甘肃省蔬菜根际寄生线虫的种类鉴定、发生分布及根结线虫rDNA-ITS-RFLP及其防治的研究
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摘要
2002年3月—8月份，对甘肃省兰州、天水、红古等地区蔬菜根际的植物寄生线虫进行了广泛调查，从番茄、辣椒、茄子、黄瓜、甘蓝、萝卜、菜豆、豇豆、苦瓜、大白菜、芹菜等的根际采集到126份土壤样品，共分离出了植物寄生线虫15个属25个种和一些待定种，包括清亮矮化线虫(Tylenchorhynchus clarus)、马舒德矮化线虫(T.mashhoodi)、网纹矮化线虫(T.aerolatus)；短小节纹线虫(Merlinius brevidens)、M.communium；落选短体线虫(Pratylenchus neglectus)、六纹短体线虫(P.hexincisus)、斯氏短体线虫(P.scribneri)；柱颈丝尾垫刃线虫(Filenchus cylindricollis)、普通丝尾垫刃线虫(F.vulgaris)、F.ditissimus；燕麦真滑刃线虫(Aphelenchus avenae)、茄真滑刃线虫(A.solani)；双宫螺旋线虫(Helicotylenchus dihystera)、热带螺旋线虫(H.tropitus)；活泼平滑垫刃线虫(Psilenchus hilarulus)；薄叶大节片线虫(Macroposthonia xenoplox)；同型伞香线虫(Boleodorus similis)；根结线虫(Meloidogyne spp.)，剑尾垫刃线虫(Malenchus spp.)，米卡垫刃线虫(Miculenchus spp.)，滑刃线虫(Aphelenchoides spp.)，伞滑刃线虫(Bursaphelenchus spp.)，滑刃线虫(Aphelenchoides spp.)，茎线虫(Ditylenchus spp.)。在这些鉴定出的线虫中，大多数种类在甘肃省都未曾报道过。其中，几种重要的线虫如清亮矮化线虫、马舒德矮化线虫、网纹矮化线虫、短小节纹线虫、落选短体线虫、六纹短体线虫和斯氏短体线虫等发生较为普遍，在许多地区的大多数蔬菜上几乎都有寄生。而兰州市安宁区吊场乡茄子、辣椒上的短小节纹线虫(M. brevidens)群体密度相对较大，并且随时间、寄主营养条件等呈现季节性变化。
用PCR技术从北京顺义甘蓝、番茄；四川简阳莴苣、花生、杂草；广西南宁香蕉；山东莒南烟草；河南洛阳烟草、海南儋州番茄、象耳豆、胡椒、番石榴；安徽桔梗；比利时番茄等15个根结线虫群体中分别扩增出一条rDNA-ITS的片段，大小约770bp。用3种限制性内切酶HaeⅢ、DraⅠ、AluⅠ分别酶切M.incognita群体和M.javanica群体的ITS产物，HaeⅢ酶切后产生约370bp、220bp、180bp片段；DraⅠ和AluⅠ酶切后分别产生约400bp、240bp、130bp和420bp、180bp的RFLP片段，另外还有大小约为770bp、520bp和770bp、600bp的多余片段。用RFLP-PCR技术揭示了由于地理条件、气候、环境等因素造成我国的M.incognita和M.javanica群体跟巴西、意大利的M.incognita和M.javanica群体的rDNA-ITS存在异质性。HaeⅢ不能酶切象耳豆根结线虫(M.enterolobii)的ITS扩增产物，DraⅠ酶切后产生约400bp的片段，AluⅠ酶切后产生约600bp、420bp和180bp的3个片段。这三种酶切结果还显示，仅靠一种限制性内切酶酶切片段的多态性分析(rDNA-ITS-RFLP)是不能将M.incognita和M.javanica两种根结线虫区分开来的。
MMrtgu4WchgurtfArt
在夏秋高温季节，采用35％威百亩水剂进行土壤熏蒸，用于防治日光温室番茄根
结线虫病。施用江苏利民化工有限公司生产的 35％威百亩（Metham sodium）水剂，
用药剂量为故／mu、4kg／mu、6kg／mu的条件下，对土壤中番茄根结线虫二龄幼虫的
防效可达80．乃％、88．m％、91．57％，番茄增产幅度分另达至5．99％、7．抡％、7．的％；
对照药剂沈阳农药厂生产的 3 5％威百亩水剂 4kg／mu用量下对二龄幼虫的防效为
80．30％，番茄的增产率为5石6％。药效试验研究表明，威百亩土壤熏蒸剂对秋季日光
温室番茄南方根结线虫有较好的控制作用，并且可以增加番茄产量，是值得在生产上
大面积推广应用的杀线虫剂。
A survey of the plant parasitic nematodes on vegetables was carried out in Lanzhou, Honggu, Tianshui district of Gansu Province,North-west of China ,from March to August, 2002. More than 120 soil samples were collected around the rhizosphere of tomato, Pepper, eggplant, cucumber, broccoli, turnip, bean, celery. 25 species belonging to 15 genera were identificated, including Tylenchorhynchus clarus, T.mashhoodi, T.aerolatus; Merlinius brevidens, M.communium-, Pratylenchus neglectus, P.hexincisus, P.scribneri; Filenchus cylindricollis, F.vulgaris, F.ditissimus , Aphelenchus avenae , A.solani ? Helicotylenchus dihystera, H.tropitw, Psilenchus hilarulus-, Macroposthonia xenoplox-, Boleodorus simiiis-, Meloidogyne spp., Malenchus spp., Miculenchus spp., Aphelenchoides spp., Bursaphelenchus spp., Aphelenchoides spp. and Ditylenchus spp.. Among them, many genera and species have not been reported in Gansu Province so far. Several species, such as T.clarus, T.mashhoodi, T.aerolatus, M.brevidens, P.neglectus, P.hexincisus and P.scribneri are the most common nematodes species on vegetables in this study. M. brevidens was frequently recovered with relatively high population density around the rhizospher of eggplant and pepper in Diaochang of Arming district, Lanzhou, it had seasonal dynamics following the host nutrition etc.
