Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

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Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars Sang Hye Ji a , Mayank Anand Gururani b , Se-Chul Chun a,∗ a b

Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea Subtropical Horticulture Research Institute, Faculty of Biotechnology, Jeju National University, Jeju 690-756, Republic of Korea

a r t i c l e

i n f o

Article history: Received 16 February 2013 Received in revised form 9 June 2013 Accepted 9 June 2013 Available online xxx Keywords: Nitrogen-fixing Plant growth-promoting bacteria (PGPB) Endophytic Diazotrophic bacteria Siderophore

a b s t r a c t We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants. © 2013 Elsevier GmbH. All rights reserved.

1. Introduction Soil is replete with microscopic life forms including bacteria, fungi, nematodes, and algae. Over 95% of the bacteria exist in the plant roots and those plants obtain many nutrients through the soil bacteria. Nitrogen is used to synthesize plant proteins and nucleic acids, including DNA. Although, it is found naturally in the atmosphere, it cannot be used by the plants in the available form (N2 ). Nitrogen can be combined chemically with oxygen or hydrogen to form various nitrogenous compounds that plants can use. These nitrogenous compounds can then be added to the soil in the form of ammonium (NH4 + ) and nitrate (NO3 + ) fertilizer. Especially, the use of nitrogen fertilizer is of great importance in rice production, as nitrogen is the major factor limiting growth under most conditions (Dawe et al. 2000). Thus, N2 fixation is a prime requisite for plant growth particularly in crops like rice. N2 fixers, also called ‘diazotrophs’ play a critical role in the plant ecosystem by reducing dinitrogen (N2 ) to ammonia (NH3 ) (Dilworth 1974). Previous reports have indicated that diazotrophs show ameliorating effects on nutrient uptake, stress tolerance, phytohormone and vitamin synthesis, inorganic phosphate solubilization and overall plant growth promotion (Sachdev et al. 2009; Martinez-Viveros et al.

∗ Corresponding author. Tel.: +82 2 450 3727; fax: +82 2 450 3726. E-mail addresses: [email protected], [email protected] (S.-C. Chun).

2010; Ali et al. 2010; Bhattacharyya and Jha 2012; Gururani et al. 2012). In addition, diazotrophs are known to counter the detrimental effects that follow with the onset of a pathogen attack. Previous reports suggest that diazotrophs are capable of synthesizing antibiotics; anti-fungal compounds etc., via competing for nutrients by producing siderophores or by triggering the induced systemic resistance (ISR) against pathogens (Dobbelaere et al. 2003). After nitrogen (N), phosphorus (P) is the most limiting nutrient for crop yields, and is particularly essential for rice growth and development. Phosphorus-deficient plants usually show inhibited stem and root development, poor flowering, lack of seed and fruit formation etc., consequently causing degradation in quality and quantity. Notably, the production of nitrogen fertilizers depends on the use of non-renewable resources such as oil, gas, or coal (Stoltzfus et al. 1997). In recent years, the use of bioinoculants composed of diazotrophic bacteria as an alternative to nitrogen fertilizers (Welbaum et al. 2004) has emerged as a promising approach. Nitrogen-fixing bacteria belonging to PGPB (Plant Growth Promoting Bacteria) can fix atmospheric nitrogen and supply it to plants. Here we use the term PGPB as bacteria including diazotrophic bacteria or plant growth-promoting rhizobacteria (PGPR). PGPB can competitively colonize plant root, promote plant growth, and reduce plant diseases. Plant growth-promoting rhizobacteria genera: Bacillus (Idriss et al. 2002), Enterobacter (Gupta et al. 1998) and Corynebacterium (El-Banana and Winkelmann 1988) have been reported to benefit plants by enhancing plant

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growth and improving plant health through various direct and indirect mechanisms. PGPB are commonly used as inoculants for improving the growth and yield of agricultural crops and offers an attractive way to replace chemical fertilizers, pesticides, and supplements (S¸tefan et al. 2008; Ashrafuzzaman et al. 2009; Saharan and Nehra 2011). These bacteria significantly affect plant growth by increasing nutrient uptake, producing biologically active phytohormones and suppressing pathogens by producing antibiotics, siderophores, and fungal cell wall-lysing enzymes (Arora et al. 2001; Persello-Cartieaux et al. 2003; Kuklinsky-Sobral et al. 2004; Frey-Klett et al. 2005; Hameeda et al. 2008). Among these, auxin is one of the most vital hormones, primarily due to its pivotal functions in the initial processes of lateral and adventitious root formation (Gaspar et al. 1996; Idris et al. 2007) and root elongation (Yang et al. 1993). Earlier reports indicate that PGPB may also enhance plant auxin synthesis (Kloepper et al. 2004; Yao et al. 2006). PGPB are known to release metal-chelating substances such as iron-chelating siderophores into the rhizosphere. These siderophore-producing PGPB then influence the uptake by plants of various metals, including Fe, Zn, and Cu (Carrillo-Castaneda et al. 2005; Egamberdiyeva 2007; Dimkpa et al. 2008, 2009; Gururani et al. 2012). Besides, siderophores as determinants of induced systemic resistance also play an important role in protection of crops from plant pathogens (Ramamoorthy et al. 2001; Kirankumar et al. 2008). PGPB could also promote plant growth by suppressing plant pathogens indirectly. This enhanced state of resistance is effective against a broad range of pathogens and parasites, including fungi, bacteria, viruses, nematodes, parasitic plants, and insects (Vauterin and Swings 1997; Murphy et al. 2003; Ryu et al. 2004). In the last few years, the number of PGPB that have been identified has seen a great increase. Species of bacteria like Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus and Serratia have been reported to enhance the plant growth (Kloepper et al. 1989; Okon and LabanderaGonzalez 1994; Glick 1995; Gururani et al. 2012). In a previous report, a nitrogen fixing bacteria, Bacillus megaterium was isolated from maize rhizosphere which did not show any antifungal activity (Liu et al. 2006). In another recent report, Bacillus subtilis was isolated from roots of banana plant and it was concluded that although, B. subtilis is not a nitrogen fixing bacterium, it can be efficiently used in a bio-organic fertilizer against Fusarium wilt (Zhang et al. 2011). To the best of our knowledge, there are no such reports available yet, where nitrogen fixing endophytic bacteria from rice root has shown both growth promotion as well as antifungal activity. In the present study, endopytic diazotrophic bacteria were isolated from various rice cultivars in Korea. The isolates were then identified and characterized for their functional traits associated with plant growth promotion and induced systemic resistance.

