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

Research of Bacillus Atrophaeus HAB-1Strain Antifungal Activity and Optimization of Culture Conditions

Author SunLiang
Tutor ZhengFuCong;ZuoWeiGuo
School Hainan University
Course Plant Pathology
Keywords Bacillus atrophaeus Promoting effect Optimization fermentation Antibacterial activity Antifungal substances composition
CLC S476.1
Type Master's thesis
Year 2013
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HAB-1is a Bacillus atrophaeus isolated by our laboratory from plant rhizosphere soil. The preliminary study found that the strain has good antibacterial activity against to a variety of plant pathogenic fungi. In order to development and utilization of this strain, in this paper, we measured the inhibitory spectrum of HAB-1and role in promoting plant growth, researched to improve the fermentation conditions, preliminary analysis of the nature of the antibacterial active ingredient and investigated the inhibitory mechanism. The results are as follows:1. HAB-1and its secretions can inhibit plant pathogenic fungi growth, such as Colletotrichum gloeosporioides on22kinds of plant pathogenic fungi. We can see HAB-1secretions affect the tested plant pathogenic fungi mycelial growth through an optical microscope, compared with CK, the treatment group mycelium with varying degrees of deformity.2. HAB-1bacterial suspension dilution factor of10times-10times, can increase the radicle length of cucumber seeds, in the pot experiment HAB-1concentration was controlled within the above range, we found that HAB-1strains can significantly improve the plant dry weight, and in cucumber seedlings before transplanting with microbial treatment in combination with bacteria liquid after transplanting treatment, its better than the other.3. We observed the pattern of HAB-1and tested its activity through to the seven kinds of different medium, we found that there are significant differences of HAB-1colony morphology in different medium plates, and colony diameter ranging from7.95-23.51mm; By plate confrontation culture method, we found that the antibcaterial activity of HAB-1showed big significance against to12kinds of plant pathogenic fungi in different medium, the differences of inhibition rate of the same kind of pathogen was up to31.33%, the smallest is2.66%. We analysis the components of seven kinds of medium, it showed that the C source and metal ions on the HAB-1inhibitory activity is more obvious, and N source have little impact.4. Using shake flask cultures method screened to the most suitable for the HAB-1growth and antibacterial substances medium ZY, the media contained:10g/L sucrose,30g/L corn flour,30g/L yeast extract,40g/L bran, and lg/L KH2PO4; on this basis, its culture conditions to optimize the test results show that the medium initial pH6, the fermentation temperature37℃, the quantity of medium60mL in a250mL flask, the inoculation volume of2%, the fermentation cycle of24h, and the rotation speed of190rpm respectively. Under this condition, a HAB-1viable cell number of the CGR-1of inhibition zone diameter respectively up2.6x1010CFU/ml and the34.0mm, increased122.81%and10.03%.5. We used different methods to deal with HAB-1supernatant, the results showed that the crude extracts of the ammonium sulfate precipitation method has no antibacterial activity, but the acid precipitation method of the precipitate and the ethyl acetate extracts have antibacterial activity, according to the size of the inhibition zone,we made preliminary analysis of HAB-1inhibitory substances which may contain lipopeptide antibiotics, but there are some other substances play a key role.Large amounts of crude extractings from preparation of ethyl acetate with column chromatography method for the coarse, and determination of the antibacterial activity of each component in the rough points, we found that the component1-19bacteria could form a zone of inhibition, the component20is not formed. The inhibition zone diameter of component1and2are26.33mm and25.92mm, the statistical analysis showed that the difference was not significant between the diameter of the inhibition zone in the two components and is not rough segmentation, and reached a very significant difference between the inhibition zone formed with other components, this showed that the active substance is mainly contained in the component1and2. The two components are eluted by small polar eluent, according to the like dissolves know the polarity of the antibacterial substance should be small. In addition, the volatile substances in the ethyl acetate extract can inhibit the growth of CGR-1,which showed that the antibacterial substance should be volatile.

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