【作者】 潘月敏；

【导师】 高智谋；

【作者基本信息】 安徽农业大学 ， 微生物学， 2008， 博士

【摘要】 串珠镰孢（Fusarium moniliforme Sheld）是寄主范围广、经济危害性大的重要植物病原真菌之一。本学位论文在对安徽、山东、江苏、湖北等省的棉花红腐病、水稻恶苗病、玉米穗腐病病原菌分离鉴定的基础上,对不同寄主来源的串珠镰孢的生物学特性、营养体亲和性、致病力分化、串珠镰孢培养滤液和毒素对棉花生长的影响、酯酶同工酶电泳以及群体遗传多样性等方面进行了系统研究,主要研究结果如下:1镰孢菌（串珠镰孢）研究进展综述对串珠镰孢的生物学特性（包括分类、分布、危害、寄主范围、形态特征等）、串珠镰孢毒素、镰孢菌营养体亲和性和分子标记技术在镰孢菌研究中的应用等方面的研究进展进行了综述。2不同寄主来源的串珠镰孢生物学特性的比较研究从安徽、山东、湖北和江苏等省采集病菌标样,通过分离培养纯化,根据形态特征与培养性状,参照Booth和Nelson的镰孢菌分类系统鉴定,获得107个串珠镰孢菌（Fusarium moniliforme）菌株。对来自不同地区、不同寄主来源的串珠镰孢菌株的菌落形态、生长速率和产孢量等性状进行了比较研究。结果表明,不同寄主的串珠镰孢菌株在菌落形态特别是色素方面、生长速率和产孢量方面存在显著差异。棉花菌株的平均生长速率最大,玉米菌株生长速率最小,水稻菌株生长速率居中,相同群体的不同菌株间生长速率有极显著差异;玉米菌株产孢量最大,棉花菌株产孢量最小,水稻菌株产孢量居中。方差分析显示,不同寄主菌株群体间产孢量存在显著差异,而同一寄主群体的不同菌株间产孢量均无显著差异,说明菌株产孢量大小主要与其寄主种类有关,而与地区来源关系不大。温度对3种不同寄主上串珠镰孢菌丝生长的影响趋势是一致的,10℃～30℃适合菌丝生长,最适生长温度在30℃左右,10℃以下和30℃以上菌丝生长明显减弱,5℃以下和40℃以上菌丝均不能正常生长;也即供试3种不同寄主来源的串珠镰孢的菌丝生长温度范围和最适温度是基本相同的。光照因子对不同寄主来源的串珠镰孢菌丝生长以及孢子形成的影响大致相同,各菌株在不同的光照条件下的菌落直径差异不显著,说明串珠镰孢菌丝生长对光照条件要求不严格。在10℃～40℃之间病菌的分生孢子均有萌发,在20℃～33℃之间孢子萌发率达到最大,在pH值为4.1～11.8之间均可以萌发,中性的环境中孢子的萌发率最高。3串珠镰孢生物学性状的遗传研究选择分离自棉花、玉米和水稻的串珠镰孢菌株Fm1、Fm19和Fm31为供试菌株,经单孢分离建立单分生孢子无性系。在实验室条件下,对来源于棉花、水稻、玉米的串珠镰孢的生物学性状在单分生孢子后代的遗传进行了研究。遗传测定结果表明,分离自棉花、玉米和水稻的串珠镰孢的菌落形态和生长速率在单分生孢子后代均可稳定遗传;产孢量性状遗传有两种情况:分离自棉花和水稻的串珠镰孢菌株Fm1和Fm31的产孢量性状在单分生孢子第1代（CG₁）和单分生孢子第2代（CG₂）均可稳定遗传;而分离自玉米的串珠镰孢菌株Fm19的产孢量性状在CG₁代发生变异。4串珠镰孢营养体亲和性研究选用107个采自安徽、山东、江苏、湖北等不同地区棉花、玉米、水稻的串珠镰孢（Fusarium moniliforme）菌株,在含KClO₃培养基上诱导筛选获得抗氯酸盐、不能还原利用硝酸盐的（nit）突变株1081株,在MM、NM、HM等3种不同氮源培养基上划分出nit A、nit B、nit C、nit D 4种突变类型,其中nit A出现频率最高,占总体76%;nit B和nit C其次,分别占12%和10%;nit D最少,占总体2%。采用nit突变体互补型配对技术,将供试菌株分别按地理来源和分离寄主进行配对培养,测得不同寄主群体内菌株的营养体亲和群（VCGs）数为56个,然后从每个VCG中随机抽取1个样本菌株,测定不同群体间菌株的VCG同一性,发现所抽取的56个菌株分属于54个VCGs,其中来自棉花的菌株Fm1、Fm2分别与来自玉米的菌株Fm19、Fm20属于同一VCG。按地理来源测定,4个群体共测得55个VCGs,其中来自山东的菌株Fm45和来自安徽的菌株Fm16发生亲和反应,属于同一VCG;107个菌株划分为54个VCGs。结果表明,串珠镰孢菌株群体内存在丰富的VCGs多样性。经多样性分析,不同地理来源的菌株平均1.9454个组成1个VCG,其P与Shannon-Wiener多样性指数分别为0.5140和1.0365;不同寄主上分离的菌株平均1.9107个形成1个VCG,P与Shannon-Wiener多样性指数分别为0.5234和0.9048。经t测验,两个群体Shannon-Wiener多样性指数H值间差异不显著（t=0.70小于t0.05=1.98）,说明两个群体的VCG多样性无显著差异,相同地区来源的菌株的遗传相似性与相同寄主来源的遗传相似性相当。5串珠镰孢nit突变体生物学性状及营养亲和性的遗传从串珠镰孢菌株Fm1（分离自棉花）、Fm19（分离自玉米）和Fm31（分离自水稻）的CG₁和CG₂代的240个单孢株中共诱变得到了1093个nit突变株。比较了各nit突变体与亲本菌株之间在菌落生长速率、产分生孢子能力和对棉苗致病性等生物学特性方面的变化。结果表明,nit突变体在PSA平板上的生长速率、产分生孢子能力和对棉苗的致病性等生物学性状与亲本菌株均没有显著性差异。因此,可用nit作为遗传标记研究串珠镰孢有关性状的遗传学。遗传测定结果表明,分离自棉花、玉米和水稻的串珠镰孢的nit突变性状在单分生孢子后代均具有较高的稳定性,每个菌株都具有自身亲和性。6串珠镰孢对棉苗致病力分化及其遗传研究采用菌丝块创伤接种法,分别测定了不同寄主来源的串珠镰孢对棉苗叶片的致病力。结果表明,所有供试菌株接种棉苗后均可引起发病,但不同菌株所致病斑的平均直径有显著差异,提示串珠镰孢菌株间对棉苗叶片的致病力存在明显的分化。总体来说,来自棉花的菌株对棉苗叶片的致病力较强,来自玉米和水稻的菌株对棉苗叶片的致病力较弱,但来自相同寄主的菌株间致病力也存在差异,菌株致病力差异与菌株地域来源无明显相关。以Fm1、Fm19和Fm31为亲本,采用菌丝块创伤接种法测定了串珠镰孢对棉苗致病力在单分生孢子后代的遗传,结果表明,串珠镰孢对棉苗的致病力在单分生孢子连续两代稳定遗传。7串珠镰孢培养滤液和毒素对棉花生长的影响探讨了串珠镰孢培养滤液对棉苗生长的影响。结果表明,不同菌株培养滤液对棉花胚根重量的影响有明显差异,大致可以分为3类:（1）在一定浓度范围内,培养滤液处理促进棉花种子胚根重量增加;随着培养滤液处理浓度的增加,胚根重量呈现先增高后降低的趋势,处理浓度为30%～50%时胚根重量达到最大,说明这类培养滤液中含有促进生长的物质;（2）不同浓度培养滤液处理种子,胚根重量波动较大,但总体趋势是胚根重量随着滤液处理浓度的增加而增加,100%培养滤液处理的棉花种子的胚根重量最大,说明培养滤液中含有促进生长的物质,且其含量在该物质的促生浓度范围内;（3）培养滤液处理对棉花种子胚根重量具有明显抑制作用,说明培养滤液中含有抑制生长的物质。在供试菌株中,以自安徽水稻上分离的菌株Fm31培养滤液促生效果最佳,自安徽棉花上分离的菌株Fm1培养滤液抑制作用最明显。测定了串珠镰孢毒素对棉花胚根的抑制作用和对棉苗的致萎性。结果表明,串珠镰孢毒素对棉花胚根生长具有明显的抑制作用,对棉苗具有显著的致萎作用;并且毒素对棉苗的致萎性,棉苗对毒素的反应,与棉苗发病症状相一致。