Study On The Endophytes In Portulaca Oleracea L. Against Bacterial Wilt

Bacterial wilt is a soil-borne disease caused by Ralstonia solanacearum,which is the main disease of tomato,eggplant,pepper,potato and other solanaceae vegetables,called as "the cancer of plant".Agricultural method,chemical agents and biological control are mainly used in production,However,specific agents and resistant varieties are still lack at present.Endophytes with anti-bacteria,anti-insect and promoting plant growth function have broad prospect as a biological agent.To find an effective biological control measure,Portulaca oleracea L.,which is known as a natural antibiotic,was used as material to isolate and identify the endophtes,and investigated their resistance mechanism on tomato bacterial wilt disease and the active effect.The main results were as follows:1.Sixty species of endophytes were isolated from P.oleracea,22 strains of endophytes were identified by ITS sequencing.2.Three endophytes,Penicillium sclerotiorum,Penicillium citrinum and Penicillium polonicum inhibited the growth of R.solanacearum.3.The mechanism of pencolide,which is the active compound in P.sclerotiorum,against R.solanacearum were carried out.First,pencolide could reduce the proliferation and rhizosphere colonization ability by inhibiting the movement of R.solanacearum.Second,pencolide(streptomycin as positive control)could inhibit the biofilm formation of R.solanacearum at different concentration and different times.Third,the concentration of pencolide(streptomycin as the control)from 30 ppm to 135 ppm could make R.solanacearum sunken,shrivel and swelling by scanning electron microscope.Fourth,pencolide could enhance the resistance of tomato by changing the activity of catalase(CAT),superoxide dismutase(SOD),peroxidase(POD),phenylalnine ammonialyase(PAL),polyphenol oxidase(PPO)in tomato and reduce its pathogenicity by inhibiting the activity of the antioxidant enzymes SOD and CAT of R.solanacearum.Fifth,real-time quantitativereverse transcription PCR(q RT-PCR)was used to test the relative expression of pathogenic genes in R.solanacearum,pencolide was found to increase the expression of the gene encoding beta-1,4-glucan and inhibit the expression of Phc A,Phc B,Peh C,Flh D,Pil T,eps E,Flh C,Pol A,Flg M,thus,the possible target of pencolide was identified.4.Pencolide at 200 ppm had 80% control efficiency in pot experiment,meanwhile P.sclerotiorum fermentation broth could delay senescence of plant leaves.5.Pencolide could promote the growth of Bacillus siamensis and had no side effect on beneficial microorganisms,such as Aspergillus oryzae,Trichoderma harzianum and Bacillus megaterium,which provided some scientific data for future applications of pencolide.6.The culture conditions of producing pencolide were optimized by Response Surface Methodology(RSM),the optimum conditions for maximum production of pencolide were fermentation time 12 days,p H of 8.00,sucrose concentration of 20 g/L,peptone concentration of 4 g/L.7.The active substance of the two endophytes Penicillium citrinum and Penicillium polonicum was identified as citrinin based on UPLC-QTQF-MS.In a word,three endophytes,P.sclerotiorum,P.citrinum and P.polonicum,which were isolated from P.oleracea,were found to have good inhibition effect on bacterial wilt.Exploring active substance pencolide anti-bacterial mechanisms,pencolide could affect R.solanacearum’s biofilm formation,morphology,motility,enzyme activity and tomato enzyme activity,and had effect on pathogenic gene expression level of R.solanacearum.Pot experiments proved that pencolide could prevent and cure tomato bacterial wilt and reduce the incidence of disease.The above studies enriched the antimicrobial function of the endophytes in P.oleracea and provided theory support for the research and development of pencolide as a biocontrol agent in agriculture.