CN110331185A - 一种葫芦抗根腐病的苗期鉴定方法 - Google Patents
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Abstract
本发明涉及一种葫芦抗根腐病的苗期鉴定方法,属于作物抗病性鉴定技术领域。首先取葫芦根腐病菌制备根腐病菌孢子悬浮液,浓度为106/mL,待葫芦幼苗子叶展平后,根部注射接种葫芦根腐病菌孢子悬浮液,20d后调查各植株的病级数并计算病情指数,评价葫芦砧木种质资源对根腐病菌的抗病性。本发明建立了葫芦砧木根腐病抗性苗期鉴定方法,该鉴定方法为苗期鉴定,操作简单,快速高效,鉴定结果准确可靠,可显著区分不同葫芦砧木材料对根腐病的抗感差异,适合对大批量的材料进行根腐病抗病性鉴定和筛选,提高了种质资源材料的筛选效率。
Description
技术领域
本发明涉及一种葫芦抗根腐病的苗期鉴定方法,属于作物抗病性鉴定技术领域。
背景技术
嫁接技术是一种有效防治由尖孢镰刀菌西瓜专化型(Fusarium oxyxporumf.sp.niveum)侵染引起的西瓜枯萎病的重要手段。目前生产上常用的西瓜砧木种类主要有葫芦、南瓜、冬瓜和野生西瓜。然而,随着嫁接西瓜设施栽培面积的不断增加,近年来在我国不同西瓜主产区,频频发生嫁接西瓜植株根部染病,叶片枯萎直至植株死亡的现象,导致西瓜产量下降,造成了严重的经济损失。该病害症状与西瓜枯萎病症状很相似,但又不同于枯萎病,其特征是发病部位及症状主要在根及根茎部,严重侵染时根茎部组织脱落,仅剩维管组织,俗称根腐病。
国内外关于嫁接西瓜根腐病的研究主要集中于病原菌鉴定、抗病砧木资源筛选方面。在嫁接西瓜枯萎病病原菌鉴定方面,林燚等(浙江农业科学,2007,1:84-86)根据菌落特征、孢子形态和分子鉴定,认为嫁接西瓜枯萎病病株上的病原菌是茄腐镰孢菌(Fusariumsolani)和尖孢镰孢菌(Fusarium oxysporum)。采用同样的鉴定方法,Armengol等(BulletinOEPP,2000,30:179-183)和Jiang等(1stISHS International SymposiumonVegetable Grafting,2014,P65)认为茄腐镰孢菌瓜类专化型生理小种1为引起嫁接西瓜根腐病的优势菌株。在抗病砧木资源筛选方面,林燚等(浙江农业科学,2007,1:84-86)用茄腐镰孢菌分别对6种葫芦砧木和“早佳”西瓜进行致病性试验,发现其对所有受试材料均具有致病性。Armengol等(Bulletin OEPP,2000,30:179-183)用2个茄腐镰孢菌瓜类专化型菌株接种于10个南瓜砧木品种进行抗病砧木筛选,但未得到理想的抗病砧木品种。Jiang等(1stISHS International SymposiumonVegetable Grafting,2014,P65)评价了26种葫芦砧木、30种南瓜砧木和20种西瓜本砧对茄腐镰孢菌生理小种1的抗病性,从中筛选出1个葫芦抗病砧木和5个南瓜抗病砧木。以上研究多集中于南瓜砧木的抗性鉴定,而以葫芦为砧木的抗性鉴定工作则非常少。同时,以上研究主要以茄腐镰孢菌进行砧木抗病性筛选,而茄腐镰孢菌在世界上被广泛报导为引起田间以南瓜为主的根颈部腐烂病的病原菌。由于病原菌与宿主的专化型识别,茄腐镰孢菌的筛选结果无法反映葫芦砧木的根腐病抗性水平。因此,针对葫芦砧木根腐病的抗性鉴定需要建立一套快速简便、鉴定结果准确的抗性鉴定方法。
发明内容
本发明的目的在于针对目前葫芦根腐病抗病育种上没有一套标准的抗性鉴定方法这一现状,提出一种操作简便、高效快速的鉴定葫芦抗根腐病的苗期鉴定方法,为葫芦砧木抗性种质资源的筛选及抗病育种提供技术支撑。
