Expressed Nifh Genes of Endophytic Bacteria Detected in Field-Grown Sweet Potatoes (Ipomoea Batatas L.)

Microbes Environ. Vol. 23, No. 1, 89–93, 2008 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.23.89

Expressed nifH Genes of Endophytic Bacteria Detected in Field-Grown Sweet Potatoes (Ipomoea batatas L.)

JUNKO TERAKADO-TONOOKA1,2*, YOSHINARI OHWAKI1, HIROMOTO YAMAKAWA3, FUKUYO TANAKA1, TADAKATSU YONEYAMA4, and SHINSUKE FUJIHARA1 1National Agricultural Research Center, Kannondai 3–1–1, Tsukuba, Ibaraki 305–8666, Japan; 2JSPS Research Fellow, Japan Society for the Promotion of Science, Ichi-ban-cho 8, Chiyoda-ku, Tokyo 102–8472, Japan; 3National Agricultural Research Center, Hokuriku Research Center, Inada 1–2–1, Jyoetsu, Nigata 943–0193, Japan; and 4Department of Applied Biological Chemistry, University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113–8657, Japan (Received November 2, 2007—Accepted December 27, 2007)

We examined the nitrogenase reductase (nifH) genes of endophytic diazotrophic bacteria expressed in field-grown sweet potatoes (Ipomoea batatas L.) by reverse transcription (RT)-PCR. Gene fragments corresponding to nifH were amplified from mRNA obtained from the stems and storage roots of field-grown sweet potatoes several months after planting. Sequence analysis revealed that these clones were homologous to the nifH sequences of Bradyrhizobium, Pelomonas, and Bacillus sp. in the DNA database. Investigation of the nifH genes amplified from the genomic DNA extracted from these sweet potatoes also showed high similarity to various α-proteobacteria including Bradyrhizobium, β-proteobacteria, and cyanobacteria. These results suggest that bradyrhizobia colonize and express nifH genes not only in the root nodules of leguminous plants but also in sweet potatoes as diazotrophic endophytes. Key words: endophyte, sweet potato, nitrogen fixation, nifH, RT-PCR

The sweet potato (Ipomoea batatas L.) is known for its identify active diazotrophic bacteria, we examined the ability to grow well in nitrogen (N)-poor soils16). It is also expression of nifH genes in sweet potato tissues by means of known that the total amount of N in sweet potato exceeds the RT-PCR targeted at the nifH gene. In a comparison with the amount of N applied as chemical fertilizer. Our previous nifH expressed in plants, we also analyzed the diversity of research found that two cultivars of sweet potatoes (cultivars the nifH genes amplified from genomic DNA isolated from Beniazuma and Ayamurasaki) grown in an upland field sweet potatoes grown under the same field conditions. (light-colored Andosol) in Tsukuba, Japan, absorbed 103 to 135 kg N ha−1 without the application of any chemical N fer- Materials and Methods tilizer (unpublished data, 2006). The sweet potato may have Sample collection special mechanisms for enhancing soil N mineralization and/ Sweet potatoes (Ipomoea batatas L, cultivars Beniazuma and or acquiring N derived from atmospheric N2. Recently, nitro- Ayamurasaki) were planted in the experimental field of the National gen fixation by endophytic diazotrophs has been observed in Agricultural Research Center, Tsukuba, Japan, from June to Octo- a wide variety of plants30). Our previous experiment using a ber. Beniazuma is a sweet and starchy cultivar that is the most pop- 15N natural abundance technique indicated that endophytic ular sweet potato in Japan. Ayamurasaki is a purple sweet potato with high anthocyanin content. Beniazuma was grown in light-col- N2 fixation contributes as much as 40% of the N intake of the 41) ored Andosol (Total N 0.54%, pH 6.1; in 2006) in 2002 and 2004 sweet potato to N nutrition . By using a cultivation tech- and Ayamurasaki was grown in gray lowland soil (Total N 0.13%, nique with an N-free culture medium, endophytic diaz- pH 6.2; in 2006) in 2005 and 2006. The applied fertilizer was N (30 −1 −1 −1 otrophic bacteria such as Klebsiella, Pantoea, and Gluconac- kg ha ), P2O5 (100 kg ha ), and K2O (100 kg ha ). The plants etobacter have been isolated from various cultivars of sweet were harvested and dissected into stems and storage roots in August 1,4,9) potatoes . Moreover, the presence of a variety of N2-fixing and/or October of each year. The samples were washed with tap bacteria in the African sweet potato has also been proven by water and surface layers of the stems and storage roots were polymerase chain reaction (PCR) amplification of the nifH removed with a sterilized peeler. Then, the remaining tissues were washed with sterilized water and frozen with liquid nitrogen. gene, indicating that many species of bacteria which have not yet been isolated from sweet potatoes by conventional cul- DNA and RNA isolation ture techniques might exist as diazotrophic endophytes29). In For the isolation of DNA and RNA, 5 g fresh weight of frozen many diazotrophs, nitrogenase activities correspond well to tissue was ground to a fine powder with a mortar and pestle in liquid the levels of nifH transcription under various nitrogen. Total DNA was isolated by cetyltrimethylammonium bro- conditions11,12,20,32). Therefore, the detection and sequence mide (CTAB) treatment followed by chloroform-isoamyl alcohol extraction and ethanol precipitation21). RNA was extracted using the analysis of nifH amplified from mRNA can provide valuable phenol-sodium dodecyl sulfate (SDS) method34,36) and further puri- information on the identification of nitrogen-fixing bacteria fied using an RNeasy plant Mini Kit (Qiagen Sciences, Inc., Ger- as well as evidence for their nitrogen fixation in situ. To mantown, MD, USA) following the manufacturer’s instructions. Purified RNA was incubated with Dnase I (Takara Bio, Inc., Otsu, * Corresponding author. E-mail: [email protected]; Tel: +81–29– Japan) at 37°C for 30 min. Reverse transcription was conducted 838–8814; Fax: +81–29–838–8814. using a Gene Amp Gold RNA PCR Core Kit (Applied Biosystems, 90 TERAKADO-TONOOKA et al.

