葡萄根际高效溶有机磷细菌的筛选及其溶磷特性初步研究
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摘要
磷是植物生长发育的必需营养元素之一,而土壤中绝大部分的磷素不能被植物直接利用。施用磷肥一直是保证农业生产增产的重要手段之一,单纯依靠大量施用磷肥来解决磷素缺乏已经出现资源浪费、环境污染等诸多弊端。利用植物根际与磷循环相关的生物学系统来解决这个问题已经越来越被人们所关注。溶磷微生物在该生物学系统中担任着重要的角色。它可将土壤中难溶性的磷酸盐转化为可溶性盐供作物生长利用,提高磷肥的利用率,减少土壤对磷酸盐的吸附固定,避免磷肥过量使用造成环境污染。因此从土壤中筛选高效溶磷微生物生产生物肥料,以减少磷肥施用量,对农业可持续发展具有重要的生态和社会意义。
本研究主要以西北几个地区葡萄根际土壤为试验材料,利用平板培养法和微生物选择培养基进行培养分离,初步筛选出具有溶有机磷能力的细菌。再通过液体摇瓶试验,利用钼锑抗比色法测定液体培养基中可溶性磷的含量,通过定量比较,得到高效溶磷菌株。利用16S rDNA序列分析对部分菌株进行鉴定。对其中几株溶磷效果较好的菌株进行溶磷能力和溶磷特性初步研究,以期为溶磷微生物肥料的开发应用提供优良菌株。得出如下主要结果:
1.从各种土壤中筛选得到溶有机磷细菌81株,从陕西泾阳和宁夏银川葡萄根际土壤中筛选到的细菌的数目较多。从宁夏银川筛选出来的菌株的总体溶磷效果较其他土样的菌株溶磷效果好。溶磷效果最好的菌株为宁夏银川葡萄园中筛得,其溶磷效果可达到62.2856mg/L。
2.对筛选出来溶磷效果较好的13株菌株进行了16S rDNA序列分析,结果表明,13株溶有机磷的细菌中9株属于根瘤菌属(Rhizobium sp.)、1株属于节杆菌属(Arthrobacter sp.)、1株属于不动杆菌属(Acinetobacter sp.)、1株属于假单胞菌属(Pseudomonas sp.)、1株属于土壤杆菌属(Agrobacterium sp.)。
3.根据统计结果和差异显著性分析,选择溶有机磷较好的5株(2-15、5-12、5-18、5-19、5-21)研究其溶磷能力受碳源、氮源的影响。选择溶磷最好的2株(5-18、5-19)检测其溶磷动态,不同培养时间溶磷量和pH之间的关系。研究结果:
在液体摇瓶条件下溶磷菌株溶磷能力受碳源、氮源的影响。总体上菌株在以葡萄糖作为唯一碳源时溶磷较好,葡萄糖还是较理想的碳源。在氮源的单因子试验中,5株菌在以硝酸钾作为氮源时,都不是最佳的溶磷效果,但是可以利用硝态氮。不同碳源、氮源对菌株的溶磷情况均有一定的影响。
两株溶有机磷细菌在整个培养过程总体溶磷趋势为先升后降,pH值是先降后升。微生物的溶磷量与培养液中的pH值存在一定的关系,但培养液pH值的下降,并不是溶磷的必要条件。溶磷机制还需要进一步研究。
Phosphorus is one of the necessary elements for the growth of the plants. Application of phosphorus fertilizer is a very crucial management to increase crop yields, because most of the phosphorus in soil can not be utilized by plants directly. In recent years, excessive phosphate fertilizer application in soil has brought a series of problems, such as mineral resource waste and environment contamination. Nowadays more and more researchers focus on solution for this problem by regulating the bio-system and micro-environment in the rizosphere of plant roots. Phosphorus solubilizing bacteria in soil plays an important role in this bio-system. Generally, insoluble phosphate in soil can be dissolved by phosphorus solublizing microorganisms and absorbed by plant, which can increase the utilizing efficiency of phosphorus fertilizer and decrease environmental pollution caused by slathering phosphorus fertilizer. Hence it is very important for agricultural sustainable development that screening high efficiency phosphate-solublizing microbial strains as inoculant to manufacture biofertilizer.