2. Materials and methods 2.1. Microorganisms and growth conditions For nitrogen fixing bacteria (NFB), Tryptic soy broth (TSB, Tryptic soy broth 3%; Difco, USA) and nitrogen-free semi-solid media agar plates were used (malic acid 4 g, KH2 PO4 0.48 g, MgSO4 ·7H2 O 0.16 g, NaCl 0.08 g, CaCl2 0.016 g, FeCl3 0.008 g, Na2 MoO4 ·2H2 O 0.0013 g, H3 BO3 0.00224 g, Cu (NO3 )· 3H2 O 0.00093 mg, ZnSO4 ·7H2 O 0.192 mg, morpholinoethane sulfonic acid (MES) 20 mM per liter (pH 6.8). The NFB was grown on TSA agar or in TSB broth medium at 30 ◦ C for 2days. All isolated strains were stored at −70 ◦ C in TSB broth containing 15% (v/v) glycerol.

2.2. Rice sample preparation and isolation of nitrogen-fixing bacteria Leaf, stem and root samples of various rice cultivars (Oryza sativa var. Japonica c.v. Chilbo, Chuchung, Haiami, Ilpum, Migwang, Nampyung, Sechuchung, Wongwang, Hwayung) were collected for isolating nitrogen-fixing bacteria. The endophytic diazotrophic microorganisms from roots, stems, and leaves of rice were isolated using nitrogen free semi-solid media. Plant tissue samples were surface sterilized with 70% ethanol for 1 min and shaken in 1.2% (w/v) NaClO solution for 15 min. Samples were then washed three times with sterile distilled water with shaking (15 min each). Surface sterilized samples were ground with sterilized mortar and pestle, and inoculated on nitrogen free semi-solid agar media. After incubation at 30 ◦ C for 2 days, the inoculants were transferred to fresh nitrogen free media and then incubated at 30 ◦ C for 2 days. The transfer procedure mentioned above was carried out 3–4 times to isolate single colonies (Choi et al. 2003; Lee et al. 2005). Nitrogen fixing bacteria were stored at −70 ◦ C in TSB broth containing 15% (v/v) glycerol. 2.3. Partial identification of nitrogen-fixing bacteria by 16S rDNA sequence analysis 2.3.1. DNA extraction Rice plants selected at the heading stage were dug out from a wetland rice field, and ground to a fine powder in a mortar and pestle. The fine powder was suspended in extraction buffer (100 mM Tris, 100 mM EDTA, 250 mM NaCl, 100 ␮g of proteinase K per ml) supplemented with sarkosyl (1% final concentration) and lysed by incubation at 55 ◦ C for 1 h. Treatment of the lysate with RNase A was followed by chloroform extraction and isopropanol precipitation. Crude DNA was purified by phenol extraction, chloroform extraction, and isopropanol precipitation. 2.3.2. PCR amplification of nifH genes and 16S rDNA sequence analysis The nitrogenase iron protein gene nifH is one of the oldest existing and functioning genes in the history of gene evolution, and the outline of the NifH tree is reported to be largely consistent with the 16S rDNA phylogeny (Young 1992, 1993). So, the existence of the nifH gene could be an indirect evidence for nitrogenase activity of some bacteria. Since nif genes are conserved among a broad spectrum of bacteria, the use of universal primers has enabled the amplification and analysis of nifH sequences from various microorganisms and environmental samples. The primers for PCR amplification used were: 19F (5 ’-GCIWTYTAYGGIAARGGIGG-3 ) and 407R (5 -AAICCRCCRCAIACIACRTC-3 ). PCR fragments (390 bp) of nifH were amplified between nucleotides 19 and 407 (Azotobacter vinelandii M20568 munbering) from rice leaf, stem and root DNA. The PCR reaction conditions with 100 ng of template DNA were: 1 min at 94 ◦ C, 1 min at 50 ◦ C, and 50 s at 72 ◦ C for 30 cycles. Sterile milliQ water was used as negative controls. Bacterial isolates were partially identified by the analysis of 16S rDNA sequence. Specific primers used for PCR were: fD1 (5 -AGAGTTTGATCCTGGCTCAG3 ) and rp2 (5 -ACGGCTACCTTGTTACGACTT-3 ) (Weisburg et al. 1991). PCR mixture contained 50 ng of template DNA, primers of 10 pmol concentration, PCR master mix containing Taq DNA polymerase, dNTPs, Tris–HCl, MgCl2 stabilizer, and tracking dye were used according to the manufacturer’s instructions (TaKaRa bio Inc., Japan). The reaction conditions were, 1 min at 94 ◦ C, 1 min at 55 ◦ C, and 1 min 50 sec at 72 ◦ C for 30 cycles. The expected size of amplicon was 1.4 kb. The amplified fragments were recovered from agarose gel using a gel extraction kit (Solgent, Korea). The nucleotide sequences were then determined through 16S rDNA gene sequencing (Macrogen, Korea). The 16S rDNA gene sequences

