野燕麦病原真菌的鉴定及其除草活性和对作物的安全性

Identification and herbicidal activity of pathogenic fungi against Avena fatua and safety to crops

[目的]旨在筛选安全性高、抗药性低、除草性强的生防菌株,为防除农田恶性杂草野燕麦提供新的生防思路。[方法]采集自然感病的瓜叶葵(Helianthus cucumerifo)、大叶藜(Chenopodium hybridum)、蜀葵(Alcea rosea)和青稞(Hordeum vulgare)植物叶片,通过组织及单孢分离法、离体叶片(初筛)和活体盆栽(复筛),获得具有生防潜力的菌株,接着采用不同浓度(10^(1)~10^(4))孢子悬浮液测定其对野燕麦活体植株的发病率、病情指数和鲜重防效,通过观察菌落形态、运用核糖体内转录间隔区(Internal transcribed spacer, ITS)和转录延伸因子(EF-1α,EF-1/EF-2)基因及MEGA 7.0软件构建系统发育树,最后选取青海地区主栽作物(油菜‘青杂5号’、小麦‘青春38号’、青稞‘昆仑18号’、蚕豆‘青海9号’、豌豆‘草原224号’、玉米‘青玉517号’)和部分蔬菜(白菜‘浙白6号’、番茄‘博玉368号’、黄瓜‘中农5号’、菠菜‘华菠1号’、辣椒‘中椒4号’、萝卜‘春白2号’)进行安全性测试,为菌株选择最适宜的应用条件。[结果]从自然感病的植株叶片中分离出7株对野燕麦具有除草活性的真菌菌株,将其命名为DT-YSB1、DT-04A2、DT-DYLC、DT-XRKA、DT-08C、DT-14A2和DT-QKBD004A。通过离体叶片和温室盆栽试验,综合评价获得1株对野燕麦具有较强除草活性的菌株DT-QKBD004A,菌落形态与镰刀菌相似,对其rDNA ITS和EF-1α(EF-1/EF-2)基因进行共同鉴定及系统发育分析,表明该菌株为燕麦镰刀菌(Fusarium avenaceum);不同浓度菌株DT-QKBD004A孢子悬浮液对野燕麦的发病率、病情指数和鲜重防效达到45.09%~89.48%、42.68~88.53和38.70%~88.42%,其中浓度9.40×10^(4)个/mL对野燕麦的防除效果最优;作物安全性测试发现,菌株DT-QKBD004A孢子浓度9.40×10^(4)个/mL对蚕豆、玉米、白菜、番茄、黄瓜和辣椒安全。[结论]燕麦镰刀菌DT-QKBD004A对野燕麦具有较强的致病性,且对部分作物安全,具有较好的应用价值,可为防除农田恶性杂草野燕麦提供良好的生防菌株。

[Objective]The study aimed to screen for biocontrol strains with high safety,low drug resistance and strong herbicidal activity,providing a new biocontrol strategy for the prevention and control of the pernicious weed Avena fatua in farmland.[Method]Leaves of natural-ly diseased Helianthus cucumerifo,Chenopodium hybridum,Alcea rosea and Hordeum vulgare plants were collected for the isolation and puri-fication of strains using tissue and single spore isolation methods.Strains with biocontrol potential were obtained through in vitro leaf assays(primary screening)and in vivo pot experiments(secondary screening).The strains were identified based on colony morphology observation and the use of the Internal Transcribed Spacer(ITS)and Elongation Factor-1 alpha(EF-la/EF-2)genes:A phylogenetic tree was con-structed using MEGA7.0 sofiware.Strains with biocontrol potential were further tested on live A.fatua plants using spore suspensions of dif-ferent concentrations(10^(1)-10^(4))to determine disease incidence,disease severity index and fresh weight efficacy.Finally,safety tests were conducted on main crops in Qinghai province Brassic anapus'Qingza5',Triticum aestivum'Qingchun38',Hordeum vulgare'Kunlun18',Vicia faba'Qinghai9',Pisum sativum'Caoyuan224',Zea mays'Qingyu517'and some vegetables(Brassia campestris'Zhebai6',Solanum lycopersicum'Boyu368',Cucum issatius'Zhongnong5',Spinacia oleracea'Huabol',Capsicum annuum'Zhongjiao4',Raphanus satius‘Chunbai2')to select the most suitable application conditions for the strains.[Result]Seven fungal strains with herbicidal activity against A.fatua were isolated from leaves of naturally diseased plants and named DT-YSB1,DT-04A2,DT-DYLC,DT-XRKA,DT-08C,DT-14A2 and DT-QKBDO04A.Through in vitro leaf assays and greenhouse pot experiments,strain DT-QKBDO04A was identified as having strong herbicidal activity against A.fatua.Its colony morphology resembled that of Fusarium species,and molecular identification using rDNA ITS and EF-lα(EF-1/EF-2)genes,along with phylogenetic analysis,confirmed it as F.avenaceum.Efficacy tests at dfferent spore concentra-tions revealed that a concentration of9.40×10^(4) spores/mL of strain DT-QKBD004A achieved the best control efficacy against A.fatua,with disease incidence,disease severity index,and fresh weight efficacy ranging from 45.09%-89.48%,42.68-88.53,and 38.70%-88.42%,respectively.Crop safety tests showed that a concentration of 9.40×10^(4) spores/mL of strain DT-QKBD004A was safe for broad bean,corn,Chinese cabbage,tomato,cucumber and pepper.[Conclusion]F.avenaceum DT-QKBDO04A has strong pathogenicity to A.fat-ua and is safe to some crops.It has good application value and can provide a good biocontrol strain for the prevention and control of malignant weeds in farmland.