葡萄卷叶病的鉴定及脱毒技术研究
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摘要
葡萄卷叶病(Grapevine leafroll disease)是葡萄上的一种重要的病毒病。现已研究表明引起该病的病毒不止一种，目前国际上承认的已有5种(GLRaⅥ—Ⅲ)，其中葡萄卷叶伴随病毒Ⅲ(GLRaⅤ—Ⅲ)是最主要的一种，它分布最广泛造成的经济损失最大，而且也是这几种病毒中唯一具有自然介体的病毒。本研究根据生产实际，对葡萄卷叶病毒的检测和脱毒技术进行了研究。
为了寻找到合适的毒源，对宁夏贺兰山东麓地区葡萄圃中的64个品种和砧木卷叶病的自然发病情况进行了调查研究，结果表明：在美洲种群、东亚种群中没有发现卷叶症状；具有优良栽培性状和古老栽培历史的欧亚种群中表现卷叶病症状的品种最多，发病程度最重；欧美杂种、欧山杂种均有表现卷叶病的品种，但发病率、严重度低于欧亚种。在欧亚种中，以鲜食品种为主的东方品种比以酿酒品种为主的西欧和黑海品种卷叶病发病率低。此外，葡萄感染卷叶病后，其叶绿素含量、光合速率、经济产量都明显地低于健康植株，同时病株浆果含糖量降低，酸度上升，直接影响了葡萄的品质。
检测是认识一种病害最基本的前提，通过对三种ELISA检测GLRaV和GFLV的比较认为，双抗夹心-ELISA(DAS-ELISA)、间接-ELISA(PTA-ELISA)和A蛋白夹心-ELISA(PAS-ELISA)都能检测GLRaV和GFLV。DAS-ELISA能缩短检测的时间，然而这项技术具有较高的株系特异性，在检测中可能产生假阴性反应，因此，必要时可用PTA—ELISA和PAS—ELISA对一些关键样品进行复检。
鉴于繁殖材料带毒是葡萄卷叶病的主要传播途径，在防治上，加强检疫措施和使用无病毒苗木是防治卷叶病最有效的措施。依据茎尖分化、伸长和生根三阶段中所需激素比例的不同，适当调节它们之间的比例，从而筛选出适合不同时期的最佳激素配比，比常规微茎尖处理缩短培养时间12-19天。在脱毒处理中，将热处理与茎尖培养相结合，使成活率和脱毒率比单纯茎尖培养提高了10.9％和9.2％，其中变温处理比恒温处理成活率提高了12.4％，而脱毒率相差不大。药剂处理与微茎尖相结合培养结果表明：药剂对葡萄病毒具有良好的钝化作用，但对茎尖的生长发育有影响。
通过对过氧化物酶活力测定结果表明：葡萄叶片中过氧化物酶活性与葡萄卷叶病有一定的关系。植株感病后，其体内过氧化物酶活性增强，其中抗病类型的过氧化物酶活性高于感病品种。同工酶谱分析表明：感病后，不同抗病品种的酶带数均增加，而感病品种各植株间过氧化物酶同工酶的变化与植株外部症状有差异，后期表现为病情越重，酶带数越少。
Grapevine leafroll disease is the most devastating disease of grapevine in many grapeving producting areas, and the pathogens of it are more than one virus . In these viruses, grapevine leafroll associated virus 3 is the most important one , and it is the only one that has a nature transmission vector .
It was investigated that the occurrence of GLRaV is more than 64 varieties and block grown in the east of Helan mountains, Ningxia . The results showed that no symptoms of GLRaV were found in the American species of Eavitis and Eastern Asian species of Eavitis while the highest percentage of infected varieties were found in V. vinifera. The results also showed that some hybrids of V.vinifera and other species had the symptom of GLRaV . In V. vinifera ,the Probes accidentalis and the Probes pantica had higher percentage of GLRaV infection than the Probes orientals. Moreover , infected grapevines are lower than healthy ones in chlorophyll content , photosynthesis and economic production, and fruit of GLRaV had less sugar and higher acide content. These resulted in a weak grapevine quality .
