CN102165914B - 用于枯萎病生物测定的黄瓜幼苗的培育方法 - Google Patents
用于枯萎病生物测定的黄瓜幼苗的培育方法 Download PDFInfo
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Abstract
本发明提供一种用于枯萎病生物测定的黄瓜幼苗的培育方法,其具体步骤:先把备好的黄瓜种子浸于盛有无菌水的容器中,并将该容器置于恒温摇床上进行振荡培养,温度设定为25℃,转速90r/min,待容器中的黄瓜种子发芽且芽长至约0.5cm后停止振荡,接着将发芽的黄瓜种子播入盛有消过毒的培育基质的育苗盘中,然后将该育苗盘置于25℃的人工气候箱中培养,培养过程中的光周期为12h光照、12h黑暗,以营养液浇灌,约20天后即可将幼苗用于枯萎病生物测定。本发明的优点在于:不仅可缩短育苗时间、出苗率高,而且采用基质培育能够不受土壤微生物及结构等因素的影响,使得幼苗生长均匀一致。
Description
【技术领域】
本发明涉及一种生物培育方法,尤其涉及一种用于枯萎病生物测定的黄瓜幼苗的培育方法。
【背景技术】
农作物的枯萎病是一种毁灭性的真菌土传病害,而尖孢镰刀菌(Fusariumoxysporum Sch1)是其主要的病原菌。该病危害范围广泛,包括:(1)经济作物:棉花、油桐、蓖麻、亚麻等;(2)茄科作物:番茄、胡椒、茄子、烟草、马铃薯等;(3)瓜类作物:黄瓜、节瓜、冬瓜、西瓜、南瓜、西葫芦、甜瓜、丝瓜、白瓜、越瓜、苦瓜、葫芦瓜、哈密瓜等;(4)豆科作物:大豆、四季豆、豇豆、猪屎豆、豌豆等;(5)花卉植物:康乃馨、郁金香、鱼尾菊、非洲菊、鸢尾、水仙、翠菊、合欢、百合、仙人掌、穿心莲、一品红、草坪草等;(6)林果植物:松树、相思树、柑桔、沙棘、水杉、铁线莲属植物、梨树等;(7)其它植物:莴苣、小麦、大蒜、荸荠、芦笋、姜、香蕉等。
在我国南方,作物枯萎病中以瓜类枯萎病发生最为严重;在我国北方,以棉花枯萎病发生最为严重。枯萎病在植株的全生育期均可发生,一旦发病就难以根除,随着连作年份的增加,发病率越发严重。20世纪90年代以来,我国设施蔬菜瓜类作物面积不断增加,由于在相对封闭的设施内形成的特殊小气候既有利于蔬菜生长,也成为各种病虫害滋生的温床,加之长年连作,使土壤中病原菌的数量不断增加,设施栽培蔬菜的枯萎病发生越来越重。有关研究结果显示:作物枯萎病一般导致减产20-30%,严重田块可达50-80%,甚至绝产,已成为限制着我国作物生产发展重要因素。
选用抗病品种是防治作物枯萎病最常用的方法之一,而抗病鉴定工作是进行抗枯萎病育种的重要环节。在枯萎病菌的致病性测定中,黄瓜苗是一种最常用的材料之一,不但所需量大,而且对幼苗的要求甚为严格。以往大多采用大田土壤育苗、栽培,采用这种方法育出的苗,接种后植株的感病情况难以准确判断或确定,因为大田土壤成份复杂,土壤结构及微生物群系易发生变化,难以确保每次试验的一致性,而且土壤中的微生物也可能参与致病菌的致病作用,导致生物测定实验失败,影响抗病育种工作的进程,另外,传统的育苗方法所需的时间较长,一般不少于30天,因此,寻找一种相对无菌且快速的育苗手段是必需的。
【发明内容】
本发明所要解决的技术问题在于提供一种用于枯萎病生物测定的黄瓜幼苗的培育方法,不仅可缩短育苗时间、出苗率高,而且采用基质培育能够不受土壤微生物及土壤结构等因素的影响,使得幼苗生长均匀一致。
本发明是通过以下技术方案解决上述技术问题的:一种用于枯萎病生物测定的黄瓜幼苗的培育方法,其包括如下步骤:先把备好的黄瓜种子浸于盛有无菌水的容器中,并将该容器置于恒温摇床上进行振荡培养,温度设定为25℃,转速90r/min,待容器中的黄瓜种子发芽且芽长至0.4-0.6cm后停止振荡,接着将发芽的黄瓜种子播入盛有消过毒的培育基质的育苗盘中,然后将该育苗盘置于25℃的人工气候箱中培养,培养过程中的光周期为12h光照、12h黑暗,以营养液浇灌,培养20-22天后即可将幼苗用于枯萎病生物测定。
进一步地,所述培育基质是由珍珠岩与泥炭按1∶3的体积比组成。
进一步地,所述营养液为市售普通花卉的营养液,其组分为:过磷酸钙800mg/L、硝酸钾700mg/L、硝酸钙700mg/L、硫酸镁280mg/L、硫酸铁120mg/L、硼酸0.6mg/L、硫酸锰0.6mg/L、硫酸锌0.6mg/L、硫酸铜0.6mg/L、硫酸铵0.6mg/L,以水配制。
本发明的有益效果在于:相比传统大田土壤育苗,不仅可缩短育苗时间、出苗率高,而且采用基质培育能够不受土壤微生物及土壤结构等因素的影响,使得幼苗生长均匀一致,进而提高后续枯萎病生物测定的可靠性和准确性。
