www.thaiagj.org Thai Journal of Agricultural Science 2010, 43(4): 217-230

Isolation, Screening of Growth-Promoting Activities and Diversity of Rhizobacteria from Vetiver Grass and Rice

C. Bhromsiri1 and A. Bhromsiri2,*

1Plant Biotechnology Program, Chiang Mai University, Thailand 2Central Laboratory, Faculty of Agriculture, Chiang Mai University, Thailand

*Corresponding author. Email: [email protected]

Abstract One hundred thirty six isolates of rhizobacteria were obtained from rhizosphere of different vetiver ecotypes and different rice varieties. Their acetylene reduction activities and indole acetic acid (IAA) producing activities were determined. A wide variation in both nitrogenase (0.01-2.15 -1 -1 -1 -1 n mole C2H4 mg h and 0.01-8.84 n mole C2H4 mg protein h for the first and second screening, respectively) and IAA (0.05-99.59 and 0.00-118.02 g IAA mg-1 protein for the first and second screening, respectively) activities of the isolates was shown. Twenty five promising isolates that have potential to be PGPR were selected on the basis of both high nitrogenase and/or IAA production activities. The diversity of the selected isolates was indicated by 16S rDNA analysis. A total of six clusters of all isolates were represented as follows, Gammaproteobacteria (Order Xanthomonadales), Betaproteobacteria (Order Burkholderiales), Gammaproteobacteria (Order Enterobateriales), Alphaproteobacteria (Order Rhodospirillales), Alphaproteobacteria (Order Rhizobiales) and Endosporobacteria (Order Bacillales).

Keywords: vetiver, rice, PGPR, ARA, IAA, 16S rDNA.

Introduction 2002; Thuler et al., 2003), Burkhoderia (Van et al., 2000; Govindarajan et al., 2006), Klebsiella (Iniquez growth-promoting rhizobacteria, PGPR are et al., 2004; Govindarajan et al., 2007), Herbaspirillium a group of that can be found in the (Barraguio et al., 1997; Elbeltagy et al., 2001) and rhizosphere, at surfaces and in association with Serratia (Gyaneshwer et al., 2001) have shown . These bacteria can improve the extent or plant growth promoting properties. quality of plant growth directly (Ahmad et al., Vetiver grass, Vetiveria zizanioides (L.) Nash, 2008) by increasing cycling such as also known as zizanioides (L.) Roberty biological nitrogen fixation, solubilization of (Adams et al., 1998; Veldkamp, 1999), is a fast phosphorus, synthesis of phytohormones or indirect growing graminaceous plant native to tropical and mechanism by synthesis of biocontrol compounds subtropical . It is a high-biomass plant having to inhibit phytopathogens (Dobbelaere et al., 2003; C4 photosynthesis efficiency (Mucciarelli et al., Lucy et al., 2004; Rothballer et al., 2009). A large 1998) with a long massive, aerenchymatous and array of bacteria including species of Pseudomonas complex root system which can easily penetrate (Ahmad et al., 2008), Azospirillum (Okon et al., into the deeper layers of acting as living dowels 1994; Malik et al., 1997), Azotobacter (Ahmad et that can pin together. Vetiver can improve the al., 2008), Azoarcus (Stein et al., 1997; Reinhold- shear strength of soil at 0.5 meters deep by as much Hurek et al., 1997), Bacillus (Cakmakci et al., 2007; as 40% (Greenfield, 2002). Due to its unique Ahmad et al., 2008), Beijerinckia (Polyanskaya et al., morphological, physiological and strong ecological

218 C. Bhromsiri and A. Bhromsiri Thai Journal of Agricultural Science adaptability, vetiver is a remedy which offers low SRI research in northern Thailand (Naveerat, 2005) cost, low maintenance and environmental-friendly were therefore included to test plant growth for soil and conservation, particularly in soil promoting (PGP) properties. control, slope stabilization (Hengchaovanich, 1998; Cazzuffi et al., 2006) and phytoremediation Isolation of Bacterial Isolates (Srivastava et al., 2007; Wong et al., 2007). Vetiver The beneficial effects, especially the nitrogen is a hardy plant by nature, but during its early stage fixation and production of plant growth promoting of growth as propagated material, it is rather weak. substances of some bacterial species such as Thus at the first establishing stage on infertile soil, Azotobacter, Azospirillum and Beijerinckia have it would be better to have beneficial micro- been well documented. Thus, all bacterial isolates organisms to support growth and development of were isolated from the roots and rhizosphere soils vetiver plants at this stage. of vetiver grass based on their growth, on three There are many papers related to the screening kinds of the respective nitrogen-free media. The and advantage of using PGPR with crop plants roots were cut into 1-cm pieces then tenfold serial particularly rice, maize and sugar cane but few on dilution of root and rhizosphere soil samples were vetiver. Little information about screening and prepared. For isolation of Azospirillum, semisolid using PGPR with vetiver grass is available. NFb medium and isolation procedure were used as Therefore, this research was conducted in order to described by Baldani et al. (2005). In the cases of isolate and screen the effective PGPR from grasses Azotobacter and Beijerinckia, the spread plate concerned with nitrogen fixation and the production method was used for isolation and 0.1 mL of each of plant growth promoting substances. The aim was diluted suspension of root or rhizosphere soil to use them for improving growth and development samples was spread on the Azotobacter and of vetiver plants used in . Beijerinckia enrichment media. The composition of Azotobacter medium (Atlas, 1993) per liter was

Materials and Methods prepared as follows, 20 g sucrose, 0.15 g KH2PO4,

0.2 g K2HPO4, 0.05 g K2HPO4, 1 mg FeCl3, 0.02

Collection of Rhizosphere Soils, Vetiver Root g CaCl2 2H2O, 1 mg Na2MoO4 2H2O. The pH was Samples and Bacterial Isolates adjusted to 6.2 and 15 g of agar was added. The Three ecotypes of Thai vetiver, Suratthani, Beijerinckia enrichment medium (Atlas, 1993)

Kuntulee and Na Phraya were collected for consisted of 20 g glucose, 0.2 g KH2PO4, 0.5 g isolation of PGPR from the roots and rhizosphere MgSO4 7H2O, 0.8 g K2HPO4, 25 mg FeCl3 6H2O, soils. The Suratthani ecotype was selected because 0.05 g CaCl2 2H2O, 5 mg Na2MoO4 2H2O it is one of the vetiver ecotypes commonly used for (quantities per liter) and the pH was adjusted to 5.0. soil conservation programs in Thailand. The site The single bacterial colony on media was selected where the roots and rhizosphere soils of the according to their phenotypic characteristics as Suratthani vetiver ecotype was obtained, was described in Bergey’s Manual of Systematic organic vegetable cultivated plots at the Ang Kang Bacteriology (Kennedy et al., 2005; Kennedy, Royal Project Research Station, ChiangMai, 2005). Finally, each of the single colonies was Northern region. In the cases of Kuntulee and Na restreaked on respective enrichment media again Phaya vetiver ecotypes, they were naturally grown for future purification. in Kuntulee and Na Phaya villages in Suratthani and Chumphon provinces in Southern region, In vitro Screening of Bacterial Isolates for Their respectively. Consideration of the relationship Plant Growth Promoting (PGP) Activities between some kinds of PGPR populations and All bacterial isolates showing the characteristic effectiveness of the System of Rice Intensification as mentioned above were tested for their PGP (SRI) (Randriamiharisoa et al., 2006; Uphoff, activities as follows, nitrogenase activity and 2006), the other 68 bacterial isolates obtained from indoleacetic acid (IAA) production.

