金刚藤叶斑病病原学、发生规律及防治技术研究
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摘要
金刚藤(Smilax china)是百合科菝葜属多年生植物,是一种民间常用中药,其根、茎、叶均可入药,具有抗菌消炎、镇痛、活血化瘀、清热利湿、强筋壮骨、消肿毒、祛风湿、补气活血、抗肿瘤和增强免疫功能等功效。湖北福人药业股份有限公司引入来自全国各地的野生品种,在通城县大坪乡建立了金刚藤种植基地。该基地最近几年叶斑病发生严重,发病严重田块叶发病率和病情指数分别达到78.9%和58.7,大大降低药材产量和质量。目前,未见对该病研究的报道。本研究从该病的病原学入手,在病原鉴定和病菌生物学特性研究的基础上,结合生产实际,根据病害发生特点和规律,探讨了该病的防治技术。研究结果如下:
从湖北省通城县大坪乡坪山村金刚藤种植基地采集典型金刚藤叶斑病的病叶标本,用常规组织分离法获得链格孢菌(Alternaria sp.),分离频率为96%,对该菌株进行了致病性测定、形态学观察及rDNA-ITS序列分析等研究。致病性测定的结果表明,链格孢菌的菌丝块接种金刚藤离体叶片、分生孢子悬浮液接种金刚藤活体植株均能产生典型的叶斑症状,发病率分别为100%,80%。其症状特点与采自田间自然发病的病叶相同,从接种的病组织再分离的病原菌与田间发病组织分离的病原菌形态一致,说明链格孢菌为其致病菌。形态学观察结果表明,金刚藤叶斑病菌在PDA培养基上,菌落圆形,菌丝初为白色,培养7d后变为淡灰色至黑褐色,边缘整齐,气生菌丝稀疏至茂密,培养12d后,菌落背面从褐色变为灰绿色至深绿色。用滤纸+PDA培养基诱导产孢,分生孢子梗簇生或单生,直或屈膝状弯曲,淡褐色,分隔,基部略膨大,20.0-58.2×3.6-5.5μm。分生孢子链生,倒棒状,具横膈膜3-7个,纵、斜隔膜0-3个,孢身27.5-48.2×10.3-17.1μm,孢子顶端略延伸,形成次生分生孢子梗(假喙),假喙柱状,顶端常略膨大,5.5-21.8×2.5-5.2μm。参考Simmons和张天宇等对链格孢种的特征描述,将该菌株鉴定为长柄链格孢(Alternaria longipes)。3个链格孢菌株的rDNA-ITS序列分析结果表明,该菌株5.8SrDNA及两侧的ITS区序列与A.longipes的同源性达100%。根据形态学和rDNA-ITS序列分析结果,确定该菌株为A.longipes。由此确定金刚藤叶斑病的病原为A.longipes。
A.longipes生物学特性研究表明:在有无光照或连续光照的条件下病菌均可生长,且无显著差异,连续黑暗培养有利于分生孢子萌发。A.longipes在5～35℃的范围内均能生长,其分生孢子在5～40℃的范围内均能萌发,25℃为菌丝生长和分生孢子萌发的最适温度。黑暗条件下,供试的9种培养基除OMA外均能使该菌产孢,产孢最好的为20%V8培养基。菌丝在pH值为:3～11的PDB培养基中均能生长,其中以pH6生长最快;分生孢子在pH3～11范围内均可萌发,萌发最适pH值为6。病菌能够有效利用多种碳源和氮源,在无碳或无氮条件下也能生长,不同碳源和氮源对菌丝生长都有显著的影响。病原菌以淀粉为碳源,以酵母膏为氮源时生长最好。供试的8种碳源溶液均可加速分生孢子的萌发,且差异不明显。分生孢子在相对湿度(RH)高于56%时方可萌发,最适RH为98%～100%。病原菌菌丝和分生孢子的致死温度分别为55℃水浴10min和60℃水浴10min。A.longipes寄住范围广泛,对供试的9科13种植物均有致病力。
2007年和2008年对金刚藤叶斑病发生规律的研究结果表明:病原菌可以菌丝在病残体上越冬,成为初侵染源。该病从5月中旬开始发生,初时病情轻,扩展缓慢,6月中旬至8月初,病情几乎直线上升,为盛发期,8月底部分病斑扩大连片,病情达到高峰,之后趋于平缓,扩展极慢。随着生长年限越长,病情越重。清除病残体后,第二年病情得到明显控制,不仅减轻病情,还能延长植物生育期,由此确定清除病残体是有效预防金刚藤叶斑病的方法之一。
在PDA平板上进行的抑菌试验结果表明,7种供试药剂在设置的不同浓度下对病原菌的生长都有一定的抑制作用,其中10%世高水分散粒剂和20.67%万兴乳油的抑菌效果最好,其次是40%福星乳油和30%爱苗乳油,抑制效果最差的是50%多菌灵硫可湿性粉剂,EC_(50)为734.94 ug/ml。2007年和2008年田间小区防治金刚藤叶斑病试验结果一致,结果表明:供试的5种杀菌剂中,10%世高水分散粒剂1500倍液和20.67%万兴乳油1500倍液对金刚藤叶斑病的防效最好,40%福星乳油8000倍液和30%爱苗乳油3000倍液的防效最差。2008年对田间防治适期的研究发现,在发病之前喷一次药并且发病后喷两次药比只在发病后喷药的防效好,说明发病前喷药能有效预防该病的发生。
Smilax china is a small perennial plant belonging to the Liliaceae frequently found in the warm temperate mountainous or hilly regions across eastern Asia.It is a commonly used Chinese herbal medicine.Leaf spot of S.china has been found to be a destructive disease in Tongcheng County,Hubei,China,where this plant is extensively cultivated. Symptoms were observed on infected leaves and fruits from 2006 to 2008 in a plantation established in 2002 on agricultural land.Up to 70%of the plants in the areas surveyed showed severe leaf spot symptoms each year.To our knowledge,no previous reports of a disease of S.china have been found.The purpose of the current research was to indentify the causal agent of leaf spot of Smilax china in China,to test the biological characteristics of the pathogen,and to determine the occurrence and the control techniques of this disease.The results were reported as followed:
