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Effects Azotobacter on the Micro-ecosystem Associated with the Rhizosphere of Maize and Wheat

Author: ChenShengNan
Tutor: GuJie
School: Northwest University of Science and Technology
Course: Environmental Science
Keywords: Azotobacter BIOLOG Nested PCR-DGGE T-RFLP Enzyme activity Microbial community diversity
CLC: S154.3
Type: PhD thesis
Year: 2012
Downloads: 367
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Abstract


In this work, RFLP, BIOLOG, PCR-T-RFLP, nested PCR-DGGE, FTIR and root scanningtechniques were employed to explore the mechanisms of interaction between Azotobacter and wheat(Triticum aestivum L.), maize (Zea mays L.) seedlings. Firstly, sixty-two Azotobacter strains wereisolated from the soils collected from Qinling Mountains, Zhouzhi County, Shaanxi Province.PCR-RFLP technology was employed to determine the fingerprinting of isolated Azotobacter strains.Nitrogenase activity was examined, and three strains with high nitrogenase activity were identified asAzotobacter chroococcum YCYS, Phyllobacterium brasssicacearum QL54and Paenibacillussabinae MX31. Whilst, we investigated the effects of Azotobacter inoculation on the growth, soilenzyme activities and microbial community diversity associated with the rhiosphere of maize (Z. maysL.) and wheat (T. aestivum L.) seedlings under pot cultural conditions. After that, we alsoinvestigated responses of Azotobacter inoculated with maize seedlings to different nitrogen levels andsalt concentrations. Finally, we examined the effects of inoculation with microbial inoculants, nitrogenaddition and utilization of enzyme preparations on the soil enzyme activities and microbialcommunities associated with the rhizosphere of wheat (T. aestivum L.) seedlings in the fieldexperimental conditions. Thus, these results providing a scientific evaluation for the Azotobacterresources utilization, microorganism inoculant development and the combined application ofAzotobacter inoculant and nitrogen fertilizer with reasonable ratio in agricultural process. The mainresults of this dissertation are as follows:1. In the present work, sixty-two strains were isolated from the soils located in Qinling Mountains,Zhouzhi County, Shaanxi Province. RFLP technology was employed to determine the fingerprintingof isolated Azotobacter strains. Nitrogenase activities were also examined, and the highest nitrogenaseactivity was observed in YCYS strain with47.74±1.02nmol/(mg·h). The three strains YCYS, QL54and MX31with higher nitrogenase activities were identified as Azotobacter chroococcum YCYS,Phyllobacterium brasssicacearum QL54and Paenibacillus sabinae MX31, respectively.2. To explore the responses of growth, nutrition status of maize (Z. mays L.), soil enzymeactivities and bacterial community functional and genetic diversity in the rhizosphere soils of maizeseedlings inoculated with Azotobacter isolates including Azotobacter chroococcum YCYS,Phyllobacterium brasssicacearum QL54and Paenibacillus sabinae MX31, the pot experiment was examined. The results showed that the inoculation of Azotobacter could improve the growth of maizeseedlings with the extension of inoculation, the shoot fresh weight was ordered as:MX31>QL54>YCYS>Control after42days inoculation. The urease activities of A. chroococcumYCYS and P. sabinae MX31treatments were increased by20.55%and9.58%compared with thecontrol after7days inoculation, respectively. The BIOLOG results showed that the Azotobacterinoculation could increase the total bacterial community metabolic activity.3. To explore the effects of Azotobacter inoculation on the growth of wheat (Triticumaestivum L.) seedlings, soil enzyme activities and functional diversity of microbialcommunities, the pot experiment was conducted. The results showed that the height ofseedlings inoculated with QL54was significantly higher than that of uninoculated control(p<0.01). The root activities of inoculated treatments were significantly higher than that ofuninoculated control in20days after inoculation process. Whilst, the hundred grain weight ofwheat inoculated with YCYS and QL54, increased22.05%and20.56%compared withcontrol(p<0.05). The nitrogen uptake of wheat grain inoculated with YCYS、QL54and MX31were higher than uninoculated control by7.58%、16.76%and1.78%, respectively. There wasno effect of Azotobacter inoculation on composition of grains among four treatments used byFTIR spectrometer. The BIOLOG principle component analysis (PCA) suggested that therewere changes in bacterial and fungal communities in the rhizosphere of wheat (T. aestivum L.)seedings inoculated with Azotobacter.4. To explore the effects of nitrogen addition on soil enzyme activities and the diversity of fungalcommunity associated with the rhizosphere of wheat (T. aestivum L.), a field experiment wasconducted. The results showed that dehydrogenase activity was decreased with the increased nitrogenaddition, the dehydrogenase activity of low nitrogen treatment was22.93±4.89μg TF (g.24h)-1, and itwas3.74times compared with high nitrogen treatment (P<0.05). Meanwhile, there was no significantdifferences of protease activity and sucrase activity among four treatments (P>0.05).58band typeswere viewed in nested PCR-DGGE profile of fungal18S rDNA V3region. Fungal Species Richness(S) and Shannon Diversity (H) index were ordered as: control>middle nitrogen>low nitrogen>highnitrogen.5. To explore the effects of Azotobacter chroococcum YCYS inoculation with differentnitrogen levels on nitrogen uptake of maize plant, soil enzyme activities and diversity ofbacterial community in the rhizosphere of maize (Z. mays L.) seedlings. The results showedthat the nitrogen uptake of Z. mays L. plant inoculation with YCYS reached59.78±0.18mg/plant at0.05nitrogen level, significantly higher than other treatments (F=237.39，P<0.01). The dehydrogenase activity in A. chroococcum treatment was significantly higherthan that of uninoculated treatment by53.16%with0.05g/kg nitrogen level. The principle component analysis (PCA) of BIOLOG and T-RFLP also revealed that A. chroococcuminocualation had no significant influence on bacterail community diversity.6. In this study, the effects of microbial fertilizer on dehydrogenase activity and microbialcommunity diversity in the rhizosphere of wheat (Triticum aestivum L.) were investigated in the fieldconditions. The results showed that significant differences were observed in dehydrogenase activityamong the tested soils, arranging from26.14±3.30to48.13±17.39TF/(g·24h). The AWCD of MI(0.77±0.05) was higher than that of other treatments, whereas the AWCD for control (0.56±0.03) wasthe lowest (P＜0.01). Principal component analysis (PCA) of BIOLOG data further revealed thatlarge differences in microbial diversity (metabolic diversity) from treatments soils.7. Effects of Azotobacter chroococcum YCYS and Phyllobacterium brasssicacearum QL54inoculation on maize (Z. mays L.) seedlings exposed to different salt concentrations wereexplored. The results showed that inoculation with Azotobacter could decrease MDA content.The activity of SOD reached23.26U/mg protein at YCYS treatment with1.0g/kg NaCl, wassignificantly higher than the treatments with no salt, and CAT activity reached12.76U/mgprotein compared with the control. The principle component analysis (PCA) of T-RFLPrevealed that salt stress reduced the diversity of soil bacterial community.

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