水稻品种抗稻曲病评价及稻曲病菌遗传多样性研究
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摘要
水稻稻曲病(Rice False Smut)是一种水稻穗期病害,在世界主要稻作区都有发生。稻曲病病原菌有性态为子囊菌门麦角菌属稻麦角菌Clavicepsoryzae-sativa Has.,无性态为半知菌门绿核菌属Ustilaginoideavirens(Cooke)Takahashi。稻曲病“菌粉”呈墨绿色或黑色,混入稻谷后影响外观,降低品质。而且稻曲病菌在代谢过程中产生有色毒素C_9H_6O_7及一种类似麦角碱的生物碱,对人、畜、家禽等造成危害,近年来稻曲病的发生有加重的趋势。仅2004年湖南因稻曲病损失稻谷1.37亿kg,直接经济损失2亿元。
2006年在衡南县对自然发病的12个品种,进行稻曲病分布型调查,调查的数据经DPS软件中的种群空间分布型聚集度指标测定进行空间分布型分析。结果表明稻曲病属聚集分布型。比较多种调查方法的调查结果,认为五点取样方法最好。
将3个主要发病组分,即病穗率、平均每穗病粒数、平均每千粒含病粒数进行主成分分析,结果得出平均每穗病粒数为主要组分,用此组分对101个品种用类平均聚类法进行聚类,在阈值取0.400时,将这101个水稻品种分成6类,其品种的田间抗性级别对应划分为6级,即0级:平均每穗病粒数为0.000～0.111;1级:平均每穗病粒数0.112～0.400;3级:平均每穗病粒数0.401～0.610;5级:平均每穗病粒数0.611～1.010;7级:平均每穗病粒数1.011～1.760;9级:平均每穗病粒数≧1.761。根据此分级标准,将水稻品种对稻曲病的抗性划分为6个类型:0为高抗品种(HR);1级为抗性品种(R);3级为中抗品种(MR);5级为中感品种(MS);7级为感病品种(S);9级为高感品种(HS)。
对15个品种每穗病粒数与千粒重、产量损失率、空秕率分别作回归分析,结果表明每穗病粒数与千粒重之间存在着极显著的负相关关系,与产量损失率、空秕率存在着极显著的正相关关系。
2007年对湖南33个县(市、区)进行了稻曲病发生情况调查,统计结果显示,稻曲病在湖南省发生比较普遍,无论是杂交稻还是常规稻,均有不同程度发生。湖南主栽水稻品种普遍表现抗病性不强,且各品种(组合)之间田间抗性差别不显著。田间表现为较抗的杂交水稻品种(组合)的母本分别来自于准S、川香28A(29A)、湘8A、珍汕97A。不同产稻区大致的发病严重度趋势是丘陵＞山区＞平川。
对12个不同抗、感病品种在抽穗前7～10d,用注射接种稻曲病菌分生孢子,自接种后1d,3d,5d,7d,测定品种的苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)活性,结果表明抗病品种的这3种酶活性在接种后迅速提高,变化幅度较大;而感病品种的则在接种后酶缓慢增加,变化幅度不大。
稻曲病菌因生长缓慢,病谷粒所带杂菌较多,常伴生大量的细菌和真菌,因而在室内分离、培养与纯化方面存在一定的难度,以往的分离成功率很低。经笔者实验比较,发现采用稻曲球不同层次组织块分离法是比较理想地得到菌株的方法。胁本哲氏培养基是目前比较适宜稻曲病菌生长的培养基。
选用10个随机引物,利用RAPD技术,分析了来自于湖南24个县(区)的53个稻曲病菌株的群体遗传结构。结果表明:在遗传相似系数0.78的水平上,将供试的53个菌株划分为5个谱系,即Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ。这5个谱系所含菌株数分别为42个、7个、2个、1个和1个,分别占总株数的79%、13%、4%、2%和2%,其谱系Ⅰ为优势谱系。由此说明湖南稻曲病菌遗传群体有分化,但分化不明显;来自同一地区和不同地区的同一品种的菌株大多数聚集在同一类。难以划分出明显的地理宗谱,据此初步认为稻曲病菌是一种遗传比较稳定的病原菌。
经2007年在株洲攸县城关镇和花垣县吉卫农场进行稻曲病田间药效试验,外资企业美国陶氏益农公司生产的应得24%悬浮剂、15%三唑醇可湿性粉剂;拜耳作物科学(中国)有限公司生产的好力克430克/升SC;瑞士先正达作物保护有限公司生产的30%苯醚甲·丙环唑EC(爱苗)平均防效均在80%以上。而国内扬州市苏灵农药化工有限公司生产的15.5%井岗霉素·三唑酮可湿性粉剂和吉林省八达农药有限公司生产的300克/升苯醚甲环唑·丙环唑乳油田间防治效果也达到75%以上。
Rice false smut is an important grain disease,caused by Ustilaginodea virens (cooke) Tak,after booting in worldwide rice.The sexual stage of the pathogens is Claviceps oryzae-sativa Hashioka,and its asexual stage is Ustilaginoidea virens(Cooke)Takahashi.It is not only greatly decrease both yield and quality of rice, but its may also cause the toxicosis incidents of human and animal from taking rice which contains high level of ustiloxins happened usually,to human and animals.Recent years,the disease has become more and more serious,such as loss 137 millions kg rice (equivalent to200 million RMB ) in Hunan Province in 2004.
