烟草靶斑病菌遗传分化、侵染特性及致病机理研究
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摘要
烟草靶斑病(tobacco target spot)是近年来我国新报道的一种烟草叶部病害,危害严重,该病害对烟草的产量和品质有显著的影响,但是国内缺乏文献记载。鉴于此,本文针对烟草靶斑病菌(Rhizoctonia solani Kiihn)的遗传分化、侵染特性和致病机理进行了系统研究,研究结果如下：
1.首次明确了我国烟草靶斑病菌的菌丝融合群为AG-3,以及病菌的致病类型,即强致病类型、中等致病类型和弱致病类型。对采自辽宁各地区的58个烟草靶斑病菌菌株进行了融合群测定,确定在我国引起烟草靶斑病的立枯丝核菌属于AG-3融合群；并对病菌的核糖体DNA内转录间隔区(ITS)序列进行了分析,通过GenBank进行序列同源性比对,结果表明,病菌的rDNA ITS序列长度为653bp,其与引起番茄叶部病害的立枯丝核菌(AG-3融合群)的ITS序列的同源性高达100%,从分子水平上也证明了烟草靶斑病菌属于AG-3融合群,从形态学和分子水平上对烟草靶斑病菌进行了准确的分类鉴定；并从中选出16个有代表性的烟草靶斑病菌菌株进行了致病力测定,根据供试菌株在烟草品种NC89、K326、云烟85和G80的抗感反应,可将这16个菌株划分为3个致病类型,即强致病类型、中等致病类型和弱致病类型,表明我国烟草靶斑病菌的致病力存在明显分化,但致病类型的分布与菌株的地理来源无明显的相关性。
2.应用RAPD和ISSR两种分子标记方法对我国烟草靶斑病菌进行遗传多样性分析,从分子水平探索烟草靶斑病菌的遗传演变和进化,并系统分析了RAPD和ISSR分组与菌株的地理来源和致病性的相互关系。利用优化的反应体系,筛选出9条RAPD引物和7条ISSR引物分别对供试的16个烟草靶斑病菌菌株进行扩增,分别扩增出111和83条带,多态条带比率分别为64.86%和68.67%；RAPD标记的菌株间的相似系数范围分别为0.63-0.93,在相似系数0.74处被划分成3个类群,ISSR标记的菌株间的相似系数范围分别为0.61-0.94,在相似系数0.75处被划分为3个类群。RAPD和ISSR分析结果均表明,我国烟草靶斑病菌群体具有丰富的遗传多样性和明显的遗传分化现象,遗传聚类组群与烟草靶斑病菌菌株的地理来源具有一定的相关性,而与菌株的致病性无明显的相关性。并利用RAPD和ISSR方法对烟草靶斑病菌菌株和引起其它病害的立枯丝核菌菌株进行了遗传多样性分析,结果表明,烟草靶斑病菌菌株间的亲缘关系最近,而与引起其它病害的立枯丝核菌菌株的亲缘关系较远,它们之间存在较大的遗传差异。
3.研究了烟草靶斑病菌的侵染特性,并首次明确了烟草靶斑病菌与重要病菌如水稻纹枯病菌和玉米纹枯病菌的交互致病性。研究结果表明,烟草靶斑病菌以菌丝体或菌丝体随病残体在土壤中越冬,或菌丝体在病残体中越冬；病菌可通过烟草叶片气孔和伤口侵入,伤口更有利于病菌侵入；病菌可侵染烟草苗期和成株期的茎部,引起烟草立枯病和茎基腐病,病菌也可以侵染烟草叶片的主脉和侧脉。对病菌的侵染过程进行观察,发现病菌在接种24h时开始侵入烟草叶片组织,接种24～48h内菌丝迅速在细胞间和细胞内扩展,受侵细胞被破坏,最终死亡。确定了病菌适宜的侵染条件,最适温度为25～30℃；保湿和接种时间越长越有利于发病；12h光照12h黑暗和连续黑暗的条件对病菌的侵染有利,而连续光照对其有明显的抑制作用。对20个烟草品种的抗性研究发现,VRG2最为抗病,发病率为25%。寄主范围测定结果表明,烟草靶斑病菌可侵染烟草、番茄、茄子、辣椒、黄瓜、冬瓜和苋色藜,其中茄子的发病率最高为97.5%。交互致病性测定结果表明,烟草靶斑病菌不能侵染水稻、玉米和白菜,但水稻纹枯病菌、玉米纹枯病菌、白菜丝核菌叶腐病菌、烟草立枯病菌和烟草茎基腐病菌均能侵染烟草叶片。通过田间试验,对该病害进行损失估计,统计结果表明产量损失率和产值损失率与病级均呈显著的正相关,病级越高,产量和产值损失越大,并建立了关系模型。选用10种药剂对烟草靶斑病菌进行室内抑菌试验,建立了各药剂的毒力回归方程并计算出EC50,结果表明,烯唑醇、菌核净、嘧肽菌净、退菌特和代森锰锌对该病菌具有较强的抑制作用,可作为防治该病害的首选药剂。
4.明确了烟草靶斑病菌产生的毒素的基本性质及其致病机理。试验通过活性炭吸附、甲醇洗脱、旋转蒸发浓缩的方法,从烟草靶斑病菌的Richard培养滤液中获得了黄褐色粗毒素,该毒素能够使烟草叶片产生与靶斑病类似的病斑,而且还能抑制种子萌发和胚根生长,导致幼苗萎蔫。明确了病菌产毒的最佳培养条件,即pH值6～7的Richard培养液,温度25℃,黑暗每12h振荡1次条件下培养15～20d。对毒素的基本性质进行研究,结果表明该毒素具有热稳定性、光稳定性和耐储藏性,它是一类易溶于水的非蛋白类的极性物质。研究探讨了毒素对烟苗叶片组织中的抗性相关生理生化指标的影响,结果表明,毒素可激活烟草叶片中POD、PPO、CAT和PAL活性,而抑制SOD活性,受害叶片的叶绿素和可溶性糖含量显著下降,而可溶性蛋白、丙二醛和游离脯氨酸含量明显升高,组织浸出液的相对电导率也显著升高,这些指标的变化表明了毒素对烟草有明显的毒害作用,同时也表明了毒素可引起烟草体内的防御反应,以抵抗毒素造成的伤害。不同致病力烟草靶斑病菌菌株的产毒素能力也不同,强致病力菌株YC-9的产毒能力比弱致病力菌株LF-1强。