技术方案
一种葫芦抗根腐病的苗期鉴定方法,包括以下步骤:
(1)取葫芦根腐病菌制备根腐病菌孢子悬浮液;
(2)将葫芦幼苗培养至子叶展平后,采用根部注射法接种根腐病菌孢子悬浮液,然后继续培养葫芦幼苗,20天后调查记录葫芦幼苗的病级,计算病情指数,进行抗病性评价;
所述病级按如下病害分级标准判断:
0级:无症状;1级:下胚轴出现轻微症状,但生长正常;2级:胚轴有明显坏死斑,且子叶轻度萎蔫,生长受到一定的影响;3级:胚轴坏死斑严重,真叶萎蔫,生长僵化;4级:植株死亡;
所述病情指数的计算公式为:病情指数%=Σ(病级值×相应病株数)/(调查总株数×4)×100;
所述抗病性评价标准为:
按病情指数评价:≤1%,高抗;1.1-10.0%,抗;10.0-30.0%,中抗;30.1-50.0%,中感;50.0-70.0%,感病;>70%,高感。
进一步,步骤(1)中,根腐病菌孢子悬浮液的制备方法:挑取葫芦根腐病菌菌丝,接种于含50ng/mL链霉素的马铃薯固体培养基上,置于25℃培养箱中,黑暗培养7d后,切取1.0×1.0cm的菌块接种于液体绿豆汤培养基中,在28℃,150-200rpm/min摇床上培养2d,收集并稀释孢子液,调节孢子液浓度至1x106个/mL,得到根腐病菌孢子悬浮液。
进一步,步骤(2)中,每株葫芦幼苗接种10mL根腐病菌孢子悬浮液。
本发明的有益效果:
1.建立了葫芦砧木根腐病抗性苗期鉴定方法。
2.本发明的鉴定方法为苗期鉴定,操作简单,快速高效,鉴定结果准确可靠,可显著区分不同葫芦砧木材料对根腐病的抗感差异。
3.本发明的鉴定方法适合对大批量的材料进行根腐病抗病性鉴定和筛选,提高了种质资源材料的筛选效率。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步说明。应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。在以下的实施例中,下述实施例中所用方法如无特别说明,均为常规方法。所用到的引物,均在首次出现时标明,其后所用相同引物,均以首次标明的内容相同。
实施例1
葫芦根腐病病原菌的分离鉴定:
采集具有典型根腐病症状(植株地上部萎蔫枯死,根部、下胚轴腐烂,只剩维管束)的病株,取病株根部及下胚轴组织,流水清洗5min,滤纸吸干水分,转移到无菌操作台中,70%无水乙醇消毒30s,10%次氯酸钠消毒4min,无菌水清洗3次,置于无菌滤纸上控干水分,接种于PDA培养基,28℃黑暗培养3~5d至菌丝长出,挑取病块周围的菌丝转接到PDA培养基上,28℃黑暗培养5d。用无菌水洗出孢子,进行单孢纯化。将获得的纯培养物在28℃黑暗培养5d后,显微镜观察菌落颜色、大小和性状,同时收集菌体,提取DNA,分别用ITS引物(ITS1:5’-TCCGTAGGTGAACCTGCGG-3’;ITS45’-TCCTCCGCTTATTGATATGC-3’)和EF1α引物(EF1-F:5’-GGAAGTTCGAGACTCCCAAGTA-3’;EF-25’-GGA(G/A)GTACCAGT(G/C)ATCATGTT-3’)从基因组中扩增544bp和500bp的条带。扩增产物割胶纯化后送南京擎科公司测序,测序结果与NCBI数据库(https://www.ncbi.nlm.nih.gov/)中的已知序列进行比对,确定为尖孢镰孢菌葫芦专化型。
实施例2
对37份葫芦砧木种质资源材料进行根腐病抗性苗期鉴定:
