Impact of Plant Genotype and Nitrogen Level on Rice Growth Response to Inoculation with Azospirillum Sp
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Soil Science and Plant Nutrition (2010) 56, 636–644 doi: 10.1111/j.1747-0765.2010.00499.x ORIGINAL ARTICLE⁄SHORT PAPER Impact of plant genotype and nitrogen level on rice growth response to inoculation with Azospirillum sp. strain B510 under paddy field conditions Kazuhiro SASAKI1,SeishiIKEDA1,2,ShimaEDA1, Hisayuki MITSUI1, Eiko HANZAWA1, Chiharu KISARA1,YuriKAZAMA1,AtsuhikoKUSHIDA2, Takuro SHINANO3,KiwamuMINAMISAWA1 and Tadashi SATO1 1Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577 Japan, 2Memuro Research Station, National Agricultural Research Center for Hokkaido Region, Hokkaido 082-0081 and 3National Agricultural Research Center for Hokkaido Region, Hokkaido 062-8555, Japan Abstract Twenty rice cultivars, including three genetically-distinct groups (japonica, indica-1,andindica-2), were evaluated for their response to inoculation with Azospirillum sp.strainB510inpaddyfieldswithstandard nitrogen (SN) and low nitrogen (LN) fertilization. In the SN field, the tiller numbers in most indica-2 cultivars, 37 days after transplanting (DAT), were significantly increased by the B510 inoculation, whereas those in 4 japonica cultivars were significantly decreased. A similar growth response was observed in the LN field, although the impacts of theB510inoculationweremorevariedthanintheSNfield.At58DAT,thetiller numbers in most cultivars were lower or unaffected by the B510 inoculation under both SN and LN condi- tions, except that the tiller number of the Nipponbare cultivar, which is classified as japonica, was signifi- cantly higher in the LN field only. These results suggest that the effects of inoculation with Azospirillum sp. strain B510 on the growth of rice plants, especially on tiller numbers at the early growth stage, vary depend- ing on the rice genotype, as well as nitrogen level. Therefore, the plant genotypes, growth stages, and fertiliza- tion managements must be considered when a plant-associated bacterium is evaluated for beneficial effects under field conditions. Key words: Azospirillum sp., nitrogen fertilization, Oryza sativa L., plant growth promotion, rice plant INTRODUCTION and Baldani 2006). Inoculation with Azospirillum sp. pro- motes plant growth, so agronomic applications of this A wide range of microorganisms, including fungi and bac- genus have been developed (Okon and Labandera-Gonz- teria, has been found in the phytosphere (Mano and Mori- alez 1994). Plant growth promotion using various strains saki 2008; Saito et al. 2007, 2008). Among these, plant of Azospirillum has been investigated on several plant spe- growth-promoting rhizobacteria (PGPR), which have ben- cies. Wheat plants treated with Azospirillum brasilense eficial effects on host plants, such as increased crop yield, Sp245 exhibited reduced root length, increased root–hair have been well characterized from both scientific and formation, and increased above-ground biomass and ear practical perspectives. numbers (Spaepen et al. 2008). In maize, seed inoculation The genus Azospirillum, characterized by spirillum- with Azospirillum lipoferum CRT1 induced changes in shaped, N2-fixing, Gram-negative alphaproteobacteria, root morphology, such as increased length and biomass contains species often found in the rhizosphere (Hartman (El Zemrany et al. 2007). In tomatoes, Azospirillum brasi- lense Sp7 and Azospirillum sp. BNM-65 exhibited posi- tive effects, such as increased shoot and root biomass Correspondence: Dr K. SASAKI, Graduate School of Life Sciences, Tohoku University, Sendai, Japan. E-mail: kazu@ige. (Romero et al. 2003). The total nitrogen content in rice tohoku.ac.jp grains was improved by inoculation with Azospirillum Received 2 January 2010. brasilense REC3 under low nitrogen (LN) fertilization Accepted for publication 14 June 2010. conditions (Pedraza et al. 2009). Treatment with a nitro- Ó 2010 Japanese Society of Soil Science and Plant Nutrition Rice response to Azospirillum sp. B510 637 gen-fixing strain of Azospirillum lipoferum to the root levels on plant growth promotion by Azospirillum sp. zone of rice plants promoted the early tillering and repro- under field conditions. Therefore, the aim of the present ductive growth of host plants, although the total dry study was to examine the cross-effects of rice genotype weight and nitrogen content in the rice plants were not and nitrogen level on the response of rice plants to inocu- markedly increased by the inoculation (Watanabe and Lin lation with Azospirillum sp. B510 under paddy field 1984). conditions. Azospirillum sp. strain B510 was isolated from a sur- face-sterilized stem of a rice plant (Oryza sativa cv. Nip- ponbare [NI]) that was grown in an experimental paddy MATERIALS AND METHODS