韋莉萍 吳凱朝 邢永秀 李楊瑞

摘要：【目的】探討不同基因型甘蔗接種內(nèi)生固氮菌A01的固氮生理特性表現(xiàn)，為內(nèi)生固氮菌在甘蔗生產(chǎn)中的應(yīng)用提供理論依據(jù)?！痉椒ā繉?nèi)生固氮菌株A01分別接種到6個(gè)不同基因型甘蔗品種（GT24、GT9、YC84/153、GT18、CP65/357和F172）中，在溫室無氮栽培條件下，于伸長期調(diào)查甘蔗固氮酶、硝酸還原酶活性及氮含量?！窘Y(jié)果】接種固氮菌A01后， 不同時(shí)期不同甘蔗品種的+2葉、+3節(jié)間和根系的固氮酶活性、硝酸還原酶活性和氮含量表現(xiàn)均有不同；GT24、F172、GT9和GT24等4個(gè)甘蔗品種的節(jié)間、葉片和根系固氮酶活性均明顯上升，GT24、GT18和F172的+2葉硝酸還原酶活性均明顯增加，其中以GT18增加最明顯，其次為F172；GT9、GT24、GT18、F172和CP65/357等5個(gè)品種的+3節(jié)間、+2葉和根的全氮含量分別比對照增加0.06%～0.20%、0.02%～0.22%和0.02%～0.15%（絕對值）。【結(jié)論】接種固氮菌A01對F172和GT18的固氮生理特性有顯著影響，可能是F172和GT18的內(nèi)部環(huán)境更適于該菌生長。

關(guān)鍵詞： 甘蔗；基因型；內(nèi)生固氮菌A01；固氮生理特性；伸長期

0 Introduction

【Research significance】Sugarcane is the most important sugar and energy crops in Guangxi， its plant area is more than 100 hectare and the yield is over 7 million tons. Being most of Guangxi sugarcane plan-

ting areas is located at sloping dry land with lower soil fertility， so the farmers are used to apply much nitrogen fertilizerto soil in order to enhance the yield； as a result， it not only couldn't increase sugarcane yield， but also enhance cost of production， decrease producing benefit， contaminate ecological environment. According to the data statistic， 500-700 kg/ha of fertilizer urea was applied to sugarcane land in Guangxi， which was 8-10 times higher than that of Brazil， but sugarcane yield （about 60.0 t/ha） of Guangxi was about 30% less than that of Byazil. Saccharum spp. is of Poaceae crop that is characterized by associated nitrogen fixation. In 1961， since Brazil Scientist D■berei-

ner found nitrogen-fixing bacteria in sugarcane firstly， many researchers for different sugarcane producing countries began to study biological nitrogen fixation of sugarcane， and different nitrogen-fixing bacteria strains with nitrogen-fixing ability were isolated from different sugarcane varieties. Therefore， to study the effects of nitrogen-fixing bacteria on nitrogen metabolism of sugarcane and know about the nitrogen-fixing mechanism of nitrogen-fixing bacteria would be of important significance on application of nitrogen-fixing bacteria on sugarcane production to reduce the application of nitrogen fertilizer and producing cost， and enhance the competitiveness of sugar industry in China. 【Research progress】In recent years， many nitrogen-fixing bacteria had been isolated from different sugarcane varieties， and were found to have nitrogenase activity and affect the growth and physiological characteristics after inoculating to sugarcane（Fan et al.， 1998； Li et al.， 1998； Xing， 2006； Su et al.， 2007）. The nitrogenase activities in root of variety ROC20 small plantlets were significantly higher than that of the control strain after inoculated with nitrogen-fixing bacteria strain MJ9703 and MJ9708， which were isolated from sugarcane variety Taitang 10 and GT16 planted in Binyang county and Nanning city of Guangxi， respectively（Fan et al.， 1998）. The nitrogen-fixing bacteria strain B16L isolated from sugarcane variety ROC16 and B8R isolated from Brazil sugarcane variety B8（Liang et al.， 2008） were inoculated into GT16， ROC16 and ROC22 plantlets， respectively； the results indicated that， ROC16 and ROC22 plantlets presented higher chlorophyll content in leaves after inoculation， and were improved on nitrogen nutrient physiological characteristics to some extent， while no effects were found to GT16 with inoculation. Lin et al. （2008） reported that， as compared to the Guangxi main cultivated varieties ROC16 and ROC22， two Brazil sugarcane varieties B1 and B8 showed higher nitrogenase activities in roots after inoculated with nitrogen-fixing bacteria strain R16SH by using sand culture in bucket， which increased by 13.22% and 12.42% as compared to the control， respectively； nitrogen-fixing bacteria strain R16SH presented stronger inducing effects on plant height of B1 and B8 and stalk growth of B1， especially at elongation stage. Huang et al.（2009） isolated nitrogen-fixing bacteria strains GGS6， LCT2 and QZT2 from Guangxi cultivated sugarcane varieties， the mixture of these strains were inoculated into Brazil sugarcane variety B8 and Guangxi cultivated variety ROC22； as a results， the carbonhydrate content and protein content in roots of two varieties were enhanced to some extent from July to September， the root activities and nitrogease activities of B8 and ROC22 were also increased at different stages， indicating that the inoculation of nitrogen-fixing bacteria was contribute to growth and metabolism of sugarcane root. 【Research breakthrough point】Nitrogen is not only the needed maximal element to sugarcane growth， but also the most common limit factor. Although the different nitrogen-fixing bacteria isolated could enhance the nitrogenase activity， improve the growth and root activity of sugarcane， it less know about the effects of them on sugarcane nitrogen fixing characteristics at present. 【Solving problems】The six genotype sugarcane varieties viz.， GT24， GT9， YC84/153， GT18， CP65/357 and F172 were inoculated with nitrogen-fixing bacteria strain A01 by using sand culture， and the nitrogenase activities， nitrogen content of +2nd leaves， +3rd internodes and roots were investigated from May to August， as well as nitrate reductase activities of +2nd leaves， in order to probe the effects of strain A01on nitrogen metabolism and provide reference for application of nitrogen-fixing bacteria on sugarcane production.