8串珠镰孢酯酶同工酶电泳分析本文采用聚丙烯酰胺凝胶电泳测定了采自不同寄主的串珠镰孢菌株的酯酶同工酶酶谱,并对同工酶谱进行聚类分析,结果表明,所有供试菌株酯酶同工酶图谱都具有Rf=0.037这条酶带（共有酶带）,菌株间有较大的同源性;但不同菌株间酯酶同工酶图谱存在明显差异。根据酯酶酶谱和聚类分析可将供试菌株分为3种类型:类型1的菌株有4条主酶带,次酶带也最多,属于棉花菌株;类型2的菌株有3条主酶带,属于水稻菌株;类型3的菌株有2条主酶带,次酶带较少,属于玉米菌株。试验还发现,供试菌株的酯酶同工酶酶谱与菌株的致病力强弱有一定相关性,类型1的菌株（棉花菌株）致病力均较强。9棉株感染串珠镰孢后SOD等3种酶活性动态变化对棉苗感染不同寄主来源的串珠镰孢后体内PPO、POD、SOD 3种酶活性的变化进行了测定,结果表明,接种串珠镰孢后,棉苗叶片PPO、POD和SOD 3种酶活性均较对照升高,说明接种串珠镰孢后,病原菌能够诱导棉花体内3种酶活性提高;其中POD,SOD与对照相比活性升高明显,PPO的活性提高效果较小。因此,PPO、POD和SOD均是棉苗抗病性有关的酶,尤以POD、SOD活性与抗病反应密切相关。结果还表明,不同寄主来源的串珠镰孢菌株在诱导棉花体内3种酶活性提高的效果上有差异:3种寄主来源菌株中,以水稻菌株Fm31诱导PPO和SOD作用最强,玉米菌株Fm19诱导POD作用最强,而作为棉花本身病原菌的菌株Fm1对3种酶活性提高效果居中。10串珠镰孢遗传多样性的RAPD分析利用随机扩增多态性DNA（RAPD）技术,对不同寄主和地理来源的串珠镰孢菌株群体进行了遗传多样性分析,筛选到8个多态性随机引物,共产生了90条RAPD带,其中93.33%具有多态性;通过聚类分析,将供试菌株分为4个RAPD组。对不同来源串珠镰孢进行遗传多样性分析,筛选到7个多态性随机引物,共产生了71条RAPD带,其中94.37%具有多态性;通过聚类分析,将供试菌株分为3个RAPD组。上述结果显示,供试串珠镰孢菌株群体具有较丰富的遗传多样性。这一结果与串珠镰孢生物学性状、营养体亲和性及其遗传的研究结果相一致。本研究从分子水平上证明了造成不同串珠镰孢生物学性状差异的原因是不同基因表达的结果;本研究还明确了串珠镰孢菌株间的亲缘关系,为串珠镰孢的鉴定及种下分类提供了有价值的分子证据。更多还原

【Abstract】 Fusarium moniliforme Sheld is an economically important plant pathogenic fungus with a wide host range. Based on the isolation and identification of the causal organism of cotton red rot, rice banakae disease, maize ear rot in Anhui, Shandong, Jiangsu and Hubei, the biological characteristics, vegetative compatibility and pathogenicity differentiation, the effect of culture filtrate and crude toxin on cotton growth, esterase isozyme electrophoresis and DNA polymorphism were studied systematically in this dissertation. The main results were as follows.1 Review of research advances on Fusarium （F. moniliforme）A review of research advances in Fusarium （F. moniliforme） was presented. The advances in the study on the biological characteristics （including geographical distribution, host range, morphology, biology, ecology, etc）, the toxins of F. moniliforme, the vegetative compatibility and the application of molecular marker technology in the study on Fusarium are reviewed to provide a useful information for the integrated management of the diseases caused by the fungus and for the related research.2 Comparison of biological characteristics of Fusarium moniliforme isolates from different hostsMore than 200 isolates in Fusarium were isolated from the diseased tissues of cotton red rot, rice banakae disease and maize ear rot in Anhui, Shandong, Jiangsu and Hubei. According to the classification system of C. Booth and Nelson, 107 of the isolates were identified as F. moniliforme based on the morphology and culture characters. The biological characters including colony morphology, growth rates and conidium production of F. moniliforme isolates from different hosts and localities were studied by comparison. The results showed they were obviously different in colony morphology （especially in pigment production）, mycelial growth rates and conidial production. The average mycelium growth rate of the isolates from cotton was the largest, next the one from rice, and the one from maize the least. There was significant difference at 0.01 levels in the mycelial growth rates among the isolates from the same population. The conidial production of the isolate from maize was the largest, next the one from rice and the one from cotton the least. The variance analysis