Pathogen Identification and Effective Fungicide for Cowpea Wilt Disease
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摘要:目的
明确引起豇豆枯萎病的病原菌种类，筛选出对豇豆枯萎病有效的防治药剂。
方法对豇豆各生长时期枯萎病的发生情况进行田间调查统计，采集发病植株进行病原菌的分离纯化，通过形态学观察、致病性测定及分子鉴定确定病原菌种类，并采用菌丝生长速率法测定10种药剂对病原菌的敏感性，同时进行10种药剂对病原真菌和病原卵菌的抑菌效果测定。
结果分离纯化得到病原真菌共计135株，病原卵菌共计128株，其中病原真菌包括尖孢镰刀菌（Fusarium oxysporum）和黑孢霉菌（Nigrospora osmanthi）等；病原卵菌包括豇豆疫霉（Phytophthora vignae）和烟草疫霉（P. nicotianae）。致病性测定结果表明，尖孢镰刀菌、黑孢霉菌、豇豆疫霉和烟草疫霉均可侵染豇豆引起豇豆枯萎病。药剂敏感性测定结果表明，10种药剂对尖孢镰刀菌菌丝生长均具有不同程度的抑制作用，其中咪鲜胺的抑菌效果最好，EC50为0.086 μg·mL−1，其次为苯醚甲环唑和多菌灵，EC50分别为0.241 μg·mL−1和0.944 μg·mL−1，王铜效果最差，EC50为
14719.994 μg·mL−1。不同药剂对病原真菌和病原卵菌的抑菌效果测定结果表明，霜脲·嘧菌酯对病原真菌尖孢镰刀菌的抑菌率为55.32%，对病原卵菌豇豆疫霉和烟草疫霉的抑菌率均为100%，可作为防治豇豆枯萎病的药剂。结论明确了豇豆枯萎病的病原菌种类，首次发现病原真菌黑孢霉菌和病原卵菌烟草疫霉可以侵染豇豆引起豇豆发病，筛选出对病原菌有较好抑制作用的防控药剂，为豇豆枯萎病的防治提供依据。
Abstract:ObjectivePathogens causing the cowpea wilt disease and an effective control agent were identified.
MethodField surveys and statistical analyses were conducted to assess the prevalence of the wilt disease on cowpea plants at various growth stages. Infected plants were collected for pathogen isolation and purification in the laboratory. Suspected microorganisms were examined for morphological characteristics, analyzed for pathogenicity, and identified based on molecular determination. Mycelial growth rate as well as inhibitory effects on the potential pathogenic fungi and oomycetes under treatments of 10 fungicides were determined.
ResultPreliminary, 135 strains of microbes and 128 of oomycetes were isolated and purified. They included fungi like Fusarium oxysporum and Nigrospora osmanthi and oomycetes like Phytophthora vignae and P. nicotianae that displayed the disease symptoms on cowpeas. The mycelial growth of F. oxysporum showed varying degrees of sensitivity toward the 10 fungicides. Prochloraz had the highest inhibitory effect of an EC50 of 0.086 μg·mL−1 followed by difenoconazole with an EC50 of 0.241 μg·mL−1 and carbendazim with an EC50 of 0.944 μg·mL−1, while copper oxychloride least effective with a dismal EC50 of 14 719.994 μg·mL−1. The inhibition rates of cymoxanil-azoxystrobin were 55.32% against F. oxysporum and 100% against P. vignae and P. nicotianae. It appeared to be the fungicide most effective for controlling, preventing, and treating the disease on cowpeas.
ConclusionThe pathogenic species of cowpea wilt were clarified, and it was first report demonstrating that the pathogenic fungus N. osmanthi and the pathogenic oomycetes P. nicotianae could infect cowpea and cause cowpea disease. Effective control agents with significant inhibitory effects against pathogens were successfully screened, providing a scientific basis for the integrated management of cowpea wilt disease .
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Keywords:
- cowpea /
- wilt disease /
- pathogenic fungi /
- pathogenic oomycetes /
- control agents
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0. 引言
