An Application Of Real-Time PCR In The Study Of Biological Control Of Tobacco Wilt Disease

The tobacco bacterial wilt, one of the main diseases of tobacco, is the soil-borne desease caused by Ralstonia solanacearum and threatens the production of tobacco. It is difficult to effectively control the disease by routine control methods, such as chemical fungicides.Compared with the routine control, rhizobacteria-mediated biocontrol is a more effective alternative way. An antagonistic bacterium strain against bacterial wilt of tobacco was identified as Brevibacillus brevis which was screened in our lab, and it was fermentted with amino acid organic fertilizer used for the prevention and control of tobacco wilt disease. In this paper, pot and field experiments were carried out to quantitatively investigate the rhizosphere microorganisms'populations by Real-time PCR after application of the bio-organic fertilizer. The main results of this work were as following:(1) Altogether 10 rhizosphere microorganisms were detected by a fluorogenic (SYBR Green I) PCR assay in this paper. And standard curves were created too. The amplification efficiency was 90%-105%(R2> 0.99) in the real-time PCR method established in our trial which was consistent with the requirements in quantitative testing.(2) The reliability of Real-time PCR was verified by comparing the results of detection of Ralstonia solanacearum with plate culture in soils. The results showed that the Real-time PCR assay was quicker and more simple, and the detected results were more close to the theoretical value, as well as the repeatability of three trials. All resluts deduced that Real-time PCR assay was suitable for detecting the soil microorganisms.(3) A pot experiment was carried out to evaluate the effect of bio-organic fertilizer on tobacco bacterial wilt. The results showed that the application of bio-organic fertilizer not only reduced the disease incidence of tobacco bacterial wilt in resistant variety K326 and susceptible variety Nanjiang No.3, but also significantly promoted the growth of tobacco with higher than 14% of biomass increment(4) A pot experiment was carried out to quantitatively investigate the dynamic change of Ralstonia solanacearum (RS) and its antibacterium in tobacco rhizosphere by the method of Real-time PCR after application of the bio-organic fertilizer.The real-time PCR results showed that the copy numbers of fliC gene in tobacco rhizosphere treated with RS was up to 107-108 copies g-1 dry soil, which was much higher than that in the treatments with BIO. The 16S rRNA copy numbers of Brevibacillus brevis was up to 106 copies g-1 dry soil in the treatments with BIO, while only 105 copies g-1 dry soil in the control. The results indicated that the bio-organic fertilizer enriched with Brevibacillus brevis can reduce the number of the pathogen in tobacco rhizosphere, thus effectively controlled the tobacco wilt disease and promoted the growth of tobacco.(5) Field plot experiments were carried out to inspect prevention and cure effect on tobacco bacterial wilt by biological organic fertilizer in Fuquan. The results showed that the disease incidence of tobacco bacterial wilt in susceptible variety Honghuadajinyuan was 100%, while its only 0.4% in resistant variety K326 before the harvest. The results indicated that the bio-organic fertilizer does no effects on tobacco bacterial wilt in the first year with both black shank and bacterial wilt of tobacco outbreak.