Preparation Of Hypersensitive Protein And Its Application In Resistance To Downy Mildew In Grape

Grapevine downy mildew is a disease with high morbidity and infection rate of grapes.Presently,the application of chemical pesticides is the effective method for preventing and curing this disease,which will not only affect the quality and safety of fruit,but also lead to environmental pollution.Therefore,an efficient and environmentally friendly new biological pesticide is urgently needed in production.The existing studies have shown that hypersensitive protein is a new type of microbial protein pesticide and are non-toxic,and it can promote plant growth and enhance plant disease resistance and effectively improve fruit quality.In this experiment,the effects of hypersensitive protein on the control of grape downy mildew was studied.The main research results are as follows:1.In this experiment,the hypersensitive proteins HRP,Hrp Z and Hrp Z＿K(the C-terminal214 aa of Hrp Z)are from pathogenic species of Pseudomonas syringae.Their homology in nucleotide and protein is 37.62 % and 16.75 %,respectively.It was found that the optimal expression conditions of Hrp Z and Hrp Z＿K were as follows: temperature,28 ℃;IPTG concentration,0.5 mmol/L;induction time,6 h.The optimal expression conditions of HRP were37 ℃,0.5 mmol/L IPTG inducer concentration and 6 h.The total concentration of the prokaryotic expression for HRP,Hrp Z and Hrp Z＿K is 0.68 mg/ml,1.07 mg/ml,and 1.18 mg/ml,respectively.2.Hypersensitive proteins Hrp Z,Hrp Z＿K and HRP generated allergic necrosis on tobacco leaves.The application of 100 times dilute of HRP,Hrp Z and Hrp Z＿K led to the largest necrotic spots;in contrast,and the application of 500 times dilute of the above hypersensitive proteins led to larger necrotic spots than the control did but smaller ones than their 100 times dilute did.Additionally,the hypersensitive protein also had obvious hypersensitive reactions on grape leaves.3.‘Ze Xiang’ grape grown in field was used to evaluate the effects of hypersensitive proteins on downy mildew.The hypersensitive proteins were diluted to 100,500 and 1000 times and applied every 15 days from the beginning of July.The hypersensitive proteins with different dilution times generated significant effects on preventing downy mildew.Particularly,the application of 100 times dilute of Hrp Z＿K lowered the disease index to 4.85 %,which was7.05 % lower than the control.Spraying hypersensitive protein increased leaf photosynthesis,and the net photosynthetic rate of the leaves treated with the100 times dilute of Hrp Z was 17μmol,which was 29.3 % higher than the control.After spraying the 100 times dilute of Hrp Z and Hrp Z＿K,the leaf Fv/Fm was increased 4.3 % and 4.25 % and Plabs was increased 12.3 %and 20.7 % than the control.After the application of the 100-fold dilute of Hrp Z,the activities of POD and SOD were significantly increased,which were 52 % and 33.1 % higher than the control,respectively.Similarly,the 100-fold dilute of Hrp Z＿K increased POD and SOD activities by 36.1 % and 28.9 %,respectively.4.The effects of hypersensitivity proteins Hrp Z,Hrp Z＿K and HRP were further evaluated by the grape leaves inoculated with downy mildew.When the leaves were sprayed with 100-fold dilute of Hrp Z and Hrp Z＿K,the disease index decreased by 33.4 % and 38.9 %,respectively,compared with the control.Hypersensitivity proteins increased the activity of antioxidant enzymes in grape leaves and inhibited the accumulation of MDA content in grape leaves inoculated with downy mildew.The application of 100-fold dilute of Hrp Z significantly increased the activity of POD and SOD,which was 66.7 % and 9.2 % higher than the control,respectively.The application of 100-fold dilute of Hrp Z＿K made the activity of POD and SOD enzyme increased 45 % and 9.2 % than those of the control,respectively.The spraying of 100-fold dilute of Hrp Z and Hrp Z＿K decreased the MDA content of leaves by 54.2 % and 58 %,respectively,compared with the control.5.The roles of the hypersensitive proteins in preventing diseases of grape berries stored in cold conditions were determined.It was found that Hrp Z and HRP decreased the rotten fruit rate.The rotten rate of the fruits treated with 100-fold dilute of Hrp Z was the lowest,only 2.67 %at 30 days after storage and approximately 20 %-25 % at 45 and 60 days after storage,was more than 20 % lower than the control.