indicated that there was significant difference in the conidial production among the isolate populations from different hosts, but no significant difference among the isolates from the same population. It was suggested that the conidial production of F. moniliforme isolates was mainly related to the host kinds, but little to the localities. The hyphae could grow in 10℃～30℃, the optimum temperature was in the range of 25℃～30℃, and the temperature lower than 5℃or higher than 40℃was not fit for the mycelial growth. The conidia spore germination was between 10～40℃, the optimum temperature was in the range of 20℃～33℃. The conidia could germinate under the condition of pH 4.1～11.8, and the optimum pH for conidial germination was about 7.0.3 Biological characters and their inheritance of Fusarium moniliformeThe isolates Fm1, Fm19, and Fm31 from cotton, maize and rice respectively were selected as tested isolates and their asexual clones of single conidia were established. The inheritance of biological characters of F. moniliforme isolates from cotton, rice and maize plants was studied in vitro. The genetic tests showed that the colony morphology and the mycelial growth rate of F. moniliforme isolates from cotton, rice and maize plants could be inherited steadily in the single conidial progenies. However, there were two exhibitions in the inheritance of the conidial production. The conidial production of F. moniliforme isolates Fm1 and Fm31, from cotton and rice respectively, could be inherited steadily in the single conidial progenies, while the character of isolate Fm19 from maize displayed variation in the first single conidium generation （CG₁）.4 Studies on vegetative compatibility of Fusarium moniliformeThe vegetative compatibility groups （VCGs） of Fusarium moniliforme were tested by applying the nitrate nonutilizing （nit） mutant complementation technique to explore the population structure of the important pathogenic fungus. The 107 strains of F. moniliforme were isolated from cotton, maize and rice diseased tissues collected from Anhui, Shandong, Jiangsu and Hubei in China. The tested single-conidium strains were then transplanted on KPS plates to mutagenize nit mutants and 1081 nitrate-nonutilizing （nit） mutants were recovered from the isolates by transferring chlorate-resistant sectors from KPS plates to MM medium. The four mutants types were identified as nit A, nit B, nit C and nit D based on the growing situation of the mutants on MM, NM and HM, the three kinds of different nitrogen media. Among the four types, nit A was the most common one, with the rate of 76%. Next were nit B and nit C, with the rates of 12% and 10% respectively. The least one was nit D, with the rate of 2%.The nit mutants then were paired on MM plates to test the vegetative compatibility of all isolates, according to their geographic location and host kinds, respectively. Fifty-six different VCGs were identified totally from all three host-origin populations of the mutants. One sample was taken randomly from each VCG to determine the identity. The result showed isolates Fm1, Fm2 from cotton and isolates Fm19, Fm20 from maize belonged to the same VCG respectively, while the other strains