5.明确了烟草靶斑病菌产生的细胞壁降解酶的致病机理,强致病力菌株的产酶能力显著高于弱致病力菌株。烟草靶斑病菌在人工Marcus培养液中能产生果胶酶(PG、PMG、PGTE、PMTE)和纤维素酶(Cx、β-葡萄糖苷酶),其中PG活性最高。明确了最佳产酶条件,Cx和β-葡萄糖苷酶的最适培养时间为10d,而PG、PMG、PGTE和PMTE为12d；培养最适温度为25℃；Cx、β-葡萄糖苷酶、PG和PMG的最适pH值为5,而PGTE和PMTE的最适pH值为6；静置培养和连续黑暗条件利于酶的产生。烟草叶片不同病斑部位酶活性不同,其中病健交界处细胞壁降解酶活性最高。烟草靶斑病菌与烟草互作过程中细胞壁降解酶活性测定结果表明,病菌侵染叶片后,PG、PMG. PMTE、PGTE、Cx、β-葡萄糖苷酶活性均显著升高,其中Cx和β-葡萄糖苷酶活性最高,其次为PG和PMG,而PMTE和PGTE活性很低。病菌产生的细胞壁降解酶液能够使烟草叶片产生病斑,混合酶对叶片的损伤作用明显高于单一酶,果胶酶的损伤作用高于纤维素酶。对不同致病力烟草靶斑病菌菌株的产酶能力进行比较,结果表明强致病力菌株YC-9的产酶能力显著高于弱致病力菌株LF-1。
6.通过同源获取基因中间片段和RACE方法克隆得到一个与烟草靶斑病菌致病性相关的基因,即病菌的内切多聚半乳糖醛酸酶(endo-PG)基因。病菌endo-PG基因的cDNA全长序列为1399 bp, CDS区长1086 bp,编码361个氨基酸。
Tobacco target spot is a newly-found and seriously harmful leaf disease of tobacco in China in recent years, and quality and yield of tobacco leaves are seriously affected, which runs short of references. Given that, the genetic differentiation, infection characteristics and pathogenic mechanism of Rhizoctonia solani were systematically studied. The results were as follows:
1. Anastomosis group of R. solani isolates from tobacco target spot was first defined to AG-3, and the isolates could be classified into three pathogenic types, including strong pathogenic type, moderate pathogenic type and weak pathogenic type. Fifty-eight isolates were isolated from tobacco target spot disease samples which were collected from Liaoning tobacco productive areas. The test of anastomosis groups (AGs) indicated that all the isolates belong to AG-3. The rDNA ITS sequence of the R. solani strain YC-9 was PCR amplified and sequenced. And then, the ITS sequence was compared and analyzed with the nucleic acid sequences of GenBank database. The result showed that the ITS sequence was 653bp in length, and the homology of ITS sequence of YC-9 and that of the R. solani(AG-3) isolate from tomato foliar blight was 100 percent. The analysis result of rDNA ITS sequence was consistent with that of morphology. Select 16 representative isolates for the