烟草根黑腐病菌Thielaviopsis basicola致病力分化与遗传多样性研究
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摘要
烟草根黑腐病是世界性烟草病害,其病原菌为根串珠霉Thielaviopsis basicola(Berk.&Br.) Ferr.,该菌寄主范围广泛,可为害多种重要的农作物。关于该病菌形态及分子遗传多样性研究方面国内未见深入系统报道,为明确我国不同烟区烟草根黑腐病菌形态、致病力及分子遗传多样性,本论文开展了以下几方面研究：(1)对我国云南、甘肃、贵州、重庆和河南5个主要产烟省(市)烟草根黑腐病样品进行病原分离,按照柯赫氏法则对得到的病原菌进行致病性测定,结合其形态特征及rDNA-ITS分子方法鉴定其所属种。(2)测定了不同菌株菌丝生长和孢子萌发的影响因子。(3)对各地代表性菌株进行致病力差异测定,并比较了7个烟草主栽品种的室内抗性水平。(4)详细观测所有菌株在V8培养基上的菌落形态和厚垣孢子特征,参照Punja&Sun的培养方法和分群鉴定依据对其进行分群鉴定。(5)利用ISSR-PCR标记技术对86株来自烟草和胡萝卜的根串珠霉菌株进行分子遗传多样性分析。(6)利用显微及超微技术探究病原菌的侵染机理。结果表明：
1.从我国云南、甘肃、贵州、重庆和河南5省(市)不同烟区采集具有典型根黑腐症状的烟草病害样本及病根处土壤,用改进的胡萝卜圆片法分离病原菌,共得到45个野生型单孢分离菌株,柯赫氏法则证病结果显示这些菌株均为该病的致病菌,另有13株白色变异型菌株为野生型菌株室内培养时产生。形态学及rDNA-ITS分子鉴定结果均显示：所有菌株包括两株来自胡萝卜的参试菌株,在分类上均为根串珠霉Thielaviopsis basicola(Berk.&Br.) Ferr.。病菌rDNA-ITS序列分析结果显示,白色变异型菌株与其野生型菌株序列一致,不同寄主来源根串珠霉rDNA-ITS序列有差异。
2.选取菌落形态不同的菌株CPW1-1、CWH-1和HLL-1',测定影响其菌丝生长和孢子萌发的培养因子。结果表明：在Czapek培养基上HLL-1'不生长,CPW1-1和CWH-1可缓慢扩展形成稀薄疏松的菌落,供试培养基中最适合三者生长的分别为PDA、PSA和PCA。温度对供试菌株菌丝生长有明显影响。3个菌株在5℃-35℃间均能生长,最适温度均为25℃。供试菌株在pH=4～12范围内均能生长,CPW1-1、CWH-1和HLL-1'分别在pH=7、pH=10和pH=6时菌丝生长速度最快,在培养基偏酸或偏碱时,CPW1-1菌落易产生白色扇状变异。除木糖外两个野生型菌株都能有效利用各种供试碳源,氮源中则是牛肉膏和酪氨酸的利用效果最好。相同光照或相同温度条件下CPW1-1孢子萌发率均高于CWH-1,在有机营养存在时,情况则相反；黑暗条件有利于孢子萌发,CPW1-1.CWH-1和HLL-1'孢子萌发的最适温度分别是20℃、15℃和20℃。
3.采用伤根接种法对15个烟草根黑腐菌株进行致病力差异测定,并比较了7个烟草主栽品种的室内抗性水平。研究结果表明：供试的15株烟草根黑腐病菌引致的病害程度不一,病情指数从19.2～62.5不等,致病力分化明显,其中4株表现强致病力,中等致病力菌株10株,白色变异体HLL-1'为弱致病力菌株,野生型菌株中未发现弱致病力菌株。不同烟草品种对同一菌株的抗性差异大,可产生高感、感病、中抗和抗病等不同抗性反应;在供试的烟草品种中,贵烟4号、秦烟96和中烟100有较强的抗病性。
4.参照Punja&Sun的分群鉴定方法,将来自烟草的45个野生型和13株白色变异型共58个根黑腐菌株进行了分群鉴定。结果表明：我国烟草根黑腐病菌分离株存在明显形态变异,共划分出6个群,其中群Ⅰ、Ⅱ、Ⅲ为野生型,群Ⅳ、Ⅴ为白色变异型；另有白色变异型HLL-1'菌落平展白色,厚垣孢子簇生或单生,有瓶梗孢子产生,形态特征明显不同于其它群,将其建为新群——群Ⅵ。群Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ及Ⅵ的分离频率分别占41.4%、32.8%、3.4%、19.0%、1.7%和1.7%。群Ⅰ主要分布重庆和云南,群Ⅱ在五个采样省(市)均有分布,群Ⅲ和群V仅分布在贵州,群Ⅳ主要分布在重庆、河南和云南。群Ⅵ仅在河南有分布。
5.以烟草根黑腐病菌基因组DNA为模板,采用单因素水平优化的方法对Taq DNA聚合酶、Mg2+、dNTP和模板等的浓度,以及退火温度等几个因素进行优化,建立了根串珠霉ISSR-PCR优化反应体系,利用该体系从20个ISSR引物中筛选出6个多态性较好的ISSR引物,PCR扩增86个不同菌株后获得大量清晰可重复的DNA指纹图谱,其平均多态位点百分率为70.91%。供试总样本的Shannon多样性指数I为0.5504,Nei's指数h为0.3726,种群间的基因多样度DST=0.1657,种群基因分化系数GST平均值为0.5405,大于0.5,表明供试种群具有较高的遗传多样性,不同寄主、不同地区来源根串珠霉存在着明显的遗传分化,聚类分析可将它们分成7个遗传聚类群。
