Ann Microbiol (2016) 66:587–595 DOI 10.1007/s13213-015-1140-z

ORIGINAL ARTICLE

Biodiversity of endophytic 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 during the long incubation times Kimura two-parameter model (Kimura 1980). The topology and incubated at 27 °C and 37 °C for up to 12 weeks. of the tree was evaluated by performing a bootstrap analysis (Felsenstein 1985) based on 1000 replications. Purification of isolates ARDRA Isolation plates were examined weekly. Emergence time of each colony was recorded, and actinobacterial isolates were Based on the sequence data, four isolates belonging to genus removed from the isolation plates every week for 12 weeks Microbispora were selected for studying ARDRA. The PCR and purified by using half-strength potato dextrose agar products of the 16S rRNA gene were singly digested with the (HPDA; Oxoid Ltd., UK). Pure cultures were maintained on restriction endonucleases HhaI, RsaIandPstI(Promega).The HPDA slants at 4 °C and in 20 % glycerol at -80 °C for further digestion reaction containing 8 μL of PCR product, 2 μLof study. restriction enzyme and 1 μL of 10x buffer (Promega) was incubated at 37 °C until complete digestion was achieved Morphological characterization (12–18 h). The digested products were separated by 1.8 % agarose gel electrophoresis. A low molecular weight-DNA All isolates were grouped based on their morphology on three ladder (Bio Labs, 25–766 bp) was loaded at both ends of the media; HPDA, International Streptomyces Project no. 2 and gel. The gel was run in 0.5x TBE (Tris/borate/EDTA) buffer at no. 3 (ISP 2 and ISP 3; Atlas 1993). Characterization included 90 V for 2.45 h. The Gene tools program (Syngene product morphological differences of their pigment or melanin pro- version 4.00, Synoptic Ltd., England) was used to compare duction, presence or absence of sporulation and mycelium the band sizes (in base pairs) from each digested product with colour following the general guidelines of the International the low molecular weight-DNA ladder. Streptomyces Project (Shirling and Gottlieb 1966). Spore morphology of cultures on agar was studied by using a slide In vitro antibacterial activity culture method (Kaewkla and Franco 2013). All endophytic actinobacteria obtained were selected for as- Partial 16S rRNA gene amplification and sequencing sessment of antimicrobial activity. used for the anti- analysis bacterial bioassay was Xanthomonas oryzae pv. oryzae KHWK 4.1_UBN_06. The antibacterial assay was modified Based on morphological study, the representative isolates of from the cross streak method described by Williston et al. non-Streptomyces genera were selected for 16S rRNA gene (1947). Briefly, actinobacteria were grown on HPDA for sequence analysis. Genomic DNA was extracted from 7 days by streaking a loop of culture at the middle of the actinobacterial cells by applying the method of Kieser et al. HPDA plate. Bacterial test strains were grown in tryptone (2000). The 16S rRNA gene was amplified using the primer soy broth (TSB; Oxoid Ltd., UK) at 37 °C by shaking at pairs 27f and 765r (R1 amplicon). Polymerase chain reaction 150 rpm for 18 h. The growth of bacteria was assessed by (PCR) amplification of the 16S rRNA gene was achieved as measuring the optical density (OD) using a spectrophotometer described previously by Coombs and Franco (2003)andPCR at OD600=0.5. Ten microlitres of bacterial suspension was products were sequenced at Macrogen Inc., Korea. 16S rRNA dropped 0.5 cm away from actinobacteria and streaked to gene sequences were analyzed and compared for pairwise the end of the Petri dish. Each actinobacterial culture was similarity with a valid name by using an EzTaxon-e server tested in triplicate. The negative control was the same as the (http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). test plate but without the actinobacteria culture. After that, the petri dishes were incubated at 37 °C for 24 h. The growth of Construction of a phylogenetic tree bacteria was measured and assessed by the percentage of in- hibition: (length of bacterial streak – length of bacterial The resultant 16S rRNA gene sequences of Microbispora growth) / length of bacterial streak x 100. Percentage of inhi- were analyzed using an EzTaxon-e server (http://eztaxon-e. bition was recorded as follow: strong inhibition (+4, ≥ 90 %), ezbiocloud.net/; Kim et al. 2012) and subsequently aligned good inhibition (+3, ≥ 75 %), moderate inhibition (+ 2, ≥ with the 16S rRNA gene sequences of representatives of all 50 %), week inhibition (+, ≥ 15 % ) and no inhibition (-). Microbispora members available from GenBank/EMBL by using CLUSTAL X (Thompson et al. 1997). The phylogenetic In vitro antifungal activity assay tree was constructed by the neighbor-joining technique (Saitou and Nei 1987) by using the software package MEGA All actinobacteria were tested for antifungal activity by modify- version 6 (Tamura et al. 2013). Pairwise distances for the ing the dual-culture methods described by Coombs et al. (2004). neighbor-joining algorithm were calculated according to the Three rice fungal pathogens, Curvularia lunata BCC 15558, 590 Ann Microbiol (2016) 66:587–595