1 Materials and methods

1. 1 Experimental materials

The six genotype sugarcane varieties viz.， GT24， GT9， YC84/153， GT18， CP65/357 and F172 were used as tested materials in the experiment， and the inoculated nitrogen-fixing bacteria was Pantoea agglomeras strain A01 isolated from sugarcane G28.

1. 2 Experimental methods

The present experiment began from February， 2009. The seed canes with single bud of each sugarcane variety（GT24， GT9， YC84/153， GT18， CP65/357 and F172） were soaked in 52 ℃ of hot water for 30 min， followed by putting into black bags to pregerminate for 2 days. The seed canes with better germinating vigor were planted by using sands in black plastic bucket. Five seed canes with single bud were planted beforehand， and 3 seedlings for each bucket were kept after emerging， each sugarcane variety was designed as 30 buckets. At 3-leaf seedlings stage， every bucket was watered with 1000 mL non-nitrogen Hoagland nutrient solution per 15 days. The water was applied normally during the experiment.

The isolated endophytic nitragen-fixing strain A01 was cultured with non-nitrogen D■bereiner liquid medium （D■bereiner， 1961） in shake culture at 28 ℃. At 3-leaf seedling stage， the strain A01 was inoculated into all seedlings of 15 buckets for all sugarcane varieties by using wound-infecting method， the pseudostems of sugarcane base were infected with 50.0 μL of strain A01 solution by using microinjector on 31 March. The 15 buckets of each sugarcane variety was used as the control without any treatment. The water management of all treatments was conducted normally.

1. 3 Index determination methods

1. 3. 1 Nitrogenase activity determination In the present， the nitrogenase activities of leaf， internode and root were assayed using the method of Fan et al.（1998）. The +2nd leaves， +3rd internodes and roots of 5 plants were sampled from the treatments and controls of all sugarcane varieties on 23 May， 24 June， 19 July and 20 August， respectively. All samples were washed thoroughly with tap water and rinsed with sterile water once. The leaves of 20-40 cm in length were taken from leaves base， internodes of 2-4 cm in length were taken from growth belt， and root of 0-15 cm in length were taken from new root tip. About 1.00 g of all samples were chopped and grounded separately with mortar and pestle in 10.0 mL SNX culture medium（containing 10% sucrose， 0.1% CaCO3， 0.02%MgSO4·7H2O， 0.02%NaCl， and 0.0005%Na2MoO4·2H2O）（Xing， 2006）， and transferred to 60 mL bottle for culture occasional shaking under 80 r/min of rapid speed at 28 ℃. After one day incubation， 0.5 mL of 10% acetylene was injected into the bottle by micro-injector and the mixture was again incubated for 24 hours continuously. After incubation， 10.0 μL of gas was extracted to assay the ethylene content by using Agilent 6890N gas chromatograph. Each treatment and control was taken with three replicates. The nitrogenase activity was expressed with nmol C2H4/gFW·h.