analysis of pathogenic type. The result showed that there were significantly different in pathogenicity among isolates by inoculation of the four tobacco varieties, including NC89, K326, Yunyan 85 and G80. They could be classified into three pathogenic types. The result suggested that the R. solani isolates of tobacco target spot had clear pathogenicity differentiation and there was no significant correlation between the distributions of the pathogenic types and geographic locations of R. solani isolates.
2. Genetic diversity of 16 R. solani isolates of tobacco target spot was analyzed by RAPD and ISSR. Heredity variation and evolution of the pathogen were explored on molecular level. The correlation of RAPD or ISSR grouping and geographic location or pathogenicity of the isolates was systematically analyzed.9 RAPD primers and 7 ISSR primers were screened to amplify 16 isolates using optimal reaction system. The results showed that the 9 RAPD primers and 7 ISSR primers obtained 111 and 83 scored bands respectively, and the ratio of polymorphic bands was 64.86% and 68.67% respectively. Similarity coefficient of RAPD among the isolates was from 0.63 to 0.93, and when similarity coefficient was 0.74, the isolates were divided into three groups. Similarity coefficient of ISSR among the isolates was from 0.61 to 0.94, and when similarity coefficient was 0.75, the isolates were divided into three groups. The analysis results of RAPD and ISSR all suggested that there were rich genetic diversity and remarkable genetic differentiation among 16 R. solani isolates of tobacco target spot, and genetic cluster groups had certain correlation with geographic location, but no significant correlation with pathogenicity of the isolates. Genetic diversity of 16 R. solani isolates of tobacco target spot and R. solani isolates of other plant diseases was analyzed by RAPD and ISSR. The results indicated that there was closest genetic relationship among 16 R. solani isolates of tobacco target spot, but they showed remote affinity to R. solani isolates of other plant diseases, and there were greater genetic differences between them.
