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Diversity of free-living nitrogen-fixing bacteria associated with Korean paddy fields

Diversity of free-living nitrogen-fixing bacteria associated with Korean paddy fields Ann Microbiol (2012) 62:1643–1650 DOI 10.1007/s13213-012-0421-z ORIGINAL ARTICLE Diversity of free-living nitrogen-fixing bacteria associated with Korean paddy fields Md. Rashedul Islam & Tahera Sultana & Jang-Cheon Cho & M. Melvin Joe & T. M. Sa Received: 21 September 2011 /Accepted: 20 January 2012 /Published online: 18 February 2012 Springer-Verlag and the University of Milan 2012 Abstract Nitrogen (N)-fixing microorganisms play a ma- Introduction jor role in maintaining soil fertility and are thereby impor- tant for sustainable rice production. Among a total of 165 The nitrogen (N) requirement of paddy rice is well known bacterial isolates recovered from seven paddy field soils and rice production today depends on the application of through an isolation process on four N-free media, 32 were large quantities of nitrogenous fertilizers (Choudhury and found to be positive for PCR amplification of nifHgene. Kennedy 2004). Unfortunately, besides increasing produc- On screening, the BOX-PCR fingerprint technique grouped tion costs, these fertilizers also cause severe environmental the nifH gene positive isolates into seven clusters. Cluster- pollution in rice-producing environments (Wartiainen et al. ing of bacteria revealed a very low level of similarity 2008). Biological nitrogen fixation (BNF) is considered as a (20%), indicating the existence of a high degree of genetic suitable alternative in the development of sustainable agri- diversity among the N-fixing isolates. Further character- culture, satisfying human needs, and conserving natural ization based on fatty acid methyl ester (FAME) showed resources (Giller and Cadisch 1995; Vance 1997). BNF is that the isolates were members of 16 different genera, with performed by phylogenetically diverse groups of bacteria maximum number belonged to the genus Burkholderia that harbor nifH genes that encode the Fe-protein subunit of followed by Sphingomonas. Our results provide evidence nitrogenase. Among nitrogen fixers, free-living N-fixing for wide diversity of free-living N-fixing bacteria that can bacteria are important contributors to soil N, reaching up −1 −1 be used in future as a feasible alternative to N fertilizers in to 60 kg ha year (Cleveland et al. 1999). It has been rice-paddy ecosystems. reported that soil N availability is linked to the presence of N- fixing organisms (Bormann and Sidle 1990;Matthews 1992), . . . Keywords Nitrogen fixation nifH gene BOX-PCR and this plays a vital role for successive microbial colonization . . Diversity FAME Paddy soil (Walker and del Moral 2003). Wartiainen et al. (2008) reported the genetic diversity of free-living N-fixing bacteria in paddy soils based on nifH gene sequences, and addressed their contribution to the N input in rice-paddy ecosystems. BOX-PCR analysis is a highly effective approach for Electronic supplementary material The online version of this article determining genetic relationships among bacterial isolates (doi:10.1007/s13213-012-0421-z) contains supplementary material, (Versalovic et al. 1991). A high correlation exists between which is available to authorized users. BOX-PCR fingerprinting and DNA-DNA homology data, : : M. R. Islam M. M. Joe T. M. Sa and this technique has been applied in numerous taxonomic Department of Agricultural Chemistry, studies on plant-associated, environmental, medical and Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea food-associated bacteria (Rademaker et al. 1998; Lanoot et al. 2004). Fatty acid methyl ester (FAME) analysis is anoth- : : M. R. Islam (*) T. Sultana J.-C. Cho er well-established method for identification and classifica- Department of Biological Sciences, Inha University, tion of bacteria based on whole cellular fatty acids Incheon 402-751, Republic of Korea e-mail: rashed@inha.ac.kr derivatized to methyl esters (Sasser 2009). Differences in 1644 Ann Microbiol (2012) 62:1643–1650 Table 1 Different paddy soil treatments used in the present study chain length, positions of double bonds and the binding of functional groups make them very useful taxonomic markers Soil sample Treatment (Dawyndt et al. 2006). Because of cheap, fast, automated and high throughput process, FAME analysis has revolutionized Control No fertilizers including rice straw bacterial identification in a way that has now increased the NPK N-P O -K O at 110:70:80 kg/ha 2 5 2 efficiency of many laboratories around the world (Kunitsky et CNPK N-P O -K O at 110:70:80 kg/ha+7.5 Mg/ha rice straw 2 5 2 al. 2005) and, as a result, libraries of fatty acid profiles for NPKC750 N-P O -K O at 110:70:80 kg/ha+7.5 Mg/ha rice straw 2 5 2 thousands of bacteria have been developed to aid in identifi- NPKC750 N-P O -K O at 110:70:80 kg/ha+15.0 Mg/ha rice 2 5 2 straw cation (Kloepper et al. 1992, Slabbinck et al. 2008). NPKC2250 N-P O -K O at 110:70:80 kg/ha+22.5 Mg/ha rice Although numerous studies have suggested that free- 2 5 2 straw living heterotrophic N-fixers are a potentially important NPKC3000 N-P O -K O at 110:70:80 kg/ha+30.0 Mg/ha 2 5 2 source of N fixation in rice fields, knowledge of the ecol- rice straw ogy of N-fixers has not been well documented and cannot be enhanced by studying only laboratory microorganisms Chemical fertilizer was applied at the rate of 75-75-75 from 1954 to (Boddey et al. 1998; Mahadevappa and Shenoy 2000). It is 1970, 100-75-75 from 1971 to 1978, 150-86-86 from 1979 to 1985 and 110:70:80 kg/ha from 1986 to present. Rice straw was used as organic therefore necessary to study the naturally occurring diver- compost by fermenting 5 months of period sity of N-fixers in a large-scale soil ecosystem in order to understand phenotypic variation (Balandreau 1986). The objective of the present study was to assess genetic diver- 2005), LGI-P (Reis et al. 1994), BAz (Estrada-De Los Santos sity among the N-fixing isolates recovered from seven et al. 2001) and JNFb (Kirchhof et al. 1997). The pure cultures different paddy soils by analyzing nifH gene and BOX- of the isolates were maintained in nutrient broth containing