茶树根际土壤氨氮转化菌的分离、鉴定及效应研究
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摘要
茶树是一种喜欢铵态氮的植物，由于北方土壤硝化作用明显，一部分铵态氮被硝化，N素损失量较大。同时，在茶树生长季节茶农重施有机肥，而有机态氮转化成无机氮的速度慢，氮素供给量不足，不能够满足茶树对氮肥的需求。因此，如何在降低化肥使用量的同时，提高氨氮微生物转氨的能力，增强土壤有机肥转化速率，同时利用固氮微生物固定氮素，从而起到提高土壤铵态氮含量的作用，成为目前生态茶园建设中的一个重要课题。本研究拟从山东茶树根际土壤中筛选氨化菌和固氮菌等促生菌，并对其特性进行研究；同时，将筛选出的高活性氨化菌（T1）、固氮菌（T2）和两者的混合菌（T3）接种茶园土壤，以不接种（CK）为对照，研究接种对根际微生物种群及多样性、土壤化学性质、茶苗生长及N代谢的影响，探讨土壤微生物与茶树生长发育的关系，为茶园土壤管理和茶树可持续生产提供依据。其主要研究结果如下：
1.从茶园根际土壤中分离纯化获得33株氨化细菌菌株。从中筛出三株不同种属的高活性菌株，经鉴定菌株SNT3属于芽孢杆菌属，菌株SNT6属于微小杆菌属，菌株SNT33属于不动杆菌属。三株菌株的最适宜生长温度均为35℃；在pH适宜范围和氨化能力方面，三株菌株存在明显差异，其中SNT3的菌株pH适宜范围最大，氨化能力最强。
2.从茶园根际土壤中分离纯化得到6株固氮菌株。从中选出两株不同种属的菌株，经鉴定菌株GD5为褐球固氮菌，菌株GD15为恶臭假单胞菌。两菌株生长最适宜的pH7，最适宜生长温度为30℃；GD5具有溶磷作用和分泌生长素的能力，而GD15仅具有分泌生长素的能力。
3.接种处理对茶园根际土壤微生物种群及土壤化学特性有显著影响。接种后微生物群落多样性指数、均匀度指数、丰富度指数上升，优势度指数下降，根际微生物多样性增加，物种之间更趋于稳定。同时接种后细菌和放线菌数量增多，真菌总量有所减少。细菌中氨化细菌和自生固氮菌的数量呈现增长趋势，硝化细菌的数量受到抑制。接种试验后根际土壤的pH都有所下降，T1和T3接种处理pH较高。在有机质方面，各处理含量变化不大，但接种处理促进了有机碳的积累。在速效养分方面，铵态氮、速效P、速效K接种处理显著高于对照处理。T1、T2、T3处理铵态氮浓度比CK分别提高40%、42%、65%；硝态氮浓度接种处理显著低于对照。在酶活性方面，接种处理酶活性高于对照。经分析细菌与铵态氮和4种土壤酶活性呈极显著正相关；真菌与铵态氮呈负相关，与硝态氮呈显著正相关，与土壤酶活性呈正相关；放线菌与脲酶和天门冬酰胺酶呈极显著正相关；特征性细菌与无机氮和土壤酶之间相关性明显且有一定的差异性。
4.接种后茶苗生物量和新梢全N量显著高于对照。叶片中硝酸还原酶、谷氨酸合成酶、谷氨酰胺合成酶的活性相似，均以处理20d最高。接种处理酶活性高于CK，枯草芽孢杆菌处理和褐球固氮菌处理无差别，与混合菌处理有差别但不显著。根系中NR活性变化不大。
The tea tree liked ammonium nitrogen. A part of ammonium nitrogen was nitrated, dueto nitrification was obvious in the the northern soil. Therefore, the absorption of ammoniumnitrogen was effected. Meanwhile, a large number of organic fertilizer was used in the teagrowing season. It could not meet the demand of tea, beacause the speed of organic nitrogeninto inorganic nitrogen was slow. It was an important subject of the ecological teaconstruction for how to reduce the amount of fertilizer, improve the ability of the ammoniamicroorganisms aminotransferase, enhance the transformation ratio of organic, fix thenitrogen by nitrogen-fixing bacteria, to increase the content of ammonium nitrogen in the soil.The ammonifier and nitrogen-fixing bacteria were planed to screen from rhizosphere soil oftea garden in Shandong province, and the characteristics were studied in this study. Then, Thehigh activity of ammonifying bacterial (T1), azotobacter (T2), mixed bacteria of the twostrains were inoculated in the rhizosphere soil of tea garden, and the CK was no inoculationtreament. The effects of inoculation on rhizosphere microbial diversity, soil chemical property,tea plant growth and the nitrogen metabolism in this study.It was aim to explore therelationship between soil microbial and growth and development of the tea tree, and toprovide a theoretical foundation for the tea garden soil management and sustainableproduction of tea.The results were as follows:
1. The33ammonifying bacterial strains were screened from the rhizosphere soil of teagarden in Shandong province, and the three different species of high activity strains wereselected. The strain SNT3was preliminary identified as Bacillus, SNT6as Exiguobacteriumand SNT33as Acinetobacter. The optimum temperature of three strains were35℃; Therewere significant differences for the optimum range of pH and ammonifying capacity. Amongwhich SNT3had the maximum optimum range of pH, and the most capacity of ammoniation.