Helminthosporium oryzae DOAC 1570 and Pyricularia grisea plants are listed in Table 1. The HVA yielded the highest 61119, were used as test fungi. Each actinobacterium was number at 58 isolates (50.4 %) with the rest of TWYA, TSA streaked onto one-third of HPDA for 7 days and incubated at andMSAat42,11and5isolates(35,10and4%)respective- 27 or 37 °C according to their isolation temperature. All test ly. There were three different genera: Streptomyces (63, fungi were grown on PDA (Oxoid Ltd. UK) at pH 6.0 for 7 days. 54.3 %), Microbispora (50, 43 %) and Kineococcus (3, An 8-mm plug of each fungi was placed one-third opposite an 2.5 %). All media yielded similar numbers of both HPDA plate containing 7 days of actinobacterial growth and Streptomyces and Microbispora except HVA. HVA gave a incubated at 27 °C for 4 days. The radius of the fungus was high number of Streptomyces at 35 isolates (31 %) while the measured toward the direction of actinobacteria. In the control number of Microbispora was 22 isolates (19 %). The two plate, the fungus was grown on HPDA without actinobacteria. nutrient-rich media, TSA and MSA, yielded the lowest num- The antagonistic activity of each strain was expressed as the bers of both Streptomyces and Microbispora.HVAand mean of three replications. Percentage of inhibition was calcu- TWYA yielded Kineococcus at one and two isolates, lated as percent inhibition of radial growth (PIRG): respectively. PIRG=(R1 - R2 x 100) / R1 (R1=radial of fungi in control There were 55 (47.4 %) and 61 (52.6 %) isolates obtained plates, R2=radial of fungi in tested plates). The evaluation of from the incubation temperatures of 27 °C and 37 °C, respec- inhibition was recorded as follows: strong inhibition (+ 4, tively. Similar numbers of both Streptomyces and ≥90 %), good inhibition (+ 3, ≥ 75 %), moderate inhibition (+ Microbispora were isolated from incubation at 37 °C while 2, ≥ 50 %), week inhibition (+, ≥ 15%)andnoinhibition(-). all isolates of Kineococcus were isolated at 27 °C. There were higher numbers of Microbispora isolated at 37 °C than at Detection of PKS-I, PKS-II, and NRPS gene sequences 27 °C. Analysis of actinobacteria from different plant parts showed that most actinobacteria were isolated from the leaf Eleven isolates which showed good antimicrobial activity were sheath at 47 isolates (40.5 %), with the stems, leaves and roots selected for detection of PKS-I and PKS-II and NRPSgenes. at 34, 18 and 17 isolates (29, 15.5, 15 %), respectively. Three sets of primers for the amplification of these genes were carried out, as recommended by other reports, and the PCR Identification to the genus level reaction was applied from Qin et al. (2009). The