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of the bacterial isolates were matched with those from NCBI BLAST search (http://blast.ncbi.nlm.nih.gov/). 2.4. Characterization of isolated endophytic diazotrophic bacteria from various rice cultivars as PGPB 2.4.1. Auxin activity test Auxin, indole-3-acetic acid (IAA), produced by the cultures was estimated by growing the isolates in King B (KB) medium supplemented with l-tryptophan as precursor of IAA (Frey-Klett et al. 2005; Gordon and Weber 1950; Leveau and Lindow 2005). The isolates were incubated in 100 ml of King B broth (Protease peptone no. 3 2%, K2 HPO4 0.115%, MgSO4 ·7H2 O 0.15%, glycerol 1.5%) supplemented with 0.1% (w/v) l-tryptophan and incubated until the OD600 reached at 0.6-0.7 at 30 ◦ C for at least 16 h. The supernatant of the culture fluid was obtained by centrifuge at 4 ◦ C for 15 min at 8000 rpm. Then, Salkowski coloring reagent (35% HClO4 50 ml, 0.5 M FeCl3 1 ml) and the supernatant were mixed in the ratio of 2:1 and left in the dark for 30 min. After the reaction, the absorbance of the mixtures was estimated at 540 nm. The IAA concentration in the culture was estimated based on the IAA standard curve (Merck, Germany) (Lee et al. 2005). 2.4.2. Plate assay for screening of siderophore-producing strains Siderophore production by various putative PGPB isolates was determined following the universal assay of Schwyn and Neilands (Schwyn and Neilands 1987). In this assay, one can identify the siderophore producing bacteria through color change of the blue media. To prepare 1 l of CAS blue agar, 4 kinds of solution were needed (Milagres et al. 1999; Schwyn and Neilands 1987). To prepare solution 1, i.e., the Fe-CAS indicator solution, 60.5 mg CAS (Sigma Aldrich, USA) was dissolved in 50 ml water and mixed with 10 ml iron (III) solution (1 mM FeCl3 • 6H2 O, 10 mM HCl). Under stirring condition, this solution was slowly added to 72.9 mg HDTMA dissolved in 40 ml water. The resultant dark blue liquid was autoclaved and cooled to 50 ◦ C. To prepare buffer solution 2, 30.24 g PIPES was dissolved in 750 ml of salt solution containing 0.3 g KH2 PO4 , 0.5 g NaCl, and 1.0 g NH4 Cl and a 50% (w/w) KOH solution was added to raise the pH to the pKa of PIPES (6.8). Solution 2 was autoclaved after adding 15 g of agar, and then cooled to 50 ◦ C. For preparing solution 3, 100 ml of 15× KB medium (proteose peptone no. 3.2%, K2 HPO4 0.115%, MgSO4 ·7H2 O 0.15%, and glycerol 1.5%) was mixed with 70 ml of a solution containing 2 g glucose, 2 g mannitol, 439 mg MgSO4 • 7H2 O, 11 mg CaCl2 , 1.17 mg MnSO4 • H2 O, 1.4 mg H3 BO3 , 0.04 mg CuSO4 • 5H2 O, 1.2 mg ZnSO4 • 7H2 O, and 1.0 mg Na2 MoO4 • 2H2 O. Solution 3 was autoclaved and cooled to 50 ◦ C. Solution 4 was a 10% (w/v) casamino acid solution, the nitrogen source. 30 ml of 10% (w/v) casamino acid solution was filter-sterilized. All those four solutions were mixed and poured into petri dishes. After solidifying, paper disk was placed on the center of the CAS-blue agar. The 40 ␮ bacterial suspensions cultured in TSB medium were dropped onto the paper disk. We used Pseudomonas aeruginosa (KACC 11085) as positive control because it is a well-known nitrogen-fixing and siderophoreproducing PGPB. Bacterial isolates inoculated on the CAS-blue agar were incubated at 30 ◦ C for 48 h. 2.4.2.1. Plate assay for phosphate-solubilizing activity. The test of relative efficiency of isolated strains for phosphate solubilization was carried out by selecting the microorganisms that are capable of producing a halo/clear zone on a plate owing to the production of organic acids into the surrounding medium (Mehta and Nautiyal 2001; Rodriguez and Fraga 1999). The ability of the bacterial isolates to solubilize phosphate was tested on the National Botanical Research Institute’s phosphate growth (NBRIP) medium [10 g glucose, 5 g Ca3 (PO4 ) 2, 5 g MgCl2 • 6H2 O, 0.25 g MgSO4 • 7H2 O,

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0.2 g KCl, and 0.1 g (NH4 ) 2SO4 (per liter), pH 7]. Agar plates and NBRI-BPB (NBRIP containing bromophenol blue) broth medium containing insoluble tricalcium phosphate as the single phosphorus source were used to detect phosphate-solubilizing activity of isolates. About 40 ␮l bacterial isolate was spread on the agar plate and after incubation at 30 ◦ C for at least one week, the clear zones around grown bacteria indicated the phosphate solubilizing activity of bacterial isolates (Frey-Klett et al. 2005; Gordon and Weber 1950). For quantitative analysis, NBRI-BPB broth composed of NBRIP supplemented with 0.025 g of bormophenol blue was used. The bacteria were cultured for 2 days in TSB broth. The pre cultured bacteria were transferred into NBRI-BPB broth and incubated at 30 ◦ C for 3 days at 250 rpm. The cultured bacteria were then harvested by centrifugation at 5000 rpm for 20 min. The optical density of the obtained culture supernatant was measured at 600 nm. P. aeruginosa (KACC 11085) was used as positive control. 2.5. Evaluation of plant growth promotion in rice treated with endophytic diazotrophic bacteria Based on the performance of PGPB in the experiments, five randomly selected PGPB isolates, KW7-S22, KW7-S06, SW521-L21, CB-R05, and HS-R01 were used on pot trails. To test the effect of these bacteria on growth promotion in rice seedlings, bacterial suspensions adjusted to an optical density of 1.0 at 660 nm were used and it was applied as a seed drench at a ratio of 10 ml per 20 Ilpum rice seeds. The plants were also incubated in 10 ml sterile distilled water under the same conditions as the untreated control. Seedlings were grown under a 14 h light/10 h dark cycle in a growth chamber at 25 ◦ C. After 4 weeks of drenching, growth parameters such as height and dry weight of the leaf and root were measured. Each experiment included 20 plants per endophytic diazotrophic bacteria treatment with three replications. 2.6. Statistical analysis Data obtained from the plant height and weight was subjected to analysis of variance general linear model (GLM). The treatment means were separated by the least significant difference (LSD) test at P = 0.05 with SAS statistical software version 9.1 (SAS Institute, Inc., Cary, NC). All treatments were in triplicate. 2.7. Test of antagonistic effects A 5 mm mycelia mat of each soil-borne fungi, Fusarium oxysporum and Rhizoctonia solani was placed on one side of a potato dextrose agar (PDA, MB Cell, USA), and each endophytic diazotrophic bacteria isolate was streaked on the other side of the medium. The PDA plate was cultured at 28 ◦ C for 7 to 14 days. During the cultivation, antagonistic effects of the bacterial isolates against the fungal isolates were confirmed by inhibition zones formed between the diazotrophic bacteria isolates and fungal isolate. The dual culture was performed in three replicates. 2.8. Screening the capacity of endophytic diazotrophic bacteria with induced systemic resistance (ISR) on rice Rice cultivar, Ilpum seeds were surface-sterilized with 70% ethanol for 1 min and shaken in 1.2% (w/v) NaClO solution for 15 min. Samples were then washed three times with sterile distilled water with shaking (15 min each). Randomly selected isolates, SW521-L21, KW7-S06 and CB-R05 bacterial suspensions were applied as a seed drench with a ratio of 10 ml per 30 seeds. Optical density of 1.0 at 660 nm was used as bacterial suspensions. Plants incubated in 10 ml of sterile distilled water under the same conditions served as the untreated controls. Tissue culture dishes