Vol. 43, No. 4, 2010 Isolation and screening of growth-promoting activities of rhizobacteria 219

Nitrogenase Activity production in each sample was measured according Nitrogenase activity was measured by the to the above procedure. acetylene reduction assay (ARA). The culture of each bacterial isolate was prepared and incubated Indoleacetic Acid (IAA) Production in 50 mL flask containing 25 mL of specific IAA production of each culture was estimated nitrogen free liquid medium supplemented with 0.5 by growing each isolate in 50 mL flask containing g l -1 yeast extract until the stationary phase was 25 mL of specific NFb medium as described above reached which took 3-5 days for Azospirillum with 0.1 g L-1 of L-tryptophan. The growth cultures and Azotobacter and 7-10 days for Beijerinckia. (stationary phase, as the previous part) were The bacterial cells were harvested by centrifugation centrifuged at 10,000 rpm for 10 min. One part of (10,000 rpm for 10 minutes). After washing twice the supernatant was mixed with two parts of by centrifugation and resuspension in sterile Salvakowski reagent. The mixture was then distilled water, the bacterial cells were resuspended incubated for 30 min for the development of pink again in 5 mL sterile distilled water. For each colour which indicated IAA production (Gordon Azospirillum isolate, 1 mL of aliquot was taken and Weber, 1950). The concentration of IAA from the cell suspension and transferred into 21 mL produced by cultures was measured by spectro- test tube containing 9 mL semisolid NFb medium photometer at 530 nm for comparison with the for further incubation for 3-5 days. The cap of each standard set of known IAA concentrations. The test tube was changed with rubber stopper and the remained bacterial cells were washed twice by head space volume was replaced with 10% v/v centrifugation and resuspended in sterile distilled acetylene which was generated from calcium water. After washing, the samples were resuspended carbide and water. The cultures were incubated for again in the same volume with sterile distilled 24 h. The ethylene production was then measured water and used for analysis of the protein by gas chromatograph (GC-14B Shimatzu) concentration according to the modified Lowry equipped with a hydrogen flame-ionization detector method. (FID) and 1 m stainless steel Porapak-N column (80-100 mesh). The gas analysis was operated at 16S rDNA sequencing of PGPR isolates isothermal 55 C with N2 as the carrier gas at a 50 The 16s rDNA sequencing was performed by a mL min -1 flow rate (Somasegaran and Hoben sequencing service (Macrogen, South Korea). All 1994). Three replications were done for each bacterial 16s rDNA was amplified in full length by isolate. The standard ethylene gas (99.9%) was PCR using two pairs of primers, 518F(CCAg- diluted with air by replacing 1 mL ethylene gas CAgCCgCggTAATACg) and 800R (TACCAggg- with air in a known volume container. One mililiter TATCTAATCC) and 27F (AgAgTTTgATCMT- of diluted ethylene was taken to inject into the GC GGCACAg) and 1429R (TACggYTACCTTgTTA- instrument for calculation of the amount of CgACTT). To evaluate the phylogenetic analysis ethylene (nanomole; nmole). After completion of of 16S rDNA sequences, the resulting sequences the ARA, the bacterial cells in the medium were were compared with the known sequences using evenly mixed. The protein concentration (modified the BLAST function of GeneBank and the National Lowry method; Lowry et al., 1951) or cell number Center Biotechnology information; NCBI website was determined to compare ethylene production of (http://www.ncbi.nlm.nih.gov/). Multiple sequence each isolate. The ARA results were expressed as alignments and consensus sequences were computed nmole of ethylene mg -1 protein h -1 or ethylene using the program CLUSTER X version 2.0 cfu -1 h -1. For Azotobacter and Beijerinckia (Thomson et al., 1997). Evolution for the data isolates, after washing and resuspending, aliquots sets were inferred from the neighbor-joining (1 mL) of each sample were incubated into 50 mL methods (Saitou and Nei, 1987) by using the flasks containing 29 mL specific NFb medium. The neighbor-joining program of Molecular Evolutionary cultures were incubated for 5 days and up to a Genetic Analysis software version 4 (MEGA 4.0) week in case of Beijerinckia. The cotton plugs were (Tamura et al., 2007) based on 1,000 reassemblings. replaced with rubber stoppers and the ethylene

220 C. Bhromsiri and A. Bhromsiri Thai Journal of Agricultural Science

Statistical Analysis tested, the bacterial isolates from Azospirillum Results of the measurements were subjected to enrichment medium showed the highest average analysis of using a completely randomized design activity in both nitrogenase and IAA producing. with three replicates. The means were compared The average nitrogenase activity was 2.76 nmole 6 -1 -1 -1 according to Least Significant Difference (LSD) at C2H4 10 cfu h and 36.10 μg IAA mg protein the 5% level. for IAA production. The isolates from Azotobacter enrichment medium showed higher average amounts Results of IAA production activity than isolates from Beijerinckia enrichment medium. On the other Isolation and Plant Growth Promoting (PGP) hand, the isolates from the Beijerinckia enrichment Activities medium had higher nitrogenase activity than those In this work two sets of the bacterial isolation from Azotobacter enrichment medium. The average were conducted. The first isolation focused on the nitrogenase and IAA production of the isolates genus Azospirillum. Based on the cultural charac- from Azotobacter enrichment medium were 0.084 6 -1 -1 -1 teristics and colony morphology on NFb medium, nmole C2H4 10 cfu h and 3.07 μg IAA mg 34 isolates were obtained from rhizosphere soils protein while those from Beijerinckia enrichment 6 -1 -1 and roots of vetiver plants. Besides the isolates medium were 0.882 nmole C2H4 10 cfu h and from vetiver grass, the other 68 isolates from the 1.39 μg IAA mg -1 protein (Figure 3). Based on different rice cultivars grown under SRI and normal selection criteria, good ability in both nitrogenase cultivation practices (Naveerat, 2005), were also activity and/or IAA production, 17 and 8 PGPR included to test plant growth promoting (PGP) isolates were screened from the first and second properties. The ARA results (Figure 1) showed a isolation, respectively (Tables 1 and 2). In addition, variation in nitrogenase activity among the 102 all 25 selected isolates were used as microbial -1 isolates tested (0.01-2.15 nmole C2H4 mg protein inoculant in our next pot and field experiments -1 h ) with the average activity at 0.20 nmole C2H4 (Bhromsiri, 2009). mg-1 protein h-1. There were 31 isolates (30.4%) having higher activity than the average activity and Morphological Characterizations and 16S rDNA one isolate from the rice showed the highest ARA Sequencing of PGPR Isolates -1 -1 activity (2.15 nmole C2H4 mg protein h ). In the Based on cell shape and Gram reaction, 25 case of IAA production (Figure 2), there was wide selected isolates showed the very similar characters variation among the 102 isolates tested (0.05-99.59 (Table 3). According to the 16S rDNA sequences, a μg IAA mg-1 protein) with an average activity of total of six clusters were represented in all isolates 15.67 μg IAA mg-1 protein. There were 40 isolates as follows (Figure 4 and Table 4), (1) Gammaproteo- or 43.2% of the total number of tested isolates, bacteria in the Order Xanthomonadales, (2) Beta- which produced higher IAA production than the proteobacteria in the Order Burkholderiales, (3) average. An isolate from rice showed the highest Gammaproteobacteria in the Order Enterobateriales, IAA production (99.59 μg IAA mg -1 protein). (4) Alphaproteobacteria in the Order Rhodospirillales, The second isolation of PGPR was conducted in (5) Alphaproteobacteria in the Order Rhizobiales order to get isolates of the other groups of PGPR and (6) Endosporobacteria in the Order Bacillales. bacteria such as Azotobacter, Beijerinckia and Thirteen isolates were classified in Order Xantho- additional Azospirillum isolates from vetiver grass. monadales that belonged to Stenotrophomonas Thirty four isolates were obtained from the roots maltophilia and uncultured bacterium. Five isolates and rhizosphere soil of Kuntulee and Na Phraya were identified as Aurantimonas altamirensis, vetiver ecotypes as follows; 10 bacterial isolates Agrobacterium tumefaciens, and Rhizobium strains from Azospirillum enrichment medium, later being in the Order Rhizobiales. Three isolates were named as Asp, 12 isolates from Azotobacter identified as Bacillus and Paenibacillus polymyxa enrichment medium, later being named as Azt and strains in the Order Bacillales. Two isolates 12 isolates from Beijerinckia enrichment medium, belonged to Serratia marcescens and Klebsiella later being referred as Bei. Among 34 isolates stains in the Order Enterobacteriales. The last

Vol. 43, No. 4, 2010 Isolation and screening of growth-promoting activities of rhizobacteria 221

two isolates were identical with Alcaligenes faecalis 2.5 Rice isolates )

-1 Vetiver isolates and Azospirillum sp.