PCR genomic DNA fingerprinting, and to identify the 50% (w/v) glycerol and refrigerated at −80°C. isolates by FAME analysis. In addition, the isolates were Physiological and biochemical characters of N-fixing bac- characterized physiologically, and the effects of different terial isolates were examined according to Bergey’sManual of fertilizer treatments on the occurrence and distribution of Determinative Bacteriology (Holt et al. 1994). Gram staining nifH gene were also investigated. was performed with a Gram stain kit (Difco, Detroit, MI). Qualitative tests for the production of oxidase and catalase were performed by using Difco strips as recommended by the Materials and methods manufacturer. The strains were also characterized biochemi- cally for the following basic reactions: nitrate reduction, gel- Site and soil sampling atin hydrolysis, starch, casein and urea hydrolysis (Stanier et al. 1966; Bossis et al. 2000). The results of these tests were Soil samples were collected from the National Institute of scored as either positive or negative (Table 2). Agricultural Science and Technology located at Suwon city of the Republic of Korea in October 2007 (after harvesting Amplification of nifH gene the rice crop), where research plots were established in 1954. Rice straw, prepared by fermenting for 5 months, The presence of the nifH gene in the N-fixing bacterial isolates was used as compost. Compost with and without nitrogen– was determined by amplifying a 390-bp fragment through phosphorus–potassium (NPK) fertilizer was applied to soil. PCR using a pair of specific primers, 19F (5′-GCIWTY Since 1986, chemical fertilizers and compost have been TAYGGIAARGGIGG-3′) and 407R (5′-AAICCRCCR applied at the rates noted in Table 1. While CNPK received CAIACIACRTC-3′) (Ueda et al. 1995) directed against con- ammonium sulfate, all the other treatments received urea as served sequences of the nifH gene. The amplified products N source. The control treatment received neither chemical were resolved on a 1% agarose gel in 1x TBE buffer and fertilizer nor compost amendments. The sampling was done visualized under UV light (Bio-Rad Laboratories, Richmond, by collecting soils (0–20 cm depth) from nine randomly CA). selected points within each plot using a 1.45 cm diameter soil core. Samples from each plot were then combined to form one composite sample and stored at 4°C. PCR amplification with specific BOX A1R primer Isolation and phenotypic characterization The DNA of the bacterial isolates was amplified by PCR using primer BOX A1R (5′-CTACGGCAAGGCGACGCTGACG- N-fixing bacteria were isolated using the serial dilution tech- 3′, Invitrogen, Life Technologies, Carlsbad, CA) (Versalovic nique on four N-free selective media viz., NFMM (Piao et al. et al. 1994). The PCR cycling protocol was as follows: 1 cycle Ann Microbiol (2012) 62:1643–1650 1645 Table 2 Biochemical character- Isolate name Soil sample Isolation medium 1 2 3 4 5 6 7 8 9 10 11 istics of the N-fixing bacteria isolated from paddy field soils. RFNB1 Control NFMM - -- -+ + + - + + - 1 Gram’s reaction; 2 motility; 3 casein hydrolysis; 4 starch RFNB2 NPK NFMM + - + + + + + + + - + hydrolysis; 5 gelatine RFNB3 CNPK NFMM - + + + + + + - - + - hydrolysis; 6 nitrate reduction; RFNB4 NPKC750 NFMM + + - - + + + - - - + 7 catalase production; 8 urease activity; 9 oxidase activity; RFNB5 NPKC750 NFMM + + - + + - - - - - - 10 V-P test; 11 polysaccharide RFNB6 NPKC2250 NFMM + - + + + + + + + - + production RFNB7 NPKC3000 NFMM - - - - + + + - + - - RFNB8 CNPK LGI-P - + + + + + + - + + - RFNB9 CNPK LGI-P - - - - + + + - - + - RFNB10 NPKC1500 LGI-P + - + + + + + + + + - RFNB11 Control BAz - - - + + - - - - - - RFNB12 Control BAz - + - + + - - - - + - RFNB13 Control BAz - + - + + - - - - + - RFNB14 Control BAz - + + + + + + + + + - RFNB15 CNPK BAz - + + + + + + - + - - RFNB16 NPKC1500 BAz - - - + + - - - - - - RFNB17 NPKC2250 BAz - + + + + + + + + - - RFNB18 NPKC2250 BAz - - - + + - - + - - - RFNB19 NPKC3000 BAz - + + + + + + + + - - RFNB20 NPKC3000 JNFb - - - - + + - - - - - RFNB21 Control JNFb - + - - + - - - - + - RFNB22 Control JNFb - + - - + - - - - - - RFNB23 Control JNFb - + - - + - + - - - - RFNB24 Control JNFb - - + + + + + + + - + RFNB25 NPK JNFb - - - + + - + - - + - RFNB26 NPK JNFb - - - - + + - - - + - RFNB27 NPK JNFb - + - + + + - - + + - RFNB28 CNPK JNFb - - - - + - - - - - - RFNB29 NPKC1500 JNFb - - - + + + - - - + - RFNB30 NPKC2250 JNFb - - - + + + + - - - - RFNB31 NPKC3000 JNFb - - - - + + - - - - - + Positive for the reaction/mo- tile, - negative for the reaction/ RFNB32 NPKC2250 JNFb - - - - + + - - - + - non-motile of denaturation at 95°C for 7 min; 35 cycles of denaturation at Identification System, Newark, NJ) (Sasser 2009). Briefly, 94°C for 1 min, annealing at 53°C for 1 min and extension at strains were grown overnight on nutrient agar plates. One 65°C for 8 min; 1 cycle of final extension at 65°C for 16 min; loopful of cells was harvested and transferred to a culture and a final soak at 4°C. The amplified fragments were sepa- tube; 1 ml saponication reagent added. Tubes were sealed rated by horizontal electrophoresis on a 1.5% agarose gel tightly with a teflon-lined screw cap and vortexed for 5– (15 cm×15 cm), at 75 V, for 5 h. Gels were stained with 10 s. The tubes were placed in water bath at 100°C for ethidium bromide, visualized under UV light and photo- 5 min. They were then taken out of the boiling water bath graphed. Cluster analyses of the BOX-PCR products were and cooled slightly, vortexed for 10 s and incubated in a performed using the method of unweighted pair grouping with water bath for an additional 25 min. Each tube received 2 ml mathematic average (UPGMA) in the Gelcompar II 3.5 soft- methylation reagent, was capped tightly and vortexed for ware package (Applied Math, Austin, Texas). 