Alternaria sp.was isolated from diseased leaves of S.china in Tongcheng country of Hubei province and pathogenicity was tested by inoculating mycelial plugs onto leaves in vitro and conidia suspension onto leaves of healthy plants in vivo respectively.Two days after inoculating mycelial plugs and twelve days after inoculating conidia suspension, brown spots were observed on inoculated leaves.Koch's postulates were fulfilled by re-isolating Alternaria sp.from diseased leaves.Alternaria sp.was determined to be the pathogen of leaf spot of S.china.Pathogens were made onto potato dextrose agar(PDA) giving initially white colonies.After 7 d on PDA,the colonies turned grey or brown,and by 12 d,the agar became blue-gray to dark green throughout.Sporulation was induced on cut filter paper following Zhang.Conidiophores were fasciculated or single,straight or knee-curved,light brown with regular septa,and 20-58×3.6-5.5μm.Each conidium was obclavate,brown,28-48×10-17μm,with 3-7 transverse and 0-3 longitudinal or oblique septa.The tops of some conidia developed into secondary conidiophores,which were cylindrical,hazel,and 5.5-22×2.5-5.2μm.The pathogen was identified as Alternaria longipes based on descriptions in Simmons and Zhang.Sequences of rDNA-ITS were obtained from three isolates,and comparisons with GenBank showed 100%similarity with A.longipes(AY751457).
Biological characteristic indicated that the pathogen can grow on PDA medium and conidiophores can germinate in water with or without light illumination,12h light and darkness alternation was the most suitable condition for the growth of mycelia,darkness faciliatated the germination of conidiophores.The range of temperature for mycelia growth was 5-35℃,that for conidia germination was 5-40℃,25℃was the optimal temperature for mycelia growth and conidia germination.20%V8 medium was best for spores production.The optimal The favorable pH value for mycelia growth and conidia germination was 3-11,with the optimal pH6.Among the tested carbon and nitrogen sources in Czapek liquid medium,amylum and yeast extract were the most favorable for mycelia growth.All the carbon sources can speed up conidia germination,and the differences were slightly.The conidia germination can only occur when relative humidity (RH) higher than 56%,with an optimum of 98%-100%.The lethal temperature for mycelia and conidia was 55℃,10min and 60℃,10 min,respectively.The host range of A. longipes was very wide.It can infect all the tested plants which belong to 13 species,9 family.
Studies on occurrence of leaf spot of S.china in 2007 and 2008.The results showed:The pathogen of this disease can overwintered in diseased leaves by mycelium. No symptom had been observed in the field until mid-May.Initially,the disease was mile and extended slowly.From mid-June to initial August,the disease index increased almost linearly and extended fast.From the end of August to October,it became stabilized gradually and extended extremely slowly.Longer the plant growed,more serious the disease was.After clearing away diseased plant debris,this disease can be controlled effectively in the next year.Therefore,clearing away diseased plant debris can effectively prevent this disease.