Foster City, CA, USA). Each 20 µl of reaction mixture contained the rhizobial species. In African sweet potatoes, about 50% 100 ng of total RNA, 1×RT-PCR buffer, 2.5 mM MgCl2, 1 mM of nifH genes derived from rhizobia, such as Shinorhizobium, dNTP mixture, 1.25 mM Random Hexamer, 10 mM DTT, 10 U Rhizobium, Mesorhizobium, or relatives belonging to the α- RNase inhibitor, and 15 U reverse transcriptase. Reverse transcrip- proteobacteria29). These results indicate that bradyrhizobia tion was carried out at 25°C for 10 min and 42°C for 12 min. and rhizobia may be the potential endophytic diazotrophs in PCR amplification sweet potatoes. The extracted DNA and transcribed cDNA were analyzed by Sequences with high similarity to β-proteobacteria nested PCR using four degenerate oligonucleotide primers, which (Herbaspirillum seropedicae, Burkholderia vietnamiensis, were designed to match the nifH gene sequences of a broad range Burkholderia unamae, Pelomonas saccharophila, and Azo- of bacteria: nifH1 (5'-TGYGAYCCNAARGCNGA-3'), nifH2 (5'- hydromonas australica) were also found in stems and storage ADNGCCATCATYTCNCC-3')44), nifH3 (5'-ATRTTRTTNGCNG- CRTA-3') and nifH4 (5'-TTYTAYGGNAARGGNGG-3')43). For roots (Table 1). H. seropedicae has been isolated as a diaz- nested PCR, the primers PolF (5'-TGCGAYCCSAARGCBGA- otrophic endophyte in many crops including rice, sugarcane, CTC-3') and PolR (5'-ATSGCCATCATYTCRCCGGA-3') were maize, sorghum, and banana24,37). The B. vietnamiensis spe- used based on the conserved sequence of nifH26). One microliter cies were isolated from rhizosphere soil of rice plants13) and (100 ng) of DNA or cDNA was added to 14 µL of the first-round confirmed as diazotrophic endophytes8). P. saccharophila × µ PCR mixture (1.5 mM MgCl2, 1 PCR Gold buffer, 800 M dNTPs, and A. australica are frequently isolated from soils as diaz- 1 µM each of primers, and 0.5 U of Amplitaq Gold DNA poly- 40) merase LD; all from Applied Biosystems). PCR was carried out otrophic bacteria . with 40 cycles of denaturation at 95°C (1 min), annealing at 53°C (1 Sequences similar to cyanobacteria (Anabaena sp., Nostoc min), and extension at 72°C (1 min). A second round of nested PCR commune, Tolypothrix sp. and Microcoleus sp.) were found was performed with 1 µL of the first-round product and 14 µL of primarily in sweet potato stems (Table 1). Cyanobacteria are the PCR mixture under the same conditions as those applied in the blue-green algae that are a diverse group of gram-negative first step of PCR. photosynthetic prokaryotes. Some filamentous bacteria Cloning and sequencing including Anabaena and Nostoc can form a symbiotic rela- The amplified DNA fragments corresponding to the anticipated tionship with certain plants such as Azolla, Gunnera and fix 22) size of approximately 360 bp were cloned into E. coli cells using N2 . TOPO TA cloning kits (Invitrogen, Carlsbad, CA, USA) following In the present study, nifH sequences similar to those the protocol recommended by the manufacturer. Sequencing was belonging to γ-proteobacteria groups such as Gluconaceto- performed by Hokkaido System Science Co., Ltd. (Sapporo, Japan) bacter, Klebsiella, and Pantoea were not recovered from the and submitted to the DNA Data Bank of Japan (DDBJ) nucleotide stems or storage roots of sweet potatoes although these bac- sequence database under accession numbers AB265689, AB365413–AB365434, AB373745–AB373747. teria have been isolated in the sweet potato using a culture- dependent technique1,4,9). In African sweet potatoes, nifH Phylogenetic analysis sequences simlar to K. pneumonia were also detected29). We A homology-based search of the GenBank-EMBL-DDBJ DNA confirmed that the gene fragments corresponding to nifH can database was performed with the BLAST program using the nifH be amplified from bacterial DNA under the same PCR condi- gene fragment. The nucleotide sequences were aligned using the tions (data not shown), indicating that these diazotrophic CLUSTAL W program and a phylogenetic tree of nifH sequences was constructed using Tree View 1.6.6 with the neighbor-joining bacteria are not predominant under the present cultivation method33). conditions. In sugarcane, the endophytic bacteria G. diazotrophicus Results and Discussion can be transferred to subsequent crops via sets (stem cut- tings) used in vegetative propagation28). To examine the pre- Representative clones of the nested PCR products, which existence of the diazotrophic endophyte in planting material, were amplified from genomic DNA or mRNA of field-grown we amplified nifH genes from sweet potato sprouts before sweet potatoes (stems and storage roots), were selected transplanting. However, we were unable to detect any nifH according to the results of the homology analysis (Tables 1 fragments from the DNA extracts of the sprouts (data not and 2), and a phylogenetic tree was constructed (Fig. 1). No shown). Reiter et al. explain that the African sweet potato, nifH fragments were amplified from DNA isolated from leaf which had an abundance of rhizobial strains, was obtained samples, indicating that endophytic bacteria exist primarily from small fields where crop rotation with leguminous plants in stems and storage roots (data not shown). The present is common29). Given that most of the nifH sequences detected homology analysis based on the nifH sequence amplified in the present study were homologous to soil bacteria, we from genomic DNA revealed that the clones could be divided hypothesize that most sweet potatoes containing endophytic into α- and β-proteobacteria as well as cyanobacteria (Table bacteria are infected from the external environment post- 1). Most of the clones showed similarity to α-proteobacteria, transplant. especially the rhizobial species (Bradyrhizobium sp., Some endophytic bacteria such as G. diazotrophicus, H. Bradyrhizobium japonicum, Rhizobium leguminosarum, Shi- seropedicae, and B. sp. can infect plants via lateral root junc- norhizobium sp. and Azorhizobium caulinodans) that are tions after inoculation5,18). B. sp. and A. caulinodans also known to nodulate and fix nitrogen in leguminous plants. infect plants through cracks at the base of the root primordia, NifH fragments similar to other α-proteobacteria including not only in the roots but also in the stems of leguminous methanotrophic bacteria (Methylosinus trichosporium) and plants. They are also known to enter the roots of non-legumi- an aerobic nitrogen fixer (Beijerinckia derxii) were also nous plants such as rice and wheat via cracks6,27,38). In the amplified, although clones were less abundant than those of present study, nifH sequences homologous to Bradyrhizo- Bacterial nifH Genes in Sweet Potatoes 91

