淡紫拟青霉异核现象、遗传多样性及不同菌株对根结线虫卵寄生率差异的比较研究
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摘要
植物线虫对许多经济作物造成严重的危害，可导致作物大幅减产。对于植物线虫的防治，历来以化学药剂为主，而化学药剂带来的负面影响，使得生物防治的研究成为热点。淡紫拟青霉(Paecilomyces lilacinus(Thom．)Samson)属于内寄生性真菌，是一些植物寄生线虫的重要天敌，能够寄生于卵，也能侵染幼虫和雌虫，从而影响正常的生长发育。利用分子生物学手段可以对形态相似的菌株进行分类，这不仅有助于了解种内遗传变异与寄主种类和地理来源相关性，而且可以解释致病性与遗传特征的关系。真菌的营养亲和群中产生的异核现象是菌株遗传变异的重要途径之一，对异核体菌株的筛选使制备高毒力生防菌成为可能。
本研究分为三部分：利用淡紫拟青霉不同分离株间自然存在的菌落特征作遗传标记，观察种内异核现象；利用RAPD对来源于我国不同省份的21株淡紫拟青霉和3株拟青霉进行种内遗传多态性的分析；用13株淡紫拟青霉，三种不同的方法处理根结线虫卵，从而筛选高致病力的菌株。其结果如下：
1．淡紫拟青霉不同分离株单孢子后代的异核现象。用来自福建、江苏、安徽的四个菌株(19、20、459、641)的单孢子培养后代，在蛋白胨马铃薯葡萄糖琼脂(PPDA)+1％活性炭培养基上进行配接试验，仅在19×459两菌株间能形成异核体，其频率为12.25％。在配接试验中两亲和菌落交界处长出致密的菌丝簇组成的实线可推测为异核体。从来自菌丝簇组线的每个单菌丝尖端培养物中分离出30个以上的单孢子，并分别移接到PDA平板上。将来源于单孢子的菌落与两亲本菌落进行比较，有三个菌丝尖端培养物(459-8×19-2，459-7×19-10，459-6×19-1)重现了两亲本类型或出现了新的菌落类型，从而异核现象得到证实。
2．对分离自根结线虫不同地理来源的21株淡紫拟青霉和3株拟青霉进行RAPD分析，从RAPD扩增结果可知淡紫拟青霉种内具有丰富的遗传多样型。RAPD指纹图谱能有效地分辨不同菌株的基因型。聚类分析结果表明淡紫拟青霉种内遗传变异大于淡紫拟青霉与桃色拟青霉和粉拟青霉得种间差异，菌株间差异和地理来源无相关性。
3．利用采自不同地域的淡紫拟青霉的13个菌株，分别以其孢子液处理南方根结线虫分散卵粒、菌丝处理分散卵粒、孢子液处理卵囊。被试所有菌株，同一菌株三种不同
河北师范大学硕士学位论文
方法对根结线虫卵的寄生率相比，抱子液处理卵囊均明显高于其他两种处理。不同菌株
对南方根结线虫卵的寄生率有很大的差异。三种处理方法结果显示618号菌株寄生率均
为最高。菌株对卵的寄生率与菌株地理来源无明显相关性，与基因型相关。
Plant parasitic nematodes are one of the important limiting factors in crop production throughout the world. Using chemical agents and nematicides cause many negative effect, while biocontrol can overcome these disadvantages. Paecilomyces lilacinus is one of nematophagous fungi which has been examined over the past two decades for potential use as agent for the control of plant pathogenic nematodes in agriculture. The facultative egg parasite is sometimes also able to infect larval and adult female nematodes, but it is most aggressive against eggs. Molecular tools can be effectively applied to discriminate biocontrol strains and assisted the selection of novel isolates for product development, which is also helpful to finding out the relationship between genetic variation and hosts and geographic distribution.
This research include three parts. The first, heterokaryosis were tested between two monoconidial progenies from different isolates of Paecilomyces lilacunus. The second, 20 strains of Paecilomyces lilacinus from different geographic origins and other 4 species of Paecilomyces were studied by random amplified polymorphic DNA(RAPD). The third, thirteen isolates of Paecilomyces lilacinus originating from difference areas was used to assess their parasitism on eggs of Meloidogyne incognita by three treatments. The results are as follows.