2.0 Discussion protein h protein -1 1.5 mg 4

H Extensive research has demonstrated the 2 1.0 potential of PGPR in plant growth improvement (Govindarajan et al., 2006; Cakmakci et al., 2007). .5 The abilities of PGPR bacterial isolates on nitrogen ARA (nmole C ARA (nmole 0.0 fixing (Malik et al., 1997; Govindarajan et al., 0 10 20 30 40 50 60 70 80 2007) and the production of plant growth Isolate Number promoting substance (Khalid et al., 2004; Ahmad et Figure 1 itrogenase (ARA) activity of PGPR isolates al., 2008) have been well documented and were from the first screening. Values were the means of focused in this study. Many varieties of PGPR triplicate measurements. which have nitrogen fixing ability, can be isolated from rhizosphere soils, rhizoplane or from inside plant tissues of Gramineae plants using an 120 Rice isolates Vetiver isolates appropriate N-free medium (Muthukumarasamy et 100 al., 2007; Park et al., 2007; Ahmad et al., 2008).

80 Identification of new isolates based on phenotypical

protein) and physiological criteria however is difficult, if the -1 60 features displayed by a particular isolate are not g mg µ 40 fully identical with a described species. Thus the

IAA ( IAA molecular based method, 16S rDNA sequence 20 analysis, was therefore chosen to identify the 0 selected isolates. The sequencing results indicated 0 10 20 30 40 50 60 70 80 that no prospective species could be recovered from Isolate Number the appropriate enrichment media and only one Figure 2 IAA production of PGPR isolates from the first Azospirillum isolate was obtained and verified. screening. Values were the means of triplicate With the limitation of culture-based isolation, non- measurements. specific microbes can be occasionally recovered

from N-free media as reported by other authors mentioned later. Since malate is a preferable

) carbon source for Azospirillum, therefore semi- -1 3.0 ARA 40

h solid malate medium is used for Azospirillum

-1 IAA 2.5 isolation. However, it is not necessarily specific for cfu 6 30 Azospirillum. van Berkum (1980) found that the 2.0 10 protein) 4 other nitrogen fixers such as Azotobacter paspapi -1 H 2 1.5 20 and Klebsiella pneumoniae are able to grow and g mg

1.0 µ reduce C2H2 in this medium. It has been reported 10 that non-nitrogen fixing bacteria could be obtained 0.5 ( IAA from N-free media also. By using N-free medium, modified Rennie medium, Elbeltagy et al., (2001) ARA ( nmole C nmole ARA ( 0.0 0 Asp Azt Bei obtained diazotroph isolates from rice which were

showed ARA and were closely related to Ideonella. Figure 3 The average nitrogenase (ARA) and IAA A bacterium in this genus is capable of growing production activities of bacterial isolates from the anaerobically with chlorate as an electron accepter individual enrichment methods in the second screening and any ability to fix nitrogen is not yet reported

(Malmqvist et al., 1994). Those reports go along

222 C. Bhromsiri and A. Bhromsiri Thai Journal of Agricultural Science

Table 1 Nitrogenase (ARA) and IAA production activities of the selected PGPR isolates from the first screening.

Nitrogenase activity1/ IAA production1/ Bacterial isolate -1 -1 -1 (nmole C2H4 mg protein h ) (μg IAA mg protein) 1 AspR1 (rice) 0.71±0.06efg 22.01±5.12f-i 2 AspR2 (rice) 1.07±0.40bcd 21.29±4.27f-i 3 AspAK6 (vetiver) 1.23±0.06b 93.96±6.55a 4 AspR3 (rice) 2.15±1.08a 99.59±41.77a 5 AspAK3 (vetiver) 0.02±0.00mn 32.14±10.59c-h 6 AspR4 (rice) 0.02±0.01mn 24.67±7.51e-i 7 AspR5 (rice) 0.84±0.58cde 18.80±6.89f-i 8 AspR6 (rice) 1.16±0.78bc 12.47±4.83f-i 9 AspR7 (rice) 0.44±0.04f-l 74.98±19.50ab 10 AspAK2 (vetiver) 0.54±0.19e-h 0.60±0.22i 11 AspAK5 (vetiver) 0.34±0.09h-n 1.29±0.14ni 12 AspR8 (rice) 0.22±0.03h-n 21.28±6.25f-i 13 AspAK1 (vetiver) 0.21±0.02h-n 24.60±14.20e-i 14 AspR9 (rice) 0.12±0.03j-n 19.60±5.96f-i 15 AspR10 (rice) 0.17±0.07i-n 19.95±4.24f-i 16 AspR11 (rice) 0.17±0.03i-n 19.64±4.96f-i 17 AspR12 (rice) 0.46±0.19f-k 81.35±20.78ab Total average 0.20 15.67 1/ Values are the means of triplicate measurements ± standard deviation. The values marked with the different letter were significantly different (P<0.05).

Table 2 Nitrogenase (ARA) and IAA production activities of the selected PGPR isolates from the second screening.

Nitrogenase activity1/ IAA production1/ Bacterial isolate -1 -1 -1 (nmole C2H4 mg protein h ) (μg IAA mg protein) 1 AspKTL1 (vetiver) 8.84±1.68 34.56±1.05 2 AspKTL2 (vetiver) 7.43±1.51 118.02±7.29 3 AztKTL (vetiver) 0.24±0.16 0.84±0.08 4 AztNPY (vetiver) 0.22±0.04 5.73±0.52 5 BeiKTL5 (vetiver) 1.74±0.49 0.22±0.07 6 BeiKTL6 (vetiver) 2.86±1.55 nd 7 BeiCHP (vetiver) 2.11±1.03 nd 8 BeiNPY (vetiver) 0.92±0.12 nd 1/ Values are the means of triplicate measurements ± standard deviation. nd = Not detected.

with our results and concern about the isolation of semisolid LGIP medium (Barraquio et al., 1997). nitrogen fixing bacteria from N-free media. Due to On the other hand, Muthukumarasamy et al. (2007) the diversity of microbial populations which were successfully screened G. diazotrophicus from dependent on the individual environment, it should Korean wetland rice using the same N-free be noticed that screening method is also medium. It has been reported that Azospirillum influencedby environment and choice of enrichment isolates have been commonly screened from media. None of the isolates of Gluconacetobacter rhizosphere of Thai vetiver root (Siripin, 2000). diazotrophicus have been isolated from wetland The contradictory result was found by Giudice et al. rice in Philippines using ARA positive culture of (2008) in which none of Azospirillum isolates were

Vol. 43, No. 4, 2010 Isolation and screening of growth-promoting activities of rhizobacteria 223

Table 3 Morphological characterization of the selected isolates.