Table 1. Similarities of sweet potato nifH gene sequences amplified from DNAs to the most similar sequences from known diazotrophic bacteria Sampling Phylogenetic Similarity Clone Representative Cultivar date Tissue Closest bacteria position (%) number clones Beniazuma Oct. 2002 Stem Anabaena sp. CPP 7120 cyanobacteria 90 8 DNA1 Herbaspirillum seropedicae β-proteobacteria 97 5 DNA2 Anabaena sp. CPP 7120 cyanobacteria 88 2 DNA3 Nostoc commune cyanobacteria 98 1 DNA4 Tuber Rhizobium leguminosarum α-proteobacteria 94 14 DNA5 Bradyrhizobium japonicum α-proteobacteria 98 6 DNA6 Aug. 2004 Stem Azohydromonas australica β-proteobacteria 96 4 DNA7 Oct. 2004 Stem Azohydromonas australica β-proteobacteria 97 7 DNA7 Bradyrhizobium sp. MAFF210318 α-proteobacteria 93 2 DNA8 Bradyrhizobium sp. IRBG230 α-proteobacteria 93 2 DNA9 Tuber Bradyrhizobium sp. MAFF210318 α-proteobacteria 93 6 DNA8 Bradyrhizobium japonicum α-proteobacteria 98 3 DNA6 Burkholderia vietnamiensis β-proteobacteria 87 2 DNA10 Beijerinckia derxii sp. venezuelae α-proteobacteria 83 2 DNA11 Ayamurasaki Oct. 2005 Stem Bradyrhizobium sp. IRBG230 α-proteobacteria 94 7 DNA9 Tuber Azorhizobium caulinodans ORS571 α-proteobacteria 91 12 DNA12 Anabaena sp. CPP 7120 cyanobacteria 89 4 DNA3 Azohydromonas australica β-proteobacteria 97 4 DNA7 Beijerinckia derxii sp. venezuelae α-proteobacteria 83 3 DNA11 Pelomonas saccharophila β-proteobacteria 94 3 DNA13 Burkholderia vietnamiensis β-proteobacteria 87 3 DNA10 Aug. 2006 Stem Tolypothrix sp. PCC 7601 cyanobacteria 91 4 DNA14 Pelomonas saccharophila β-proteobacteria 94 3 DNA13 Burkholderia unamae β-proteobacteria 92 2 DNA15 Azohydromonas australica β-proteobacteria 94 2 DNA19 Tuber Bradyrhizobium sp. IRBG230 α-proteobacteria 94 3 DNA9 Burkholderia unamae β-proteobacteria 91 2 DNA15 Pelomonas saccharophila β-proteobacteria 93 4 DNA13 Azohydromonas australica β-proteobacteria 93 2 DNA19 Microcoleus sp. PC8701 cyanobacteria 93 1 DNA16 Oct. 2006 Stem Pelomonas saccharophila β-proteobacteria 93 3 DNA13 Tuber Bradyrhizobium sp. IRBG230 α-proteobacteria 93 4 DNA9 Shinorhizobium sp. JT170 α-proteobacteria 92 3 DNA17 Azohydromonas australica β-proteobacteria 93 2 DNA19 Pelomonas saccharophila β-proteobacteria 94 3 DNA13 Methylosinus trichosporium α-proteobacteria 90 2 DNA18 Clones that exceeded 99% base sequence similarity with each other were divided into different groups and a representative clone was chosen from each group.

