我国20个梨品种(种质)对国外梨火疫病菌的抗病性评价
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摘要
【目的】选取Erwinia amylovora国外菌株及国内梨品种(种质),经室内接种,评价不同菌株的致病力及梨品种的抗病性,为研究梨火疫病防控技术提供技术资料。【方法】对供试的梨品种(种质)枝条采用离体接种、梨幼果半果接种法,比较3个不同来源的国外菌株E.a 0001、E.a 0017、E.a 0055的致病力。通过离体枝条接种强致病力菌株E.a0017,结合实时荧光PCR定量检测病原菌数量,制定梨品种抗病性分级指标,综合评估梨品种的抗病水平。【结果】3个菌株对供试梨品种枝条均具有致病力,对‘库尔勒香梨‘’砀山梨‘’黑酸梨’、杜梨具有强致病力,对‘库尔勒香梨’的致病力最强;综合对梨枝条接种的病情指数均值,其致病力的强弱依次为E.a 0001>E.a 0055>E.a 0017。E.a 0017菌株对梨幼果的致病力较E.a 0001和E.a 0055强,而E.a 0001和E.a 0055对幼果的致病力相近。供试的20个梨品种(种质)对Erwinia amylovora的抗性水平普遍较低,其中14个品种都不同程度感病(占70%),没有发现高抗品种,2个表现出抗病性和4个具有耐病性的品种均为我国的地方品种。【结论】3个E. amylovora菌株对供试梨品种(种质)均具有强和较强的致病力。供试梨品种(种质)中‘,晋酥‘’绿梨’表现抗病性‘,霍城冬黄梨‘’八月酥‘’库车阿木特‘’棉梨’表现出耐病性,其他均为感病品种。
【Objective】Fire blight, caused by the bacterium Erwinia amylovora, is one of the most destructive disease affecting plants of family Rosaceae. The bacterium can destroy blossoms, shoots and stems, and may seriously damage an entire pear or apple orchard within a growing season under optimal conditions. The disease has currently spread more than 60 countries, and is a serious concern to growers of pears or apples worldwide. Up to now, fire blight and Erwinia amylovora have not yet recorded in China, but have been found in neighbouring countries like Kazakhstan, Kyrgyzstan and Russia during last decade. The control measure consists of removal of diseased host plants or their parts, cultural practices and application of chemical sprays. However, these measures are not always satisfactory. In all the ways, growing relatively resistant cultivars and rootstocks can be one of the most efficient control methods for fire blight. The disease could be better controlled by understanding virulence of Erwinia amylovora strains and responses of host-plant cultivars. The aim of this study was to test pathogenicity of exotic E. amylovora strains and resistance of domestic pear varieties to these strains, so as to provide the foundation for the prevention and treatment of the disease.【Methods】The experiment was conducted in a screen house using in vitro culture assay. The relative virulence of three different E.amylovora strains,E.a 0001 isolated from apple of Kyrgyzstan, E.a 0017 from pear of Kyrgyzstan and E.a 0055 from pear of USA, was examined by both in vitro artificial inoculation twigs and young fruits of testing pear cultivars. The plant materials for testing were obtained from Luntai National Pomology Germplasm Garden,Xinjiang. The shoots collected from healthy pear trees were cut into the sections of 25 cm in length,then sterilized with 75% alcohol, and later 1/3 of the branches were inserted into the triangular bottles containing sterilize-distilled water. Sterilization surgery knife was used to cut a wound of about 5 mm on the shoots surface. On the wound was placed a small degreasing cotton piece saturated with 2 mL tested bacteria suspension at the concentration of approximately 1 × 109 CFU · mL-1, and then wrapped with plastic wrap to prevent desiccation. The inoculated branches were cultivated in an artificial climate chamber at 28.5 ℃, 75% RH and 12 h light. Twenty shoots from each species were inoculated with each bacterium in the experiment. The length of resulting necrosis was measured 7 days after inoculation, and statistics on the incidence and index were carried out. The virulence of the E. amylovora strains was divided into the strongest, stronger, medium, and