rDNA-ITS RFLP was used for identification and characterisation of fifteen population of Meloidogyne on broccoli, tomato from Shunyi of Beijing; lettuce, peanut, weed from Jianyang of Sichuan; banana from Manning of Guangxi; tobacco from Junan of Shandong; tobacco from Luoyang of Henan; tomato, enterolobius, pepper, guava from Danzhou of Hainan; graudiflorum from Anhui; tomato from Belgium. The ITS regions were amplified using F195 and V5367 primers and yielded a single fragment of about 770bp for all samples. No PCR products were obtained in the negetive control. A total 10 scored fragments were obtained with 3 restriction enzymes digestion. Intraspecific polymorphism was revealed by Haelll Dral and AM digestion. The Haelll digested ITS products of M.incognita and M.javanica ,yielded three fragments having lengths approximately 370bp 220bp 180bp; The Dral digestion yielded three fragments having lengths approximately 400bp 240bp 130bp and the AM digestion yielded two fragments having lengths approximately 420bp 180bp. At the same time, additional bands were also obtained, the sizes of them were about 770bp 520bp and 770bp 600bp by Haelll and Dral degistion, respectively. This showed heterogenecity existed in the ITS regions of
M. incognita and M.javanica from China. Haelll did not digest ITS product of M.enterolobii , one brand about 400bp was obtained by the Dral digestion and three fragments having lengths approximately 600bp 420bp 180bp were obtained by the Alul digestion. The results showed that none of three enzymes differentiated M. incognita and M.javanica from each other.
Soil fumigation with Metham sodium gave better effects to root-knot nematodes disease on tomato. 35% Metham sodium ,which was produced by Liming Chemical-industry Ltd. Jiangsu province. When application dosage were 2kg/mu 4kg/mu 6kg/mu, it decreased population of second-stage juveniles of Meloidogyne incognita up to 80.76% 88.10% 91.57%, compard with untreatment, respectively, it increased tomato yield 5.99% 7.29% 7.46%, respectively. Comparing nematicide-35% Metham sodium, which was produced by Agrichemical Ltd. Shenyang, When application dosage was 4kg/mu, its Controlling effect to second-stage juveniles of M.incognita on tomato was 80.30%, compared with untreatment, it increased tomato yield 5.66%. The experiment results showed that 35% Metham sodium was a effective soil fumigation for controlling root-knot nematodes.
