Mass ectrometr Y ActIvatSIPon oF NItrogen-F IxIng EnD o PHY tes DOI: 10.5702/massspectrometry.A0032
Vol. 3 (2014), A0032
Original Article
Activation of Nitrogen-Fixing Endophytes Is Associated with the Tuber Growth of Sweet Potato Koyo Yonebayashi,* Naoya Katsumi, Tomoe Nishi, and Masanori Okazaki Ishikawa Prefectural University, Suematsu, Nonoichi, Ishikawa 921–8836, Japan
Endophytic nitrogen-fixing organisms have been isolated from the aerial parts of field-grown sweet potato (Ipomoea batatas). The 15N dilution method, which is based on the differences in stable nitrogen isotope ratios, is useful for measuring nitrogen fixation in the field. In this study, seedlings of two sweet potato cultivars, ‘Beniazuma’ and ‘Benikomachi,’ were transplanted into an alluvial soil that had been treated with organic improving material in advance. Whole plants were sampled every 2 or 3 weeks. After separating plants into tuberous roots and leaves, the fresh weights of the samples were measured, and the nitrogen content and natural 15N content of leaves were determined with an elemental analyzer and an isotope ratio mass spectrometer linked to an elemental analyzer, respectively. The contribution of nitrogen fixation derived from atmospheric N2 in sweet potato was calculated by assuming that leaves at 2 weeks after transplanting were in a non-nitrogen-fixing state. The contribution ratios of nitrogen fixation by nitrogen-fixing endophytes in leaves of both sweet potato cultivars increased rapidly from 35 to 61 days after transplanting and then increased gradually to 55–57% at 90 days after transplanting. Over the course of the sweet potato growing season, the activity of nitrogen-fixing endophytes in leaves began to increase at about 47 days after transplanting, the weight of leaves increased rapidly, and then growth of tuberous roots began a few weeks later. Our findings indicate that nitrogen-fixing endophytes will be activated under inorganic nitrogen-free sweet potato cultivation, allowing for growth of the tuberous roots. Please cite this article as: K. Yonebayashi, et al., Activation of Nitrogen-Fixing Endophytes Is Associated with the Tuber Growth of Sweet Potato, Mass Spectrom (Tokyo) 2014; 3(1): A0032; DOI: 10.5702/ massspectrometry.A0032 Keywords: contribution ratio, Ipomoea batatas, legumes
15
N dilution method, nitrogen stable isotope ratio, non-
(Received September 24, 2014; Accepted October 18, 2014)
INTRODUCTION Recent evidence of significant biological nitrogen fixation in sugarcane (Saccharum spp.), rice (Oryza sativa), sweet potato (Ipomoea batatas), kallar grass (Leptochloa fusca),1) and sago palm (Metroxylon sagu)2) has generated a lot of interest in nitrogen fixation by non-legumes.1) Nitrogen-fixing bacteria (Azospirillum spp.) have been isolated from sweet potato roots.3,4) Acetobacter diazotrophicus, an endophytic nitrogen-fixing bacterium, has been isolated from aerial parts of field-grown sweet potato, sugarcane, and sweet sorghum (Sorghum vulgare),3,5) and various nitrogen-fixing bacteria were shown to colonize the sago palm extensively.6) In a recent study by Khan and Doty,7) endophytic bacteria associated with sweet potato plants were isolated, identified, and tested for their ability to fix nitrogen and exhibit stress tolerance. The 15N dilution method, which is based on the differences in stable nitrogen isotope ratios, is very useful for measuring nitrogen fixation in the field. In this method, the proportion of fixed nitrogen in nitrogen-fixing plants is calculated from the difference in δ15N values between nitrogen-fixing and neighboring non-nitrogen-fixing plants.8) For example, Yoneyama et al. compared the δ15N values of