3. Study on the infection characteristics of R. solani of tobacco target spot, and first define the relationship of interactive pathogenicity between R. solani isolate of tobacco target spot and R. solani isolates of corn sheath blight and rice sheath blight. The results showed that mycelium of R. solani or mycelium with host residue overwintered in the soil, or mycelium in host residue overwintered. R. solani invaded through the stoma and wound of tobacco leaves, and wound was beneficial to the invasion. R. solani could infect tobacco stems of seedling and adult plant stage, and main and lateral veins of tobacco leaves. The study of infection process indicated that R. solani invaded tobacco leaves after inoculation for 24h, hyphae of R. solani expanded rapidly between and within cells after inoculation for 24-48h, and infected cells were damaged and died finally. Optimum infection conditions of R. solani were determined, the optimum temperature was 25-30℃, the longer moist and inoculation time, the more benefit to the infection of R. solani,12h illumination 12h darkness and continuous darkness were good for the infection, but continuous illumination had significant inhibition. The twenty tested tobacco varieties had different resistance to R. solani, VRG2 had stronger disease resistance and the morbidity was 25 percent. The test result of the host range of R. solani demonstrated that R. solani could infect tobacco, tomato, eggplant and capsicum, cucumber, wax gourd and chenopodium amaranticolor, and eggplant had the highest disease rate (97.5 percent). The test result of interactive pathogenicity demonstrated that R. solani of tobacco target spot could not infect rice, corn and cabbage, but R. solani of rice sheath blight, corn sheath blight and cabbage Rhizoctonia leaf rot could infect tobacco leaves. Field experiment was conducted to investigate damage assessment of the disease, and the statistical result showed that there was significantly positive relationship between the loss rate of yield and value and the disease degree. The higher the disease degree, the greater the yield and value loss, and the relational mode was established. The fungistatic actions of ten fungicides to R. solani were determined. The toxicity regression equations of the fungicides were established and EC50 was computed. The results showed that Diniconazole, dimethachlon, Mitaijunjing, Tuzet and Mancozeb could strongly inhibit the colony growth, so they could be used to control the disease.
4. The study defined the basic feature and pathogenic mechanism of toxin of R. solani. The tan crude toxin was obtained from Richard culture filtrate of R. solani by the methods of activated carbon adsorption, methanol elution and rotating evaporation. The biological activity assay of the crude toxin showed that it could induce the characteristic symptom of tobacco target spot, inhibit seed germination and radicle growth, and make seeding wilting. The optimum conditions of producing toxin were as follows:Richard culture filtrate, pH value 6 to 7,25℃,15 to 20 days under dark and vibration 2 times every day. The test result of basic feature of toxin showed that toxin was of thermostability, photostability and reservation, and it was a kind of nonprotein polar substance. Toxin had effect on physiological and biochemical indexes related with resistance of tobacco leaves. The results demonstrated that toxin activated POD, PPO, CAT and PAL, but inhibited activity of SOD. The contents of chlorophyll and soluble sugar of injured tobacco leaves declined remarkably, but the contents of soluble protein, MDA and free proline increased significantly, and the relative conductivity of the leaching solution of leaf tissue also had a sharp increase. The changes of those indexes illustrated that toxin had poisonous effect on tobacco. Meanwhile, it also showed that toxin could trigger defensive reactions of tobacco. R. solani isolates of different pathogenicity also had different abilities of producing toxin. The ability of strong pathogenic isolate YC-9 was stronger than weak pathogenic isolate LF-1.
5. The study defined pathogenic mechanism of cell wall degrading enzyme of R. solani, and the ability of producing enzyme of strong pathogenic isolate was significantly stronger than weak pathogenic isolate. R. solani could produce pectinase (PG, PMG, PGTE and PMTE) and cellulase (Cx andβ-glucosidase) in Marcus culture solution, and the activity of PG was the highest. The optimum conditions of producing enzyme were determined. The optimum culture time of Cx andβ-glucosidase was 10d, and that of PG, PMG, PGTE and PMTE was 12d; the optimum culture temperature was 25℃; the optimum pH of Cx,β-glucosidase, PG and PMG was 5, and that of PGTE and PMTE was 6; and static culture and continuous darkness were good for the production of enzyme. The enzyme activities of different disease spot parts were also different, and the activities of enzymes in the junction parts of disease and health were the highest. Activity of cell wall degrading enzyme was determined during interaction process of R. solani and tobacco. The result showed that the activities of PG, PMG, PGTE, PMTE, Cx andβ-glucosidase of tobacco leaves infected by R. solani increased markedly. Among these enzymes, the activities of Cx andβ-glucosidase were the highest, followed by PG and PMG, but the activities of PMTE and PGTE were the lowest. Cell wall degrading enzyme of R. solani could make tobacco leaf produce disease spot, the damage effect of mixed enzymes was notably higher than single enzyme, and the damage effect of pectinase was higher than cellulase. Moreover, the abilities of producing enzyme of isolates of different pathogenicity were compared. The result showed that the ability of strong pathogenic isolate YC-9 was stronger than weak pathogenic isolate LF-1.