weak virulent class according to the disease index. The gathered pear fruitlets were soaked with 75% ethanol for 10 minutes. After the ethanol was volatilized, they were cut in half with sterile scalpel. The sterilization bamboo stick was dipped in bacterial colony to drip on the sarcocarp, and 2 to 4 inoculation points were selected on each section,and the inoculation sterile water was used for negative control. After inoculation, the fruits were put into a sterile Petri dish, which was placed in a germination box with absorbent paper in an incubator. The pathogenicity of the strains was analyzed by recording the appearance time of initial bacterial oozes and the ratio of bacterial oozes area to fruitlet area. Twenty-one pear varieties were chosen for testing their relative resistance to E. amylovora 0017 strain that was aggressive in the above test. Through in vitro shoot culture inoculation method, the level of resistance to E. amylovora was evaluated according to the extent of lesion development on the shoot and the population of the bacteria in the tissue with real-time fluorescence quantitative PCR assay. The level of resistance was converted into six class evaluation scales comprehensively.【Results】The result of pathogenicity test revealed that all strains had different virulence, and were highly strong virulent to the‘Kuerlexiangli'‘Dangshanli'‘Heisuanli'and Pyrus betulifolia, being most virulent to‘Kuerlexiangli'. All strains had strong virulence to‘Xinli No.7',‘Heisuanli', and‘Hesejujuli', while they had medium virulence to‘UChe Amute'and‘Huochengdonghuangli'. Fruitlets were most sensitive to fire blight and fruitlet inoculation was a common method to identify the pathogenicity of E.amylovora. The results indicated that three tested stains(E.a 0001, E.a0017 and E.a 0055) all had pathogenicity to the measured fruitlets. After inoculation, the bacterial pus generated within 10-24 hours. According to the time of appearance and amount of bacterial pus on the fruitlets, it was inferred that E.a 0017 strain was more virulence to fruitlets than the additional two strains, but it was hard to compare the pathogenicity between E.a 0001 and E.a 0055. The level of resistance of the twenty-one pear varieties to E.amylovora wasn't higher generally. The majority of the pear cultivars tested was susceptible to E.amylovora, 14 pear cultivars(70%) were evaluated as varying degrees susceptible, including 4 highly susceptible, 7 susceptible and 3 moderately susceptible cultivars.None of them was highly resistant to the pathogen, while 2 cultivars performed as resistant, and 4 cultivars as disease-tolerant, which were all local varieties in our country.【Conclusion】A system for testing virulence and host plant resistance to Erwinia amylovora have been developed in laboratory. The variability in virulence among the strains was found, with E.a 0001> E.a 0055> E.a 0017. Evaluation of obtained results proved the varieties with a higher level of resistance were‘Lüli'and‘Jinsu'.‘Bayuesu',‘Huochengdonghuangli'and‘UChe Amute'and‘Mianli'were evaluated as disease-tolerant.‘Huangsuanli'‘Jinchuanxue'and‘Hesejujuli'were evaluated as moderately susceptible.‘Xuehuali'‘Pyrus Betulifolia'‘Hongxiangsu'‘Qipan'‘Hongxiangli'‘Zaosu'and‘Xinli No.7'were evaluated as susceptible.‘Kuerlexiangli'‘Dangshanli'‘Jinhuali'and‘Huangsuanli'were evaluated as highly susceptible.