10 s. The tubes were placed in a water bath at 80±1°C for 10 min. Then, 1.25 ml extraction reagent was added to the Fatty acid methyl ester profiles cooled tubes followed by gentle tumbling on a rotator for 10 min. The bottom phase was removed by using a pipette. FAME profiles were obtained by saponification, methyla- Finally, 3 ml base reagent was added to upper phase remain- tion and extraction following the MIDI system (Microbial ing in the tubes and tumbled for 5 min. The upper solvent 1646 Ann Microbiol (2012) 62:1643–1650 phase was transferred to vials for fatty acid analysis. FAME However, when comparison was made based on various soil data were analyzed with Sherlock 6.0 MIDI TSBA60 library treatments, the highest number (28%) of nifH-gene-positive (see Sasser 2009), and bacteria were identified based on isolates was obtained in control plots (Table 3). This is not similarity index value. surprising, because the control plots never received any fertilizer, and thus might allow a higher number of N- fixing bacteria to grow. However, we found no differentia- tion between the occurrence of nifH isolates in plots Results and discussion amended with different levels of fertilizers, supporting the observations of Ogilvie et al. (2008) and Wakelin et al. Biochemical characterization (2007) that long-term application of N fertilizers does not have much impact on nifH abundance within agricultural In the present study, the composition of the N-fixing soils. bacteria associated with paddy soils was assessed by phenotypic characterization and by genomic DNA finger- printing analysis to evaluate genetic variation among iso- Genomic finger printing by BOX-PCR lates. With the exception of RFNB2, RFNB4, RFNB5, RFNB6 and RFNB10, all tested isolates were Gram neg- The BOX-PCR technique is a multilocus analysis used to ative; 14 isolates exhibited motility and the remaining 18 elucidate the phylogenetic relatedness among different iso- isolates were non-motile. RFNB10 and RFNB14 were lates (Martin et al. 1992; Cottyn et al. 2001). To access positive for all biochemical tests including casein, starch, genetic relationships, the N-fixing bacterial isolates were gelatine, nitrate reduction, catalase, urease, oxidase and V- submitted to DNA analysis with the BOX A1R-PCR meth- P test. The isolates RFNB2, RFNB4, RFNB6 and od to amplify the DNA with primers (BOX A1R) of repet- RFNB24 were able to produce polysaccharide (Table 2). itive and evolutionary conserved regions. BOX sequences are dispersed in the genomes of diverse bacteria (Lupski and Amplification of nifH gene and its distribution Weinstock 1992) and the primers amplify genomic regions between the two BOX elements. Successful amplification Out of a total of 165 N-fixing isolates, 32 were positive for was achieved from isolates with high polymorphism in the amplification of a 390-bp nifH gene fragment (Supplemental well resolved bands, with 3–10 amplification products rang- Fig. S1). Amplicons of the nifH gene were obtained to ing in the size from 200 to 1,350 bp. Information about the evaluate the diversity of potential N-fixing bacteria in the FAME analysis (Table 4) was used for the description of rice-paddy system. Among isolates containing the nifH BOX-PCR results (Fig. 1). gene, most were isolated from JNFb medium (41%), fol- Based on the BOX-PCR banding pattern, the isolates lowed by BAz (28%), NFMM (22%) and LGI-P (9%), were grouped into seven clusters (Fig. 1); 34% of the iso- respectively. It should be noted that the isolates obtained lates were grouped into cluster III with 45% similarity from different media represent only the culturable fraction. within the group. This cluster was most diverse, comprising Table 3 Distribution of the isolates showing nifH gene amplifications to various paddy soils and the four types of N-free medium used for isolation purposes Soil sample Medium Type of bacteria identified NFMM LGI-P BAz JNFb Control 1 (8) 0 (0) 4 (5) 4 (9) Methylobacterium, Burkholderia, Roseomonas, Klebsiella, Sphingomonas, Stenotrophomonas NPK 1 (7) 0 (4) 0 (6) 3 (8) Mycobacterium, Grimontia CNPK 1 (9) 2 (2) 1 (1) 1 (4) Paucimonas, Enterobacter, Novosphingobium NPKC750 2 (10) 0 (5) 0 (3) 0 (6) Paenibacillus, Brochothrix NPKC1500 0 (5) 1 (6) 1 (3) 1 (8) Novosphingobium, Burkholderia NPKC2250 1 (6) 0 (6) 2 (6) 2 (7) Bacillus, Burkholderia, Pseudomonas, Sphingomonas NPKC3000 1 (9) 0 (8) 1 (6) 2 (8) Methylobacterium, Burkholderia, Pseudomonas, Phyllobacterium Values in parenthesis indicate the total number of N-fixing bacteria obtained on the respective medium from seven soil samples Ann Microbiol (2012) 62:1643–1650 1647 five different genera viz. Burkholderia (RFNB11, RFNB12, (RFNB25) and Phyllobacterium (RFNB31) formed part of RFNB16, RFNB17, RFNB18), Mycobacterium (RFNB2), this group. Five isolates of cluster I, comprising different Novosphingobium (RFNB10), Klebsiella (RFNB14) and genera, viz. Methylobacterium (RFNB1, RFNB7), Brocho- Enterobacter (RFNB15). While isolate RFNB11 shared an thrix (RFNB5) and Stenotrophomonas (RFNB24), shared a identical electrophoretic profile with RFNB12, isolate low level of similarity (29%). Group II has three isolates, RFNB2 was only 45% similar with other members of this viz. Paucimonas (RFNB9), Pseudomonas (RFNB30), with group. Isolates RFNB17 and RFNB18 also shared indistin- 42% similarity within the group and approximately 40% guishable electrophoretic profiles. Cluster IV formed the similarity with group III. Group VII clustered with all the second largest group, with 22% of isolates sharing approx- other groups with a very low level similarity (20%). These imately 43% similarity among them. Sphingomonas results confirmed the existence of a wide genetic diversity (RFNB22, RFNB23), Burkholderia (RFNB19), Grimontia among N-fixing bacterial isolates recovered from paddy soils. Specific primers of the nifH genes and BOX-PCR were used in the present study, which allowed the genetic struc- Table 4 Taxonomic identification of N-fixing isolates as determined ture of N-fixers in paddy fields to be assessed through the by fatty acid methyl ester (FAME) profiles diversity of nifH genes. Our result revealed that many N- fixing bacteria isolated from different fertilization treatments Isolate code FAME identification Similarity index shared identical BOX-PCR patterns, indicating similarity RFNB1 Methylobacterium mesophilicum 0.457 among isolates. However, the fingerprinting pattern was RFNB2 Mycobacterium aichiense 0.152 complex, with seven major clusters joining with a very RFNB3 Not matched - low final level of similarity (20%) indicating a degree of diversity among N-fixers. This fingerprinting technique has RFNB4 