Comparision of three ELISA methods for detection of GLRaV showed that DAS-ELISA,PTA-ELISA and PAS-ELISA could detect GLRaV and GFLV. DAS-ELISA could shorten the detection time. The techniques , however, have higher strain specificity and the false negatives can be caused . Therefore, PTA-ELISA and PAS-ELISA must repeatedly dectect some important samples.
A series of broad spectrum media for stem tip tissue Virus- ree culture and propagation of grapevine was prepared. These media could shorten the culture time by 12?9 days as compared with the media used commonly. With heat treaement and stem tip tissue Virus梖ree culture, the survival rate of the plant and elimination rate of GLRaV increased more 10.9% and 9.2%, respectively, than singlar stem tip tissue Virus梖ree culture. Moreover, in the survival rate, the changable heat treatment increased more 12.4% than contant heat treatment while the elimination effect of the virus in grapevine showed little difference. The addition of WCT and FenYuanBao in medium exhibited the inhibitory effect on virus multipliction. It, however, had negative effect to the rate of plantlet.
Activity of peroxidase (POD) was analyzed in leaves of grapevine after inoculation of GLRaV. The infection of GLRaV resulted in increase in activities of POD in the resistant and susceptible varieties, and activity of enzyme was significantly greater in resistant varieties than in susceptible ones. Moreover, analysis of POD isoenzyme showed that the number of enzyme bands in resistant varieties varied directly with symptom of plant while in susceptible ones ,varied versely with it,especially mid- late period.
为了寻找到合适的毒源，对宁夏贺兰山东麓地区葡萄圃中的64个品种和砧木卷叶病的自然发病情况进行了调查研究，结果表明：在美洲种群、东亚种群中没有发现卷叶症状；具有优良栽培性状和古老栽培历史的欧亚种群中表现卷叶病症状的品种最多，发病程度最重；欧美杂种、欧山杂种均有表现卷叶病的品种，但发病率、严重度低于欧亚种。在欧亚种中，以鲜食品种为主的东方品种比以酿酒品种为主的西欧和黑海品种卷叶病发病率低。此外，葡萄感染卷叶病后，其叶绿素含量、光合速率、经济产量都明显地低于健康植株，同时病株浆果含糖量降低，酸度上升，直接影响了葡萄的品质。
检测是认识一种病害最基本的前提，通过对三种ELISA检测GLRaV和GFLV的比较认为，双抗夹心-ELISA(DAS-ELISA)、间接-ELISA(PTA-ELISA)和A蛋白夹心-ELISA(PAS-ELISA)都能检测GLRaV和GFLV。DAS-ELISA能缩短检测的时间，然而这项技术具有较高的株系特异性，在检测中可能产生假阴性反应，因此，必要时可用PTA—ELISA和PAS—ELISA对一些关键样品进行复检。
鉴于繁殖材料带毒是葡萄卷叶病的主要传播途径，在防治上，加强检疫措施和使用无病毒苗木是防治卷叶病最有效的措施。依据茎尖分化、伸长和生根三阶段中所需激素比例的不同，适当调节它们之间的比例，从而筛选出适合不同时期的最佳激素配比，比常规微茎尖处理缩短培养时间12-19天。在脱毒处理中，将热处理与茎尖培养相结合，使成活率和脱毒率比单纯茎尖培养提高了10.9％和9.2％，其中变温处理比恒温处理成活率提高了12.4％，而脱毒率相差不大。药剂处理与微茎尖相结合培养结果表明：药剂对葡萄病毒具有良好的钝化作用，但对茎尖的生长发育有影响。
通过对过氧化物酶活力测定结果表明：葡萄叶片中过氧化物酶活性与葡萄卷叶病有一定的关系。植株感病后，其体内过氧化物酶活性增强，其中抗病类型的过氧化物酶活性高于感病品种。同工酶谱分析表明：感病后，不同抗病品种的酶带数均增加，而感病品种各植株间过氧化物酶同工酶的变化与植株外部症状有差异，后期表现为病情越重，酶带数越少。
Grapevine leafroll disease is the most devastating disease of grapevine in many grapeving producting areas, and the pathogens of it are more than one virus . In these viruses, grapevine leafroll associated virus 3 is the most important one , and it is the only one that has a nature transmission vector .