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containing water agar medium and seedlings were incubated in a growth chamber at 25 ◦ C under a 14 h light/10 h dark cycle. One week after bacterial treatments, a mycelium piece of F. oxysporum or R. solani was placed on the center of the seedlings, respectively. Degree of disease symptoms of rice plants were determined by visual inspections 2–3 weeks after inoculation. Each experiment included 10 plants per endophytic diazotrophic bacteria treatment with three replications. 2.9. Elicitation of ISR by the endophytic diazotrophic bacterial isolates in rice Rice (Oryza sativa var. Japonica c.v. ilpum) seeds were surfacesterilized by soaking in 12% sodium hypochlorite (NaClO) for 15 min. Seeds were rinsed several times with sterile distilled water. Three bacteria isolates (SW521-L21, KW7-S06 and CB-R05) found effective on plant growth in previous observation, were selected. Bacterial suspensions were diluted with sterile distilled water to yield 108 cfu/ml based on optical density and it was applied as a seed drench at a ratio of 10 ml per 50 seeds. Seeds incubated in 10 ml of sterile distilled water under the same conditions served as the untreated controls. These seeds were placed to plug tray (5 × 10 holes, 5 cm in diameter, 57 × 37 mm) and grown in chamber at 25 ◦ C under a 14 h light/10 h dark cycle. Soil pathogens F. oxysporum and R. solani were grown in 250 ml Erlenmeyer flasks containing 10 g sterilized barley and 15 ml sterile distilled water, respectively. 2 weeks after treating with endophytic diazotrophic bacteria on rice seeds, barley seeds infected with the F. oxysporum and R. solani were used as inoculums, respectively. Two barley seeds per hole were planted in the soil around the roots. Degree of disease symptoms were determined by visual inspections, two weeks after inoculation. Disease severity was determined on the following scale; 0 = no symptom, 1 = less than 10% of leaf area with lesions, 2 = 10–25% of leaf area with lesions, 3 = 25–50% of leaf area with lesions, 4 = 50–75% of leaf area with lesions, and 5 = 75% or more severe lesion or dead leaves. Each experiment included 50 plants per treatment with three replications. 2.10. Biochemical characteristics of CB-R05, KW7-S06 and SW521-L21 The biochemical characteristics of endophytic diazotrophic bacterial strains (KW7-S06, CB-R05, SW521-L21) were identified by API 20E, API ZYM and API 50 CHB systems (BioMerieux, France). The API ZYM kit is it to determine the enzymatic activity of microorganism. This system consisted of 19 enzymatic reactions for rapid detection. The API 50 CHB kit is it to isolate of Bacillus and Enterobacteria. This system is used to confirm the degree of the 49 carbohydrate fermentation. These bacterial strains were pre-cultured overnight in LB agar medium at 30 ◦ C. The cultured bacteria were diluted to 0.85% NaCl medium until the OD600 reached to 0.2 to 0.3. The tests using the commercial systems API 20E, API ZYM and API 50 CHB were generally performed according to the manufacturer’s instructions. The API Web program (http://apiweb.biomerieux.com) was used to analyze the data. 3. Results and discussion 3.1. Isolation of nitrogen-fixing bacteria We isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars. All bacterial isolates were identified by nifH gene PCR, 16S rDNA PCR and sequence analysis. The 390 bp

Fig. 1. PCR amplification with chromosomal DNAs of nitrogen-fixing of bacteria. A, PCR amplification of nifH gene: (A) Approximately 390 bp fragments of nifH were expected to be amplified between nucleotides 19 and 407 (Azotobacter vinelandii M20568 numbering), (B) Amplification of 16S rDNA gene was performed. The amplification product was 1.4 kb. Lane M, 1 kb DNA plus ladder: NC, negative control.

fragments were obtained from only 12 isolates among all 576 endophytic bacteria, through the nifH gene PCR (Fig. 1A). Through 16S rDNA sequence analysis, these 12 endophytic diazotrophic bacteria were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella (Table 1). 12 isolates showed 99–100% of high similarity with the corresponding strains. The amino acid sequence based phylogenetic relationship between these isolates demonstrated that the 12 diazotrrophic bacteria genes were confirmed by genus grouping (Fig. 2). 3.2. Characterization of endophytic diazotrophic bacteria as PGPB 3.2.1. Production of auxin IAA is the main auxin in plants, controlling many important physiological processes including cell enlargement and division, tissue differentiation, and responses to light (Frey-Klett et al. 2005; Gordon and Weber 1950; Khalid et al. 2004; Leveau and Lindow Table 1 Identification of isolated strains from various wild type rice cultivars by 16S rDNA sequence analysis. Strain

Homologous microorganism (% identity) Genebank accession no.

HS-R01 HS-R14 HS-S05 KW7-R08 KW7-S06 KW7-S22 KW7-S27 KW7-S33 CB-R05 CB-S18 SW521-L21 SW521-L37

Paenibacillus kribbensis (99%) Paenibacillus kribbensis (99%) Bacillus aryabhattai (100%) Bacillus megaterium (99%) Klebsiella pneumoniae (99%) Klebsiella pneumoniae (99%) Klebsiella pneumoniae (99%) Klebsiella pneumoniae (99%) Bacillus subtilis (99%) Microbacterium binotii (99%) Microbacterium trichotecenolyticum (99%) Microbacterium trichotecenolyticum (99%)

NR025169 NR025169 JQ799103 JX997394 GU373625 GU373625 GU373625 GU373625 AY030331 JQ291602 EU714362 EU714362

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Fig. 2. Phenogram expressing the relationships of identified bacterial endophytes to taxonomically similar microorganisms based on the 16S rDNA gene sequences. The GenBank accession number is given for each organism. The positions of the endophyte strains are based on the best match for genus and species. This analysis was done using Unweighted Pair Grouping method based on arithmetic averages (UPGMA) and the numbers at the nodes are bootstrap values based on 1000 replications. The rice endophytic diazotrophic bacteria group was divided into Actinobacteria, Bacilli and Gammaproteobacteria class.

2005). The auxin production depending on growth phase was estimated and it was found that maximal production of auxin was usually in the stationary phase. Among 12 isolates, 10 were able to produce high levels of auxin. The isolates produced variable amount of auxins ranging from 11 to 24.6 ␮g/ml (Table 2) while 2 isolates (KW7-R08 and CB-S18) produced relatively less auxin (3.1and

Table 2 Estimation of IAA production by 12 endopytic diazotrophic bacterial isolates. Strain

Concentration of IAA(␮/ml)a

B.subtilisb P. aeruginosac S. marcescensd HS-R01 HS-R14 HS-S05 KW7-S06 KW7-S22 KW7-S22 KW7-S33 KW7-S33 KW7-R08 CB-R05 CB-S18 SW521-L21 SW521-L37 SW521-L37

8.8 15.9 17.4 24.0 24.6 21.6 18.2 21.4 10.4 11.0 11.1 3.1 13.7 4.1 18.0 17.8 18.8

a IAA was estimated more than 16 h after the bacterial growth in King’s B medium supplemented with 0.1% tryptophan with absorbance at 540 nm. b Bacillus subtilis (KACC 17047) was used as type strain for the control of potential strain CB-R05 as a endopytic diazotrophic bacteria and PGPB. c Pseudomonas aeruginosa (KACC 11085) was used as positive control for IAA producer. d Serratia marcescens (KACC 11961) was used as positive control for IAA producer.