【Objective】Fire blight, caused by the bacterium Erwinia amylovora, is one of the most destructive disease affecting plants of family Rosaceae. The bacterium can destroy blossoms, shoots and stems, and may seriously damage an entire pear or apple orchard within a growing season under optimal conditions. The disease has currently spread more than 60 countries, and is a serious concern to growers of pears or apples worldwide. Up to now, fire blight and Erwinia amylovora have not yet recorded in China, but have been found in neighbouring countries like Kazakhstan, Kyrgyzstan and Russia during last decade. The control measure consists of removal of diseased host plants or their parts, cultural practices and application of chemical sprays. However, these measures are not always satisfactory. In all the ways, growing relatively resistant cultivars and rootstocks can be one of the most efficient control methods for fire blight. The disease could be better controlled by understanding virulence of Erwinia amylovora strains and responses of host-plant cultivars. The aim of this study was to test pathogenicity of exotic E. amylovora strains and resistance of domestic pear varieties to these strains, so as to provide the foundation for the prevention and treatment of the disease.【Methods】The experiment was conducted in a screen house using in vitro culture assay. The relative virulence of three different E.amylovora strains,E.a 0001 isolated from apple of Kyrgyzstan, E.a 0017 from pear of Kyrgyzstan and E.a 0055 from pear of USA, was examined by both in vitro artificial inoculation twigs and young fruits of testing pear cultivars. The plant materials for testing were obtained from Luntai National Pomology Germplasm Garden,Xinjiang. The shoots collected from healthy pear trees were cut into the sections of 25 cm in length,then sterilized with 75% alcohol, and later 1/3 of the branches were inserted into the triangular bottles containing sterilize-distilled water. Sterilization surgery knife was used to cut a wound of about 5 mm on the shoots surface. On the wound was placed a small degreasing cotton piece saturated with 2 mL tested bacteria suspension at the concentration of approximately 1 × 109 CFU · mL-1, and then wrapped with plastic wrap to prevent desiccation. The inoculated branches were cultivated in an artificial climate chamber at 28.5 ℃, 75% RH and 12 h light. Twenty shoots from each species were inoculated with each bacterium in the experiment. The length of resulting necrosis was measured 7 days after inoculation, and statistics on the incidence and index were carried out. The virulence of the E. amylovora strains was divided into the strongest, stronger, medium, and weak virulent class according to the disease index. The gathered pear fruitlets were soaked with 75% ethanol for 10 minutes. After the ethanol was volatilized, they were cut in half with sterile scalpel. The sterilization bamboo stick was dipped in bacterial colony to drip on the sarcocarp, and 2 to 4 inoculation points were selected on each section,and the inoculation sterile water was used for negative control. After inoculation, the fruits were put into a sterile Petri dish, which was placed in a germination box with absorbent paper in an incubator. The pathogenicity of the strains was analyzed by recording the appearance time of initial bacterial oozes and the ratio of bacterial oozes area to fruitlet area. Twenty-one pear varieties were chosen for testing their relative resistance to E. amylovora 0017 strain that was aggressive in the above test. Through in vitro shoot culture inoculation method, the level of resistance to E. amylovora was evaluated according to the extent of lesion development on the shoot and the population of the bacteria in the tissue with real-time fluorescence quantitative PCR assay. The level of resistance was converted into six class evaluation scales comprehensively.【Results】The result of pathogenicity test revealed that all strains had different virulence, and were highly strong virulent to the‘Kuerlexiangli'‘Dangshanli'‘Heisuanli'and Pyrus betulifolia, being most virulent to‘Kuerlexiangli'. All strains had strong virulence to‘Xinli No.7',‘Heisuanli', and‘Hesejujuli', while they had medium virulence to‘UChe Amute'and‘Huochengdonghuangli'. Fruitlets were most sensitive to fire blight and fruitlet inoculation was a common method to identify the pathogenicity of E.amylovora. The results indicated that three tested stains(E.a 0001, E.a0017 and E.a 0055) all had pathogenicity to the measured fruitlets. After inoculation, the bacterial pus generated within 10-24 hours. According to the time of appearance and amount of bacterial pus on the fruitlets, it was inferred that E.a 0017 strain was more virulence to fruitlets than the additional two strains, but it was hard to compare the pathogenicity between E.a 0001 and E.a 0055. The level of resistance of the twenty-one pear varieties to E.amylovora wasn't higher generally. The majority of the pear cultivars tested was susceptible to E.amylovora, 14 pear cultivars(70%) were evaluated as varying degrees susceptible, including 4 highly susceptible, 7 susceptible and 3 moderately susceptible cultivars.None of them was highly resistant to the pathogen, while 2 cultivars performed as resistant, and 4 cultivars as disease-tolerant, which were all local varieties in our country.【Conclusion】A system for testing virulence and host plant resistance to Erwinia amylovora have been developed in laboratory. The variability in virulence among the strains was found, with E.a 0001> E.a 0055> E.a 0017. Evaluation of obtained results proved the varieties with a higher level of resistance were‘Lüli'and‘Jinsu'.‘Bayuesu',‘Huochengdonghuangli'and‘UChe Amute'and‘Mianli'were evaluated as disease-tolerant.‘Huangsuanli'‘Jinchuanxue'and‘Hesejujuli'were evaluated as moderately susceptible.‘Xuehuali'‘Pyrus Betulifolia'‘Hongxiangsu'‘Qipan'‘Hongxiangli'‘Zaosu'and‘Xinli No.7'were evaluated as susceptible.‘Kuerlexiangli'‘Dangshanli'‘Jinhuali'and‘Huangsuanli'were evaluated as highly susceptible.
