辣椒疫霉菌遗传多样性分析及致病机理研究
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摘要
辣椒疫病自首次报道后,迅速蔓延,已成为一种分布广、危害许多国家辣椒生产的严重病害之一。本研究收集了13份来自不同地区的辣椒疫霉菌菌株,用SRAP技术对其DNA进行遗传多样性分析;利用辣椒疫霉菌标准鉴别寄主,对其进行生理小种鉴定;比较了辣椒疫霉菌在不同抗性辣椒茎中的侵染过程;并初步建立了辣椒疫霉菌复壮及单孢子囊快速分离的技术体系。其结果如下:
1.把13份来自于不同地区的辣椒疫霉菌经单孢子囊分离后,用SRAP技术对其进行遗传多样性分析,143对SRAP引物中可筛选出33对重复性好的多态性引物,其共扩增出606条DNA条带,其中多态性条带有587条,占总条带的96.86%,特异条带有75条,占总条带的12.38%,占多态性条带的12.78%,说明供试辣椒疫霉菌具有较丰富的遗传多样性。通过对SRAP扩增结果进行聚类分析表明,可将供试菌株分为三大类:第一类有7个菌株,其中P15来自于陕西省,P3来自青海省,ZLT来自广州,YN-9, YN-10来自云南省,PC来自韩国,PG-1来自北京;第二大类为有4个菌株PG-3来自北京,HX-5,HX-9来自河北省,YN-1来自云南省,第三大类有2个菌株YN-7,N-1分别来自云南省,内蒙。供试辣椒疫霉菌的相似系数在0.24~0.66之间以上。结果表明,来自同一地区的菌株可以聚在一起,表现出很近的亲缘关系,来自不同地区之间的菌株也可以交叉聚类,说明不同地区菌株的亲缘关系与其地理分布无直接关系。
2.利用灌根法分别对感病和抗病两个辣椒材料进行接种,于接种后0、12、24、48、72、144h,取样观察疫霉菌在不同抗性辣椒中的侵染过程。结果发现,抗病材料在接种后一直未表现出发病症状而且在其茎部内也没有发现菌丝,而感病品种在接种后第三天就出现了发病症状,经透射电镜观察,菌丝72h后侵入皮层细胞,144h进入次生木质部,使其细胞内的细胞质膜解体,细胞发生质壁分离,线粒体等细胞器消失。以上结果说明,辣椒疫霉菌在接种2天后开始与辣椒互作,并发生侵染现象,先进入细胞间隙再扩展到细胞内。抗病品种的根系可能会阻止病原菌的侵染,使疫霉菌不能到达其茎部。
3.对13份来自不同国家和地区的辣椒疫霉菌菌株进行生理小种鉴定。结果表明:来自韩国菌株PC和云南菌株YN-1为ph1;北京菌株PG-1、PG-3,云南菌株YN-9、YN-10和青海菌株P3为ph2;内蒙古菌株N-1、陕西菌株P15,云南菌株YN-1,河北菌株HX-5、HX-9和广州菌株ZLT为ph3。生理小种1、2、3分别占供试菌株的比例依次为,15.4%,38.5%,46.2%,可推测出生理小种3为供试菌株的优势小种。生理小种划分与地理来源无直接关。
4.通过研究马铃薯葡萄糖琼脂培养基(PDA)和PDA含药选择性培养基(PDAPR)对辣椒疫霉菌单个孢子囊分离的影响,筛选出适合单个孢子囊分离的最佳培养基;利用一对抗、感品种作为寄主,通过常规抗病性鉴定技术,对陕西辣椒疫霉菌复壮前后的致病力进行了测定。结果表明:辣椒疫霉菌单个孢子囊分离的最佳培养基是PDA含药选择性培养基,其分离率高达95%,其中污染率只有15.8%;复壮前后菌株对感病品种的致病力有明显的差异,复壮后菌株的致病力比于复壮前提高27.2%,且与刚分离后菌株的致病力相当,这说明通过复壮能够恢复辣椒疫霉菌菌株的致病力。
Pepper Phytophthora blight spreaded quickly and has become one of the serious widely distributed diseases that jeopardize the production of pepper in many countries since it's first being reported. This research has collected 13 Phytophthora capsici strain from different regions, analysis of genetic diversity of DNA by SRAP. We identified the host by phytophthora sp standards, and identified their physiological strains. and we studied the infection process in item of peppers with different resistances. we initially established the technology of the rejuvenescence of Phytophthora capsici and the separation of single spores, The results are as follows:
1. We analyzed the genetic diversity of the physiological strains from 13 different regions after their single spores have been separated, Among the screened out priming with 143 pairs SRAP primers it was observed that 33 pairs primer has shown promising results regarding repeatability and polymorphism and have cloned 606 DNA bands, among them, 587 of a total of 96.86% bands were depicted polymorphism bands, 75 of a total of 12.38% are special bands, which takes up 12.78% of polymorphism bands, it means that pepper Phytophthora blight have a wealth of genetic diversity. According to the clustering analysis of the SRAP results, the strains we were studying can be divided into three categories, the first class has seven strains, namely as P15 from Shanxi, ph3 from Qinghai, ZLT from Guangzhou, YN -9, YN-10 from Yunnan province, PC from Korea and PG-1 from Beijing; four strains can be classified as second type, namely as PG -3 from Beijing, HX-5, HX-9 from Hebei province, YN-1 from Yunnan province; only two strains can be referred as third category. The similarity coefficients were ranged from 0.24 to 0.66. The isolates from the same area were clustered together and showed close genetic relationships. Some isolates from different areas were also clustered together with similarity. We found that the clustering of the strains has no obvious relationship with the place no matter where they came from.
