CN115380824B - 一种铁皮石斛茎腐病组培苗抗性鉴定方法 - Google Patents
一种铁皮石斛茎腐病组培苗抗性鉴定方法 Download PDFInfo
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Abstract
本发明涉及抗茎腐病铁皮石斛种质鉴定技术,本发明铁皮石斛茎腐病组培苗抗性鉴定的方法,包括如下步骤:(1)病害部位的子实体分离纯化培养,病原菌分子鉴定;(2)配制组培苗培养基,转入组培苗培养;(3)配制接种菌液液体培养基,制备1×106个/mL的孢子悬浮液;(4)无菌棉签头蘸取孢子悬浮液,紧贴铁皮石斛组培苗放到培养基上接种;(5)接种后进行病情调查,依据病情指数评价铁皮石斛种质资源抗病性。本发明弥补了铁皮石斛种质抗病性鉴定技术的空白,提供一种操作简单、准确、快速的铁皮石斛茎腐病组培苗抗性鉴定方法。
Description
技术领域
本发明涉及一种铁皮石斛茎腐病组培苗抗性鉴定方法,属于植物病理学研究技术领域。
背景技术
铁皮石斛(Dendrobium officinale Kimura et Migo)为兰科石斛属植物,是我国重要的药用植物,具有“养阴生津、润喉护嗓、温胃明目、补肾益力、延年益寿”等功效,道家医学经典《道藏》把铁皮石斛称为九大仙草之首,被列入《中华人民共和国药典》(2020版),也具有重要的观赏价值。随着组培技术的快速发展和栽培水平不断提高,人工种植的铁皮石斛种植面积不断增加,铁皮石斛茎腐病成为了主要的病害,其主要在夏季、秋季两季高温高湿的环境中发生,具有较明显的发病中心,叶片呈水渍状,假鳞茎软化枯萎,假鳞茎基部可见淡黄色霉状物,发病严重时,叶片全部失绿或脱落,整株失绿枯萎。铁皮石斛茎腐病主要采用化学药剂防治,不同铁皮石斛品种对于茎腐病的抗病和感病程度不同,利用抗病品种防治铁皮石斛茎腐病也是一种重要的防治手段,抗病品种的选育和利用离不开铁皮石斛茎腐病病害抗性鉴定。目前常用的植物抗病鉴定方法是种子侵染接种、幼苗期喷雾接种、灌根接种、离体叶片接种等,尚未见到铁皮石斛茎腐病抗性鉴定的方法,严重制约了铁皮石斛茎腐病抗病性品种的选育,因此,探索一种操作简单、准确、快速的铁皮石斛茎腐病组培苗抗性鉴定方法,采用科学有效的方法评价和鉴定不同铁皮石斛品种的抗病性,为筛选抗病铁皮石斛种质资源和新品种提供科学方法。
发明内容
本发明根据上述领域存在的空白和技术需求,提供一种操作简单、准确、快速的铁皮石斛茎腐病组培苗抗性鉴定方法。
本发明是以如下技术方案实现的,一种铁皮石斛茎腐病组培苗抗性鉴定方法,按如下步骤进行:
(1)病原菌分离与鉴定:观察并用无菌解剖针挑取铁皮石斛茎腐病病害部位的子实体,用无菌的滤纸吸干其表面水分,将子实体移至PDA培养基上,25℃避光培养,再将培养物进行划线纯化培养,即可获得纯培养铁皮石斛茎腐病病原菌菌株,提取菌株的DNA进行PCR扩增,将得到的PCR扩增产物进行凝胶电泳分析,PCR产物进行测序,测得序列向NCBI数据库提交并进行DNA序列对比,铁皮石斛茎腐病病原菌为尖孢镰刀菌(Fusarium oxysporum),铁皮石斛茎腐病病害部位的子实体为尖孢镰刀菌无性阶段的分生孢子座。
(2)组培苗及培养基的准备:优选组培苗培养基,挑选不同铁皮石斛种质资源生根壮苗培养后生长一致的组培苗,转入盛有组培苗培养基的650ml组培瓶中,每瓶接3株,9h光照15h黑暗25℃培养15d。
(3)接种菌液的培养及配制:优选接种菌液液体培养基,将铁皮石斛茎腐病病原菌菌株转接至PDA培养基上25℃恒温避光培养7d,用打孔器在菌落边缘打取菌饼,挑取3个菌饼放置于装有接种菌液液体培养基的锥形瓶中,于12h光照12h黑暗,25℃恒温,130r/min振荡培养3d,用4层纱布过滤得到分生孢子滤液,加入无菌水稀释并采用血球计数板调整孢子浓度,得到浓度为1×106个/mL的孢子悬浮液。