Paenibacillus azotofixans 0.515 RFNB5 Brochothrix campestris 0.417 proven valuable in studies of N-fixing bacteria and has been used extensively in ecology, genetic and taxonomic studies, RFNB6 Bacillus megaterium 0.388 as well as for identification of N-fixing bacterial isolates (e.g., RFNB7 Methylobacterium rhodesianum 0.631 Versalovic et al. 1994;Chen et al. 2000; Kaschuk et al. 2006). RFNB8 Not matched - From our results, it appears that BOX analysis of nifHPCR RFNB9 Paucimonas lemoignei 0.729 products from environmental sample is a powerful tool for RFNB10 Novosphingobium capsulatum 0.291 assessing the presence and diversity of N-fixing microorgan- RFNB11 Burkholderia gladioli 0.471 isms in ecosystems (Sato et al. 2009), and the technique is RFNB12 Burkholderia gladioli 0.224 rapid and simple. The number and positions of the BOX RFNB13 Roseomonas sp. 0.594 fragments reflect the diversity and heterogeneity of the bacte- RFNB14 Klebsiella pneumoniae 0.308 ria in a sample. Although this approach does not directly allow RFNB15 Enterobacter hormaechei 0.631 evaluation of functional aspects of the N-fixers in a sample, RFNB16 Burkholderia gladioli 0.173 genetic information on the gene pool and the potential for N RFNB17 Burkholderia gladioli 0.214 fixation may be assessed. RFNB18 Burkholderia cenocepacia 0.518 RFNB19 Burkholderia gladioli 0.214 Identification of isolates by FAME analysis RFNB20 Pseudomonas pertucinogena 0.221 RFNB21 Not matched - The MIDI-FAME technique was used to determine the RFNB22 Sphingomonas paucimobilis 0.590 whole-cell cellular fatty acid profiles of N-fixing bacterial RFNB23 Sphingomonas paucimobilis 0.475 isolates (Table 4). In the present work, this analysis iden- RFNB24 Stenotrophomonas maltophilia 0.179 tified more than 81% of the bacteria with varied levels of RFNB25 Grimontia hollisae (Vibrio) 0.175 confidence value, for a total of 16 different genera, viz., RFNB26 Not matched - Burkholderia sp., Sphingomonas sp., Methylobacterium RFNB27 Not matched - sp., Pseudomonas sp., Novosphingobium sp., Bacillus RFNB28 Novosphingobium capsulatum 0.130 sp., Paenibacillus sp., Enterobacter sp., Klebsiella sp., RFNB29 Not matched - Mycobacterium sp., Roseomonas sp., Brochothrix sp., RFNB30 Pseudomonas pertucinogena 0.213 Paucimonas sp., Stenotrophomonas sp., Phyllobacterium RFNB31 Phyllobacterium myrsinacearum 0.579 sp., Grimontia sp., etc. The highest number of isolates RFNB32 Sphingomonas paucimobilis 0.369 was assigned to Burkholderia sp. and Sphingomonas sp. Isolates RFNB3, RFNB8, RFNB21, RFNB26, RFNB27 Isolates not matched with information available in MIDI Aerobic Bacteria Library TSBA60 and RFNB29did notgive a matchwiththe MIDI Aerobic 1648 Ann Microbiol (2012) 62:1643–1650 Fig. 1 BOX-PCR Genetic Distance (%) BOX Isolate Cluster fingerprinting gel and unweighted pair grouping with RFNB5 Brochothrix mathematic average (UPGMA) RFNB24 Stenotrophomonas dendrogram showing genetic RFNB7 Methylobacterium I relationship as estimated by the RFNB1 Methylobacterium cluster analysis of products of RFNB21 N-fixing isolates obtained from RFNB9 Paucimonas different paddy soil samples. RFNB8 II Scale bar Percent similarity RFNB30 Pseudomonas RFNB18 Burkholderia RFNB17 Burkholderia RFNB16 Burkholderia RFNB10 Novosphingobium RFNB15 Enterobacter III RFNB14 Klebsiella RFNB12 Burkholderia RFNB11 Burkholderia RFNB26 RFNB29 RFNB2 Mycobacterium RFNB27 RFNB25 Grimontia RFNB19 Burkholderia IV RFNB23 Sphingomonas RFNB22 Sphingomonas RFNB3 RFNB31 Phyllobacterium RFNB13 Roseomonas RFNB20 Pseudomonas RFNB6 Bacillus VI RFNB28 Novosphingobium RFNB4 Paenibacillus VII RFNB32 Sphingomonas Bacteria Library TSBA60 and therefore we were not able 2004). On the other hand, FAME profiles are used routinely to to identify them. Earlier studies by McInroy and Kloepper identify genera, species and strains of bacteria (Cavigelli et al. (1995) and Lilley et al. (1996) found that MIDI-FAME 1995; Ibekwe and Kennedy 1999). This analysis was devel- systems could identify 95% and 80% of their isolates, oped to identify bacterial species more quickly and easily, and respectively. These differences may be due to the different is currently able to identify accurately over 1,500 species of similarity standards used: 0.1 in McInroy and Kloepper bacteria, many to the subspecies or strain level (Kunitsky et al. (1995), 0.3 in Poonguzhali et al. (2006) and unknown in 2005). Earlier, Cottyn et al. (2001) used FAME analysis Lilley et al. (1996). successfully to classify phenotypic diversity among bacterial DNA-based technology, which typically uses only the 16S isolates. rRNA gene as the basis for microbial identification, has the In conclusion, the classification of N-fixing isolates accord- advantage of identifying difficult-to-cultivate strains and is ing to biochemical analyses and generated BOX-PCR finger- growth independent. As the 16S rRNA gene is highly con- prints supported the observation that there is a high degree of served at the species level, speciation is normally quite good, nifH gene diversity in paddy field soils. The information on but subspecies and strain level differences are not shown the N-fixing bacteria of paddy soils obtained in the present (Kunitsky et al. 2005). Some other problems with 16S rRNA report provide a framework that will aid in their DNA signa- technology include the requirement for a high level of techni- ture sequences for further ecological and taxonomic studies. cal proficiency, and the cost per sample, as well as equipment costs, are high (Slabbinck at al. 2010). Therefore, this tech- Acknowledgments This work was supported by grants from the nology is not well suited for routine microbial quality control Korea Research Foundation (KRF), Republic of Korea. We thank the in the pharmaceutical and other sectors (Sutton and Cundell anonymous reviewers for their thorough evaluation and constructive Ann Microbiol (2012) 62:1643–1650 1649 suggestions for improving the quality of the original manuscript. The Sherlock Microbial Identification System. 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Methods Mol Cell Biol 5:25–40 39:65–75 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Diversity of free-living nitrogen-fixing bacteria associated with Korean paddy fields