2. Root pouring method to inoculate susceptible and resistant pepper. After the vaccination on the interval of 0, 12, 24, 48, 72, 177h, samples were observed for infectionprocess by Phytophthora capsici in different resistant cultivars of pepper. The results shown that Resistant varieties have shown no symptom either in their appearance and hyphae after being vaccinated, But the susceptible cultivar have shown quite vivid symptoms on the third day of inoculation, Under evaluating transmission electron microscopy, Phytophthora capsici penetrated into cortical cells after 72h, entered secondary xylem after 144h, and made the cell membrane disintegrated and cell plasmolysis, mitochondria and other organelles disappeared. These results indicated that Phytophthora capsici interacted with pepper two days later after the inoculation and subsequently infection occurred. Mycelium first enters into the intercellular space and then enters the cell. Resistant varieties of root can inhibit Phytophthora capsici infection, so that their stems can not be infected.
3. We have identified the physiological strains of 13 pepper Phytophthora blights from different regions, experiments indicate that: strain PC from Korea and strain YN-1 from Yunnan are ph1, strains of PG-1 and PG-3 from Beijing and strains of YN-9Y and YN-10 from Yunnan and strain ph3 from Qinghai are ph2, N-1 from Inner Mongolia, P15 from Shannxi, YN-1 from Yunnan, HX-5 and HX-9 from Hebei and ZLT from Guangzhou are ph3. The proportion of 1、2 and 3 of the total population are 15.4%,38.5%,46.2%, respectively, it can be predict that physiological strain 3 is the preponderant strain, and there is no direct relationship between the physical separation and sources of geography.
4. By studying the effect of the potato dextrose agar (PDA) and PDA drug-containing selective culture medium (PDAPR) on the separation of individual zoosporangium to obtain the most effective culture for the separation of single Phytophthora capsici sporangium. We compared the pathogenicity of Phytophthora capsici before and after rejuvenation by using conventional techniques with one disease resistant host plant and a susceptible one. The results showed that the best culture for a single sporangium separation is the PDAPR, with a separation rate of 95%, where the pollution rate is only 15.8%.The pathogenicity of Phytophthora capsici after rejuvenation to susceptible plant is significant higher than that of before rejuvenation, The virulence of Phytophthora capsici after rejuvenation increased by 27.2%, which shows that the strain pathogenicity can be restored back through rejuvenation.