field (Elbeltagy et al. 2001). The B510 strain is closely related to Azospirillum oryzae COC8 (97.7% identity in Plant materials and growth conditions their 16S rRNA gene sequences), which was reported as a Plant materials were obtained from the National Institute paddy soil bacterium (Xie and Yokota 2005). In addition of Agrobiological Sciences (NIAS) Genebank (Tsukuba, to being a diazotroph under free-living conditions, B510 Japan). In total, 20 rice cultivars (10 cultivars each for was found to have positive motility and to be capable of japonica and indica types) were used (Table 1). The 10 degrading plant cell walls (Elbeltagy et al. 2001). Inocula- indica-type cultivars were classified into two subgroups tion with Azospirillum sp. B510 was shown to promote (indica-1 and indica-2), based on a principal coordinate plant growth under both laboratory and field conditions analysis using 179 restriction fragment length polymor- (Isawa et al. 2010). Noticeably, the field experiment in phism data (Ebana et al. 2008; Kanemura et al. 2007; Hokkaido, Japan indicated that inoculation with B510 Kojima et al. 2005; Uga et al. 2009). increases tiller number, resulting in an increase in seed Seeds from each cultivar were placed on two layers of yield at commercial levels (Isawa et al. 2010). Moreover, filter paper (Toyo Roshi Kaisha, Tokyo, Japan) in a Petri the B510 inoculation enhanced disease resistance to fun- dish (6-cm diameter) containing 4 mL tap water. The gal and bacterial pathogens (Yasuda et al. 2009). A gen- Petri dishes were placed in an incubator at 30°C. After ome analysis of B510 has been completed and revealed 2 days (30 April 2009), the germinated seeds were sown that the B510 strain possesses putative plant hormone- in a commercial soil (Mitsui-Toatsu no. 3, Tokyo, Japan) related genes, which could promote plant growth (Kaneko in a 60 · 30 cm cell tray (cell diameter, 1.5 cm; depth, et al. 2010). Thus, Azospirillum sp.strainB510isa 3 cm) and grown in a greenhouse under natural light con- likely beneficial plant-associated bacterium for agronomic ditions for 4 weeks. After 3 weeks in the greenhouse, applications. selected seedlings of each cultivar were inoculated with Plant genotype is known to influence the plant growth- Azospirillum sp. strain B510, as described in the next sec- promotion effects of inoculation with Azospirillum (Bashan and Holguin 1997). For example, significant Table 1 Rice plant materials used this study effectsongrowthandyieldofmaizeplantswereobserved in one bacterium–plant combination and absent from Cultivar name Origin Variety ⁄ group another combination, indicating an unknown interaction Nipponbare Japan Japonica between plant genotype and bacterial strain (Garcia De Koshihikari Japan Japonica Salomone and Dobereiner 1996). Similar phenomena Fukkurinko Japan Japonica were reported when local Nepalese wheat varieties and Nanatsuboshi Japan Japonica mustardwereinoculatedwithlocalAzospirillum spp. Akage Japan Japonica (Bhattarai and Hess 1993; Kesava Rao et al. 1990). Sekiyama Japan Japonica It is also known that fertilization management can Habataki Japan Japonica Dianyu 1 China Japonica affect the community structures of plant-associated bacte- Khao Nam Jen Laos Japonica ria (Coelho et al. 2009; Ikeda et al. 2010; Roesti et al. Khau Mac Kho Vietnam Japonica 2006). There is debate regarding the impacts of fertiliza- Tupa 121–3 Bangladesh Indica-1 tion strategies on the effects of PGPR, such as Azospiril- Kasalath India Indica-1 lum species. The plant growth promotion was observed Muha Indonesia Indica-1 only under LN conditions in some studies (Dobbelaere Basilanon Philippines Indica-1 et al. 2001; Fallik and Okon 1996), whereas other studies Bei Khe Cambodia Indica-2 showed that the use of nitrogen in combination with Ryou Suisan Koumai China Indica-2 Azospirillum produced significantly higher green and Deng Pao Zhai China Indica-2 dry-matter yields (Chela et al. 1993; Okon and Labander- Naba India Indica-2 Milyang 23 Korea Indica-2 a-Gonzalez 1994). Furthermore, little is known about the Bleiyo Tailand Indica-2 cross-effects of rice genotypes and nitrogen fertilization Ó 2010 Japanese Society of Soil Science and Plant Nutrition 638 K. Sasaki et al. Table 2 Characteristics of field soils pH Total C Total N Truog phosphorous )1 Field (H2O) content (%) content (%) content (mg P2O5 kg ) Standard nitrogen 5.2 4.3 0.10 74.0 Low nitrogen 5.5 3.3 0.08 62.5 tion. A total of 100 seedlings of each cultivar were planted grown under SN and LN conditions using the following in an experimental field (37°28¢N, 141°06¢E) in a square equation: pattern (5 · 5 plants). Hills were spaced 30 cm apart. IEI (%) ¼ (tiller number or shoot length of rice The seedlings inoculated with B510 and those that were plants inoculated with B510)/(tiller not inoculated were cultivated nearby in the