【具体实施方式】
本发明用于枯萎病生物测定的黄瓜幼苗的培育方法包括如下步骤:先把备好的黄瓜种子浸于盛有无菌水的容器中,并将该容器置于恒温摇床上进行振荡培养,温度设定为25℃,转速90r/min,待容器中的黄瓜种子发芽且芽长至0.4-0.6cm(约振荡培养24h)后停止振荡,接着将发芽的黄瓜种子播入盛有消过毒的培育基质的育苗盘中,然后将该育苗盘置于25℃的人工气候箱中培养,培养过程中的光周期为12h光照、12h黑暗,以营养液浇灌,培养20-22天后即可将幼苗用于枯萎病生物测定。
其中,所述培育基质是由珍珠岩与泥炭按1∶3的体积比组成;所述营养液为市售普通花卉的营养液,其组分为:过磷酸钙800mg/L、硝酸钾700mg/L、硝酸钙700mg/L、硫酸镁280mg/L、硫酸铁120mg/L、硼酸0.6mg/L、硫酸锰0.6mg/L、硫酸锌0.6mg/L、硫酸铜0.6mg/L、硫酸铵0.6mg/L,以水配制。
以下是本发明的一个实施例及利用该实施例培育得到的黄瓜幼苗进行枯萎病的生物测定。
1.用于枯萎病生物生物测定的黄瓜幼苗的培育
(1)材料:黄瓜种子,培育基质,营养液
(2)培育方法的具体步骤
先把备好的黄瓜种子浸于盛有20ml无菌水的150ml三角瓶中,并将三角瓶置于恒温摇床上进行振荡培养,以促进种子发芽及提高种子萌发率,且温度设定为25℃,转速90r/min,待容器中的黄瓜种子发芽且芽长至约0.5cm(约振荡培养24h)后停止振荡,接着将发芽的黄瓜种子播入盛有消过毒的培育基质的育苗盘中,然后将该育苗盘置于25℃的人工气候箱中培养,培养过程中的光周期为12h光照、12h黑暗,并以市售普通花卉的营养液浇灌,22天后即可培育出可用于枯萎病生物测定的黄瓜幼苗,相比传统的大田土壤育苗方法,本发明不仅在培育时间上缩短了1/3左右,且获得的黄瓜幼苗苗状一致,易于挑选。
2.枯萎病的生物测定
(1)供试枯萎病病原菌孢子悬浮液的制备:先把供试枯萎病病原菌(Fusarium oxysporum f.sp.cucumerinum)置于PS液体培养基(马铃薯200g,蔗糖20g)中,且将该培养基置于摇床上培养7天,培养温度设定为28℃,转速170r/min,接着把培养后所得的菌液用双层纱布滤去菌丝,最后将滤液配制成1×107CFU/ml的孢子悬浮液备用。
(2)枯萎病的生物测定一般采取以下两种接种方法:a.浇灌接菌法:挑选长势均一的黄瓜幼苗,轻轻拔出后将其主根及2根须根截断后移栽入塑料钵头,且每株根部浇入10ml枯萎病病原菌孢子悬浮液;b.浸根接菌法:挑选长势均一的黄瓜幼苗,轻轻拔出后用无菌水洗净根部,接着将其主根及2根须根截断后浸入孢子悬浮液中,2h后移栽入盛有已浇清水的无菌基质的塑料钵头中。
每一种接种方法均同时设置对照组和实验组,实验组采用1中所得成活后的黄瓜幼苗,而对照组采用传统大田育苗所获得的黄瓜幼苗,且实验组及对照组的苗期管理措施相同。逐日观察两组中黄瓜幼苗的生长与感染情况,并比较30天后各组中黄瓜幼苗的感病情况。
结果表明,接种病原菌30天后,两对照组中黄瓜幼苗的枯萎病发病率均在60%以下,并且发病表现相当不一致,难以给出统一评价;而实验组无论是采用浇灌接种法还是浸根接种法,其黄瓜幼苗的枯萎病发病率均达到100%,不仅高度感病,而且感病表现一致,进而表明按照本发明方法获得的黄瓜幼苗能完全满足生物测定和抗病育种的要求。
Claims (3)
1.一种用于枯萎病生物测定的黄瓜幼苗的培育方法,其特征在于:包括如下步骤:先把备好的黄瓜种子浸于盛有无菌水的容器中,并将该容器置于恒温摇床上进行振荡培养,温度设定为25℃,转速90r/min,待容器中的黄瓜种子发芽且芽长至0.4-0.6cm后停止振荡,接着将发芽的黄瓜种子播入盛有消过毒的培育基质的育苗盘中,然后将该育苗盘置于25℃的人工气候箱中培养,培养过程中的光周期为12h光照、12h黑暗,以营养液浇灌,培养20-22天后即可将幼苗用于枯萎病生物测定。
2.如权利要求1所述的用于枯萎病生物测定的黄瓜幼苗的培育方法,其特征在于:所述培育基质是由珍珠岩与泥炭按1∶3的体积比组成。
3.如权利要求1所述的用于枯萎病生物测定的黄瓜幼苗的培育方法,其特征在于:所述营养液为市售普通花卉的营养液,其组分为:过磷酸钙800mg/L、硝酸钾700mg/L、硝酸钙700mg/L、硫酸镁280mg/L、硫酸铁120mg/L、硼酸0.6mg/L、硫酸锰0.6mg/L、硫酸锌0.6mg/L、硫酸铜0.6mg/L、硫酸铵0.6mg/L,以水配制。
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