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Vegetable soil ecosystem Molecular Microbial Ecology Research

Author: LeiJuanLi
Tutor: YuJingQuan
School: Zhejiang University
Course: Olericulture
Keywords: Vegetable Soil ecosystem Microbial diversity PCR-RFLP PCR-DGGE
CLC: S154.3
Type: PhD thesis
Year: 2006
Downloads: 1102
Quote: 12
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Abstract


Microbial organisms play a key role in energy and nutrient cycling, plant litter decomposition, soil productivity maintenance, plant growth and plant disease protection within the agricultural ecosystems. Due to the selectivity of the traditional methods, only 1% of the total population of soil microbial organisms can be cultured and limited data is available concerning how closely they reflect the actual composition of these communities. In this study, molecular methods were used to examine the microbial diversity relationships in the vegetable ecosystems. The results are as follows:1. To study the influence of cropping system on bacterial community structure, PCR-RFLP analysis, a culture-independent method, was used to compare the bacterial community structure between two contrasting types of soils: vegetable soil and paddy rice soil. Bacterial 16S rRNA gene (16S rDNA) libraries of the two types of soils were built and the 16S rDNA were then reamplified and digested with restriction endonuclease (Hinf I). In the vegetable soil, there were total 33 16S rDNA genotypes in 75 recombinants, and in paddy soil there were total 17 16S rDNA genotypes in 55 recombinants. Compared to the paddy soil, the vegetable soil showed higher bacterial diversity index, and most of the clones belonged to Proteobacteria gamma, alpha and beta subdivisions while a few clones were affiliated with high G+C bacteria, Chloroflexus and Nitrospina bacteria. For the paddy soil, half of the clones belonged to Proteobacteria gamma and beta subdivisions, with the remaining half belonging to high G+C bacteria, Prosthecobacter and Sphingobacterium.2. PCR-RFLP analysis, a culture-independent method, was used to examine the fungal diversity in the two contrasting types of soils: vegetable soil and rice paddy soil. Fungal 18S rDNA libraries of the two types of soils were built and the 18S rDNA were then reamplified and digested with restriction endonuclease (Hinf 1). In the vegetable soil there were a total of 26 18S rDNA genotypes in 82 recombinants, and in paddy soil there were a total of 23 18S rDNA genotypes in 84 recombinants. Compared to the paddy soil, the vegetable soil showed higher fungal community diversity and most of them belongedto Ascomyta division, while other clones were affiliated with Basidimycota, Chytridiomycota and Zygomycota divisions. In comparison, for the paddy soil, most clones belonged to unclassified fungi, with the remaining clones belonging to Chytridimycota, Ascomycota and Zygomycota divisions, and no Basidiomycota was found.3. Soil bacterial communities were examined by culture-independent means, namely, 16S rDNA-nested PCR-DGGEprofiling. Crude DNA was extracted from soil used to grow different vegetables for different periods, amplified with bacterial specific primers of 16S rDNA by PCR, and separated by denaturing gradient gel electrophoresis (DGGE). Subsequently, bands on DGGE gels were cloned sequenced and the resultant information was used to infer the diversity of bacteria in different soils. The results showed that soil samples collected from same area had a high identity in DGGE bands patterns. Bacterial community structure was significantly influenced by continuous cropping system. The effects were closely related to the kind of vegetables and the history of continuous cropping. The sequence results showed that most of the bacteria belong to Proteobacteria;Acidobacteria, Sphingobacteria and Actinobacteria also appeared in the continuously cropped soils.4. The vegetable soil bacteria community structure in a field with long-term fertilizer use was studied by PCR, clone, RFLP and sequence of the bacterial 16S rRNA gene. The results showed that the soil bacterial diversity under long-term organic fertilizer was higher than that of under long-term no fertilizer control, and the soil bacterial diversity under long-term chemical fertilizer was lower than that of under long-term no fertilizer control. At the same time, the bacterial community changed differently under long-term organic or chemical fertilizer compared to no fertilizer control: In the long-term organic fertilizer soil, a-Proteobacteria were increased compared to the control by 15%, y-Proteobacteria and Sphingobacteria were decreased by 8% respectively, and there were no 8-Proteobacteria found (the control is 5%);In the long-term chemical fertilizer soil, had increased Acidobacteria compared to the control by 8%, the differences of the other bacterial division compared to control was less than 3%.5. Using the bacterial 16S rDNA PCR amplification, DGGE, cloning, RFLP and sequencing methods, resistant and susceptible tomato varieties as material to study the rhizoplane bacteria with inoculation or without inoculation, and the of different levels of rhizosphere bacteria of diseased tomatoes. The results showed: (1) There are different bacterial DGGE bands for diseased and healthy tomato rhizoplane;(2) There are different DGGE band patterns for different level of diseased tomato rhizoshpere bacteria. The closer the level of diseased plants, the higher the similarity of the DGGE band patterns;(3) There are different dominant bacterial genotypes and bacterial divisions for resistant and susceptible tomato varieties. There Y -proteobacteria were more in susceptibe tomato variety, whereas P -proteobacteria and Sphigobacteria were more common in resistant tomato variety. The distribution unevenness and diversity of rhizoplane bacterial of resistant tomato variety was higher than that of the susceptible tomato variety.6. Using DNA directly extracted, bacterial 16S rDNA PCR amplification, DGGE, cloning and sequencing methods, to study the bacterial diversity in tomato hydroponic culture with different element Ca and P level, indicated interesting results. The results showed that each hydroponic culture of element level had the similar DGGE bands patterns. Compared with the control, every DGGE band patterns of the hydroponic culture changed differently. The bands distribution in the control was more evenly than that of the treatments. Sequencing results showed that the main bacterial divisons in tomato hydroponic culture were Proteobacteria (Alpha, Beta, Gamma and Unclassified), Bacteroidetes and Gram Positive Bacteria.

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CLC: > Agricultural Sciences > Agriculture as the foundation of science > Soil > Soil Biology > Soil microbiology
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