(1)制备葫芦根腐病菌制备根腐病菌孢子悬浮液:取实施例1纯化鉴定后的葫芦根腐病菌菌株接种于马铃薯斜面培养基上,4℃保存,用灭菌的接种环挑取根腐病菌菌丝,接种于含50ng/mL链霉素的马铃薯固体培养基上,置于25℃培养箱中,黑暗培养7d,切取1.0×1.0cm的菌块接种于液体绿豆汤培养基(绿豆20g、去离子水1000mL)中,在28℃,150-200rpm/min摇床上培养2d,收集并稀释孢子液,调节孢子液浓度至1x106个/mL,得到根腐病菌孢子悬浮液;
(2)葫芦砧木种质资源材料共37份,采用50孔穴盘(穴深6cm,单穴口径6cm,单穴底径3cm)育苗,穴盘中装入品氏育苗基质,1粒每穴,每份材料种植30株,设置3次重复,共90株。播种后将穴盘置于温室,常规管理,待葫芦幼苗子叶展平后,采用根部注射法接种根腐病菌孢子悬浮液,每株葫芦幼苗接种10mL根腐病菌孢子悬浮液,然后继续培养葫芦幼苗,20天后调查记录葫芦幼苗的病级,计算病情指数,进行抗病性评价;
所述病级按如下病害分级标准判断:
0级:无症状;1级:下胚轴出现轻微症状,但生长正常;2级:胚轴有明显坏死斑,且子叶轻度萎蔫,生长受到一定的影响;3级:胚轴坏死斑严重,真叶萎蔫,生长僵化;4级:植株死亡;
所述病情指数的计算公式为:病情指数%=Σ(病级值×相应病株数)/(调查总株数×4)×100;
所述抗病性评价标准为:
按病情指数评价:≤1%,高抗;1.1-10.0%,抗;10.0-30.0%,中抗;30.1-50.0%,中感;50.0-70.0%,感病;>70%,高感。
37份葫芦砧木种质资源根腐病抗性鉴定结果见表1:
表1 37份葫芦砧木种质资源材料的根腐病抗性评价结果
由表1可以看出,在37份不同葫芦砧木种质资源材料中,1份材料(编号为16s-71)病情指数为27%,属于中抗材料,1份材料(编号为16s-82)病情指数为43.37%,属于中感材料,有12份材料病情指数在50.0-70.0%之间,属于感病材料,有23份材料病情指数在>70.0%,属于高感材料。
本发明的葫芦抗根腐病苗期鉴定方法接种效率高、结果准确,可操作性强,能够显著区分不同葫芦砧木材料对根腐病的抗感差异,可广泛用于葫芦砧木种质资源筛选和品种抗病性鉴定。
Claims (3)
1.一种葫芦抗根腐病的苗期鉴定方法,其特征在于,包括以下步骤:
(1)取葫芦根腐病菌制备根腐病菌孢子悬浮液;
(2)将葫芦幼苗培养至子叶展平后,采用根部注射法接种根腐病菌孢子悬浮液,然后继续培养葫芦幼苗,20天后调查记录葫芦幼苗的病级,计算病情指数,进行抗病性评价;
所述病级按如下病害分级标准判断:
0级:无症状;1级:下胚轴出现轻微症状,但生长正常;2级:胚轴有明显坏死斑,且子叶轻度萎蔫,生长受到一定的影响;3级:胚轴坏死斑严重,真叶萎蔫,生长僵化;4级:植株死亡;
所述病情指数的计算公式为:病情指数%=Σ(病级值×相应病株数)/(调查总株数×4)×100;
所述抗病性评价标准为:
按病情指数评价:≤1%,高抗;1.1-10.0%,抗;10.0-30.0%,中抗;30.1-50.0%,中感;50.0-70.0%,感病;>70%,高感。
2.如权利要求1所述葫芦抗根腐病的苗期鉴定方法,其特征在于,步骤(1)中,根腐病菌孢子悬浮液的制备方法:挑取葫芦根腐病菌菌丝,接种于含50ng/mL链霉素的马铃薯固体培养基上,置于25℃培养箱中,黑暗培养7d后,切取1.0×1.0cm的菌块接种于液体绿豆汤培养基中,在28℃,150-200rpm/min摇床上培养2d,收集并稀释孢子液,调节孢子液浓度至1x106个/mL,得到根腐病菌孢子悬浮液。
3.如权利要求1或2所述葫芦抗根腐病的苗期鉴定方法,其特征在于,步骤(2)中,每株葫芦幼苗接种10mL根腐病菌孢子悬浮液。
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