1. 3. 2 Nitrate reductase activity determination The nitrate reductase activity of all treatments was assayed by using method of Zhou and Zheng（1985）. The leaves of 20-50 cm in length were taken from +2nd leaf ring and got out midrib， and was cut into 0.5 cm of section. Four samples（one control and 3 replicates） for each treatment and control were weighted as 2.000 g， followed by put into triangular flask and added 10.0 mL of 0.1 mol/L phosphate buffer（pH 7.5） and 5.0 mL of 0.2 mol/L KNO3 solution（5.0 mL distilled water for control）. The mixture was shaken up and exhausted air time and again under vacuum drier till to all samples sinking thoroughly. After put in dark for 60 min， the mixture was taken out to terminate reaction by adding 0.5 mL of 30% trichloracetic acid. After sha-

king for 3 min， 2.0 mL of mixture was put into testtube and added with 2.0 mL of 1.0% sulfanilamide and 2.0 mL of 0.2% α-naphthylamine to shake up and stand for 20 min. The absorbance of mixture was determined at 540 nm， the nitrate reducatse activity was expressed with total content of NO2-N（μgNO2--N/gFW·h）.

1. 3. 3 Total nitrogen content determination The 5 plants of each treatment and control for all sugarcane varieties were sampled to separate +2nd leaves， +3rd internode and roots， followed by cleaning out with distill water and putting into oven for 10 min at 105 ℃， and dried out till to constant weight. All samples were smashed with pulverizer and put into dryer， about 0.19 g sample was put into 50 mL digestive tract and digested with H2SO4-H2O2 to measure total nitrogen content with micro kjeldahl method（Bao， 2000）.

1. 4 Data statistics and analysis

All the data of all treatments for 6 sugarcane varieties were processed and assayed with soft system DPS V9.50.

2 Results and anlysis

2. 1 Effects of nitrogen-fixing bacteria strain A01 on nitrogenase activities of different sugarcane varieties

From the results listed in Tab.1， it found that the nitrogenase activities in +3rd internodes， +2nd leaves and roots of different sugarcane varieties were diffe-

rently affected by inoculating P. agglomeras strain A01 at different stages. After inoculated with strain A01， the nitrogenase activities in +3rd internode and +2nd leaf of sugarcane variety YC84/153 were slightly decreased from 23 May to 20 August as compared to the control， but the nitrogenase activities in its root were 3.61-9.18 nmol C2H4/gFW·h higher than the control at different stages. While the sugarcane variety CP65/357 showed the contrary results to YC84/153 because of higher nitrogenase activities in internode and leaf of treated sugarcane and lower nitrogenase activities in root of the control excepting for 23 May. As to the other 4 sugarcane varieties viz.， GT24， F172， GT9 and GT24， the nitrogenase activities in their internodes， leaves and roots were found to increase significantly at different stages after inoculated strain A01， indicating that strain A01 could adapt to the growing environment of different tissues for these sugarcane varieties.

The results from Tab.1 showed that， an increa-

sing trend in nitrogenase activities at different stages were found in +3rd internode of sugarcane varieties F172 and CP65/357 for the treatment and the control of F172， as well as in +2nd leaf of sugarcane variety F172 inoculated treatment and the control of CP65/357. The change trends of increase following by decrease in nitrogenase activities were found in +3rd internode of the other 4 varieties and the control of CP65/357， as well as in +2nd leaf of the treatments and the controls of other 5 sugarcane varieties， and most of them presented the highest nitrogenase activities on 19 July. As to the root， it showed that no significant change regulars were found in nitrogenase activities of the treatments and the controls for different sugarcane varieties at different stages. Whatever the inoculated treatment or the control， the nitrogenase activity in root of GT24 was changed from decreasing to increasing， and the highest one（17.75 nmol C2H4/gFW·h） was found on 24 June； that of inoculated treatment for F172 presented an increase trend from 3.99 to 24.39 nmol C2H4/gFW·h.

As to different sugarcane varieties， we found that F172 presented the highest nitrogenase activity in +3rd internode as 39.11-87.56 nmol C2H4/gFW·h of the treatment and 28.26-38.89 nmol C2H4/gFW·h of the control， followed by GT18. The variety GT18 was found to has higher nitrogenase activity （15.61-19.30 nmolC2H4/gFW·h） in +2nd leaf from May to June， but it was lower than that （36.76.61-39.24 nmol C2H4/gFW·h） of F172 from July to August， indicating that these two sugarcane varieties might be of stronger nitrogen-fixing ability and were easy to utilize strain A01 for conducting nitrogen-fixed， and F172 was more stronger than GT18.

