苜蓿根和根颈腐烂病的病原及种质抗病性研究
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摘要
1．苜蓿根和根颈腐烂病的症状。苜蓿根和根颈腐烂病是一种危害严重的病害，在全世界所有苜蓿栽培地区一般都有发生。一般来说，病害的症状因病原物的不同而略有差异。在甘肃定西发生的病害，田间四月初苜蓿返青季节即可表现症状，发病植株比健康植株发芽延迟20天左右，分蘖茎数明显减少，植株稀疏，生长缓慢，田间感病品种德宝发病率达20％—40％。根部典型症状是在根颈中心处出现楔形黑褐色坏死斑，并由根颈处通过维管系统向根尖扩散，严重时根颈全部死亡，腐烂、中空。据五月中下旬，调查田间发病率严重时可达43％。室内苗期接种发现，幼苗感染该病，初期根颈处形成水浸状坏死斑点，后逐渐出现缢缩变黑坏死，茎叶逐渐枯死。
2．苜蓿根和根颈腐烂病的病原研究。通过对定西九华沟第二年生苜蓿(Medicago sativa L.)根和根颈腐烂病的156个样品分离鉴定，幼苗接种致病性测定，采用Booth的分类标准和系统，确识发生在甘肃中部干旱地区定西的苜蓿根和根颈腐烂病优势病原菌为三种镰刀菌，即尖孢镰刀菌(F.oxysporum)、锐顶镰刀菌(F.acuminatum)和半裸镰刀菌(F.semitectum)。尖孢镰刀菌(F.oxysporum Schlecht．)：在PSA培养基上25℃4天时生长速度为2.35cm，气生菌丝繁茂，初白色，后变玫瑰红色，Bilais和WA培养基上菌丝少，白色，米饭培养基上产生典型玫瑰红色。小型分生孢子数量多，杆状。大型分生孢子少见。产孢细胞短，单瓶梗，在菌丝上分散生长。厚垣孢子多，间生或顶生，多串生，球形；锐顶镰刀菌(F.acumitectum Ellis&Everhert)：在PSA培养基上25℃4天后生长速度为3.13cm，气生菌丝繁茂，绒状，色素枣红色，在Bilai's和WA培养基上菌丝少，白色，在米饭培养基上产生枣红色色素。无小型分生孢子，大型分生孢子形态较为一致，镰刀型，弯曲，中部细胞明显膨大，顶胞延长呈锥形，腹背双曲线弯曲不明显，3～6隔，多数4～5隔，产孢细胞单瓶梗。厚垣孢子单生或串生，多串生，间生，也有顶生；半裸镰刀菌(F.semitectum Brek & Rav.)：在PSA培养基上25℃ 4天后生长速度为3.35cm，气生菌丝繁茂，初为白色，后至浅驼色，在Bilai's和WA培养基上菌丝稀疏，白色。在米饭培养基上表现为浅驼色。小型分生孢子少见，大型分生孢子两种形态，一为纺锤型，两端均为楔形，多为3～5隔，二为镰刀型，顶端楔形，基胞有明显足跟，3～8隔，多为5隔，产孢细
胞有两种，分别是多芽生和复瓶梗，厚垣抱子球形，多串生，间或顶生。
这是国内关于该病病原的首次研究报道。锐顶镰刀菌（厂 acumitectum
Ellis＆Everhert）和半裸镰刀菌（F semitectum Brek＆Ray）是中国盲淆上的
新记录。
3．首适根和板颈腐烂病病原致病性和种质抗病性研究。用室内试管苗人工
接种方法对首稽根和根颈腐烂病的 3个致病镰刀菌（Fsarium p）的致病性
及20个首蒂品种的抗病性进行了测定。接种时将活化培养7天的病原菌分离
物，每M加水45ail，连同培养基切碎，制成悬浮液，将供试小苗浸根5分钟，
同时每试管接种悬浮液3ml，每天观察记载发病情况，以幼苗存活率表示品
种抗病性的大小，实验结果表明，尖抱镰刀菌（厂p orum）的7个分离物，
锐顶镰刀菌（F acuminatum）的 4个分离物和半裸镰刀菌（F semitectum）的
5个分离物都对首蒲具有致病性，但致病性有差异。20个首桔品种幼茵测定，
各品种对根和根颈腐烂病的抗病性存在差异，其中抗性较强的有维拉
（Verla）、德福（Derful）、巨人 201（AIneristand 201）、草原 2号（Caowan 2）、
塞特（Sitel）、阿尔冈金（Algongum）、图牧 2号（TUrnu 2）、甘农 2号（Gannong
2）。
4．过氧化物酶活性与种质的根和根颈腐烂病抗病性的关系研究。通过对盲
稽16个品种，采用接种和不接种尖抱镰刀菌两种处理，研究了过氧化物酶活
性的变化，结果表明：在首蓉幼苗叶片和幼苗根部的过氧化物酶活性，不论
接种与否，品种间均存在显著差异。另外，通过实验发现受侵染植株的幼苗
叶片中过氧化物酶活性在抗性品种中降低，在感病品种中升高。经过对过氧
化物酶活性与品种的根和根颈腐烂病抗病性的相关性分析，首稽幼苗叶片中
过氧化物酶，不论接种与否，均与抗病性存在显著正相关，酶活性变量亦达
到显著正相关。因此可以用幼苗叶片中的酶活性，或接种病原菌后的酶活性
或其变量来鉴定和评价品种间对根和根颈腐烂病的抗病性，目前尚未见到过
首蓉上的同类研究报道。
5．过氧化物醇同工酶与种质根和根颈腐烂病抗病性研究。采用聚丙烯酞胺凝
胶电泳技术，研究了17个首淆品种，经人工接种和不接种病原尖抱镰刀菌的
情况下，过氧化物酶同工酶的组成及其变化，并探讨了同工酶与品种抗病性
之间的相关性，结果表明：不?
