Percentage of nitrogen derived from fertilizer (%Ndff), from stalk (%NDfs), and atmospheric nitrogen (%Ndfa) at 12 WAT and 20WAT.
1.1. Biological nitrogen fixation by endophytic diazotrophic bacteria
Nitrogen (N) is a major essential element for all organisms, and generally the amount of available N (mainly inorganic nitrogen such as nitrate or ammonia) in soil is limiting factor for natural and agricultural plant production . Biological nitrogen fixation (BNF) is a process by which atmospheric dinitrogen (N2) is reduced into 2 molecules of ammonia (NH3) by the enzyme nitrogenase with 8H+, 8e- and 16 Mg ATP. BNF have important role in N cycle in both global ecosystem and agro-ecosystem. Based on the data compiled by Bezdicek and Kennedy in 1988 , about 175 million metric tons of nitrogen per year is estimated to be fixed in global ecosystems, in which 90 million metric tones in agricultural land, 50 million metric tones in forest and non-agricultural land, and 35 million metric tones in sea. At that time, non-biological nitrogen fixation was estimated about 50 million metric tones per year by industrial nitrogen fixation mainly for the synthesis of ammonia fertilizer, and about 20 million metric tones by combustion, and about 10 million metric tones by lightening. In 2009, the production of N fertilizers increased to 106 million metric tones (FAOSTAT), but the amount of BNF still exceeds over non-biological nitrogen fixation.
Historically, the first experimental data to show the BNF by legumes was obtained by Boussingault (from ). He began crop rotation experiment including clover (legumes) in 1837, and the result clearly indicated the increase in nitrogen content was associated with cultivation of clover. Hellriegel and Wilfarth demonstrated the bacterial nitrogen fixation in association with legumes in 1888 (from ). Beijerinck isolated and cultivated the bacteria from legume nodules, and then he succeeded to inoculate the isolated bacteria to the uninfected host legume plant. In 1893, Winogradsky found that the free-living strictly anaerobic bacterium,
Several types of nitrogen fixing bacteria are recognized. First is “symbiotic bacteria”, such as
2. Characteristics of sugarcane
In 2011, world production of sugarcane was 1,794 million tons (FAOSTAT). This is much greater than for the other major crops such as maize (883 million tons), paddy rice (723 million tons), wheat (704 million tons) and potatoes (374 million tons). Sugarcane production is highest in Brazil (734 million tons), followed by India (342 million tons), and China (115 million tons). In 2011, sugarcane was cropped over an area of 25 million hectares. Sugarcane is a C4 plant, which has an efficient photosynthetic system, and it can convert up to 2% of incident solar energy into biomass. In 2011, the average yield was 70.5 tons per hectare. It grows up to 2-6 m in height (Figure 1) and the thick stalks (stems) store a high concentration of sucrose accumulated in stalk internodes, and the expressed stalk juice contains sucrose concentration at between 12 and 20% (W/V).
3. Contribution of biological nitrogen fixation in sugarcane cultivation
In Brazil, sugarcane crops accumulate N between 100 and 200 kg N per hectare per year, while N fertilization rates are relatively low, usually less than 60 kg N per hectare . Also, the response of sugarcane crops to N fertilizers is usually very weak [12,47]. The use of low N fertilizer input was justified by the results of 135 field experiments in all of main cane-growing areas of Brazil, with only 19% of the studies showing a significant increase in yield owing to the application of N fertilizer . In some sites of Brazil, sugarcane has been grown continuously for more than 100 years without any N fertilizer being applied at all . This circumstantial evidence suggests a high potential for BNF in sugarcane.
4. Estimation of contribution of biological nitrogen fixation by 15N dilution technique and 15N natural abundance method
Using a 15N dilution technique involving the supply of a 15N-labeled fertilizer, Lima
In Niigata, a pot experiment was conducted to estimate the contribution of nitrogen fixation in sugarcane (
Figure 2 shows the changes in the shoot length and leaf number of each treatment for 12 weeks after transplanting (WAT). The N0 plants grew very poor, and sole nitrogen fixation is not enough to support vigorous growth of sugarcane NiF8 cultivated in Niigata. The N100 plants and N100N0 plants showed relatively similar shoot length and leaf number, but the N100N0 plants leaves were pale compared with the N100 plants. At 12 WAT, total N content was 408 mgN and 286 mgN per plant in N100 treatment and N100N0 treatment, respectively. At 20 WAT, total N content was 569 mgN (N100) and 292mgN (N100N0).