did not. Similarly, 55 different VCGs were identified totally from all four locality-origin populations of the mutants. Only did isolate Fm45 from Shandong and isolate Fm16 from Anhui belong to the same VCG, while the other strains from different geographic origins belonged to different VCGs. The 107 tested isolates were divided into 54 VCGs, suggesting there was abundant diversity of VCGs in the population of Fusarium moniliforme isolates. The diversity analysis indicated that on the average, 1.9454 isolates from locality-origin population made up 1 VCG; the P and Shannon-Wiener diversity indices were 0.5140 and 1.0365, respectively. And 1.9107 isolates from host-origin population composed 1 VCG on an average; the P and Shannon-Wiener diversity indices were 0.5234 and 0.9048, respectively. The Shannon-Wiener diversity indices （H values） of the two populations were not significantly different from each other based on t test (t = 0.70, below t_0.05= 1.98). It was suggested that the VCG diversity of the two populations was not significantly different from each other, and that the genetic similarity among the isolates from the same locality-origin was equal to that from the same host-origin.5 Studies on the biological characters of nit mutants and the inheritance of vegetative compatibility in Fusarium moniliformeThe 240 single-conidium strains of F. moniliforme were isolated from Fm1 （from cotton）, Fml9 （from maize）, and Fm31 （from rice） to establish CG₁ and CG₂ populations. The tested single-conidium strains were then transplanted on KPS plates to mutagenize nit mutants and 1093 nitrate-nonutilizing （nit） mutants were recovered from the isolates by transferring chlorate-resistant sectors from KPS plates to MM medium. Some biological characters, such as growth rate, conidial production ability, and pathogenicity were compared between nit mutants and their parental isolates. Results showed that there were no significant differences in mycelial growth rate, conidial production ability and pathogenicity between nit mutants and their parental isolates. The genetic tests showed that the vegetative compatibility of F. moniliforme isolates from cotton, rice and maize plants could be inherited steadily in the single conidium progenies. Therefore, the nit character and vegetative compatibility can be used as genetic markers for the study of F. moniliforme. 6 Studies on differentiation of pathogenicity of Fusarium Moniliforme from different hosts to cotton and the inheritanceThe pathogenicity of the isolates of F. Moniliforme from different hosts to the leaves of cotton was investigated by applying mycelial block wound inoculation, respectively. The results showed that all of the tested isolates caused occurrence of red rot lesions on the leaves of cotton, but there was significant difference in the average diameters of the lesions caused by different isolates, suggesting that there was significant differentiation in pathogenicity of F. moniliforme to the leaves of cotton among isolates. Strongly pathogenic to the leaves of cotton, the isolated from cotton, maize and rice were weakly pathogenic to the leaves of cotton. However, there was difference in pathogenicity among