成都市龙泉驿区桃树黄化病调查及初步研究
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摘要
桃树原产于我国，其色、香、味具佳及营养丰富等优点深受人们的青睐。近年来，四川省成都市龙泉驿区桃树黄化病日趋严重，已成为桃生产上的制约因子。本研究以龙泉驿区的黄化病桃树为材料，对龙泉驿区桃树黄化病的症状特点和危害、病原的形态和检测、病原在树体内的分布、消长和传播介体昆虫及龙泉驿区桃树黄化病同工酶等4部分内容进行了研究。得出以下结论：
龙泉驿区桃树黄化病的症状及危害。桃树黄化病5月中旬开始严重发生，发病盛期在6月至9月下旬发病初期桃树叶脉保持绿色，叶肉变黄，继而叶脉变黄，叶片质地变脆，形成穿孔，易脱落；桃树黄化病危害经调查发现：黄化病病株率26％～45％，黄叶率达85％，黄化病情指数36.4，严重削弱树势及影响品质和产量。
运用组织化学技术、PCR技术、抗生素治疗试验以及透射电镜技术对桃树黄化病病原的检测和形态进行研究。(1)经组织化学技术发现，光学显微镜下病株叶脉韧皮部可被迪纳氏试剂染成天蓝色，并且荧光显微镜也观察到病叶叶脉韧皮部存在4′，6-二脒基-2-苯基-6-吲哚(4′，6-diamidino-2-phenylindole，DAPI)荧光点；利用Lee等设计的引物对桃树黄化病DNA样品进行PCR扩增，结果获得1.5kb特异性片段，与目的片段大小一致，并且枣疯病阳性对照也扩增出改片段；盐酸土霉素输液式灌注治疗结果表明龙泉驿区桃树黄化病病原对盐酸土霉素敏感，病株出现明显地复绿。(2)通过透射电镜观察到桃树黄化病病原以近球形和椭圆形为主，还有哑铃形、丝状体和大量100nm以下不规则的形状，表明该病原具有多型性现象；桃树黄化病植原体大小在40～850nm，存在电子云密度高低两种。电子云密度低的植原体形成空泡型，可以清晰地看到植原体两暗一明的双膜结构，膜间距约为12～22nm，膜宽为10～16nm；而电子云密度高的形成实心球型。
利用组织化学技术和透射电镜对桃树黄化病病原的分布、树体内周年消长及传病介体昆虫进行研究。组织化学技术研究发现桃树黄化病病原在树体内的分布具有普遍性和不均匀性等特点；病原常年存在于病株体内的韧皮部组织中，冬季的病原浓度低于其他季节；利用透射电镜技术发现植原体的菌体存在于桃小绿叶蝉(Empoasca flavescens)及梨冠网蝽(Stephanitis nashi)的唾液腺内，多为不规则形，椭圆形的哑铃形。大小为150-840nm，单位膜厚度8nm。
桃树黄化病病株的同工酶研究。酶活性测定结果表明，发病初期各品种内健康叶片、病株的健叶及病叶的酶活性存在显著差异，即桃树感染植原体黄化病后过氧化物酶活性呈先升高后降低的趋势；而多酚氧化酶的活性呈降低的趋势；同工酶聚丙烯酰胺凝胶电泳结果表明：发病初期除北京24外的3个品种的过氧化物酶同工酶和酯酶同工酶的酶谱都表现为病株叶片的酶带减少，颜色降低。
Peach, a native fruit tree of China, is popular for its beauty, good taste, fruit rich in nutrition and so on. In recent, peach yellow disease becomes more and more serious, and presents up-trend year by year. In the paper, the healthy peach, diseased peach were employed to study the symptom, the modality and detecting of the pathogen; And the distribution and annual variation of the pathogen in peach tissue were studied by staining with DAPI, and transmission electron microscope was used for research transmission insect vectors; In addition to, the pathophysiology including activity of POD and PPO, POD isozyme and esterase isozyme were researched by using spectrophotometer and polyaerylamicle electrophoretic methods. The results were showed as follow.
The results of investigation of the peach yellow disease are: peach leaves become yellow little by little except for leaves vein at first; Then, leaves vein become yellow, too; Finally, the texture of leaves is friable and, and many little pore form in leaves. It is easy to fall off peach tree; The incident of peach yellow disease is to 26%～45%; yellow leaf's rate amounts to 85%; yellow disease index is 36.4. It is serious to impair tree vigor class, quality and yield.