6. A homologous cDNA fragment encoding the endo-polygalacturonase gene from R. solani was obtained. The method of RACE was used to clone and generate full-length cDNA. The cDNA full length of endo-PG gene of R. solani was 1399 bp. The length of CDS was 1086 bp and it encoded 361 amino acids.
1.首次明确了我国烟草靶斑病菌的菌丝融合群为AG-3,以及病菌的致病类型,即强致病类型、中等致病类型和弱致病类型。对采自辽宁各地区的58个烟草靶斑病菌菌株进行了融合群测定,确定在我国引起烟草靶斑病的立枯丝核菌属于AG-3融合群；并对病菌的核糖体DNA内转录间隔区(ITS)序列进行了分析,通过GenBank进行序列同源性比对,结果表明,病菌的rDNA ITS序列长度为653bp,其与引起番茄叶部病害的立枯丝核菌(AG-3融合群)的ITS序列的同源性高达100%,从分子水平上也证明了烟草靶斑病菌属于AG-3融合群,从形态学和分子水平上对烟草靶斑病菌进行了准确的分类鉴定；并从中选出16个有代表性的烟草靶斑病菌菌株进行了致病力测定,根据供试菌株在烟草品种NC89、K326、云烟85和G80的抗感反应,可将这16个菌株划分为3个致病类型,即强致病类型、中等致病类型和弱致病类型,表明我国烟草靶斑病菌的致病力存在明显分化,但致病类型的分布与菌株的地理来源无明显的相关性。
2.应用RAPD和ISSR两种分子标记方法对我国烟草靶斑病菌进行遗传多样性分析,从分子水平探索烟草靶斑病菌的遗传演变和进化,并系统分析了RAPD和ISSR分组与菌株的地理来源和致病性的相互关系。利用优化的反应体系,筛选出9条RAPD引物和7条ISSR引物分别对供试的16个烟草靶斑病菌菌株进行扩增,分别扩增出111和83条带,多态条带比率分别为64.86%和68.67%；RAPD标记的菌株间的相似系数范围分别为0.63-0.93,在相似系数0.74处被划分成3个类群,ISSR标记的菌株间的相似系数范围分别为0.61-0.94,在相似系数0.75处被划分为3个类群。RAPD和ISSR分析结果均表明,我国烟草靶斑病菌群体具有丰富的遗传多样性和明显的遗传分化现象,遗传聚类组群与烟草靶斑病菌菌株的地理来源具有一定的相关性,而与菌株的致病性无明显的相关性。并利用RAPD和ISSR方法对烟草靶斑病菌菌株和引起其它病害的立枯丝核菌菌株进行了遗传多样性分析,结果表明,烟草靶斑病菌菌株间的亲缘关系最近,而与引起其它病害的立枯丝核菌菌株的亲缘关系较远,它们之间存在较大的遗传差异。
3.研究了烟草靶斑病菌的侵染特性,并首次明确了烟草靶斑病菌与重要病菌如水稻纹枯病菌和玉米纹枯病菌的交互致病性。研究结果表明,烟草靶斑病菌以菌丝体或菌丝体随病残体在土壤中越冬,或菌丝体在病残体中越冬；病菌可通过烟草叶片气孔和伤口侵入,伤口更有利于病菌侵入；病菌可侵染烟草苗期和成株期的茎部,引起烟草立枯病和茎基腐病,病菌也可以侵染烟草叶片的主脉和侧脉。对病菌的侵染过程进行观察,发现病菌在接种24h时开始侵入烟草叶片组织,接种24～48h内菌丝迅速在细胞间和细胞内扩展,受侵细胞被破坏,最终死亡。确定了病菌适宜的侵染条件,最适温度为25～30℃；保湿和接种时间越长越有利于发病；12h光照12h黑暗和连续黑暗的条件对病菌的侵染有利,而连续光照对其有明显的抑制作用。对20个烟草品种的抗性研究发现,VRG2最为抗病,发病率为25%。寄主范围测定结果表明,烟草靶斑病菌可侵染烟草、番茄、茄子、辣椒、黄瓜、冬瓜和苋色藜,其中茄子的发病率最高为97.5%。交互致病性测定结果表明,烟草靶斑病菌不能侵染水稻、玉米和白菜,但水稻纹枯病菌、玉米纹枯病菌、白菜丝核菌叶腐病菌、烟草立枯病菌和烟草茎基腐病菌均能侵染烟草叶片。通过田间试验,对该病害进行损失估计,统计结果表明产量损失率和产值损失率与病级均呈显著的正相关,病级越高,产量和产值损失越大,并建立了关系模型。选用10种药剂对烟草靶斑病菌进行室内抑菌试验,建立了各药剂的毒力回归方程并计算出EC50,结果表明,烯唑醇、菌核净、嘧肽菌净、退菌特和代森锰锌对该病菌具有较强的抑制作用,可作为防治该病害的首选药剂。
4.明确了烟草靶斑病菌产生的毒素的基本性质及其致病机理。试验通过活性炭吸附、甲醇洗脱、旋转蒸发浓缩的方法,从烟草靶斑病菌的Richard培养滤液中获得了黄褐色粗毒素,该毒素能够使烟草叶片产生与靶斑病类似的病斑,而且还能抑制种子萌发和胚根生长,导致幼苗萎蔫。明确了病菌产毒的最佳培养条件,即pH值6～7的Richard培养液,温度25℃,黑暗每12h振荡1次条件下培养15～20d。对毒素的基本性质进行研究,结果表明该毒素具有热稳定性、光稳定性和耐储藏性,它是一类易溶于水的非蛋白类的极性物质。研究探讨了毒素对烟苗叶片组织中的抗性相关生理生化指标的影响,结果表明,毒素可激活烟草叶片中POD、PPO、CAT和PAL活性,而抑制SOD活性,受害叶片的叶绿素和可溶性糖含量显著下降,而可溶性蛋白、丙二醛和游离脯氨酸含量明显升高,组织浸出液的相对电导率也显著升高,这些指标的变化表明了毒素对烟草有明显的毒害作用,同时也表明了毒素可引起烟草体内的防御反应,以抵抗毒素造成的伤害。不同致病力烟草靶斑病菌菌株的产毒素能力也不同,强致病力菌株YC-9的产毒能力比弱致病力菌株LF-1强。
5.明确了烟草靶斑病菌产生的细胞壁降解酶的致病机理,强致病力菌株的产酶能力显著高于弱致病力菌株。烟草靶斑病菌在人工Marcus培养液中能产生果胶酶(PG、PMG、PGTE、PMTE)和纤维素酶(Cx、β-葡萄糖苷酶),其中PG活性最高。明确了最佳产酶条件,Cx和β-葡萄糖苷酶的最适培养时间为10d,而PG、PMG、PGTE和PMTE为12d；培养最适温度为25℃；Cx、β-葡萄糖苷酶、PG和PMG的最适pH值为5,而PGTE和PMTE的最适pH值为6；静置培养和连续黑暗条件利于酶的产生。烟草叶片不同病斑部位酶活性不同,其中病健交界处细胞壁降解酶活性最高。烟草靶斑病菌与烟草互作过程中细胞壁降解酶活性测定结果表明,病菌侵染叶片后,PG、PMG. PMTE、PGTE、Cx、β-葡萄糖苷酶活性均显著升高,其中Cx和β-葡萄糖苷酶活性最高,其次为PG和PMG,而PMTE和PGTE活性很低。病菌产生的细胞壁降解酶液能够使烟草叶片产生病斑,混合酶对叶片的损伤作用明显高于单一酶,果胶酶的损伤作用高于纤维素酶。对不同致病力烟草靶斑病菌菌株的产酶能力进行比较,结果表明强致病力菌株YC-9的产酶能力显著高于弱致病力菌株LF-1。
6.通过同源获取基因中间片段和RACE方法克隆得到一个与烟草靶斑病菌致病性相关的基因,即病菌的内切多聚半乳糖醛酸酶(endo-PG)基因。病菌endo-PG基因的cDNA全长序列为1399 bp, CDS区长1086 bp,编码361个氨基酸。