1. Heterokaryosis were tested between two monoconidial progenies from different isolates of Paecilomyces lilacinus. Heterokaryotic hyphae appeared as a solid line of white mycelial tufts where contact occurred between colonies of compatible isolates when they were paired on a modified peptone potato dextrose agar add medium containing 1% active carbon. A small mycelial fragment from the tufted zone was transferred to Petri dishes containing water agar and hyphal tip isolates were obtained from the resulting colonies.Thirty or more monoconidial isolates were further obtained from each hyphal tip
isolate and plated on PDA in Petri dishes. The resulting cultures were compared with the two parental isolates that produced the tufted reaction. Heterokaryosis was demonstrated when different monoconidial isolates from hyphal tip cultures 459-8X 19-2, 459-7X 19-10, 459-6 X 19-1 were representative of both parental types and/or when new colony morphologies were recovered. One of 6 different combinations involving 4 different field isolates produced heterokaryons. Heterokaryons were formed at a frekuency of 12.25% between the monoconidial progeny of isolates 459 and 19.
2. Tweety-one strains of Paecilomyces lilacinus from different geographic origins and other three species of Paecilomyces were studied by random amplified polymorphic DNA(RAPD). Distinct genetic diversity among 20 strains of Paecilomyces lilacinus were found. DNA fringerprinting with RAPD-PCR provides high resolution of genotype distribution. The cluster analysis showed that the genetic variations among different strains of Paecilomyces lilacinus are very distinct, intra-specific variation of P.lilacinus is larger the inter-specific variation between Paecilomyces farinosus and Paecilomyces fumosorseus. The results also indicated that genetic variations of different strians of Paecilomyces lilacinus are not related to geographic distributions.
3. Thirteen isolates of Paecilomyces lilacinus originating from difference areas was used to assess their parasitism on eggs of Meloidogyne incognita by three treatments including individual eggs treated by spore suspension, individual eggs treated by fungus hypha and egg masses treated by spore suspension respectively. The results showed that egg parasitic rate differed significantly among 13 isolates and parasitic rate of the same one of all isolates differed in 3 treatments, in which parasitic rate of egg masses treated by spore suspension was more higher than that in other two treatments. In the three treatments, parasitic rate of isolate 618 among 13 isolates on egg masses of Meloidogyne incognita was the highest. Parasitic rates of different isolates are related to genoty
本研究分为三部分：利用淡紫拟青霉不同分离株间自然存在的菌落特征作遗传标记，观察种内异核现象；利用RAPD对来源于我国不同省份的21株淡紫拟青霉和3株拟青霉进行种内遗传多态性的分析；用13株淡紫拟青霉，三种不同的方法处理根结线虫卵，从而筛选高致病力的菌株。其结果如下：
1．淡紫拟青霉不同分离株单孢子后代的异核现象。用来自福建、江苏、安徽的四个菌株(19、20、459、641)的单孢子培养后代，在蛋白胨马铃薯葡萄糖琼脂(PPDA)+1％活性炭培养基上进行配接试验，仅在19×459两菌株间能形成异核体，其频率为12.25％。在配接试验中两亲和菌落交界处长出致密的菌丝簇组成的实线可推测为异核体。从来自菌丝簇组线的每个单菌丝尖端培养物中分离出30个以上的单孢子，并分别移接到PDA平板上。将来源于单孢子的菌落与两亲本菌落进行比较，有三个菌丝尖端培养物(459-8×19-2，459-7×19-10，459-6×19-1)重现了两亲本类型或出现了新的菌落类型，从而异核现象得到证实。
2．对分离自根结线虫不同地理来源的21株淡紫拟青霉和3株拟青霉进行RAPD分析，从RAPD扩增结果可知淡紫拟青霉种内具有丰富的遗传多样型。RAPD指纹图谱能有效地分辨不同菌株的基因型。聚类分析结果表明淡紫拟青霉种内遗传变异大于淡紫拟青霉与桃色拟青霉和粉拟青霉得种间差异，菌株间差异和地理来源无相关性。
3．利用采自不同地域的淡紫拟青霉的13个菌株，分别以其孢子液处理南方根结线虫分散卵粒、菌丝处理分散卵粒、孢子液处理卵囊。被试所有菌株，同一菌株三种不同
河北师范大学硕士学位论文
方法对根结线虫卵的寄生率相比，抱子液处理卵囊均明显高于其他两种处理。不同菌株
对南方根结线虫卵的寄生率有很大的差异。三种处理方法结果显示618号菌株寄生率均
为最高。菌株对卵的寄生率与菌株地理来源无明显相关性，与基因型相关。
Plant parasitic nematodes are one of the important limiting factors in crop production throughout the world. Using chemical agents and nematicides cause many negative effect, while biocontrol can overcome these disadvantages. Paecilomyces lilacinus is one of nematophagous fungi which has been examined over the past two decades for potential use as agent for the control of plant pathogenic nematodes in agriculture. The facultative egg parasite is sometimes also able to infect larval and adult female nematodes, but it is most aggressive against eggs. Molecular tools can be effectively applied to discriminate biocontrol strains and assisted the selection of novel isolates for product development, which is also helpful to finding out the relationship between genetic variation and hosts and geographic distribution.