The investigation for rice false smut spatial distributing of 12 cultivars was carried out in rice area of Hengnan County in 2006.DPS software was used to analyse the spatial distributing aggregation indexes determination of the survey data.The data The results showed that the spatial distributing of rice false smut subjected to aggregation distributing pattern and the five-point sampling is the best method through compared with the results of the other investigation methods.
The main components analysis among the three components viz.disease grain number of the average each panicle(X1),percentage of infected panicle(X2),disease grain number in1000 grains(X3),displayed that X1 was the main component,.Then X1 used as index of clustering analysis in 101 varieties with cluster-average method.The result clearly indicated the 101 rice varieties were divided into 6 groups at 0.40 threshold level,which the matching scale value(X1) of field resistance of the 6 group verities were 0:0.000～0.111(height resistance,HR);1:0.112～0.400(resistance,R);3: 0.401～0.610(moderately resistant,MR);5:0.611～1.010(moderately susceptible,SR); 7:1.011～1.760(susceptible,S);9:＞1.761(highly susceptible),separately.
The regression analysis was based on X1 with 1000-grain weighty(g),yield loss rate and sterile grain rate of 15 cultivars.The results showed that relationship between X1 and 1000-grain weighty was obviously negative,and between X1 and both sterile grain and yield loss rate was significantly positive.
Rice false smut of different cultivars of 33 counties and districts in Hunan in 2007 was investigated.The results showed that both the disease on hybrid rice and conventional cultivars are common occurrence,but severity level was obviously variation.As usual,the main cultivated cultivars in Hunan illustrate low level resistance. Besides,the hybrid cultivars using Zhun-S,Chuanxiang28A(29A),Xiang8A, Zhenshan97A as sterile line suggested having better resistant to the disease.The severity of rice false smut in different rice cultured areas displayed that the mountainous area was more serious than hills and the plain area.
PAL,POD,PPO activity of 12 cultivars,1d,3d,5d,7d after inoculating conidia of rice false smut with the injection at 7～10d before heading was detected.The results showed that these enzyme's activity quickly enhanced and changed in the resistant cultivars after inoculating,but slowly in the susceptibility cultivars.
The pure false smut fungus is very difficult isolated in laboratory because it slowly grows and is usually associated with a lot of bacteria and fungi on the spore balls.The tissue isolation in the study was the most convenient and effective method of isolates obtained of Ustilaginodea virens in several isolation methods and Xieben medium in the several media was the best one to get pure culture.
Genomic fingerprints of 53 strains of U.virens from Hunan 24 counties and districts were analyzed by using 10 RAPD primers.The results showed that:all strains tested were divided into five genetic lineages(Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ) at 0.78 level of the genetic similarity through the clustering method of UPGMA.The five genetic lineages separately held 42,7,2,1 and 1 strains and they accounted for the rates of 79%,13%, 4%,2%and 2%in total strains,respectively.It was also obviously that the lineage I was the dominant one with the rates of 79%.The resultt showed that the genetic diversity of U.virens in Hunan was not significant.Besides,most strains obtained from the same cultivar in same and the different regions were grouped in the same lineage.The preliminary analysis showed that the genetic diversity of U.virens in Hunan was not significant.