2. 2 Effects of nitrogen-fixing bacteria strain A01 on nitrate reductase activity in leaf of different sugarcane varieties

The nitrate reductase is one of the key enzymes for the nitrogen metabolism in plant， it is related to absorption and utilization of nitrogen fertilizer for crops， which could affect the yield and quality of crops. The results seen from Tab.2 indicated that， without any inoculation of strain A01， the nitrate reductase activities in +2nd leaf of 6 sugarcane varieties were found to be only 0.14-1.10 μg NO2--N/gFW·h on 23 May， followed by increasing to 3.87-39.48 μg NO2--N/gFW·h significantly on 24 June； of which， variety F172 was found to has the highest nitrate reductase activity（32.06 μg NO2--N/gFW·h） and be extremely significantly higher than that of the other varieties（P<0.01， the same as below）. Excepting for sugarcane varieties GT24 and GT9， the nitrate reductase activities in +2nd leaf of other varieties viz.， YC84/153， GT18， F172 and CP65/357 were found to decrease as ranging from 6.06 to 24.63 μg NO2--N/gFW·h on 19 July； of which， YC84/153 presented the highest nitrate reductase activity and was extremely significantly higher than those of the other varieties. On 20 August， it found that 5 sugarcane varieties showed an increasing trend of nitrate reductase activities in +2nd leaf excepting for YC84/153， and GT18 was the highest one as 41.94 μg NO2--N/gFW·h.

After inoculated with strain A01， the nitrate reductase activities in +2nd leaves of variety YC84/153 showed a change trend of increase following by a decrease， but those of the other 5 varieties were changed from increase to decrease， followed by an increasing trend， and it reached the highest as 8.47-86.49 μg NO2--N/gFW·h on 24 June. Amongst all sugarcane varieties， GT18 could get the highest nitrate reductase activity in +2nd leaf（86.49 μg NO2--N/gFW·h）. As compared to the control without any inoculation， strain A01 inoculated could enhance the nitrate reductase activities in +2nd leaves of GT24， GT18， F172 and CP65/357 significantly， and GT18 was found to has the highest increment， followed by F172， indicating that this variety maybe has stronger nitrogen metabolism ability. As to GT9 and YC84/153， the nitrate reductases activities in +2nd leaf were found to sharply increase from May to June， and reached the highest as 35.33 and 33.62 μg NO2--N/gFW·h； while nitrate reductases activities lasted decreasing to lower than that of the control from July to August， but no significant difference were found between them.

2. 3 Effects of nitrogen-fixing bacteria strain A01 on total nitrogen content of different sugarcane varieties

The results showed in Tab.3 indicated that no significant difference in total nitrogen contents in different parts of YC84/153 were found between the control and treatment with strain A01 inoculation， and only its root had 0.15% increment in nitrogen content. The +3rd internode， +2nd leaf and root other 5 sugarcane varieties were found to present an increment of 0.06%-0.20%， 0.02%-0.22% and 0.02%-0.15% in nitrogen content after inoculation， respectively. On the whole， F172 showed higher nitrogen content and increment after inoculation being significantly higher than those of the other varieties， which was according with its nitrogenase activity， indicating that nitrogen-fixing bacterium strain A01 could play evident enhancing role on nutrient utilization or nitrogen metabolism of F172.

3 Discussion

Nitrogen deficiency could make sugarcane suffer destruction at some extent on a series of physiological processes such as protein synthesis， photosynthesis and respiration， and so on. As a result， the formation of sugarcane yield was affected by these unnormal physiological changes. The endophytic nitrogen-fixing bacteria could colonize at different tissues of crops to play associated nitrogen fixation at different organs after inoculation， and the cell would transmit the fixed nitrogen to leaf. The results of 15N isotope tracer technique proved that， endophytic nitrogen-fixing bacteria could provide 60%-80% of needed nitrogen nutrient for sugarcane growth， even nitrogen fertilizer was avoided to apply again（Boddey et al.， 1991）.

Nitrogen-fixing bacteria live among cells of su-

garcane root， stalk and leaf， and are of certain specificity with sugarcane； they could play nitrogen fixation under suitable growing conditions in sugarcane tissue to promote sugarcane growth. Endophytic nitrogen-fixing bacteria strain A01 was isolated from root of su-

garcane variety GT28； under non-nitrogen culture condition， inoculating strain A01 could promote sugarcane growth， increase stalk weight（Wu et al.， 2010）， improve photosynthetic physiology（Wu et al.， 2011）， but significant difference were affected by different varieties， indicating that nitrogen-fixing bacteria might be of selectivity to internal environment of sugarcane plants. The results from this study suggested that， both different sugarcane varieties and three different organs（+2nd leaf， +3rd stalk and root） showed different nitrogenase activities at different stages after inocula-