4.1 ␮g/ml, respectively). Especially CB-R05, was found highly efficient IAA producer and showed higher IAA than the values earlier reported by Khalid et al. (2004), Yasmin et al. (2007) and Ng et al. (2012). We expect that the growth effect on other crops such as wheat and maize could be increased through the enhancement of IAA by the CB-R05 treatment, for which we are currently performing detailed investigations. 3.2.2. Detection of siderophore production Several reports from the past have confirmed that siderophoreproducing bacteria significantly influence the uptake of various metals, including Fe, Zn, and Cu by plants (Carrillo-Castaneda et al. 2005; Egamberdiyeva 2007; Dimkpa et al., 2008, 2009; Gururani et al. 2012). Siderophores directly stimulate the biosynthesis of other antimicrobial compounds by increasing the availability of these minerals to the bacteria, that suppresses the growth of pathogenic organisms viz., F. oxysporum and R. solani, function as stress factors in inducing host resistance (Haas and Défago 2005; Joseph et al. 2007; Wahyudi et al. 2011). Among 12 isolates selected, 6 isolates (HS-R01, HS-R14, HS-S05, KW7-S06, CB-R05, SW521L21) were confirmed to produce siderophores by CAS -blue agar assay. Six isolates were able to produce the color change from blue to orange (Fig. 3) B. subtilis KACC 17047 as the comparative control for B. subtilis CB-R05 was also confirmed to produce siderophres (data not shown). The production of siderophores is indicated by the typical color described in the literature for the reaction involving removal of iron from CAS by the siderophores (Schwyn and Neilands 1987). The manner of expressing the rate of color-change reaction is not exactly a quantification of the reaction in solid media. Siderophore produced by Pseudomonas living on the surface of plant roots as PGPB, absorb the iron ions (Fe3+ ) and inhibits pathogens

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ARTICLE IN PRESS S.H. Ji et al. / Microbiological Research xxx (2013) xxx–xxx Table 3 Estimation of phosphate solubilization by endophytic diazotrophic bacterial isolates. Strain b

PC HS-R01 HS-R14 HS-S05 KW7-R08 KW7-S06 KW7-S22 KW7-S27 KW7-S33 CB-R05 CB-S18 SW521-L21 SW521-L37

Solubilized phosphate (␮g/ml)a 3.3 0.3 0.5 0.6 0.6 3.3 2.5 1.3 2.3 0.8 1.0 0.6 0.7

a Phosphate-solubilizing abilitities of the isolates were tested by inoculating each bacterium on NBRIP agar plate and estimated with absorbance at 600 nm. b Pseudomonas aeruginosa (KACC 11085) was used as positive control (PC).

(Seong and Shin 1996). The PGPB isolates showing high siderophore producing activity can be further studied for its ability to confer disease resistance in higher plants. Fig. 3. Confirmation of siderophore production by color change on CAS-blue agar: (A) HS-S05. The blue color of CAS-blue agar was changed to orange. Similarly, six isolates (HS-R01, HS-R14, HS-S05, KW7-S06, CB-R05, SW521-L21) were confirmed to produce siderophores by CAS-blue agar assay, (B) HS-R15 (negative control; Rhizobium sp.). (For interpretation of references to colur in this figure legend, the reader is referred to the web version of this article.)

3.2.3. Phosphate-solubilizing activity The release of insoluble and fixed forms of phosphorus is an important aspect of increasing soil phosphorus availability (Rodriguez and Fraga 1999). The use of phosphate-solubilizing bacteria as inoculants simultaneously increase phosphorus uptake by the plant and crop yield (Mehta and Nautiyal 2001). Bacteria belonging to genera Bacillus, Pseudomonas, Serratia, Enterobacter,

Fig. 4. Confirmation of phosphate-solubilizing activity by production of clear zones on NBRIP agar. KW7-S06, KW7-S22, KW7-S27, KW7-S33 and PC (positive control; Pseudomonas aeruginosa KACC 11085). The turbid NBRIP agar became clear.

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Table 4 Effect of 5 endophytic diazotrophic bacterial strains on the growth of rice seedlings. Treatmenta c

Control KW7-S22 KW7-S06 SW521-L21 CB-R05 HS-R01 LSD (P = 0.05)

fresh leaf length (cm)b d

33.98 39.11 41.29 37.99 50.25 43.21 1.2012

fresh root length (cm)b

dry leaf weight (g)b

dry root weight (g)b

13.16 21.98 25.56 19.41 33.73 28.11 1.0689

0.18 0.36 0.39 0.31 0.49 0.42 0.023

0.10 0.24 0.27 0.18 0.39 0.33 0.0186

a Endophytic diazotrophic bacterial strain identifications: KW7-S22 and KW7-S06 = Klebsiella pneumonia, SW521-L21 = Microbacterium trichotecenolyticum, CBR05 = Bacillus subtilis, HS-R01 = Paenibcillus kribbensis; bacteria were applied as seed coat treatments. b Fresh leaf, root length and dry leaf, root weight was measured 4 weeks after planting. c Rice cultivar, Ilpum seeds were used as untreated control. d Mean with different letters are significantly different at P = 0.05 according to LSD test procedure using GLM in SAS. There were three replications per treatment (20 plants per replication).

Fig. 5. Effect of endophytic diazotrophic bacteria KW7-S22, KW7-S06, SW521-L21, CB-R05, HS-R01 on fresh height and dry weight in rice seedlings: (A) four-week-old plants of control rice cultivar (Oryza sativa L.cv. ‘Ilpum’), SW521-L21, KW7-S22, KW7-S06, HS-R01, CB-R05, (B) comparison of root growth; Ilpum (control), SW521-L21, KW7-S22, KW7-S06, HS-R01, CB-R05. We treated rice seeds with five endophytic diazotrophic bacteria by immersing rice seeds in bacteria suspension to determine the effect of plant growth promotion. These strains increased the number of surviving plants in the soil without any fertilization. (C) and (D) data obtained by SAS statistics program. Differences between treatment means were determined using the least significant difference (LSD) test at a probability level of 0.05 (P = 0.05), (C) the effect of endophytic diazotrophic bacteria on fresh height in rice plants, (D) the effect of endophytic diazotrophic bacteria on dry weight in rice plants.

