The Rhizosphere Effect Of Mature Stage Tobacco (Nicotiana Tabacum)

In order to understand the rhizosphere effect of tobacco(Nicotiana tabacum) at the mature stage, the rhizosphere and bulk soil samples were collected from representative sites in the tobacco-growing regions in Yanbian, China. The main soil physical and chemical properties of the samples were analyzed. Chemical analysis method was employed to determine the soil microbial biomass carbon, enzyme activities and fungal biomass. The plate count method was used to determine the viable counts of culturable microorganisms.16S rDNA-RFLP was used to study the diversity and community structure of culturable bacteria, actinobacteria, fungi and azotobacteria. Two culture independent mehods, denaturing gradient gel electrophoresis (DGGE) and length heterogeneity polymerase chain reaction (LH-PCR), were combined to study bacterial community structure and diversity. The community structure and diversity of actinobacteria, fungi (including18S and ITS gene), nifH gene, nitrobacteria and arbuscular mycorrhizal fungi (AMF) were analyzed by DGGE. The canonical correspondence analysis (CCA) and multiple regression tree analysis (MRT) were used to explore the microbiological characteristics associated with environmental variables. The results were combined to explore the rhizosphere effect of tobacco at the mature stage.1. The results of soil physical and chemical properties indicated that rhizosphere soils were acidified and the organic matter contents were higher than that of bulk soils. Total and available phosphorus and available potassium were significantly higher in the rhizosphere than in the bulk soil samples, possibly due to mycorrhizal symbionts and acidification of the rhizosphere soils. However, the decrease of total nitrogen indicated competition for nutrients between the plant roots and microbes.2. The results of soil microbial biomass carbon, enzyme activities, fungal biomass, viable microbial counts and the microbial diversity and community structure indicated that a systematical rhizosphere effect on soil microbial characteristics was not found, although typical rhizosphere effect was evident in some of the samples and characteristics tested, due to the accumulation of autotoxins in the tobacco rhizosphere.3. Among the culturable microorganisms, bacteria showed the richest genetic diversity, followed by actinobacteria, fungi and azotobacteria. The results of DGGE analysis indicated that among the microbes bacteria were genetically most diverse, followed by actinobacteria, fungal ITS gene, nifH gene, AMF, nitrobacteria and fungal18S gene.4. In the comparative experiment, the results of comparative analysis of DGGE and LH-PCR showed that the bacterial diversity index detected by DGGE analysis was significantly higher than the bacterial diversity detected by LH-PCR analysis. At the same time, the DGGE revealed more information on the microbial community structure than LH-PCR. However, the LH-PCR method was more convenient, faster, more stable and cheaper than DGGE. The results of comparative analysis of fungal18S gene and ITS gene showed that the fungal diversity index of ITS gene was significantly higher than that of18S gene.5. The results of sequencing of culturable microbes indicated that the dominant species in rhizosphere soil samples were significantly different to that of bulk soil samples. The dominant bacterial groups in tobacco rhizosphere soil samples were Chryseobacterium, Flavobacterium, Agrobacterium, Pseudomonas, Stenotrophomonas, Enterobacter and Acinetobacter; The dominant actinobacterial group was Streptomyces; The dominant fungal groups were Rhizopus, Absidia, Gibberella, Fusarium, Penicillium and Aspergillus; The dominant azobacterial groups were Rhizobium, Arthrobacter and Agrobacterium. However, the dominant bacterial groups in bulk soil samples were Arthrobacter, Brevibacillus and Acinetobacter; The dominant actinobacterial group was Streptomyces; The dominant fungal groups were Fusarium, Eupenicillium, Chaetomium, Paecilomyces and Cerrena; The dominant azobacterial groups were Enterobacter and Arthrobacter.6. According to the culture independent analyses, the dominant species of microorganisms in bulk soil were more specific than in rhizosphere soil. For example, some groups like Pseudonocardia, Oerskovia, Promicromonospora, Lecanicillium, Pseudomonas, Penicillium, some uncultured fungi and uncultured Ascomycota fungi were only present in bulk soil, while Trametes and some uncultured fungi were only present in the rhizosphere soils.7. The sequencing of microbes detected with culture independent methods indicated that the dominant groups in rhizosphere soil samples were significantly different to those of bulk soil samples. The dominant bacterial groups in rhizosphere soil were Steroidobacter, Nitrospira and some genus of Rhizobiales and a-Proteobacteria; The dominant actinobacterial groups were Streptomyces and uncultured actinobacteria; The dominant fungal groups were Malassezia, Glomus, Fusarium, Ceratobasidiaceae, Agaricomycetes, Eurotiomycetes, uncultured fungi and uncultured Ascomycota fungi; The dominant groups of nifH gene were Azonexus, Rhodocyclaceae and uncultured bacterium; The dominant nitrobacterial groups were Nitrospira and uncultured bacteria; The dominant genus of AMF were Glomus and uncultured fungi. However, the dominant bacterial groups in bulk soil samples were Steroidobacter, Actinomycetales and Chitinophagaceae; The dominant actinobacterial groups were Streptomyces, Mycobacterium, Oerskovia and uncultured actinobacteria; The dominant fungal groups were Lecanicillim, Fusarium, Ceratobasidiaceae, Agaricomycetes, uncultured fungi and uncultured Ascomycota fungi; The domiant species of nifH gene were Azonexus, Rhodocyclaceae and uncultured bacterium; The dominant nitrobacterial groups were Nitrospira and uncultured bacteria; The dominant genus of AMF was Glomus.8. The results of soil microbial diversity associated with environmental variables indicated that total phosphorus and available phosphorus were the major environmental factors affecting the soil microbial biomass carbon, soil enzyme activities and soil fungal biomass. Available potassium and available phosphorus were the main environmental factors affecting the soil viable microbial counts. Available potassium was the most important environmental factor affecting the microbial diversity detected with the culture independent methods, and available phosphorus, available nitrogen and total nitrogen also had definite effects. Total and available phosphorus were the main environmental factors affecting the bacterial diversity detected with the culture independent methods. Available potassium affected actinobacterial and fungal diversity, available nitrogen and organic matter affected nifH gene diversity, and available phosphorus affected the diversity of nitrobacteria and AMF.