This research include three parts. The first, heterokaryosis were tested between two monoconidial progenies from different isolates of Paecilomyces lilacunus. The second, 20 strains of Paecilomyces lilacinus from different geographic origins and other 4 species of Paecilomyces were studied by random amplified polymorphic DNA(RAPD). The third, thirteen isolates of Paecilomyces lilacinus originating from difference areas was used to assess their parasitism on eggs of Meloidogyne incognita by three treatments. The results are as follows.
1. Heterokaryosis were tested between two monoconidial progenies from different isolates of Paecilomyces lilacinus. Heterokaryotic hyphae appeared as a solid line of white mycelial tufts where contact occurred between colonies of compatible isolates when they were paired on a modified peptone potato dextrose agar add medium containing 1% active carbon. A small mycelial fragment from the tufted zone was transferred to Petri dishes containing water agar and hyphal tip isolates were obtained from the resulting colonies.Thirty or more monoconidial isolates were further obtained from each hyphal tip
isolate and plated on PDA in Petri dishes. The resulting cultures were compared with the two parental isolates that produced the tufted reaction. Heterokaryosis was demonstrated when different monoconidial isolates from hyphal tip cultures 459-8X 19-2, 459-7X 19-10, 459-6 X 19-1 were representative of both parental types and/or when new colony morphologies were recovered. One of 6 different combinations involving 4 different field isolates produced heterokaryons. Heterokaryons were formed at a frekuency of 12.25% between the monoconidial progeny of isolates 459 and 19.
2. Tweety-one strains of Paecilomyces lilacinus from different geographic origins and other three species of Paecilomyces were studied by random amplified polymorphic DNA(RAPD). Distinct genetic diversity among 20 strains of Paecilomyces lilacinus were found. DNA fringerprinting with RAPD-PCR provides high resolution of genotype distribution. The cluster analysis showed that the genetic variations among different strains of Paecilomyces lilacinus are very distinct, intra-specific variation of P.lilacinus is larger the inter-specific variation between Paecilomyces farinosus and Paecilomyces fumosorseus. The results also indicated that genetic variations of different strians of Paecilomyces lilacinus are not related to geographic distributions.
3. Thirteen isolates of Paecilomyces lilacinus originating from difference areas was used to assess their parasitism on eggs of Meloidogyne incognita by three treatments including individual eggs treated by spore suspension, individual eggs treated by fungus hypha and egg masses treated by spore suspension respectively. The results showed that egg parasitic rate differed significantly among 13 isolates and parasitic rate of the same one of all isolates differed in 3 treatments, in which parasitic rate of egg masses treated by spore suspension was more higher than that in other two treatments. In the three treatments, parasitic rate of isolate 618 among 13 isolates on egg masses of Meloidogyne incognita was the highest. Parasitic rates of different isolates are related to genoty
引文
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Joaquim T. R. and R. C. Rowe. Reasssesment of vegetative compatibility relationship among strains of Ⅴ. erticillium dahliae using nitrate～nonutilizing mutants. Phytopathology 1990,80:1160～1166.