ted with nitrogen-fixing bacteria strain A01， that was the same with the results reported by Lin et al.（2008） and Luo et al.（2010）. In the present study， the leave and internodes of four sugarcane varieties viz.， F172， GT9， GT18 and CP65/357 in inoculated treatment and the control were found to present higher nitrogenase activities than GT24 and YC84/153， while their roots presented no any regular at different stages. As compared to the control， inoculating strain A01 could enhance the nitrogenase activities in internodes and leaves of F172， GT9， GT18 and CP65/357 and in roots of F172， GT9， GT18， GT24 and YC84/153； as to different sugarcane varieties， we found that F172 presented the highest nitrogenase activities in the inoculated treatment and the control， followed by GT18， indicating that strain A01 might be suitable for the internal environment of these two sugarcane and could play nitrogen fixation well. Furthermore， +3rd inter-

node had the highest nitrogenase activity， followed by leaf and root， suggesting that internode environment might be beneficial to colonization of nitrogen-fixing bacteria and playing nitrogen fixation.

The nitrate reductase play important role in the process of plant nitrogen metabolism. Inoculating nitrogen-fixing bacteria strains（G， R and B） or their mixture could enhance nitrate reductase activities of B8， ROC22 and GT28， and different strains showed different effects on different sugarcane varieties， but their mixture was found to has the higher nitrate reductase activities in leaves at initial growing stage（Ouyang et al.， 2010）. In the present study， the nitrate reductase activities in +2nd leaves of the control for 6 sugarcane varieties were changed differently at different stages， the highest nitrate reductase activity was found in variety GT18， F172， YC84/153 and GT18 in May， June， July and August， respectively. After inoculated with strain A01， all sugarcane varieties were found to have the highest nitrate reductase（8.47-86.49 μg NO2--N/gFW·h） on 24 June， and GT18 was found to be the highest one （86.49 μg NO2--N/gFW·h）. As compared to the control without any inoculation， strain A01 inoculated could enhance the nitrate reductase activities in +2nd leaves of GT24， GT18， F172 and CP65/357 significantly， and GT18 was found to has the highest increment， followed by F172， indicating that this variety maybe has stronger nitrogen metabolism ability.

It is reported that， nitrogen-fixing bacteria strains viz.， MJ9702， MJ9703， MJ9704， MJ9705， MJ9708 and MJ9709 could enhance nitrogen content in plantlets of ROC20（Fan et al.， 1998）. Sevilla et al.（2001） suggested that， nitrogen-fixing bacteria could accumulate nitrogen element on the balance of nitrogen for a long time， but its effect was changed with different crops， organs and environmental conditions， the possible reasons was that the promoting effects of nitrogen-fixing bacteria on plants were the synthetic function of nitrogen-fixing， phosphorus dissolving， zinc dissolving， and secretion of plant hormones（Mirz et al.，2000；Munoz-Rojas et al.，2003；Loiret et al.，2009）. We found that， the +3rd internode， +2nd leaf and root of other 5 sugarcane varieties（GT24， GT18， GT9， F172 and CP65/357） were found to present an increment of nitrogen content as compared to the control after inoculation. On the whole， F172 showed higher nitrogen content and increment after inoculation， followed by GT18， which were the same with the result of nigrogenase activity， indicating that nitrogen-fixing bacterium strain A01 could play evident enhancing role on nutrient utilization or nitrogen metabolism of F172. That might be caused that， strain A01 could enhance chlorophyll content in leaf of F172， improve all parameters of photosynthesis and chlorophyll fluorescence， resulting in enhancement of photosynthesis for F172 and providing enough nitrogen to its growth（Wu et al.，2011）.

On the research basis of the predecessor， this study was conducted to probe the effects of inoculating strain 01 on nitrogen-fixing physiological characteristics of different genotype sugarcane varieties. As a result， strain 01 showed different nitrogen-fixing physiology in different tissues of different sugarcane varieties， indicating that this nigrogen-fixing bacterium was of selectivity to different genotypes of sugarcane. In the future research of sugarcane associate nitrogen fixation， the host selectivity of nitrogen-fixing bacteria should be reinforced， especially for the adaptive endophytic nitrogen-fixing bacteria to mainly cultivated sugarcane varieties in Guangxi. Furthermore， the effecting mechanism of sugarcane varieties to nitrogenase activity and the interactions between nitrogen-fixing bacteria and sugarcane host are also needed to study deeply.

4 Conclusion

It is concluded that， the nitrogen-fixing bacteria strain A01 could affect nitrogen-fixing physiological characteristics of sugarcane varieties F172 and GT18， might be the internal environment of F172 and GT18 were suitable for its growth.

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（責(zé)任編輯 韋莉萍）