It was investigated that the occurrence of GLRaV is more than 64 varieties and block grown in the east of Helan mountains, Ningxia . The results showed that no symptoms of GLRaV were found in the American species of Eavitis and Eastern Asian species of Eavitis while the highest percentage of infected varieties were found in V. vinifera. The results also showed that some hybrids of V.vinifera and other species had the symptom of GLRaV . In V. vinifera ,the Probes accidentalis and the Probes pantica had higher percentage of GLRaV infection than the Probes orientals. Moreover , infected grapevines are lower than healthy ones in chlorophyll content , photosynthesis and economic production, and fruit of GLRaV had less sugar and higher acide content. These resulted in a weak grapevine quality .
Comparision of three ELISA methods for detection of GLRaV showed that DAS-ELISA,PTA-ELISA and PAS-ELISA could detect GLRaV and GFLV. DAS-ELISA could shorten the detection time. The techniques , however, have higher strain specificity and the false negatives can be caused . Therefore, PTA-ELISA and PAS-ELISA must repeatedly dectect some important samples.
A series of broad spectrum media for stem tip tissue Virus- ree culture and propagation of grapevine was prepared. These media could shorten the culture time by 12?9 days as compared with the media used commonly. With heat treaement and stem tip tissue Virus梖ree culture, the survival rate of the plant and elimination rate of GLRaV increased more 10.9% and 9.2%, respectively, than singlar stem tip tissue Virus梖ree culture. Moreover, in the survival rate, the changable heat treatment increased more 12.4% than contant heat treatment while the elimination effect of the virus in grapevine showed little difference. The addition of WCT and FenYuanBao in medium exhibited the inhibitory effect on virus multipliction. It, however, had negative effect to the rate of plantlet.
Activity of peroxidase (POD) was analyzed in leaves of grapevine after inoculation of GLRaV. The infection of GLRaV resulted in increase in activities of POD in the resistant and susceptible varieties, and activity of enzyme was significantly greater in resistant varieties than in susceptible ones. Moreover, analysis of POD isoenzyme showed that the number of enzyme bands in resistant varieties varied directly with symptom of plant while in susceptible ones ,varied versely with it,especially mid- late period.
引文
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69． Hacluiger L. A, Schuochau . M. E, Host resistance responses on induction hypothesis. Phytopathol. 1969,59:223-227
70． Harris,R.E. et al :Tissue culture of grapes, 《Canade Agriculture》,1982,No.1
71． Hu. H.Y&Ling K.S. et al, 1998,Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus , the closterovirus type member, Journal of General Virology 79:1289-1298
72． Hu, J. S., Gons Alves D.et al 1991． , Comparison of rapid detection assays for grapevine leafroll disease associated closteroviruses. Vitis 30:87-95
73． Hu,J.S., Gonsalves , D. et al 1090b, Use of monoclonal antibodies to characterize grapevine leafroll associated clostero-viruses . Phytopathology 80, 920-925
74． Lider, L. A. et al , Resistance to the grapevine fanleaf virus ,Xiphinema index complex Vitis species , The 4th International Symposium on Grape-Vine Breeding Verona, Italy, 1985
75． Ling K.S..Zhu.H.Y. et al .Nucleotide sequence of 3'-terminal two thirds of grapevine leafroll-associated virus-3 genome reveals a typital monopartite closterovirus. 1998． 1299-1307
76． Martelli . G.P., Galea Souchet, D. et al ,1992, Viruses of grapevine in Malta . Bulletin OEPP/EPPO 22:607-612
77． Martelli Q.P., ed, Grafe-Transmissible pisease of Grapevine. Hand book for Detection and Diagnosis. ICVG, FAO,Rome.l993