用PCR技术从北京顺义甘蓝、番茄；四川简阳莴苣、花生、杂草；广西南宁香蕉；山东莒南烟草；河南洛阳烟草、海南儋州番茄、象耳豆、胡椒、番石榴；安徽桔梗；比利时番茄等15个根结线虫群体中分别扩增出一条rDNA-ITS的片段，大小约770bp。用3种限制性内切酶HaeⅢ、DraⅠ、AluⅠ分别酶切M.incognita群体和M.javanica群体的ITS产物，HaeⅢ酶切后产生约370bp、220bp、180bp片段；DraⅠ和AluⅠ酶切后分别产生约400bp、240bp、130bp和420bp、180bp的RFLP片段，另外还有大小约为770bp、520bp和770bp、600bp的多余片段。用RFLP-PCR技术揭示了由于地理条件、气候、环境等因素造成我国的M.incognita和M.javanica群体跟巴西、意大利的M.incognita和M.javanica群体的rDNA-ITS存在异质性。HaeⅢ不能酶切象耳豆根结线虫(M.enterolobii)的ITS扩增产物，DraⅠ酶切后产生约400bp的片段，AluⅠ酶切后产生约600bp、420bp和180bp的3个片段。这三种酶切结果还显示，仅靠一种限制性内切酶酶切片段的多态性分析(rDNA-ITS-RFLP)是不能将M.incognita和M.javanica两种根结线虫区分开来的。
MMrtgu4WchgurtfArt
在夏秋高温季节，采用35％威百亩水剂进行土壤熏蒸，用于防治日光温室番茄根
结线虫病。施用江苏利民化工有限公司生产的 35％威百亩（Metham sodium）水剂，
用药剂量为故／mu、4kg／mu、6kg／mu的条件下，对土壤中番茄根结线虫二龄幼虫的
防效可达80．乃％、88．m％、91．57％，番茄增产幅度分另达至5．99％、7．抡％、7．的％；
对照药剂沈阳农药厂生产的 3 5％威百亩水剂 4kg／mu用量下对二龄幼虫的防效为
80．30％，番茄的增产率为5石6％。药效试验研究表明，威百亩土壤熏蒸剂对秋季日光
温室番茄南方根结线虫有较好的控制作用，并且可以增加番茄产量，是值得在生产上
大面积推广应用的杀线虫剂。
A survey of the plant parasitic nematodes on vegetables was carried out in Lanzhou, Honggu, Tianshui district of Gansu Province,North-west of China ,from March to August, 2002. More than 120 soil samples were collected around the rhizosphere of tomato, Pepper, eggplant, cucumber, broccoli, turnip, bean, celery. 25 species belonging to 15 genera were identificated, including Tylenchorhynchus clarus, T.mashhoodi, T.aerolatus; Merlinius brevidens, M.communium-, Pratylenchus neglectus, P.hexincisus, P.scribneri; Filenchus cylindricollis, F.vulgaris, F.ditissimus , Aphelenchus avenae , A.solani ? Helicotylenchus dihystera, H.tropitw, Psilenchus hilarulus-, Macroposthonia xenoplox-, Boleodorus simiiis-, Meloidogyne spp., Malenchus spp., Miculenchus spp., Aphelenchoides spp., Bursaphelenchus spp., Aphelenchoides spp. and Ditylenchus spp.. Among them, many genera and species have not been reported in Gansu Province so far. Several species, such as T.clarus, T.mashhoodi, T.aerolatus, M.brevidens, P.neglectus, P.hexincisus and P.scribneri are the most common nematodes species on vegetables in this study. M. brevidens was frequently recovered with relatively high population density around the rhizospher of eggplant and pepper in Diaochang of Arming district, Lanzhou, it had seasonal dynamics following the host nutrition etc.
rDNA-ITS RFLP was used for identification and characterisation of fifteen population of Meloidogyne on broccoli, tomato from Shunyi of Beijing; lettuce, peanut, weed from Jianyang of Sichuan; banana from Manning of Guangxi; tobacco from Junan of Shandong; tobacco from Luoyang of Henan; tomato, enterolobius, pepper, guava from Danzhou of Hainan; graudiflorum from Anhui; tomato from Belgium. The ITS regions were amplified using F195 and V5367 primers and yielded a single fragment of about 770bp for all samples. No PCR products were obtained in the negetive control. A total 10 scored fragments were obtained with 3 restriction enzymes digestion. Intraspecific polymorphism was revealed by Haelll Dral and AM digestion. The Haelll digested ITS products of M.incognita and M.javanica ,yielded three fragments having lengths approximately 370bp 220bp 180bp; The Dral digestion yielded three fragments having lengths approximately 400bp 240bp 130bp and the AM digestion yielded two fragments having lengths approximately 420bp 180bp. At the same time, additional bands were also obtained, the sizes of them were about 770bp 520bp and 770bp 600bp by Haelll and Dral degistion, respectively. This showed heterogenecity existed in the ITS regions of
M. incognita and M.javanica from China. Haelll did not digest ITS product of M.enterolobii , one brand about 400bp was obtained by the Dral digestion and three fragments having lengths approximately 600bp 420bp 180bp were obtained by the Alul digestion. The results showed that none of three enzymes differentiated M. incognita and M.javanica from each other.
Soil fumigation with Metham sodium gave better effects to root-knot nematodes disease on tomato. 35% Metham sodium ,which was produced by Liming Chemical-industry Ltd. Jiangsu province. When application dosage were 2kg/mu 4kg/mu 6kg/mu, it decreased population of second-stage juveniles of Meloidogyne incognita up to 80.76% 88.10% 91.57%, compard with untreatment, respectively, it increased tomato yield 5.99% 7.29% 7.46%, respectively. Comparing nematicide-35% Metham sodium, which was produced by Agrichemical Ltd. Shenyang, When application dosage was 4kg/mu, its Controlling effect to second-stage juveniles of M.incognita on tomato was 80.30%, compared with untreatment, it increased tomato yield 5.66%. The experiment results showed that 35% Metham sodium was a effective soil fumigation for controlling root-knot nematodes.
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