CN110643517A - 致病疫霉菌培养基myeb及检测致病疫霉菌对植物致病力的方法 - Google Patents
致病疫霉菌培养基myeb及检测致病疫霉菌对植物致病力的方法 Download PDFInfo
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Abstract
本发明属于农业生物领域,具体涉及致病疫霉菌培养基MYEB及检测致病疫霉菌对植物的致病力的方法。本发明的致病疫霉菌培养基,包括酵母浸粉、牛肉浸膏、蛋白胨、琼脂粉等组分。本发明的检测致病疫霉菌对植物的致病力包括利用植物无菌苗检测替代培养基所培养致病疫霉菌致病力的步骤。本发明的培养基组分容易获得、配制流程简单,且具有良好菌丝生长速度与产孢能力,以及长期储存恢复能力;本发明的检测方法可以在植物组织培养条件下更加精准地呈现植物与病原菌的互作关系,为番茄致病疫霉菌培养的简化、番茄与致病疫霉菌互作的分子机制研究提供稳定的研究系统和验证平台。
Description
技术领域
本发明属于农业生物领域,具体涉及致病疫霉菌培养基MYEB及检测致病疫霉菌对植物的致病力的方法。
背景技术
致病疫霉菌(Phytophthora infestans(Mont.)de bary)是对茄科农作物危害最大的病原菌,比稻瘟病和小麦锈病更加难以控制,可引起茄科晚疫病,主要危害马铃薯、番茄等粮食蔬菜作物的生产,造成严重经济损失。致病疫霉菌是卵菌的模式菌种,但是卵菌侵染宿主植物的机制目前还不清楚。致病疫霉菌的部分基因组测序工作已于2009年完成,现有技术开发了一种农杆菌介导的致病疫霉菌遗传转化系体系。然而,构建一种配制流程简单、培养基组分容易获得的致病疫霉菌培养方法用于开发更加高效的致病疫霉菌遗传转化方法,显得尤为必要。马铃薯块茎和切片常用来培养致病疫霉菌,但是需要频繁的转移材料,并且所分离病原菌常受到其他微生物的干扰、污染。现有技术还应用一些半合成或有机培养基,以满足致病疫霉菌病原菌的生长和孢子囊形成,其中包括小麦、黑麦、青豆、马铃薯、豌豆、四季豆、酵母粉、甜玉米、玉米、嘴豆、燕麦、谷物、V8果汁、黑豆、红芸豆、大豆和胡萝卜。然而,以上培养基均具有不同程度的配制复杂、培养效率较低等缺陷。对病原体的系统研究需要更加简单、更加高效的培养基,特别是能够为建立高效的致病疫霉菌遗传转化体系奠定良好的培养系统。
对于叶传病害,宿主植物发病叶片常被用于对病原菌的致病性测定,然而致病性检测实验常常在开放环境中进行,由于受到其他微生物的污染,结果往往出现较大误差。为了提高受试植物致病性分析的准确性,需要在没有其他微生物污染的独立环境中进行致病性分析。目前现有技术尚无对致病疫霉菌新型简易培养及植物病原菌瓶内互作方法的报道。
发明内容
本发明的目的在于提供一种致病疫霉菌培养基MYEB。
本发明的再一目的在于提供检测致病疫霉菌对植物的致病力的方法。
根据本发明具体实施方式的致病疫霉菌培养基,所述培养基包括以下组分:酵母浸粉0.5-1.5g/L、牛肉浸膏4-6g/L、蛋白胨4-6g/L、NaNO3 0.4-0.6g/L、MgSO4·7H2O 0.4-0.5g/L、琼脂粉10-20g/L。
根据本发明具体实施方式的致病疫霉菌培养基,所述培养基包括以下组分:酵母浸粉1g/L、牛肉浸膏5g/L、蛋白胨5g/L、NaNO3 0.5g/L、MgSO4·7H2O 0.493g/L、琼脂粉15g/L。
根据本发明具体实施方式的检测致病疫霉菌对植物的致病力的方法,所述方法包括以下步骤:
(1)培养寄主植物无菌苗;
(2)将由上述致病疫霉菌培养基制成的致病霉菌孢子囊悬液接种于步骤(1)培养的无菌苗叶片上,于20℃进行培养,光周期为120μmol.m-2s-1强度光照16h和8h黑暗;
(3)观察叶片上的致病疫霉菌,确定寄主植物的抗感病性。
根据本发明具体实施方式的检测致病疫霉菌对植物的致病力的方法,步骤(1)中,无菌苗培养包括以下步骤:将寄主植物种子消毒后,在4℃低温催芽2-3d;然后将发芽的种子播于MS培养基中,在16h光照/8h黑暗,光照度为150μmol.m-2s-1的组培条件下进行培养。
根据本发明具体实施方式的检测致病疫霉菌对植物的致病力的方法,步骤(1)中,所述寄主植物包括NC89烟草、W38烟草、珊西烟草、本生烟草、番茄(MM和MT)、里基茄、矮牵牛、白蛋茄、枸杞和拟南芥。
本发明的有益效果:
