| Understanding the chronological changes in soil microbial and biochemical properties of tea orchard ecosystems after wasteland has been reclaimed is important from ecological, environmental and management perspectives. In the present study, soil samples of tea orchards with different ages, adjacent wasteland and 90-year-old forest derived from the same parent material were collected from Meijiawu tea area, one of the famous regions of green tea production, located in West Lake district of Hangzhou, Zhejiang Province in Southeast China. A series of laboratory experiments and field investigation were conducted to study soil microbial community structure and diversity, the effect of nitrogen and lime application on soil microbial biochemical properties, and soil nitrification and nitrifiers in relation to land use and tea orchard age. The main results were summarized as follows:1. With the succeeding development of tea orchard ecosystems, the primary microbial biochemical properties in tea orchard soils, i.e., microbial biomass C, basal respiration, microbial quotient (microbial biomass C as a percent of soil organic C), invertase, urease, proteinase, acid phosphatase activity showed a significantly (p<0.05) increasing trend from 8- to 50-year-old tea orchard and then a significantly decreasing trend from 50- to 90-year-old tea orchard, and the highest value was found in the 50-year-old tea orchard. The primary chemical properties in tea orchard soils, i.e., soil acidity, organic C, total N, available P accumulation increased significantly (p<0.05) with increasing tea orchard age, with the largest value occurring in the 90-year-old tea orchard. Soil urease, proteinase, acid phosphatase activity and inorganic N in the tea orchards were significantly (p<0.05) higher than those in the wasteland and forest.2. Soil microbial community structure and diversity were investigated in the wasteland, tea orchards and forest. Biolog analysis showed that soil microbial functional diversity index decreased significantly (p<0.05) with the increase of tea orchard age, and changed in the following order: wasteland > forest > tea orchard. For the DGGE analysis, soil microbial genetic diversity index showed an increasing trend from the 8- to the 50-year-old tea orchard, then a decreasing trend from the 50- to 90-year-old tea orchard, and changed in the following: wasteland > tea orchard > forest. The results based on PLFA analysis indicated that both tea orchard age and land use significantly affected soil microbial community composition and characterizing microbial PLFAs (Gram positive bacteria, Gram negative bacteria, bacteria, fungi, actimomycete, the ratio of Gram positive bacteria to Gram negative bacteria, the ratio of fungi to bacteria). Soil microbial structure diversity index based on PLFA analysis had no significant change in response to tea orchard age and land use. Cluster analysis based on Biolog data, DGGE data, and PLFA data accordantly showed that tea orchard soils with different ages were linked together at a low distance scale, whereas three different land use soils (wasteland, tea orchards, forest) were linked at a high distance scale. The result suggested that different land use had a greater effect on soil microbial community structure compared to tea orchard age.3. Based on laboratory incubation experiment, soil microbial biomass, invertase, urease, proteinase, acid phosphatase in the tea orchards, wasteland, and forest rapidly increased after urea application, and came to maximum points within first five days. These microbial biochemical properties in the tea orchard soils decreased significantly (p<0.05) and became stable at 45 days of incubation. Urea application caused significant (p<0.05) decreases in soil microbial functional diversity index of the tea orchards, and the decline extent was in the order of 8-year-old tea orchard > 50-year-old tea orchard > 90-year-old tea orchard. The soil microbial genetic diversity index increased in the 8-year-old tea orchard and decreased significantly (p<0.05) in the 50- and 90-year-old tea orchard due to urea application. Urea application had no significant effect on soil microbial structure diversity index of the tea orchard soils, but significantly (p<0.05) affected the characterizing microbial PLFAs (Gram positive bacteria, Gram negative bacteria, bacteria, fungi, actimomycete, the ratio of fungi to bacteria). Urea application caused significant increases in soil microbial functional, genetic, and structure diversity indices of the wasteland and forest. Principal component analysis based on Biolog, DGGE, and PLFA data accordantly showed that the effect of urea application on soil microbial community structure in the tea orchards was different from the effect in the wasteland and forest.4. The effect of lime application on soil microbial community structure and diversity was investigated. Biolog analysis showed that both the average well color development (AWCD) of all carbon sources and the functional diversity index increased with the increasing lime quantity in the tea orchards and forest, and decreased with the lime quantity in the wasteland. Principal component analysis based on Biolog data showed that the response of soil microbial communities structure to lime application was significantly different among the tea orchards, wasteland and forest. PLFA analysis showed that soil microbial community structure diversity index increased with the increasing lime quantity in all the tea orchards, wasteland and forest. Lime application increased soil bacterial PLFA content in the tea orchards, wasteland and forest. The tea orchard soil fungal and actinomycete PLFAs showed an increasing trend from 0, 1.6 to 3.2 g CaCO3kg-1 application and then a decreasing trend from 3.2 to 6.4 g CaCO3kg-1 application. For the wasteland and forest, soil fungal PLFA decreased and actinomycete PLFA increased with the increasing lime quantity. Principal component analysis based on PLFA data showed that the effect of lime application on soil microbial community structure in the tea orchards was similar with that in the forest, but different from the effect in the wasteland.5. Two strains of heterotrophic nitrifiers, named strain N1 and N2, were isolated from the acid 50-year-old tea orchard soil. According to the morphological characteristics and the phylogenetic analysis based on 16S rDNA sequence, strain N1 and N2 was identified to be Pseudomonas and Achromobacter, respectively. Strain N2 was the closest relative of Achromobacter xylosoxidans with 98%-99% 16S rDNA sequence identity. For strain N1 and N2, the optimal pH for ammonia oxidation was 7.5 and 7.0, and the optimal temperature for ammonia oxidation was 32℃and 30℃, respectively. Both strain N1 and N2 had the ability to oxidize ammonium on the inorganic medium of pH 3.5, and caused significant decrease in pH of the inorganic medium when they grew on the inorganic medium.6. Soil net N mineralization and nitrification showed an increasing trend from the 8- to 50-year-old tea orchard and then a decreasing trend from the 50- to 90-year-old tea orchard, and were significantly (p<0.05) higher in the tea orchards compared to the wasteland and forest. Tea leaf litter, urea and lime application could improve soil nitrification rate, and the increased percentage of soil nitrification rate was larger in the tea orchards than in the wasteland and forest. For the tea orchard soil ecosystems, the increased percentage of soil nitrification rate in response to urea application was larger in the 8- and 50-year-old tea orchard compared to the 90-year-old tea orchard. As for the tea leaf litter and lime application, the increased percentage of soil nitrification rate was larger in the 50-year-old tea orchard than in the 8-year-old and 90-year-old tea orchard.7. Soil nitrification potential was assessed by the method of soil incubation and suspension incubation. The results indicated that the number of ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, and nitrification potential increased significantly (p<0.05) from the 8- to 50-year-old tea orchard and then decreased significantly from the 50- to 90-year-old tea orchard, and were significantly higher in the tea orchards compared to the wasteland and forest. Urea application stimulated soil nitrification potential in all the soils with different land use, and the change in the order of the tea orchard > forest > wasteland. The comparative analysis based on the method of soil incubation and suspension incubation in determining soil nitrification potential indicated that the method of suspension incubation was much better than the method of soil incubation. The pH 7.2 liquid culture medium could not buffer the highly acid soils enough to neutralize. This may be the reason why the modified suspension incubation method showed higher nitrification potential. |
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