Bacterial Colony morphology Cell shape Gram reaction isolate The first isolation AspR1 Opaque, circular, convex with entire margin Short rods - AspR2 Opaque, circular, convex with entire margin Short rods - AspR3 White, circular, convex with entire margin Short rods - AspR4 White, circular, convex with entire margin Short rods - AspR5 Opaque, circular, convex with entire margin Short rods - AspR6 White, circular, convex with entire margin Short rods - AspR7 White, circular, convex with entire margin Short rods - AspR8 Opaque, circular, convex with entire margin Short rods - AspR9 Opaque, circular, convex with entire margin Short rods - AspR10 Opaque, circular, convex with entire margin Short rods - AspR11 Opaque, circular, convex with entire margin Short rods - AspR12 Opaque, circular, convex with entire margin Short rods - AspAK1 Opaque, circular, convex with entire margin Short rods - AspAK2 Opaque, irregular, convex with entire margin Short rods - AspAK3 White, irregular, flat with undulate margin Short rods - AspAK5 Opaque, circular, convex with entire margin Short rods - AspAK6 White, irregular, flat with undulate margin Short rods - The second isolation AspKTL1 White circular, rise with entire margin Short rods - AspKTL2 Pink, circular, rise with entire margin Short rods - AztKTL White, irregular, mucoid, convex with entire margin Short rods - (positive in very young culture) AztNPY White, circular, mucoid, convex with entire margin Short rods - (positive in very young culture) BeiKTL5 Opaque, circular, mucoid, convex with entire margin Short rods - BeiKTL6 White, circular, mucoid, convex with entire margin Short rods - BeiCHP White, circular, mucoid, convex with entire margin Short rods - BeiNPY White, circular, mucoid, convex with entire margin Short rods -

recovered from culture-based analysis of microbial Jardin, 1989; Berg et al., 1999; Suckstorff and community of vetiver roots conducted in Italy. Berg, 2003; Park et al., 2005; Chowdhury et al., This result is the same as our findings, Azospirillum 2007) and also from cyanobacteria-deprived was not the major microbial group and only one lichenized fungi (Liba et al., 2007). It is not Azospirillum isolate was obtained and identified. defined as nitrogen fixing bacteria despite that their Utilizing molecular approach, the diversity nitrogen fixing activities have been investigated within bacterial isolates was shown and many taxa and confirmed (Park et al. 2005; Liba et al. 2007). were represented. The major bacterial group in this Our findings might be the first report about research belonged to Stenotrophomonas maltophillia, nitrogen fixing and IAA production abilities of S. previously called Pseudomonas maltophillia and maltophillia isolated from vetiver grass (AspAK5) Xanthomonas maltophillia (Palleroni and Bradbury, in Thailand. Moreover, much evidence has 1993). S. maltophillia is ubiquitous in the environment, indicated a potential role of such species in plant often associated with plants and has been isolated growth promotion properties as fungal biocontrol from rhizosphere of grass, wheat, oat , cucumber, (Kai et al., 2007; Wang et al., 2007) and IAA maize, oilseed rape, potato, lettuce (Juhnke and production (Park et al., 2005; Liba et al., 2007).

224 C. Bhromsiri and A. Bhromsiri Thai Journal of Agricultural Science

Table 4 Most related reference sequences with the selected bacterial isolates.

Bacterial Most related reference isolate Alignment isolate (% identity)

The first isolation AspR1 Stenotrophomonas maltophilla (100%) 1309/1309 AspR2 Stenotrophomonas maltophilla (99%) 1357/1361 AspR3 Serratia marcescens strain P1 (99%) 1379/1381 AspR4 Stenotrophomonas maltophilla (99%) 1286/1292 AspR5 Klebsiella sp. Strain zmmo (100%) 1379/1379 AspR6 Agrobacterim tumefaciens strain 2001023242 (100%) 1316/1316 AspR7 Alcaligenes faecalis strain SP03 (99%) 1377/1379 AspR8 Stenotrophomonas maltophilla (99%) 1371/1375 AspR9 Stenotrophomonas maltophilla strain IAM 12423 1284/1305 (98%) AspR10 Stenotrophomonas maltophilla (99%) 1376/1378 AspR11 Stenotrophomonas maltophilla (99%) 1376/1379 AspR12 Aurantimonas altamirensis strain NML 070723 (99%) 1312/1317 AspAK1 Stenotrophomonas maltophilla (99%) 1374/1378 AspAK2 Stenotrophomonas sp. FB206 (99%) 1371/1377 AspAK3 Stenotrophomonas sp. FB206 (99%) 1368/1374 AspAK5 Stenotrophomonas sp. FB206 (99%) 1238/1244 AspAK6 Stenotrophomonas maltophilla (99%) 1284/1287 The second isolation AspKTL1 Agrobacterium tumefaciens AMF2 (100%) 1235/1235 AspKTL2 Azospirillum sp. TS19 (97%) 1194/1220 AztKTL Bacillus subtilis strain BCB19 (99%) 1371/1373 AztNPY Bacillus subtilis strain BCB19 (100%) 1324/1324 BeiKTL5 Rhizobium sp. NY11 (99%) 1241/1246 BeiKTL6 Paenibacillus polymyxa strain GBR-465 (99%) 1397/1407 BeiCHP Uncultured bacterium clone P5D15-621 (98%) 1365/1379 BeiNPY Rhizobium tropici strain CAF344 (99%) 1231/1233

The isolate from rice, AspR5 is phylogenetically et al. (2008) reported that non-pigmented S. close to Alcaligenes faecallis. A. faecallis is common marcescens was isolated and verified from vetiver. in paddy soil and colonize rice root endophytically Furthermore, the phosphate solubilization property (You and Zhou, 1989). It has been shown to express and the presence of the nitrogen fixation gene of S. nifH and recently reclassified as Pseudomonas marcescens were demonstrated (Liba et al., 2007). stuzeri (Vermeiren et al., 1999). Nitrogen fixing Both previous reports were similar to this research activity of A. faecallis isolate was observed also by which found that non-pigmented S. marcescens was ARA in this work. The AspAK6 and AspAK3 isolated from vetiver and its the nitrogen fixing isolates belong to Serratia marcescens and Klebsiella ability was confirmed. Due to chitinase activity pneumoniae within the order Enterobateriales, (McInroy et al., 1995; Kalbe et al., 1996) inducing respectively. Diverse species of Serratia have been systemic resistance in plants (Press et al., 1997), S. isolated from cotton and sweet corn (McInroy et al., marcescens has been also used as biocontrol agent. 1995), rice rhizosphere (Rosales et al., 1993) and Klebsiella pneumoniae was reported as an rice seed (Mukhopadhyay et al., 1996). It has been endophytic bacteria in maize (Chelius and Triplett, also found as an endophytic colonizer of rice 2000). In wheat, Iniquez et al. (2004) demonstrated (Gyaneshwer et al., 2001; Tan et al., 2001). Giudice and confirmed the nitrogen fixing activity of

Vol. 43, No. 4, 2010 Isolation and screening of growth-promoting activities of rhizobacteria 225

AspR7 100 Stenotrophomonas maltophilia strain IAM 12423 (AB294553) Stenotrophomonas maltophilia strain M5-1 (AY880274) Stenotrophomonas maltophilia (DQ777866) AspR5 AspR12

84 AspAK3 AspR6

AspR2 γ proteobacteria AspAK1 Order Xanthomonadales 100 AspAK2 AspR1 Xanthomonas sp. BJQ-H4 (FJ600362) 100 BeiCHP 95 Uncultured bacterium clone P5D15-621 (EF511543) Stenotrophomonas sp. FB206 (AY259519) 77 AspR10 98 AspR11 91 AspR9 Burkholderia vietnamiensis (AF043302) β proteobacteria AspR5 100 Order Burkhoderiales 99 Uncultured beta proteobacterium clone 20F1 (DQ366010) 100 Alcaligenes faecalis strain SP03 (EF427887) 76 Pseudomonas stutzeri (AF143245) γ proteobacteria 100 Pseudomonas fluorescens strain P21(FJ605510) Order Pseudomonales Azotobacter chroococcum strain AM12A (AB430880) 100 AspAK6 94 Serratia marcescens strain P1 (EU031439) γ proteobacteria 100 AspAK3 Order Enterobateriales Klebsiella pneumoniae (AB368777) 100 Klebsiella sp. strain zmmo (U31075) AztNPY