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Copyright © 2012 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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Abstract

Ann Microbiol (2012) 62:1643–1650 DOI 10.1007/s13213-012-0421-z ORIGINAL ARTICLE Diversity of free-living nitrogen-fixing bacteria associated with Korean paddy fields Md. Rashedul Islam & Tahera Sultana & Jang-Cheon Cho & M. Melvin Joe & T. M. Sa Received: 21 September 2011 /Accepted: 20 January 2012 /Published online: 18 February 2012 Springer-Verlag and the University of Milan 2012 Abstract Nitrogen (N)-fixing microorganisms play a ma- Introduction jor role in maintaining soil fertility and are thereby impor- tant for sustainable rice production. Among a total of 165 The nitrogen (N) requirement of paddy rice is well known bacterial isolates recovered from seven paddy field soils and rice production today depends on the application of through an isolation process on four N-free media, 32 were large quantities of nitrogenous fertilizers (Choudhury and found to be positive for PCR amplification of nifHgene. Kennedy 2004). Unfortunately, besides increasing produc- On screening, the BOX-PCR fingerprint technique grouped tion costs, these fertilizers also cause severe environmental the nifH gene positive isolates into seven clusters. Cluster- pollution in rice-producing environments (Wartiainen et al. ing of bacteria revealed a very low level of similarity 2008). Biological nitrogen fixation (BNF) is considered as a (20%), indicating the existence of a high degree of genetic suitable alternative in the development of sustainable agri- diversity among the N-fixing isolates. Further character- culture, satisfying human needs, and conserving natural ization based on fatty acid methyl ester (FAME) showed resources (Giller and Cadisch 1995; Vance 1997). BNF is that the isolates were members of 16 different genera, with performed by phylogenetically diverse groups of bacteria maximum number belonged to the genus Burkholderia that harbor nifH genes that encode the Fe-protein subunit of followed by Sphingomonas. Our results provide evidence nitrogenase. Among nitrogen fixers, free-living N-fixing for wide diversity of free-living N-fixing bacteria that can bacteria are important contributors to soil N, reaching up −1 −1 be used in future as a feasible alternative to N fertilizers in to 60 kg ha year (Cleveland et al. 1999). It has been rice-paddy ecosystems. reported that soil N availability is linked to the presence of N- fixing organisms (Bormann and Sidle 1990;Matthews 1992), . . . Keywords Nitrogen fixation nifH gene BOX-PCR and this plays a vital role for successive microbial colonization . . Diversity FAME Paddy soil (Walker and del Moral 2003). Wartiainen et al. (2008) reported the genetic diversity of free-living N-fixing bacteria in paddy soils based on nifH gene sequences, and addressed their contribution to the N input in rice-paddy ecosystems. BOX-PCR analysis is a highly effective approach for Electronic supplementary material The online version of this article determining genetic relationships among bacterial isolates (doi:10.1007/s13213-012-0421-z) contains supplementary material, (Versalovic et al. 1991). A high correlation exists between which is available to authorized users. BOX-PCR fingerprinting and DNA-DNA homology data, : : M. R. Islam M. M. Joe T. M. Sa and this technique has been applied in numerous taxonomic Department of Agricultural Chemistry, studies on plant-associated, environmental, medical and Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea food-associated bacteria (Rademaker et al. 1998; Lanoot et al. 2004). Fatty acid methyl ester (FAME) analysis is anoth- : : M. R. Islam (*) T. Sultana J.-C. Cho er well-established method for identification and classifica- Department of Biological Sciences, Inha University, tion of bacteria based on whole cellular fatty acids Incheon 402-751, Republic of Korea e-mail: rashed@inha.ac.kr derivatized to methyl esters (Sasser 2009). Differences in 1644 Ann Microbiol (2012) 62:1643–1650 Table 1 Different paddy soil treatments used in the present study chain length, positions of double bonds and the binding of functional groups make them very useful taxonomic markers Soil sample Treatment (Dawyndt et al. 2006). Because of cheap, fast, automated and high throughput process, FAME analysis has revolutionized Control No fertilizers including rice straw bacterial identification in a way that has now increased the NPK N-P O -K O at 110:70:80 kg/ha 2 5 2 efficiency of many laboratories around the world (Kunitsky et CNPK N-P O -K O at 110:70:80 kg/ha+7.5 Mg/ha rice straw 2 5 2 al. 2005) and, as a result, libraries of fatty acid profiles for NPKC750 N-P O -K O at 110:70:80 kg/ha+7.5 Mg/ha rice straw 2 5 2 thousands of bacteria have been developed to aid in identifi- NPKC750 N-P O -K O at 110:70:80 kg/ha+15.0 Mg/ha rice 2 5 2 straw cation (Kloepper et al. 1992, Slabbinck et al. 2008). NPKC2250 N-P O -K O at 110:70:80 kg/ha+22.5 Mg/ha rice Although numerous studies have suggested that free- 2 5 2 straw living heterotrophic N-fixers are a potentially important NPKC3000 N-P O -K O at 110:70:80 kg/ha+30.0 Mg/ha 2 5 2 source of N fixation in rice fields, knowledge of the ecol- rice straw ogy of N-fixers has not been well documented and cannot be enhanced by studying only laboratory microorganisms Chemical fertilizer was applied at the rate of 75-75-75 from 1954 to (Boddey et al. 1998; Mahadevappa and Shenoy 2000). It is 1970, 100-75-75 from 1971 to 1978, 150-86-86 from 1979 to 1985 and 110:70:80 kg/ha from 1986 to present. Rice straw was used as organic therefore necessary to study the naturally occurring diver- compost by fermenting 5 months of period sity of N-fixers in a large-scale soil ecosystem in order to understand phenotypic variation (Balandreau 1986). The objective of the present study was to assess genetic diver- 2005), LGI-P (Reis et al. 1994), BAz (Estrada-De Los Santos sity among the N-fixing isolates recovered from seven et al. 2001) and JNFb (Kirchhof et al. 1997). The pure cultures different paddy soils by analyzing nifH gene and BOX- of the isolates were maintained in nutrient broth containing PCR genomic DNA fingerprinting, and