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Table 5 Effect of CB-R05 endophytic diazotrophic bacteria on the growth of rice seedlings. Treatmenta c

Control B. subtilis CB-R05 LSD (P = 0.05)

Fresh leaf length (cm)b d

33.68 35.09 50.84 1.1328

Fresh root length (cm)b

Dry leaf weight (g)b

Dry root weight (g)b

15.65 12.50 23.01 1.5251

0.40 0.34 0.51 0.0232

0.29 0.22 0.42 0.0194

a Endopytic diazotrophic bacterial strain identifications: B. subtilis = Bacillus subtilis (KACC 17047); type strain for CB-R05, CB-R05 = Bacillus subtilis, bacteria were applied as seed coat treatments. b Fresh leaf, root length and dry leaf, root weight was measured at 4 weeks after planting. c Rice cultivar, Ilpum seeds were used as untreated control. d Means with different letters are significantly different at P = 0.05 according to LSD test procedure using GLM in SAS. There were three replications per treatment (20 plants per replication).

Fig. 6. Effect of endophytic diazotrophic bacteria CB-R05 height and dry weight in rice seedlings: (A) four-week-old plants of control rice cultivar (Oryza sativa L.cv. ‘Ilpum’), B. subtilis (KACC 17047) and CB-R05, (B) comparison of root growth; Ilpum (control), B. subtilis (KACC 17047) and CB-R05. We treated rice seeds with endophytic diazotrophic bacteria CB-R05 and its type strain, B. subtilis by immersing rice seeds in bacteria suspension to determine the effect of plant growth promotion, (C) and (D) data obtained by SAS statistics program. Differences between treatment means were determined using the least significant difference (LSD) test at a probability level of 0.05 (P = 0.05), (C) the effect of endophytic diazotrophic bacteria on fresh height in rice plants, (D) the effect of endophytic diazotrophic bacteria on dry weight in rice plants.

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Fig. 7. Antifungal activities of endophytic diazotrophic bacteria against Fusarium oxysporum and Rhizoctonia solani: (A) seven isolates showed the highest antifungal activity in the F. oxyporum, (B) four isolates showed the highest antifungal activity in the R. solani. All endophytic diazotrophic bacteria isolates showed antifungal activities against F. oxyporum. All endophytic diazotrophic bacteria isolates showed antifungal activities against R. solani, except for 2 strains (HS-S05, KW7-R08; data not shown). In particular, KW7-S06, CB-R05 and SW521-L21 showed highest antifungal activities on both fungal isolates. These antifungal activity tests were repeated three times (n = 3).

and so on are reported to solubilize the insoluble phosphate compounds and aid in plant growth (Frey-Klett et al. 2005; Hameeda et al. 2008). Among 12 isolates, 4 isolates exhibited the phosphatesolubilizing activity by forming clear zones on NBRIP agar plates (Fig. 4). B. subtilis did not exhibit the phosphate-solubilizing activity on NBRIP agar plate (data not shown). P. aeruginosa (KACC 11085) was used as positive control (PC). The 4 isolates solubilized variable amount of phosphates ranging from 1.3 to 3.3 ␮g/ml (Table 3). In particular, KW7-S06 isolate exhibited similar phosphate-solubilizing activity to the positive control, P. aeruginosa (KACC 11085). Similar PGPB isolates belonging to Pseudomonas and Bacillus genera were recently reported to induce the plant growth and resistance to water stress in green gram plants (Saravanakumar et al. 2011).

3.2.4. Effect of endophytic diazotrophic bacteria as PGPB PGPB have been applied to a wide range of agricultural species for the purposes of growth enhancement, including increased seed emergence, crop yields, stress tolerance and disease control (Kloepper et al. 1991, 1980; Gururani et al. 2012). For example, emergence increases of 10–40% resulted for canola when seeds were coated with PGPR (Plant Growth Promoting Rhizobacteria) before planting, and plant weight of tuber-treated potatoes increased by 80% on average by midseason (Kloepper and Schroth 1981). Yield increases between 10% and 20% with PGPR applications have been documented for several agricultural crops (Kloepper et al. 1991; Dimkpa et al. 2008, 2009). To determine the plant growth promotion by endophytic diazotrophic bacteria, bacterial suspensions of SW521-L21, KW7-S22, KW7-S06, HS-R01 and

Table 6 Estimation of various biological activities of endophytic diazotrophic bacteria. Biological activity Strain

Auxin productiona

Siderophore productionb

Phosphate solubilizationc

HS-R01 HS-R14 HS-S05 KW7-R08 KW7-S06 KW7-S22 KW7-S27 KW7-S33 CB-R05 CB-S18 SW521-L21 SW521-L37

+ + + − + + + + + − + +

+ + + + + + + + + + + +

− − − − + + + + − − − −

a b c d

Antifungal activityd F.oxyporum

R.solani

++ ++ + ++ ++ + + + ++ + ++ ++

++ ++ − − ++ + + + ++ + ++ ++

High concentration of IAA (+); low concentration of IAA (−). Siderophore producing (+); non producing (−). The strains that solubilize phosphates (+); strains unable to solubilize phosphates (−). High antagonistic effect (++); normal antagonistic effect (+); non antagonistic effect (−).

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Table 7 Physiological characteristics of KW7-S06, SW521-L21 and CB-R05. Characterisitcs

KW7-S06a

Klebsiella sp.a

Characterisitcs

KW7-S06a

Klebsiella spa .

␤-Galactosidase Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization H2 S production UREase Tryptophanedeaminase Indole production Acetoin production Gelatinase Fermentation/oxidation (glucose) Fermentation/oxidation (mannitol) Fermentation/oxidation (inositol) Fermentation/oxidation (sorbitol) Fermentation/oxidation (rhamnose) Fermentation/oxidation (sacdcharose) Fermentation/oxidation (melibiose) Fermentation/oxidation (amygdalin) Fermentation/oxidation (arabinose)

+ + − + − + − − + − +

+ − + − + − + − − + − −

+++ + + − + − − − − +++ +

++ + + − + − − − − ++ +

+

+

++ + −

+ + −

+

+

+

+

+

+

Alkaline phosphatase Esterase Esterase lipase Lipase Leucinearylamidase Valinearylamidase Crystinearylamidase Trypsin ␣-Chymotrypsin Acid phospatase Naphtol-AS-BI-phosphohydrolase ␣-Galactosidase ␤-Glucuronidase ␤-Glucosidase ␣-Glucosidase ␤-Glucosidase N-acetyl-␤-glucosaminidase ␣-Mannosidase ␣-Fucosidase