2. The6azotobacter strains were screened from the soil. The two different speciesstrains were selected. The strain GD5was preliminary identified as Azotobacter chrococcum,GD15as Pseudomonas putida. The optimum pH for GD5,GD15were7; the optimumtemperature of two strains were30℃;The GD5showed the phosphate solubilizing power andauxin production, and the GD15could produce auxin only.
3. There were significant differences for the microbial population and soil chemicalproperty in rhizosphere soil after inoculating. The Shannon-Wiener index, Brillouin index,Margalef index rise while Simpson index fall through Bio-Dap software analysis. Themicrobial diversity was enhanced and the total biomass tend to stability.Meanwhile, thenumber of bacteria and actinomycetes increased, but the fungus decreased. Theammonifilation bacteria and abiogenous azotobacter had the growth trend among the bacteria,but the number of nitrifying bacteria was inhibited.After inoculating, although soil pHdecreased, T1and T3treatment showed the higher soil pH. The soil organic matter have noeffects among them, but T1, T2and T3treatment promote the accumulation of organic carbon.The content of ammonium nitrogen, available P, available K of inoculation treatments weresignificantly higher than CK. The content of ammonium nitrogen were increased separatelyby40%,42%and65%compared with the CK, while the nitrate nitrogen was vice versa. Theactivities of urease, catalase, protease and L-asparainase of in T1, T2and T3treatment waswas higher than CK. According to correlative analysis, bacteria was positively correlated withammonium nutrition and4types of enzyme activities; fungus was negative correlation withammonium, but had positive correlation with nitrate nitrogen and4types of enzyme activities;Actinomycetes were significantly and positively correlated with urease and L-asparainase inboth indexes. There were significant differences in the characteristic bacteria, soil nitrogenmineralization and soil enzyme.
4The biomass and total N content of inoculation treatments were significantly higherthan CK. The activities of NR, GS, GOGAT were similitude. The enzyme activity ofinoculation treatments were significantly higher than CK. There were no differences betweenT1and T2, whie the differences between T1, T2and T3were significantly. The change of NRin root wre not significantly.
1.从茶园根际土壤中分离纯化获得33株氨化细菌菌株。从中筛出三株不同种属的高活性菌株，经鉴定菌株SNT3属于芽孢杆菌属，菌株SNT6属于微小杆菌属，菌株SNT33属于不动杆菌属。三株菌株的最适宜生长温度均为35℃；在pH适宜范围和氨化能力方面，三株菌株存在明显差异，其中SNT3的菌株pH适宜范围最大，氨化能力最强。
2.从茶园根际土壤中分离纯化得到6株固氮菌株。从中选出两株不同种属的菌株，经鉴定菌株GD5为褐球固氮菌，菌株GD15为恶臭假单胞菌。两菌株生长最适宜的pH7，最适宜生长温度为30℃；GD5具有溶磷作用和分泌生长素的能力，而GD15仅具有分泌生长素的能力。
3.接种处理对茶园根际土壤微生物种群及土壤化学特性有显著影响。接种后微生物群落多样性指数、均匀度指数、丰富度指数上升，优势度指数下降，根际微生物多样性增加，物种之间更趋于稳定。同时接种后细菌和放线菌数量增多，真菌总量有所减少。细菌中氨化细菌和自生固氮菌的数量呈现增长趋势，硝化细菌的数量受到抑制。接种试验后根际土壤的pH都有所下降，T1和T3接种处理pH较高。在有机质方面，各处理含量变化不大，但接种处理促进了有机碳的积累。在速效养分方面，铵态氮、速效P、速效K接种处理显著高于对照处理。T1、T2、T3处理铵态氮浓度比CK分别提高40%、42%、65%；硝态氮浓度接种处理显著低于对照。在酶活性方面，接种处理酶活性高于对照。经分析细菌与铵态氮和4种土壤酶活性呈极显著正相关；真菌与铵态氮呈负相关，与硝态氮呈显著正相关，与土壤酶活性呈正相关；放线菌与脲酶和天门冬酰胺酶呈极显著正相关；特征性细菌与无机氮和土壤酶之间相关性明显且有一定的差异性。
4.接种后茶苗生物量和新梢全N量显著高于对照。叶片中硝酸还原酶、谷氨酸合成酶、谷氨酰胺合成酶的活性相似，均以处理20d最高。接种处理酶活性高于CK，枯草芽孢杆菌处理和褐球固氮菌处理无差别，与混合菌处理有差别但不显著。根系中NR活性变化不大。
The tea tree liked ammonium nitrogen. A part of ammonium nitrogen was nitrated, dueto nitrification was obvious in the the northern soil. Therefore, the absorption of ammoniumnitrogen was effected. Meanwhile, a large number of organic fertilizer was used in the teagrowing season. It could not meet the demand of tea, beacause the speed of organic nitrogeninto inorganic nitrogen was slow. It was an important subject of the ecological teaconstruction for how to reduce the amount of fertilizer, improve the ability of the ammoniamicroorganisms aminotransferase, enhance the transformation ratio of organic, fix