to identify the 50% (w/v) glycerol and refrigerated at −80°C. isolates by FAME analysis. In addition, the isolates were Physiological and biochemical characters of N-fixing bac- characterized physiologically, and the effects of different terial isolates were examined according to Bergey’sManual of fertilizer treatments on the occurrence and distribution of Determinative Bacteriology (Holt et al. 1994). Gram staining nifH gene were also investigated. was performed with a Gram stain kit (Difco, Detroit, MI). Qualitative tests for the production of oxidase and catalase were performed by using Difco strips as recommended by the Materials and methods manufacturer. The strains were also characterized biochemi- cally for the following basic reactions: nitrate reduction, gel- Site and soil sampling atin hydrolysis, starch, casein and urea hydrolysis (Stanier et al. 1966; Bossis et al. 2000). The results of these tests were Soil samples were collected from the National Institute of scored as either positive or negative (Table 2). Agricultural Science and Technology located at Suwon city of the Republic of Korea in October 2007 (after harvesting Amplification of nifH gene the rice crop), where research plots were established in 1954. Rice straw, prepared by fermenting for 5 months, The presence of the nifH gene in the N-fixing bacterial isolates was used as compost. Compost with and without nitrogen– was determined by amplifying a 390-bp fragment through phosphorus–potassium (NPK) fertilizer was applied to soil. PCR using a pair of specific primers, 19F (5′-GCIWTY Since 1986, chemical fertilizers and compost have been TAYGGIAARGGIGG-3′) and 407R (5′-AAICCRCCR applied at the rates noted in Table 1. While CNPK received CAIACIACRTC-3′) (Ueda et al. 1995) directed against con- ammonium sulfate, all the other treatments received urea as served sequences of the nifH gene. The amplified products N source. The control treatment received neither chemical were resolved on a 1% agarose gel in 1x TBE buffer and fertilizer nor compost amendments. The sampling was done visualized under UV light (Bio-Rad Laboratories, Richmond, by collecting soils (0–20 cm depth) from nine randomly CA). selected points within each plot using a 1.45 cm diameter soil core. Samples from each plot were then combined to form one composite sample and stored at 4°C. PCR amplification with specific BOX A1R primer Isolation and phenotypic characterization The DNA of the bacterial isolates was amplified by PCR using primer BOX A1R (5′-CTACGGCAAGGCGACGCTGACG- N-fixing bacteria were isolated using the serial dilution tech- 3′, Invitrogen, Life Technologies, Carlsbad, CA) (Versalovic nique on four N-free selective media viz., NFMM (Piao et al. et al. 1994). The PCR cycling protocol was as follows: 1 cycle Ann Microbiol (2012) 62:1643–1650 1645 Table 2 Biochemical character- Isolate name Soil sample Isolation medium 1 2 3 4 5 6 7 8 9 10 11 istics of the N-fixing bacteria isolated from paddy field soils. RFNB1 Control NFMM - -- -+ + + - + + - 1 Gram’s reaction; 2 motility; 3 casein hydrolysis; 4 starch RFNB2 NPK NFMM + - + + + + + + + - + hydrolysis; 5 gelatine RFNB3 CNPK NFMM - + + + + + + - - + - hydrolysis; 6 nitrate reduction; RFNB4 NPKC750 NFMM + + - - + + + - - - + 7 catalase production; 8 urease activity; 9 oxidase activity; RFNB5 NPKC750 NFMM + + - + + - - - - - - 10 V-P test; 11 polysaccharide RFNB6 NPKC2250 NFMM + - + + + + + + + - + production RFNB7 NPKC3000 NFMM - - - - + + + - + - - RFNB8 CNPK LGI-P - + + + + + + - + + - RFNB9 CNPK LGI-P - - - - + + + - - + - RFNB10 NPKC1500 LGI-P + - + + + + + + + + - RFNB11 Control BAz - - - + + - - - - - - RFNB12 Control BAz - + - + + - - - - + - RFNB13 Control BAz - + - + + - - - - + - RFNB14 Control BAz - + + + + + + + + + - RFNB15 CNPK BAz - + + + + + + - + - - RFNB16 NPKC1500 BAz - - - + + - - - - - - RFNB17 NPKC2250 BAz - + + + + + + + + - - RFNB18 NPKC2250 BAz - - - + + - - + - - - RFNB19 NPKC3000 BAz - + + + + + + + + - - RFNB20 NPKC3000 JNFb - - - - + + - - - - - RFNB21 Control JNFb - + - - + - - - - + - RFNB22 Control JNFb - + - - + - - - - - - RFNB23 Control JNFb - + - - + - + - - - - RFNB24 Control JNFb - - + + + + + + + - + RFNB25 NPK JNFb - - - + + - + - - + - RFNB26 NPK JNFb - - - - + + - - - + - RFNB27 NPK JNFb - + - + + + - - + + - RFNB28 CNPK JNFb - - - - + - - - - - - RFNB29 NPKC1500 JNFb - - - + + + - - - + - RFNB30 NPKC2250 JNFb - - - + + + + - - - - RFNB31 NPKC3000 JNFb - - - - + + - - - - - + Positive for the reaction/mo- tile, - negative for the reaction/ RFNB32 NPKC2250 JNFb - - - - + + - - - + - non-motile of denaturation at 95°C for 7 min; 35 cycles of denaturation at Identification System, Newark, NJ) (Sasser 2009). Briefly, 94°C for 1 min, annealing at 53°C for 1 min and extension at strains were grown overnight on nutrient agar plates. One 65°C for 8 min; 1 cycle of final extension at 65°C for 16 min; loopful of cells was harvested and transferred to a culture and a final soak at 4°C. The amplified fragments were sepa- tube; 1 ml saponication reagent added. Tubes were sealed rated by horizontal electrophoresis on a 1.5% agarose gel tightly with a teflon-lined screw cap and vortexed for 5– (15 cm×15 cm), at 75 V, for 5 h. Gels were stained with 10 s. The tubes were placed in water bath at 100°C for ethidium bromide, visualized under UV light and photo- 5 min. They were then taken out of the boiling water bath graphed. Cluster analyses of the BOX-PCR products were and cooled slightly, vortexed for 10 s and incubated in a performed using the method of unweighted pair grouping with water bath for an additional 25 min. Each tube received 2 ml mathematic average (UPGMA) in the Gelcompar II 3.5 soft- methylation reagent, was capped tightly and vortexed for ware package (Applied Math, Austin, Texas). 