6.分析以上几个结果间的关系后发现,烟草根黑腐病原菌致病力分化与其培养特性、形态分群之间有明显的相关性,ISSR标记揭示的遗传聚类组群与菌株致病力、形态分群相关性不明显,但与不同寄主来源关系密切。
7.烟草根黑腐病菌侵染烟苗的显微观察结果显示：该菌以直接侵入的方式侵染寄主,病菌培养时间影响分生孢子萌发时间、萌发后芽管的形态和侵染力。幼龄孢子易萌发,侵染根毛和表皮细胞形成的结构不一。老龄分生孢子侵染烟草根部表皮细胞时,萌发需要的时间长,产生的芽管短,在顶端膨大成类似附着胞的结构后再行侵染。厚垣孢子萌发亦可直接侵入,侵染时芽管顶端略有膨大。
Tobacco black root rot is a worldwide disease, caused by Thielaviopsis basicola (Berk.&Br.) Ferr., the pathogen is associated with the roots of wide range of plants in nature and is recognized as a pathogen that causes black root rot on many important agricultural crops worldwide. However, until now little is known about the extent of morphological variation within T. basicola or about the level of genetic diversity within this fungal species in China.
In order to explore the morphological, pathogenicity differentiation and molecular diversity of T. basicola, the following studies were carried out.(1) The pathogen was isolated from diseased tobacco tissues and soil samples of tobacco root rot were collected from5different Chinese geographic regions, including Yunnan, Gansu, Guizhou, Chongqing and Henan provinces. Pathogenicity of the all isolates was tested according to Koch's postulates. Then using traditional morphological classification and rDNA-ITS sequences analysis methods, the T. basicola isolates were further identified.(2) The culture characteristics of three isolates with different colony characteristics that obtained from tobacco black root rot were studied.(3)Tested the pathogenicity differentiation of some T. basicola isolates, and the disease resistances of7main tobacco cultivars against different T. basicola isolates were also evaluated.(4) According to Punja&Sun's grouping identification standard, grouping identification of all isolates was studied.(5)The molecular genetic diversity of all T. basicola isolates including32isolates from carrots was analyzed by the inter-simple sequence repeat (ISSR) technique.(6) The initial interactions between T. basicola and root hairs of tobacco were examined microscopically to make the infection mechanism of T. basicola clear. The results were as followings.