Tobacco target spot is a newly-found and seriously harmful leaf disease of tobacco in China in recent years, and quality and yield of tobacco leaves are seriously affected, which runs short of references. Given that, the genetic differentiation, infection characteristics and pathogenic mechanism of Rhizoctonia solani were systematically studied. The results were as follows:
1. Anastomosis group of R. solani isolates from tobacco target spot was first defined to AG-3, and the isolates could be classified into three pathogenic types, including strong pathogenic type, moderate pathogenic type and weak pathogenic type. Fifty-eight isolates were isolated from tobacco target spot disease samples which were collected from Liaoning tobacco productive areas. The test of anastomosis groups (AGs) indicated that all the isolates belong to AG-3. The rDNA ITS sequence of the R. solani strain YC-9 was PCR amplified and sequenced. And then, the ITS sequence was compared and analyzed with the nucleic acid sequences of GenBank database. The result showed that the ITS sequence was 653bp in length, and the homology of ITS sequence of YC-9 and that of the R. solani(AG-3) isolate from tomato foliar blight was 100 percent. The analysis result of rDNA ITS sequence was consistent with that of morphology. Select 16 representative isolates for the analysis of pathogenic type. The result showed that there were significantly different in pathogenicity among isolates by inoculation of the four tobacco varieties, including NC89, K326, Yunyan 85 and G80. They could be classified into three pathogenic types. The result suggested that the R. solani isolates of tobacco target spot had clear pathogenicity differentiation and there was no significant correlation between the distributions of the pathogenic types and geographic locations of R. solani isolates.
2. Genetic diversity of 16 R. solani isolates of tobacco target spot was analyzed by RAPD and ISSR. Heredity variation and evolution of the pathogen were explored on molecular level. The correlation of RAPD or ISSR grouping and geographic location or pathogenicity of the isolates was systematically analyzed.9 RAPD primers and 7 ISSR primers were screened to amplify 16 isolates using optimal reaction system. The results showed that the 9 RAPD primers and 7 ISSR primers obtained 111 and 83 scored bands respectively, and the ratio of polymorphic bands was 64.86% and 68.67% respectively. Similarity coefficient of RAPD among the isolates was from 0.63 to 0.93, and when similarity coefficient was 0.74, the isolates were divided into three groups. Similarity coefficient of ISSR among the isolates was from 0.61 to 0.94, and when similarity coefficient was 0.75, the isolates were divided into three groups. The analysis results of RAPD and ISSR all suggested that there were rich genetic diversity and remarkable genetic differentiation among 16 R. solani isolates of tobacco target spot, and genetic cluster groups had certain correlation