1.把13份来自于不同地区的辣椒疫霉菌经单孢子囊分离后,用SRAP技术对其进行遗传多样性分析,143对SRAP引物中可筛选出33对重复性好的多态性引物,其共扩增出606条DNA条带,其中多态性条带有587条,占总条带的96.86%,特异条带有75条,占总条带的12.38%,占多态性条带的12.78%,说明供试辣椒疫霉菌具有较丰富的遗传多样性。通过对SRAP扩增结果进行聚类分析表明,可将供试菌株分为三大类:第一类有7个菌株,其中P15来自于陕西省,P3来自青海省,ZLT来自广州,YN-9, YN-10来自云南省,PC来自韩国,PG-1来自北京;第二大类为有4个菌株PG-3来自北京,HX-5,HX-9来自河北省,YN-1来自云南省,第三大类有2个菌株YN-7,N-1分别来自云南省,内蒙。供试辣椒疫霉菌的相似系数在0.24~0.66之间以上。结果表明,来自同一地区的菌株可以聚在一起,表现出很近的亲缘关系,来自不同地区之间的菌株也可以交叉聚类,说明不同地区菌株的亲缘关系与其地理分布无直接关系。
2.利用灌根法分别对感病和抗病两个辣椒材料进行接种,于接种后0、12、24、48、72、144h,取样观察疫霉菌在不同抗性辣椒中的侵染过程。结果发现,抗病材料在接种后一直未表现出发病症状而且在其茎部内也没有发现菌丝,而感病品种在接种后第三天就出现了发病症状,经透射电镜观察,菌丝72h后侵入皮层细胞,144h进入次生木质部,使其细胞内的细胞质膜解体,细胞发生质壁分离,线粒体等细胞器消失。以上结果说明,辣椒疫霉菌在接种2天后开始与辣椒互作,并发生侵染现象,先进入细胞间隙再扩展到细胞内。抗病品种的根系可能会阻止病原菌的侵染,使疫霉菌不能到达其茎部。
3.对13份来自不同国家和地区的辣椒疫霉菌菌株进行生理小种鉴定。结果表明:来自韩国菌株PC和云南菌株YN-1为ph1;北京菌株PG-1、PG-3,云南菌株YN-9、YN-10和青海菌株P3为ph2;内蒙古菌株N-1、陕西菌株P15,云南菌株YN-1,河北菌株HX-5、HX-9和广州菌株ZLT为ph3。生理小种1、2、3分别占供试菌株的比例依次为,15.4%,38.5%,46.2%,可推测出生理小种3为供试菌株的优势小种。生理小种划分与地理来源无直接关。
4.通过研究马铃薯葡萄糖琼脂培养基(PDA)和PDA含药选择性培养基(PDAPR)对辣椒疫霉菌单个孢子囊分离的影响,筛选出适合单个孢子囊分离的最佳培养基;利用一对抗、感品种作为寄主,通过常规抗病性鉴定技术,对陕西辣椒疫霉菌复壮前后的致病力进行了测定。结果表明:辣椒疫霉菌单个孢子囊分离的最佳培养基是PDA含药选择性培养基,其分离率高达95%,其中污染率只有15.8%;复壮前后菌株对感病品种的致病力有明显的差异,复壮后菌株的致病力比于复壮前提高27.2%,且与刚分离后菌株的致病力相当,这说明通过复壮能够恢复辣椒疫霉菌菌株的致病力。
Pepper Phytophthora blight spreaded quickly and has become one of the serious widely distributed diseases that jeopardize the production of pepper in many countries since it's first being reported. This research has collected 13 Phytophthora capsici strain from different regions, analysis of genetic diversity of DNA by SRAP. We identified the host by phytophthora sp standards, and identified their physiological strains. and we studied the infection process in item of peppers with different resistances. we initially established the technology of the rejuvenescence of Phytophthora capsici and the separation of single spores, The results are as follows:
1. We analyzed the genetic diversity of the physiological strains from 13 different regions after their single spores have been separated, Among the screened out priming with 143 pairs SRAP primers it was observed that 33 pairs primer has shown promising results regarding repeatability and polymorphism and have cloned 606 DNA bands, among them, 587 of a total of 96.86% bands were depicted polymorphism bands, 75 of a total of 12.38% are special bands, which takes up 12.78% of polymorphism bands, it means that pepper Phytophthora blight have a wealth of genetic diversity. According to the clustering analysis of the SRAP results, the strains we were studying can be divided into three categories, the first class has seven strains, namely as P15 from Shanxi, ph3 from Qinghai, ZLT from Guangzhou, YN -9, YN-10 from Yunnan province, PC from Korea and PG-1 from Beijing; four strains can be classified as second type, namely as PG -3 from Beijing, HX-5, HX-9 from Hebei province, YN-1 from Yunnan province; only two strains can be referred as third category. The similarity coefficients were ranged from 0.24 to 0.66. The isolates from the same area were clustered together and showed close genetic relationships. Some isolates from different areas were also clustered together with similarity. We found that the clustering of the strains has no obvious relationship with the place no matter where they came from.