【研究意义】豇豆（Vigna unguiculata L. Walp）因其适应性强、生育周期短且经济价值高，深受消费者和种植户青睐[1−2]。我国作为豇豆的次生起源和多样性中心，豇豆种植范围广，除高寒地区外均有种植[3−4]。近年来，由于复种指数大[5]，豇豆根部病害频发，其中豇豆枯萎病和豇豆根腐病是豇豆种植过程中发生最普遍、危害最严重的根部病害[6−7]，其引起的产量损失和农药残留等问题严重制约豇豆产业的健康发展。同时，由于豇豆是花果同期作物，采摘间隔期短[8]，枯萎病的发生呈现南高北低的趋势，我国南方豇豆种植区春冬季温、湿度高，病虫害多发，用药频率高，进而导致农残问题突出，阻碍豇豆的健康生产[9]。因此，明确豇豆枯萎病病原菌的种类并筛选出有效的防控药剂可以更好地指导豇豆枯萎病的防治。【前人研究进展】目前认为豇豆枯萎病的病原菌主要是尖孢镰刀菌菜豆专化型（Fusarium oxysporum f. sp. phaseolus）、尖孢镰刀菌嗜导管专化型（F. oxysporum f.sp. tracheiphilum）、尖孢镰刀菌（F. oxysporum）、裂口镰刀菌（F. lacertarum）和层生镰孢菌（F. proliferatum）[10−15]，发病症状主要为叶脉两侧变黄萎蔫，最终叶片枯死脱落，主、侧根及茎基部变色腐烂，维管束变为黑褐色[16]；豇豆根腐病的病原菌主要有尖孢镰刀菌（F. oxysporum）、茄腐镰刀菌（F. solani）、层生镰孢菌（F. proliferatum）和立枯丝核菌（Rhizoctonia solani）等[17−19]，发病症状为植株下部叶片发黄，叶缘枯萎，拔出病株，可见主根及茎基部变为黑褐色，病部凹陷，维管束变色，当主根全部腐烂时，病株即枯萎死亡[20]。二者在田间均会出现叶片失绿变黄枯萎，且根和茎基部变色等症状，根据病株的田间症状较难区分具体病害，并且病原菌的描述也存在重叠。【本研究切入点】目前关于豇豆枯萎病虽已有较多相关报道，但随着研究发现，对其防治仍存在较大难度，且其病原菌的种类仍需进一步明确和完善。【拟解决的关键问题】明确引起豇豆枯萎病的病原菌种类，筛选出对豇豆枯萎病病原菌防效良好的药剂，为防治豇豆枯萎病提供有效的化学药剂和科学的理论依据。
1. 材料与方法
1.1 试验材料
豇豆各生长时期的枯萎病病害样本采自海南省三亚市、海南省乐东黎族自治县、河南省焦作市、湖南省长沙市和贵州省安顺市的豇豆种植区，无菌采集袋密封，带回实验室进行病原菌的分离与纯化。
供试药剂：75%百菌清可湿性粉剂（山东百农思达生物科技有限公司）、96%咪鲜胺原药（生工生物工程股份有限公司）、80%代森锰锌原药（生工生物工程股份有限公司）、70%甲基硫菌灵原药（生工生物工程股份有限公司）、96%苯醚甲环唑原药（生工生物工程股份有限公司）、98%多菌灵原药（生工生物工程股份有限公司）、99%恶霉灵原药（生工生物工程股份有限公司）、84%王铜可湿性粉剂（宁波三江益农化学有限公司）、30%唑醚·啶酰菌悬浮剂（河南金田地农化有限责任公司）、70%霜脲·嘧菌酯水分散粒剂（美国世科姆公司）。
1.2 病原菌的分离与纯化
采用组织分离法，超净台内切取病株病健交界处组织置于75%酒精溶液中90 s，无菌水洗涤3次，晾干后分别接种至PDA和V8培养基上，25 ℃恒温黑暗培养，待菌丝长出后尽快挑取菌落边缘菌丝进行纯化培养，获得分离菌株。
1.3 病原菌的致病性测定
黄化苗期采用下胚轴接种法[21]对分离所得菌株进行致病性测定，每个处理6株，重复3次，统计各处理发病情况；幼苗期和抽蔓期时，病原真菌采用浸根接种法[22]进行致病性测定，病原卵菌采用菌土法[23]进行致病性测定，每个处理6株，重复3次，统计各处理发病情况。
1.4 病原菌的形态观察
病原真菌接种至PDA培养基上，病原卵菌接种至V8培养基上，25 ℃恒温黑暗培养，观察菌落的大小、形状和颜色，在显微镜下观察病原真菌的菌丝、分生孢子等以及病原卵菌的菌丝、藏卵器、雄器和孢子囊等显微结构的大小与形状。
1.5 病原菌的分子鉴定
使用CTAB法[24]提取病原菌的基因组DNA，分别用通用引物ITS[25]、β-tublin[26]和LSU[27]以基因组DNA为模板进行PCR扩增，扩增所得产物经1%琼脂糖凝胶电泳后进行测序，测序所得序列于NCBI的NCBI GenBank核酸序列库进行同源性比对分析，确定病原菌的分类地位。进一步使用MEGA11.0软件采用邻接法（Neighbour-Joining, NJ）构建多基因联合（顺序串接ITS-β-tublin-LSU基因序列）系统发育树。
1.6 不同药剂对病原真菌的敏感性测定
各药剂的浓度分别设置如下：75%百菌清可湿性粉剂0.1、1、10、20、40 μg·mL−1；96%咪鲜胺原药0.001、0.005、0.05、0.1、0.5 μg·mL−1；80%代森锰锌原药5、10、15、20、30 μg·mL−1；70%甲基硫菌灵原药10、20、30、40、60 μg·mL−1；96%苯醚甲环唑原药0.05、0.1、0.5、1、5 μg·mL−1；98%多菌灵原药0.1、0.2、0.5、0.8、1 μg·mL−1；99%恶霉灵原药25、50、100、200、400 μg·mL−1；84%王铜可湿性粉剂
1250 、2500 、5000 、10000 、20000 μg·mL−1；30%唑醚·啶酰菌悬浮剂0.01、0.1、0.5、1、125 μg·mL−1；70%霜脲·嘧菌酯水分散粒剂0.001、0.01、0.1、1、100 μg·mL−1；王铜、唑醚·啶酰菌以及霜脲·嘧菌酯的溶剂为水，其余药剂的溶剂均为DMSO。病原真菌在PDA培养基上生长5 d后，切取2 mm × 2 mm的菌丝块接种于PDA培养基，对照为不加药剂的PDA培养基，每个浓度3个皿，重复3次，25 ℃恒温黑暗培养7 d，十字交叉法测定各处理菌落直径，计算各药剂的毒力回归方程及抑制中浓度EC50值。1.7 不同药剂对病原真菌和病原卵菌的抑菌效果测定
各药剂浓度设置分别为百菌清4 μg·mL−1、咪鲜胺1 μg·mL−1、代森锰锌15 μg·mL−1、甲基硫菌灵40 μg·mL−1、苯醚甲环唑2.5 μg·mL−1、多菌灵2.5 μg·mL−1、恶霉灵50 μg·mL−1、王铜
1250 μg·mL−1、唑醚·啶酰菌500 μg·mL−1和霜脲·嘧菌酯500 μg·mL−1；王铜、唑醚·啶酰菌以及霜脲·嘧菌酯的溶剂为水，其余药剂的溶剂均为DMSO。病原真菌在PDA培养基上生长5 d后，切取2 mm × 2 mm的菌丝块接种于PDA培养基上，病原卵菌在V8培养基上培养生长5 d后，切取2 mm × 2 mm的菌丝块接种于V8培养基上，对照为不加药剂的PDA或V8培养基，25 ℃恒温黑暗培养，每个浓度3个皿，重复3次，十字交叉法测定各处理菌落直径，计算抑菌率，比较供试杀菌剂对病原真菌和病原卵菌的抑菌效果。2. 结果与分析
2.1 豇豆枯萎病田间调查
田间调查发现，豇豆枯萎病在豇豆整个生长时期，包括苗期、抽蔓期和开花结荚期均有发生，不同年间、不同地块以及不同生长时期的发病程度存在差异。苗期发病重的地块发病率超过40%，症状表现为叶片萎蔫，严重时幼苗呈倒伏状态，根系皱缩变为褐色（图1A、B）；抽蔓期发病率在发病重的地块约为30%～40%，发病症状表现为植株矮小，自下而上的叶片变黄，主根及茎基部变为深褐色，发病严重时主根腐烂（图1C、D）；开花结荚期即使临近地块也常出现发病率差异显著的现象，发病重的地块发病率超过70%（图1G），且自豇豆经历第一次采收之后，发病率常呈上升趋势，症状表现为整株叶片萎蔫，或是从底部自下而上的叶片黄化脱落，茎基部变褐色，根部腐烂，严重时甚至成片枯死导致绝收（图1E、F、H）。对上年度发病较重的地块进行系统调查发现，次年继续在该地块种植豇豆，会出现幼苗期发病率显著升高，甚至出现幼苗全部发病枯死的现象（图1I、J）。对海南省三亚市、海南省乐东黎族自治县、河南省焦作市、湖南省长沙市和贵州省安顺市豇豆种植区枯萎病发病情况的调查结果显示，相较于河南省、湖南省和贵州省，海南省豇豆枯萎病发病情况最为严重。