100 AztKTL Bacillus subtilis strain BCB19 (EU826024) Endosporobateria 100 Bacillus amyloliquefaciens strain SB177 (FJ608706) Order Bacillales Paenibacillus polymyxa strain JSa-9 (EU882855) BeiKTL6 100 Paenibacillus polymyxa strain GBR-465 (AY359623) Azospirillum sp. TS19 (AB114197) α proteobacteria 100 Azospirillum brasilense strain ATCC 29145 Order Rhodospirillales (AspKTL2AY324110 )

100 Beijerinckia indica subsp. indica ATCC 9039 (NC_010581) 100 Beijerinckia mobilis (AB119200) 100 AspR8

93 Aurantimonas altamirensis strain NML 070723 (EU442517) 99 AspR4 α proteobacteria 100 Agrobacterium tumefaciens strain 2001025242 (AY513492) 90 Order Rhizobiales AspKTL1

96 Agrobacterium tumefaciens strain AFM2 (EU592041) BeiNPY 91 100 Rhizobium tropici strain CAF344 (FJ405376) Rhizobium tropici strain rif200849 (FJ527674) 100 BeiKTL5 99 Rhizobium sp. NY11 (EU826645)

0.02

Figure 4 Phylogenetic relationship based on neighbor-joining analysis of 16S rDNA sequence data from the selected isolates. Bootstrap analysis was made with 1000 cycles and only bootstrap values of 50% are showed. The scale bar represents percentage of substitutions.

K. pneumoniae 342. Besides that the production of The BeiNPY and BeiKTL5 isolates are identified IAA and related compound by K. pneumoniae in as Rhizobium. The beneficial effect of inoculating culture media supplement with tryptophan was legumes with rhizobia is well known and the found (El-Khawas and Adachi, 1999). The research has shown that these bacteria have the nitrogen fixing and IAA production activities of K. potential to be used as plant growth promoting pneumoniae isolates were again confirmed by our rhizobacteria with non-legume plants as rice also work. (Yanni et al. 1997). Besides rice, rhizobia have

226 C. Bhromsiri and A. Bhromsiri Thai Journal of Agricultural Science been isolated as natural endophytes from other non- plants. This bacterium has been isolated as an legumes species such as cotton, sweet corn endophytes from many species of agronomic crops (McInroy and Kloepper, 1995), maize (Gutierrez- and Gramineae plants (Zinniel et al., 2002; Zamora and Martinez-Romero, 2001), banana Chowdhury et al., 2007). Beside its phytopatho- (Martinez et al., 2003) perennial grass (Chowdhury genicity, the potential benefit as PGPR of et al., 2007) and this work that might be the first Agrobacterium species was indicated. The phosphate report of the rhizobia isolated from vetiver grass. solubilization (Hameed et al., 2004) and IAA Rhizobia also had the growth promoting activities production of Agrobacterium strains (Barazani and for example the production of phytohormone and/or Friedman, 1999; Hameed et al., 2004) have been phosphate solubilizing activities (Chabot et al. reported. The later property was also found in this 1996; Antoun et al. 1998). Furthermore, the study. The species A. altamirensis was first isolated inhibition of fungal growth (Nautiyal, 1997; Akhtar in the subterranean environment of the Altamira and Siddiqui, 2008) and the antagonism of cave (Cantabria, Spain) from a white microbial indigenous soil microflora (Schloter et al., 1997) growth on the walls of the cave (Jurado et al., abilities of them were shown. 2006). Recently, it was recovered from human The AztNPY and AztKTL isolates were clinical material (Luong et al., 2008) and in this phylogenetically shown to be Bacillus subtilis and study one isolate of A. altamirensis from rice BeiKTL6 is Paenibacillus polymyxa. Bacillus and rhizosphere soil was identified. Paenibacillus have been commonly found in Although the standard practice of the ARA has rhizosphere of many types of grass (Ding et al., been established, slightly different conditions of 2005; Beneduzi et al., 2008). The nitrogen fixation measurement technique have been adopted for ability of some Paenibacillus species have been individual usage. The differences of media and described and confirmed (von der Weid et al., incubation time have led to the contradictory results 2002; Ma et al., 2008). On the other hand, the nifH on nitrogenase activity in pure culture under genes have been detected in some species of non- various conditions. Therefore, it is rather difficult to fixing bacilli also such as B. cereus, B. marisflavi compare the nitrogenese activity values among the and B. megaterium (Ding et al., 2005). Those studies. For example, Han and New (1998) reported works go along with our results which found that that Azospirillum isolates had a wide range of -1 the low value of ARA activities could be detected nitrogenase activities (0.0-154.9 nmole C2H4 mg in non-fixing bacilli, our two B. subtilis isolates. protein h-1) while that Malik et al. (1997) found a -1 The further test as nifH genes detection should be high nitrogenase activity (686 nmole C2H4 mg done and confirmed about this. In addition, due to protein h-1). In this work, a low nitrogenase activity 6 -1 -1 phytohormone production (Timmusk et al., 1999), (7.43 nmole C2H4 10 cfu h ) of Azospirillum phosphate solubilizing (Vazquez et al., 2000; isolate was examined. In another case with the Canbolat et al., 2006) and biocontrol properties studies of S. maltophilia isolates, the differences (Kloepper et al., 2004; McSpadden-Gardener, between the slightly high ARA values (153.33 and -1 -1 2004) many strains of Bacillus have also been 167.07 C2H4 mg protein h ) (Park et al., 2005) widely used as microbial inoculum for improving and the low nitrogenase activities (0.44-0.71 C2H4 plant growth and/or biocontrol agents in trials with mg-1 protein h-1) (Liba et al., 2007) and also this various plant species such as wheat and spinach study with the low nitrogenase activities (0.21-2.15 -1 -1 (Cakmakci et al., 2007), strawberry (Vestberg et al., C2H4 mg protein h ) were shown. The ARA of 2004) and tomato (Mena-Violante and Olalde- pure culture is commonly performed with a Portugal, 2007). favorable carbon source and the evidences in this Two isolates, AspR4 and AspKTL1 showed study which showed that most isolates were close similarity with Agrobacterium tumefaciens obtained from inappropriate media. The low and the AspR8 was shown to be Aurantimonas nitrogenase activity presence in this work thus altamirensis. A. tumefaciens is a soil bacterium might be caused by using an inappropriate carbon known for its phytopathogenicity, causing crown source. Furthermore, much evidence has shown gall disease in a wide variety of dicotyledonous that the nitrogen fixing ability of PGPR does not