The screening of fungicide against rice false smut was conducted in Chengguan Township,YouCounty and Jiwei farm Huayuan.the results showed that efficacys of 24%Fenbuconazole SC and 15%triadimenol WP produced by Dow AgroSciences U.S.,430g/L Tebuconazole SC produced by Bayer CropScience(China) Co.Ltd.and 30%Difenoconazole·propiconazol EC,produced by Syngenta,against rice false smut, were all at above 80%.While,impacts of 15.5%Jianggangmeisu·triadimefon WP produced by Suling agricultural chemicals Co.Ltd in Yangzhou.,and 300g/L Difenoconazole·propiconazol EC produced by Hengda Co.Ltd.in Jilin,on the diseas possessed about 75%.
2006年在衡南县对自然发病的12个品种,进行稻曲病分布型调查,调查的数据经DPS软件中的种群空间分布型聚集度指标测定进行空间分布型分析。结果表明稻曲病属聚集分布型。比较多种调查方法的调查结果,认为五点取样方法最好。
将3个主要发病组分,即病穗率、平均每穗病粒数、平均每千粒含病粒数进行主成分分析,结果得出平均每穗病粒数为主要组分,用此组分对101个品种用类平均聚类法进行聚类,在阈值取0.400时,将这101个水稻品种分成6类,其品种的田间抗性级别对应划分为6级,即0级:平均每穗病粒数为0.000～0.111;1级:平均每穗病粒数0.112～0.400;3级:平均每穗病粒数0.401～0.610;5级:平均每穗病粒数0.611～1.010;7级:平均每穗病粒数1.011～1.760;9级:平均每穗病粒数≧1.761。根据此分级标准,将水稻品种对稻曲病的抗性划分为6个类型:0为高抗品种(HR);1级为抗性品种(R);3级为中抗品种(MR);5级为中感品种(MS);7级为感病品种(S);9级为高感品种(HS)。
对15个品种每穗病粒数与千粒重、产量损失率、空秕率分别作回归分析,结果表明每穗病粒数与千粒重之间存在着极显著的负相关关系,与产量损失率、空秕率存在着极显著的正相关关系。
2007年对湖南33个县(市、区)进行了稻曲病发生情况调查,统计结果显示,稻曲病在湖南省发生比较普遍,无论是杂交稻还是常规稻,均有不同程度发生。湖南主栽水稻品种普遍表现抗病性不强,且各品种(组合)之间田间抗性差别不显著。田间表现为较抗的杂交水稻品种(组合)的母本分别来自于准S、川香28A(29A)、湘8A、珍汕97A。不同产稻区大致的发病严重度趋势是丘陵＞山区＞平川。
对12个不同抗、感病品种在抽穗前7～10d,用注射接种稻曲病菌分生孢子,自接种后1d,3d,5d,7d,测定品种的苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)活性,结果表明抗病品种的这3种酶活性在接种后迅速提高,变化幅度较大;而感病品种的则在接种后酶缓慢增加,变化幅度不大。
稻曲病菌因生长缓慢,病谷粒所带杂菌较多,常伴生大量的细菌和真菌,因而在室内分离、培养与纯化方面存在一定的难度,以往的分离成功率很低。经笔者实验比较,发现采用稻曲球不同层次组织块分离法是比较理想地得到菌株的方法。胁本哲氏培养基是目前比较适宜稻曲病菌生长的培养基。
选用10个随机引物,利用RAPD技术,分析了来自于湖南24个县(区)的53个稻曲病菌株的群体遗传结构。结果表明:在遗传相似系数0.78的水平上,将供试的53个菌株划分为5个谱系,即Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ。这5个谱系所含菌株数分别为42个、7个、2个、1个和1个,分别占总株数的79%、13%、4%、2%和2%,其谱系Ⅰ为优势谱系。由此说明湖南稻曲病菌遗传群体有分化,但分化不明显;来自同一地区和不同地区的同一品种的菌株大多数聚集在同一类。难以划分出明显的地理宗谱,据此初步认为稻曲病菌是一种遗传比较稳定的病原菌。
经2007年在株洲攸县城关镇和花垣县吉卫农场进行稻曲病田间药效试验,外资企业美国陶氏益农公司生产的应得24%悬浮剂、15%三唑醇可湿性粉剂;拜耳作物科学(中国)有限公司生产的好力克430克/升SC;瑞士先正达作物保护有限公司生产的30%苯醚甲·丙环唑EC(爱苗)平均防效均在80%以上。而国内扬州市苏灵农药化工有限公司生产的15.5%井岗霉素·三唑酮可湿性粉剂和吉林省八达农药有限公司生产的300克/升苯醚甲环唑·丙环唑乳油田间防治效果也达到75%以上。
Rice false smut is an important grain disease,caused by Ustilaginodea virens (cooke) Tak,after booting in worldwide rice.The sexual stage of the pathogens is Claviceps oryzae-sativa Hashioka,and its asexual stage is Ustilaginoidea virens(Cooke)Takahashi.It is not only greatly decrease both yield and quality of rice, but its may also cause the toxicosis incidents of human and animal from taking rice which contains high level of ustiloxins happened usually,to human and animals.Recent years,the disease has become more and more serious,such as loss 137 millions kg rice (equivalent to200 million RMB ) in Hunan Province in 2004.
