Study On The Growth-promoting Effects Of Three Bacillus Strains And Their GFP-taging

The Bacillus brevis ch2-22、Bacillus amyloliquefaciens D1 and Bacillus subtilis BSD-2 all have a wide antibacterial spectrum.They not only have a significant inhibitory effect on a variety of plant pathogens but also can promote growth to plant to some extent.They all have a good prospect in development and application.In order to make them be a soil microbial agent that have the function like medical-fertilizer,the following aspects were studied,the best concentration of three bacteria on the fermentation medium,combined effect,growth-promoting effects,and in the soil colonization.In order to study the best growth-promoting concentration and the combined effect of the three strains,a plate test was conducted.The fermentation broth was diluted.Combined ratio of three strains is 1:1:1.In this test,the seedlings bud,root length,root number and dry weight of cucumber were measured.The results showed that the best growth-promoting concentration of the ch2-22,D1 and BSD-2 is 50,200,200 fold dilution respectively.At the same time,complex effect of the three strains is better than other two treatments.They have a synergistic effect.The results of plate test were applied to initial applications.Greenhouse pot planting was employed to study the promoting effects through by measuring its plant index and physiological indexes such as plant height,stem diameter,leaf area,soluble sugars,etc.Results showed that three strains of bacteria all have a significant role in the growth of cucumber.From an overall perspective,the effect of plant growth-promoting was the best by ch2-22 treatment.It made stem diameter 1.22 times as much as D1 treatment.Dry weight of roots,stems and leaves of the plant were 1.52,1.86 and 1.43 times than the BSD-2 treatment group.The seedlings of cucumber treated with BSD-2 fermentation have more and larger leaves,longer root hair than the water treatment.The strain of D1 plays an important role in promoting stem length and the number of flowers.In this study,the plasmid of pGFP4412 that contains green fluorescent protein gene(GFP)was transformed into the B.brevis ch2-22,B.amyloliquefaciens D1 and B.subtilis BSD-2 by modified electroporation.The plasmid stability,growth curve and the inhibition activity of GFP-labeled strain of BSD-2 were measured.The results showed that the three strains were all tagged by GFP.They all can emit green fluorescence successfully.The tagged strain of B.subtilis BSD-2 nearly has the same trend with the wild type strain in growth.The stability of GFP-marked in engineering B.subtilis BSD-2 strain was 86% after transference of culture 56 hours continuously without selective pressure.Inhibition activity showed that the GFP-marked strain of B.subtilis BSD-2 exhibits the comparable ability as the wild type strain to inhibit Botrytis cinerea.The fermentation of the GFP-marked strain of B.subtilis BSD-2 were irrigated the cucumber plants root for the purpose of exploring the colonization on the cucumber.Observation by fluorescence microscope indicated that the GFP-tagged BSD-2 could colonize on the root tissue after inoculated 1 d.It could be seen largely on the root,stem,stipe and leaf veins after 11,13,15 and 19 days respectively.It still could be observed on the leaf veins after 50 days.The above data showed marked strain can colonize in the plant roots strongly.While the strains can transport and migrate to the stem and leaves of the plant with water and nutrients.