In this study, different rhizosphere soils of grapevine from vineyards located in northwestern areas in China were taken as samples. The method of plate culture and selective medium were used to culture and separate soil microbes, and then organic-phosphorus solubilizing bacteria was screened preliminary. In liquid medium, ability of dissolving organic phosphorus of organic-phosphoru solubilizing bacteria (OSB) was investigated via Molybdenum-antimony spectrophotometric method. Phosphorus solublizing bacteria was isolated from soil samples, and then identified by 16S rDNA sequence analysis. Several isolates were studied on their phosphorus solubilizing ability and mechanism to develop excellent phosphate solubilizing strains for bettering phosphorus utilization. The main results are as follows:
1. There are 81 OSB strains with better effect of solubilizing organic phosphorus in liquid medium (NBRIP), and the number of OSB in Jingyang(Shannxi) and Yinchuan (Ningxia) was more than other areas.The phosphorus solubilizing ability of the strains isolated from Ningxia area were better than the strains isolated from other areas. The best performance of dissolving organic phosphorus was found on the OSB isolated from Ningxia with 62.2856mg/L.
2. 13 strains of OSB with better performance were identified by 16S rDNA sequence analysis. The results showed that 9 strains belong to the Rhizobium sp., other four strains belong to the Arthrobacter sp., the Acinetobacter sp., the Pseudomonas sp.and the Agrobacterium sp., respectively.
3. An experiment was conducted with 5 chosen OSB strains(2-15、5-12、5-18、5-19、5-21) to figure out their phosphorus solubilizing ability under different carbon sources and nitrogen sources.
It indicated that phosphate-solubilizing abilities of these 5 strains were affected by carbon source and nitrogen source. Glucose looks like the best candidate when it treated as the only carbon source. All the strains can not show the best phosphorus solubilizing ability when KNO3 was the only nitrogen source, but the result showed that the strains can use the nitrate nitrogen source.
Other 2 strains (5-18 and 5-19) were chosen to study the dynamic of phosphate solubilization and the relationship between phosphorus content and pH value at different time. It was indicated that the phosphorus content in the fermentation liquid of the two strains increased at first and then decreased, while the pH Value decreased at first and then increase during the culture process. At last, the conclusion which can be obtained was that the phosphorus content had some relation with the pH value, but the decline of the pH value was not the necessary condition for the microorganism to solubilize the phosphorus.
本研究主要以西北几个地区葡萄根际土壤为试验材料,利用平板培养法和微生物选择培养基进行培养分离,初步筛选出具有溶有机磷能力的细菌。再通过液体摇瓶试验,利用钼锑抗比色法测定液体培养基中可溶性磷的含量,通过定量比较,得到高效溶磷菌株。利用16S rDNA序列分析对部分菌株进行鉴定。对其中几株溶磷效果较好的菌株进行溶磷能力和溶磷特性初步研究,以期为溶磷微生物肥料的开发应用提供优良菌株。得出如下主要结果:
1.从各种土壤中筛选得到溶有机磷细菌81株,从陕西泾阳和宁夏银川葡萄根际土壤中筛选到的细菌的数目较多。从宁夏银川筛选出来的菌株的总体溶磷效果较其他土样的菌株溶磷效果好。溶磷效果最好的菌株为宁夏银川葡萄园中筛得,其溶磷效果可达到62.2856mg/L。
2.对筛选出来溶磷效果较好的13株菌株进行了16S rDNA序列分析,结果表明,13株溶有机磷的细菌中9株属于根瘤菌属(Rhizobium sp.)、1株属于节杆菌属(Arthrobacter sp.)、1株属于不动杆菌属(Acinetobacter sp.)、1株属于假单胞菌属(Pseudomonas sp.)、1株属于土壤杆菌属(Agrobacterium sp.)。
3.根据统计结果和差异显著性分析,选择溶有机磷较好的5株(2-15、5-12、5-18、5-19、5-21)研究其溶磷能力受碳源、氮源的影响。选择溶磷最好的2株(5-18、5-19)检测其溶磷动态,不同培养时间溶磷量和pH之间的关系。研究结果:
在液体摇瓶条件下溶磷菌株溶磷能力受碳源、氮源的影响。总体上菌株在以葡萄糖作为唯一碳源时溶磷较好,葡萄糖还是较理想的碳源。在氮源的单因子试验中,5株菌在以硝酸钾作为氮源时,都不是最佳的溶磷效果,但是可以利用硝态氮。不同碳源、氮源对菌株的溶磷情况均有一定的影响。
两株溶有机磷细菌在整个培养过程总体溶磷趋势为先升后降,pH值是先降后升。微生物的溶磷量与培养液中的pH值存在一定的关系,但培养液pH值的下降,并不是溶磷的必要条件。溶磷机制还需要进一步研究。
