2014 Volume 3 Issue 1 Pages A0032
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.
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:
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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 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.
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, K2O=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 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:
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The contribution of nitrogen fixation (%) derived from the atmosphere was calculated as 1−(δ15N of the leaf of sweet potato at time t/δ15N of the leaf of sweet potato at 2 weeks after transplanting), where we assumed that the leaf of sweet potato at 2 weeks after transplanting was in a non-nitrogen-fixing state.
The growth of both sweet potato cultivars was normal, and no disease symptoms were evident. The nitrogen concentrations in leaves of the sweet potato cultivars ‘Beniazuma’ and ‘Benikomachi’ at 14 days were 31 and 35 g kg−1, respectively, but gradually decreased to 20 and 23 g kg−1 at 75 days and were maintained at similar values until 103 days after transplanting (Figs. 1a, b). The fresh weight of leaves of the two cultivars increased rapidly from 47 to 90 days after transplanting (Figs. 1a, b). The leaf weight of ‘Benikomachi’ increased gradually after 90 days, and it was 5 kg vine−1 at 120 days after transplanting, whereas that of ‘Beniazuma’ continued to increase after 90 days and reached 6 kg vine−1 by 120 days. The fact that the weight of the leaves increased rapidly and the nitrogen concentration declined from 47 and 90 days after transplanting suggests that the soil nitrogen supply did not meet the nitrogen demand of the plants.
The standard deviation of each measurement of δ15N was <0.2‰.
The δ15N values of the leaves of ‘Benikomachi’ and ‘Beniazuma’ were 5.5 and 5.9‰, respectively, at 14 days, gradually decreased to 2.9 and 2.7‰ at 61 days, and were 2.3 and 2.6‰ at 90 days after transplanting (Figs. 1a, b). The δ15N values of the leaves of ‘Benikomachi’ increased to 3.4‰ whereas that of ‘Beniazuma’ decreased to 1.3‰ at 120 days after transplanting. During the early growth stage, the δ15N value of the leaves was higher than that of soil (3.95‰), but it was similar to that of organic improving material (5.26‰). The δ15N value of the sweet potato leaves decreased during growth, which indicates the fixation of atmospheric N2 by nitrogen-fixing endophytes. Thus, during the early growth of the sweet potato plants, the nitrogen source was available soil nitrogen and organic improving material nitrogen and the leaves were in a non-nitrogen-fixing state. Our data suggest that nitrogen-fixing endophytes in the leaves of sweet potato enhanced their activity in response to the nitrogen requirement of the plants.
The contribution ratios of nitrogen fixation by nitrogen-fixing endophytes in leaves of sweet potato increased rapidly from 35 to 61 days and then gradually increased to 55–57% at 90 days after transplanting (Fig. 2). The subsequent increase or decrease of the nitrogen fixation contribution ratio depended on the cultivars, and the contribution ratios of ‘Beniazuma’ and ‘Benikomachi’ were 77% and 36%, respectively, at 120 days after transplanting. Thus, the nitrogen fixation contribution ratio and the weight of leaves both increased at approximately the same time.
The standard deviation of each measurement of δ15N was <0.2‰.
Yoneyama et al.3) calculated the percentage of plant nitrogen derived from atmospheric N2 (%Ndfa) of sweet potato cultivars (‘Beniazuma’ and ‘Chili’) relative to neighboring pumpkin, a non-nitrogen-fixing plant. The average %Ndfa calculated for each plot at 95 days after transplanting was 44% for ‘Beniazuma’ and 34% for ‘Chili.’ In this study, the %Ndfa measured at 103 days after transplanting was 57% for ‘Beniazuma’ and 29% for ‘Benikomachi.’
The contribution of nitrogen fixation in sugarcane plants grown in fields at various sites in Brazil was examined using the difference in natural 15N content in the leaves of sugarcane compared to those of neighboring plants.9) The sugarcane plants showed an average contribution of nitrogen fixation to total plant nitrogen of 30% (ranging from 0 to 72%).9) In the case of sago palm, the contribution of nitrogen fixation was less than 30% until 10 years after transplanting, when it rose to about 90%.2)
Nitrogen uptake of leaves of ‘Beniazuma’ and ‘Benikomachi’ increased almost linearly from 40 days onward, reaching 116 and 122 g vine−1, respectively, by 120 days after transplanting (Figs. 3a, b). Likewise, the amount of fixed nitrogen in leaves of both cultivars increased almost linearly from 35 to 90 days after transplanting, reaching 46 and 54 g vine−1, respectively. The amount of fixed nitrogen of ‘Benikomachi’ then declined to 44 g vine−1 and that of ‘Beniazuma’ increased to 90 g vine−1 at 120 days after transplanting (Figs. 3a, b). Further work is needed to examine whether the difference in the amount of fixed nitrogen is due to cultivar differences.
The standard deviation of each measurement of δ15N was <0.2‰.
The fresh weight of tuberous roots of both sweet potato cultivars increased rapidly from 75 to 120 days, reaching 6.1 and 7.2 kg vine−1 at 120 days after transplanting (Figs. 3a, b). This increase in tuberous root weight occurred a few weeks after the increase in leaf weight (Figs. 1a, b). Thus, over the course of the growing season of sweet potato, 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. Likewise, Yoneyama and Akao12) estimated that the activity of nitrogen-fixing endophytes in sugarcane was higher during the sugar integration phase late in the growing season.
Both marketable and non-marketable sweet potato yields were negatively affected by nitrogen fertilizer.13) Despite the negative effect of nitrogen on sweet potato tuber yield, I. batatas plants have a high nitrogen-use efficiency, which is used for increasing the size of leaves rather than tuberous roots. Our findings indicate that under inorganic nitrogen-free cultivation, nitrogen-fixing endophytes will be activated, allowing for growth of the tuberous roots.