Figure 3 shows the amount of N derived from three sources of N; Ndfa (N derived from nitrogen fixation), Ndfs (N derived from a mother stalk), and NDff (N derived from fertilizer). The amount of Ndfa in N100 and N100N0 was 87 mgN (21%Ndfa) and 48 mgN (17%Ndfa) per plant respectively. At 20 WAT, the amount of Ndfa was 87 mg (15%Ndfa) in N100 and 57 mgN (20%Ndfa) in N100N0 treatment. Among organs, the estimated %Ndfa tended to be higher in old leaves and stalk, and lower in green leaves and stems (Table 1). From this experiment, the continuous supply of N fertilizer did not inhibit nitrogen fixation in sugarcane compared with N deficient plants.
5. 15N2 fixation studies in sugarcane
Both 15N dilution method and 15N natural-abundance method are indirect methods for estimating nitrogen fixation. Direct evidence can be obtained by 15N2 tracer experiment. In earlier studies, Ruschel
The plants exposed to 15N2 for 7 days were grown in normal air for a further chase period. After 21 days, about half of the N originating in the stem cutting had been transported to the shoot and roots, suggesting that the cutting played a role in supplying N for the growth of shoot and roots (Figure 7). Most of the fixed N was distributed in the 80 % ethanol-insoluble fractions in each plant part, and the 15N fixed either in the roots or in the stem cutting remained there and was not appreciably transported to the shoot (Figure 8). The results were quite different from the fate of fixed N in soybean nodules, which is rapidly transported from nodules to roots and shoots.
From the results obtained in this experiment with young sugarcane plants, it is confirmed that the roots are the most active site of N2 fixation followed by the stem cutting. The sugarcane cuttings were initially cultured in water not in soil, so the N2-fixing endophytes in the roots might originate from the stem cutting or root primordia. If this is the case, to support active N2 fixation, nitrogen-fixing bacteria may move into the developing roots, and colonize the intercellular space in the roots.
In an earlier study with NiF8 sugarcane using a 15N dilution technique , after 5 months of cultivation in a pot supplied with 15N-labeled mineral fertilizer, it was estimated that the roots contributed greater proportions of BNF (26%) than the stem (14%) and leaves (21%). Compared with the stem and shoot, the roots offer certain advantages as sites of N2 fixation. These are first that the host plant provides carbohydrates to the root endophytes, and second that oxygen concentrations are usually lower in the roots and soil than in the atmosphere (
The fixation of nitrogen in stem cuttings of sugarcane is probably due to the activity of endophytes. Because very young sugarcane plants were used in our experiments, the finding of extremely limited N2 fixing activity in the shoots should not necessarily be taken to imply an insignificant stem endophytic contribution to the N economies of mature field-grown sugarcane plants. Many sugarcane endophytes (e.g.
The absence of any significant translocation of fixed 15N, most of it remaining in the 80% ethanol-insoluble fraction, in young sugarcane plants supports the possibility that fixed N may be used after the disintegration of dead endophytes, rather than being rapidly transported to the host plant as in the case of soybean nodules [44, 45]. However, it does not mean that there is no contribution of N2 fixation in the roots to shoot growth. When the N2-fixing bacteria in the roots eventually die, the products of their decomposition may well contribute to the growth of the roots and shoots of the plant. Even if this does not occur, then the N fixed in the roots will at least contribute to soil fertility in the field after the natural processes of root turnover and decomposition. Ando
6. Diazotrophic endophytes in sugarcane
As for the presence of N2-fixing bacteria in sugarcane, diazotrophic bacteria belonging to the
It is known that endophytic diazotrophic bacteria colonize in the vascular tissues or intercellular spaces and of sugarcane organs. For the presence of endophytic diazotrophs in sugarcane juice, and Bellone and Bellone  concluded that in the mature region of the sugarcane stem
Recently, complete genome sequence of the sugarcane nitrogen-fixing endophyte
A broad proteomic description of
The mechanisms for the association between sugarcane plants and diazotrophic endophytes are as yet poorly understood.
