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核桃(Juglans regia Linn.)是世界四大干果之一,果实营养丰富,含有人体所需的多种微量元素,以及不饱和脂肪酸,被认为是有益于健康的食品,也是我国重要的“木本粮油”战略树种[1]。四川省是我国核桃生产大省,资源极其丰富,产量居全国第二。近年来四川核产业发展迅猛,全省核桃栽培面积和产量由20世纪50年代的4 600多hm2及年产606 t发展到2000年的6.5万hm2及年产29 834.39 t[2],到2016年底核桃种植面积已达1 300万hm2,而其病害发生也呈现爆发趋势。核桃褐斑病的侵染造成核桃果实黑果、烂果、提前掉果,对核桃产业造成巨大的经济损失,该病害是目前四川地区阻碍核桃产业发展的最重要问题。为此,本文就核桃褐斑病的病原进行分离鉴定,对该病害的发生规律进行了调查,为该病害的防治奠定基础。
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样品采集: 供试植物样品采自四川省中江县双龙镇核桃园自然发病的核桃果实。
PDA培养基:马铃薯200 g,葡萄糖20 g,琼脂18 g,自来水1 000 mL。
病原的分离:采用组织分离法[3], 将采回的病果用自来水清洗表面,吸水纸擦干。用无菌手术刀在病健交界处取0.5 cm×0.5 cm的组织块,浸泡于70%酒精中,表面消毒30 s,组织块转入5%次氯酸钠溶液消毒1 min,无菌水冲洗3次,用无菌吸水纸吸干残留水分,再将组织块置于PDA平板上,28℃、12 h/12 h光照-黑暗交替培养7 d。
病原的纯化:分离得到的真菌,用无菌水将菌孢子洗脱、稀释到平均每视野只有一个孢子,在显微镜下直接将孢子挑取到PDA平板上,28℃、12 h/12 h光照-黑暗交替培养7 d、备用。
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根据柯赫氏法则,将分离纯化得到的真菌回接到健康的核桃果实上。核桃果实表面用酒精、无菌水清洗后晾干,用无菌打孔器把纯化好的真菌打成菌饼,核桃绿皮上打孔,用无菌镊子将真菌菌饼转入核桃果皮圆孔内,用保鲜膜封闭接种孔,将核桃置于28℃人工气候箱保湿培养3 d,观察记录发病情况。每种真菌接种三个核桃,用空白PDA培养基接种做对照。田间接种方法同室内接种方法相同。接种后发病的果实再用组织分离法进行分离培养病原菌。
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将接种发病后再分离得到的病原菌,用PDA培养基28℃、14 d遮光培养,得到的菌孢子用于显微观察。
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本研究选择了ITS和gdp (3-磷酸甘油醛脱氢酶)基因序列对分离到的致病菌株进行扩增,所需引物见表 1,PCR产物克隆到pMD18后送样测序。测序结果通过Blast比对,根据比对结果选取与研究菌株序列接近的种的代表菌株序列,用软件Clustal X1.81与研究菌株的序列进行比对,用PAUP* 4.0 Bata10制作系统进化树。
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根据四川省核桃病害发生特点,在2014年2月—2015年2月,对德阳市中江县双龙镇核桃果园核桃褐斑病进行病害调查。调查随机选择5年生以上盛果期核桃树,在果园内随机选取5个样地,每个样地大小为20 m×20 m,每样地随机选择10棵核桃树,每株树随机选取30个果实进行调查。在果园树枝上悬挂野外温度湿度记录仪,记录每次采样的温度和湿度。
核桃褐斑病病原菌的分离鉴定和发病规律的调查
Identification of Pathogen of Walnut Brown Spot and Investigation of the Disease Occurrence
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摘要:
目的 为了明确四川核桃褐斑病的病原菌及其发病规律。 方法 采用组织分离法,对四川中江核桃果园自然发病的核桃病原菌进行分离,并回接检测分离物的致病性;结合孢子形态学、ITS序列和gdp序列分析对病原菌进行鉴定;同时对核桃褐斑病的发病规律进行了调查。 结果 分离得到6种真菌,回接只有菌株ZJ5能使核桃发病;孢子形态和多序列比较分析将菌株ZJ5鉴定为链格孢菌;调查明确了核桃褐斑病的发病规律。 结论 本文首次报道了四川核桃褐斑病病原菌为链格孢菌,并揭示了该病原菌的发病规律。 Abstract:Objective The purpose of this study is to identify the pathogen of walnut brown spot disease and reveal its pathogenesis in Sichuan. Method To isolate the pathogenic bacteria through tissue separation in diseased walnut from walnut orchard in Zhongjiang of Sichuan Province, and its pathogenicity was tested by inoculation back to walnut. The isolated strain was identified by the combination of spore morphology and the multiple sequence analysis of ITS and gdp sequence. And the pathogenesis regularity of walnut brown spot disease was investigated. Result 6 fungi were isolated, and just the strain ZJ5 could cause walnut brown spot disease. The strain ZJ5 was identified as Alternaria alternata according to the spore morphology and multiple sequence analysis. Beside, the pathogenesis regularity of walnut brown spot disease was revealed by investigation. Conclusion This is the first report that A. alternata is the pathogen of walnut brown spot disease in Sichuan and its pathogenesis regularity was studied. -
Key words:
- walnut
- / brown spot disease
- / pathogen
- / pathogenesis regularity
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