引文
[1]ZHU J Y,LIAO X L,GAO B D,ZHU S F,CHEN H Y,DAI FQ.The establishment of detection of real-time fluorescent PCRand capture-PCR-ELISA for detection of Fire Blight Erwinia amylovora[J].Plant Quarantine,2003,17(1):7-10.
[2]赵友福.梨火疫病的传播扩散与检疫处理[J].中国进出境动植检,1996(3):39-40.ZHAO Youfu.Spread and quarantine of Fire Blight[J].China Entry-Exit Animal and Plant Quarantine,1996(3):39-40.
[3]赵友福.梨火疫病的世界性传播现状及检疫对策[J].中国进出境动植检,1995(4):38-39.ZHAO Youfu.Present situation of worldwide spread of Fire Blight and quarantine ountermeasures[J].China Entry-Exit Animal and Plant Quarantine,1995(4):38-39.
[4]戴芳澜.中国经济植物病原目录[M].北京:科学出版社,1958.DAI Fanglan.Chinese economic plant pathogen directory[M].Beijing:Science Press,1958.
[5]PM 7/20(2)Erwinia amylovora[J].Eppo Bulletin,2013,43(1):21-45.
[6]MARINOVATODOROVA M,RANTA J,HANNUNEN S.The suitability of Finnish climate for Fire Blight(Erwinia amylovora)epidemics on apple[J].Agricultural&Food Science,2015,24(1):59-66.
[7]DOOLOTKELDIEVA T,BOBUSHEVA S.Fire blight disease caused by Erwinia amylovora on Rosaceae plants in kyrgyzstan and biological agents to control this disease[J].Advances in Microbiology,2016,6(11):831-851.
[8]胡白石,许志刚,周国梁,易建平,焦国尧.梨火疫病的进境风险分析[J].植物保护学报,2001,28(4):303-308.HU Baishi,XU Zhigang,ZHOU Guoliang,YI Jianping,JIAOGuoyao.Analysis on the risk of entry of Fire Blight[J].Journal of Plant Protection,2001,28(4):303-308.
[9]于海伦.重大林果外来有害生物入侵新疆的风险分析[D].乌鲁木齐:新疆农业大学,2012.YU Hailun.Pest risk analysis of invasion on Xinjiang features fruit[D].Urumqi:Xinjiang Agricultural University,2012.
[10]张乐,石秀丽.用离体巴梨枝条测定梨火疫病菌的致病性[J].植物检疫,1993(2):104-105.ZHANG Le,SHI Xiuli.Determination of Fire Blight with isolated Bartlett branches pathogenicity[J].Plant Quarantine,1993(2):104-105.
[11]李颖章,韩碧文,OGNJANOV V.苹果组培苗和温室盆载苗对炎疫病的感病性研究[J].中国农业大学学报,2000,5(1):25-30.LI Yingzhang,HAN Biwen,OGNJANOV V.Susceptibility of apples to Fire Blight grown in vitro and in greenhouse[J].Journal of China Agricultural University,2000,5(1):25-30.
[12]PAPRSTEIN F,SEDLAK J,KORBA J,SILLEROVA J.Testing of resistance to Erwinia amylovora in an in vitro culture assay[J].Acta Horticulturae,2011,896:381-384.
[13]刘华威,王晓鸣,郭庆元,韩丽娟,曹玉芬,张薇田,路明.梨种质对梨火疫病的抗性研究[J].植物遗传资源学报,2008,9(2):195-200.LIU Huawei,WANG Xiaoming,GUO Qingyuan,HAN Lijuan,CAO Yufen,ZHANG Weitian,LU Ming.Identification of resistance to Fire Blight in pear germplasm[J].Journal of Plant Genetic Resources,2008,9(2):195-200.
[14]吴晓芳.万州猕猴桃溃疡病病原菌的分离鉴定及环介导等温扩增快速检测[D].重庆:重庆大学,2015.WU Xiaofang.Isolation and identification of the causal agent for kiwifruit bacterial canker in Wanzhou district of Chongqing and the detection method construction of loop-mediated isothermal amplification[D].Chongqing:Chongqing University,2015.