the different isolates from the same host, and the pathogenicity difference was not obviously related to the localities of isolates. The pathogenicity was compared between nit mutants and their parental isolates. Results showed that there were no significant differences in pathogenicity between nit mutants and their parental isolates. The genetic tests showed that the pathogenicity of F. moniliforme isolates from cotton, rice and maize plants to cotton seeling leaves could be inherited steadily in the single conidium progenies.7 Studies on effects of culture filtrate and toxin of Fusarium moniliforme isolates on cotton growthEffects of culture filtrate of Fusarium moniliforme on growth of cotton radicles were tested. The results showed that there was obvious difference in the effect among different isolates. There were three types as follows: （1） The culture filtrate of this type of isolate（s） promoted growth of the radicles in a certain range of concentration. The weight of the radicles of the cotton seeds increased earlier and decreased later with the rise of the concentration of culture filtrate. The weight of the radicles got to the peak value when the concentration of culture filtrate was 30%～50%. The results indicated that the culture filtrate contains certain substance promoting growth. （2） The growth- promoting effect of the culture filtrate of this type was instable, but in general, the weight of the radicles increased with the rise of the concentration of culture filtrate. The weight of the radicles was the greatest when the concentration of culture filtrate was 100%. It was suggested that the culture filtrate contains also certain growth- promoting substance, of which the concentration was in the range of promoting growth. （3） The culture filtrate of this type of isolate（s） inhibited obviously growth of the radicles, suggesting the culture filtrate contains certain growth-inhibiting substance. Among the isolates tested, the culture filtrate of Fm31 isolated from rice in Anhui was the best for the promotion of plant growth, while the culture filtrate of Fm1 isolated from cotton in Anhui was the most evident for the inhibition of plant growth.The inhibition of the crude toxin of F. moniliforme agaist cotton radicle growth and the wilting effect on cotton seedlings were measured, respectively. The results showed that the crude toxin of F. moniliforme could obviously inhibit the growth of cotton radicles and caused the wilting of cotton seedlings. The wilting effect of the crude toxin was equal to the symptom of cotton red rot.8 Analysis of esterase isozymes in Fusarium moniliforme isolatesThe polyacrylamicide gel electrophoresis analysis of esterase （EST） isozymes of 10 isolates of F. morniliforme from cotton, rice and maize were carried out to compare their differences in EST isoenzyme zymogram. The results showed that EST isoenzyme patterns had the common band with Rf of 0.037, among all the tested strains. And there was greater genetic similarity among the isolates. But there was a significant difference in the patterns of EST isozyme among different strains. Based on EST isozyme and cluster analysis, those 10 strains were divided into 3 groups. There