7. Mechanism by which N is transferred to the host plant from endophytic nitrogen-fixing bacteria
To promote sugarcane growth and high yield of sugar, the transport of N from diazotrophic endophytes to the host plant is important in addition to the occurrence of high nitrogen fixation activity. The mechanism by which N is transferred to the host sugarcane plant from N2-fixing endophytes has not yet been fully elucidated. There are two possible ways for this transfer to occur. The first is that living bacteria actively excrete fixed N into the apoplast of the host tissue and the plant cells then absorb the released N compounds. This is an analogous to legume-rhizobia symbiosis, in which fixed ammonia is rapidly excreted from bacteroid (a symbiotic state of rhizobia) to cytosol of infected cells in soybean root nodules . The second is that bacteria proliferate and colonize in the host tissue and the fixed N is released to the host cells only after their death and disintegration. No direct evidence has yet been obtained.
There is little direct evidence on how N2 fixed is supplied from endophyte to sugarcane plants. When an amylolytic yeast was used to mimic the plant,
The characteristics of nitrogen fixation and transport in endophytic bacteria isolated from sugarcane stem were investigated . The strains JA1 and JA2 were putatively identified as
The growth and ARA of the strain JA1 cultivated with liquid LGIP medium were measured every day for 10 days (Figure12). Bacteria growth increased rapidly at day-1 after inoculation, and continued to increase until day-6, then the increase was almost stopped thereafter. The strain might regulate bacterial density, possibly by quorum sensing mechanism. The ARA increased rapidly from day-1 to day-5, but it decreased rapidly after day-6. Very low ARA was detected after day-7 to day-10.
This result suggests that nitrogen fixation is active only during early stage of proliferation of JA1. After the bacterium growth stops, nitrogen fixation activity is inactivated. If the situation is the same inside the sugarcane organs, the continuous proliferation should be essential to keep nitrogen fixation activity of the diazotrophic endophyte.
The N release mechanism from endophytes to sugarcane plant is very important to support N nutrition for sugarcane growth. From the experiment with JA1 strain, 15N fixed during 24 hours was mainly distributed in bacteria fraction and only a little portion (about 4%) was released to the medium. However, at 10 days after one day feeding of 15N2, a significant portion of fixed 15N was distributed to the medium, especially the percentage of released 15N was highest at 40% under 20% O2 conditions (Figure 13). This result indicates that the cultured JA1 released N after stopping growth and nitrogen fixation, possibly by their death and degradation. Lethbridge and Davidson  suggested that endophytic bacteria only transferred fixed N to the plant when they died and were eventually decomposed.
In our studies, a 15N2 tracer experiment was conducted to investigate the sites of N2 fixation and the possible translocation of the fixed N in young sugarcane plants. Most active nitrogen fixation was observed in the roots, followed by planted stalk. The young shoot showed very little nitrogen fixation. Although many diazotrophic endophytic bacteria are reported in all parts of sugarcane plants (Figure 14), the roots may be a principal part of nitrogen fixation.
The fixed 15N either in roots or stalk were not readily transported to the shoots. In addition, the fixed N was mainly located in the 80% ethanol insoluble fractions, which contains high molecular weight compounds such as protein. The isolated diazotrophic endophyte fixed 15N2 in N free liquid and solid cultures. They fixed 15N2 only during their active growth period, and nitrogen fixation stopped thereafter. It is possible that the endophytic bacteria can fix N during proliferation stage, and they will release N after their death and decomposition.
The translocation of fixed N is quite different from legume-rhizobia symbiosis. In soybean nodules, N2 is fixed by bacteroids (N2-fixing rhizobia) in the infected cells and ammonia or ammonium is readily excreted from bacteroid to the cytosol of the infected cells. On short-term (5 min) exposure to 15N2, 97% of the fixed 15N in the 80% ethanol-soluble fraction, which contain low molecular weight compounds such as amino acids, in the nodules was distributed in the cytosol of the nodule plant cells, while only 3% remained within the bacteroids . The ammonium is then assimilated via the glutamine synthetase/glutamate synthase (GS/GOGAT) system [41,43], and used mainly to produce ureides, allantoin and allantoic acids, and the ureides are transported to the various plant organs via the xylem vessels [42,45].
Other than in Brazil, 150-250 kg of urea-N per hectare per year is usually applied to sugarcane, the actual amount depending on soil fertility, on genotype and on target yield . By promoting BNF through endophytic or associative diazotrophs, the cost associated with N fertilizer usage in sugarcane production can be reduced and environmental problems such as NO3- leaching or N2O gas emission, consequent upon the use of excessive chemical fertilizers, can be avoided. Further research will be important for promoting more efficient N2 fixation rates in sugarcane production.
We greatly appreciate the late Prof. Dr. Shoichiro Akao for giving us the chance to do the experiment on sugarcane-endophyte association. This research was supported by a grant from a Research Project of MAFF, Japan.
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