川芎根腐病病原鉴定、生物学特性和同工酶研究
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摘要
川芎为伞形科(Umbelliferae)藁本属植物川芎(Ligusticum chuanxiong Hort)的干燥根茎,是著名的川产道地药材,具有近两千年的栽种和使用历史。
川芎根腐病[Fusarium solani(Mart) Sacc]是四川省都江堰市川芎种植基地的一种重要病害,2006年,该病田间的发病株率为6.08%～45.47%,平均为23.61%,重病田达68.46%,造成严重危害,已成为该片区川芎上的主要病害。本试验是对川芎根腐病的症状、病原鉴定、生物学特性、室内药剂筛选和田间药剂防治及同工酶活性进行的研究,结果如下:
1川芎根腐病的症状
川芎根腐病是一种土传病害,在川芎的整个生长期都会发生。发病初期,地上部从外围的叶片开始褪色发黄,并逐渐向心叶扩展。地下块茎的病部初呈褐色至红褐色,部分变为水渍状,随着病情的发展,病株叶尖、叶缘开始焦枯。感病块茎髓部变黑,并且向上发展,褐色渐浅。后期感病块茎内部腐烂,呈黄褐色浆糊状,有特殊臭味,呈软腐状;最后整个植株停止生长、枯死。
2川芎根腐病的分离和培养性状
经组织分离得到川芎根腐病菌纯培养。所有分离菌株在PDA平板上的形态特征基本一致,2d后出现白色至浅灰色菌丝,3d后开始产孢,菌丝有隔,絮状,菌落正面有同心轮纹,菌落反面呈淡紫色。
3分离菌的回接与病原鉴定
在室温条件下,用分生孢子悬浮液浸根接种川芎的根部,回接植株15d后全部发病,症状与田间症状相同,而CK不发病。从发病的部位进行常规分离再次分离到相同的病菌。由此证明该分离菌是川芎根腐病的病原菌。
根据病原菌的形态特征、培养性状观察和rDNA-ITS的序列分析。参照张中义等《植物病原真菌学》和Booth的镰刀菌分类系统,将该病原菌鉴定为茄腐镰孢菌[Fusarium solani(Mart)Sacc],属于半知菌亚门(Deuteromycotina)、丝孢纲(Hyphomycetes),瘤座孢目(Tuberculariales),瘤座孢科(Tuberculariaceae),,镰刀菌属(Fusarium)。由[Fusarium solani(Mart)Sacc]引起的川芎根腐病是国内外首次报道。
4川芎根腐病菌的生物学特性
本研究明确了不同培养基、碳源、氮源、温度、pH值以及光照等对川芎根腐病菌菌丝生长、产孢以及分生孢子萌发的影响。在不同的培养基中,病原菌在PSA上菌丝生长和产孢最好;可溶性淀粉培养基上菌丝生长最差;在PS培养基上产孢最差;温度在25～30℃间适于菌丝的生长,最适温28℃,低于10℃或高于40℃均会对孢子产生及萌发产生抑制作用,高温会造成孢子畸形;pH 5～8之间适于病原菌的生长,最适生长pH6,pH过低或过高均会造成对病原菌生长的影响;不同光照条件处理表明:各处理对菌丝生长和产孢的影响无显著性差异;菌丝生长最适碳源为蔗糖,产孢最适碳源为果糖,乳糖最差;菌丝生长在硝酸钾上生长最好,在硫酸铵生长最差,牛肉膏产孢最好,氯化铵产孢最差。
分生孢子萌发适温20℃～35℃;最适28℃,RH从90%～100%和水滴中均可萌发,水滴最好,低于90%不萌发;适宜pH 5～7,最好pH6,萌发率71.73%,高于pH 8不萌发;分生孢子致死温度为53℃。
5室内药剂筛选和田间药剂防治
8种药剂(根腐菌、卡菌丹、根必治、甲基托布津、代森锰锌、百菌清、敌克松一号、地菌统克)筛选结果表明,对病原菌菌丝生长和产孢抑制效果最好的是根必治、甲基托布津,达到极显著水平(菌落直径为2.20cm和1.75cm,产孢量为0.92×10~7个/皿和0.68×10~7个/皿);百菌清和根腐灵对病原菌的抑制次之(菌落直径为2.93cm和3.30cm,产孢量为0.52×10~7个/皿和1.8×10~7个/皿);敌克松一号和卡菌丹对病原菌无抑制作用。
对病原菌分生孢子萌发抑制效果最好的是百菌清、代森锰锌,抑制萌发率达到100%;根必治、地菌统克、根腐灵次之,抑制萌发率分别为99.8%、99.5%和98.7%;敌克松一号和卡菌丹对分生孢子萌发抑制较差,仅为30.4%和14.6%。
选用4种药剂进行田间药效试验,药剂防治试验表明:百菌清、代森锰锌、甲基托布津和根必治的病株率和防效均达到极显著水平。
6茄腐镰刀菌对川芎同工酶活性的影响
同工酶试验表明:接种后,POD酶活性表现为先上升而后下降的趋势,在接种后第7 d时达到最大值,峰值为274.335,为对照的5.43倍,而后缓慢下降,到第12 d时下降到对照的水平,而对照的变化一直较为平稳。PPO酶活性表现为先上升而后下降的趋势,在接种后第4d时活性开始增大,到第7 d时,峰值为为对照的2.82倍,而后缓慢下降,到第9 d达到最大值出现第二个峰值,为76,而后逐渐下降,到第12 d时下降到对照的水平,而对照的变化一直较为平稳。
Ligusticum chuanxiong belongs to Umbelliferae of Ligusticum L.It is famouse mainland medicinal materials in Si-chuan province,it has two thousands growed and used history.