were 4 main isoenzyme bands and more minor isoenzyme bands in in EST zymogram of the strains in Group 1, which were from cotton. There were 3 main isoenzyme bands in in EST zymogram of the strains in Group 2, which were from rice. There were 2 main isoenzyme bands and a few minor isoenzyme bands in in EST zymogram of the strains in Group 3, which were from maize. It was also found that the pathogenicity difference was obviously related to esterase isozymes. Group 1 strains, cotton strain, have strong pathogenicity to cotton seedlings.9 Analysis of the dynamic activity of resistant enzymes in cotton plants inoculated with different Fusarium moniliforme isolatesThe activities of PPO, POD, and SOD in tested cotton plants inoculated with F. moniliforme isolates from different hosts were tested and analyzed in 10 days after inoculation respectively. The results indicated that PPO, POD and SOD activities in leaves all rose obviously compared to the control, which suggesting the isolates could induce the rising of the activities of the 3 enzymes in the plants by inoculating with them. Meanwhile, the activities of POD and SOD rose obviously after inoculation, while the increase of PPO activity was not significant. Therefore, PPO, POD and SOD all were related to the disease-resistance of cotton seedlings, especially POD and SOD with greater potential. The results also show that there was difference in the inducing effect of the activity of 3 enzymes among the strains from different host sources. Fm31 （rice strain） has strong ability of induction of PPO and SOD. Fm19 （maize strain） has strong ability of induction of POD. However, as an original pathogen, Fm1 （cotton strain） displayed a moderate ability of induction of the activity of 3 enzymes related to defence.10 RAPD-PCR analyses of genetic diversity in Fusarium moniliforme populationRandom amplified polymorphic DNA （RAPD） markers were used to assess genetic diversity among the strains of Fusarium moniliforme. A total of 18 strains isolated from different hosts were amplified with 8 RAPD primers. As the result, 90 RAPD fragments were obtained and 93.33 % were polymorphic found in the tested strains. 18 isolates tested were divided into 4 groups of RAPD and the genetic relationships among the strains were determined by hierarchical cluster method. Alike, RAPD markers were used to assess genetic diversity among the strains from different areas. 71 RAPD bands were obtained and 94.37 % were polymorphic in the tested strains. Eighteen strains tested were classified as 3 groups of RAPD and the genetic relationships among the strains were determined by cluster analysis. The results indicated that there was abundant genetic diversity in the population of F. moniliforme, which was consistent with the research results of biological characters, vegetative compatibility and their inheritance of the fungus. The study demonstrated that the differences in biological characters of different F. moniliforme isolates were resulted from the expression of different genotypes. The results elucidated the genetic relations among the strains and provided valuable molecular evidences for the idetifiction and classification of F. moniliforme.更多还原