1. The specimens of diseased tobacco plants with typical syptoms of black root rot and its soil was collected, the geographic origins of the specimens were Yunnan, Gansu, Guizhou, Chongqing and Henan provinces in China. Using the improved carrot disks methods more than20isolates were collected. After further purification and single spore isolation,45wild types and13white aberrant phenotypes of the pathogen were obtained. The results of experiments accoding Koch's postulates revealed that these wild type isolates were the pathogen of the disease. The morphology and rDNA-ITS molecular identification results show that:all the isolates including two from carrot were Thielaviopsis basicola (Berk.&Br.) Ferr. in classification. The rDNA-ITS sequence analysis suggested that the white aberrant phenotypes were identical to it's wild types in sequences, but completely different from the isolates growing on other host plants.
2. Biological characteristics of three T. basicola isolates CPW1-1, CWH-1and HLL-1' with different colony characteristics were studied. The results showed:HLL-1'could not develop conoly on Czapek media, CPW1-1and CWH-1could but conoly is very thin. PSA, PCA and PDA were the most favorable media of three isolates respectively. The temperature has a significant effect on the mycelial growth of the tested isolates, they could grow in5℃～35℃, and the optimum temperature was25℃. The tested strains could grow under the pH=4-12, while under pH=7,10and6were optimum pH values for the mycelial growth of CPW1-1, CWH-1and HLL-1', respectively. CPW1-1and CWH-1could use all of the tested carbon sources except xylose, beef ertract and casein was their favorable nitrogen sources. Under the same culture conditions, the conidia germination rate of CPW1-1was higher than that of CWH-1, however under the organic nutrition, CWH-1was higher than CPW1-1. That showed that nutrition enhanced the conidia germination rate of CWH-1. Dark conditoin was favorable for the germination. The optimum conidia germination temperature of CPW1-1, CWH-1and HLL-1'were20℃,15℃and20℃, respectively.
3. Pathogenicity differentiation against black root rot was compared by root cutting inoculation method within15T. basicola isolates, and their pathogenicity showed significant difference. The disease index of15isolates ranged from19.2to62.5, in which4isolates showed high pathogenicity,10isolates were at moderate level, and HLL-1'the albino shouwed weak pathogenicity, and no weak pathogenicity was found in wild type isolates. The disease resistances were significant difference among the7cultivars when different cultivars inoculated by a certain T. basicola isolate, the disease resistance changed from resistant, moderate resistant, susceptible and highly susceptible. Based on the experimental results, it was showed that the resistance of Guiyan4, Qinyan96and Zhongyan100was higher than those of other tested cultivars.
4. According to Punja &Sun's Grouping identification standard of T. basicola, the extent of grouping identification was studied in those58T. basicola isolates from tobacco including45wild types and13albino isolates. The results showed that six general morphological groups could be distinguished among the isolates, in which group I, group II and group III were wild types, group Ⅳ and group V were aberrant phenotypes (albino and mycelial). The characteristics of colony and chlamydospores of another aberrant phenotype HLL-1'was completely different from that of the five groups above, colony of it was flat and white, the chlamydospores were borne either in clusters or singly, phialospores could be borne, so it was assigned to be the new group——group Ⅵ. The isolating ratios of groups Ⅰ, Ⅱ, Ⅲ, IV, V and VI were41.4%,32.8%,3.4%,19.0%,1.7%and1.7%respectively. Group I was mainly distributed in Chongqing and Yunnan, group III and V in Guizhou, group IV mainly in Chongqing, Henan and Yunnan, group VI only in Henan, and group II was distributed in all investigating provinces.
5. The ISSR amplification system of T. basicola was optimized and established with the parameters, such as the concentrations of Taq DNA polymerase, Mg2+, dNTP and DNA templates, the annealing temperature. A total of6specific ISSR primers were screened from20ISSR primers, and the average percentage of polymorphic loci of populations was70.91%, which suggested all tested isolates of T. basicola had high genetic diversity (the average value of Shannon index I is0.5504). The genetic variation was significant among populations (average Nei's index h was0.3726, and average GST is0.1657). The tested isolates were clustered into7genetic lineages.