T124和T12两种致病疫霉菌在MYEB中培养9天,径向生长能力表现良好,其中,T124气生菌丝生长半径可达41.10mm、T12气生菌丝生长半径可达41.05mm;T124产孢个数可达3175/cm2、T12产孢个数可达3155/cm2;两种致病疫霉菌的菌丝颜色均为灰白色。
本发明的检测致病疫霉菌对植物的致病力的方法,将封闭无菌的培养瓶作为研究植物与病原菌相互作用的良好微室,可有效避免其他微生物的污染,其成本低于气候室内操作低,且实验结果较为准确。
附图说明
图1显示本发明培养基所培养致病疫霉菌T124和T12对番茄MM的致病性和毒性,其中,A和A1为T124;B和B1为T12;A-A1和B-B1为MYEB;
图2显示致病疫霉菌对多种茄科植物和拟南芥致病性分析情况,其中,A-A2为W38烟草;B-B2为珊西烟草;C-C2为NC89烟草;D-D2为本生烟草;E-E2为番茄MT;F-F2为里基茄;G-G2为矮牵牛;H-H2为白蛋茄;I-I2为枸杞;J-J2为拟南芥,A-E和F-J为不同植物接种致病疫霉菌T124(源于)后整株症状;A1-E1和F1-J1为被接种叶片的局部症状;A2-E2和F2-J2为被接种叶片的显微症状。
具体实施方式
实施例1配制致病疫霉菌培养基MYEB
1.本发明的培养基MYEB包括以下组分:酵母浸粉0.5g/L、牛肉浸膏4g/L、蛋白胨4g/L、NaNO3 0.4g/L、MgSO4·7H2O 0.4g/L、琼脂粉10g/L。
2.本发明的培养基MYEB包括以下组分:酵母浸粉1g/L、牛肉浸膏5g/L、蛋白胨5g/L、NaNO3 0.5g/L、MgSO4·7H2O 0.493g/L、琼脂粉15g/L。
3.本发明的培养基MYEB包括以下组分:酵母浸粉1.5g/L、牛肉浸膏6g/L、蛋白胨6g/L、NaNO3 0.6g/L、MgSO4·7H2O 0.5g/L、琼脂粉20g/L。
实施例2考察培养基的菌丝生长速度、产孢能力、长期储存恢复能力
配制黑麦培养基及其他35种对比培养基,用于与本发明的培养基进行效果对比,具体如下:
表1 35种对比培养基的序号、名称缩写、名称与组分
2.1培养致病疫霉菌
采用液体培养法制备致病疫霉菌T124、T12的孢子囊悬液(8.0×103孢子囊/ml)。在90mm培养皿中分别添加25mL的本发明培养基及上述对照培养基,配制成固体平板,在培养基中央滴加10μl病原菌孢子囊悬液。每种培养基重复10次。在黑暗条件下,所有接种的培养基于20℃的恒温培养箱中倒扣培养。
2.2测定菌丝半径
接种致病疫霉菌后,测定T124和T12在上述37种培养基(黑麦培养基为对照培养基)上生长9天后的菌丝生长半径,结果如表2所示:
表2致病疫霉菌T124和T12在37种培养基的菌丝生长半径数据
注:大写字母和小写字母分别表示1%和5%概率水平上的显著差异。差异显著性采用SPSS 16.0进行Duncan检验。
由结果可知,MYEB培养基的培养效果与黑麦培养基效果相当,二者不存在显著性差异,其生长半径均在41mm以上,MYEB培养基可作为黑麦培养基(对照)的理想替代。
2.3统计孢子产量
接种致病疫霉菌后15d,利用打孔器从每个培养皿中分别获取3个培养的致病疫霉菌圆盘(0.5cm2),将孢子囊悬浮于1ml蒸馏水中,用血球计数板统计孢子囊的产量。每种培养基均处理5次,整个实验重复3次。
表3致病疫霉菌T124和T12在37种培养基上生长15天后的孢子囊产量
注:大写字母和小写字母分别表示1%和5%概率水平上的显著差异。差异显著性采用SPSS 16.0进行Duncan检验。
由结果可知,MYEB培养基与黑麦培养基(对照)的培养效果相当,其孢子囊产量均在3150以上,可作为黑麦培养基(对照)的理想替代。
2.4长期储存恢复能力评价
利用打孔器从每个培养皿中分别获取致病疫霉菌圆盘(0.5cm2),将它们转接到同一种培养基上,以进一步评估其生长能力。接种后每天记录菌丝生长情况。并对不同候选培养基培养的致病疫霉菌的恢复能力进行评价。在接种后的14d后,从每种候选培养基所培养的菌落边缘共取40个致病疫霉菌圆盘,转移到40个小瓶(10mL)中,每个小瓶中含有5mL相对应的液体培养基。小瓶在4℃与20℃的黑暗环境中保存。每种培养基每月取样5瓶,取样时间段分别为1个月、3个月、5个月、7个月。将培养瓶中的真菌转移到黑麦培养基,对其长期储存恢复能力评价。
表4 4℃和20℃保存1-7个月后MYEB培养基对T124孢子囊数量的影响
