Identification, Detection And Infection Characteristics Of Viral And Bacterial Diseases And Virus-Free Tissue Culture In Pinellia Ternata

Pinellia ternata (Thunb) Breit is a perennial herb belonging to the family Araceae.Its dried tubers are common traditional Chinese medicinal materials used for morethan 2000 years. Its original habitats include those provinces along Yangtze River,northeast and north parts of China. Among 558 Chinese medical prescriptions, it isranked as the twenty second most frequently used one.Because of the decreasing of wild resources, P. ternata was cultivated in some areas.In the growth Season of P. ternata, viruses and soft-rot causing bacteria caused yieldloss. In this research, viruses and bacteria infected in P. ternata were identified.Infection characteristics, detection methods and virus-free tissue culture techniquewere discussed as follows:1,Study on viral diseases in Pinellia ternata(1),Identification of viruses. Field symptoms such as molttle, mosaic, savoy, littleleaf, vein-banding, istorting leaf and stunt in viruses infected Pinellia ternata can beshown. Electron microscopic examination has shown spherical virions at diameter of35nm and linear virons at length of 750nm. Detective results of extraction andelectrophoresis of dsRNA, RT-PCR and ELISA have proved that the two virusesbelong to Cucumber mosaic virus(CMV) and Soybean mosaic virus(SMV).(2),Investigation of viruses infection rate. Results of ELISA test have shownCMV infection rates of cultivated P. ternata from Ningbo, Xiaoshan, Hebei, Anhui,Nanchong, Beijing, Feng County are 75%,100%,63%,30%,5%,0% and 30%; SMVinfection rates are 35%,30%,40%,25%,0%,25% and 30%; and complex infectionrates of both viruses are 25%, 10%, 20%, 25%, 0%, 0% and 5%. The infection ratesof CMV, SMV and CMV/SMV in 25 samples of wild P. ternata from different areasare 20%, 20% and 16%. The results have shown that CMV and SMV infection incultivated and wild P. ternata are ubiquitous and the virus infection rate of cultivatedP. ternata is obviously higher.(3),Infection characteristics of viruses. CMV from P. ternata can not infectP.ternata, Nicotiana tobacum, Chenopodium amarantocolor, C.quinoa andN.clevilendii by friction inoculate, while CMV from tomato and N. tobacum can infectP.ternata by friction inoculate. SMV from P.ternata and Giycine max can infectP.ternata by friction inoculate.2,Study on soft-rot of P. ternata.(1),Identification of pathogen. Soft-rot is the most commonly appeared symptomoccurring on tubes, and leaf rot-away and plant death are ready to happen under hotand humidity conditions. Ten bacteria isolates distigushed as RF1～10, obtained fromsoft-rotted P. ternata as dominating pathogenic organisms, were characterized bypathogenicity, morphlogy, bacteriological characteristics. Among them RF1 wasanalyzed by 16S rDNA sequence. The results have showed that all the ten isolatescaused soft rot disease on P. ternata, as well as on carrot, potato, tomato, cucumberand Chinese cabbage. Short bacillary with 4-6 flagella was found for the isolatesunder scan electron microscopy. All the dominating isloates were found beingfacultative anaerobes and gram negative. The G+C mol% were 51% of their genomicDNA. 16S rDNA genomic sequencing and phylogenetic analysis of RF1 have shownthat RF1 shared 100% identity to that of Pectobacterium carotovorum subsp. Carotovorum M1 strain, and 97～98.5% to those Strains E161 and 441. The resultshave confirmed that the pathogenic bacteria causing soft-rot disease belong to P.carotovorum subsp, carotovorum.(2),Detection of exoenzyme for RF1. After inoculated by P. carotovorum subsp.Carotovorum RF1 for 24hrs, pectase activity in cell extracts of P. ternata increased 8times compared with the contrasts, protase activity increased more than 12 times, andcellulose activity was approximately 0. The same high activities of pectase andprotease were detected during in vitro culture of RF1.3,Study on virus-free tissue culture of P.ternata.(1) Identification and elimination of bacteria polluted in tissue culture. 3 strains ofbacillus were separated from polluted tissue cultured P. ternata. They were identifiedas Bacillus circulans, B. megaterium and B. cereus through morphlogy, physiologicaland biochemical tests. The antibiotics tests have shown that pollution can beeliminated while tissue cultured P. ternata are cultured on media containing 30mg/Lof penicillin or 30mg/L of penbritin. The growth of plants is not influenced under thisculture condition.(2) Detection methods comparison for viruses-carried tissue cultured plants. With18S rDNA as endogenous reference, relative quantification detection of CMV in P.Ternata and its tissue cultured plantlets was carried by using Real-Time PCR, andcombined with methods of DAS-ELISA and RT-PCR. The results have Shown thatCMV quantity in leaf of tissue cultured plantlets coming from explant of CMVpositive P. ternata is relatively higher than that of tubercles and callus, whileconcentration of CMV in tissue cultured plantlets is decreased greatly compared withthe parent plantlet. There were fake positive results while using ELISA method todetect samples of low concentration virus. RT-PCR method is sensitive but notquantified exactly to detected samples. Real Tune PCR method has better definitionand ration. The results has also showed that concentration of CMV decreases with theprocess of tissue culture. More accurate detection methods such as Real Time PCRshould be used.(3),Cluster buds of P. ternata eliminating virus by meristem tip culture can besuccessive transfer cultured for at least 20 generations. The suitable successivetransfer culture cycle is about 20 days. Growth rate comparison tests have shown thatthe growth speed of virus-free tissue cultured tubes of P. ternata is 25% faster thancultivated virus-carried tubes. The in vitro tubes formed straightly from tissuecultured plants have nearly 100% survival rate when sown in the field.