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Identification of endophytic diazotrophs habitating sweetpotato stems and the approach to utilize the endophytes for crop production
Katsuki Adachi
National Agricultural Research Center for Kyushu Okinawa (KONARC), Department of Upland Farming Research, Laboratory of Crop Production Management, Miyakonojo, Miyazaki 885-0091, Japan.
Sweetpotato is one of the important crops in the southern Kyushu, Okinawa, and Kanto regions in Japan. In general, relatively low amount of N fertilizer is applied for sweetpotato cultivation. Yoneyama et al.14, 15) suggested biological nitrogen fixation (BNF) in sweetpotato plants by 15N natural abundance method. Recently the evaluation of BNF in sweetpotato plants has been conducted and is preceding10). Hence, our laboratory of Crop Production Management, Department of Upland Farming Research, conducted isolation and identification of diazotrophic bacteria habitating sweetpotato stems in Japan.
Adachi et al.1) isolated an endophytic diazotroph, Klebsiella oxytoca strain BO-1, from surface-sterilized stem of cultivar Beniotome by using semi-solid nitrogen-free medium. Asis and Adachi4) isolated the other endophytic diazotroph, Pantoea agglomerans strain MY1 from cultivar Koganesengan by using semi-solid modified Rennie medium. Figure 1 shows photographs of the microscopic observation of K. oxytoca strain BO-1 (A) and P. agglomerans strain MY1 (B). Then we examined isolation of the endophytic diazotrophs habitating sweetpotato stems from different areas, i.e. Miyakonojo, Tanegashima, Okinawa, and Tsukuba, in Japan. Diazotrophic strains of Klebsiella spp. and Pantoea agglomerans have been isolated from these areas.
A B C
Figure 1. Optical microphotographs of diazotrophic Klebsiella oxytoca strain BO-1 (A, left), diazotrophic Pantoea agglomerans strain MY1 (B, center), and non-diazotrophic Enterobacter asburiae strain MY2 (C, right). Bars indicate 10ƒΚm. (Referred from Adachi et al.1) and Asis and Adachi4))
Among the endophytic strains from sweetpotato stems, non-diazotrophic endophytes were also isolated, one of which, strain MY2 isolated from Koganesengan, was identified as Enterobacter asburiae. Figure 1 (C) shows the photograph of E. asburiae strain MY2. Because they, diazotrophic and non-diazotrophic endophytes, are habitating the same niches of sweetpotato stems and may have certain close relation, we conducted co-culture experiment of diazotrophic P. agglomerans and non-diazotrophic E. asburiae in test tubes. Figure 2 shows the N2-fixing activity of co-culture and mono-culture of P. agglomerans with and without E. asburiae, respectively, after 36 h incubation in test tube experiment3). The N2-fixing activity of P. agglomerans under co-culture conditions with E. asburiae was enhanced approximately twice compared to that under mono-culture conditions of P. agglomerans. It is suggested that they may have interaction linkage in sweetpotato stems to enhance N2-fixing activity.
Figure 2. Enhancement of N2-fixing activity of Pantoea agglomerans strain MY1 by co-culture with non-diazotrophic Enterobacter asburiae strain MY2 compared with mono-culture of P. agglomerans MY1. Aliquots of each inocula of cell suspensions of P. agglomerans MY1 and E. asbriae MY2 were inoculated to mono-culture tubes (P. agglomerans only) and co-culture tubes (both of P. agglomerans and E. asburiae) by using a semi-solid modified Rennie medium. The N2-fixing activity (acetylene reduction activity (ARA) value) was examined after 36 h incubation. (Referred from Adachi and Asis3))
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In order to confirm the occurrence and to investigate the localization of endophytic diazotrophs in@different portions of sweetpotato stems, stem-pieces-incubation (SPI) and acetylene-reduction-activity (ARA) method was conducted using a semi-solid modified Rennie medium2). Figure 3 shows the sketch of sampled portions (portions 1-5) of the stem and sliced stem-pieces (pieces 1-5) from each sampled internode. Two stem samples were taken two months after planting (2 MAP, July 30-31, 2002) and 4 MAP (September 18-20, 2002). Positive responses of the stem-pieces to ARA were detected, confirming the occurrence of diazotrophic endophytes in the stems of Japanese sweetpotato cultivars. Figure 4 shows the ARA in five stem-pieces from different internode portions of cultivars
Figure 3. Sampled portions of sweetpotato stem (portions 1-5) and sliced stem-pieces (pieces 1-5) from each sampled internode. (Referred from Adachi et al.2))
Figure 4. Acetylene reduction activity in five culture tubes inoculated with neighboring thinly sliced stem-pieces from different stem portions of cultivars Shiroyutaka and Shirosatsuma at 4 MAP. (Referred form Adachi et al.2))