with geographic location, but no significant correlation with pathogenicity of the isolates. Genetic diversity of 16 R. solani isolates of tobacco target spot and R. solani isolates of other plant diseases was analyzed by RAPD and ISSR. The results indicated that there was closest genetic relationship among 16 R. solani isolates of tobacco target spot, but they showed remote affinity to R. solani isolates of other plant diseases, and there were greater genetic differences between them.
3. Study on the infection characteristics of R. solani of tobacco target spot, and first define the relationship of interactive pathogenicity between R. solani isolate of tobacco target spot and R. solani isolates of corn sheath blight and rice sheath blight. The results showed that mycelium of R. solani or mycelium with host residue overwintered in the soil, or mycelium in host residue overwintered. R. solani invaded through the stoma and wound of tobacco leaves, and wound was beneficial to the invasion. R. solani could infect tobacco stems of seedling and adult plant stage, and main and lateral veins of tobacco leaves. The study of infection process indicated that R. solani invaded tobacco leaves after inoculation for 24h, hyphae of R. solani expanded rapidly between and within cells after inoculation for 24-48h, and infected cells were damaged and died finally. Optimum infection conditions of R. solani were determined, the optimum temperature was 25-30℃, the longer moist and inoculation time, the more benefit to the infection of R. solani,12h illumination 12h darkness and continuous darkness were good for the infection, but continuous illumination had significant inhibition. The twenty tested tobacco varieties had different resistance to R. solani, VRG2 had stronger disease resistance and the morbidity was 25 percent. The test result of the host range of R. solani demonstrated that R. solani could infect tobacco, tomato, eggplant and capsicum, cucumber, wax gourd and chenopodium amaranticolor, and eggplant had the highest disease rate (97.5 percent). The test result of interactive pathogenicity demonstrated that R. solani of tobacco target spot could not infect rice, corn and cabbage, but R. solani of rice sheath blight, corn sheath blight and cabbage Rhizoctonia leaf rot could infect tobacco leaves. Field experiment was conducted to investigate damage assessment of the disease, and the statistical result showed that there was significantly positive relationship between the loss rate of yield and value and the disease degree. The higher the disease degree, the greater the yield and value loss, and the relational mode was established. The fungistatic actions of ten fungicides to R. solani were determined. The toxicity regression equations of the fungicides were established and EC50 was computed. The results showed that Diniconazole, dimethachlon, Mitaijunjing, Tuzet and Mancozeb could strongly inhibit the colony growth, so they could be used to control the disease.