Khan H. U.; A. Riaz, S. M. Khan. Evaluation of culture filtrates of different fungi on the hatching of Meloidogyne incognita. Pakistan Journal of Phytopathology. 2001, 13(1), 58-60
Khan M. R., B. K. Goswami. Evaluation of Paecilomyces lilacinus isolate 6 against Meloidogyne incognita infecting tomato. International Journal of Nematology. 2002, 12(1): 111-114.
Kim W. K., W. Mauthe, G. Hausner. Isolation of high molecular weight DNA and double stranded RNAs from fungi. Can J Bot,1990,68: 1 898-902
Kohn, L. M., E. Stasovski, I. Carbone et al.. Mycelial compatibility and molecular markers iden tify gentic variability in field populations of Sclrrotina sclerotiorum, Phytopathology, 1991, 81: 480～485
Leal S. C. M, D. J. Bertioli, T.M.Butt et al.. Amplification and restriction endonuclease digestion of the Pr1 gene for the detection and charactization of Metarhizium strains. Mycol Res, 1997,101:257-265.
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Maurer P., Y. Couteaudier, P. A. Girard et al.. Genetic of Beauveria bassianaand relatedness to host insect range. Mycol Res, 1997, 101(2): 159～164
Meijer G., B. Megnegneau, E. G. A. Linders. Varriability for isozyme,vegetative compatibility and RAPD markers in natural population of Phomopsis subordinaria. Mycoll.Res., 1994,98:167-276
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N. vijaya, et al.. Effect of iocontrol agents and Furadon 3G on the juvenile mortality of root knot nematode Meloidogyne incognita. Acta Agriculturae Zhejiangensis, 2002, 14(3): 159-162.
Neuveglis C, Y. Brygoo, B. Vercambre, et al..Comparative analysis of molecular and biology characteristics of strains of Beavueria brongniartii isolated from insects. Mycol Res,1994,98:322～328.
Pankaj, H. K. Sharma, S. D. Mishra, et al.. Integrated management of the root-knot nematode, Meloidogyne incognita in spinach(Spinacia oleracea L.). Indian Journal of Nematology. 2001, 31(2): 165-166.
Paris,S.. Heterokaryosis in Beauveria tenella.Mycopathologia, 1977,61(2):67-76.
Proffer T. J. and J. H. Hart. Vegetative compatibility groups in Leucoytospora kunzei. Phytopathology 1988,78:256-260
Proffer T. J. and F. M. Shokes. Evidence for homothallism and vegetative compativility in Southern Diaporthe phaseolorum. Can.J.Bot. 1986,64:2197-2200
Puhalla J. E., M. Hummel. Vegetative compatibility groups within Verticillium dahliae. Phytopathology, 1983,73:1305-1308
Puhalla J. H.. Classification of strains of Fusarium oxysporum on the base of vegetative compatibility. Gan.J.Bot.1985,63:179-185.
Raeder, U. and P. Broda. Rapid preparation of DNA from filamentous fungi. Lett.Appl.
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Riba, G.. Recombination after heterokaryosis in the entomogenous fungi Paecilomyces fumosorseus. Entomophaga, 1978, 23(4):417-421.
Sidhu G. S.. Genetics of Gibberella fujikuroi V Ⅱ.vegetative compatibility groups. Gan.J.bot. 1986,64:117-121.
Sosamma V K, Koshy P K. Biological control of Meloidogyne incognita on black pepper by Pasteuria penetrans and Paecilomyces lilacinus. Journal of Plantation Crops, 1997, 25(1), 72-76.
St Leger R. J., L. L. Alee, B. May, et al. World-wide distribution of genetic variation among isolates of Beauveria spp. Mycol. Res., 1992a, 96(12): 1007-1015.
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Tigano-Milani, M., R. J. Honeycutt, L. Lacey, et al.. Genetic variability of Paecilomyces fumosoroseus strains revealed by molecular markers. J. Inv. Pathol., 1995, 65:274～282.
Tigano-Milani, M., R. Samson, B. W. S. Sobral. DNA markers for differentiating isolates of Paecilomyces lilacinus. Microbiology 1995.141:239～245.