sugarcane and neighboring plants to assess the contribution of nitrogen fixation in field-grown sugarcane plants.9) In another study, Yoneyama et al. used the δ15N method to estimate the N2-derived nitrogen in sweet potato.3) They assumed that the only source of nitrogen for neighboring pumpkin plants was available soil nitrogen and that a reduction of the δ15N value in sweet potato indicated the input of nitrogen by biological nitrogen fixation without isotope fractionation.10) They were able to calculate the percentage of plant nitrogen derived from atmospheric N2 (%Ndfa) by using the following equation: %Ndfa = [1 − (δ 15Nsweet potato / δ 15N pumpkin )] × 100 Yonebayashi et al. estimated nitrogen fixation by endophytes in sago palm by using the 15N method.2) The decline in δ15N values with age implied that atmospheric N2 fixation was occurring in sago palm. The δ15N value of the youngest leaf was assumed to be the value for the non-nitrogen-fixing state, such that the contribution of nitrogen fixation of each leaf could be calculated based on this value.2) In a previous study, we found that the δ15N value of sweet potato leaves at the initial stage of cultivation (i.e., 15 days after transplanting) was the same as that of organic fertilizer, whereas the δ15N value of leaves later in the growing
* Correspondence to: Koyo Yonebayashi, Ishikawa Prefectural University, Suematsu, Nonoichi, Ishikawa 921–8836, Japan, e-mail: [email protected]
© 2014 The Mass Spectrometry Society of Japan
Page 1 of 4 (page number not for citation purpose)
ActIvatIon oF NItrogen-FIxIng EnD oPHY tes
season was reduced.11) Thus, it appears that the leaves in the early cultivation stage were in a non-nitrogen-fixing state, and nitrogen-fixing endophytes were activated during the later stages of leaf growth. We proposed a method to calculate the contribution of nitrogen fixation in reference to that during the initial cultivation stage. In addition, the contribution of endophytes to nitrogen fixation in leaves of sweet potato increased over the growing season, and it increased particularly rapidly with growth of tuberous root. The present study examines the relationship of this activation of nitrogen-fixing endophytes to starch accumulation in sweet potato tubers.
MATERIALS AND METHODS The seedlings of two sweet potato cultivars, ‘Beniazuma’ and ‘Benikomachi,’ were transplanted on 4 June 2009 in an alluvial soil (Entisol; Ishikawa Prefectural University farm located at 36°30′36″ north latitude and 136°35′48″ east longitude). The size of field was 10×15 m. The ridge width was 50 cm, the distance between ridges was 90 cm, and the spacing of the seedlings was 35 cm. Three ridges were used for each cultivar. Prior to transplanting, a soil sample was collected from the 0- to 15-cm layer, air-dried, and passed through a 2-mm-mesh sieve. Fine roots and litter were removed from the soil sample with tweezers. The chemical characteristics of the soil were as follows: clay-loam texture, pH (H2O)=5.51, TOC=19.0 g kg−1, TN=1.90 g kg−1, exchangeable Ca=5.79 cmolc kg−1, exchangeable Mg=1.32 cmolc kg−1, exchangeable K=0.14 cmolc kg−1, Truog P2O5 =1042 mg kg−1, and δ15N=3.95‰. Organic improving material (cow dung and bark compost) was applied (30 t per ha) 3 weeks prior to transplanting of the sweet potato seedlings. A sample of organic improving material was air-dried and ground into fine powder. The chemical traits of the organic improving material were as follows: pH(H2O)=6.29, TOC=418 g kg−1, TN=11.7 g kg−1, C/N=35.8, P2O5 =4.12 g kg−1, K 2O=2.60 g kg−1, CaO=2.99 g kg−1, MgO=19.2 g kg−1, and δ15N=5.26‰. Two whole plants were sampled every 2 or 3 weeks from 25 June onward. The plants were separated into tuberous roots (if present) and shoots (leaves, stems, and petioles). The leaf fraction was analyzed by mixing the leaves and
Fig. 1.
