葡萄炭疽病菌对4种杀菌剂的敏感性分析

Sensitivity analysis of Colletotrichum gloeosporioides to four fungicides

【目的】了解辽宁地区葡萄炭疽病菌(Colletotrichum gloeosporioides)对代森锰锌、戊唑醇、咪鲜胺和苯醚甲环唑4种常用杀菌剂的抗药性现状,为其有效的化学防治提供依据和指导。【方法】采用区分剂量法和菌丝生长速率法,对辽宁的6个葡萄产区的120个葡萄炭疽菌株进行敏感性测定。【结果】辽宁地区葡萄炭疽病菌对不同药剂的敏感性检测结果表明,戊唑醇对供试菌株的EC50值为0.6771~2.6910 mg·L^-1,平均EC50值为1.4749 mg·L^-1,29.17%的菌株对戊唑醇表现出抗药性,沈阳地区葡萄炭疽抗性频率最低为16.67%,葫芦岛地区葡萄炭疽最高抗性频率为45%;代森锰锌对供试菌株的EC50值为11.1565~65.9343 mg·L^-1,平均EC50值为19.8346 mg·L^-1,28.33%的菌株对代森锰锌表现出抗药性,其中,葫芦岛地区葡萄炭疽菌对代森锰锌抗性频率最高,达45%;葡萄炭疽病菌供试群体对咪鲜胺和苯醚甲环唑的敏感性较强,平均EC50值分别为0.0115 mg·L^-1和0.0611 mg·L^-1,不同地区的EC50值差异并不明显,且未在试验中发现抗性菌株。【结论】葡萄炭疽病菌对戊唑醇和代森锰锌表现出中低的抗药性;对咪鲜胺和苯醚甲环唑敏感性较强。

【Objective】Colletotrichum gloeosporioides is the causal agent of grapevine anthracnose,causing a sharp decline in grape growth and yield.Infection of grape industry by Colletotrichum gloeosporioides has led to big yield losses in recent years.The disease will break out in warm and rainy period just before and during maturing time and the conidia are transferred by wind or rain and glues itself to the grape fruit surfaces.After falling on grape fruit,it produces germ tubes and differentiates into specialized appressorium structure.After infection,Colletotrichum gloeosporioides propagates into neighboring cells,causing lesions on grape.In practice,the grapevine anthracnose is generally controlled through fungicides.In order to investigate the sensitivity of C.gloeosporioides to mancozeb,tebuconazole,prochloraz and difenoconazole,diseased berries were collected from different grape vineyards,and the obtained pathogen isolates were tested for their resistance to mancozeb,tebuconazole,prochloraz and difenoconazole.This study could offer a theoretical direction for scientific preventing of grape anthracnose effectively.【Methods】Collected diseased berries were first surface disinfected with 75%ethanol for 30 seconds and then washed with sterile water,two millimeter sized tissues from junction of healthy and diseased grape anthracnose were placed on PDA including chloramphenicol and cultured at 25℃for four days.For getting purified strains,disk containing mycelium picked from medium edge and inoculated to new PDA and cultured at 25℃for eight 8 days,collected conidia were diluted into 10^5 per mL conidial suspensions.Thirty microlitre conidial suspensions were evenly spread on agar medium plates and incubated for twenty hours,and single conidium was selected under a microscope.The strains collected by single spore isolation were observed after culturing on PDA medium for 8 days at 25℃under a light microscope.Furthermore,a droplet of 5μL fresh conidial suspensions was inoculated on slightly wound grape berry made by toothpick prick,and then placed in a box chamber under 25℃.The pathogenecity of grape anthracnose was examined through lesions condition after three or four days after inoculation.Each assay was repeated four times with four duplications for every time.Finally,120 Colletotrichum gloeosporioides isolates from 6 regions of Liaoning were indentified through cultivation and pathogenicity characteristic observation.Distinguishing dosages and the mycelium growth rate method were used to detect the resistance of 120 grape anthracnose isolates.Hyphae were transferred into PDA plates covering different dosages of fungicides,and the diameter of the colony was measured after eight days.The effective inhibiting medium concentrations(EC50)value was calculated according to colony diameter through a software.【Results】EC50 of tebuconazole ranged from 0.6771 to 2.6910 mg·L^-1 with an average value of 1.4749 mg·L-1;29.17%of the 120 isolates showed resistance to tebuconazole in sensitive assay;no strain showed high resistance and thirty strains showed low resistance to tebuconazole.The resistant frequency of grape anthracnose was from 16.67%to 45.00%in tebuconazole plate from six different regions.The lowest resistant frequency of grape anthracnose was 16.67%from Shenyang and the highest resistance of grape anthracnose was 45.00%from Huludao area.However,the EC50 values of different strains from different regions were varied.The EC50 of mancozeb ranged from 11.1565 to 65.9343 mg·L^-1 with a mean value of 19.8346 mg·L-1;28.33%of the 120 isolates showed certain resistance to mancozeb in sensitive test.The grape anthracnose from Huludao area was the most resistant to mancozeb-resistant strains,reaching 45%.From the analysis of EC50 distribution values,there were higher resistant strains from Dalian and Benxi areas,reaching 65.9343 mg·L^-1 and 50.0091 mg·L-1,respectively;There was no colony on PDA plates containing 1 mg·L^-1 prochloraz and resistant prochloraz strain was not found.The EC50 value of the strain was less than 5 times of the sensitive baseline in difenoconazole resistant experiment and studies showed that grape anthracnose from different regions had lower resistance to difenoconazole.It was found that EC50 values of different strains from different regions were less different in prochloraz and difenoconazole plate.All 120 isolates were highly sensitive to prochloraz and difenoconazole with an average EC50 of 0.0115 mg·L^-1 and 0.0611 mg·L-1,respectively【.Conclusion】In this study it was showed that C.gloeosporioides had certain resistance to mancozeb and tebuconazole,and a high sensitivity to prochloraz and difenoconazole.Because mancozeb was a multi-acting site fungicide and had a long history of application,it was very effortless to produce some low resistant strains.It was single site of action that may be an important reason for the development of resistant strains like tebuconazole fungicide.Therefore,growers need to consider the condition of the resistance situation in different regions when tebuconazole and mancozeb will be used in manufacture.Although strains with a certain resistance to prochloraz and difenoconazole fungicide were not found in this test,it was recommended to use fungicides with different target sites in order to avoid the emergence of resistant strains.