6. The above results showed that the pathogen virulence differentiation had obvious correlation with its cultural characteristics and morphological grouping, whereas the ISSR clustering groups was insignificant correlation with the virulence and morphological grouping of the isolates, but they were closely related to the different host sources.
7. The ultrastructure of initial interactions between T. basicola and tobacco was examined microscopically. It showed that the pathogen invaded the tobacco directly; the culture time of T. basicola affects germination time, germ tube morphology and infectivity of the conidial. The structure that formed is different when the young endoconidia invade into the root hairs from into the epidermic cells. No appressoria were formed when invade into the root hair. The maturity conidial germination time is a little longer and appressoria formed on the top of germtubes. And the chlamydospore could invaded directly, and the top of germtube swollen slightly.
1.从我国云南、甘肃、贵州、重庆和河南5省(市)不同烟区采集具有典型根黑腐症状的烟草病害样本及病根处土壤,用改进的胡萝卜圆片法分离病原菌,共得到45个野生型单孢分离菌株,柯赫氏法则证病结果显示这些菌株均为该病的致病菌,另有13株白色变异型菌株为野生型菌株室内培养时产生。形态学及rDNA-ITS分子鉴定结果均显示：所有菌株包括两株来自胡萝卜的参试菌株,在分类上均为根串珠霉Thielaviopsis basicola(Berk.&Br.) Ferr.。病菌rDNA-ITS序列分析结果显示,白色变异型菌株与其野生型菌株序列一致,不同寄主来源根串珠霉rDNA-ITS序列有差异。
2.选取菌落形态不同的菌株CPW1-1、CWH-1和HLL-1',测定影响其菌丝生长和孢子萌发的培养因子。结果表明：在Czapek培养基上HLL-1'不生长,CPW1-1和CWH-1可缓慢扩展形成稀薄疏松的菌落,供试培养基中最适合三者生长的分别为PDA、PSA和PCA。温度对供试菌株菌丝生长有明显影响。3个菌株在5℃-35℃间均能生长,最适温度均为25℃。供试菌株在pH=4～12范围内均能生长,CPW1-1、CWH-1和HLL-1'分别在pH=7、pH=10和pH=6时菌丝生长速度最快,在培养基偏酸或偏碱时,CPW1-1菌落易产生白色扇状变异。除木糖外两个野生型菌株都能有效利用各种供试碳源,氮源中则是牛肉膏和酪氨酸的利用效果最好。相同光照或相同温度条件下CPW1-1孢子萌发率均高于CWH-1,在有机营养存在时,情况则相反；黑暗条件有利于孢子萌发,CPW1-1.CWH-1和HLL-1'孢子萌发的最适温度分别是20℃、15℃和20℃。
3.采用伤根接种法对15个烟草根黑腐菌株进行致病力差异测定,并比较了7个烟草主栽品种的室内抗性水平。研究结果表明：供试的15株烟草根黑腐病菌引致的病害程度不一,病情指数从19.2～62.5不等,致病力分化明显,其中4株表现强致病力,中等致病力菌株10株,白色变异体HLL-1'为弱致病力菌株,野生型菌株中未发现弱致病力菌株。不同烟草品种对同一菌株的抗性差异大,可产生高感、感病、中抗和抗病等不同抗性反应;在供试的烟草品种中,贵烟4号、秦烟96和中烟100有较强的抗病性。
4.参照Punja&Sun的分群鉴定方法,将来自烟草的45个野生型和13株白色变异型共58个根黑腐菌株进行了分群鉴定。结果表明：我国烟草根黑腐病菌分离株存在明显形态变异,共划分出6个群,其中群Ⅰ、Ⅱ、Ⅲ为野生型,群Ⅳ、Ⅴ为白色变异型；另有白色变异型HLL-1'菌落平展白色,厚垣孢子簇生或单生,有瓶梗孢子产生,形态特征明显不同于其它群,将其建为新群——群Ⅵ。群Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ及Ⅵ的分离频率分别占41.4%、32.8%、3.4%、19.0%、1.7%和1.7%。群Ⅰ主要分布重庆和云南,群Ⅱ在五个采样省(市)均有分布,群Ⅲ和群V仅分布在贵州,群Ⅳ主要分布在重庆、河南和云南。群Ⅵ仅在河南有分布。
5.以烟草根黑腐病菌基因组DNA为模板,采用单因素水平优化的方法对Taq DNA聚合酶、Mg2+、dNTP和模板等的浓度,以及退火温度等几个因素进行优化,建立了根串珠霉ISSR-PCR优化反应体系,利用该体系从20个ISSR引物中筛选出6个多态性较好的ISSR引物,PCR扩增86个不同菌株后获得大量清晰可重复的DNA指纹图谱,其平均多态位点百分率为70.91%。供试总样本的Shannon多样性指数I为0.5504,Nei's指数h为0.3726,种群间的基因多样度DST=0.1657,种群基因分化系数GST平均值为0.5405,大于0.5,表明供试种群具有较高的遗传多样性,不同寄主、不同地区来源根串珠霉存在着明显的遗传分化,聚类分析可将它们分成7个遗传聚类群。
6.分析以上几个结果间的关系后发现,烟草根黑腐病原菌致病力分化与其培养特性、形态分群之间有明显的相关性,ISSR标记揭示的遗传聚类组群与菌株致病力、形态分群相关性不明显,但与不同寄主来源关系密切。
7.烟草根黑腐病菌侵染烟苗的显微观察结果显示：该菌以直接侵入的方式侵染寄主,病菌培养时间影响分生孢子萌发时间、萌发后芽管的形态和侵染力。幼龄孢子易萌发,侵染根毛和表皮细胞形成的结构不一。老龄分生孢子侵染烟草根部表皮细胞时,萌发需要的时间长,产生的芽管短,在顶端膨大成类似附着胞的结构后再行侵染。厚垣孢子萌发亦可直接侵入,侵染时芽管顶端略有膨大。