thenitrogen by nitrogen-fixing bacteria, to increase the content of ammonium nitrogen in the soil.The ammonifier and nitrogen-fixing bacteria were planed to screen from rhizosphere soil oftea garden in Shandong province, and the characteristics were studied in this study. Then, Thehigh activity of ammonifying bacterial (T1), azotobacter (T2), mixed bacteria of the twostrains were inoculated in the rhizosphere soil of tea garden, and the CK was no inoculationtreament. The effects of inoculation on rhizosphere microbial diversity, soil chemical property,tea plant growth and the nitrogen metabolism in this study.It was aim to explore therelationship between soil microbial and growth and development of the tea tree, and toprovide a theoretical foundation for the tea garden soil management and sustainableproduction of tea.The results were as follows:
1. The33ammonifying bacterial strains were screened from the rhizosphere soil of teagarden in Shandong province, and the three different species of high activity strains wereselected. The strain SNT3was preliminary identified as Bacillus, SNT6as Exiguobacteriumand SNT33as Acinetobacter. The optimum temperature of three strains were35℃; Therewere significant differences for the optimum range of pH and ammonifying capacity. Amongwhich SNT3had the maximum optimum range of pH, and the most capacity of ammoniation.
2. The6azotobacter strains were screened from the soil. The two different speciesstrains were selected. The strain GD5was preliminary identified as Azotobacter chrococcum,GD15as Pseudomonas putida. The optimum pH for GD5,GD15were7; the optimumtemperature of two strains were30℃;The GD5showed the phosphate solubilizing power andauxin production, and the GD15could produce auxin only.
3. There were significant differences for the microbial population and soil chemicalproperty in rhizosphere soil after inoculating. The Shannon-Wiener index, Brillouin index,Margalef index rise while Simpson index fall through Bio-Dap software analysis. Themicrobial diversity was enhanced and the total biomass tend to stability.Meanwhile, thenumber of bacteria and actinomycetes increased, but the fungus decreased. Theammonifilation bacteria and abiogenous azotobacter had the growth trend among the bacteria,but the number of nitrifying bacteria was inhibited.After inoculating, although soil pHdecreased, T1and T3treatment showed the higher soil pH. The soil organic matter have noeffects among them, but T1, T2and T3treatment promote the accumulation of organic carbon.The content of ammonium nitrogen, available P, available K of inoculation treatments weresignificantly higher than CK. The content of ammonium nitrogen were increased separatelyby40%,42%and65%compared with the CK, while the nitrate nitrogen was vice versa. Theactivities of urease, catalase, protease and L-asparainase of in T1, T2and T3treatment waswas higher than CK. According to correlative analysis, bacteria was positively correlated withammonium nutrition and4types of enzyme activities; fungus was negative correlation withammonium, but had positive correlation with nitrate nitrogen and4types of enzyme activities;Actinomycetes were significantly and positively correlated with urease and L-asparainase inboth indexes. There were significant differences in the characteristic bacteria, soil nitrogenmineralization and soil enzyme.
4The biomass and total N content of inoculation treatments were significantly higherthan CK. The activities of NR, GS, GOGAT were similitude. The enzyme activity ofinoculation treatments were significantly higher than CK. There were no differences betweenT1and T2, whie the differences between T1, T2and T3were significantly. The change of NRin root wre not significantly.
引文
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