| Tea plant [Camellia sinensis(L.)O.Kuntze] is an important perennial cash crop with Chinese characteristics,and its growing environment plays an important role in the growth and development of tea plants and the formation of physicochemical components related to tea quality.However,at present,most tea plantations use a single pure tea plantation cultivation pattern,resulting in soil degradation and soil microecological environment changes,the intensification of diseases and insect pests,the declining of biodiversity and productivity in tea plantations.Rational planting of crop diversity can not only enhance the utilization of nutrient resources,crop yield and quality,but also increased the diversity of soil microbial community and promote the nutrient cycle of soil ecosystem.As an important way to increase plant diversity,intercropping,especially with legumes as the main functional group,promotes the diversity of plant communities,increases the utilization rate of nutrients,and be benefits to the construction of agricultural ecosystem.Intercropping leguminous green manure in tea plantation is one of the main ways to construct ecological tea plantation system.Many studies have described the effects of intercropping on soil physicochemical properties,soil microbial community,growth and metabolism of tea plants and tea quality.However,the mechanism of the effects of intercropping leguminous green manure on soil environment of tea plantation is not clear,and the formation of tea chemical components has not been further studied.In this study,tea cultivar ’Huangshan Quntizhong’,soybean cultivar ’Nannong 48’ and Chinese milk vetch cultivar ’Minzi 5’ were selected as trial materials.The experimental design included monocultured tea plants as the blank control,the tea plants-soybean + Chinese milk vetch intercropping,as well as the tea plants-soybean intercropping,continuously monitor for immovable position.Soil microbiome and metagenomics were used to analyze the effects of intercropping leguminous green manure on the composition and function of soil microbial community,and combined with the physicochemical properties of soil,soil enzyme activities,changes in the quality constituents of fresh leaves of tea plants and the distribution of amino acids in soil,roots and leaves of tea plants.To explore the effects of intercropping leguminous green manure on soil environment and the formation of amino acids in tea,at the same time,the gene of amino acid permeability enzyme was studied to further explore the mechanism of the amino acid absorption and transport in tea plants.The main researches and conclusions were as follows.1.Intercropping leguminous green manure in tea plantation for one year and three years,in the profuse flowering of Chinese milk vetch was turned over and returned to the soil for one month,intercropping leguminous green manure in tea plantation could improve soil physicochemical properties and tea quality effectively.For intercropping tea plants-soybean,the tea plantation soil total and available nutrients,soil p H,and the related carbon,nitrogen and phosphorus cycle of extracellular enzyme activity were significantly higher than other treatments,and the EGCG,amino acids and soluble sugar content of fresh tea leaves were also significantly higher than monoculture tea plants.In the intercropping tea plants-soybean + Chinese milk vetch,except the soil nitrogen nutrient,the other soil available nutrients were significantly increased for one year,but soil total and available nutrients,as well as the extracellular enzyme activity with carbon nitrogen and phosphorus cycle were significantly lower than monoculture tea plants,but the amino acids and soluble sugar content in fresh tea leaves were significantly increased for three years.In the profuse flowering of Chinese milk vetch was turned over and returned to the soil for one month,for leguminous green manure intercropping,the total and available nutrients and soil enzyme activity related to soil carbon and nitrogen cycle,the content of EGCG,amino acids and soluble sugar in tea plants fresh leaves were significantly higher,and the effect of intercropping tea plants-soybean + Chinese milk vetch was more significant.In addition,the soil p H value was increased in intercropping tea plants-soybean + Chinese milk vetch treatment,which could effectively alleviate soil acidification.2.Intercropping leguminous green manure in tea plantation could increase the diversity and richness of soil microbial community,and change the relative abundance of soil microbial community.Compared with monoculture tea plants,the relative abundance of Acidobacteria,Proteobacteria,Gemmatimonadetes and Chloroflexi were changed in the tea plantation with intercropping leguminous green manure,and Ascomycota and Basidiomycota were the most abundant fungi,followed by Mortierellomycota with the significantly increased relative abundance.The soil microbial community were asynchronous in different intercropping pattern.In the profuse flowering period of Chinese milk vetch,the relative abundance of Acidobacteria,Planctomycetes,Armatimonadetes and Gemmatimonadetes was increased with intercropping tea plants-soybean + Chinese milk vetch,while the relative abundance of Proteobacteria and Chloroflexi in the intercropping tea plants-soybean was higher than that of other treatments.In the profuse flowering of Chinese milk vetch was turned over and returned to the soil for one month,the relative abundance of other soil bacteria in the intercropping tea plants-soybean + Chinese milk vetch was increased,except for Acidobacteria,Planctomycetes and Armatimonadetes.The complexity of the soil bacteria and the interaction of soil fungi were increased in the tea plantation with intercropping leguminous green manure,the complexity and interaction of the total network in different intercropping patterns were different,and the soil p H and nitrate nitrogen are the key factors affecting soil microbial community,and soil bacteria community was more easily affected