[15]TAGHDAREH G,BAGHAEE-RAVARI S,MOSLEMKHANIC,MAHDIKHANI-MOGHADDAM E.Evaluation of repeat sequences on plasmid pEA29 of Erwinia amylovora from Iran[J].European Journal of Plant Pathology,2014,140(4):1-10.
[16]Korba J,?illerováj,K dela v.Resistance of apple varieties and selections to Erwinia amylovora in the Czech Republic[J].Plant Protection Science,2008,44(3):91-96.
[17]ATANASOVA I,STEFANOVA K,KABADHOVA P,TISHKOVS,DIMITROV Z,BOGATZEVSKA N,MONCHEVA P.Phenotypic diversity of Erwinia amylovora in Bulgaria[J].Zeitschrift Fur Naturforschung C,2007,62(12):857-868.
[18]胡白石.梨火疫病菌的风险分析及检测技术研究[D].南京:南京农业大学,2000.HU Baishi.Pest risk analysis and detection techniques of Ewinia amylovora.[D].Nanjing:Nanjing Agricultural University,2000.
[19]SAN S P,CULLUM J,THOMIDIS T.An assessment of the relative resistance of three hawthorn species to three strains of Erwinia amylovora using three different inoculation methods[J].Phytoparasitica,2009,37(4):371-373.
[20]KIM J H,BEER S V,TANII A,ZUMOFF C H,LABY R J,GUSTAFSON H L,ALDWINCKLE H S.Characterization of Erwinia amylovora strains from different hosts and geographical areas[J].Acta Horticulturae,1996,411(1):183-185.
[21]BELL R L,ZWET T V D,MILLER D D.‘SHENANDOAH’:A new Fire Blight resistant pear cultivar[J].Hortscience,2004,39:805.
[22]LAUX P,ZELLER W.Fire blight resistance in extensive pome fruit production in Germany[J].Acta Horticulturae,2006,704:531-534.
[23]SILLEROVA J,KORBA J,PAPRSTEIN F,SEDLAK J.Testing of resistance of pear cultivars after artificial inoculation with Erwinia amylovora in field conditions[J].Acta Horticulturae,2011,896:353-355.
[24]BOGS J,RICHTER K,KIM W S,JOCK S,GEIDER K.Alternative methods to describe virulence of Erwinia amylovora and host-plant resistance against Fire Blight[J].Plant Pathology,2004,53(1):80.
[2]赵友福.梨火疫病的传播扩散与检疫处理[J].中国进出境动植检,1996(3):39-40.ZHAO Youfu.Spread and quarantine of Fire Blight[J].China Entry-Exit Animal and Plant Quarantine,1996(3):39-40.
[3]赵友福.梨火疫病的世界性传播现状及检疫对策[J].中国进出境动植检,1995(4):38-39.ZHAO Youfu.Present situation of worldwide spread of Fire Blight and quarantine ountermeasures[J].China Entry-Exit Animal and Plant Quarantine,1995(4):38-39.
[4]戴芳澜.中国经济植物病原目录[M].北京:科学出版社,1958.DAI Fanglan.Chinese economic plant pathogen directory[M].Beijing:Science Press,1958.
[5]PM 7/20(2)Erwinia amylovora[J].Eppo Bulletin,2013,43(1):21-45.
[6]MARINOVATODOROVA M,RANTA J,HANNUNEN S.The suitability of Finnish climate for Fire Blight(Erwinia amylovora)epidemics on apple[J].Agricultural&Food Science,2015,24(1):59-66.
[7]DOOLOTKELDIEVA T,BOBUSHEVA S.Fire blight disease caused by Erwinia amylovora on Rosaceae plants in kyrgyzstan and biological agents to control this disease[J].Advances in Microbiology,2016,6(11):831-851.
[8]胡白石,许志刚,周国梁,易建平,焦国尧.梨火疫病的进境风险分析[J].植物保护学报,2001,28(4):303-308.HU Baishi,XU Zhigang,ZHOU Guoliang,YI Jianping,JIAOGuoyao.Analysis on the risk of entry of Fire Blight[J].Journal of Plant Protection,2001,28(4):303-308.
[9]于海伦.重大林果外来有害生物入侵新疆的风险分析[D].乌鲁木齐:新疆农业大学,2012.YU Hailun.Pest risk analysis of invasion on Xinjiang features fruit[D].Urumqi:Xinjiang Agricultural University,2012.