The Ligusticum chuanxiong root rot was severity disease in Du-Jiang Yan basement of Si chuan province.In 2006,the percentage of the disease reached 6.08%～45.47%,the average was 23.61%,which caused the decline and death of the Ligusticum chuanxiong.The dissertation included the symptom characteristics,the identification of pathogen,the biological characteristics,the fungicides screening in Laboratory for Ligusticum chuanxiong root rot and the POD,PPO isozyme activity.
1 The sympyom characteristics of Ligusticum chuanxiong root rot
The original symptoms indicated aboveground leaf fading and yellowing,underground tuber presented brown and mahogany,The later symptoms indicated tuber rot inside,presented filemot,finally,the whole plant stoped growing and die.
2 Isolation and cultural characteristics of Ligusticum chuanxiong root rot
Isolate the diseased root,and gain the pathogen.The pathogen is cultured on PDA medium,the colony is white,circular,and purple later.The spores begin to be produced after 3 days and the pile of spores is white.
3 Inoculation and identification
The diseased symptom and the pathogen characteristics of inoculated plant,were similarly to the diseased plant in field.It is improved that the isolating was the pathogen of Ligusticum chuanxiong root rot.After the morphological characteristics and the sequence of ribosomal DNA-ITS,the pathogen was identified as Fusarium solani(Mart)Sacc.
3 Biological characteristics
The pathogen[Fusarium solani(Mart) Sacc.]of Ligusticum chuanxiong root rot.can produce many spores and grow best on PSA from different media.There are 7 kinds of carbon and nitrogen sources,which are best for the mycelial growth and spore production,the optimum C source were sucrose,spores occurr on fruit sugar media.Among nitrogen sources, potassium nitrate is the best for mycelial growth.The spore formation is best on beef extract. For the growth and ascospore production of it,the best medium was PSA,the optimum temperature was 28℃and the optimum pH was 6.Whole light promote spore formation.
For spore germination of the pathogen,the optimum temperature and relative humidity(RH) are 20～35℃and 90%～100%,respectively,and the best condition is 28℃and in drops of water.The spores faile to germinated at temperatures higher than 40℃or lower than 10℃and at an RH of＜90%.The optimum pH for spore germination is in the range of 5～7,and the spores faile to germinate at pH higher than 8.The spores will be killed at or over 53℃.
5 Select fungicides and field trial
The inhibiting effects of 8 fungicides on Fusarium solani(Mart) Sacc.,the results demonstrated that the best fungicides of inhibiting the the mycelia growth and spores formation are Genbizhi and thiophanate methyl,the second are chlorothalonil and fenmaminofulf,the worst are fenmaminofulf-fulphuy and Kajundan.
The results demonstrats that the best fungicides of inhibiting conidial germination are chlorothalonil and mancozeb,the percentage of spore germination are both 100%;the second are Genbizhi,bactericide and fenmaminofulf,the percentage of spore germination are 99.8%、99.5%and 98.7%;the worst are fenmaminofulf-fulphuy and Kajundan.,the percentage of spore germination are 30.4%and 14.6%.
Field control had demonstrated that 4 fungicides are all markedness,thiophanate methyl and Genbizhi are lower it.
6 Isozyme activity's variety of Ligusticum chuanxiong damaged by Fusarium solani(Mart) Sacc
Isozyme activity dissertation showed that:After inoculability,POD isozyme activity's variety ascended firstly then descended,it reached max after 7 days,the peak value was 274.335,5.43 multiple than antitheses,it came down as antitheses after 12 days,the antitheses calmly during those period.PPO isozyme activity's variety ascend firstly then descend,it became accretion after 4 days,reach max at 7th day,the peak value was 2.82 multiple than antitheses,it appeared second peak value at 9th day,12 days later it came down as antitheses. the antitheses calmly also.
