| Pinellia ternate(Thunb.)Breit.is a dry tuber of Pinellia ternata in Araceae,which has attracted much attention due to its pharmacological effects such as dampness,phlegm,anti-ulcer,hypolipidemic and detoxification.However,with the continuous expansion of Pinellia ternata planting scale,the virus disease of Pinellia ternata has become more and more serious,which has seriously affected it's yield and quality.At present,spraying chemical pesticides and breeding resistant varieties are considered as two effective ways of disease resistance,but they have the disadvantages of environmental pollution and heavy workload.Studies have shown that Harpin protein secreted by Gram-negative plant pathogenic bacteria has the effect of improving broad-spectrum resistance.In our Preliminary experiments,sprayed Hpa1(a kind of Harpin protein)on Pinellia ternata leaves showed that Hpa1 can significantly alleviate the mosaic symptoms of Pinellia ternata,indicating that Hpa1 can stimulate effective resistance,but the molecular mechanism of disease resistance is still unclear.In this study,on the basis of defining the optimum effective dose of Hpa1,the resistance mechanism of Pinellia ternata was analyzed from the aspects of resistance-related defense enzyme activity,content of resistance-related substances,plant signal transduction pathway and inhibition of virus replication.At the same time,the stability of its field application was explored by statistical of the incidence and the virus content of Pinellia ternata in different induction periods.The main results of this study are as follows:1.Virus-free cultivation of Pinellia ternata was carried out by plant tissue culture technique in vitro.Using Pinellia ternata shoot tip as explant,the optimum medium for callus induction was determined by designing L9(32)orthogonal experiment based on single factor experiment,and the virus content of virus-free seedlings was detected by molecular method.Orthogonal test showed that the best medium for callus induction was MS+1.5 mg/L 6-BA+2.0 mg/L 2,4-D+7 g/L Agar+30 sucrose g/L,and seedlings could be formed at one time.After detection by Dot-ELASA and RT-PCR,the virus-free rates of SMV,TMV and CMV reached 100%.2.The anti-TMV activity and the optimal effective dose of Hap1 were determined by ELASA in terms of protection and therapeutic effect.The results showed that Hap1 had significant anti-TMV activity compared with the control group,and its protective effect was stronger than the cure effect,indicating that Hap1 canprevent TMV from infecting Pinellia ternata in a stronger way.Secondly,when the concentration of Hap1 was15 g/m L,the control effect of Hap1 was the best,the protection effect was as high as 71.4%,and the control effect was as high as 55.4%.3.Using 15 ?g/m L Hap1 to induce resistance,TMV was inoculated 1 day after induction.the activities of defense-related enzymes and some disease-related substances in Pinellia ternata at different time points after inoculation were measured.The results showed that the activities of PPO,POD,CAT and SOD and the content of H2O2 in Pinellia ternata leaves increased significantly after Hap1 treatment,while the content of MDA decreased significantly.4.Using 15 ?g/m L Hap1 to induce resistance,TMV was inoculated 1 day after induction.Histopathological examination of Pinellia ternata was carried out by fluorescence microscopy at different time points after inoculation.The results showed that the accumulation patterns of H2O2,callose and phenolic substances at1 ininfection sites were basically the same in different treatments,which increased first and then decreased.However,compared with the control group,the four substances of Pinellia ternata treated with Hap1 and Ningnanmycin accumulated in a faster and more versatile way,which could enhance the resistance of Pinellia ternata to TMV.5.The results showed that PR1 and PR5(two genes related to SA pathway)and PR3 and PDF1.2(two genes related to JA/ET pathway)were significantly increased after treatment with Hap1 and Ningnanmycin,and there was no significant difference between them.The relative expression of PR1 in Pinellia ternata treated with Hap1 reached its peak after inoculation with TMV for 4 days,which was 4.3 times higher than that in the control group.The relative expression of PR3 reached its peak after inoculation with TMV for 4 days,which was 2.9 times higher than that in the control group.PR5 and PDF1.2 induced resistance in a faster mode,and the relative expression reached a maximum after 2 days of inoculation,which was 2.8 and 2.4 times higher than that in the control group,respectively.6.Based on the uncertainty of the time when the virus infects plants in the field,the stability and the feasibilityof TMV resistance induced by Hpa1 were analyzed by the statistical of the incidence and determination of the virus content in the infected leaves of Pinellia ternata in the field.The results showed that after spraying Hpa1 on the leaves of Pinellia ternata for two days,TMV was used to attack the virus.The symptoms of Pinellia ternata leaves were the lightest,and the control effect was as high as 85%.Real-time PCR results also showed that when the interval was 2 days,the relative expression of TMV-CP in the system leaveswas the least,which was 11.52 times lower than that of the control group.In addition,the control effect of Hpa1 protein in the whole test cycle was significantly higher than that of the control group,and the high protective effect could last for 10 days. |
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