Tinline R.D. and C.Noviello. Heterokaryosis in the entomogenous Metarrhizum anisopliae. Mycologia, 1971,63:701-712.
Vest,G. and N.A.Anderson. Studies on heterokaryosis and virulence of Rhizoctonia solani isolate from flax. Phytopathology, 1968,58:802-807.
Vyas R.V., N. B. Patel, D. J. Patel. Management of root-knot nematodes in banana. Indian Journal of Nematology. 2001, 31(1):86-87.
Welsh J. and M. McClelland. Fingerprinting genomes using PCR with arbitrary primers. Nucl.Acids
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Whithey H. S. and J. R. Parmeter. Synthesis of heterokaryons in Rhizoctonia solani kuhn. Can.J.Bot. 1963,41:879～886.
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Jatala P.. Biological control of plant-parasific nematodes. Ann. Rev. Phytopathol.,1986,24: 435-439.
Jean C. C., C. Djian, L. Pijarowski. Study of the nematocidal properties of the culture filtrate of the nematophagous fugus Paecilomyces lilacinus. Revus Nematol, 1989,12(4):331～336.
Joaquim T. R. and R. C. Rowe. Reasssesment of vegetative compatibility relationship among strains of Ⅴ. erticillium dahliae using nitrate～nonutilizing mutants. Phytopathology 1990,80:1160～1166.
Khan H. U.; A. Riaz, S. M. Khan. Evaluation of culture filtrates of different fungi on the hatching of Meloidogyne incognita. Pakistan Journal of Phytopathology. 2001, 13(1), 58-60
Khan M. R., B. K. Goswami. Evaluation of Paecilomyces lilacinus isolate 6 against Meloidogyne incognita infecting tomato. International Journal of Nematology. 2002, 12(1): 111-114.
Kim W. K., W. Mauthe, G. Hausner. Isolation of high molecular weight DNA and double stranded RNAs from fungi. Can J Bot,1990,68: 1 898-902
Kohn, L. M., E. Stasovski, I. Carbone et al.. Mycelial compatibility and molecular markers iden tify gentic variability in field populations of Sclrrotina sclerotiorum, Phytopathology, 1991, 81: 480～485
Leal S. C. M, D. J. Bertioli, T.M.Butt et al.. Amplification and restriction endonuclease digestion of the Pr1 gene for the detection and charactization of Metarhizium strains. Mycol Res, 1997,101:257-265.
Leal S.C.M., Bertioli D J, Butt TM et al., 1994 Charaterization of isolates of the Entomopathogenic fungus Metarhizium anisopliae by RAPD-PCR.Mycol Res, 98:1077-1081.
Leal S. C. M., D. J. Bertioli, T. M. Butt et al.. Characterization of isolates of the entomopathogenic fungus Metarhizium anisopliae by RAPD—PCR. Mycol.Res,1994.98(9):1077-1081.
Maurer P., Y. Couteaudier, P. A. Girard et al.. Genetic of Beauveria bassianaand relatedness to host insect range. Mycol Res, 1997, 101(2): 159～164
Meijer G., B. Megnegneau, E. G. A. Linders. Varriability for isozyme,vegetative compatibility and RAPD markers in natural population of Phomopsis subordinaria. Mycoll.Res., 1994,98:167-276
Murry H. G., W. F. Thompson. Rapid extraction of high molecular weight DNA. Nucl Acids Res,1980,8:4 321—4 326
N. vijaya, et al.. Effect of iocontrol agents and Furadon 3G on the juvenile mortality of root knot nematode Meloidogyne incognita. Acta Agriculturae Zhejiangensis, 2002, 14(3): 159-162.
Neuveglis C, Y. Brygoo, B. Vercambre, et al..Comparative analysis of molecular and biology characteristics of strains of Beavueria brongniartii isolated from insects. Mycol Res,1994,98:322～328.
Pankaj, H. K. Sharma, S. D. Mishra, et al.. Integrated management of the root-knot nematode, Meloidogyne incognita in spinach(Spinacia oleracea L.). Indian Journal of Nematology. 2001, 31(2): 165-166.