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图 1 豇豆枯萎病不同时期田间发病情况A～B，幼苗期豇豆枯萎病田间症状；C～D，抽蔓期豇豆枯萎病田间症状；E～F，开花结荚期豇豆枯萎病田间症状；G，开花结荚期临近两地发病率差异显著，左为发病严重地块，右为发病轻微地块；H，开花结荚期豇豆枯萎病田间发病情况；I，上年度发病较重地块，次年继续种植豇豆一周时的苗期生长情况；J，上年度发病较重地块，次年继续种植豇豆两周时的苗期生长情况。Figure 1. Disease incidence on cowpea plants at different time periods in the fieldA-B: symptoms of cowpea wilt disease at seedling stage in the field; C-D: disease symptoms at cowpea vining stage in the field; E-F: disease symptoms at cowpea flowering and podding stages in the field; G: significantly different disease incident of cowpea plants at flowering and podding stages in two neighboring lots—severely infected lot on left and mildly on right; H: appearance of lot with wilt, flowering and podding cowpea plants; I: 1-week-old cowpea seedling on lot severely infected by disease in previous year; J: 2-week-old cowpea seedling on lot severely infected by disease in previous year.2.2 病原菌的分离
对采集得到的285株豇豆各生长时期的病株进行病原菌的分离，共分离得到菌株744株。其中，从苗期采集的157株病样中分离获得200个菌株，包括真菌160株，占比80%，卵菌40株，占比20%。在抽蔓期采集的98株病样中分离获得316个菌株，包括真菌269株，占比85%，卵菌47株，占比15%。从开花结荚期采集的30株病样中分离获得228个菌株，包括真菌187株，占比82%，卵菌41株，占比18%（图2A）。
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图 2 分离菌株数量统计及豇豆黄化苗致病性测定A，各生长期豇豆枯萎病分离菌株的数量统计；B，部分病原菌接种豇豆黄化苗发病情况，CK为接种空白培养基3 d的黄化苗下胚轴，病原真菌的编号为1~49（F. oxysporum）、50~54（Nigrospora osmanthi）、55（F. solani）和56（R. solani）；病原卵菌的编号为57~74（Phytophthora vignae）和75（P. nicotianae）；病原真菌接种3 d后统计结果，病原卵菌接种2 d后统计结果。Figure 2. Statistics on number of isolated strains and determination of infected yellowing cowpea seedlingsA: statistics on number of pathogens isolated from cowpea plants at various stages of growth; B: disease incidence on yellowing cowpea seedlings inoculated with pathogens; CK: hypocotyl of yellowing cowpea seedlings inoculated with blank media for 3 d; codes of fungi: 1-49 for F. oxysporum, 50-54 for N. osmanthi, 55 for F. solani, and 56 for R. solani; codes of oomycetes: 57-74 for P. vignae and 75 for P. nicotianae; results were taken 3d after inoculation on fungi, and 2 d on oomycetes.2.3 致病性测定
对分离得到的真菌和卵菌通过形态学特征进行归类编码，分别从每一类中挑选5株作为代表性菌株进行致病性测定。结果表明，共有4类真菌和2类卵菌可侵染豇豆。进一步对这6类菌中包含的全部菌株进行致病性测定。统计结果显示，共有可侵染豇豆的真菌菌株135株，可侵染豇豆的卵菌菌株128株。对上述发病的豇豆再一次进行病原菌分离鉴定，得到的分离菌株与接种菌株一致（图2B）。
2.4 病原菌鉴定
上述可侵染豇豆的病原菌每类中选取一半数量的菌株提取其基因组，利用ITS、β-tublin和LSU引物进行PCR扩增，并将获得的基因测序结果于NCBI GenBank核酸序列库中进行同源性比对分析，综合形态学分类结果、致病性测定结果及分子生物学鉴定结果进行归类统计，确定具有致病性的菌株种类及数目。结果显示，263株病原菌中病原真菌135株，包括尖孢镰刀菌（F. oxysporum）86株、黑孢霉菌（Nigrospora osmanthi）25株、茄腐镰刀菌（F. solani）14株和立枯丝核菌（R. solani）10株；病原卵菌128株，包括豇豆疫霉（Phytophthora vignae）80株、烟草疫霉（P. nicotianae）48株。
根据初步的鉴定结果，进一步选取1株致病性强的优势病原真菌尖孢镰刀菌（编号Fo-24）、1株在豇豆上未报道过的病原真菌黑孢霉菌（编号Nl-71）、1株致病性强的优势病原卵菌豇豆疫霉（编号Pv-11）和1株在豇豆上未报道过的病原卵菌烟草疫霉（编号Pn-10），并对其进行形态学鉴定。菌株Fo-24于PDA培养基上培养7 d可长满90 mm直径的培养皿，菌落形态呈圆形，菌丝淡粉色或淡紫色，气生菌丝发达，大型分生孢子镰刀形，顶细胞弯曲，多数为3隔，少数为4～7隔，大小为（18.33～63.33） μm×（3.33～5.00）μm，小型分生孢子卵圆形或椭圆形，大小为（8.33～20.00）μm×（2.50～5.03）μm，符合尖孢镰刀菌的形态特征；多基因系统发育树的结果显示，菌株Fo-24与F. oxysporum聚类于同一支，因此将菌株Fo-24鉴定为尖孢镰刀菌。菌株Nl-71于PDA培养基上培养4 d可长满90 mm直径的培养皿，菌落形态呈圆形，菌丝前期白色，后期变为褐色，气生菌丝不发达，分生孢子单孢，亮黑色，椭球形或近球形，大小为（13.33～15.37） μm×（10.63～15.84） μm，符合黑孢霉菌的形态特征；多基因系统发育树的结果显示，菌株Nl-71与N. osmanthi聚类于同一支，因此将菌株Nl-71鉴定为黑孢霉菌。菌株Pv-11于V8培养基上培养9 d可长满90 mm直径的培养皿，菌落形态呈圆形白色，气生菌丝不发达，孢子囊多为卵形、椭圆形或倒梨形，大小为（34.57～60.78 ）μm×（30.63～42.78） μm，无明显乳突结构，成熟后不脱落，具内层出现象；藏卵器球形，内含卵孢子，直径35.34～43.45 μm，雄器短棒状形或球形，围生，大小为（16.66～23.33 ）μm×（16.66～25.00） μm，符合豇豆疫霉的形态特征；多基因系统发育树的结果显示，菌株Pv-11与P. vignae聚类于同一支，因此将菌株Pv-11鉴定为豇豆疫霉。菌株Pn-10于V8培养基上培养6 d可长满90 mm直径的培养皿，菌落形态呈圆形白色，气生菌丝发达呈棉絮状，孢子囊梨形、卵形或球形，大小为（22.91～53.33） μm×（18.33～45.00） μm，顶端具有明显乳突结构，成熟后不脱落，不存在明显内层出现象，符合烟草疫霉的形态特征；多基因系统发育树的结果显示，菌株Pn-10与P. nicotianae聚类于同一支，因此将菌株Pn-10鉴定为烟草疫霉（图3）。