78． Martelli.G.P.. 1986, Virus and virus-like diseases of the grapevine in the mediterranean area , FAO-PI.Protection. 34(1) :25-42
79． Merkuri. J., Martelli G.P., 1994． Viruses of grapevine in Albania. Bulletin OEPP/EPPO 24． 215-220
80． Milne .R.G. et al 1984,Closterovirus-like particles of two types associated with disease grapevine Phytopathology . Z.110． 360-368
81． Monette. P. L. 1966． Elimination in vitro of twe grapevine Nepovirnses by an alternating temperature regime., J. phytopathology 116:88-91
82． Namba,S. et al . 1979． A small spherical virus associated with the A jinashika disease of Foshu grapevine . Ann , phytopathol. Soc. Jpn.45,70-73
83． Petersen C. L.& Charles J.G. 1997． Transmission of grapevine leafroll associated closteroviruses by Pseudococcus longispinus and P. calceolariae . Plant Pathology 46:509-515
84． Petersen.C.L&Jordan D.T.. 1992． ELISA works well for the identification of leafroll. Zn:Jordan K.T. ed . Proceedings of the New Zealand Grape and Wine Symposium . New Zealand Society of Viticulture and Oenology, Christchurch, New Zealand :NZSVO,54-55
85． Pollini C.P, Giunchedi, L. et al A chemiluminescent immunoassay for the disgnosis of grapevine closteroviruses on nitrocellulose membrone. J. Virol. Methods, 1993,42(1) :107-116
86． Raski , D.J. et al : Strategies against grapevine fanleaf virus and its nematode vector , Plant Diseases, 1983March:335-339
87． Reisch, .B.I. Influence of genotype anb cytokinins n in vitro shoot proliferation of grape.J.Amer.Soc.Hortic. Sci,1986,111:138-141
88． Saldarelli P., Minafra A. et al 1994, Detection of grapevine leafroll associated closterovirus III by molecular by bridization plant pathology 43,91-96
89． Scheu, G., 1936 , Mein Winzerbush . Reichsnahrstands-Verlags, G.M.B.H., Berlin.274pp
90． Shannox L. M. Plant isoenzymes . Annker Plant Physical. 1968,19:187-210
91． Stahmann M. A. The new fiontiers in planot biochemist. Japaan Scientific Press-Tokyo ,1983:233-250
92． Tanne, E. et al, 1977,葡萄卷叶病毒有关的病毒提纯及其特性, phytopathology 67(4) .442-447
93． Tanne,E. et al 1989, Serological and molecular evidence for the complexity of the leafroll disease of grapevine . Plant Pathology 38:183-189
94． Valvat, C. et al, Thermotherapie in vitro .Vignes et vins
95． Van Huystee R. B. Ann Rev. plant. 1987, 38:205-219
96． Walker, M. A. et al, Resistance to the grapevine fanleaf virus(GFLV) in Vitis species, Vitis, 1985,24:218-228
97． Yu Danhua and Meredith ,C.P ,The influence of explant origin on tissue browning and shoot production in shoot tip culutures of Vitis vinifera L.. J. Amer. Soc Horti. Sci,1986
98． Zee F. et al : Isolation of virus-like particles from leafroll-infected grapevines, APS annual meeting abstract form, 1985． pp.3
99． Zee, F. et al , Cytopathology of leafroll diseased grapevines and the purification and serology of associated closterovirus-like particles.Phytopathology, 1987, 77: 1427-1434
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67． HabiIi N., Fazeli, C. F. et al . 1997, Identification of a cDNA clone specific to grapevine leafroll-associated virus 1 , and occurrunce of the virus in Australia. Plant Pathology 46,516-522
68． Habili N., Fazeli, C.F. et al . Natural spread and molecular analysis of grapevine leafroll-associated virus 3 in Australia. Phytopathology ,1995,85:1418-1422
69． Hacluiger L. A, Schuochau . M. E, Host resistance responses on induction hypothesis. Phytopathol. 1969,59:223-227
70． Harris,R.E. et al :Tissue culture of grapes, 《Canade Agriculture》,1982,No.1
71． Hu. H.Y&Ling K.S. et al, 1998,Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus , the closterovirus type member, Journal of General Virology 79:1289-1298
