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.