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图 3 病原菌尖孢镰刀菌、黑孢霉菌、豇豆疫霉和烟草疫霉的形态学及分子生物学鉴定A，菌株Fo-24、Nl-71、Pv-11和Pn-10的菌落形态；B，菌株Fo-24的分生孢子、菌株Nl-71的分生孢子、菌株Pv-11的孢子囊、藏卵器和雄器以及菌株Pn-10的孢子囊。比例尺为20 μm；C，菌株Fo-24和Nl-71基于ITS、β-tublin和LSU基因序列用邻接法构建的多基因系统发育树；D，菌株Pv-11和Pn-10基于ITS、β-tublin和LSU基因序列用邻接法构建的多基因系统发育树。Figure 3. Morphological and molecular biological identifications of F. oxysporum, N. osmanthi, P. vignae, and P. nicotianaeA: colony morphology of Fo-24, Nl-71, Pv-11, and Pn-10; B: conidia of Fo-24 and Nl-71, sporangia, oogonium, and male organelles of Pv-11, and sporangia of Pn-10; scale=20 μm; C: phylogenetic trees of Fo-24 and Nl-71 based on ITS, andβ-tublin, and LSU constructed by neighbor-joining method; D: phylogenetic trees of Pv-11 and Pn-10 based on ITS, and β-tublin, and LSU constructed by neighbor-joining method.2.5 不同生长时期的致病性测定
2.5.1 黄化苗致病性
接种菌株Fo-24和Nl-71的黄化苗下胚轴接种部位出现褐色病斑并且上下扩展，接种菌株Pv-11和Pn-10的黄化苗下胚轴接种部位先出现水渍状凹陷，继而出现褐色绕茎病斑（图4A）。结果表明，在黄化苗时期，菌株Fo-24、Nl-71、Pv-11和Pn-10均具有致病性。
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图 4 尖孢镰刀菌、黑孢霉菌、豇豆疫霉和烟草疫霉在豇豆各生长时期的致病性测定A，豇豆黄化苗致病性测定，CK为接种空白培养基3 d的黄化苗；B，豇豆幼苗期致病性测定，CK为接种无菌水的幼苗；C，豇豆抽蔓期致病性测定，CK为接种无菌水的幼苗。Figure 4. Pathogenicity ofF. oxysporum, N. osmanthi, P. vignae, and P. nicotianae on cowpea plants at various growth stagesA: determination of infected yellowing cowpea seedlings; CK: seedlings inoculated with blank media for 3 d; B: determination of infected cowpea seedlings; CK: seedlings inoculated with sterile water; C: determination of infected vining cowpea plants; CK: seedlings inoculated with sterile water.2.5.2 幼苗期致病性
豇豆幼苗期接种菌株Fo-24 7～8 d后植株开始出现发病症状，发病时可见叶片黄化脱落，剖开植株根茎部可见维管束变褐色。接种菌株Pv-11植株于5～8 d后出现发病症状，叶片呈水渍状萎蔫，拔出植株可见植株根茎部有褐色绕茎病斑。接种菌株Pn-10的植株于11 d后出现发病症状，发病植株叶片萎蔫变黄，根系皱缩变褐色。接种菌株Nl-71 10 d后植株出现发病症状，发病时可见植株下部叶片萎蔫变黄，拔出植株可见根系变色（图4B）。结果表明，菌株Fo-24、Nl-71、Pv-11和Pn-10均可使豇豆幼苗发病，发病症状与田间一致。
2.5.3 抽蔓期致病性
豇豆抽蔓期接种菌株Fo-24、Pv-11和Pn-10植株均于8～10 d开始出现发病症状，接种菌株Nl-71的植株于10 d后出现发病症状，发病时病株叶片萎蔫变黄脱落，拔出植株可见根茎皱缩变色（图4C）。结果表明，抽蔓期接种Fo-24、Nl-71、Pv-11和Pn-10植株的发病症状与田间一致。
综上所述，尖孢镰刀菌、黑孢霉菌、豇豆疫霉和烟草疫霉对不同生长时期的豇豆植株具有强且稳定的致病能力，并且发病症状与田间豇豆枯萎病症状一致。
2.6 不同药剂对尖孢镰刀菌的敏感性测定
为筛选出对豇豆枯萎病病原真菌尖孢镰刀菌有效的防治药剂，进行10种药剂对尖孢镰刀菌的敏感性测定。结果表明，10种药剂对尖孢镰刀菌都有一定的抑制作用，比较10种药剂的EC50值，从大到小依次为
14719.994 μg·mL−1（王铜）＞65.674 μg·mL−1（恶霉灵）＞38.208 μg·mL−1（代森锰锌）＞24.365 μg·mL−1（唑醚·啶酰菌）＞24.204 μg·mL−1（甲基硫菌灵）＞15.408 μg·mL−1（霜脲·嘧菌酯）＞1.762 μg·mL−1（百菌清）＞0.944 μg·mL−1（多菌灵）＞0.241 μg·mL−1（苯醚甲环唑）＞0.086 μg·mL−1（咪鲜胺），其中咪鲜胺、苯醚甲环唑和多菌灵的EC50值小于1 μg·mL−1，综上所述，尖孢镰刀菌对咪鲜胺、苯醚甲环唑和多菌灵最为敏感，百菌清、霜脲·嘧菌酯次之，EC50值小于20 μg·mL−1，王铜EC50最大，抑菌效果最差（表1）。表 1 不同药剂对尖孢镰刀菌的敏感性测定Table 1. Sensitivity to fungicides of F. oxysporum杀菌剂
Fungicide毒力回归方程
Virulent regression equation相关系数r
Correlation coefficient rEC50值
EC50/(μg·mL−1)百菌清 Chlorothalonil y=0.5413x− 0.1332 0.983 1.762 咪鲜胺 Prochloraz y=1.1652x+ 1.2415 0.991 0.086 代森锰锌 Mancozeb y=2.7541x− 4.3575 0.985 38.208 甲基硫菌灵Thiophanate-methyl y=3.4767x− 4.8113 0.995 24.204 苯醚甲环唑 Dioxoconazole y=0.7250x+ 0.4477 0.983 0.241 多菌灵 Carbendazim y=3.0965x+ 0.7771 0.987 0.944 恶霉灵 Hymexazol y=2.7704x− 5.0348 0.997 65.674 王铜 Copper oxychloride y=1.4458x− 6.0258 0.991 14719.994 唑醚·啶酰菌 Pyraclostrobin-boscalid y=0.6124x− 0.8493 0.964 24.365 霜脲·嘧菌酯 Cymoxanil-azoxystrobin y=0.7184x− 0.8459 0.965 15.408 2.7 不同药剂对病原真菌和病原卵菌的抑菌效果测定
由于在豇豆各个生长时期均能分离得到病原卵菌，为筛选到防治豇豆枯萎病的高效杀菌剂，进一步测定10种药剂对尖孢镰刀菌、豇豆疫霉和烟草疫霉的抑菌效果。结果表明，对尖孢镰刀菌抑菌率大于50%的药剂有咪鲜胺、多菌灵、甲基硫菌灵、苯醚甲环唑、霜脲·嘧菌酯和百菌清。对豇豆疫霉抑菌率大于50%的药剂有霜脲·嘧菌酯、王铜、代森锰锌和恶霉灵。对烟草疫霉抑制率大于50%的药剂有霜脲·嘧菌酯、王铜和代森锰锌。其中，当浓度为500 μg·mL−1时，霜脲·嘧菌酯对豇豆疫霉和烟草疫霉的抑菌率为100%，对尖孢镰刀菌的抑菌率为55.32%（表2）。