Vol. 3 (2014), A0032
petioles. The fresh weights were measured, and then samples were oven-dried at 70°C for 3 days, weighed, and ground into fine powder with a grinder (WB-1, Osaka Chemicals Co., Osaka, Japan). Ground subsamples were used to determine the nitrogen content and natural 15N content. The nitrogen contents in the samples were determined with an elemental analyzer (2400 Series II, Perkin-Elmer Japan, Yokohama, Japan). The δ15N values of the plant (or soil) samples were determined with an isotope-ratio mass spectrometer (IsoPrime IRMS, GV Instruments, Manchester, UK) linked to an elemental analyzer (EA3000, EuroVector, Milan, Italy). The results are expressed as relative δ values calculated as follows:
δ 15N (‰) = [(Rs ample / Rstandard ) − 1]× 1000 where Rsample and Rstandard represent the 15N/14N ratios of the sample and the standard, respectively. An L-glutamic acid reference material (USGS40, U.S. Geological Survey, Reston, VA, USA) with a δ15N value of −4.5‰ was used as a secondary standard, calibrated relative to the primary standards for N2 (atmospheric N2). The experimental errors in the δ15N values were ≤0.1‰. Each sample was analyzed in triplicate. If the standard deviation was >0.2‰, measurements were repeated until the standard deviation of all the replicates was
Vol. 3 (2014), A0032
Original Article
Activation of Nitrogen-Fixing Endophytes Is Associated with the Tuber Growth of Sweet Potato Koyo Yonebayashi,* Naoya Katsumi, Tomoe Nishi, and Masanori Okazaki Ishikawa Prefectural University, Suematsu, Nonoichi, Ishikawa 921–8836, Japan
Endophytic nitrogen-fixing organisms have been isolated from the aerial parts of field-grown sweet potato (Ipomoea batatas). The 15N dilution method, which is based on the differences in stable nitrogen isotope ratios, is useful for measuring nitrogen fixation in the field. In this study, seedlings of two sweet potato cultivars, ‘Beniazuma’ and ‘Benikomachi,’ were transplanted into an alluvial soil that had been treated with organic improving material in advance. Whole plants were sampled every 2 or 3 weeks. After separating plants into tuberous roots and leaves, the fresh weights of the samples were measured, and the nitrogen content and natural 15N content of leaves were determined with an elemental analyzer and an isotope ratio mass spectrometer linked to an elemental analyzer, respectively. The contribution of nitrogen fixation derived from atmospheric N2 in sweet potato was calculated by assuming that leaves at 2 weeks after transplanting were in a non-nitrogen-fixing state. The contribution ratios of nitrogen fixation by nitrogen-fixing endophytes in leaves of both sweet potato cultivars increased rapidly from 35 to 61 days after transplanting and then increased gradually to 55–57% at 90 days after transplanting. Over the course of the sweet potato growing season, the activity of nitrogen-fixing endophytes in leaves began to increase at about 47 days after transplanting, the weight of leaves increased rapidly, and then growth of tuberous roots began a few weeks later. Our findings indicate that nitrogen-fixing endophytes will be activated under inorganic nitrogen-free sweet potato cultivation, allowing for growth of the tuberous roots. Please cite this article as: K. Yonebayashi, et al., Activation of Nitrogen-Fixing Endophytes Is Associated with the Tuber Growth of Sweet Potato, Mass Spectrom (Tokyo) 2014; 3(1): A0032; DOI: 10.5702/ massspectrometry.A0032 Keywords: contribution ratio, Ipomoea batatas, legumes
15
N dilution method, nitrogen stable isotope ratio, non-
(Received September 24, 2014; Accepted October 18, 2014)
INTRODUCTION Recent evidence of significant biological nitrogen fixation in sugarcane (Saccharum spp.), rice (Oryza sativa), sweet potato (Ipomoea batatas), kallar grass (Leptochloa fusca),1) and sago palm (Metroxylon sagu)2) has generated a lot of interest in nitrogen fixation by non-legumes.1) Nitrogen-fixing bacteria (Azospirillum spp.) have been isolated from sweet potato roots.3,4) Acetobacter diazotrophicus, an endophytic nitrogen-fixing bacterium, has been isolated from aerial parts of field-grown sweet potato, sugarcane, and sweet sorghum (Sorghum vulgare),3,5) and various nitrogen-fixing bacteria were shown to colonize the sago palm extensively.6) In a recent study by Khan and Doty,7) endophytic bacteria associated with sweet potato plants were isolated, identified, and tested for their ability to fix nitrogen and exhibit stress tolerance. The 15N dilution method, which is based on the differences in stable nitrogen isotope ratios, is very useful for measuring nitrogen fixation in the field. In this method, the proportion of fixed nitrogen in nitrogen-fixing plants is calculated from the difference in δ15N values between nitrogen-fixing and neighboring non-nitrogen-fixing plants.8) For example, Yoneyama et al. compared the δ15N values of
sugarcane and neighboring plants to assess the contribution of nitrogen fixation in field-grown sugarcane plants.9) In another study, Yoneyama et al. used the δ15N method to estimate the N2-derived nitrogen in sweet potato.3) They assumed that the only source of nitrogen for neighboring pumpkin plants was available soil nitrogen and that a reduction of the δ15N value in sweet potato indicated the input of nitrogen by biological nitrogen fixation without isotope fractionation.10) They were able to calculate the percentage of plant nitrogen derived from atmospheric N2 (%Ndfa) by using the following equation: %Ndfa = [1 − (δ 15Nsweet potato / δ 15N pumpkin )] × 100 Yonebayashi et al. estimated nitrogen fixation by endophytes in sago palm by using the 15N method.2) The decline in δ15N values with age implied that atmospheric N2 fixation was occurring in sago palm. The δ15N value of the youngest leaf was assumed to be the value for the non-nitrogen-fixing state, such that the contribution of nitrogen fixation of each leaf could be calculated based on this value.2) In a previous study, we found that the δ15N value of sweet potato leaves at the initial stage of cultivation (i.e., 15 days after transplanting) was the same as that of organic fertilizer, whereas the δ15N value of leaves later in the growing
* Correspondence to: Koyo Yonebayashi, Ishikawa Prefectural University, Suematsu, Nonoichi, Ishikawa 921–8836, Japan, e-mail: [email protected]
© 2014 The Mass Spectrometry Society of Japan
Page 1 of 4 (page number not for citation purpose)
ActIvatIon oF NItrogen-FIxIng EnD oPHY tes
season was reduced.11) Thus, it appears that the leaves in the early cultivation stage were in a non-nitrogen-fixing state, and nitrogen-fixing endophytes were activated during the later stages of leaf growth. We proposed a method to calculate the contribution of nitrogen fixation in reference to that during the initial cultivation stage. In addition, the contribution of endophytes to nitrogen fixation in leaves of sweet potato increased over the growing season, and it increased particularly rapidly with growth of tuberous root. The present study examines the relationship of this activation of nitrogen-fixing endophytes to starch accumulation in sweet potato tubers.