reaction mixture contained 2.5 units of Taq DNA polymerase, 1 mM MgCl2, One hundred and sixteen isolates were obtained over the 0.4 mM deoxynucleoside triphosphates, 2 μM of each primer, 12 weeks of incubation. The prefixes SBP1, SBP4 and and 5 % dimethyl sulfoxide (DMSO) in a 50-μL reaction vol- SBP6 refer to isolates from samples of paddy field sites 1, 4 ume. Control reaction mixtures had no DNA template. and 6, respectively. Thirteen groups were distinguished based Thermocycling conditions consisted of one denaturation step on cultural and morphological characteristics on three differ- of: 94 °C for 5 min; 30 amplification cycles of 94 °C for ent media. Based on their aerial mycelium colour, 1min;57°Cfor1min,foramplificationofPKS-Iand Streptomyces (63, 56 %) were classified to 4 groups; grey, NRPS genes or 58 °C for 1 min for amplification of PKS- green, white and dark grey at 23, 23, 10 and 7 isolates, respec- II and 72 °C for 2 min; and a final extension at 72 °C for tively. There were three types of Streptomyces spore morphol- 5 min. Primers for PKS-I gene (1200-1400 bp) were K1F ogies: spiral, rectiflexible and retinaculiaperti types at 33, 25 (5′-TSAAGTCSAACATCGGBCA-3′)andM6R(5′- and 5 isolates, respectively. Based on these morphological CGCAGGTTSCSGTACCAGTA-3′) (Ayuso-Sacido and characterizations, it was obvious that they belonged to the Genilloud 2005). Primers for PKS-II gene (600 bp) were genus Streptomyces. Therefore, it was not necessary to iden- KSα (5′-TSGCSTGCTTGGAYGCSATC-3′)andKSβ (5′- tify them by using 16S rRNA gene sequencing. Other mor- TGGAANCCG CCGAABCCTCT-3′) (Metsä-Ketelä et al. phological groups were classified into 8 groups (50, 44 %) on 1999). Primers for the NRPS gene (700-800 bp) were three different media, but all isolates contained spore pairs A3F (5′-GCSTACSYSATSTACACSTCSGG-3′)andA7R whichwerelikelytobelongtothegenusMicrobispora. (5′-SASGTCVCCSGTSCGGTAS-3′) (Ayuso-Sacido and Then, the representative of each group was selected for 16S Genilloud 2005). rRNA gene sequence analysis. The results showed that all of them belonged to the genus Microbispora and the closest spe- cies for all isolates was Microbispora thailandensis NN 276T, Results ranging from 99.08 - 99.38 percent. The pairwise similarity of SBP1L6 (KP967691), SBP1S12 (KP967689), SBP4LS15 Actinobacterial diversity (KP967688), SBP4LS16 (KP967692), SBP4S1(KP967694), SBP6L4(KP967693), SBP6R5 (KP967695) and SBP6R6 There were 116 isolates obtained. Numbers of isolates from (KP967690) was 99.38 %, 99.22 %, 99.37 %, 99.36 %, each isolation medium, different temperatures and each part of 99.31 %, 99.38 %, 99.37 %, and 99.08 %, respectively. The Ann Microbiol (2016) 66:587–595 591