Inhibition of mycelia growth of A.longipes by seven fundicides was assayed on amended PDA medium.The inhibiting effects of 10%Score WG and 20.67%Charisma EC were higher than any others.Their 50%effective concentration(EC50) values were less than 2 ug/ml.50%Carbendazim-Sulphur WP had the lowest mycelial inhibition with EC_(50) values of 56.2914 ug/ml.The result of fungicide tests in the field in 2007 were consistent with that in 2008.The results indicated that effect of 10%Score WG 1500x and 20.67%Charisma EC 1500x on control of the disease were the best,40%Nustar EC 8000x and 30%Armure EC 3000x were the worst.In addition,spraying one time before the disease appeared and twice while the disease spreading was better than that spraying only while the disease spreading.From this,it can be seen that spraying before the disease appeared can effectively prevent this disease.
从湖北省通城县大坪乡坪山村金刚藤种植基地采集典型金刚藤叶斑病的病叶标本,用常规组织分离法获得链格孢菌(Alternaria sp.),分离频率为96%,对该菌株进行了致病性测定、形态学观察及rDNA-ITS序列分析等研究。致病性测定的结果表明,链格孢菌的菌丝块接种金刚藤离体叶片、分生孢子悬浮液接种金刚藤活体植株均能产生典型的叶斑症状,发病率分别为100%,80%。其症状特点与采自田间自然发病的病叶相同,从接种的病组织再分离的病原菌与田间发病组织分离的病原菌形态一致,说明链格孢菌为其致病菌。形态学观察结果表明,金刚藤叶斑病菌在PDA培养基上,菌落圆形,菌丝初为白色,培养7d后变为淡灰色至黑褐色,边缘整齐,气生菌丝稀疏至茂密,培养12d后,菌落背面从褐色变为灰绿色至深绿色。用滤纸+PDA培养基诱导产孢,分生孢子梗簇生或单生,直或屈膝状弯曲,淡褐色,分隔,基部略膨大,20.0-58.2×3.6-5.5μm。分生孢子链生,倒棒状,具横膈膜3-7个,纵、斜隔膜0-3个,孢身27.5-48.2×10.3-17.1μm,孢子顶端略延伸,形成次生分生孢子梗(假喙),假喙柱状,顶端常略膨大,5.5-21.8×2.5-5.2μm。参考Simmons和张天宇等对链格孢种的特征描述,将该菌株鉴定为长柄链格孢(Alternaria longipes)。3个链格孢菌株的rDNA-ITS序列分析结果表明,该菌株5.8SrDNA及两侧的ITS区序列与A.longipes的同源性达100%。根据形态学和rDNA-ITS序列分析结果,确定该菌株为A.longipes。由此确定金刚藤叶斑病的病原为A.longipes。
A.longipes生物学特性研究表明:在有无光照或连续光照的条件下病菌均可生长,且无显著差异,连续黑暗培养有利于分生孢子萌发。A.longipes在5～35℃的范围内均能生长,其分生孢子在5～40℃的范围内均能萌发,25℃为菌丝生长和分生孢子萌发的最适温度。黑暗条件下,供试的9种培养基除OMA外均能使该菌产孢,产孢最好的为20%V8培养基。菌丝在pH值为:3～11的PDB培养基中均能生长,其中以pH6生长最快;分生孢子在pH3～11范围内均可萌发,萌发最适pH值为6。病菌能够有效利用多种碳源和氮源,在无碳或无氮条件下也能生长,不同碳源和氮源对菌丝生长都有显著的影响。病原菌以淀粉为碳源,以酵母膏为氮源时生长最好。供试的8种碳源溶液均可加速分生孢子的萌发,且差异不明显。分生孢子在相对湿度(RH)高于56%时方可萌发,最适RH为98%～100%。病原菌菌丝和分生孢子的致死温度分别为55℃水浴10min和60℃水浴10min。A.longipes寄住范围广泛,对供试的9科13种植物均有致病力。
2007年和2008年对金刚藤叶斑病发生规律的研究结果表明:病原菌可以菌丝在病残体上越冬,成为初侵染源。该病从5月中旬开始发生,初时病情轻,扩展缓慢,6月中旬至8月初,病情几乎直线上升,为盛发期,8月底部分病斑扩大连片,病情达到高峰,之后趋于平缓,扩展极慢。随着生长年限越长,病情越重。清除病残体后,第二年病情得到明显控制,不仅减轻病情,还能延长植物生育期,由此确定清除病残体是有效预防金刚藤叶斑病的方法之一。
在PDA平板上进行的抑菌试验结果表明,7种供试药剂在设置的不同浓度下对病原菌的生长都有一定的抑制作用,其中10%世高水分散粒剂和20.67%万兴乳油的抑菌效果最好,其次是40%福星乳油和30%爱苗乳油,抑制效果最差的是50%多菌灵硫可湿性粉剂,EC_(50)为734.94 ug/ml。2007年和2008年田间小区防治金刚藤叶斑病试验结果一致,结果表明:供试的5种杀菌剂中,10%世高水分散粒剂1500倍液和20.67%万兴乳油1500倍液对金刚藤叶斑病的防效最好,40%福星乳油8000倍液和30%爱苗乳油3000倍液的防效最差。2008年对田间防治适期的研究发现,在发病之前喷一次药并且发病后喷两次药比只在发病后喷药的防效好,说明发病前喷药能有效预防该病的发生。
Smilax china is a small perennial plant belonging to the Liliaceae frequently found in the warm temperate mountainous or hilly regions across eastern Asia.It is a commonly used Chinese herbal medicine.Leaf spot of S.china has been found to be a destructive disease in Tongcheng County,Hubei,China,where this plant is extensively cultivated. Symptoms were observed on infected leaves and fruits from 2006 to 2008 in a plantation established in 2002 on agricultural land.Up to 70%of the plants in the areas surveyed showed severe leaf spot symptoms each year.To our knowledge,no previous reports of a disease of S.china have been found.The purpose of the current research was to indentify the causal agent of leaf spot of Smilax china in China,to test the biological characteristics of the pathogen,and to determine the occurrence and the control techniques of this disease.The results were reported as followed:
