Screening Of Biocontrol Bacteria Strains And Study On Biocontrol Mechanism Of Watermelon Wilt

Watermelon fusarium wilt caused by Fusarium oxysporum f.sp.niveum(FON)that is a worldwide soil-borne disease.The use of biocontrol bacteria to control this disease is an environmentally friendly prevention measure and has attracted much attention from the academic community.This experiment aims to isolate and screen out the biocontrol bacteria that can control watermelon wilt,explore its live-defense mechanism,build a real-time fluorescent quantitative PCR(Quantitative Real-Time PCR,qPCR)detection system for FON and target strains to explore and clarify the live stain of the target strain to provide the diseased biological control of the disease and theoretical basis.The results of the study are as follows:1.Strains PJ3 and HY5 with strong antagonism to FON were obtained by plate confrontation culture;Through morphology,molecular biology and physiological and biochemical property identification to use explicitly Bacillus subtilis PJ3 and Bacillus atrophaeus HY5.Strains PJ3 and HY5 entered the logarithmic growth phase at the 4th h of culture and the stable growth phase at the 20th and 16th h,respectively.The most suitable fermentation medium for both strains was beef paste peptone medium,and there was no antagonism between the two.PJ3/HY5 and other bacterial mass fermentation broth combined with FON inhibition effect is stronger than that of single strain fermentation broth.2.Clarified the biocontrol mechanism of strains PJ3 and HY5 on FON.The crude extract of bacteriostatic substances of the two strains can rupture FON spores,increase the permeability of mycelium,increase electrical conductivity,and the effect of the combination of the two strains is stronger than that of single bacteria.The crude extract of bacteriostatic substances of strains PJ3 and HY5 can significantly reduce the content of ergosterol and mycelium protein in FON cell membrane,damage the integrity of cell membrane,increase cell membrane permeability,and increase the MDA content in FON culture medium.It shows that both biocontrol bacteria have the effect of destroying cell membranes.3.Build a qPCR detection system for biocontrol bacteria and FON.The qPCR detection method established by primers PJ3-F/R.HY5-F/R and FON-F/R has good specificity and sensitivity,and when the initial DNA concentration of the strain is in the range of 0.001～100 ng/μL,the logarithmic value of the initial DNA concentration of the strain has a good linear relationship with the Ct value,and the qPCR detection system can effectively detect the DNA content in the roots of watermelon seedlings and their rhizosphere soil in pot tests.4.The results of pot experiments showed that the incidence of watermelon seedlings vaccinated only with pathogenic bacteria FON was 100%,and the disease index reached 90.67,compared with the vaccination of PJ3,HY5 and PJ3/HY5,the incidence of plants was reduced to less than 60%,and the disease index was reduced by 75.00%-83.46%.The control effect of the combination of two biocontrol bacteria was as high as 71.43%,which was significantly higher than that of single biocontrol bacterial inoculation.5.Clarified the effects of biocontrol bacteria strains PJ3 and HY5 on the colonization capacity of FON.The results showed that strains PJ3 and HY5 reduced the amount of DNA of FON in the rhizosphere soil and root of melon seedlings by 52.98%-92.42%and 50.52%-94.75%,respectively,and the colonization effect of PJ3/HY5 was statistically different from that of single strains(P<0.05).During the experiment,the amount of FON colonization in the rhizosphere soil and root of melon seedlings with biocontrol bacteria decreased with time,and by the 30th d,the DNA quantity of FON decreased by 71.41%-78.49%and 74.65%-86.40%,respectively,compared with the 10th d.The biocontrol bacteria Bacillus subtilis PJ3 and Bacillus atrophaeus HY5 isolated and screened in this experiment had good control effect on watermelon wilt,and the effect of the combination of the two bacteria was better than that of a single strain.Therefore,the biocontrol bacteria strains PJ3 and HY5 have good biocontrol potential and development prospects.