Table 1 Type of media, temperature and part of plants Type of media, Number of isolates from each genus Total number employed to isolate temperature and * actinobacterial endophyte to parts of plants Streptomyces Microbispora Kineococcus different genera HVA 35 22 1 58 (50.4 %) TWYA 20 20 2 42 (35.3 %) TSA 5 6 − 11 (9.7 %) MSA 3 2 − 5 (4.42 %) Temperature 27 °C 31 21 3 55 (46 %) 37 °C 32 29 − 61 (54 %) Part of plants Leaf sheath 33 13 1 47 (40.5 %) Stem 17 16 1 34 (29 %) Leaf 9 8 1 18 (15.5 %) Root 4 13 − 17 (15 %)

*The full name of the isolation medium is presented in the materials and methods

GenBank accession no. of the 16S rRNA gene sequence is in the conclusion that they were closely related to this species brackets. which was isolated from the cave soil in Thailand. The other morphological characters of three isolates had a unique colony morphology which was a reddish orange color ARDRA and mucous-like without spore production. The representative isolate; SBP1S2 was sequenced and the result showed that the Based on 16S rRNA gene sequences, four isolates belonging closest match of SBP1S2 (KR0077964) was the genus to the genus Microbispora were classified using ARDRA. Kineococcus, of which the closest species was Kineococcus HhaI digestion yielded three ARDRA patterns and RsaI endophytica KLBMP 1274T (JQ819257) with 81.6 % similar- yielded two patterns. PstI yielded only one pattern (Table 2). ity of the 16S rRNA gene sequence. A neighbor-joining tree Microbispora had a high divergence in their 16S rRNA genes of all Microbispora isolates was constructed with all members as they presented three different ARDRA patterns by of Microbispora spp. The result revealed that 6 isolates were digesting with HhaI(Fig.2). in the same clade with high bootstrap support at 72 % while two isolates, SBP1LS15 and SBP1LS16, formed a distinct Antagonistic activity of actinobacteria against rice clade. Both these isolates were in different clades but all eight pathogens isolates belonged to the same clusters. The results showed that eight isolates were in the same cluster of the closest species, The overall antagonistic activity of all isolates against rice Microbispora thailandensis NN 276T (Fig. 1). This supported pathogens is shown in Table 3. No isolates could inhibit any

Fig. 1 16S rRNA gene-based neighbor-joining tree showing the phylogenetic relationships between eight isolates belonging to the genus Microbispora and all members of Microbispora and Herbidospora yilanensis 0351M- 12PT as the outgroup. The sequence length was 626 bp. The numbers on the branches indicate the percentage bootstrap values of 1000 replicates. Bootstrap numbers higher than 50 percent are presented. The scale bar represents 0.002 changes per nucleotide 592 Ann Microbiol (2016) 66:587–595

Table 2 The differentiation of isolates belonging to genus Enzyme Pattern Isolate Fragment size Microbispora by ARDRA using three enzymes HhaI 1A SBP4LS15, SBP1S12 385, 175, 124 and 106 1B SBP6R6 730, 620, 480, 390, 335, 247, 225, 175, 160 and 106 1C SBP6R5 730, 620, 480, 390, 335, 225, 175, 160 RsaI 2A SBP4LS15, SBP1S12 365, 300 and 83 2B SBP6R6,SBP6R5 770,690,620,475,380,300,142and83 PstI 3A SBP4LS15, SBP1S12 350 SBP6R6, SBP6R5 test microorganism with a strong activity. Most isolates Discussion showed activity against bacteria of which there were 22 iso- lates (19 %) showing good inhibition against Xanthomonas The aims of this study were to investigate endophytic oryzae pv. oryzae. There were four isolates (4 %) showing actinobacteria from rice and their antimicrobial activity good inhibition against P.grisea 61119 while there were seven against rice pathogens. A total of 116 isolates of endophytic (6 %) and nine (8 %) isolates showing moderate inhibition actinobacteria were obtained from roots, stems, leaves and against H. oryzae DOAC 1570 and C. lunata BCC 15558. leaves sheaths of rice. The majority of endophytic actinobacteria were isolated from the leaf sheaths. This study was different from the others (Tian et al. 2004, 2007;Gangwar Detection of PKS-I, PKS-II, and NRPS gene sequences et al. 2012;Runginetal.2012) who reported that most endo- phytic actinobacteria were isolated from rice root. The previ- Eleven isolates which showed good or moderate activity ous study showed that there were endophytic actinobacteria against test microorganisms were selected for detection of isolated from root (53.4 %), leaf (33 %) and stem of rice PKS-I, PKS-II and NRPS genes. None of these genes was (13.3 %) (Gangwar et al. 2012). In this study, the sampling detected in any of the selected isolates (Table 4). site was a paddy field which used inorganic fertilizers and applied a variety of pesticides. The accumulation of pesticides 1 2 3 4 5 6 in soil may have a negative effect on the actinobacteria in rice roots. The results showed that there were high numbers of isolates 766 bp from only three rice samples. Although it did not yield a good biodiversity of different genera, there were high numbers of non-Streptomyces genera: Microbispora (43 %) and 500 bp Kineococcus (2 %). Most isolates were Streptomyces spp. at 64 isolates (55 %) and this result conformed with other reports (Tian et al. 2004, 2007; Gangwar et al. 2012). Interestingly, 350 bp there were high numbers of Microbispora spp. which prefer