Tobacco black root rot is a worldwide disease, caused by Thielaviopsis basicola (Berk.&Br.) Ferr., the pathogen is associated with the roots of wide range of plants in nature and is recognized as a pathogen that causes black root rot on many important agricultural crops worldwide. However, until now little is known about the extent of morphological variation within T. basicola or about the level of genetic diversity within this fungal species in China.
In order to explore the morphological, pathogenicity differentiation and molecular diversity of T. basicola, the following studies were carried out.(1) The pathogen was isolated from diseased tobacco tissues and soil samples of tobacco root rot were collected from5different Chinese geographic regions, including Yunnan, Gansu, Guizhou, Chongqing and Henan provinces. Pathogenicity of the all isolates was tested according to Koch's postulates. Then using traditional morphological classification and rDNA-ITS sequences analysis methods, the T. basicola isolates were further identified.(2) The culture characteristics of three isolates with different colony characteristics that obtained from tobacco black root rot were studied.(3)Tested the pathogenicity differentiation of some T. basicola isolates, and the disease resistances of7main tobacco cultivars against different T. basicola isolates were also evaluated.(4) According to Punja&Sun's grouping identification standard, grouping identification of all isolates was studied.(5)The molecular genetic diversity of all T. basicola isolates including32isolates from carrots was analyzed by the inter-simple sequence repeat (ISSR) technique.(6) The initial interactions between T. basicola and root hairs of tobacco were examined microscopically to make the infection mechanism of T. basicola clear. The results were as followings.
1. The specimens of diseased tobacco plants with typical syptoms of black root rot and its soil was collected, the geographic origins of the specimens were Yunnan, Gansu, Guizhou, Chongqing and Henan provinces in China. Using the improved carrot disks methods more than20isolates were collected. After further purification and single spore isolation,45wild types and13white aberrant phenotypes of the pathogen were obtained. The results of experiments accoding Koch's postulates revealed that these wild type isolates were the pathogen of the disease. The morphology and rDNA-ITS molecular identification results show that:all the isolates including two from carrot were Thielaviopsis basicola (Berk.&Br.) Ferr. in classification. The rDNA-ITS sequence analysis suggested that the white aberrant phenotypes were identical to it's wild types in sequences, but completely different from the isolates growing on other host plants.