10 s. The tubes were placed in a water bath at 80±1°C for 10 min. Then, 1.25 ml extraction reagent was added to the Fatty acid methyl ester profiles cooled tubes followed by gentle tumbling on a rotator for 10 min. The bottom phase was removed by using a pipette. FAME profiles were obtained by saponification, methyla- Finally, 3 ml base reagent was added to upper phase remain- tion and extraction following the MIDI system (Microbial ing in the tubes and tumbled for 5 min. The upper solvent 1646 Ann Microbiol (2012) 62:1643–1650 phase was transferred to vials for fatty acid analysis. FAME However, when comparison was made based on various soil data were analyzed with Sherlock 6.0 MIDI TSBA60 library treatments, the highest number (28%) of nifH-gene-positive (see Sasser 2009), and bacteria were identified based on isolates was obtained in control plots (Table 3). This is not similarity index value. surprising, because the control plots never received any fertilizer, and thus might allow a higher number of N- fixing bacteria to grow. However, we found no differentia- tion between the occurrence of nifH isolates in plots Results and discussion amended with different levels of fertilizers, supporting the observations of Ogilvie et al. (2008) and Wakelin et al. Biochemical characterization (2007) that long-term application of N fertilizers does not have much impact on nifH abundance within agricultural In the present study, the composition of the N-fixing soils. bacteria associated with paddy soils was assessed by phenotypic characterization and by genomic DNA finger- printing analysis to evaluate genetic variation among iso- Genomic finger printing by BOX-PCR lates. With the exception of RFNB2, RFNB4, RFNB5, RFNB6 and RFNB10, all tested isolates were Gram neg- The BOX-PCR technique is a multilocus analysis used to ative; 14 isolates exhibited motility and the remaining 18 elucidate the phylogenetic relatedness among different iso- isolates were non-motile. RFNB10 and RFNB14 were lates (Martin et al. 1992; Cottyn et al. 2001). To access positive for all biochemical tests including casein, starch, genetic relationships, the N-fixing bacterial isolates were gelatine, nitrate reduction, catalase, urease, oxidase and V- submitted to DNA analysis with the BOX A1R-PCR meth- P test. The isolates RFNB2, RFNB4, RFNB6 and od to amplify the DNA with primers (BOX A1R) of repet- RFNB24 were able to produce polysaccharide (Table 2). itive and evolutionary conserved regions. BOX sequences are dispersed in the genomes of diverse bacteria (Lupski and Amplification of nifH gene and its distribution Weinstock 1992) and the primers amplify genomic regions between the two BOX elements. Successful amplification Out of a total of 165 N-fixing isolates, 32 were positive for was achieved from isolates with high polymorphism in the amplification of a 390-bp nifH gene fragment (Supplemental well resolved bands, with 3–10 amplification products rang- Fig. S1). Amplicons of the nifH gene were obtained to ing in the size from 200 to 1,350 bp. Information about the evaluate the diversity of potential N-fixing bacteria in the FAME analysis (Table 4) was used for the description of rice-paddy system. Among isolates containing the nifH BOX-PCR results (Fig. 1). gene, most were isolated from JNFb medium (41%), fol- Based on the BOX-PCR banding pattern, the isolates lowed by BAz (28%), NFMM (22%) and LGI-P (9%), were grouped into seven clusters (Fig. 1); 34% of the iso- respectively. It should be noted that the isolates obtained lates were grouped into cluster III with 45% similarity from different media represent only the culturable fraction. within the group. This cluster was most diverse, comprising Table 3 Distribution of the isolates showing nifH gene amplifications to various paddy soils and the four types of N-free medium used for isolation purposes Soil sample Medium Type of bacteria identified NFMM LGI-P BAz JNFb Control 1 (8) 0 (0) 4 (5) 4 (9) Methylobacterium, Burkholderia, Roseomonas, Klebsiella, Sphingomonas, Stenotrophomonas NPK 1 (7) 0 (4) 0 (6) 3 (8) Mycobacterium, Grimontia CNPK 1 (9) 2 (2) 1 (1) 1 (4) Paucimonas, Enterobacter, Novosphingobium NPKC750 2 (10) 0 (5) 0 (3) 0 (6) Paenibacillus, Brochothrix NPKC1500 0 (5) 1 (6) 1 (3) 1 (8) Novosphingobium, Burkholderia NPKC2250 1 (6) 0 (6) 2 (6) 2 (7) Bacillus, Burkholderia, Pseudomonas, Sphingomonas NPKC3000 1 (9) 0 (8) 1 (6) 2 (8) Methylobacterium, Burkholderia, Pseudomonas, Phyllobacterium Values in parenthesis indicate the total number of N-fixing bacteria obtained on the respective medium from seven soil samples Ann Microbiol (2012) 62:1643–1650 1647 five different genera viz. Burkholderia (RFNB11, RFNB12, (RFNB25) and Phyllobacterium (RFNB31) formed part of RFNB16, RFNB17, RFNB18), Mycobacterium (RFNB2), this group. Five isolates of cluster I, comprising different Novosphingobium (RFNB10), Klebsiella (RFNB14) and genera, viz. Methylobacterium (RFNB1, RFNB7), Brocho- Enterobacter (RFNB15). While isolate RFNB11 shared an thrix (RFNB5) and Stenotrophomonas (RFNB24), shared a identical electrophoretic profile with RFNB12, isolate low level of similarity (29%). Group II has three isolates, RFNB2 was only 45% similar with other members of this viz. Paucimonas (RFNB9), Pseudomonas (RFNB30), with group. Isolates RFNB17 and RFNB18 also shared indistin- 42% similarity within the group and approximately 40% guishable electrophoretic profiles. Cluster IV formed the similarity with group III. Group VII clustered with all the second largest group, with 22% of isolates sharing approx- other groups with a very low level similarity (20%). These imately 43% similarity among them. Sphingomonas results confirmed the existence of a wide genetic diversity (RFNB22, RFNB23), Burkholderia (RFNB19), Grimontia among N-fixing bacterial isolates recovered from paddy soils. Specific primers of the nifH genes and BOX-PCR were used in the present study, which allowed the genetic struc- Table 4 Taxonomic identification of N-fixing isolates as determined ture of N-fixers in paddy fields to be assessed through the by fatty acid methyl ester (FAME) profiles diversity of nifH genes. Our result revealed that many N- fixing bacteria isolated from different fertilization treatments Isolate code FAME identification Similarity index shared identical BOX-PCR patterns, indicating similarity RFNB1 Methylobacterium mesophilicum 0.457 among isolates. However, the fingerprinting pattern was RFNB2 Mycobacterium aichiense 0.152 complex, with seven major clusters joining with a very RFNB3 Not matched - low final level of similarity (20%) indicating a degree of diversity among N-fixers. This fingerprinting technique has RFNB4 Paenibacillus azotofixans 0.515 RFNB5 