocontrol.2008.03.009. Buchenauer H. 1998. Biological control of soil-borne disease by rhizobacteria. J Plant Dis Protec. 105 (4):329-348. Bell CR, Dickie GA, Harvey WLG, Chan JWYF. 1995. Endophytic bacteria in grapevine. Can J Microbiol. 41(1):46-53. doi: 10.1139/m95-006. Fischer PJ, Petrini O, Scott HML. 1992. The distribution of some fungal and bacterial endophytes in maize (Zea mays L.) New Phytol. 122(2): 299-309. Fuchs J, Defago G. 1990. Protection of cucumber plant against black root rot caused by Phomopsis sclerotioides with rhizobacteria. Proc 2th International Workshop on Plant Growth Promoting Rhizobacteria, Interlaken, Switzerland. p: 57-92. Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW. 1997. Bacterial endophytes in agricultural crops. Can J Microbiol. 43(10): 895-914. doi: 10.1139/m97- 131. He RI, Wang GP, Liu XH, Zhang CL, Lin FC. 2009. Antagonistic bioactivity of an endophytic bacterium isolated from Epimidium brevicornu Maxim. Afr J Biotechnol. 8(2):191-195. Mahaffee WF, Kloepper JW. 1997. Temporal changes in the bacterial communities of soil, rhizosphere, and endorhiza associated with field-grown cucumber (Cucumis sativus L.). Microb Ecol. 34(3): 210-223. doi: 10.1007/s002489900050. Mclnroy JA, Kloepper JW. 1995. Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil. 173(2): 337-342. Munif A, Hallmann J, Sikora RA. 2000. Evaluation of the biocontrol activity of endophytic bacteria from tomato against Meloidogyne incognita. Med Fac Landbouww Univ Gent. 65(2b): 471-480. Misaghi I, Donndelinger LR. 1990. Endophytic bacteria in symptom-free catton plants. Phytopathol. 80:808-811. Quadt-Hallmann A, Kloepper JW. 1996. Immunological detection and localization of the cotton endophyte Enterobacter asburiae JM22 in different plant species. Can J Microbiol. 42:1144-1154. Quadt-Hallmann A, Benhamou N, Kloepper JW. 1997. Bacterial endophytes in cotton: mechanisms of entering the plant. Can J Microbiol. 43(6): 577-582. Rocha R, da Luz DE, Engels C, Pileggi SAV, Filho DSJ, Matiello RR, Pileggi M. 2009. Selection of endophytic fungi from comfrey (Symphytum officinale L.) for in vitro biological control of the phytopatogen Sclerotia sclerotium (Lib.). Brazilian J Microbiol. 40(1): 73-78. doi: 10.1590/S1517-83822009000100011. Sarr PS, Yamakawa T, Asatsuma S, Fujimoto S, Sakai M. 2010. Investigation of endophytic and symbiotic features of Rasltonia sp. TSC1 isolated from cowpea nodules. Afr J Microbiol Res. 4(19): 1959-1963. Sasser M. 1990. Identification of bacteria through fatty acid analysis. In: Klement Z, Rudolp K, Sand S, edistors. Methods in Phytobacteriology. Budapest: Akademiai Kiado. p 199-204. Sturz AV, Christie BR, Matheson, BG, Nowak J. 1997. Biodiversity of endophytic bacteria Potential rol which colonize red clover nodules, root stems and foliage and their influence on host growth. Biol Fertil Soil. 25: 13-19. . Sessitsch A, Reiter B, Berg G. 2004. Endophytic bacterial communities of field-grown potato plants and their plant growth-promoting abilities. Can J Microbiol. 50(4): 239-249. doi: 10.1139/w03-118. Volume 6, 2012 Microbiol Indones 155 Van Buren AM, Andre C, Ishimaru CA. 1993. Biological control of the ring rot pathogen by endophytic bacteria isolated from potato. Phytopathol. 83:1406. Vetrivelkalai P, Sivakumar M, Jonathan FI. 2010. Biocontrol potential of endophytic bacteria on Meloidogyne incognita and its effect on plant growth in bhendi. J Biopest. 3(2): 452-457. Yang CJ, Zhang XG, Shi GY, Zhao HY, Chen L, Tao K, Hou TP. 2011. Isolation and Identification of endophytic bacterium W4 against tomato Botrytis cinerea and antagonistic activity stability. Afr J Microbiol Res. 5(2): 131-136. Yasuda M, Isawa T, Shinozaki S, Minamisawa K, Nakashita H. 2009. Effects of Colonization of bacterial endophyte, Azospirilum sp. B510, on disease resistance in rice. Biosci Biotechnol Biochem. 73 (12): 2595- 2599. doi: 10.1271/bbb.90402. 156 MUNIF ET AL. Microbiol Indones 1: 148 2: 149 3: 150 4: 151 5: 152 6: 153 7: 154 8: 155 9: 156