Table 2. Similarities of sweet potato nifH gene sequences amplified from RNAs to the most similar sequences from known diazotrophic bacteria

Cultivar Sampling date Tissue Closest bacteria Phylogenetic Similarity Clone Representative position (%) number clones Beniazuma Oct. 2002 Stem Bradyrhizobium sp. IRBG228 α-proteobacteria 95 4 RNA1 Bacillus sp. BT97 Firmicutes 98 14 RNA2 Tuber Bradyrhizobium japonicum α-proteobacteria 99 2 RNA3 Bacillus sp. BT97 Firmicutes 98 11 RNA2 Aug. 2004 Stem Bradyrhizobium sp. MAFF 210318 α-proteobacteria 93 4 RNA5 Bradyrhizobium sp. IRBG230 α-proteobacteria 93 6 RNA6 Oct. 2004 Stem Not detected Tuber Not detected Ayamurasaki Oct. 2005 Stem Not detected Tuber Not detected Aug. 2006 Stem Pelomonas saccharophila β-proteobacteria 97 4 RNA7 Tuber Bradyrhizobium sp. IRBG230 α-proteobacteria 94 5 RNA6 Oct. 2006 Stem Not detected Tuber Not detected Clones that exceeded 99% base sequence similarity with each other were divided into different groups and a representative clone was chosen from each group. 92 TERAKADO-TONOOKA et al.