Vol. 43, No. 4, 2010 Isolation and screening of growth-promoting activities of rhizobacteria 227 correlate well with nitrogen uptake, growth or rhizobacteria on non-legumes: effect on radishes yields of host plants (Lindberg et al., 1985; Will (Raphanus sativus L.). Plant Soil 204: 57-67. Atlas, R.M. 1993. Handbook of Microbiology Media. and Sylvia, 1990). The plant growth promotion CRC Press, Boca Raton. effects of PGPR might pass though the Baldani, J.I., N.R. Krieg, V.L.D. Badani, A. Hartmann, phytohormone producing property rather than and J. Dobereiner. 2005. Genus Azospirillum, pp. 7- nitrogenase activity as described by several 26. In: D.J. Brenner, N.R. Krieg, and J.T. Staley, eds. publications (Biswas et al., 2000a; Biswas et al., Bergey’s Manual of Systematic Bacteriology Second Edition Volume Two, The Proteobacteria. Part C The 2000b). In this work, IAA production was another Alpha-, Beta-, Delta- and Epsilonproteobacteria. expected feature for screening the PGPR isolates Springer, New York. and most of selected isolates were found to have Barazani, O. and J. Friedman. 1999. Is IAA the major this property. root growth factor secreted from plant-growth- mediating bacteria? J. Chem. Ecol. 25: 2397-2406. In summary, even though the prospective Barraquio, W.L., L. Revilla and J.K. Ladha. 1997. species could not be recovered from enrichment Isolation of endophytic diazotrophic bacteria from methods, nevertheless with the potential of 16S wetland rice. Plant Soil 194: 15-24. rDNA sequencing analysis, the diversity of bacteria Beneduzi, A., D. Peres, P.R. de Costa, Zanettini, M.H.B. isolated from rice and vetiver rhizosphere soils was and L.M.P. Passaqlia. 2008. Genetic and phenotypic diversity of plant-growth-promoting bacilli isolated shown. Their nitrogen fixing and/or phytohormone from wheat fields in southern Brazil. Res. Microbiol. producing activities were also found. Moreover, the 159: 244-250. other plant growth promotion properties such as Berg, G., N. Roskot and K. Smalla. 1999. Genotype and phosphate solubilizing and biocontrol properties of phenotype relationships between clinical and environmental isolates of Stenotrophomonas maltophilia. almost all isolates have been well documented. It is J. Clin. Microbiol. 37: 3594-3600. therefore probable that they can be used as bacterial Bhromsiri, C. 2009. Use of soil microbial inoculation for inocula to support growth and development of improving the effectiveness of vetiver grass and the vetiver plants in the further pot and field effect on natural soil microbial ecology. Ph.D. Thesis experiments (Bhromsiri, 2009). Chiang Mai University. Biswas, J.C., J.K. Ladha and F.B. Dazzo. 2000a. Rhizobia inoculation improves nutrient uptake and growth of Acknowledgments lowland rice. Soil. Sci. Soc. Am J. 64: 1644-1650. Biswas, J.C., J.K. Ladha, F.B. Dazzo, Y.G. Yanni and This work was supported by The Thailand B.G. Rolfe. 2000b. Rhizobial inoculation influences seedling vigor and yield of rice. Agron J. 92: 880- Research Fund through the Royal Golden Jubilee 886. Ph.D. Program (Grant no. PHD/0079/2546) and Cakmakci, R., M. Erat, U. Erdogan and M.F. Donmez. Petroleum Authority of Thailand (PTT) Public 2007. The influence of plant growth-promoting Company Limited through Royal Project Foundation. rhizobacteria on growth and enzyme activities in wheat and spinach plant. J. Plant. Nutr. Soil Sc. 170: 288-295. References Canbolat, M.Y., S. Bilen, R. Cakmakci, F. Sahin and A. Aydin. 2006. Effect of plant growth-promoting Adams, R.P., M. Zhong, Y. Turaspekov, M.R. Dafforn, bacteria and on barley seedling, and J.F. Veldkamp, 1998. DNA fingerprinting reveals growth, nutrient uptake, soil properties and clonal nature of Vetiveria zizanioides (L.) Nash rhizosphere microflora. Biol. Fert. Soils. 42: 350- graminaeae and sources of potential new germplasm. 357. Mol. Ecol. 7: 813-818. Cazzuffi, P., A. Cornor and E. Crippa. 2006. Stabilization Ahmad, F., I. Ahmad and M.S. Khan. 2008. Screening of by perennial “graminese” in Southern Italy: plant free-living rhizosphereric bacteria for their multiple growth and temporal performance. Geotech. Geol. growth promoting activities. Microbiol. Res. 163: Eng. 21: 429-447. 173-181. Chabot, R., H. Antoun and M.P. Cescas. 1996. Growth Akhtar, M.S. and Z.A. Siddiqui. 2008. Biocontrol of a promotion of maize and lettuce by phosphate- root-rot disease complex of chickpea by Glomus solubilizing Rhizobium leguminosarum biovar. intraradices, Rhizobium sp. and Pseudomonas phaseoli. Plant Soil. 184: 311-321. straita. Crop Prot. 27: 410–417. Chelius, M.K. and E.W. Triplett. 2000. Immunolocali- Antoun, H., C.J. Beauchamp, N. Goussard, R. Chabot, zation of dinitrogenase reductase produced by and R. Lalande. 1998. Potential of Rhizobium and Klebsiella pneumoniae in association with Zea may Bradyrizobium species as plant growth promoting L. Appl. Environ. Microbiol. 66: 783-787.

228 C. Bhromsiri and A. Bhromsiri Thai Journal of Agricultural Science

Chowdhury, S.P., M. Schmid, A. Hartmann, and K. Hengchaovanich, D. 1998. Vetiver grass technology for Tripathi. 2007. Identification of diazotrophs in the mine stabilization and . Pacific Rim culturable bacterial community associated with roots Vetiver Network Technical Bulletin No.1998/2. of Lasiurus sindicus, a perential grass of Thur desert, Office of the Royal Development Projects Board, India. Microb. Ecol. 54: 82-90. Bangkok. Ding, Y., J. Wang and S. Chen. 2005. Isolation and Iniguez, A.L., Y. Dong and E.W. Triplett. 2004. Nitrogen identification of nitrogen-fixing bacilli from plant fixation in wheat provided by Klebsiella pneumoniae rhizospheres in Beijing region. J. Appl. Microbiol. 342. Mol Plant Microbe In. 17: 1078-1085. 99: 1271-1281. Juhnke, M.E. and E.D. Jardin. 1989. Selective Medium Dobbelaere, S., J. Vanderleyden and Y. Okon. 2003. for isolation of Xanthomonas maltophilia from soil Plant growth-promoting effects of diazotrophs in the and rhizosphere environments. Appl. Environ. rhizosphere. Crit. Rev. Plant Sci. 22: 107-149. Microbiol. 55: 747-750. El-Khawas, H. and K. Adachi. 1999. Identification and Jurado, V., J.M. Gonzalez, L. Laiz and C. Saiz-Jimenez. quantification of auxins in culture media of 2006. Aurantimonas altamirensis sp. nov., a member Azospirillum and Klebsiella and their effect on rice of the order Rhizobiales isolated from Altamira cave. roots. Biol. Fert. Soils 28: 377-381. Int. J. Syst. Evol. Micr. 56: 2583-2585. Elbeltagy, A., K. Nishioka, T. H. Suzuki, Sato, B. Ye, T. Kai, M., U. Effmert, G. Berg and B. Piechulla. 2007. Hamada, T. Isawa, H. Mitsui and K. Minamisawa. Volatiles of bacterial antagonists inhibit mycelial 2001. Endophytic colonization and in planta nitrogen growth of the plant pathogen . fixation by a Herbaspirillum sp. isolated from wild Arch. Microbiology.187: 351–360. rice species. Appl. Environ. Microbiol. 67: 5285- Kalbe, C., P. Marten and G. Berg. 1996. Strain of genus 5293. Serratia as beneficial rhizobacteria of oilseed rape Giudice, L.D., D.R. Massardo, P. Pontier, C.M. Bertea, with antifungal properties. Microbiol. Res. 151: 433- D. Mombello, E. Carata, S.M. Tredici, A. Tala, M. 439. Mucciarelli, V.I. Groudeva, M.D. Stefano, G. Kennedy, C. 2005. Genus Beijerinckia, pp. 423-432. In: Vigliotta, M.E. Maffei and P. Alifano. 2008. The D.J. Brenner, N.R. Krieg and J.T. Staley, eds. microbial community of vetiver root and its Bergey’s Manual of Systematic Bacteriology, Second involvement into essential biogenesis. Environ. Edition, Volume Two. The Proteobacteria. Springer, Microbiol. 10: 2824-2841. New York. Gordon, S.A. and R.P. Weber. 1950. Colorimetric Kennedy, C., P. Rundnick, M.L. MacDonald, and T. estimation of indoleacetic acid. Plant Physiol. 26: Meton. 2005. Genus Azotobacter, pp. 384-402. In: 192-195. D.J. Brenner, N.R. Krieg and J.T. Staley, eds. Govindarajan, M., S.W. Kwon and H.Y. Weon. 2007. Bergey’s Manual of Systematic Bacteriology Second Isolation, molecular characterization and growth- Edition, Volume Two. The Proteobacteria, Part B promoting activities of endophytic sugarcane The Gammaproteobacteria. Springer, New York. diazotroph Klebsiella sp. GR9. World J. Microbiol. Khalid, A., M. Arshad and Z.A. Zahir. 2004. Screening Biotechnol. 23: 997-1006. plant growth-promoting rhizobacteria for improving Govindarajan, M., G. Revathi and C. Lakshminara- growth and yield of wheat. J Appl Microbiol. 96: simhan. 2006. Improved yield of micropropagated 473-480. sugarcane following inoculation by endophytic Kloepper, J.W., C.M. Ryu, and S. Zhang. 2004. Induced Burkhoderia vietnamiensis. Plant Soil 280: 239-252. systemic resistance and promotion of plant growth by Greenfield, J.C. 2002. Vetiver Grass An Essential Grass Bacillus spp. Phytopathology 94: 1259-1266. for The Conservation of Planet Earth. Infinity Liba, C.M., F.I.S. Ferraral, G.P. Manfio, F. Fantinatti- Publishing, Pennsylvania. Garboggini, R.C. Albuquerque, Gutierrez-Zamora, M.L. and E. Matinez-Romero. 2001. Lindberg, T., Granhall, U. and K. Tomenius. 1985. Natural endophytic association between Rhizobium Infectivity and acetylene reduction of diazotrophic etli and maize (Zea may L.). J. Biotechnol. 91: 117- rhizosphere bacteria in wheat (Triticum aesivum) 126. seedlings under gnotobic conditions. Biol. Fert. Soil. Gyaneshwar, P., E.K. James, N. Mathan, P.M. Reddy, B. 1: 123-129. Reihold-Hurek and J.K. Ladha. 2001. Endophytic Lowry, O.H., Rosbrough, N.J., Farr, A.L and R.J. colonization of rice by a diazotrophic strains of Randall. 1951. Protein measurement with the Folin Serratia marcescens. J. Bacteriol. 183: 2634-2645. Phenol Reagent. J. Biol. Chem. 193: 267-275. Hameed, S., S. Yasmin, K.A. Malik, Y. Zafar and F.Y. Lucy, M., Reed, E. and B.R. Glick. 2004. Applications Hafeez. 2004. Rhizobium, Bradyrhizobium and of free living plant growth-promoting rhizobacteria. Agrobacterium strains isolated from cultivated A Van Leeuw J. Microb. 86: 1-25. legumes. Biol. Fert. Soils 39: 179-185. Luong, M-L., Bekal, S., Vinh, D.C., Lauzon, D., Leung, Han, S.O. and P.B. New. 1998. Variation of nitrogen V., Al-Rawahi, G.N., Ng, B., Bursz, T. and K. fixing ability among natural isolates. Microb. Ecol. Bernard. 2008. First report of isolation and 36: 193-201. characterization of Aurantimonas altamirensis from clinical samples. J. Clin. Microbiol. 46: 2435-2437.