(4)接种:剪下无菌棉签的棉签头,蘸取孢子悬浮液,然后将棉签头紧贴铁皮石斛组培苗放到培养基上,每份铁皮石斛种质资源接种5瓶,3次重复,棉签头蘸取无菌水的铁皮石斛组培苗为对照,9h光照15h黑暗25℃培养。
(5)病情调查与评价:接种14d后进行病情调查,观察和记录每株组培苗的发病情况,统计各病级株数,计算病情指数,依据病情指数评价铁皮石斛种质资源抗病性。
组培苗受害分级标准:
按病害严重度将病级分为以下级别,以株为单位
0级:整株无发病症状;
1级:1/4以下叶片变黄,假鳞茎变褐色;
2级:1/4-1/2叶片变黄,假鳞茎轻度腐烂;
3级:1/2-3/4叶片变黄,假鳞茎中度腐烂;
4级:3/4以上叶片变黄,假鳞茎严重腐烂;
5级:假鳞茎完全腐烂,整株倒伏。
病情指数(T)按公式进行计算:,式中:T—病情指数;XI—各级株数;I—相对级数值;Y—调查总株数。
抗病分级标准:
免疫(I):病情指数(T)=0;
高抗(HR):0<病情指数(T)≤15;
抗病(R):15<病情指数(T)≤30;
中抗(MR):30<病情指数(T)≤45;
感病(S):45<病情指数(T)≤75;
高感(HS):病情指数(T)>75。
本发明的有益效果:
一是优选的组培苗培养基能确保铁皮石斛组培苗正常生长,优选的接种菌液液体培养基及培养方法可以快速获得大量高浓度的孢子悬浮液,保障了铁皮石斛茎腐病组培苗的抗性鉴定。人工模拟铁皮石斛的生长环境,温度为铁皮石斛和茎腐病菌的最适生长温度,在无其他影响因素的培养条件下评测活体铁皮石斛种质资源的茎腐病抗性。
二是弥补了铁皮石斛茎腐病抗性鉴定方法的缺失,铁皮石斛茎腐病组培苗抗性鉴定方法在组培苗阶段就能鉴定,不受铁皮石斛生长季节的限制,材料易得,操作简单、准确、快速,通过统计病情指数即可评价铁皮石斛茎腐病抗病性,通过上述鉴定方法对不同铁皮石斛种质资源进行茎腐病抗性鉴定,可有效筛选出抗性品种。
具体实施方式
实施例
(1)病原菌分离与鉴定:配制PDA培养基,PDA培养基的配方为:200g/L马铃薯浸出液,20.0g/L葡萄糖,8g/L琼脂。按照PDA培养基的配方定容,pH自然,121℃高压灭菌20min后冷却备用。在体式显微镜下观察并用无菌解剖针挑取铁皮石斛茎腐病病害部位的子实体,在超净工作台上,用无菌的滤纸吸干其表面水分,将子实体移至PDA培养基上,25℃避光培养,再将培养物进行划线纯化培养,即可获得纯培养菌株,备用。用DNA提取试剂盒提取菌株的DNA进行PCR扩增,将得到的PCR扩增产物进行凝胶电泳分析,将准备好PCR产物进行测序,测得序列向NCBI数据库提交,用Blast在已知序列中进行同源性比对,与序列号为KM231809.1、ON003566.1、MK842095.1等尖孢镰刀菌(Fusarium oxysporum)的同源性均为100%,将铁皮石斛茎腐病病原菌鉴定为尖孢镰刀菌(F. oxysporum),铁皮石斛茎腐病病害部位的子实体为尖孢镰刀菌无性阶段的分生孢子座。
(2)组培苗及培养基的准备:优选组培苗培养基,组培苗培养基为:MS培养基+5g/L琼脂,具体配方为:170 mg/L KH2PO4,370 mg/L MgSO4·7H2O,440 mg/L CaCl2·2H2O,1900mg/L KNO3,1650 mg/L NH4NO3,0.83 mg/L KI,6.2 mg/L H3BO3,0.25 mg/L Na2MoO4·2H20,8.6 mg/L ZnSO4·7H20,0.025 mg/L CuSO4·5H20,22.3 mg/L MnSO4·4H20,0.025 mg/LCoCl2·6H2O,37.25 mg/L Na2-EDTA·2H20,27.85 mg/L FeSO4·7H2O,100 mg/L肌醇,2.0mg/L甘氨酸,0.5 mg/L VB6,0.5 mg/L VB5,0.1 mg/L VB1,5g/L琼脂。按照组培苗培养基的配方定容后,用NaOH和HCl调整pH为5.8-6.0,倒入650ml组培瓶中,每瓶倒入100ml,121℃高压灭菌20min后冷却备用。在超净工作台上,选择不同铁皮石斛种质资源生根壮苗培养后的组培苗,挑选高8-10cm,叶6-7片,假鳞茎6-7节,生长一致的组培苗,转入盛有组培苗培养基的650ml组培瓶中,每瓶接3株,9h光照15h黑暗25℃培养15d。