1.Symptoms of Crown and Root Rot of Alfalfa. Crown and root rot of Alfalfa (CRRA) is a chronic disease throughout the world. Although there were some differences in symptoms caused by different pathogens, the symptoms were not always specific to any one of the pathogens. In Dingxi of Gansu province, infected plants displayed a 20 days late germination and decreased tillering capacity as well as slow growth. The disease is characterized by the presence of dark brown to black, wedge shaped necrotic lesions that spread from the affected crowns downward into root top. According to the investigation in mid-late May in Dingxi of Gansu province, field incidence of CRRA was between 20% and 40%.
2. Identification of Pathogens of Crown and Root Rot of Alfalfa .The distribution, pathogenicity of Fusarium species associated with crown and root rot of alfalfa (Medicago saliva L.) were examined in DingXi, Gansu Province, China. Three dominant species have been discovered: (1) Fusarium oxysporum, average growth rate of cultures on PSA in 4 days was 2.35 cm. Myceleium was abundant with white color at first and became violet late on. Microconidia were generally abundant, cylindrical. Chlamydosporses were generally abundant and terminal or intercalary, formed in pairs or in chains. (2) E acuminaium, cultures had growth rate of 3.13 cm, Culture pigmentation was carmine red. Only macroconidia were produced. They were 3-7 septate often with an incurved elongation of the apical cell , and were produced from phialides. Chlamydospores were intercalary in knots. (3) E semitectum, growth rate was 3.35 cm. Culture pigmentation was peach changing to buff brown. Macroconidia displayed wedge shape foot cell , 0-5 septate, formed blastospores from polyblstic sympodial cell. Secondary macroconidia were typical heeled foot cell, 3-7 septate, formed from phialides usually grouped in sporodochia. Chlamydospores were often sparse, globose, intercalary, single or in chains. All of the species were the pathogenic to the disease.
This paper happened to be the original study on pathogens of crown and root rot on Alfalfa in China. F. acuminatum and F. semitectum were the first record in alfalfa in China.
3. Comparison of Pathogenicity among isolates from Fusarium spp and resistance among cultrvars in alfalfa. Twenty cultivars of alfalfa were artificially inoculated with seven isolates from Fusarium oxysporum, four isolates from F. acuminatum and five isolates from F.semitectum using a test tubes nutritive
solution method. All of the isolates originated from alfalfa in Dingxi of Gansu province, China. Inoculum was prepared from cultures by growing single conidia from each petri dish of potato sucrose agar (PSA) at 24 癈 for 10 days . Then myceleium and conidia spensions were inoculated into tubes that were culturing the alfalfa seedling in. Each test tube was inoculated by equal pathogen suspension. Numbers of surviving seedling was recorded after 7, 9, 11 days. Isolates from different species displayed different virulence. Different alfalfa cultivars differed hi resistance also. Some cultivars such as Verla, Derful , Ameristand 201,Caoyuan2 ,Sitel, Algongum, Tumu 2 , Gannong 2 expressed resistance to crown and root of alfalfa.