表 2 不同药剂对病原真菌和病原卵菌的抑菌效果测定Table 2. Inhibitory effects of fungicides on fungi and oomycetes杀菌剂
Fungicide质量浓度
Concentration/(μg·mL−1)抑菌率 Inhibitory rate/% 尖孢镰刀菌
F. oxysporum豇豆疫霉
P vignae烟草疫霉
P. nicotianae咪鲜胺 Prochloraz 1 100.00±0.00 a 19.26±2.35 b 17.42±1.93 b 多菌灵 Carbendazim 2.5 87.05±1.95 a 4.04±2.70 b 4.27±1.55 b 甲基硫菌灵 Thiophanate-methyl 40 69.51±1.78 a 25.99±0.31 b 13.13±1.41 c 苯醚甲环唑 Dioxoconazole 2.5 63.46±1.53 a 8.36±1.76 b 13.71±1.18 b 霜脲·嘧菌酯 Cymoxanil-azoxystrobin 500 55.32±3.43 b 100.00±0.00 a 100.00±0.00 a 百菌清 Chlorothalonil 4 53.86±0.85 a 11.80±0.28 c 22.85±2.29 b 王铜 Copper oxychloride 1250 21.50±2.66 b 100.00±0.00 a 100.00±0.00 a 代森锰锌 Mancozeb 15 28.18±1.82 b 100.00±0.00 a 100.00±0.00 a 恶霉灵 Hymexazol 50 39.08±2.00 b 73.89±2.07 a 15.37±2.57 c 唑醚·啶酰菌 Pyraclostrobin-boscalid 500 44.17±0.81 a 45.70±2.59 a 36.77±1.31 b 抑菌率数据为平均数±标准差，同行不同字母表示经单因素方差分析法检验差异显著（P＜0.05）。
Data on fungal inhibition rate are mean ± standard error; those with different letters indicate significant difference at P＜0.05 by one-way ANOVA.3. 讨论与结论
本研究通过对豇豆各种植区的豇豆枯萎病的发病情况进行田间调查，发现在不同年间、不同地区、不同地块以及豇豆生长的不同时期，枯萎病的发病率差异显著，相较于河南省、贵州省和湖南省，海南省豇豆枯萎病的发病情况最为严重。豇豆枯萎病从苗期到开花结荚期均有发生，开花结荚期时，由于此时豇豆已从营养生长转为营养和生殖生长并存时期，更易受到病原菌的侵害，自豇豆经历第一次采收后，随着豇豆生长发病率逐渐上升，到采收末期，发病严重地块甚至出现绝收现象，严重影响豇豆的产量和经济效益。
明确病原菌是防治病害的关键，相关研究报道显示，豇豆枯萎病属于土传性真菌病害，其病原菌为尖镰孢[28−29]，也有研究指出，裂口镰刀菌和层生镰刀菌同样能够引发豇豆枯萎病[14−15]，此外，色二孢（Diplodia seriata）可致使豇豆出现枯萎和坏死症状[30]。除病原真菌外，病原卵菌豇豆疫霉也是导致豇豆死藤和萎蔫的重要病原菌[31]。本研究采集大量豇豆枯萎病病株分离得到可侵染豇豆的病原真菌共计135株（占比51.3%），包括尖孢镰刀菌86株，占比32.7%，黑孢霉菌25株，占比9.5%，茄腐镰刀菌14株，占比5.3%和立枯丝核菌10株，占比3.8%；病原卵菌共计128株（占比48.7%），包括豇豆疫霉80株，占比30.4%，烟草疫霉48株，占比18.3%。其中占比最高的是尖孢镰刀菌，其次是豇豆疫霉和烟草疫霉。不同时期的致病性测定结果显示，尖孢镰刀菌、黑孢霉菌、豇豆疫霉以及烟草疫霉均为引发豇豆枯萎病的病原菌。在本研究中，首次发现病原真菌黑孢霉菌与病原卵菌烟草疫霉能够引起豇豆枯萎病的发生。针对豇豆枯萎病的防治除了要重视病原真菌尖孢镰刀菌外，还应密切关注新发现的病原真菌黑孢霉菌和病原卵菌豇豆疫霉和烟草疫霉的防控。
目前市面上防治豇豆枯萎病的药剂种类多，田间用药防控时，对病原卵菌的忽视可能是造成豇豆枯萎病难以防治的原因之一。本研究选取了10种常用防治枯萎病的药剂进行对病原真菌尖孢镰刀菌的敏感性测定，结果表明，病原真菌尖孢镰刀菌对咪鲜胺、苯醚甲环唑和多菌灵敏感性较高，EC50值均小于1 μg·mL−1，尖孢镰刀菌对百菌清的敏感性次之，EC50值小于10 μg·mL−1。田间生产中，针对尖孢镰刀菌引起的病害可以选取上述这4种药剂进行防治。不同药剂对病原真菌和病原卵菌的抑菌试验表明，咪鲜胺、多菌灵、甲基硫菌灵、苯醚甲环唑和百菌清虽然对病原真菌尖孢镰刀菌抑菌作用较好，但对烟草疫霉和豇豆疫霉抑菌效果甚微；王铜和代森锰锌虽然对病原卵菌豇豆疫霉和烟草疫霉的抑菌作用好，但对病原真菌尖孢镰刀菌抑菌效果不好；供试的10种药剂中，霜脲·嘧菌酯对尖孢镰刀菌、豇豆疫霉和烟草疫霉均有较好的抑制效果。霜脲·嘧菌酯是霜脲氰和嘧菌酯的混配药剂，霜脲氰属于氰基乙酰胺类杀菌剂，广泛应用于卵菌纲病害的防治[32]，嘧菌酯是甲氧基丙烯酸酯类杀菌剂，具有渗透、保护、清除和内吸作用等，被广泛应用于子囊菌（真菌）、卵菌病害的防治[33]。2 000倍稀释浓度（田间使用浓度）下，即500 μg·mL−1时，霜脲·嘧菌酯对尖孢镰刀菌的抑菌率为55.32%，对豇豆疫霉和烟草疫霉的抑制率达100%，说明霜脲·嘧菌酯对尖孢镰刀菌、豇豆疫霉和烟草疫霉抑菌效果较强，可以作为田间防治豇豆枯萎病的防治药剂。
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图 1 豇豆枯萎病不同时期田间发病情况
A～B，幼苗期豇豆枯萎病田间症状；C～D，抽蔓期豇豆枯萎病田间症状；E～F，开花结荚期豇豆枯萎病田间症状；G，开花结荚期临近两地发病率差异显著，左为发病严重地块，右为发病轻微地块；H，开花结荚期豇豆枯萎病田间发病情况；I，上年度发病较重地块，次年继续种植豇豆一周时的苗期生长情况；J，上年度发病较重地块，次年继续种植豇豆两周时的苗期生长情况。
Figure 1. Disease incidence on cowpea plants at different time periods in the field
A-B: symptoms of cowpea wilt disease at seedling stage in the field; C-D: disease symptoms at cowpea vining stage in the field; E-F: disease symptoms at cowpea flowering and podding stages in the field; G: significantly different disease incident of cowpea plants at flowering and podding stages in two neighboring lots—severely infected lot on left and mildly on right; H: appearance of lot with wilt, flowering and podding cowpea plants; I: 1-week-old cowpea seedling on lot severely infected by disease in previous year; J: 2-week-old cowpea seedling on lot severely infected by disease in previous year.