川芎根腐病[Fusarium solani(Mart) Sacc]是四川省都江堰市川芎种植基地的一种重要病害,2006年,该病田间的发病株率为6.08%～45.47%,平均为23.61%,重病田达68.46%,造成严重危害,已成为该片区川芎上的主要病害。本试验是对川芎根腐病的症状、病原鉴定、生物学特性、室内药剂筛选和田间药剂防治及同工酶活性进行的研究,结果如下:
1川芎根腐病的症状
川芎根腐病是一种土传病害,在川芎的整个生长期都会发生。发病初期,地上部从外围的叶片开始褪色发黄,并逐渐向心叶扩展。地下块茎的病部初呈褐色至红褐色,部分变为水渍状,随着病情的发展,病株叶尖、叶缘开始焦枯。感病块茎髓部变黑,并且向上发展,褐色渐浅。后期感病块茎内部腐烂,呈黄褐色浆糊状,有特殊臭味,呈软腐状;最后整个植株停止生长、枯死。
2川芎根腐病的分离和培养性状
经组织分离得到川芎根腐病菌纯培养。所有分离菌株在PDA平板上的形态特征基本一致,2d后出现白色至浅灰色菌丝,3d后开始产孢,菌丝有隔,絮状,菌落正面有同心轮纹,菌落反面呈淡紫色。
3分离菌的回接与病原鉴定
在室温条件下,用分生孢子悬浮液浸根接种川芎的根部,回接植株15d后全部发病,症状与田间症状相同,而CK不发病。从发病的部位进行常规分离再次分离到相同的病菌。由此证明该分离菌是川芎根腐病的病原菌。
根据病原菌的形态特征、培养性状观察和rDNA-ITS的序列分析。参照张中义等《植物病原真菌学》和Booth的镰刀菌分类系统,将该病原菌鉴定为茄腐镰孢菌[Fusarium solani(Mart)Sacc],属于半知菌亚门(Deuteromycotina)、丝孢纲(Hyphomycetes),瘤座孢目(Tuberculariales),瘤座孢科(Tuberculariaceae),,镰刀菌属(Fusarium)。由[Fusarium solani(Mart)Sacc]引起的川芎根腐病是国内外首次报道。
4川芎根腐病菌的生物学特性
本研究明确了不同培养基、碳源、氮源、温度、pH值以及光照等对川芎根腐病菌菌丝生长、产孢以及分生孢子萌发的影响。在不同的培养基中,病原菌在PSA上菌丝生长和产孢最好;可溶性淀粉培养基上菌丝生长最差;在PS培养基上产孢最差;温度在25～30℃间适于菌丝的生长,最适温28℃,低于10℃或高于40℃均会对孢子产生及萌发产生抑制作用,高温会造成孢子畸形;pH 5～8之间适于病原菌的生长,最适生长pH6,pH过低或过高均会造成对病原菌生长的影响;不同光照条件处理表明:各处理对菌丝生长和产孢的影响无显著性差异;菌丝生长最适碳源为蔗糖,产孢最适碳源为果糖,乳糖最差;菌丝生长在硝酸钾上生长最好,在硫酸铵生长最差,牛肉膏产孢最好,氯化铵产孢最差。
分生孢子萌发适温20℃～35℃;最适28℃,RH从90%～100%和水滴中均可萌发,水滴最好,低于90%不萌发;适宜pH 5～7,最好pH6,萌发率71.73%,高于pH 8不萌发;分生孢子致死温度为53℃。
5室内药剂筛选和田间药剂防治
8种药剂(根腐菌、卡菌丹、根必治、甲基托布津、代森锰锌、百菌清、敌克松一号、地菌统克)筛选结果表明,对病原菌菌丝生长和产孢抑制效果最好的是根必治、甲基托布津,达到极显著水平(菌落直径为2.20cm和1.75cm,产孢量为0.92×10~7个/皿和0.68×10~7个/皿);百菌清和根腐灵对病原菌的抑制次之(菌落直径为2.93cm和3.30cm,产孢量为0.52×10~7个/皿和1.8×10~7个/皿);敌克松一号和卡菌丹对病原菌无抑制作用。
对病原菌分生孢子萌发抑制效果最好的是百菌清、代森锰锌,抑制萌发率达到100%;根必治、地菌统克、根腐灵次之,抑制萌发率分别为99.8%、99.5%和98.7%;敌克松一号和卡菌丹对分生孢子萌发抑制较差,仅为30.4%和14.6%。
选用4种药剂进行田间药效试验,药剂防治试验表明:百菌清、代森锰锌、甲基托布津和根必治的病株率和防效均达到极显著水平。
6茄腐镰刀菌对川芎同工酶活性的影响
同工酶试验表明:接种后,POD酶活性表现为先上升而后下降的趋势,在接种后第7 d时达到最大值,峰值为274.335,为对照的5.43倍,而后缓慢下降,到第12 d时下降到对照的水平,而对照的变化一直较为平稳。PPO酶活性表现为先上升而后下降的趋势,在接种后第4d时活性开始增大,到第7 d时,峰值为为对照的2.82倍,而后缓慢下降,到第9 d达到最大值出现第二个峰值,为76,而后逐渐下降,到第12 d时下降到对照的水平,而对照的变化一直较为平稳。
Ligusticum chuanxiong belongs to Umbelliferae of Ligusticum L.It is famouse mainland medicinal materials in Si-chuan province,it has two thousands growed and used history.