Paris,S.. Heterokaryosis in Beauveria tenella.Mycopathologia, 1977,61(2):67-76.
Proffer T. J. and J. H. Hart. Vegetative compatibility groups in Leucoytospora kunzei. Phytopathology 1988,78:256-260
Proffer T. J. and F. M. Shokes. Evidence for homothallism and vegetative compativility in Southern Diaporthe phaseolorum. Can.J.Bot. 1986,64:2197-2200
Puhalla J. E., M. Hummel. Vegetative compatibility groups within Verticillium dahliae. Phytopathology, 1983,73:1305-1308
Puhalla J. H.. Classification of strains of Fusarium oxysporum on the base of vegetative compatibility. Gan.J.Bot.1985,63:179-185.
Raeder, U. and P. Broda. Rapid preparation of DNA from filamentous fungi. Lett.Appl.
Microbiol., 1985,1:17-20.
Riba, G.. Recombination after heterokaryosis in the entomogenous fungi Paecilomyces fumosorseus. Entomophaga, 1978, 23(4):417-421.
Sidhu G. S.. Genetics of Gibberella fujikuroi V Ⅱ.vegetative compatibility groups. Gan.J.bot. 1986,64:117-121.
Sosamma V K, Koshy P K. Biological control of Meloidogyne incognita on black pepper by Pasteuria penetrans and Paecilomyces lilacinus. Journal of Plantation Crops, 1997, 25(1), 72-76.
St Leger R. J., L. L. Alee, B. May, et al. World-wide distribution of genetic variation among isolates of Beauveria spp. Mycol. Res., 1992a, 96(12): 1007-1015.
St Leger R. J., B. May, L. L. Allee et al.. Genetic differences in allozymes and in formation of infection structures among isolates of entomopathogenic fungus Metarhiziurn anisopliae. J Inverteber Pathol,1992b,60:89-101.
Stephan Z. A., M. S. Hassan, I. K. Hassoon. Efficacy of Fenamiphos, Trichoderma harzianum, Paecilomyces lilacinus and some organic soil amendments in the control of root-knot and root-rot/wilt disease complex of eggplant. Arab Journal of Plant Protection. 2002, 20(1), 1-5.
Taylor B., A. Powell. Isolation of plant DNA and RNA. Focus, 1982,4:4—6
Tigano-milan M. S. et al..Isozyme characterization and Pathogenicity of Paecilomyces lilacinus and P. lilacinus to Diabrotica speciosa(Coleoptera, Chtysomelicdae) and Meloidogyne javanica (Nematoda,tylenchidae).Biological control, 1995, 5: 378—382.
Tigano-Milani, M., R. J. Honeycutt, L. Lacey, et al.. Genetic variability of Paecilomyces fumosoroseus strains revealed by molecular markers. J. Inv. Pathol., 1995, 65:274～282.
Tigano-Milani, M., R. Samson, B. W. S. Sobral. DNA markers for differentiating isolates of Paecilomyces lilacinus. Microbiology 1995.141:239～245.
Tinline R.D. and C.Noviello. Heterokaryosis in the entomogenous Metarrhizum anisopliae. Mycologia, 1971,63:701-712.
Vest,G. and N.A.Anderson. Studies on heterokaryosis and virulence of Rhizoctonia solani isolate from flax. Phytopathology, 1968,58:802-807.
Vyas R.V., N. B. Patel, D. J. Patel. Management of root-knot nematodes in banana. Indian Journal of Nematology. 2001, 31(1):86-87.
Welsh J. and M. McClelland. Fingerprinting genomes using PCR with arbitrary primers. Nucl.Acids
Res. 1990.18(24):7213—7218.
Whithey H. S. and J. R. Parmeter. Synthesis of heterokaryons in Rhizoctonia solani kuhn. Can.J.Bot. 1963,41:879～886.
Williams,J. G. K., A. R. Kubelik, K. J. Livak.et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl.Acids Res. 1990.18(22):6531～6535.
Yurchenko L., I. A. Zakharov. Genetique et selection du chamipignon entomopathogene Beauveria bassiana(BALS)Vuill Etude de Theterocaryose. Genetika, 1974,10:95-101.