[10]张乐,石秀丽.用离体巴梨枝条测定梨火疫病菌的致病性[J].植物检疫,1993(2):104-105.ZHANG Le,SHI Xiuli.Determination of Fire Blight with isolated Bartlett branches pathogenicity[J].Plant Quarantine,1993(2):104-105.
[11]李颖章,韩碧文,OGNJANOV V.苹果组培苗和温室盆载苗对炎疫病的感病性研究[J].中国农业大学学报,2000,5(1):25-30.LI Yingzhang,HAN Biwen,OGNJANOV V.Susceptibility of apples to Fire Blight grown in vitro and in greenhouse[J].Journal of China Agricultural University,2000,5(1):25-30.
[12]PAPRSTEIN F,SEDLAK J,KORBA J,SILLEROVA J.Testing of resistance to Erwinia amylovora in an in vitro culture assay[J].Acta Horticulturae,2011,896:381-384.
[13]刘华威,王晓鸣,郭庆元,韩丽娟,曹玉芬,张薇田,路明.梨种质对梨火疫病的抗性研究[J].植物遗传资源学报,2008,9(2):195-200.LIU Huawei,WANG Xiaoming,GUO Qingyuan,HAN Lijuan,CAO Yufen,ZHANG Weitian,LU Ming.Identification of resistance to Fire Blight in pear germplasm[J].Journal of Plant Genetic Resources,2008,9(2):195-200.
[14]吴晓芳.万州猕猴桃溃疡病病原菌的分离鉴定及环介导等温扩增快速检测[D].重庆:重庆大学,2015.WU Xiaofang.Isolation and identification of the causal agent for kiwifruit bacterial canker in Wanzhou district of Chongqing and the detection method construction of loop-mediated isothermal amplification[D].Chongqing:Chongqing University,2015.
[15]TAGHDAREH G,BAGHAEE-RAVARI S,MOSLEMKHANIC,MAHDIKHANI-MOGHADDAM E.Evaluation of repeat sequences on plasmid pEA29 of Erwinia amylovora from Iran[J].European Journal of Plant Pathology,2014,140(4):1-10.
[16]Korba J,?illerováj,K dela v.Resistance of apple varieties and selections to Erwinia amylovora in the Czech Republic[J].Plant Protection Science,2008,44(3):91-96.
[17]ATANASOVA I,STEFANOVA K,KABADHOVA P,TISHKOVS,DIMITROV Z,BOGATZEVSKA N,MONCHEVA P.Phenotypic diversity of Erwinia amylovora in Bulgaria[J].Zeitschrift Fur Naturforschung C,2007,62(12):857-868.
[18]胡白石.梨火疫病菌的风险分析及检测技术研究[D].南京:南京农业大学,2000.HU Baishi.Pest risk analysis and detection techniques of Ewinia amylovora.[D].Nanjing:Nanjing Agricultural University,2000.
[19]SAN S P,CULLUM J,THOMIDIS T.An assessment of the relative resistance of three hawthorn species to three strains of Erwinia amylovora using three different inoculation methods[J].Phytoparasitica,2009,37(4):371-373.
[20]KIM J H,BEER S V,TANII A,ZUMOFF C H,LABY R J,GUSTAFSON H L,ALDWINCKLE H S.Characterization of Erwinia amylovora strains from different hosts and geographical areas[J].Acta Horticulturae,1996,411(1):183-185.
[21]BELL R L,ZWET T V D,MILLER D D.‘SHENANDOAH’:A new Fire Blight resistant pear cultivar[J].Hortscience,2004,39:805.
[22]LAUX P,ZELLER W.Fire blight resistance in extensive pome fruit production in Germany[J].Acta Horticulturae,2006,704:531-534.
[23]SILLEROVA J,KORBA J,PAPRSTEIN F,SEDLAK J.Testing of resistance of pear cultivars after artificial inoculation with Erwinia amylovora in field conditions[J].Acta Horticulturae,2011,896:353-355.
[24]BOGS J,RICHTER K,KIM W S,JOCK S,GEIDER K.Alternative methods to describe virulence of Erwinia amylovora and host-plant resistance against Fire Blight[J].Plant Pathology,2004,53(1):80.