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Shiroyutaka and Shirosatuma at 4 MAP. Although we detected ARA-positive tubes in certain stem portions, ARA-negative tubes were also detected in tubes that were inoculated with five neighboring thinly sliced pieces from the same internode portions. The frequency of ARA-positive tubes varied among the portions. These results suggest that the culturable diazotrophic endophytes were discretely localized in sweetpotato stems. Table 1 shows the average ARA values in five culture tubes that were inoculated with neighboring thinly sliced stem-pieces from each portion of cultivar Shiroyutaka, Koganesengan, and Shirosatsuma. The site of ARA-positive portions and level of ARA in the portions varied between replicated stems in each cultivar. The average ARA values in all portions and cultivars was higher at 4 MAP than at 2 MAP. Moreover, the frequency of ARA-positive portions was higher at 4 MAP than at 2 MAP. Among the internode portions, ARA values ware higher in ground-side portions (portions 4 and 5) than in upper portions (portion 1) especially at 4 MAP. The higher ARA values and frequency of ARA-positive portions at 4 MAP than at 2 MAP indicate that the endophytic microbial community or activity in the stems may have changed gradually from early to later growth stages.
Table 1. Average of acetylene reduction activity (ARA) values of five culture tubes inoculated with neighboring thinly sliced stem-pieces in each portion of three cultivars.
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Recently we have conducted inoculation experiments of P. agglomerans to in vitro grown sweetpotato plants (in large culture tubes and mayonnaise bottles) and examined N2-fixing activity under low chemical nitrogen conditions (15N dilution method). How to inoculate and how to let the diazotrophic bactgeria infect and establish effectively in plants are quite important research targets for development of techniques utilizing the symbiotic BNF of the crop-microbe-interaction.
Endophytic diazotrophs are habitating sweetpotato stems, and sweetpotato plants have a specific characteristic in nitrogen nutrition. On the other hand, Masuhara et al.9) observed mycorrhizal infection and formation of high density of arbuscular organs in sweetpotato roots, indicating that mycorrhizal fungi symbiotically infect sweetpotato plants. We are also interested in studying phosphorus nutrition of sweetpotato plants in relation to sweetpotato roots-mycorrhizal fungi-interaction. Paula et al.11) conducted co-inoculation experiment of a mycorrhizal fungus and an endophytic diazotroph to sweetpotato plants. Co-inoculation of mycorrhizal fungi may support the infection of endophytic diazotrophs into sweetpotato plants.
Pantoea agglomerans, isolated from sweetpotato stems endophytically, was also detected from maize and winter wheat as endophytic bacteria12, 13). In addition, P. agglomerans was recognized as phylloplane bacteria5, 8) and its antagonistic function was investigated for biological disease control6, 7, 13). Leaf-surface spray-inoculation may be one of the practical choices for inoculation experiments to utilize P. agglomerans for both possible aspects of endophytic BNF and certain biological disease control by the infection and settlement on and within plants.
Recently and in future, objectives of our studies on endophytic diazotrophs are 1) to develop effective inoculation techniques utilizing more promising endophytic diazotrophic bacteria into sweetpotato plants, and 2) to expand the useful biological function of BNF to other non-leguminous crops by the inoculation techniques. However, the ecological information about the endophytic diazotrophs in sweetpotato plants is still limited. Hence, further studies are required to elucidate the biological N2-fixing ability in sweetpotato plants by using several methodologies in pots and the field, and to clarify the colonization mechanism of the bacteria in sweetpotato plants.
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Acknowledgements
The author expresses his gratitude to Drs. C.A. Asis, Jr., Y. Takahata, M. Nakatani, and H. Mochida for their collaboration in this study on sweetpotato endophytic diazotrophs at Department of Upland Farming Research, KONARC. The author also thanks Drs. T. Yoneyama, The University of Tokyo, S. Akao, Miyazaki University, and O. Yamakawa, Director-General, KONARC, for their valuable comments and support in this study.
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References
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