The Ligusticum chuanxiong root rot was severity disease in Du-Jiang Yan basement of Si chuan province.In 2006,the percentage of the disease reached 6.08%～45.47%,the average was 23.61%,which caused the decline and death of the Ligusticum chuanxiong.The dissertation included the symptom characteristics,the identification of pathogen,the biological characteristics,the fungicides screening in Laboratory for Ligusticum chuanxiong root rot and the POD,PPO isozyme activity.
1 The sympyom characteristics of Ligusticum chuanxiong root rot
The original symptoms indicated aboveground leaf fading and yellowing,underground tuber presented brown and mahogany,The later symptoms indicated tuber rot inside,presented filemot,finally,the whole plant stoped growing and die.
2 Isolation and cultural characteristics of Ligusticum chuanxiong root rot
Isolate the diseased root,and gain the pathogen.The pathogen is cultured on PDA medium,the colony is white,circular,and purple later.The spores begin to be produced after 3 days and the pile of spores is white.
3 Inoculation and identification
The diseased symptom and the pathogen characteristics of inoculated plant,were similarly to the diseased plant in field.It is improved that the isolating was the pathogen of Ligusticum chuanxiong root rot.After the morphological characteristics and the sequence of ribosomal DNA-ITS,the pathogen was identified as Fusarium solani(Mart)Sacc.
3 Biological characteristics
The pathogen[Fusarium solani(Mart) Sacc.]of Ligusticum chuanxiong root rot.can produce many spores and grow best on PSA from different media.There are 7 kinds of carbon and nitrogen sources,which are best for the mycelial growth and spore production,the optimum C source were sucrose,spores occurr on fruit sugar media.Among nitrogen sources, potassium nitrate is the best for mycelial growth.The spore formation is best on beef extract. For the growth and ascospore production of it,the best medium was PSA,the optimum temperature was 28℃and the optimum pH was 6.Whole light promote spore formation.
For spore germination of the pathogen,the optimum temperature and relative humidity(RH) are 20～35℃and 90%～100%,respectively,and the best condition is 28℃and in drops of water.The spores faile to germinated at temperatures higher than 40℃or lower than 10℃and at an RH of＜90%.The optimum pH for spore germination is in the range of 5～7,and the spores faile to germinate at pH higher than 8.The spores will be killed at or over 53℃.
5 Select fungicides and field trial
The inhibiting effects of 8 fungicides on Fusarium solani(Mart) Sacc.,the results demonstrated that the best fungicides of inhibiting the the mycelia growth and spores formation are Genbizhi and thiophanate methyl,the second are chlorothalonil and fenmaminofulf,the worst are fenmaminofulf-fulphuy and Kajundan.
The results demonstrats that the best fungicides of inhibiting conidial germination are chlorothalonil and mancozeb,the percentage of spore germination are both 100%;the second are Genbizhi,bactericide and fenmaminofulf,the percentage of spore germination are 99.8%、99.5%and 98.7%;the worst are fenmaminofulf-fulphuy and Kajundan.,the percentage of spore germination are 30.4%and 14.6%.
Field control had demonstrated that 4 fungicides are all markedness,thiophanate methyl and Genbizhi are lower it.
6 Isozyme activity's variety of Ligusticum chuanxiong damaged by Fusarium solani(Mart) Sacc
Isozyme activity dissertation showed that:After inoculability,POD isozyme activity's variety ascended firstly then descended,it reached max after 7 days,the peak value was 274.335,5.43 multiple than antitheses,it came down as antitheses after 12 days,the antitheses calmly during those period.PPO isozyme activity's variety ascend firstly then descend,it became accretion after 4 days,reach max at 7th day,the peak value was 2.82 multiple than antitheses,it appeared second peak value at 9th day,12 days later it came down as antitheses. the antitheses calmly also.
引文
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