Impact Of Long Term Fertilization On The Calcareous Purplish Soil Microbial Characteristics

In order to address and monitor the effect of long-term fertilization on the soil quality, the Soil and Fertilizer Institute of Sichuan Academy of Agricultural Sciences established a 'N, P, K long-term fertilization field experiment'on the Calcareous Purplish Soil in 1982 located in Chuanshan district, Suining city, Sichuan province, China. The impact of the long term fertilization on soil physiochemical properties and crop yields were studied extensively. Based on the previous works, the pour plate count method and most probable number method were used to study the impact of different long term fertilizers on soil microbes' quantity. Chemical analysis was employed to determine the influence of long-term application of fertilizers on soil microbial biomass carbon and nitrogen, soil respiration, nitrification rates and enzymes activity. And finally, the Denaturing gradient gel electrophoresis (DGGE) molecule fingerprint method was applied to study the community structure of bacteria, AM fungi, archaea, ammonium oxidizing bacterial and nitrobacteria. The results were listed as following.(1) The results of long-term application of different fertilizers on soil microbial characteristics and activity in calcareous purple paddy soil showed that the treatments receiving any fertilizer application tended to increase the number of soil microbes compared with CK (no fertilizer treatment). The contents of SMBC and SMBN were among 10.8-91.4 mg.kg^-1 and 10.8-37.2 mg.kg^-1, respectively. Among the 8 fertilizer treatments, the highest amounts of SMBC and SMBN were the soil amended with NPKM. Different fertilizer treatments made varied impact on the soil enzyme activity. Fertilization could improve the activities of invertases, urease and phosphatase, but decrease the activities of polyphenoloxidase. Soil respiration was also improved with fertilization. The respiration in the soil amended with NM was the highest while it in the soil with CK was the lowest. The soil nitrification rates of the same soil varied substantially after long-term treatments with various fertilizers. Generally, soil amended with any fertilizers tended to increase nitrification rates compared with the control. The nitrification rates of the soil appeared to be lower under rice cultivation than those of the same soil under wheat cultivation.(2) Soil bacterial community structure was analyzed by PCR-DGGE targeting bacterial 16S rRNA genes. The results showed that higher diversity of the soil bacterial community was found in soil amended with farmyard manure plus mineral fertilizer than in other fertilizer treatments. Some specific band emerged in the soil amended with farmyard manure. The highest diversity of bacterial communities was found in the NPKM treated soil. The bacterial community structures differed in rice and wheat plots. Sequencing of PCR products separated in DGGE showed that some of the common and dominant bands were closely related to Aquicella lusitana and Acidobacteria.(3) The results about the impact of long-term fertilization on the AM fungi community showed that the diversity of AM fungi was affected by different fertilization treatments and different crop cultivation. The Shannon diversity index of the soil AM fungi community under different long term fertilization with wheat cultivation was higher than that in the soil with rice cultivation. Among the eight long term fertilizer treatments, the highest Shannon diversity index appeared in the soil amended with NM, the diversity index of AM fungi was improved as the soil exposed to the fertilizer treatments with manure (M, NM, NPM and NPK), while the application of phosphor fertilizer would decrease the diversity index. Cluster analysis showed that when the coefficient index was 0.53, in the soil after rice culitivation, the AM fungi communitiy in soil amended with N, CK, M, NM, NPKM was in the first cluster, fertilizers was in the first cluster. AM fungi communities exposed to the NP, NPK and NPM fell into the other three clusters, respectively. In the soil after wheat ciltivation, AM fungi communities in N, NP, M, NM, NPM and NPKM fertilizer treatments were clustered into one group, that in NPK to another group, and that in CK as the third cluster.(4) Archaea community structure in Calcareous Purplish Paddy soil under a long term fertilization experiment was studied. The results showed long term fertilizer could make great impact on the soil archaea community. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that wheat and rice cropping had different impact on the soil archaea community structure, the richness and diversity of archaea community in the soil under NM, NP and NPKM were lower than those under the other fertilizer treatments (as M, NPM, CK, N, and NPK). The sequences analysis of cloned 16S ribosomal DNA showed that the archaea in this study fell into two groups within the terrestrial cluster of Crenarchaeota. One group was closely related to clones from freshwater reservoir, while the second group was similar to clones found in terrestrial habitats. Cluster analysis of the DGGE profiles showed that archaea communities under 8 fertilizer treatments with rice and wheat cultivation were all clustered into 3 groups. In the soil with rice cultivation, the archaea communities in soil amended with M and NPM were in the same cluster. Archaea communities exposed to NP fell into the second group, while those exposed to the NPKM, NM, CK, N and NPM treatments were in the third group. In the soil with wheat cultivation, archaea community under NP was clustered into a single cluster. Those under NPKM and M were fallen into the second cluster, while those in N, NPK, NM, NPM and CK fertilizer treatments were in the third cluster.(5) Soil ammonium oxidizing bacterial community structure was analyzed by using DGGE technique. The treatments receiving any fertilizer application tended to alter the ammonium oxidizing bacterial community compared with the control. Among the eight fertilizer treatments, soil samples from the treatments of mineral fertilizers in combination with farmyard manure acquired more complex ammonium oxidizing bacterial community structure than those receiving mineral fertilizers alone. The principal component analyses (PCA) for ammonium oxidizing bacterial community structure showed that the eight fertilizer treatments were grouped into two PCAs. In the soil after rice harvested, PCA1 was including NP, NM, NPM and NPKM fertilizer treatments, PCA2 were consisted of CK, N, M and NPK fertilizer treatments. In the soil after wheat harvested, PCA1 was formed by M, NM, NPM and NPKM fertilizer treatments. PCA2 was composed of CK, N, NP and NPK fertilizer treatments. The richness of ammonium oxidizing bacterial community in PCA1 was higher than that in PCA2 and also higher in the soil after rice harvested than that after wheat harvested.(6) DGGE method was employed to determine the influence of long-term application of fertilizers on nitrobacteria community in calcareous purple paddy soil. The results showed that fertilizer amendment altered the nitrobacteria community structures. As compared to the mineral fertilizer treatments, the soil amended with the combination of the mineral fertilizers and farmyard manure increased the diversity of the nitrobacteria community as revealed by Unweighted Pair Group Method Clustering Analysis (UPGMA) of he DGGE banding patterns and cluster analysis of the DGGE profiles. In the soil after rice culitivation, the nitrobacteria communitiy in soil amended with nitrogen-phosphor (NP) fertilizers was in the first cluster. Nitrobacteria communities exposed to the fertilizer treatments with manure [manure only (M), nitrogen plus manure (NM), nitrogen and phosphor plus manure (NPM), and nitrogen, phosphor and potassium (NPK)] fell into the second group while those exposed to the CK, N fertilzer treatments were in the third group. In the soil after wheat ciltivation, nitrobacteria communities in CK, M, NM, NPM and NPKM fertilizer treatments were clustered into one group, those in NP, NPK to another group, and those in N as a single cluster.