+++

+

+

+

+

+++ − − −

+

+

+

+

+

+

− − −

Characterisitcs

SW521-L21a

Microbacteriumsp.a

CB-R05a

Bacillus

Alkaline phosphatase Esterase Esterase lipase Lipase Leucinearylamidase Valinearylamidase Crystinearylamidase Trypsin ␣-Chymotrypsin Acid phospatase Naphtol-AS-BI-phosphohydrolase ␣-Galactosidase ␤-Glucuronidase ␤-Glucosidase ␣-Glucosidase ␤-Glucosidase N-acetyl-␤-glucosaminidase ␣-Mannosidase ␣-Fucosidase

− + + − − − − − − − + − − − + − + − −

− + ++ − − − − − − − +++ − − − + − ++ − −

− ++ + − − − − − − − − − − − + − − − −

− +++ + − − − − − − − − − − − ++ − − − −

Characterisitcs

CB-R05a

Bacillus sp.a

Characterisitcs

CB-R05a

Bacillus sp.a

Glycerol Erythritol d-Arabinose l-Arabinose d-Ribose d-Xylose l-Xylose d-Adonitol Methyl-␤ d-xylopyranoside d-Galactose d-Glucose d-Fructose d-Mannose l-Sorbose l-Rhamnose Dulcitol Inositol d-Mannitol d-Sorbitol Methyl-␣ d−mannopyranoside Methyl-␣ d-glucopyranoside N-acetylglucosamine Amygdalin Arbutin Esculin

+ − + + + + − − − + + + + + + + + + + + + + − + +

+ − − − + − − − − − + + − − − − − − − − − + − + +

Salicin d-Cellobiose d-Maltose d-Lactose(bovine origin) d-Melibiose d-Saccharose(sucrose) d-Trehalose Inulin d-Melezitose d-Raffinose Amidon (starch) Glycogen Xylitol Gentiobiose d-Turanose d-Lyxose d-Tagatose d-Fucose l-Fucose d-Arabitol l-Arabitol Potassium gluconate Potassium 2-ketogluconate Potassium 2-ketogluconate

+ + + + + + + + + + + + − + − − + − + + − − − +

+ + + − − + + − − − + + − + − − − + − − − − − −

a

+++, strongactivity; ++, moderate activity; +, low activity; −, no activity.

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Fig. 8. Effect of isolates on induced systemic resistance of rice against Rhizoctonia solani and Fusarium oxysporum: (A) Antifungal activity of rice plants treated with endophytic diazotrophic bacteria SW521-L21, KW7-S06, CB-R05 against R. solani, (B) antifungal activity of rice plants treated with endophytic diazotrophic bacteria SW521-L21, KW7S06, CB-R05 against F. oxysporum, and (C) three endophytic diazotrophic bacteria SW521-L21, KW7-S06 and CB-R05, elicited ISR against R. solani and F. oxysporum on water agar medium.

CB-R05 were applied to rice seeds by drenching applications. Fourweeks after drenching, growth parameters such as height and dry weight of the leaf and root were measured. Interestingly, these strains increased the number of surviving plants in the soil without any fertilization. Plants treated with CB-R05 isolate were taller compared to the untreated control (Table 4 and Fig. 5) with significantly enhanced dry weight of leaf and root. These results indicated that the five isolates, particularly CB-R05 could be used to facilitate an effective plant growth promotion in rice plants. In Table 5 and Fig. 6, Bacillus subtilis (KACC 17047) isolated from rhizosphere used as a type strain for the CB-R05 (B. subtilis) and in corroboration with the previous results, CB-R05 showed more significant growth effect than the type strain B. subtilis (KACC 17047). The type strain B. subtilis (KACC 17047) even reduced rice growth rather than promotion, regarding the dry weights of leaves and roots, although leaf length was increased compared to the untreated control plants. Further, we evaluated the growth-promoting effect of these PGPB on the Dongjin plants. Dongjin plants treated with CB-R05 isolate showed significant growth-promoting effect than the untreated control. In contrast, B. subtilis; a type strain for the CB-R05, did not show

growth promotion on the Dongjin plants (Supplementary Fig. S1). The growth-promoting effects of these isolates were slightly different on other rice cultivars as observed in our preliminary studies (data not shown). Further detailed investigations are underway to determine the growth promoting ability of these isolates in other rice cultivars as well as in other crops. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micres. 2013.06.003. 3.3. Antagonistic effect of diazorophic bacteria in vitro A dual culture test is extensively used as one of in vitro tests for preliminary screening of biological control agents (Desai et al. 2002). According to the previous reports, dual culture tests have shown that many Bacillus isolates from livestock manure composts and cotton-waste composts have antagonistic effects against the isolates of soilborne fungi, F. oxysporum, P. capsici, R. solani AG-4, and S. sclerotiorum (Kim et al. 2008). Similarly, in this study, all 12 endophytic diazotrophic bacteria isolates were tested for

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Fig. 9. Comparison of ISR effect on B.subtilis and CB-R05 against Rhizoctonia solani and Fusarium oxysporum. Degree of disease symptoms of rice plants were determined by visual inspections 6 weeks after inoculation: (A) antifungal activity of rice plants treated with bacteria B. subtilis (KACC 17047) and endophytic diazotrophic bacteria CB-R05 against R. solani, (B) antifungal activity of rice plants treated with B. subtilis (KACC 17047) and edophytic diazotrophic bacteria CB-R05 against F. oxysporum, (C) B. subtilis (KACC 17047) and CB-R05, elicited ISR against R. solani and F. oxysporum on water agar medium.

antagonistic effects in vitro against soil pathogenic fungi, F. oxysporum and R. solani. Antagonistic effects were confirmed by the formation of inhibition zones between the bacteria isolates and the fungal isolates (Fig. 7). All 12 endophytic diazotrophic bacterial isolates had antagonistic effects on mycelial growth of all the isolates of F. oxysporum tested. Among them, 7 isolates HS-R01, HS-R14, KW7-R08, KW7-S06, CB-R05, SW521-L21 and SW521-L37 showed

highest antifungal activities (Fig. 7A). Ten isolates of endophytic diazotrophic bacteria showed antagonistic effects with R. solani, except HS-S05 and KW7-R08. KW7-S06, CB-R05, SW521-L21 and SW521-L37 isolates were most effective in terms of formation of large inhibition zones against the fungal isolates (Fig. 7B). Moreover, these isolates exhibited the antagonistic effects even after a long period of time.