表5 4℃和20℃保存1-7个月后本发明培养基对致病疫霉菌(T12)孢子囊数量的影响
注:大写字母和小写字母分别表示1%和5%概率水平上的显著差异。差异显著性采用SPSS 16.0进行Duncan检验。
由结果可知,MYEB培养基的长期储存恢复能力相对较佳,可作为黑麦培养基(对照)的理想替代。
实施例3检测致病疫霉菌对植物的致病力
检测致病疫霉菌对植物的致病力的方法,包括以下步骤:
(1)培养寄主植物无菌苗;
选择健康饱满的烟草W38(珊西、NC89、本生烟草、番茄MT、里基茄、矮牵牛、白蛋茄、枸杞或拟南芥种子),先用75%(v/v)乙醇表面消毒45s-1min,无菌水冲洗3-5遍;再用2.5%次氯酸钠处理8-10min,无菌水冲洗3-5遍;将消毒后的种子至于无菌1.5ml离心管中,4℃低温催芽2-3d;然后将之播于含有MS培养基(添加30mg/L蔗糖和7.8g/L琼脂,pH 5.8)的玻璃组培瓶,至于25℃,16h光照/8h黑暗,光照度为150μmol.m-2s-1组培条件下进行培养;
(3)将致病疫霉菌培养基制成的致病霉菌孢子囊悬液接种于4周龄大的无菌苗叶片上,于20℃的光照培养箱中进行培养,光周期为16h强度为120μmol.m-2s-1光照和8h黑暗;
(4)H2O2积累检测与抗/感病性检测:采用DAB染色观察H2O2的累积,在显微镜下,采用台盼蓝染色法观察不同植物叶片上的致病疫霉菌的形态,从而确定不同植物的抗/感病性,进而筛选出新的茄科抗性材料。
3.1分析致病疫霉菌T124和T12对番茄MM的致病性和毒性
于4周龄大的番茄MM无菌苗叶片上,分别接种少量致病疫霉菌(T124、T12)孢子囊悬液,然后置于20℃的光照培养箱中进行培养,光周期为16h光照(120μmol.m-2s-1)和8h黑暗;统计发病症状与发病率。
如图1所示,MYEB培养基所培养出的致病疫霉菌T124和T12,均可使感病番茄MM正常发病,发病率为100%,与对照培养基(黑麦培养基)相比,RIS培养基所培养的致病疫霉菌具有正常的致病性和毒性。
3.2对多种茄科植物和拟南芥致病性分析
在4周龄大的烟草W38、珊西、NC89和本生烟草,番茄MT,矮牵牛,里基茄,白蛋茄,枸杞和拟南芥无菌苗,接种少量致病疫霉菌(T124、T12)孢子囊悬液,然后置于20℃的光照培养箱中进行培养,光周期为16h光照(120μmol.m-2s-1)和8h黑暗。
H2O2积累检测与抗/感病性检测:采用DAB染色观察H2O2的累积。在显微镜下,采用台盼蓝染色法观察不同植物叶片上的致病疫霉菌的形态,从而确定不同植物的抗/感病性,进而筛选出新的茄科抗性材料。
结果如图2所示,四种烟草(烟草W38、珊西、NC89和本生烟草)、矮牵牛、白蛋茄表现为抗性反应,番茄MT,里基茄、枸杞和拟南芥均可感病。
Claims (5)
1.致病疫霉菌培养基,其特征在于,所述培养基包括以下组分:酵母浸粉0.5-1.5g/L、牛肉浸膏4-6g/L、蛋白胨4-6g/L、NaNO3 0.4-0.6g/L、MgSO4·7H2O 0.4-0.5g/L、琼脂粉10-20g/L。
2.根据权利要求1所述的致病疫霉菌培养基,其特征在于,所述培养基包括以下组分:酵母浸粉1g/L、牛肉浸膏5g/L、蛋白胨5g/L、NaNO3 0.5g/L、MgSO4·7H2O 0.493g/L、琼脂粉15g/L。
3.检测致病疫霉菌对植物的致病力的方法,其特征在于,所述方法包括以下步骤:
(1)培养寄主植物无菌苗;
(2)将由权利要求1或2所述致病疫霉菌培养基制成的致病疫霉菌孢子囊悬液接种于步骤(1)培养的无菌苗叶片上,于20℃进行培养,光周期为120μmol.m-2s-1强度光照16h和8h黑暗;
(3)观察叶片上的致病疫霉菌,确定寄主植物的抗感病性。
4.根据权利要求3所述的检测致病疫霉菌对植物的致病力的方法,其特征在于,步骤(1)中,无菌苗培养包括以下步骤:将寄主植物种子消毒后,在4℃低温催芽2-3d;然后将发芽的种子播于MS培养基中,在16h光照/8h黑暗,光照度为150μmol.m-2s-1的组培条件下进行培养。
5.根据权利要求4所述的检测致病疫霉菌对植物的致病力的方法,其特征在于,步骤(1)中,所述寄主植物包括NC89烟草、W38烟草、珊西烟草、本生烟草、番茄、里基茄、矮牵牛、白蛋茄、枸杞或拟南芥的组。
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