genes expressed were not simply reflected by the population of microorganisms; rather, specific nifH genes were expressed under certain environmental conditions. In experi- ments similar to the present ones, only one alternative nitrogenase gene was found to be preferentially expressed in spite of the existence of remarkably diverse nifH sequences in the termite gut microbial community23). Many environmental 12,42) factors such as the O2 concentration and nitrogen sources might limit or regulate nifH gene expression in plants. Some Bradyrhizobium, including B. spp., fix nitrogen in the free-living state under low oxygen concentrations2,10). B. sp. IRBG228 and sp. IRBG230 are also known to be photosynthetic strains having bacteriochlorophyll, that may provide energy for nitrogen fixation and allow bacteria to sur- vive inside the plant14). These endophytic bradyrhizobia might fix nitrogen in sweet potatoes under free-living conditions. Rhizobia change to a symbiotic form, a bacteroid in nodules, which is surrounded by a peribacteroid membrane of plant origin. Endophytic bacteria, Azoarcus spp. are also known to form an intracytoplasmic membrane (diazosomes) under microaerobic conditions17). It is suggested that this morphological change of Azoarcus sp. is analogous to the differentiation of rhizobia to bacteroids. Investigating the morphology of bradyrhizobia colonized in plants using microscopic techniques may help to define the mechanism of endophytic nitrogen fixation. Fig. 1. Phylogenetic relationships among the sequences of representative clones listed in Tables 1 and 2 and the nifH sequence from the DNA In endophytic association, carbohydrates also limit the 32) database. Rhodobacter capsulatus bchL was used as the outgroup. The growth and N2-fixation of diazotrophic bacteria . N2-fixing scale bar denotes 0.1 substitutions per site. activities have been found only when carbohydrates such as sucrose7), malate15,39) and citrate31) were fed to the plant. In bium, Azorhydromonas, Pelomonas, and Burkholderia were the present experiment, the expression of the nifH gene was frequently detected in Beniazuma and Ayamurasaki. On the detected in August, two months after planting (Table 2), but other hand, sequences similar to A. caulinodans, H. seropedi- was rarely detected in October. In the case of soybeans, cae, and R. leguminosarum were not always detected. Envi- nitrogen fixation depends on the amount of photosynthetic ronmental conditions can influence the bacterial flora in products, and maximal nitrogenase activity is usually associ- fields and this may be reflected in the variety of bacteria ated with the highest level of photosynthetic activity. Previ- inside the sweet potatoes. ous reports have shown that the amount of chlorophyll, one We successfully amplified a fragment of the nifH gene of the indicators of the photosynthetic rate, reaches maxi- from RNA extracted from stems and storage roots of the mum at 3 months after transplantation in the sweet potato cv. field-grown sweet potatoes (Table 2). In the absence of Beniazuma in Japan25). The expression of the nifH gene reverse transcriptase, no amplification was detected, indicat- might also be correlated with the photosynthetic rate in sweet ing that the RT-PCR specifically detects nifH mRNA. potatoes. Although the existence of nifH genes in sweet potatoes has In the present experiment, we demonstrated that nifH been reported previously29), to the best of our knowledge, the genes are expressed in field-grown sweet potatoes. This find- present study is the first to show the expression of nifH genes ing strongly supports the results of our previous study, which in field-grown sweet potatoes. indicated the possible input of atmospheric nitrogen into Sequence analysis revealed the clones expressed in sweet sweet potatoes41). However, to the best of our knowledge, potatoes to be homologous to nifH gene fragments of most of the bacteria detected in the present study have not bradyrhizobia (B. sp. IRBG228, sp. IRBG230, sp. MAFF yet been isolated from sweet potatoes by any culture-depen- 210318 and B. japonicum), Pelomonas, and Bacillus sp. dent technique although endophytic bacteria, Azoarcus spp., BT97. Bacillus has been isolated from many crops such as are known to fix nitrogen in an unculturable state17). To con- rice, citrus plants, maize, and carrots as a diazotrophic firm the presence of diazotrophic bacteria, further studies endophyte3,19,35). with improved culturing methods are necessary, and the tran- Phylogenetic analysis showed that the nifH sequences scription of nifH in endophytic bacteria should be monitored derived from DNA clustered in various taxonomic groups, under various environmental conditions. though the diversity of the nifH sequence in RNAs was less significant than that in DNAs (Tables1 and 2, Fig. 1). More- Acknowledgements over, the nifH sequences, which were amplified in RNAs, The authors would like to thank Dr. T. Kuranouchi of the were not always detected in DNAs, indicating that the nifH National Institute of Crop Science, Japan, for supplying the various Bacterial nifH Genes in Sweet Potatoes 93 sweet potato lines. This work was carried out as part of a research 23) Noda, S., M. Ohkuma, R. Usami, K. Horikoshi, and T. 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