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Fig. 10. Resistance induced in ilpum rice plants treated with endophytic diazotrophic bacteria CB-R05, KW7-S06 and SW521-L21: (A) and (B) growth promotion of rice plants treated with endophytic diazotrophic bacteria against soilborn plant pathogenic fungi Rhizoctonis solani and Fusarium oxysporum, and (C) ISR against R. solani and F. oxysporum by inoculated endophytic diazotrophic bacteria SW521-L21, KW7-S06 and CB-R05 in the soil without any fertilization.

3.4. Screening of endophytic diazotrophic bacteria ISR activity on rice Systemic resistance induced by PGPR has been termed ‘induced systemic resistance’ (ISR) (Kloepper and Beauchamp 1992). A reduction of plant growth after triggering of ISR might be explained as a trade-off in terms of the cost in conditions in which nutrient or mineral resources for growth and plant defense against pathogen atttack are limited (Heil and Baldwin 2002). Rice seedlings were exposed to the 3 isolates for 7 days and injected with soil pathogenic fungi. All 3 isolates protected rice seedlings against F. oxysporum and R. solani. These isolates exhibited the lowest number of symptomatic plants with pathogen inoculation as compared to the untreated control (Fig. 8). Also, CB-R05 isolate had survived for more than four weeks in the fresh state (data not shown). Disease severity against endophytic diazotrophic bacteria treatments is shown in Fig. 8C. In accordance with the previous results, CB-R05 isolate exhibited the lowest number of symptomatic plants with pathogen inoculation as compared to the untreated control and B. subtilis (KACC 17047) (Fig. 9). Our results clearly indicate that the application of KW7-S06 and CB-R05 suspensions was significantly effective on plant growth promotion and induction of ISR. Moreover, KW7-S06 isolates were also able to produce auxin, siderophore and solubilize phosphate. Also, the CB-R05 showed a significantly high ISR effect compared to type strain B. subtilis.

These isolates also showed higher antifungal activity against F. oxysporum and R. solani suggesting that these isolates could be used as a multiple PGPB and endophytic diazotrophic bacteria (Table 6). 3.5. Assessment of ISR against pathogen F. oxysporum and R. solani on rice. Bioassays for elicitation of ISR in rice by the three isolates of SW521-L21, KW7-S06 and CB-R05 showed that they significantly reduced the disease symptom in rice plants compared to the untreated control (Fig. 10). Severity of disease caused by F. oxysporum and R.solani was visually determined on a scale of 0–5, 2 weeks after the pathogen challenge. Treatment with 3 isolates results in reduction of disease severity compared to the untreated control plants. The CB-R05 isolate was the most effective for fungal resistance in the soil without fertilizer. In particular, the elicitation of ISR was found best in plants treated with the CB-R05 isolate than B.subtilis (KACC 17047) type strain for CB-R05 (Fig. 11). Generally, rice plants treated with all the 3 endophytic diazotrophic bacteria showed both induced ISR and plant growth promotion. ISR is dependent on colonization of the root system by sufficient numbers of PGPR. PGPR-ISR protects cucumber against bacterial wilt not only by reducing beetle feeding and pathogen transmission, but also through the induction of other plant defense

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Fig. 11. Resistance induced in ilpum rice plants treated with B. subtilis (KACC 17047) and endophytic diazotrophic bacteria CB-R05, (A) and (B) growth promotion of rice plants treated with endophytic diazotrophic bacteria against soilborn plant pathogenic fungi Fusarium oxysporum and Rhizoctonis solani, and (C) ISR against F. oxysporum and R. solani by inoculated B.subtilis (KACC 17047) and endophytic diazotrophic bacteria CB-R05 in the soil without any fertilization.

mechanisms after the pathogen attack (Zehnder et al. 2001). The two endophytic strains, PS4 and PS27, showed significant pepper growth-promoting effects, especially on root growth, and the strains also elicited ISR against a bacterial pathogen, Xav (Kang et al. 2007).

an important role in reducing disease symptoms in Arabidopsis (Ryu et al. 2004). The enzyme activity tests revealed that the KW7-S06 isolate produces the acetoin, which acts as an elicitor of ISR. Hence, we concluded that KW7-S06 isolate might play an important role in inducing the resistance of plants.

3.6. Biochemical characteristics of SW521, KW7-S06 and CB-R05 4. Conclusion We determined the biochemical characteristic of PGPB isolates through the enzyme activity tests, API 20E, CHB, ZYM as described in the materials and methods section. The enzyme activity tests revealed that there was no significant difference between KW7-S06 and Klebsiella sp., SW521-L21 and Microbacterium sp., CB-R05 and Bacillus sp (Table 7). Further, we were interested in determining the production of acetoin that is known to play an important role in elicitation of ISR. Acetoin is referred to as the 3-hydroxy-2-butanone, 2, 3-butanolone, acetyl methyl carbinol, dimethylketol. Many bacteria are able to survive on acetoin, however, little is known about its catabolism. In Bacillus subtilis and Alcaligenes eutrophus, this 2,3-butanediol cycle is not present since mutants lacking 2,3-butanediol dehydrogenase, which is one of the key enzymes of the cycle, grew on acetoin (Lopez et al. 1973; Steinbuchel et al. 1987). Microbial volatile organic compounds (VOCs) are involved in regulating plant growth and development, although it has been observed earlier that bacterial volatile components can serve as agents for triggering growth promotion in Arabidopsis (Ryu et al. 2003). Previous studies suggest that VOCs associated with rhizobacteria can initiate ISR. Bacillus subtilis GB03 produced the VOCs, such as 2,3-butanediol and acetoin, which play

A total of 576 endophytic bacteria isolated from different organs of Korean rice cultivars, were screened for their putative beneficial characteristics as PGPB. Following the nucleic acid analysis and in silico nucleotide similarity search analysis, 12 endophytic diazotrophic bacteria were selected for further investigation. Further, experiments were performed with these 12 isolates to determine their auxin producing activities, phosphate solubilizing activities, siderophore producing activities and enzymatic activities. Our results revealed that 10 bacterial isolates belonging to various species produced high levels of auxin, while 6 isolates showed high siderophore producing activity, and 4 isolates showed remarkable phosphate solubilizing activity. Morphological differences among rice cultivars treated with these PGPB isolates clearly demonstrated their positive effects on plant growth and development. Furthermore, the estimation of antagonistic effects as well as the biochemical and enzymatic activities of these PGPB isolates against two common soil pathogenic fungi, F. oxysporum and R. solani confirmed that these isolates could well be used to induce fungal resistance in higher plants. However, further studies at the molecular and biochemical levels are warranted to

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Please cite this article in press as: Ji SH, et al. Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.003