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Biodiversity of Endophytic Actinobacteria from Jasmine Rice (Oryza Sativa L. KDML 105) Grown in Roi-Et Province, Thailand and Th

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Biodiversity of Endophytic Actinobacteria from Jasmine Rice (Oryza Sativa L. KDML 105) Grown in Roi-Et Province, Thailand and Th Ann Microbiol (2016) 66:587–595 DOI 10.1007/s13213-015-1140-z ORIGINAL ARTICLE Biodiversity of endophytic actinobacteria from jasmine rice (Oryza sativa L. KDML 105) grown in Roi-Et Province, Thailand and their antimicrobial activity against rice pathogens Dudcheewan Kampapongsa1 & Onuma Kaewkla1 Received: 1 June 2015 /Accepted: 5 August 2015 /Published online: 27 August 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract The aims of this study were to investigate endo- pathogens in pot experiments. The effective strains can be phytic actinobacteria from jasmine rice (Oryza sativa)L. further studied for development as biocontrol agents. KDML 105 and their antimicrobial activity against rice path- ogens. Three rice samples were collected from the rice paddy Keywords Endophyte . Actinobacteria . Rice . Biodiversity . fields in Suvannabhum District, Roi-Et Province, Thailand. Biological control Roots, stems, leaves and leaf sheaths were surface-sterilized prior to incubation on four different isolation media at 27 and 37 °C. One hundred and sixteen isolates were obtained. Introduction Colony and spore morphology accompanied with 16S rRNA gene sequencing were used to identify all the isolates. There Actinobacteria are a group of Gram-positive, filamentous bac- were 63 Streptomyces isolates (54.3 %), 50 Microbispora iso- teria with a high guanine (G) plus cytosine (C) content in their lates (43 %) and 3 Kineococcus isolates (2 %). All the isolates DNA. Actinobacteria are found in natural habitats such as were tested for antimicrobial activity against rice pathogens; soils, fresh water basins, composts, the atmosphere and plant Xanthomonas oryzae KHWK 4.1_UBN_06, Pyricularia tissues. Actinobacteria produce a great variety of natural prod- grisea 61119, Helminthosporium oryzae DOAC 1570 and ucts such as enzymes, antibiotics, anticancer and anti-parasitic Curvularia lunata BCC 15558 by using a dual-culture tech- compounds and immunosuppressors (Chin et al. 2006;Qin nique. The results showed that twenty-two and four isolates et al. 2009, 2011). Most actinobacteria reported to produce (18.8 and 3.4 %) showed good activity against X. oryzae antibiotics are Streptomyces which produced about 70 % KHWK 4.1_UBN_06 and P. grisea 61119, respectively. (8000 compounds) of known antibiotics (Demain and Most of the active isolates belonged to the genus Sanchez 2009), while an uncommon genus such as Streptomyces. Selected isolates which showed good activity Micromonospora was reported to produce 5 % or 740 types against test microorganisms were screened for PKS-I, PKS-II of known antibiotics (Raja and Prabakarana 2011). and NRPS genes. None of the isolates contained these func- Endophytic actinobacteria are microorganisms living in the tional genes, which revealed that antimicrobial activity did not intercellular space of plant tissues without causing apparent correlate to these genes. In conclusion, as endophytic disease symptoms. On the other hand, they can produce anti- actinobacteria from rice can inhibit rice pathogens in vitro they biotics to inhibit plant pathogens or produce plant growth can be further evaluated to select for activity in suppressing hormones, auxins, cytokinins and gibberellins and also pro- mote drought tolerance (Igarashi et al. 2002;Naiketal.2009). Rice (Oryza sativa) is the most important economic crop in Thailand, utilizing about 11.3 % of the cultivated land. The * Onuma Kaewkla major regions of rice production are central and northeastern [email protected] Thailand. However, critical problems are pest management, low yields and the high cost of inorganic fertilizers and pesti- 1 Department of Biology, Faculty of Science, Mahasarakham cides. The major rice pathogen of jasmine rice, Oryza sativa University, Maha Sarakham, Thailand KDML 105, is Pyricularia grisea, a fungus which causes blast 588 Ann Microbiol (2016) 66:587–595 disease, and Xanthomonas oryzae pv. oryzae, a bacterial leaf Materials and methods blight disease (Priya and Kalaichelvan 2011). Endophytic actinobacteria which live inside plant tissues may produce Sampling of rice plants antibiotics or plant growth-promoting compounds to support plant growth, and, therefore, are a good choice for a beneficial Jasmine rice plant samples were collected from three paddy microorganism screening program. To successfully screen ef- fields located in the Surin Rice Research Center, fective actinobacteria for biological control, it is necessary to Suvannabhum District, Roi-Et Province, Thailand on study their diversity, species richness and distribution in rice July 2011. Rice plants