Effects Of Interplanting With Cassia Rotundifolia Pers And Fertilization On Soil Nitrogen-fixing Microbial Communities In Tea Plantations

In tea plantations, the long-term cultivation of tea plants and the interplanting of other plants lead to the formation of unique soil microbial communities. Among these, nitrogen-fixing microbes are a very important special functional group. Nitrogen-fixing microbes play an irreplaceable role in the nitrogen cycle, and their diversity directly affects the processes of the nitrogen cycle and the utilization of nitrogen-fixing resources. Until now, the research on interplanting in tea plantations has mainly focused on the arthropod community, the growth of tea plants and the quality and yield of tea and so on. However, research on the diversity of nitrogen-fixing microbes has rarely been reported. This thesis studied the effects of interplanting with Cassia rotundifolia Pers and fertilization in tea plantations on the diversity of nitrogen-fixing microbes. The results were as follows:(1) There were two peaks in the abundance of soil microorganisms in tea plantations every year. The amount of soil microorganisms gradually ascended from February until its first peak in June. It then slightly declined in August before climbing to its second peak in October. After that, it began to fall until the next February. The ratio between bacteria, fungi, actinomycetes and nitrogen-fixing bacteria was broadly maintained in the soil of tea plantations under different treatments. According to their amounts, the order of different groups was bacteria, actinomycetes, nitrogen-fixing bacteria and fungi. The amount of microorganisms in 0~20 cm soil layer was obviously greater than that in 20~40 cm soil layer. In comparison with CK, both interplanting with Cassia rotundifolia Pers and fertilization significantly increased the amount of bacteria and total microorganisms in different soil layers. Fertilization obviously increased the amount of fungi and decreased the amount of actinomycetes and nitrogen-fixing bacteria in different soil layers. Interplanting with Cassia rotundifolia Pers significantly decreased the amount of fungi and increased the amount of actinomycetes and nitrogen-fixing bacteria in different soil layers.(2) The acquisition of high quality, large-fragment sized and unbiased DNA is an important basis for the molecular ecology studies on soil microbes in tea plantations. Five DNA extraction methods for soil microbes were compared: a SDS high-salt method, a denaturant plus SDS high-salt method, a removal of humic acids and SDS high-salt method, a CTAB method and a Krsek-improved method. The results indicated that the Krsek-improved method was an efficient, reliable DNA extraction method for molecular ecology studies on soil microbes in tea plantations. Using this method, the DNA obtained was >23 kb of fragment, >1.70 in OD260/OD280, >1.35 in OD260/OD230, and >34.50μg·g-1 dry soil of yield and could be directly used for the following molecular ecology studies without further purification.(3) The OTU (operational taxonomic unit) types, H', D and dMa of the nifH gene in the 0~20 cm soil layer under different treatments were greater than their counterparts in the 20~40 cm soil layer. In comparison with CK, interplanting with Cassia rotundifolia Pers increased the OTU types, H', D and dMa of the nifH gene in different soil layers, whereas fertilization had few effects. The similarity between the 23 sequences of nifH genes cloned and the nifH gene sequences published in the GenBank database ranged from 85%~90%, and 91.3% of the cloned sequences shared their highest identity with the uncultured bacteria in the environment. Nitrogen-fixing microorganisms distributed in theα-,β-,γ-Proteobacteria and the Cyanobacteria with the rations of 69.6%, 8.7%, 13% and 8.7%, respectively, which represented the diversity of the soil nitrogen-fixing microbes in tea plantations.