by these soil factors than soil fungal community.3.The metagenomic analysis of the tea plantation soil with intercropping leguminous green manure showed that intercropping leguminous green manure in tea plantation could maintain the balance of soil microorganisms,but also significantly improve the number,metabolic activity and diversity of soil microorganism.The relative abundance of Proteobacteria,Acidobacteria and Gemmatimonadetes in the intercropping tea plants-soybean + Chinese milk vetch was higher,and the relative abundance of soil Actinobacteria was significantly increased with intercropping tea plants-soybean,while the relative abundance of Chloroflexi was significantly decreased in the treatment of intercropping leguminous green manure.And compared with the monoculture tea plants,the relative abundance of Burkholderia,Mycobacterium and Paraburkholderia was significantly increased in the treatment of intercropping leguminous green manure.KEGG annotated metabolic pathways and KO functional categories of soil microbial community were significantly different in leguminous green manure intercropping,and tea plants-soybean + Chinese milk vetch intercropping showed more significant functional differences,and the number of metabolic pathways and functional categories significantly increased.The abundance of soil environmental information processing was increased with tea plants-soybean + Chinese milk vetch treatment,and the abundance of carbohydrate metabolism and carbon metabolic function significantly increased.Compared with the monoculture tea plantation,soil carbon and nitrogen metabolism,alanine,glutamic acid and aspartic acid metabolism,amino sugar and starch and sucrose metabolism and nucleic acid metabolism of sugar and other metabolic pathways associated with carbon and nitrogen in the treatment of intercropping leguminous green manure were significantly increased.Besides the metabolism of starch and sucrose and amino sugars and nucleic sugars was increased in the treatment of intercropping tea plants-soybean,the relative abundance of other metabolic pathways was higher in the intercropping tea plants-soybean + Chinese milk vetch.At KO level,there were also significant differences in the functional categories of carbon metabolism,carbohydrate metabolism,nitrogen metabolism and various amino acid metabolic pathways.The abundance of soil carbon metabolism and carbohydrate metabolism(K01114,K00163 and K00688)in intercropping leguminous green manure treatment were significantly increased.The functional categories involved in nitrogen metabolism and amino acid metabolism showed no steady trend in tea plant-soybean +Chinese vetch intercropping treatment,but the functional categories involved in alanine,aspartic acid and glutamate metabolism was increased(K01953 and K01915).In addition,the soil available nitrogen(AN)and amino acid nitrogen(ANN)on soil microbial community composition and functions plays an important role,and the soil nutrient(AN and AAN)and extracellular enzyme activity related to carbon and nitrogen cycle(β-1,4-glucosidase and β-1,4-N-acetyl-glucosaminidase)were influenced with intercropping leguminous green manure,further to change the soil microbial community composition and functions,in particular,carbon and nitrogen cycling related metabolism,and thus changed soil ecosystem functions.4.The soil amino acid nitrogen content with intercropping leguminous green manure were significantly higher than that of monoculture tea plantation,and the all kinds of free amino acids content were increased.While the total free amino acid in the roots and leaves of tea plants were not significantly increased,especially the contents of glutamate and theanine in the root of tea plants intercropping with soybean decreased significantly,but the theanine content in fresh leaves of tea plants were significantly higher than that of monoculture tea plants,especially in the treatment of intercropping tea plants-soybean +Chinese milk vetch,which was beneficial to the formation of tea quality.5.The members of the CsAAPs gene family were identified and the expression specificity of CsAAPs genes was determined.It was found that CsAAP2 and CsAAP5 genes located on the endoplasmic reticulum membrane,and could be induced expression by the treatments of different nitrogen and intercropping leguminous green manure,and could absorbed amino acids.RT-q PCR was used to analyze the expression abundance of CsAAPs gene family members in different tissues and varieties of tea plants.Except for CsAAP9,and the expression level of the CsAAPs gene family members was significantly increased in tea varieties with high amino acids,especially in ’Huangjinya’ varieties.In addition,the expression abundance of CsAAP2,CsAAP12 and CsAAP13 was increased in tea plants with inorganic nitrogen and glutamic acid as the only nitrogen source,and CsAAP5 was induced in roots with ammonium nitrogen as the nitrogen source.CsAAP5 was induced in both roots and leaves of tea plants with intercropping leguminous green manure,and CsAAP2 was only induced in roots of tea plants with intercropping tea plants-soybean + Chinese milk vetch.Meanwhile,though CsAAP2 and CsAAP5 were expressed in the amino acid deficient yeast mutant 22Δ10α.CsAAP2 and CsAAP5 were found that could transported most acid and neutral amino acids in yeast cells,and CsAAP5 had a higher affinity for amino acids absorption.The results of this study showed that intercropping leguminous green manure in tea plantation were regulated the soil microbial community structure,improved soil productivity and ecological function,achieved sustainable utilization of soil,and benefited the growth and metabolism of tea plants.In this paper,the soil microbial community structure and functional response mechanism of intercropping leguminous green manure in tea plantation were explored in depth,and the theoretical basis for the construction of modern ecological composite cultivation tea plantation was provided. |
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