Alternaria sp.was isolated from diseased leaves of S.china in Tongcheng country of Hubei province and pathogenicity was tested by inoculating mycelial plugs onto leaves in vitro and conidia suspension onto leaves of healthy plants in vivo respectively.Two days after inoculating mycelial plugs and twelve days after inoculating conidia suspension, brown spots were observed on inoculated leaves.Koch's postulates were fulfilled by re-isolating Alternaria sp.from diseased leaves.Alternaria sp.was determined to be the pathogen of leaf spot of S.china.Pathogens were made onto potato dextrose agar(PDA) giving initially white colonies.After 7 d on PDA,the colonies turned grey or brown,and by 12 d,the agar became blue-gray to dark green throughout.Sporulation was induced on cut filter paper following Zhang.Conidiophores were fasciculated or single,straight or knee-curved,light brown with regular septa,and 20-58×3.6-5.5μm.Each conidium was obclavate,brown,28-48×10-17μm,with 3-7 transverse and 0-3 longitudinal or oblique septa.The tops of some conidia developed into secondary conidiophores,which were cylindrical,hazel,and 5.5-22×2.5-5.2μm.The pathogen was identified as Alternaria longipes based on descriptions in Simmons and Zhang.Sequences of rDNA-ITS were obtained from three isolates,and comparisons with GenBank showed 100%similarity with A.longipes(AY751457).
Biological characteristic indicated that the pathogen can grow on PDA medium and conidiophores can germinate in water with or without light illumination,12h light and darkness alternation was the most suitable condition for the growth of mycelia,darkness faciliatated the germination of conidiophores.The range of temperature for mycelia growth was 5-35℃,that for conidia germination was 5-40℃,25℃was the optimal temperature for mycelia growth and conidia germination.20%V8 medium was best for spores production.The optimal The favorable pH value for mycelia growth and conidia germination was 3-11,with the optimal pH6.Among the tested carbon and nitrogen sources in Czapek liquid medium,amylum and yeast extract were the most favorable for mycelia growth.All the carbon sources can speed up conidia germination,and the differences were slightly.The conidia germination can only occur when relative humidity (RH) higher than 56%,with an optimum of 98%-100%.The lethal temperature for mycelia and conidia was 55℃,10min and 60℃,10 min,respectively.The host range of A. longipes was very wide.It can infect all the tested plants which belong to 13 species,9 family.
Studies on occurrence of leaf spot of S.china in 2007 and 2008.The results showed:The pathogen of this disease can overwintered in diseased leaves by mycelium. No symptom had been observed in the field until mid-May.Initially,the disease was mile and extended slowly.From mid-June to initial August,the disease index increased almost linearly and extended fast.From the end of August to October,it became stabilized gradually and extended extremely slowly.Longer the plant growed,more serious the disease was.After clearing away diseased plant debris,this disease can be controlled effectively in the next year.Therefore,clearing away diseased plant debris can effectively prevent this disease.
Inhibition of mycelia growth of A.longipes by seven fundicides was assayed on amended PDA medium.The inhibiting effects of 10%Score WG and 20.67%Charisma EC were higher than any others.Their 50%effective concentration(EC50) values were less than 2 ug/ml.50%Carbendazim-Sulphur WP had the lowest mycelial inhibition with EC_(50) values of 56.2914 ug/ml.The result of fungicide tests in the field in 2007 were consistent with that in 2008.The results indicated that effect of 10%Score WG 1500x and 20.67%Charisma EC 1500x on control of the disease were the best,40%Nustar EC 8000x and 30%Armure EC 3000x were the worst.In addition,spraying one time before the disease appeared and twice while the disease spreading was better than that spraying only while the disease spreading.From this,it can be seen that spraying before the disease appeared can effectively prevent this disease.
引文
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