2. Biological characteristics of three T. basicola isolates CPW1-1, CWH-1and HLL-1' with different colony characteristics were studied. The results showed:HLL-1'could not develop conoly on Czapek media, CPW1-1and CWH-1could but conoly is very thin. PSA, PCA and PDA were the most favorable media of three isolates respectively. The temperature has a significant effect on the mycelial growth of the tested isolates, they could grow in5℃～35℃, and the optimum temperature was25℃. The tested strains could grow under the pH=4-12, while under pH=7,10and6were optimum pH values for the mycelial growth of CPW1-1, CWH-1and HLL-1', respectively. CPW1-1and CWH-1could use all of the tested carbon sources except xylose, beef ertract and casein was their favorable nitrogen sources. Under the same culture conditions, the conidia germination rate of CPW1-1was higher than that of CWH-1, however under the organic nutrition, CWH-1was higher than CPW1-1. That showed that nutrition enhanced the conidia germination rate of CWH-1. Dark conditoin was favorable for the germination. The optimum conidia germination temperature of CPW1-1, CWH-1and HLL-1'were20℃,15℃and20℃, respectively.
3. Pathogenicity differentiation against black root rot was compared by root cutting inoculation method within15T. basicola isolates, and their pathogenicity showed significant difference. The disease index of15isolates ranged from19.2to62.5, in which4isolates showed high pathogenicity,10isolates were at moderate level, and HLL-1'the albino shouwed weak pathogenicity, and no weak pathogenicity was found in wild type isolates. The disease resistances were significant difference among the7cultivars when different cultivars inoculated by a certain T. basicola isolate, the disease resistance changed from resistant, moderate resistant, susceptible and highly susceptible. Based on the experimental results, it was showed that the resistance of Guiyan4, Qinyan96and Zhongyan100was higher than those of other tested cultivars.
4. According to Punja &Sun's Grouping identification standard of T. basicola, the extent of grouping identification was studied in those58T. basicola isolates from tobacco including45wild types and13albino isolates. The results showed that six general morphological groups could be distinguished among the isolates, in which group I, group II and group III were wild types, group Ⅳ and group V were aberrant phenotypes (albino and mycelial). The characteristics of colony and chlamydospores of another aberrant phenotype HLL-1'was completely different from that of the five groups above, colony of it was flat and white, the chlamydospores were borne either in clusters or singly, phialospores could be borne, so it was assigned to be the new group——group Ⅵ. The isolating ratios of groups Ⅰ, Ⅱ, Ⅲ, IV, V and VI were41.4%,32.8%,3.4%,19.0%,1.7%and1.7%respectively. Group I was mainly distributed in Chongqing and Yunnan, group III and V in Guizhou, group IV mainly in Chongqing, Henan and Yunnan, group VI only in Henan, and group II was distributed in all investigating provinces.
5. The ISSR amplification system of T. basicola was optimized and established with the parameters, such as the concentrations of Taq DNA polymerase, Mg2+, dNTP and DNA templates, the annealing temperature. A total of6specific ISSR primers were screened from20ISSR primers, and the average percentage of polymorphic loci of populations was70.91%, which suggested all tested isolates of T. basicola had high genetic diversity (the average value of Shannon index I is0.5504). The genetic variation was significant among populations (average Nei's index h was0.3726, and average GST is0.1657). The tested isolates were clustered into7genetic lineages.
6. The above results showed that the pathogen virulence differentiation had obvious correlation with its cultural characteristics and morphological grouping, whereas the ISSR clustering groups was insignificant correlation with the virulence and morphological grouping of the isolates, but they were closely related to the different host sources.
7. The ultrastructure of initial interactions between T. basicola and tobacco was examined microscopically. It showed that the pathogen invaded the tobacco directly; the culture time of T. basicola affects germination time, germ tube morphology and infectivity of the conidial. The structure that formed is different when the young endoconidia invade into the root hairs from into the epidermic cells. No appressoria were formed when invade into the root hair. The maturity conidial germination time is a little longer and appressoria formed on the top of germtubes. And the chlamydospore could invaded directly, and the top of germtube swollen slightly.
引文
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