MATERIALS AND METHODS The seedlings of two sweet potato cultivars, ‘Beniazuma’ and ‘Benikomachi,’ were transplanted on 4 June 2009 in an alluvial soil (Entisol; Ishikawa Prefectural University farm located at 36°30′36″ north latitude and 136°35′48″ east longitude). The size of field was 10×15 m. The ridge width was 50 cm, the distance between ridges was 90 cm, and the spacing of the seedlings was 35 cm. Three ridges were used for each cultivar. Prior to transplanting, a soil sample was collected from the 0- to 15-cm layer, air-dried, and passed through a 2-mm-mesh sieve. Fine roots and litter were removed from the soil sample with tweezers. The chemical characteristics of the soil were as follows: clay-loam texture, pH (H2O)=5.51, TOC=19.0 g kg−1, TN=1.90 g kg−1, exchangeable Ca=5.79 cmolc kg−1, exchangeable Mg=1.32 cmolc kg−1, exchangeable K=0.14 cmolc kg−1, Truog P2O5 =1042 mg kg−1, and δ15N=3.95‰. Organic improving material (cow dung and bark compost) was applied (30 t per ha) 3 weeks prior to transplanting of the sweet potato seedlings. A sample of organic improving material was air-dried and ground into fine powder. The chemical traits of the organic improving material were as follows: pH(H2O)=6.29, TOC=418 g kg−1, TN=11.7 g kg−1, C/N=35.8, P2O5 =4.12 g kg−1, K 2O=2.60 g kg−1, CaO=2.99 g kg−1, MgO=19.2 g kg−1, and δ15N=5.26‰. Two whole plants were sampled every 2 or 3 weeks from 25 June onward. The plants were separated into tuberous roots (if present) and shoots (leaves, stems, and petioles). The leaf fraction was analyzed by mixing the leaves and
Fig. 1.
Vol. 3 (2014), A0032
petioles. The fresh weights were measured, and then samples were oven-dried at 70°C for 3 days, weighed, and ground into fine powder with a grinder (WB-1, Osaka Chemicals Co., Osaka, Japan). Ground subsamples were used to determine the nitrogen content and natural 15N content. The nitrogen contents in the samples were determined with an elemental analyzer (2400 Series II, Perkin-Elmer Japan, Yokohama, Japan). The δ15N values of the plant (or soil) samples were determined with an isotope-ratio mass spectrometer (IsoPrime IRMS, GV Instruments, Manchester, UK) linked to an elemental analyzer (EA3000, EuroVector, Milan, Italy). The results are expressed as relative δ values calculated as follows:
δ 15N (‰) = [(Rs ample / Rstandard ) − 1]× 1000 where Rsample and Rstandard represent the 15N/14N ratios of the sample and the standard, respectively. An L-glutamic acid reference material (USGS40, U.S. Geological Survey, Reston, VA, USA) with a δ15N value of −4.5‰ was used as a secondary standard, calibrated relative to the primary standards for N2 (atmospheric N2). The experimental errors in the δ15N values were ≤0.1‰. Each sample was analyzed in triplicate. If the standard deviation was >0.2‰, measurements were repeated until the standard deviation of all the replicates was