300 bp high temperatures for growth. The result of this study was similar to that of Widiantini (2012) who studied biodiversity 250 bp of endophytic actinobacteria from rice in South Australia and obtained a high number of Microbispora. It was likely that 200 bp Microbispora may tolerate the pesticides that contaminate the paddy fields. This was in agreement with reports showing 150 bp Microbispora could tolerate toxic chemicals by application of new selective method of Microbispora by sample pretreat- 100 bp ment using 1.5 % phenol and 0.03 % chlorhexidine gluconate (CG) (Hayakawa et al. 1991). 75 bp The best medium for isolation was HVAwhich yielded the highest number of isolates (50 %) and 3 different genera. The Fig. 2 Visualization of the 16S rRNA PCR-restriction fragment length second best medium was TWYA, yielding 38 % of isolates polymorphism (RFLP) with the HhaI enzyme digestion on 1.8 % agarose gel electrophoresis: lane 1, 6 low MW marker, lane 2 SBP4LS15, lane 3 and 3 different genera. HVAwas a selective and low nutrient- SBP1S12, lane 4 SBP6R6, and lane 5 SBP6R5 medium and TWYA was a low nutrient-medium. They were Ann Microbiol (2016) 66:587–595 593

Table 3 Antagonistic activity of endophytic actinobacteria isolates Potential Number of endophytic actinobacterial isolates against rice pathogens against rice pathogens antagonistic activity X. oryzae H. oryzae P. grisea C. lunata pv. oryzae DOAC 1570 61119 BCC 15558

+4 −− −− + 3 22 (19 %) − 4(4%) − + 2 9 (8 %) 7 (6 %) 18 (15 %) 9 (8 %) + 1 10 (9 %) 26 (22 %) 39 (33 %) 40 (34 %) − 75 (64 %) 83 (72 %) 55 (48 %) 67 (58 %)

Symbols: −, no activity; + 1, + 2, + 3 and+4, weak activity, moderate activity, good activity and strong activity, respectively suitable for use as isolation media of actinobacteria which grow Microbispora (26 and 25 %). There were 49 percent of slowly and required some vitamin B and some amino acid to actinobacteria isolated from 27 °C which belonged to the gen- germinate (Inderiati and Franco 2008; Qin et al. 2009, 2011). era Streptomyces, Microbispora and Kineococcus (28, 18 and There are many reports showing that HVA is the best medium 3%,respectively).MostStreptomyces are mesophilic bacteria for isolating actinobacteria (Hayakawa et al. 1991; Coombs and which grow well at 30 °C. Streptomyces from this study may Franco 2003; Widiantini 2012; Kaewkla and Franco 2013). have evolved to a high-temperature environment and may be Also, some studies successfully isolated high numbers of different from other sources. The best temperature for isola- actinobacteria by using TWYA (Coombs and Franco 2003; tion of Microbispora was 37 °C. This result corresponded to Inderiati and Franco 2008). The other two media, TSA and other reports that applied a high temperature for isolation of MSA, which contained high concentration of nutrients, were this genus (Hayakawa et al. 1991). Also, Microbispora has not suitable to isolate actinobacteria. There was contamination many strains which produce a variety of bioactive compounds of fast growing bacteria which could inhibit growth of such as novel antibiotics, anti-cancer, and anti-tumor agents actinobacteria and this was consistent with results from pre- (Chin et al. 2006; Demain and Sanchez 2009). Kineococcus vious research (Qin et al. 2009; Kaewkla and Franco 2013). was the rare genus which currently contains eight valid spe- This study applied two different temperatures for cies. The closest match of isolates SBP1S2 was Kineococcus actinobacterial isolation. Interestingly, most isolates emerged endophytica (Bian et al. 2012) which was isolated from plant from 37 °C (51 %) with similar numbers of Streptomyces and tissue. The similarity of 16S rRNA gene sequences of