Brochothrix campestris 0.417 proven valuable in studies of N-fixing bacteria and has been used extensively in ecology, genetic and taxonomic studies, RFNB6 Bacillus megaterium 0.388 as well as for identification of N-fixing bacterial isolates (e.g., RFNB7 Methylobacterium rhodesianum 0.631 Versalovic et al. 1994;Chen et al. 2000; Kaschuk et al. 2006). RFNB8 Not matched - From our results, it appears that BOX analysis of nifHPCR RFNB9 Paucimonas lemoignei 0.729 products from environmental sample is a powerful tool for RFNB10 Novosphingobium capsulatum 0.291 assessing the presence and diversity of N-fixing microorgan- RFNB11 Burkholderia gladioli 0.471 isms in ecosystems (Sato et al. 2009), and the technique is RFNB12 Burkholderia gladioli 0.224 rapid and simple. The number and positions of the BOX RFNB13 Roseomonas sp. 0.594 fragments reflect the diversity and heterogeneity of the bacte- RFNB14 Klebsiella pneumoniae 0.308 ria in a sample. Although this approach does not directly allow RFNB15 Enterobacter hormaechei 0.631 evaluation of functional aspects of the N-fixers in a sample, RFNB16 Burkholderia gladioli 0.173 genetic information on the gene pool and the potential for N RFNB17 Burkholderia gladioli 0.214 fixation may be assessed. RFNB18 Burkholderia cenocepacia 0.518 RFNB19 Burkholderia gladioli 0.214 Identification of isolates by FAME analysis RFNB20 Pseudomonas pertucinogena 0.221 RFNB21 Not matched - The MIDI-FAME technique was used to determine the RFNB22 Sphingomonas paucimobilis 0.590 whole-cell cellular fatty acid profiles of N-fixing bacterial RFNB23 Sphingomonas paucimobilis 0.475 isolates (Table 4). In the present work, this analysis iden- RFNB24 Stenotrophomonas maltophilia 0.179 tified more than 81% of the bacteria with varied levels of RFNB25 Grimontia hollisae (Vibrio) 0.175 confidence value, for a total of 16 different genera, viz., RFNB26 Not matched - Burkholderia sp., Sphingomonas sp., Methylobacterium RFNB27 Not matched - sp., Pseudomonas sp., Novosphingobium sp., Bacillus RFNB28 Novosphingobium capsulatum 0.130 sp., Paenibacillus sp., Enterobacter sp., Klebsiella sp., RFNB29 Not matched - Mycobacterium sp., Roseomonas sp., Brochothrix sp., RFNB30 Pseudomonas pertucinogena 0.213 Paucimonas sp., Stenotrophomonas sp., Phyllobacterium RFNB31 Phyllobacterium myrsinacearum 0.579 sp., Grimontia sp., etc. The highest number of isolates RFNB32 Sphingomonas paucimobilis 0.369 was assigned to Burkholderia sp. and Sphingomonas sp. Isolates RFNB3, RFNB8, RFNB21, RFNB26, RFNB27 Isolates not matched with information available in MIDI Aerobic Bacteria Library TSBA60 and RFNB29did notgive a matchwiththe MIDI Aerobic 1648 Ann Microbiol (2012) 62:1643–1650 Fig. 1 BOX-PCR Genetic Distance (%) BOX Isolate Cluster fingerprinting gel and unweighted pair grouping with RFNB5 Brochothrix mathematic average (UPGMA) RFNB24 Stenotrophomonas dendrogram showing genetic RFNB7 Methylobacterium I relationship as estimated by the RFNB1 Methylobacterium cluster analysis of products of RFNB21 N-fixing isolates obtained from RFNB9 Paucimonas different paddy soil samples. RFNB8 II Scale bar Percent similarity RFNB30 Pseudomonas RFNB18 Burkholderia RFNB17 Burkholderia RFNB16 Burkholderia RFNB10 Novosphingobium RFNB15 Enterobacter III RFNB14 Klebsiella RFNB12 Burkholderia RFNB11 Burkholderia RFNB26 RFNB29 RFNB2 Mycobacterium RFNB27 RFNB25 Grimontia RFNB19 Burkholderia IV RFNB23 Sphingomonas RFNB22 Sphingomonas RFNB3 RFNB31 Phyllobacterium RFNB13 Roseomonas RFNB20 Pseudomonas RFNB6 Bacillus VI RFNB28 Novosphingobium RFNB4 Paenibacillus VII RFNB32 Sphingomonas Bacteria Library TSBA60 and therefore we were not able 2004). On the other hand, FAME profiles are used routinely to to identify them. Earlier studies by McInroy and Kloepper identify genera, species and strains of bacteria (Cavigelli et al. (1995) and Lilley et al. (1996) found that MIDI-FAME 1995; Ibekwe and Kennedy 1999). This analysis was devel- systems could identify 95% and 80% of their isolates, oped to identify bacterial species more quickly and easily, and respectively. These differences may be due to the different is currently able to identify accurately over 1,500 species of similarity standards used: 0.1 in McInroy and Kloepper bacteria, many to the subspecies or strain level (Kunitsky et al. (1995), 0.3 in Poonguzhali et al. (2006) and unknown in 2005). Earlier, Cottyn et al. (2001) used FAME analysis Lilley et al. (1996). successfully to classify phenotypic diversity among bacterial DNA-based technology, which typically uses only the 16S isolates. rRNA gene as the basis for microbial identification, has the In conclusion, the classification of N-fixing isolates accord- advantage of identifying difficult-to-cultivate strains and is ing to biochemical analyses and generated BOX-PCR finger- growth independent. As the 16S rRNA gene is highly con- prints supported the observation that there is a high degree of served at the species level, speciation is normally quite good, nifH gene diversity in paddy field soils. The information on but subspecies and strain level differences are not shown the N-fixing bacteria of paddy soils obtained in the present (Kunitsky et al. 2005). Some other problems with 16S rRNA report provide a framework that will aid in their DNA signa- technology include the requirement for a high level of techni- ture sequences for further ecological and taxonomic studies. cal proficiency, and the cost per sample, as well as equipment costs, are high (Slabbinck at al. 2010). Therefore, this tech- Acknowledgments This work was supported by grants from the nology is not well suited for routine microbial quality control Korea Research Foundation (KRF), Republic of Korea. We thank the in the pharmaceutical and other sectors (Sutton and Cundell anonymous reviewers for their thorough evaluation and constructive Ann Microbiol (2012) 62:1643–1650 1649 suggestions for improving the quality of the original manuscript. The Sherlock Microbial Identification System. 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Methods Mol Cell Biol 5:25–40 39:65–75

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Annals of Microbiology – Springer Journals

Published: Feb 18, 2012

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