were harvested after 7 weeks of growth. plants. There are some reports which studied the biodiversity At each sampling field, a total of 25 plants were dug out of actinobacteria from rice and their antimicrobial activity randomly from 5 spots to ensure that maximal amounts of against rice pathogens in other countries, including Thailand plant material were collected. The Surin Rice Research (Tian et al. 2004, 2007;Naiketal.2009;Priyaand Center is characterized by loamy sand and alkaline soil and Kalaichelvan 2011; Gangwar et al. 2012; Rungin et al. 2012; relatively low rainfall (average, 1490 mm/yr, pH 4.5-6.0). Widiantini 2012). Previous studies on endophytic This research center is the major organization which produces actinobacterial communities in rice based on cultivation indi- quality jasmine rice seed for distribution to farmers. cated that 50 % of the most frequently isolated Streptomyces were antagonistic to rice pathogens such as Fusarium Test microorganisms moniliforme, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae (Tian et al. 2004, 2007). The biodiver- Xanthomonas oryzae KHWK 4.1_UBN_06 and Pyricularia sity of endophytic actinobacteria from rice has not been stud- grisea 61119 were kindly provided by the Ubon Ratchatani ied in much detail and this study contributes to microbial Rice Research Centre, Thailand. Helminthosporium oryzae ecology by demonstrating methods to obtain high numbers DOAC 1570 was kindly contributed by the Department of of the population that would not normally be isolated. This Agriculture, Ministry of Agriculture and Cooperative, is the first report of endophytic actinobacterial biodiversity Thailand, and Curvularia lunata BCC 15558 was provided from the Thung Gura Rong Hai area, Thailand. Thung Gura by the National Centre for Genetic Engineering and Rong Hai is the biggest rice paddy field in northeastern Biotechnology, Thailand. Thailand and it is a major area for good quality jasmine rice production. The area is about 3372 square km and covers five provinces; Buri Rum, Maha Sarakham, Roi-Et, Surin and Surface sterilization of rice plants and isolation method Yasotorn. Three-fifths of this rice paddy field belongs to Roi-Et Province. The plants were thoroughly washed to remove all soil from the The aims of this project were to investigate biodiversity of root mass. Leaves, leaf sheathes, stems and roots were sepa- endophytic actinobacteria isolated from rice (Oryza sativa) rated. Samples were cut to approximately 5 cm and subjected KDML 105 which were grown in Thung Gura Rong Hai, to a three-step surface sterilization procedure by modifying Suvannabhum District, Roi-Et Province and screen for bene- the method of Coombs and Franco (2003); a 60-s wash in ficial microorganisms to inhibit rice pathogens. The presence 70 % ethanol, followed by a 6-min wash in 3 % sodium hy- of polyketide synthases (PKSs) and nonribosomal peptide pochlorite, and a final rinse in sterile reverse osmosis (RO)- synthetase (NRPS) genes was screened to determine correla- treated water. The surface-sterilized samples were then asep- tion with their antimicrobial activity. The rationale to select tically sectioned into 1-cm fragments. Approximately 5-10 g these three genes is that PKS-I, PKS-II and NRPS genes en- of each surface-sterilized root, stem, leaf and leaf sheath sam- code to type I polyketide synthases, type II polyketide ple of each plant was plated onto four isolation media in trip- synthases and NRPSs, respectively. They are multi- licate for each medium. The isolation media were: 1) mannitol enzymatic and multi-domain megasynthases involved in the mung bean yeast extract mineral salt agar (MSA; Kaewkla and biosynthesis of polyketides and nonribosomal peptides. These Franco 2013); 2) tap water-yeast extract agar (TWYA; secondary metabolites reveal a remarkable range of biological Coombs and Franco 2003); 3) humic acid vitamin B agar activity and many of them are clinically valuable as anti-mi- (HVA; Hayakawa and Nonomura 1987); and 4) tryptone crobial, anti-fungal, anti-parasitic, anti-tumor and immuno- soytone agar (TSA; Oxoid Ltd., UK). The pH of all media suppressive agents (Ansari et al. 2004). If any of these genes was adjusted to 7.2. Each medium was supplemented with was detected, actinobacteria were likely to have biosynthetic 100-IU/mL nystatin as an antifungal agent. The efficacy of capabilities of polyketides or nonribosomal peptides. the surface sterilization procedure was assessed by aseptically Identification of all isolates was based on culture morphology rolling surface-sterilized plant tissue onto each of the isolation accompanied by 16S rRNA gene sequencing and amplified media and then incubated at 27 °C and 37 °C. Plates were kept ribosomal DNA restriction analysis (ARDRA). in small sealable plastic boxes lined with wet paper towels to Ann Microbiol (2016) 66:587–595 589 maintain the moisture levels
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