4 The relationship between the peroxidase (POD) activity and cultivars resistance to crown and root rot in alfalfa. Peroxidase activities in seedling leaves and seedling roots of 16 alfalfa cultivars with different resistance to crown and root rot were determined. The results showed that there were significant difference hi peroxidase activity among 16 alfalfa cultivars both in the leaves and roots. The same results were obtained after inoculated seedling with Fusarium oxysporum. The POD activity decreased in the resistance cultivars but increased in susceptible cultivars after infected. Statistics analysis showed that the POD activity in leaves was positively correlated with their resistance. Perhaps the POD activity in seedling leaves of alfalfa can be used to evaluating the resistance to crown and root rot in alfa
2．苜蓿根和根颈腐烂病的病原研究。通过对定西九华沟第二年生苜蓿(Medicago sativa L.)根和根颈腐烂病的156个样品分离鉴定，幼苗接种致病性测定，采用Booth的分类标准和系统，确识发生在甘肃中部干旱地区定西的苜蓿根和根颈腐烂病优势病原菌为三种镰刀菌，即尖孢镰刀菌(F.oxysporum)、锐顶镰刀菌(F.acuminatum)和半裸镰刀菌(F.semitectum)。尖孢镰刀菌(F.oxysporum Schlecht．)：在PSA培养基上25℃4天时生长速度为2.35cm，气生菌丝繁茂，初白色，后变玫瑰红色，Bilais和WA培养基上菌丝少，白色，米饭培养基上产生典型玫瑰红色。小型分生孢子数量多，杆状。大型分生孢子少见。产孢细胞短，单瓶梗，在菌丝上分散生长。厚垣孢子多，间生或顶生，多串生，球形；锐顶镰刀菌(F.acumitectum Ellis&Everhert)：在PSA培养基上25℃4天后生长速度为3.13cm，气生菌丝繁茂，绒状，色素枣红色，在Bilai's和WA培养基上菌丝少，白色，在米饭培养基上产生枣红色色素。无小型分生孢子，大型分生孢子形态较为一致，镰刀型，弯曲，中部细胞明显膨大，顶胞延长呈锥形，腹背双曲线弯曲不明显，3～6隔，多数4～5隔，产孢细胞单瓶梗。厚垣孢子单生或串生，多串生，间生，也有顶生；半裸镰刀菌(F.semitectum Brek & Rav.)：在PSA培养基上25℃ 4天后生长速度为3.35cm，气生菌丝繁茂，初为白色，后至浅驼色，在Bilai's和WA培养基上菌丝稀疏，白色。在米饭培养基上表现为浅驼色。小型分生孢子少见，大型分生孢子两种形态，一为纺锤型，两端均为楔形，多为3～5隔，二为镰刀型，顶端楔形，基胞有明显足跟，3～8隔，多为5隔，产孢细
胞有两种，分别是多芽生和复瓶梗，厚垣抱子球形，多串生，间或顶生。
这是国内关于该病病原的首次研究报道。锐顶镰刀菌（厂 acumitectum
Ellis＆Everhert）和半裸镰刀菌（F semitectum Brek＆Ray）是中国盲淆上的
新记录。
3．首适根和板颈腐烂病病原致病性和种质抗病性研究。用室内试管苗人工
接种方法对首稽根和根颈腐烂病的 3个致病镰刀菌（Fsarium p）的致病性
及20个首蒂品种的抗病性进行了测定。接种时将活化培养7天的病原菌分离
物，每M加水45ail，连同培养基切碎，制成悬浮液，将供试小苗浸根5分钟，
同时每试管接种悬浮液3ml，每天观察记载发病情况，以幼苗存活率表示品
种抗病性的大小，实验结果表明，尖抱镰刀菌（厂p orum）的7个分离物，
锐顶镰刀菌（F acuminatum）的 4个分离物和半裸镰刀菌（F semitectum）的
5个分离物都对首蒲具有致病性，但致病性有差异。20个首桔品种幼茵测定，
各品种对根和根颈腐烂病的抗病性存在差异，其中抗性较强的有维拉
（Verla）、德福（Derful）、巨人 201（AIneristand 201）、草原 2号（Caowan 2）、
塞特（Sitel）、阿尔冈金（Algongum）、图牧 2号（TUrnu 2）、甘农 2号（Gannong
2）。
4．过氧化物酶活性与种质的根和根颈腐烂病抗病性的关系研究。通过对盲
稽16个品种，采用接种和不接种尖抱镰刀菌两种处理，研究了过氧化物酶活
性的变化，结果表明：在首蓉幼苗叶片和幼苗根部的过氧化物酶活性，不论
接种与否，品种间均存在显著差异。另外，通过实验发现受侵染植株的幼苗
叶片中过氧化物酶活性在抗性品种中降低，在感病品种中升高。经过对过氧
化物酶活性与品种的根和根颈腐烂病抗病性的相关性分析，首稽幼苗叶片中
过氧化物酶，不论接种与否，均与抗病性存在显著正相关，酶活性变量亦达
到显著正相关。因此可以用幼苗叶片中的酶活性，或接种病原菌后的酶活性
或其变量来鉴定和评价品种间对根和根颈腐烂病的抗病性，目前尚未见到过
首蓉上的同类研究报道。
5．过氧化物醇同工酶与种质根和根颈腐烂病抗病性研究。采用聚丙烯酞胺凝
胶电泳技术，研究了17个首淆品种，经人工接种和不接种病原尖抱镰刀菌的
情况下，过氧化物酶同工酶的组成及其变化，并探讨了同工酶与品种抗病性
之间的相关性，结果表明：不?