Vol. 43, No. 4, 2010 Isolation and screening of growth-promoting activities of rhizobacteria 229

Ma, W.K., Siciliano, S.D. and J.J. Germida. 2005. A Palleroni, N.J. and J.F. Bradbury. 1993. Stenotrophomonas, PCR-DGGE methods for detection arbuscular a new bacterial genus for Xanthomonas maltophilia mycorrhizal fungi in cultivated soils. Soil. Biol. (Huge 1980) Swing et al. 1983. Int. J. Syst. Bacteriol. Biochem. 37:1589-1597. 43: 606-609. Malik, K.A., Bilal, R., Mezhnez, S., Rasul, G., Mirza, Park, M., C. Kim, J. Yang, H. Lee, W. Shin, S. Kim and M.S. and S. Ali. 1997. Association of nitrogen- T. Sa. 2005. Isolation and characterization of diazo- fixing, plant-growth-promoting rhizobacteria (PGPR) trophic growth promoting bacteria from rhizosphere with kallar grass and rice. Plant Soil. 194: 37-44. of agricultural crops of Korea. Microbiol. Res. 160: Malmqvist, A., Welander, T., Moore, E., Ternstrom, A., 127-133. Molin, G. and L. Stenstrom. 1994.Ideonella Polyanskaya, L.M., O.T. Vedina, L.V. Lysak and D.G. dechloratans, gen. nov., sp. nov., a new bacterium Zvyagintsev. 2002. The growth-promoting effect of capable of growing anaerobically with chlorate as an Beijerinckia mobillis and Clostridium sp. cultures on electron acceptor. Syst. Appl. Microbiol. 17: 58-64. some agricultural crops. Microbiology. 71: 109-115. Martinez, L., Caballero-Mellado, J., Orozco, J. and E. Press, C.M., M. Wilson, S. Tuzun and J.W. Kloepper. Martinez-Romero. 2003. Diazotrophic bacteria 1997. Salicylic acid produced by Serratia marcescens associated with banana (Musa spp.). Plant Soil. 90-166 is not the primary determinant of induced 257:35-47. systematic resistance in cucumber of tobacco. Mol. McInroy, J.A. and J.W. Kloepper. 1995. Survey of Plant. Microbe. In. 10: 761-768. indigenous bacterial endophyes from cotton and Randriamiharisoa, R., J. Barison and N. Uphoff. 2006. sweet corn. Plant Soil. 173: 337-342. Soil biological contributions to the System of Rice McSpadden-Gardener, B.B. 2004. The nature and Intensification, pp. 409-424. In: N. Uphoff, A. Ball, application of biocontrol microbes:Bacillis spp. E.C.M. Fernandes, H. Herren, O. Husson, M. Laing, ecology of Bacillus and Paenibacillus spp. in C.A. Palm, J. Pretty, P.A. Sanchez, N. Sanginga and agricultural systems. Phytopathology. 94: 661-664. J. Thies, eds. Biological Approaches to Sustainable Mena-Violante, H.G. and V. Olalde-Portugal. 2007. Soil System. CRC Press, Boca Raton. Alteration of tomato fruit quality by root inoculation Reinhold-Hurek, B. and T. Hurek. 1997. Azoarcus spp. with plant growth-promoting rhizobateria (PGPR): and their interaction with grass roots. Plant Soil 194: Bacillus subtilis BEB-13bs. Sci Hortic-Amsterdam. 57-64. 113: 103-106. Rosales, A.M., R. Vantomme, J. Swings, J. de Lay and Mukhopadhyay, K., N.K. Garrison, D.M. Hinton, C.W. T.W. Mew. 1993. Identification of some bacteria Bacon, G.S. Khush, H.D. Peck and N. Datta. 1996. from paddy antagonistic to several rice fungal Identification and characterization of bacterial pathogen. J. Phytopathol. 138: 189-208. endophytes of rice. Mycopathologia. 134: 151-159. Rothballer, M., M. Schmid and A. Hartmann. 2009. Mucciarelli, M., C.M. Bertea, M. Cozzo, S. Scannerin Diazotrophic bacterial endophytes in Gramineae and and M. Gallino. 1998. Vetiveria zizaniodes as a tool other plants, pp. 273-302. In: A. Steinbuchel, ed. for environmental engineering. Acta Hortic. 457: Microbiology Monographs volume 8, Prokaryotic 261-270. symbionts in plants. Springer-Verlag, Berlin. Muthukumarasamy, R., U.G. Kang, K.D. Park, W.T. Saitou, N. and M. Nei, 1987. The neighbor-joining Jeon, C.Y. Park, Y.S. Cho, S.W. Kwon, J. Song, method: a new method for reconstructing phylogenetic D.H. Roh and G. Revathi. 2007. Enumeration, trees. Mol. Biol. Evol. 4: 406-425. isolation and identification of diazotrophs from Schloter, W., W. Wiehe, B. Assmus, H. Steindl, H. Korean wetland rice varieties grown with long-term Becke, G. Hoflich and A. Hartmann. 1997. Root application of N and compost and their short-term colonization of different plants by plant-growth- inoculation effect on rice plants. J. Appl. Microbiol. promoting Rhizobium leguminosarum bv. trifolii R39 102: 981-991. studied with monospecific polyclonal antisera. Appl. Nautiyal, C.S. 1997. Rhizosphere competence of Environ. Microbiol. 63: 2038-2046. Pseudomonas sp. NBRI9926 and Rhizobium sp. Siripin, S. 2000. Microbiology associated with the NBI9513 involved in the suppression of chickpea vetiver plant, pp. 374-376. In: N. Chomchalow, and (Cicer arietinum L.) phathogenic fungi. FEMS M. Barang, eds. Proceedings of the Second Microb. Ecol. 23: 145-158. International Conference on Vetiver (ICV–2). Office Naveerat, P. 2005. Availabilities of primary plant of the Royal Development Projects Board, Bangkok. in soil and plant nutrient uptake by some Somasegaran, P. and H.J. Hoben. 1994. The acetylene rice varieties under system of rice intensification and reduction assay for measuring nitrogenase activity, different fertilizer management. M.S. Thesis. Chiang pp. 392-398. In: P. Somasegaran and H.J. Hoben, Mai University (in Thai). eds. Handbook for Rhizobia. Springer-Verlag, New Okon, Y. and C.A. Labandrera-Gonzalez. 1994. Agronomic York. applications of Azospirillum : an evaluation of 20 Srivastava, J., H. Chandra and N. Singh. 2007. Allelopathic years of worldwide field inoculation. Soil. Biol. response of Vetiveria zizaniodes (L.) Nash on Biochem. 26: 1591-1601. member of the family Enterobacteriaceae and Pseudomonas spp. Environmentalist. 27: 253-260.