Table 4 Antimicrobial activities and PKS/NRPS genes of selected actinobacteria

Isolate Genus Activity againsta Presenceb of gene

X. oryzae H. oryzae P. g ris ea 61119 C. lunata PKS-I PKS-II NRPS pv. oryzae DOAC 1570 BCC 15558

SBP1L6 Microbispora +++ − + −−−− SBP4L3 Streptomyces +++ + − + −− − SBP4L6 Streptomyces −− +++ ++ −− − SBP4LS20 Streptomyces +++ + −−−−− SBP4LS21 Streptomyces +++ − ++−− − SBP6LS4 Streptomyces +++ + + + −− − SBP6LS8 Streptomyces +++ − ++−− − SBP6LS18 Streptomyces − + +++ + −− − SBP6S7 Streptomyces +++ + − + −− − SBP6S10 Streptomyces +++ ++ − ++ −− − SBP6S11 Streptomyces +++ + ++ ++ −− − a Evaluated by measuring the percentage of inhibition. Symbols: −, no activity; +, ++, and +++, weak activity, moderate activity, and good activity, respectively b +, present; −,absent 594 Ann Microbiol (2016) 66:587–595

Microbispora showed that some isolates are likely to be novel of which is in the major area of rice production, in species. Also, the closest match of isolate SBP1S2 with low Northeastern Thailand. Currently, none of antibiotics can in- 16S rRNA gene similarity at 81.63 % is K. endophytica hibit X. oryzae pv. oryzae . This study successfully isolated a KLBMP 1274T. Then, this isolate is probably a novel genus high number of rare Microbispora which are likely to produce but it requires detailed polyphasic to capture this novel bioactive compounds, according to reports of others information. This showed that rice is a good source for novel (Kizuka et al. 1998; Igarashi et al. 1998).Thelackofdetection microorganisms, which was related to other studies of PKS-I, PKS-II and NRPS genes of eleven isolates which (Indananda et al. 2010, 2011). There were diverse morpholog- showed good activity against test microorganisms revealed ical patterns of Microbispora on three different media. that antimicrobial activity did not correlate to these genes. However, 16S rRNA gene sequences of all representatives Therefore, we can conclude that the active compounds are of each morphological group showed the closest match of produced by other biosynthetic pathways, such as the amino- selected isolates was Microbispora thailandensis NN 276T. glycoside pathway (Huang et al. 2005). Also, the number of The neighbor-joining tree of 16S rRNA genes of all these selected isolates for detecting these genes was low. This re- isolates and all members of genus Microbispora showed that sults were similar to other reports (Li et al. 2008; Qin et al. they were in a same cluster of the closest type strain. The 2009;Zhaoetal.2011) in which strains showing antimicro- nearest neighbor was Microbispora thailandensis NN 276T bial activity did not contain these genes, but strains that pos- which was isolated from cave soil in Thailand (Duangmal sessed these functional genes showed no antimicrobial et al. 2012). For these eight isolates, SBP4LS15 and activity. SBP4LS16 formed a different clade from the other six iso- In conclusion, the actinobacteria from this study can be lates. Then, these two isolates were likely to be different spe- evaluated via in planta experiments to select strains that can cies and will be selected for a polyphasic study into novel suppress microbial pathogens of rice plants, and also promote species in further study. plant growth. The effective strains can be developed as inoc- ARDRA results revealed that Microbispora contained di- ulum for biocontrol agents or plant growth promoters but it vergence in their 16S rRNA gene. Although, four isolates had would require further research to demonstrate whether this a 16S rRNA gene similarity range from 99.5 to 100 % might be possible. (pairwise similarity of 626 bp), there were 3 patterns when they were digested with HhaI. HhaI was the best enzyme which could distinguish closely related Microbispora. These Acknowledgments This work was financially supported by a research results conformed to other results (Kaewkla and Franco 2013) grant from the faculty of science and the postgraduate fund at successfully distinguishing different genera of actinobacteria Mahasarakham University. We would like to thank Prof. Chris Franco for his suggestions and for editing the manuscript. We would like to thank by using this enzyme, and this enzyme could yield more than the Surin Rice Research Center for giving rice samples and the Ubon one pattern for one genus. 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