2. Root pouring method to inoculate susceptible and resistant pepper. After the vaccination on the interval of 0, 12, 24, 48, 72, 177h, samples were observed for infectionprocess by Phytophthora capsici in different resistant cultivars of pepper. The results shown that Resistant varieties have shown no symptom either in their appearance and hyphae after being vaccinated, But the susceptible cultivar have shown quite vivid symptoms on the third day of inoculation, Under evaluating transmission electron microscopy, Phytophthora capsici penetrated into cortical cells after 72h, entered secondary xylem after 144h, and made the cell membrane disintegrated and cell plasmolysis, mitochondria and other organelles disappeared. These results indicated that Phytophthora capsici interacted with pepper two days later after the inoculation and subsequently infection occurred. Mycelium first enters into the intercellular space and then enters the cell. Resistant varieties of root can inhibit Phytophthora capsici infection, so that their stems can not be infected.
3. We have identified the physiological strains of 13 pepper Phytophthora blights from different regions, experiments indicate that: strain PC from Korea and strain YN-1 from Yunnan are ph1, strains of PG-1 and PG-3 from Beijing and strains of YN-9Y and YN-10 from Yunnan and strain ph3 from Qinghai are ph2, N-1 from Inner Mongolia, P15 from Shannxi, YN-1 from Yunnan, HX-5 and HX-9 from Hebei and ZLT from Guangzhou are ph3. The proportion of 1、2 and 3 of the total population are 15.4%,38.5%,46.2%, respectively, it can be predict that physiological strain 3 is the preponderant strain, and there is no direct relationship between the physical separation and sources of geography.
4. By studying the effect of the potato dextrose agar (PDA) and PDA drug-containing selective culture medium (PDAPR) on the separation of individual zoosporangium to obtain the most effective culture for the separation of single Phytophthora capsici sporangium. We compared the pathogenicity of Phytophthora capsici before and after rejuvenation by using conventional techniques with one disease resistant host plant and a susceptible one. The results showed that the best culture for a single sporangium separation is the PDAPR, with a separation rate of 95%, where the pollution rate is only 15.8%.The pathogenicity of Phytophthora capsici after rejuvenation to susceptible plant is significant higher than that of before rejuvenation, The virulence of Phytophthora capsici after rejuvenation increased by 27.2%, which shows that the strain pathogenicity can be restored back through rejuvenation.
引文
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