Dynamic Differences In Root Colonization Of Fusarium Oxysporum F.sp.niveum And Biocontrol Trichoderma Harzianum In Watermelon And Their Response To Soil PH And Salt Concentration

Watermelon fusarium wilt caused by Fusarium oxysporum f.sp.niveum(FON)is a soil-borne disease occurring all over the world.In biological control,T.harzianum has been proved to be a feasible green control method for watermelon fusarium wilt.Understanding the colonization ability of T.harzianum and FON in watermelon rhizosphere is the key to control watermelon fusarium wilt.Therefore,the protoplast mediated by PEG-CaCl2 was applied to integrate the pCT74 plasmid carrying the green fluorescent protein(GFP)gene with the genomes of FON and T.harzianum M3 to obtain the corresponding transformants,respectively.The transformants were inoculated in soil,and the infection dynamic process of watermelon seedling roots by the transformants of the two strains was traced and compared using LSCM.Construct quantitative real-time PCR(real-time PCR)detection system for FON and M3.The effects of pH and salt mass fraction on the colonization of watermelon root were studied by using this system,in order to provide theoretical basis for the control of watermelon fusarium wilt.The results are as follows:1.GFP labeling system of T.harzianum M3 and FON was constructed.The tolerance concentrations of M3 and FON to HmB were 1000 μg/mL and 150 μg/mL,respectively.The protoplast yield of M3 was the highest after 2.5 h of collapse enzyme treatment,and that of FON was the highest after 2 h of collapse enzyme treatment.It can be indicated that the transformants could still emit green fluorescence with good fluorescence intensity for six consecutive generations and could be inherited stably.The transformants could still emit green fluorescence for six consecutive generations and could be inherited stably,and the fluorescence intensity was stable.The GFP gene was successfully transferred by PCR.2.To clarify the difference in colonization dynamics between T.harzianum M3 and FON.The biological characteristics of transformants M3-GFP and FON-GFP were not significantly different from those of wild type.The biocontrol ability of transformants M3-GFP and pathogenicity of transformants FON-GFP did not change significantly.Both transformants were successfully colonized in the watermelon seedling roots in pot trials.In the early stage of colonization,the FON spread rapidly in the roots than the T.harzianum M3.3.Real-time PCR system for detection of T.harzianum M3 and FON was established.The minimum detection amounts of primer 2F2/2R2 and Fon-1/Fon-2 were 1×10-5ng/μL for M3 and FON DNA by gradient dilution under real-time PCR,which were 1000 times higher than those of conventional PCR.The standard curves of M3 and FON DNA were y1=17.12-3.27×(R2=0.99857)and y2=16.75-3.17x(R2=0.99484),respectively.There is a good linear relationship between the cyclic threshold of standard curve and template concentration.Real-time PCR system can effectively detect the DNA content of M3 and FON in watermelon seedling roots.4.To investigate the effects of different soil pH and salt mass fractions on the colonization of T.harzianum M3 and FON in the roots of watermelon seedlings.The soil with pH 7 and salt mass fraction 0.12%was the most suitable for colonization of M3.The soil with pH 6 and salt mass fraction 0.12%was the most suitable for colonization of FON.M3 had strong inhibitory effect on FON colonization in watermelon roots,and it increased significantly with the increase of colonization time.The biocontrol effect of T.harzianum M3 was the best in soil with pH 6-7 and salt mass fraction 0.08%-0.12%,and the effect of soil pH was more significant than salt mass fraction.