72． Hu, J. S., Gons Alves D.et al 1991． , Comparison of rapid detection assays for grapevine leafroll disease associated closteroviruses. Vitis 30:87-95
73． Hu,J.S., Gonsalves , D. et al 1090b, Use of monoclonal antibodies to characterize grapevine leafroll associated clostero-viruses . Phytopathology 80, 920-925
74． Lider, L. A. et al , Resistance to the grapevine fanleaf virus ,Xiphinema index complex Vitis species , The 4th International Symposium on Grape-Vine Breeding Verona, Italy, 1985
75． Ling K.S..Zhu.H.Y. et al .Nucleotide sequence of 3'-terminal two thirds of grapevine leafroll-associated virus-3 genome reveals a typital monopartite closterovirus. 1998． 1299-1307
76． Martelli . G.P., Galea Souchet, D. et al ,1992, Viruses of grapevine in Malta . Bulletin OEPP/EPPO 22:607-612
77． Martelli Q.P., ed, Grafe-Transmissible pisease of Grapevine. Hand book for Detection and Diagnosis. ICVG, FAO,Rome.l993
78． Martelli.G.P.. 1986, Virus and virus-like diseases of the grapevine in the mediterranean area , FAO-PI.Protection. 34(1) :25-42
79． Merkuri. J., Martelli G.P., 1994． Viruses of grapevine in Albania. Bulletin OEPP/EPPO 24． 215-220
80． Milne .R.G. et al 1984,Closterovirus-like particles of two types associated with disease grapevine Phytopathology . Z.110． 360-368
81． Monette. P. L. 1966． Elimination in vitro of twe grapevine Nepovirnses by an alternating temperature regime., J. phytopathology 116:88-91
82． Namba,S. et al . 1979． A small spherical virus associated with the A jinashika disease of Foshu grapevine . Ann , phytopathol. Soc. Jpn.45,70-73
83． Petersen C. L.& Charles J.G. 1997． Transmission of grapevine leafroll associated closteroviruses by Pseudococcus longispinus and P. calceolariae . Plant Pathology 46:509-515
84． Petersen.C.L&Jordan D.T.. 1992． ELISA works well for the identification of leafroll. Zn:Jordan K.T. ed . Proceedings of the New Zealand Grape and Wine Symposium . New Zealand Society of Viticulture and Oenology, Christchurch, New Zealand :NZSVO,54-55
85． Pollini C.P, Giunchedi, L. et al A chemiluminescent immunoassay for the disgnosis of grapevine closteroviruses on nitrocellulose membrone. J. Virol. Methods, 1993,42(1) :107-116
86． Raski , D.J. et al : Strategies against grapevine fanleaf virus and its nematode vector , Plant Diseases, 1983March:335-339
87． Reisch, .B.I. Influence of genotype anb cytokinins n in vitro shoot proliferation of grape.J.Amer.Soc.Hortic. Sci,1986,111:138-141
88． Saldarelli P., Minafra A. et al 1994, Detection of grapevine leafroll associated closterovirus III by molecular by bridization plant pathology 43,91-96
89． Scheu, G., 1936 , Mein Winzerbush . Reichsnahrstands-Verlags, G.M.B.H., Berlin.274pp
90． Shannox L. M. Plant isoenzymes . Annker Plant Physical. 1968,19:187-210
91． Stahmann M. A. The new fiontiers in planot biochemist. Japaan Scientific Press-Tokyo ,1983:233-250
92． Tanne, E. et al, 1977,葡萄卷叶病毒有关的病毒提纯及其特性, phytopathology 67(4) .442-447
93． Tanne,E. et al 1989, Serological and molecular evidence for the complexity of the leafroll disease of grapevine . Plant Pathology 38:183-189
94． Valvat, C. et al, Thermotherapie in vitro .Vignes et vins
95． Van Huystee R. B. Ann Rev. plant. 1987, 38:205-219
96． Walker, M. A. et al, Resistance to the grapevine fanleaf virus(GFLV) in Vitis species, Vitis, 1985,24:218-228
97． Yu Danhua and Meredith ,C.P ,The influence of explant origin on tissue browning and shoot production in shoot tip culutures of Vitis vinifera L.. J. Amer. Soc Horti. Sci,1986
98． Zee F. et al : Isolation of virus-like particles from leafroll-infected grapevines, APS annual meeting abstract form, 1985． pp.3
99． Zee, F. et al , Cytopathology of leafroll diseased grapevines and the purification and serology of associated closterovirus-like particles.Phytopathology, 1987, 77: 1427-1434