The paper detected pathogen of peach yellow disease by histochemistry technology, polymerase chain reaction and treatment by acheomycin. (1) The sky-blue phloem was observed by staining with denese under optical microscope; Fluorescence spots were found in the phloem of leaf midrib which was stained by DAPI. The pathogen was confirmed by analysis with polymerase chain reaction using a pair of universal primer, which was designed by Lee according to phytoplasma 16SrDNA. An amplified product of the 1.5kb particularity DNA fragment was acquired in samples from infected peach, but not in healthy peach. The result of treatment by acheomycin showed that the pathogen of peach yellow disease was susceptive to acheomycin, peach yellow leaves resumed green incoordinately. (2) The phytoplasmas of peach yellow disease are mainly globose, oval, and some is like dumbbell, protonema and erose shape, which showed the phytoplasma of peach yellow disease has a polymorphism. The phytoplasmas are 40～850nm or so. There are two kinds of phytoplasmas having high and low electron cloud density. Phytoplasmas having low electron cloud density are like vacuole with two unit membranes which
龙泉驿区桃树黄化病的症状及危害。桃树黄化病5月中旬开始严重发生，发病盛期在6月至9月下旬发病初期桃树叶脉保持绿色，叶肉变黄，继而叶脉变黄，叶片质地变脆，形成穿孔，易脱落；桃树黄化病危害经调查发现：黄化病病株率26％～45％，黄叶率达85％，黄化病情指数36.4，严重削弱树势及影响品质和产量。
运用组织化学技术、PCR技术、抗生素治疗试验以及透射电镜技术对桃树黄化病病原的检测和形态进行研究。(1)经组织化学技术发现，光学显微镜下病株叶脉韧皮部可被迪纳氏试剂染成天蓝色，并且荧光显微镜也观察到病叶叶脉韧皮部存在4′，6-二脒基-2-苯基-6-吲哚(4′，6-diamidino-2-phenylindole，DAPI)荧光点；利用Lee等设计的引物对桃树黄化病DNA样品进行PCR扩增，结果获得1.5kb特异性片段，与目的片段大小一致，并且枣疯病阳性对照也扩增出改片段；盐酸土霉素输液式灌注治疗结果表明龙泉驿区桃树黄化病病原对盐酸土霉素敏感，病株出现明显地复绿。(2)通过透射电镜观察到桃树黄化病病原以近球形和椭圆形为主，还有哑铃形、丝状体和大量100nm以下不规则的形状，表明该病原具有多型性现象；桃树黄化病植原体大小在40～850nm，存在电子云密度高低两种。电子云密度低的植原体形成空泡型，可以清晰地看到植原体两暗一明的双膜结构，膜间距约为12～22nm，膜宽为10～16nm；而电子云密度高的形成实心球型。
利用组织化学技术和透射电镜对桃树黄化病病原的分布、树体内周年消长及传病介体昆虫进行研究。组织化学技术研究发现桃树黄化病病原在树体内的分布具有普遍性和不均匀性等特点；病原常年存在于病株体内的韧皮部组织中，冬季的病原浓度低于其他季节；利用透射电镜技术发现植原体的菌体存在于桃小绿叶蝉(Empoasca flavescens)及梨冠网蝽(Stephanitis nashi)的唾液腺内，多为不规则形，椭圆形的哑铃形。大小为150-840nm，单位膜厚度8nm。
桃树黄化病病株的同工酶研究。酶活性测定结果表明，发病初期各品种内健康叶片、病株的健叶及病叶的酶活性存在显著差异，即桃树感染植原体黄化病后过氧化物酶活性呈先升高后降低的趋势；而多酚氧化酶的活性呈降低的趋势；同工酶聚丙烯酰胺凝胶电泳结果表明：发病初期除北京24外的3个品种的过氧化物酶同工酶和酯酶同工酶的酶谱都表现为病株叶片的酶带减少，颜色降低。
Peach, a native fruit tree of China, is popular for its beauty, good taste, fruit rich in nutrition and so on. In recent, peach yellow disease becomes more and more serious, and presents up-trend year by year. In the paper, the healthy peach, diseased peach were employed to study the symptom, the modality and detecting of the pathogen; And the distribution and annual variation of the pathogen in peach tissue were studied by staining with DAPI, and transmission electron microscope was used for research transmission insect vectors; In addition to, the pathophysiology including activity of POD and PPO, POD isozyme and esterase isozyme were researched by using spectrophotometer and polyaerylamicle electrophoretic methods. The results were showed as follow.
The results of investigation of the peach yellow disease are: peach leaves become yellow little by little except for leaves vein at first; Then, leaves vein become yellow, too; Finally, the texture of leaves is friable and, and many little pore form in leaves. It is easy to fall off peach tree; The incident of peach yellow disease is to 26%～45%; yellow leaf's rate amounts to 85%; yellow disease index is 36.4. It is serious to impair tree vigor class, quality and yield.
The paper detected pathogen of peach yellow disease by histochemistry technology, polymerase chain reaction and treatment by acheomycin. (1) The sky-blue phloem was observed by staining with denese under optical microscope; Fluorescence spots were found in the phloem of leaf midrib which was stained by DAPI. The pathogen was confirmed by analysis with polymerase chain reaction using a pair of universal primer, which was designed by Lee according to phytoplasma 16SrDNA. An amplified product of the 1.5kb particularity DNA fragment was acquired in samples from infected peach, but not in healthy peach. The result of treatment by acheomycin showed that the pathogen of peach yellow disease was susceptive to acheomycin, peach yellow leaves resumed green incoordinately. (2) The phytoplasmas of peach yellow disease are mainly globose, oval, and some is like dumbbell, protonema and erose shape, which showed the phytoplasma of peach yellow disease has a polymorphism. The phytoplasmas are 40～850nm or so. There are two kinds of phytoplasmas having high and low electron cloud density. Phytoplasmas having low electron cloud density are like vacuole with two unit membranes which
引文
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