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图 2 分离菌株数量统计及豇豆黄化苗致病性测定
A，各生长期豇豆枯萎病分离菌株的数量统计；B，部分病原菌接种豇豆黄化苗发病情况，CK为接种空白培养基3 d的黄化苗下胚轴，病原真菌的编号为1~49（F. oxysporum）、50~54（Nigrospora osmanthi）、55（F. solani）和56（R. solani）；病原卵菌的编号为57~74（Phytophthora vignae）和75（P. nicotianae）；病原真菌接种3 d后统计结果，病原卵菌接种2 d后统计结果。
Figure 2. Statistics on number of isolated strains and determination of infected yellowing cowpea seedlings
A: statistics on number of pathogens isolated from cowpea plants at various stages of growth; B: disease incidence on yellowing cowpea seedlings inoculated with pathogens; CK: hypocotyl of yellowing cowpea seedlings inoculated with blank media for 3 d; codes of fungi: 1-49 for F. oxysporum, 50-54 for N. osmanthi, 55 for F. solani, and 56 for R. solani; codes of oomycetes: 57-74 for P. vignae and 75 for P. nicotianae; results were taken 3d after inoculation on fungi, and 2 d on oomycetes.
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图 3 病原菌尖孢镰刀菌、黑孢霉菌、豇豆疫霉和烟草疫霉的形态学及分子生物学鉴定
A，菌株Fo-24、Nl-71、Pv-11和Pn-10的菌落形态；B，菌株Fo-24的分生孢子、菌株Nl-71的分生孢子、菌株Pv-11的孢子囊、藏卵器和雄器以及菌株Pn-10的孢子囊。比例尺为20 μm；C，菌株Fo-24和Nl-71基于ITS、β-tublin和LSU基因序列用邻接法构建的多基因系统发育树；D，菌株Pv-11和Pn-10基于ITS、β-tublin和LSU基因序列用邻接法构建的多基因系统发育树。
Figure 3. Morphological and molecular biological identifications of F. oxysporum, N. osmanthi, P. vignae, and P. nicotianae
A: colony morphology of Fo-24, Nl-71, Pv-11, and Pn-10; B: conidia of Fo-24 and Nl-71, sporangia, oogonium, and male organelles of Pv-11, and sporangia of Pn-10; scale=20 μm; C: phylogenetic trees of Fo-24 and Nl-71 based on ITS, andβ-tublin, and LSU constructed by neighbor-joining method; D: phylogenetic trees of Pv-11 and Pn-10 based on ITS, and β-tublin, and LSU constructed by neighbor-joining method.
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图 4 尖孢镰刀菌、黑孢霉菌、豇豆疫霉和烟草疫霉在豇豆各生长时期的致病性测定
A，豇豆黄化苗致病性测定，CK为接种空白培养基3 d的黄化苗；B，豇豆幼苗期致病性测定，CK为接种无菌水的幼苗；C，豇豆抽蔓期致病性测定，CK为接种无菌水的幼苗。
Figure 4. Pathogenicity ofF. oxysporum, N. osmanthi, P. vignae, and P. nicotianae on cowpea plants at various growth stages
A: determination of infected yellowing cowpea seedlings; CK: seedlings inoculated with blank media for 3 d; B: determination of infected cowpea seedlings; CK: seedlings inoculated with sterile water; C: determination of infected vining cowpea plants; CK: seedlings inoculated with sterile water.
表 1 不同药剂对尖孢镰刀菌的敏感性测定
Table 1 Sensitivity to fungicides of F. oxysporum
杀菌剂
Fungicide毒力回归方程
Virulent regression equation相关系数r
Correlation coefficient rEC50值
EC50/(μg·mL−1)百菌清 Chlorothalonil y=0.5413x− 0.1332 0.983 1.762 咪鲜胺 Prochloraz y=1.1652x+ 1.2415 0.991 0.086 代森锰锌 Mancozeb y=2.7541x− 4.3575 0.985 38.208 甲基硫菌灵Thiophanate-methyl y=3.4767x− 4.8113 0.995 24.204 苯醚甲环唑 Dioxoconazole y=0.7250x+ 0.4477 0.983 0.241 多菌灵 Carbendazim y=3.0965x+ 0.7771 0.987 0.944 恶霉灵 Hymexazol y=2.7704x− 5.0348 0.997 65.674 王铜 Copper oxychloride y=1.4458x− 6.0258 0.991 14719.994 唑醚·啶酰菌 Pyraclostrobin-boscalid y=0.6124x− 0.8493 0.964 24.365 霜脲·嘧菌酯 Cymoxanil-azoxystrobin y=0.7184x− 0.8459 0.965 15.408 
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表 2 不同药剂对病原真菌和病原卵菌的抑菌效果测定
Table 2 Inhibitory effects of fungicides on fungi and oomycetes
杀菌剂
Fungicide质量浓度
Concentration/(μg·mL−1)抑菌率 Inhibitory rate/% 尖孢镰刀菌
F. oxysporum豇豆疫霉
P vignae烟草疫霉
P. nicotianae咪鲜胺 Prochloraz 1 100.00±0.00 a 19.26±2.35 b 17.42±1.93 b 多菌灵 Carbendazim 2.5 87.05±1.95 a 4.04±2.70 b 4.27±1.55 b 甲基硫菌灵 Thiophanate-methyl 40 69.51±1.78 a 25.99±0.31 b 13.13±1.41 c 苯醚甲环唑 Dioxoconazole 2.5 63.46±1.53 a 8.36±1.76 b 13.71±1.18 b 霜脲·嘧菌酯 Cymoxanil-azoxystrobin 500 55.32±3.43 b 100.00±0.00 a 100.00±0.00 a 百菌清 Chlorothalonil 4 53.86±0.85 a 11.80±0.28 c 22.85±2.29 b 王铜 Copper oxychloride 1250 21.50±2.66 b 100.00±0.00 a 100.00±0.00 a 代森锰锌 Mancozeb 15 28.18±1.82 b 100.00±0.00 a 100.00±0.00 a 恶霉灵 Hymexazol 50 39.08±2.00 b 73.89±2.07 a 15.37±2.57 c 唑醚·啶酰菌 Pyraclostrobin-boscalid 500 44.17±0.81 a 45.70±2.59 a 36.77±1.31 b 抑菌率数据为平均数±标准差，同行不同字母表示经单因素方差分析法检验差异显著（P＜0.05）。
Data on fungal inhibition rate are mean ± standard error; those with different letters indicate significant difference at P＜0.05 by one-way ANOVA.