4. The study defined the basic feature and pathogenic mechanism of toxin of R. solani. The tan crude toxin was obtained from Richard culture filtrate of R. solani by the methods of activated carbon adsorption, methanol elution and rotating evaporation. The biological activity assay of the crude toxin showed that it could induce the characteristic symptom of tobacco target spot, inhibit seed germination and radicle growth, and make seeding wilting. The optimum conditions of producing toxin were as follows:Richard culture filtrate, pH value 6 to 7,25℃,15 to 20 days under dark and vibration 2 times every day. The test result of basic feature of toxin showed that toxin was of thermostability, photostability and reservation, and it was a kind of nonprotein polar substance. Toxin had effect on physiological and biochemical indexes related with resistance of tobacco leaves. The results demonstrated that toxin activated POD, PPO, CAT and PAL, but inhibited activity of SOD. The contents of chlorophyll and soluble sugar of injured tobacco leaves declined remarkably, but the contents of soluble protein, MDA and free proline increased significantly, and the relative conductivity of the leaching solution of leaf tissue also had a sharp increase. The changes of those indexes illustrated that toxin had poisonous effect on tobacco. Meanwhile, it also showed that toxin could trigger defensive reactions of tobacco. R. solani isolates of different pathogenicity also had different abilities of producing toxin. The ability of strong pathogenic isolate YC-9 was stronger than weak pathogenic isolate LF-1.
5. The study defined pathogenic mechanism of cell wall degrading enzyme of R. solani, and the ability of producing enzyme of strong pathogenic isolate was significantly stronger than weak pathogenic isolate. R. solani could produce pectinase (PG, PMG, PGTE and PMTE) and cellulase (Cx andβ-glucosidase) in Marcus culture solution, and the activity of PG was the highest. The optimum conditions of producing enzyme were determined. The optimum culture time of Cx andβ-glucosidase was 10d, and that of PG, PMG, PGTE and PMTE was 12d; the optimum culture temperature was 25℃; the optimum pH of Cx,β-glucosidase, PG and PMG was 5, and that of PGTE and PMTE was 6; and static culture and continuous darkness were good for the production of enzyme. The enzyme activities of different disease spot parts were also different, and the activities of enzymes in the junction parts of disease and health were the highest. Activity of cell wall degrading enzyme was determined during interaction process of R. solani and tobacco. The result showed that the activities of PG, PMG, PGTE, PMTE, Cx andβ-glucosidase of tobacco leaves infected by R. solani increased markedly. Among these enzymes, the activities of Cx andβ-glucosidase were the highest, followed by PG and PMG, but the activities of PMTE and PGTE were the lowest. Cell wall degrading enzyme of R. solani could make tobacco leaf produce disease spot, the damage effect of mixed enzymes was notably higher than single enzyme, and the damage effect of pectinase was higher than cellulase. Moreover, the abilities of producing enzyme of isolates of different pathogenicity were compared. The result showed that the ability of strong pathogenic isolate YC-9 was stronger than weak pathogenic isolate LF-1.
6. A homologous cDNA fragment encoding the endo-polygalacturonase gene from R. solani was obtained. The method of RACE was used to clone and generate full-length cDNA. The cDNA full length of endo-PG gene of R. solani was 1399 bp. The length of CDS was 1086 bp and it encoded 361 amino acids.
引文
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