Phosphorus is one of the necessary elements for the growth of the plants. Application of phosphorus fertilizer is a very crucial management to increase crop yields, because most of the phosphorus in soil can not be utilized by plants directly. In recent years, excessive phosphate fertilizer application in soil has brought a series of problems, such as mineral resource waste and environment contamination. Nowadays more and more researchers focus on solution for this problem by regulating the bio-system and micro-environment in the rizosphere of plant roots. Phosphorus solubilizing bacteria in soil plays an important role in this bio-system. Generally, insoluble phosphate in soil can be dissolved by phosphorus solublizing microorganisms and absorbed by plant, which can increase the utilizing efficiency of phosphorus fertilizer and decrease environmental pollution caused by slathering phosphorus fertilizer. Hence it is very important for agricultural sustainable development that screening high efficiency phosphate-solublizing microbial strains as inoculant to manufacture biofertilizer.
In this study, different rhizosphere soils of grapevine from vineyards located in northwestern areas in China were taken as samples. The method of plate culture and selective medium were used to culture and separate soil microbes, and then organic-phosphorus solubilizing bacteria was screened preliminary. In liquid medium, ability of dissolving organic phosphorus of organic-phosphoru solubilizing bacteria (OSB) was investigated via Molybdenum-antimony spectrophotometric method. Phosphorus solublizing bacteria was isolated from soil samples, and then identified by 16S rDNA sequence analysis. Several isolates were studied on their phosphorus solubilizing ability and mechanism to develop excellent phosphate solubilizing strains for bettering phosphorus utilization. The main results are as follows:
1. There are 81 OSB strains with better effect of solubilizing organic phosphorus in liquid medium (NBRIP), and the number of OSB in Jingyang(Shannxi) and Yinchuan (Ningxia) was more than other areas.The phosphorus solubilizing ability of the strains isolated from Ningxia area were better than the strains isolated from other areas. The best performance of dissolving organic phosphorus was found on the OSB isolated from Ningxia with 62.2856mg/L.
2. 13 strains of OSB with better performance were identified by 16S rDNA sequence analysis. The results showed that 9 strains belong to the Rhizobium sp., other four strains belong to the Arthrobacter sp., the Acinetobacter sp., the Pseudomonas sp.and the Agrobacterium sp., respectively.
3. An experiment was conducted with 5 chosen OSB strains(2-15、5-12、5-18、5-19、5-21) to figure out their phosphorus solubilizing ability under different carbon sources and nitrogen sources.
It indicated that phosphate-solubilizing abilities of these 5 strains were affected by carbon source and nitrogen source. Glucose looks like the best candidate when it treated as the only carbon source. All the strains can not show the best phosphorus solubilizing ability when KNO3 was the only nitrogen source, but the result showed that the strains can use the nitrate nitrogen source.
Other 2 strains (5-18 and 5-19) were chosen to study the dynamic of phosphate solubilization and the relationship between phosphorus content and pH value at different time. It was indicated that the phosphorus content in the fermentation liquid of the two strains increased at first and then decreased, while the pH Value decreased at first and then increase during the culture process. At last, the conclusion which can be obtained was that the phosphorus content had some relation with the pH value, but the decline of the pH value was not the necessary condition for the microorganism to solubilize the phosphorus.
引文
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