1.Symptoms of Crown and Root Rot of Alfalfa. Crown and root rot of Alfalfa (CRRA) is a chronic disease throughout the world. Although there were some differences in symptoms caused by different pathogens, the symptoms were not always specific to any one of the pathogens. In Dingxi of Gansu province, infected plants displayed a 20 days late germination and decreased tillering capacity as well as slow growth. The disease is characterized by the presence of dark brown to black, wedge shaped necrotic lesions that spread from the affected crowns downward into root top. According to the investigation in mid-late May in Dingxi of Gansu province, field incidence of CRRA was between 20% and 40%.
2. Identification of Pathogens of Crown and Root Rot of Alfalfa .The distribution, pathogenicity of Fusarium species associated with crown and root rot of alfalfa (Medicago saliva L.) were examined in DingXi, Gansu Province, China. Three dominant species have been discovered: (1) Fusarium oxysporum, average growth rate of cultures on PSA in 4 days was 2.35 cm. Myceleium was abundant with white color at first and became violet late on. Microconidia were generally abundant, cylindrical. Chlamydosporses were generally abundant and terminal or intercalary, formed in pairs or in chains. (2) E acuminaium, cultures had growth rate of 3.13 cm, Culture pigmentation was carmine red. Only macroconidia were produced. They were 3-7 septate often with an incurved elongation of the apical cell , and were produced from phialides. Chlamydospores were intercalary in knots. (3) E semitectum, growth rate was 3.35 cm. Culture pigmentation was peach changing to buff brown. Macroconidia displayed wedge shape foot cell , 0-5 septate, formed blastospores from polyblstic sympodial cell. Secondary macroconidia were typical heeled foot cell, 3-7 septate, formed from phialides usually grouped in sporodochia. Chlamydospores were often sparse, globose, intercalary, single or in chains. All of the species were the pathogenic to the disease.
This paper happened to be the original study on pathogens of crown and root rot on Alfalfa in China. F. acuminatum and F. semitectum were the first record in alfalfa in China.
3. Comparison of Pathogenicity among isolates from Fusarium spp and resistance among cultrvars in alfalfa. Twenty cultivars of alfalfa were artificially inoculated with seven isolates from Fusarium oxysporum, four isolates from F. acuminatum and five isolates from F.semitectum using a test tubes nutritive
solution method. All of the isolates originated from alfalfa in Dingxi of Gansu province, China. Inoculum was prepared from cultures by growing single conidia from each petri dish of potato sucrose agar (PSA) at 24 癈 for 10 days . Then myceleium and conidia spensions were inoculated into tubes that were culturing the alfalfa seedling in. Each test tube was inoculated by equal pathogen suspension. Numbers of surviving seedling was recorded after 7, 9, 11 days. Isolates from different species displayed different virulence. Different alfalfa cultivars differed hi resistance also. Some cultivars such as Verla, Derful , Ameristand 201,Caoyuan2 ,Sitel, Algongum, Tumu 2 , Gannong 2 expressed resistance to crown and root of alfalfa.
4 The relationship between the peroxidase (POD) activity and cultivars resistance to crown and root rot in alfalfa. Peroxidase activities in seedling leaves and seedling roots of 16 alfalfa cultivars with different resistance to crown and root rot were determined. The results showed that there were significant difference hi peroxidase activity among 16 alfalfa cultivars both in the leaves and roots. The same results were obtained after inoculated seedling with Fusarium oxysporum. The POD activity decreased in the resistance cultivars but increased in susceptible cultivars after infected. Statistics analysis showed that the POD activity in leaves was positively correlated with their resistance. Perhaps the POD activity in seedling leaves of alfalfa can be used to evaluating the resistance to crown and root rot in alfa
引文
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