Dissertation
Dissertation > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens

Identification、Diversity and Phylogenetic Analysis of Antagonistic Bacillus Isolated from Different Chinese Areas

Author LiuFang
Tutor GaoXueWen
School Nanjing Agricultural College
Course Plant Pathology
Keywords antagonistic Bacillus biodiversity ERIC-PCR fingerprinting 16S rDNA and gyrB gene sequences growth promotion and seeds germination low-temperature adapted Bacillus
CLC S476.1
Type Master's thesis
Year 2010
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Sutdies on taxonomy and resource of Bacillus isolated from traditional medieine, cheese, Luzhou-flavor fermentation grain, oarl durgs, extreme environments and soil have been carried out in the two last decades, but reports of antagonistic Bacillus from soil haven’t been published. Biodivesrity and phylogenic status of antagonistic Bacillius by performing taxonomy, is significant for finding consevring, utilizing of the Bacillius resoueres. In this study 28 soil samples were collected from Jiangsu province, Heilongjiang province, Hainan province and Xinjiang Uygur Autonomous Region in China, using traditional isolated and identified motheds,1593 isolates were abained from these samples. Among those stains,545 isolates were found to have inhibitory activity on plant pathogenic fungi Sclerotinia sclerotiorum and pathogenic bacteria Xanthomonas oryzae pv. oryzae.330 Bacillus strains with good inhibition effect were selected from different province and regions, using of ERIC-PCR amplification, after the fingerprints analysis, the results showed that in the similarity of 78%, the antagonistic Bacillus strains were divided into a number of groups, the largest one was 16 groups in Aksu prefecture, Xinjiang; the small one was 10 groups in Hainan area. All indicated that the distribution of antagonistic Bacillus had good diversity in the areas. Further comparison suggested that the majority of antagonistic Bacillus selected were focused on B.amyloliquefaciens group, B.subtilis group, B.pumilus group and B.megatrium group. For different province and regions, in the southern parts mainly dominated by B.megatrium groups, in the northern and western maily B.pumilus group, but in the eastern did not find antagonistic Bacillus because of fewer isolates. For the different regions in the same provinces and autonomous regions of Xinjiang Uygur Autonomous Region, the dominant species were inequality, but a certain law was showed, the less distribution group in Southern Xinjiang is B. amyloliquefaciens group, while the less distribution group in the northern Xinjiang is B. megatrium group. Different habitats for the distribution of antagonistic Bacillus have no special effects. 24 isolates with higher antibacterial activity were selected from the 330 Bacillus strains with good inhibitory activity; the inhibition zone on pathogenic fungi reached more than 25mm and on pathogenic bacteria was above 30mm. After morphological, physiological and biochemical determination, then their 16S rDNA and gyrB gene sequences were amplified, combined with their ERIC-PCR fingerprinting analysis, we found that there was good consistency of the results between the fingerprint analysis and gyrB gene sequences, which demonstrates that the use of ERIC-PCR fingerprinting for diversity analysis had high reliability.In this study, the survived suitable at low-temperature strains were screened from antagonistic Bacillus in Tibet, finally we found two strains RJGP41 and CJLC2 could grow at 4℃,25 strains could grow well under 10℃, which were all called low-temperature adapted Bacillus. Through the growth promotion of Arabidopsis thaliana and corn seeds germination experiments, the strains RJGP41, CJLC2, GBSC56 and NMSL88 were proved having better effect on both of them, furthermore they could also promote the growth of corn at low-temperature (15℃) stabily. Using of 16S rDNA sequencing, they were respectively identified to species:RJGP41 belonged to B. simplex; CJLC2 was B.megaterium; GBSC56 was B. atropheus and NMSL88 was B.cereus. The results of determination all the 27 strains’antibacterial activity showed that the strain LSSC3 (B.subtilis) has strong inhibitory effect on all testing pathogen(including plant pathogenic fungi(Sclerotinia sclerotiorum, Fusarium graminearum and Rhizoctonia solani)and pathogenic bacteria (Xanthomonas oryzae pv. oryzae, Erwinia amyloyora and E. carotovora), particular against pathogenic fungi, the inbibiton zone was reached 45mm. Thus, these strains had the dual characteristics of biocontrol and adaption to low temperature, which would have an application prospect in Agriculture.

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