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[1] 刘琴,黄伟康,符启位,等. 99份长荚豇豆种质资源及其枯萎病抗性相关分析研究[J]. 种子,2022,41(4) :70−80,85. LIU Q,HUANG W K,FU Q W,et al. Study on 99 long podded cow pea germplasm reasources and correlation analysis of their resistance to Fusarium wilt[J]. Seed,2022,41(4) :70−80,85. (in Chinese)
[2] 袁伟方,王硕,张曼丽,等. 三亚市豇豆重大病虫发生动态监测[J]. 中国植保导刊,2017,37(7) :39−41,65. DOI: 10.3969/j.issn.1672-6820.2017.07.008 YUAN W F,WANG S,ZHANG M L,et al. Dynamic monitoring of major diseases and pests of cowpea in Sanya City[J]. China Plant Protection,2017,37(7) :39−41,65. (in Chinese) DOI: 10.3969/j.issn.1672-6820.2017.07.008
[3] 潘磊,李依,余晓露,等. 豇豆分子遗传学研究进展[J]. 长江蔬菜,2014(24) :1−13. DOI: 10.3865/j.issn.1001-3547.2014.24.001 PAN L,LI Y,YU X L,et al. Research progress of molecular genetics in cowpea (Vigna unguiculata) [J]. Journal of Changjiang Vegetables,2014(24) :1−13. (in Chinese) DOI: 10.3865/j.issn.1001-3547.2014.24.001
[4] 汪宝根,吴新义,徐沛,等. 豇豆新品种之豇618的选育[J]. 中国蔬菜,2020(5) :88−91. WANG B G,WU X Y,XU P,et al. A new cowpea cultivar:'Zhijiang 618'[J]. China Vegetables,2020(5) :88−91. (in Chinese)
[5] 肖敏,曾向萍,严婉荣,等. 海南豇豆枯萎病病原鉴定及生物学特性初步研究[J]. 基因组学与应用生物学,2015,34(2) :345−349. XIAO M,ZENG X P,YAN W R,et al. Pathogenic identification of cowpea Fusarium wilt and preliminary research of its biological characteristics in Hainan[J]. Genomics and Applied Biology,2015,34(2) :345−349. (in Chinese)
[6] 张衍荣,李桂花,何自福,等. 豇豆枯萎病抗病性鉴定技术研究[J]. 华南农业大学学报,2005,26(3) :22−25. DOI: 10.3969/j.issn.1001-411X.2005.03.006 ZHANG Y R,LI G H,HE Z F,et al. Studies on techniques for evaluating the resistance in yardlong bean to Fusarium wilt[J]. Journal of South China Agricultural University,2005,26(3) :22−25. (in Chinese) DOI: 10.3969/j.issn.1001-411X.2005.03.006
[7] 吴仁锋,杨绍丽,万鹏,等. 豇豆根腐病病原分离鉴定及其核糖体rDNA-ITS序列分析[J]. 湖北农业科学,2011,50(24) :5107−5110. DOI: 10.3969/j.issn.0439-8114.2011.24.026 WU R F,YANG S L,WAN P,et al. Pathogeny isolation and identification of cowpea root rot disease and sequencing of ribosomal rDNA-ITS[J]. Hubei Agricultural Sciences,2011,50(24) :5107−5110. (in Chinese) DOI: 10.3969/j.issn.0439-8114.2011.24.026
[8] 饶荣莲. 豇豆全生育期病虫害非化学防治技术探索[J]. 中国植保导刊,2024,44(11) :61−66. RAO R L. Exploration on non-chemical control technology of Vigna unguiculata pests during the whole growth period[J]. China Plant Protection,2024,44(11) :61−66. (in Chinese)
[9] 姜鹏,聂继云,刘明雨,等. 我国豇豆农药登记产品和残留限量分析及相关建议[J]. 中国瓜菜,2024,37(1) :11−17. JIANG P,NIE J Y,LIU M Y,et al. Analysis and suggestions of Chinese pesticide registration products and residue limits for cowpea[J]. China Cucurbits and Vegetables,2024,37(1) :11−17. (in Chinese)
[10] DING C P,ZHANG Y F,CHEN C B,et al. Hollow mesoporous silica nanoparticles as a new nanoscale resistance inducer for Fusarium wilt control:size effects and mechanism of action[J]. International Journal of Molecular Sciences,2024,25(8) :4514. DOI: 10.3390/ijms25084514
[11] OMOIGUI L O,DANMAIGONA C C,KAMARA A Y,et al. Genetic analysis of Fusarium wilt resistance in cowpea (Vigna unguiculata Walp. ) [J]. Plant Breeding,2018,137(5) :773−781. DOI: 10.1111/pbr.12628
[12] 李祖莅,袁伟方. 海南豇豆枯萎病病原菌的药敏性分析[J]. 中国热带农业,2017(6) :40−42. DOI: 10.3969/j.issn.1673-0658.2017.06.011 LI Z L,YUAN W F. Drug sensitivity analysis of pathogen of cowpea Fusarium wilt in Hainan[J]. China Tropical Agriculture,2017(6) :40−42. (in Chinese) DOI: 10.3969/j.issn.1673-0658.2017.06.011
[13] HAO Y L,LIU R,MAO Z C,et al. Identification and analysis of WRKY transcription factors in response to cowpea Fusarium wilt in cowpea[J]. Plants,2024,13(16) :2273. DOI: 10.3390/plants13162273
[14] DO AMARAL A C T,KOROIVA R,DA COSTA A F,et al. First report of Fusarium lacertarum as the causal agent of wilt in Vigna unguiculata[J]. Journal of Plant Pathology,2022,104(3) :1173. DOI: 10.1007/s42161-022-01146-7
[15] FARIAS O R,CRUZ J M F L,VELOSO J S,et al. Occurrence of Fusarium proliferatum causing vascular wilt on cowpea (Vigna unguiculata) in Brazil[J]. Plant Disease,2022,106(7) :1992.