The investigation for rice false smut spatial distributing of 12 cultivars was carried out in rice area of Hengnan County in 2006.DPS software was used to analyse the spatial distributing aggregation indexes determination of the survey data.The data The results showed that the spatial distributing of rice false smut subjected to aggregation distributing pattern and the five-point sampling is the best method through compared with the results of the other investigation methods.
The main components analysis among the three components viz.disease grain number of the average each panicle(X1),percentage of infected panicle(X2),disease grain number in1000 grains(X3),displayed that X1 was the main component,.Then X1 used as index of clustering analysis in 101 varieties with cluster-average method.The result clearly indicated the 101 rice varieties were divided into 6 groups at 0.40 threshold level,which the matching scale value(X1) of field resistance of the 6 group verities were 0:0.000～0.111(height resistance,HR);1:0.112～0.400(resistance,R);3: 0.401～0.610(moderately resistant,MR);5:0.611～1.010(moderately susceptible,SR); 7:1.011～1.760(susceptible,S);9:＞1.761(highly susceptible),separately.
The regression analysis was based on X1 with 1000-grain weighty(g),yield loss rate and sterile grain rate of 15 cultivars.The results showed that relationship between X1 and 1000-grain weighty was obviously negative,and between X1 and both sterile grain and yield loss rate was significantly positive.
Rice false smut of different cultivars of 33 counties and districts in Hunan in 2007 was investigated.The results showed that both the disease on hybrid rice and conventional cultivars are common occurrence,but severity level was obviously variation.As usual,the main cultivated cultivars in Hunan illustrate low level resistance. Besides,the hybrid cultivars using Zhun-S,Chuanxiang28A(29A),Xiang8A, Zhenshan97A as sterile line suggested having better resistant to the disease.The severity of rice false smut in different rice cultured areas displayed that the mountainous area was more serious than hills and the plain area.
PAL,POD,PPO activity of 12 cultivars,1d,3d,5d,7d after inoculating conidia of rice false smut with the injection at 7～10d before heading was detected.The results showed that these enzyme's activity quickly enhanced and changed in the resistant cultivars after inoculating,but slowly in the susceptibility cultivars.
The pure false smut fungus is very difficult isolated in laboratory because it slowly grows and is usually associated with a lot of bacteria and fungi on the spore balls.The tissue isolation in the study was the most convenient and effective method of isolates obtained of Ustilaginodea virens in several isolation methods and Xieben medium in the several media was the best one to get pure culture.
Genomic fingerprints of 53 strains of U.virens from Hunan 24 counties and districts were analyzed by using 10 RAPD primers.The results showed that:all strains tested were divided into five genetic lineages(Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ) at 0.78 level of the genetic similarity through the clustering method of UPGMA.The five genetic lineages separately held 42,7,2,1 and 1 strains and they accounted for the rates of 79%,13%, 4%,2%and 2%in total strains,respectively.It was also obviously that the lineage I was the dominant one with the rates of 79%.The resultt showed that the genetic diversity of U.virens in Hunan was not significant.Besides,most strains obtained from the same cultivar in same and the different regions were grouped in the same lineage.The preliminary analysis showed that the genetic diversity of U.virens in Hunan was not significant.
The screening of fungicide against rice false smut was conducted in Chengguan Township,YouCounty and Jiwei farm Huayuan.the results showed that efficacys of 24%Fenbuconazole SC and 15%triadimenol WP produced by Dow AgroSciences U.S.,430g/L Tebuconazole SC produced by Bayer CropScience(China) Co.Ltd.and 30%Difenoconazole·propiconazol EC,produced by Syngenta,against rice false smut, were all at above 80%.While,impacts of 15.5%Jianggangmeisu·triadimefon WP produced by Suling agricultural chemicals Co.Ltd in Yangzhou.,and 300g/L Difenoconazole·propiconazol EC produced by Hengda Co.Ltd.in Jilin,on the diseas possessed about 75%.
引文
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