230 C. Bhromsiri and A. Bhromsiri Thai Journal of Agricultural Science

Stein, T., N. Hayen-Schneg and I. Fendrik. 1997. Veldkamp, J.F. 1999. A revision of Chrysoposon Trin. Contribution of BNF by Azoarcus sp. BH72 in including Vetiveria Bory () in Thailand a vulgare. Soil. Biol. Biochem. 29: 969-971. Malesia with notes some other species from Africa Suckstorff, I. and G. Berg. 2003. Evidence for dose- and Australia. Austrobaileya. 5. dependent effects on plant growth by Stenotropho- Vermeiren, H., A. Willlems, G. Schoofs, R. de Mot, V. monas strains from different origins. J. Appl. Keijer, W. Hei and J.Venderleyden. 1999. The rice Microbiol. 95: 656–663. inoculant strain Alcaligenes faecalis A15 is a Tamura, K., Dudley, J., Nei, M. and S. Kumar. 2007. nitrogen-fixing Pseudomonas stutzeri. Syst. Appl. MEGA4: molecular evolution genetics analysis Microbiol. 22: 215-224. (MEGA) software version 4.0. Mol. Biol. Evol. 24: Vestberg, M., S. Kukkonen, K. Saari, P. Parikka, J. 1596-1599. Huttunen, L. Tainio, N. Devos, F. Weekers, C. Tan, Z., T. Hurek, P. Gyaneshwar, J.K. Ladha and B. Kevers, P. Thonart, M.C. Lemonine, C. Cordier, C. Reinhold-Hurek. 2001. Novel endophytes of rice Alabouvette and S. Gianinazzi. 2004. Microbial form a taxonomically distinct subgroup of Serratia inoculation for improving the growth and health of marcescens. Syst. Appl. Microbiol. 24: 245-251. micropropagated strawberry. Appl Soil Ecol. 27: Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin 243-258. and D.G. Higgins. 1997. The ClustalX windows Von Der Weid, I., G.F. Duarte, J.D. Van Elsas and L. interface: flexible strategies for multiple sequence Seldin. 2002. Paenibacillus brasilensis sp. nov. a alignment aided by quality analysis tools. Nucleic novel nitrogen-fixing species isolated from maize Acids Res. 25: 4876-4882. rhizophere in Brazil. Int. J. Syst. Evol. Micr. 52: Thuler, D.S., E.I.S. Floh, W. Handro and H.R. Barbosa. 2147-2153. 2003. Beijerinckia derxii releases plant growth Wang, Y.S., Y.G. Zheng, and Y.C. Sen. 2007. Isolation regulators an amino acids in synthetic media and identification of a novel valienamine producing independent of nitrogenase activity. J. Appl. bacterium. J. Appl. Microbiol. 102: 838-844. Microbiol. 95: 799-806. Will, M.E. and D.M. Sylvia. 1990. Interaction of Timmusk, S., B. Nicander, U. Granhall and E. Tillberg. rhizosphere bacteria, fertilizer and vesicular- 1999. Cytokinin production by Paenibacillus polymyxa. arbuscular mycorrhizal fungi with sea oats. Appl. Soil. Biol. Biochem. 31: 1847-1852. Environ. Microbiol. 56: 2073-2079. Uphoff, N. 2006. The system of rice intensification Wong, C.C., S.C. Wu, C. Kuek, A.G. Khan and M.H. (SRI) as a methodology for reducing water Wong. 2007. The role of mycorrhizae associated with requirement in irrigated rice production. Paper for vetiver grown in Pb- / Zn- contaminated soils : green International Dialogue on Rice and Water: Exploring house study. Restor Ecol. 15: 60-67. Options for Food Security and Sustainable Yanni, Y.G., R.Y. Rizk, V. Corich, A. Squartini, K. Environments, Los Banos, Philippines; Available via Ninke, S. Phillip-Hollingsworth, G. Orgambide, F. de DIALOG http: //ciifad.cornell.edu/sri/conferences/ Bruijn, J. Stoltzfus, D. Buckley, T.M. Schmidt, P.F. index.html#wwfirri. Mateos, J.K. Ladha and F.B. Dazzo. 1997. Natural Van Berkum, P. 1980. Evaluation of acetylene reduction endophytic association between Rhizobium legumi- by excised roots for the determination of nitrogen nosarum bv. trifolii and rice and assessment of its fixation in grasses. Soil. Biol. Biochem. 12: 141-145. potential to promote rice growth. Plant Soil 194: 99- Van, V.T., O. Berge, S.N. Ke, J. Balandreau and T. 114. Heulin. 2000. Repeated beneficial effects of rice You, C. and F. Zhou. 1989. Non-nodular endorhizo- inoculation with a strain of Burkhoderia vietnamiensis sphereic nitrogen fixation in wetland rice. Can. J. on early and late yield components in low fertility Microbiol. 35: 403-408 sulphate acid soils of Vietnam. Plant Soil 218: 273- Zinniel, D.K., P. Lambrecht, N.B. Harris, Z. Feng, D. 284. Kuczmarski, P. Higley, C.A. Ishimaru, A. Arunakumari, Vasquez, M.M., S. Cesar, R. Azcon and J.M. Barea. R.G. Barletta and K. Vidaver. 2002. Isolation and 2000. Interactions between arbuscular mycorrhizal characterization of endophytic colonizing bacteria fungi and other microbial inoculants (Azospirillum, from agronomic crops and prairie plants. Appl. Pseudomonas, Trichoderma) and other effects on Environ. Microbiol. 68: 2198-2208. microbial population and enzyme activities in the rhizosphere of maize plants. Appl. Soil. Ecol. 15: 261-272. Manuscript received 5 October 2010, accepted 25 January 2011

Now online at http://www.thaiagj.org