[16] 吴仁锋,杜凤珍,罗惠玲. 豇豆枯萎病的识别与防治[J]. 长江蔬菜,2011(15) :45−46,67. DOI: 10.3865/j.issn.1001-3547.2011.15.028 WU R F,DU F Z,LUO H L. Identification and control of cowpea wilt[J]. Journal of Changjiang Vegetables,2011(15) :45−46,67. (in Chinese) DOI: 10.3865/j.issn.1001-3547.2011.15.028
[17] 陈玲,向娟,张河庆,等. 成都平原豇豆根腐病菌的分离与鉴定[J]. 西南大学学报(自然科学版) ,2021,43(9) :21−29. CHEN L,XIANG J,ZHANG H Q,et al. Isolation and identification of the pathogenic Fusarium spp. causing cowpea root rot in Chengdu Plain[J]. Journal of Southwest University (Natural Science Edition) ,2021,43(9) :21−29. (in Chinese)
[18] ABBAS A,ALI A,HUSSAIN A,et al. Assessment of genetic variability and evolutionary relationships of Rhizoctonia solani inherent in legume crops[J]. Plants,2023,12(13) :2515. DOI: 10.3390/plants12132515
[19] LAMINI S,KUSI F,CORNELIUS E W,et al. Identification of sources of resistance in cowpea lines to Macrophomina root rot disease in Northern Ghana[J]. Heliyon,2022,8(12) :e12217. DOI: 10.1016/j.heliyon.2022.e12217
[20] 郭玉珍,李庆杰,刘和吉,等. 豇豆根腐病症状及防治措施[J]. 河北农业科技,2008(15) :18. GUO Y Z,LI Q J,LIU H J,et al. Cowpea root rot symptoms and control measures[J]. Journal of Hebei Agricultural Sciences,2008(15) :18. (in Chinese)
[21] WANG S C,ZHANG X Y,ZHANG Z C,et al. Fusarium-produced vitamin B6 promotes the evasion of soybean resistance by Phytophthora sojae[J]. Journal of Integrative Plant Biology,2023,65(9) :2204−2217. DOI: 10.1111/jipb.13505
[22] 张衍荣,王小菁,张晓云,等. 水杨酸对豇豆枯萎菌的抑制作用[J]. 华中农业大学学报,2006,25(6) :610−613. DOI: 10.3321/j.issn:1000-2421.2006.06.008 ZHANG Y R,WANG X J,ZHANG X Y,et al. The effects of salicylic acid on yardlong bean Fusarium wilt fungus[J]. Journal of Huazhong Agricultural University (Natural Science Edition) ,2006,25(6) :610−613. (in Chinese) DOI: 10.3321/j.issn:1000-2421.2006.06.008
[23] 沙洪林,薛丽静,金哲宇,等. 蓖麻枯萎病抗病性鉴定方法及抗病资源筛选研究[J]. 吉林农业科学,2002,27(S1) :27−29. DOI: 10.3969/j.issn.1003-8701.2002.z1.012 SHA H L,XUE L J,JIN Z Y,et al. Study on identification method of Castor Fusarium wilt resistance and screening of resistance resources[J]. Journal of Northeast Agricultural Sciences,2002,27(S1) :27−29. (in Chinese) DOI: 10.3969/j.issn.1003-8701.2002.z1.012
[24] 吴多桂,林栖凤,李冠一. 红树DNA的十六烷基三甲基溴化铵法提取及其随机扩增多态DNA反应[J]. 中国生物化学与分子生物学报,1999,15(1) :71−74. WU D G,LIN Q F,LI G Y. Extraction of mangrove DNA by cetyltrimethylammonium bromide method and its random amplification polymorphic DNA reaction[J]. Chinese Journal of Biochemistry and Molecular Biology,1999,15(1) :71−74. (in Chinese)
[25] 苏晓梅,王梦蕊,吕宏君,等. 番茄颈腐根腐病病原菌鉴定及抗性品种筛选[J]. 山东农业科学,2024,56(4) :145−150. SU X M,WANG M R,LYU H J,et al. Identification of pathogen causing Fusarium crown and root rot and screening resistant varieties in tomato[J]. Shandong Agricultural Sciences,2024,56(4) :145−150. (in Chinese)
[26] O’DONNELL K,CIGELNIK E. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous[J]. Molecular Phylogenetics and Evolution,1997,7(1) :103−116. DOI: 10.1006/mpev.1996.0376
[27] 陈丹,张岩,杨华,等. 湖南宜章县金刚藤锈病病原鉴定[J]. 生物灾害科学,2024,47(4) :574−580. CHEN D,ZHANG Y,YANG H,et al. Identification of the pathogen of Smilax China in Yizhang County of Hunan Province[J]. Biological Disaster Science,2024,47(4) :574−580. (in Chinese)
[28] GAN Y D,TU Z W,YANG Y X,et al. Enhancing cowpea wilt resistance:Insights from gene coexpression network analysis with exogenous melatonin treatment[J]. BMC Plant Biology,2024,24(1) :599. DOI: 10.1186/s12870-024-05289-w
[29] LIU Z,XU N,PANG Q Y,et al. A salt-tolerant strain of Trichoderma longibrachiatum HL167 is effective in alleviating salt stress,promoting plant growth,and managing Fusarium wilt disease in cowpea[J]. Journal of Fungi,2023,9(3) :304. DOI: 10.3390/jof9030304
[30] SWILLING K J,SHRESTHA U,YODER C E,et al. First report of wilt and necrosis caused by Diplodia seriata on cowpea in Tennessee,USA[J]. New Disease Reports,2020,42(1) :12. DOI: 10.5197/j.2044-0588.2020.042.012
[31] 黄健坤,戚佩坤. 长豇豆疫病的初步研究[J]. 植物病理学报,1984,14(4) :3−9,68. HUANG J K,QI P K. A preliminary study on Phytophthora of yard-long bean (Vigna sesquipedalis (L.) Fruw) [J]. Acta Phytopathologica Sinica,1984,14(4) :3−9,68. (in Chinese)
[32] 曹爽,尹晓,马金龙,等. 葡萄霜霉菌对五种主要杀菌剂的抗药性研究进展[J]. 农业科学研究,2023,44(3) :64−70. DOI: 10.3969/j.issn.1673-0747.2023.03.013 CAO S,YIN X,MA J L,et al. Research progress on resistance of Plasmopara viticola to five major fungicides[J]. Journal of Agricultural Sciences,2023,44(3) :64−70. (in Chinese) DOI: 10.3969/j.issn.1673-0747.2023.03.013
[33] 苗建强,蔡萌,张灿,等. 植物病原卵菌对重要抑制剂的抗性分子机制研究进展[J]. 农药学学报,2019,21(S1) :736−746. MIAO J Q,CAI M,ZHANG C,et al. Molecular resistance mechanism of phytopathogenic oomycete to several important fungicides[J]. Chinese Journal of Pesticide Science,2019,21(S1) :736−746. (in Chinese)
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