(3)接种菌液的培养及配制:优选接种菌液液体培养基,接种菌液液体培养基的配方为:200g/L马铃薯浸出液,20g/L蔗糖,4g/L酵母浸膏,2g/L MgSO4·7H2O,2g/L K2HPO4,0.002g/L MnSO4。按照接种菌液液体培养基的配方定容,pH自然,121℃高压灭菌20min后冷却备用。在超净工作台上,将铁皮石斛茎腐病病原菌菌株转接至PDA培养基上,25℃恒温避光培养7d,用打孔器在菌落边缘打取菌饼,挑取3个菌饼放置于装有接种菌液液体培养基的锥形瓶中,于12h光照12h黑暗,25℃恒温,130r/min振荡培养3d。在超净工作台上,用4层无菌纱布过滤得到分生孢子滤液,加入无菌水稀释并采用血球计数板调整孢子浓度,得到浓度为1×106个/mL的孢子悬浮液,备用。
(4)接种:在超净工作台上,剪下无菌棉签的棉签头,蘸取孢子悬浮液,然后将棉签头紧贴铁皮石斛组培苗放到培养基上,每份铁皮石斛种质资源接种5瓶,3次重复,棉签头蘸取无菌水的铁皮石斛组培苗为对照,9h光照15h黑暗25℃培养。
(5)病情调查与评价:接种14d后进行病情调查,观察和记录每株组培苗的发病情况,统计各病级株数。按组培苗受害严重度将病级分为0-5级,0级为整株无发病症状;1级为1/4以下叶片变黄,假鳞茎变褐色;2级为1/4-1/2叶片变黄,假鳞茎轻度腐烂;3级为1/2-3/4叶片变黄,假鳞茎中度腐烂;4级为3/4以上叶片变黄,假鳞茎严重腐烂;5级为假鳞茎完全腐烂,整株倒伏。根据病害评价病级带入公式计算病情指数(T):,式中:T—病情指数;Xi—各级株数;I—相对级数值;Y—调查总株数。依据病情指数评价铁皮石斛种质资源抗病性:免疫(I):病情指数(T)=0;高抗(HR):0<病情指数(T)≤15;抗病(R):15<病情指数(T)≤30;中抗(MR):30<病情指数(T)≤45;感病(S):45<病情指数(T)≤75;高感(HS):病情指数(T)>75。
铁皮石斛种质资源抗性鉴定结果见表1:
表1 铁皮石斛茎腐病组培苗抗性鉴定结果
铁皮石斛种质资源圃存编号 | 对照病情指数(均值) | 处理病情指数(均值) | 抗病级别 |
NYBRLSH099 | 0.00 | 51.56 | S |
NYBRLSH100 | 0.00 | 37.33 | MR |
NYBRLSH101 | 0.00 | 70.22 | S |
NYBRLSH102 | 0.00 | 65.78 | S |
NYBRLSH103 | 0.00 | 52.44 | S |
NYBRLSH104 | 0.00 | 42.22 | MR |
NYBRLSH105 | 0.00 | 26.67 | R |
NYBRLSH111 | 0.00 | 61.33 | S |
NYBRLSH112 | 0.00 | 78.22 | HS |
NYBRLSH113 | 0.00 | 74.22 | S |
NYBRLSH114 | 0.00 | 62.22 | S |
NYBRLSH115 | 0.00 | 48.89 | S |
NYBRLSH116 | 0.00 | 80.00 | HS |
NYBRLSH117 | 0.00 | 31.56 | MR |
NYBRLSH118 | 0.00 | 76.89 | HS |
NYBRLSH119 | 0.00 | 55.11 | S |
NYBRLSH120 | 0.00 | 65.78 | S |
NYBRLSH121 | 0.00 | 72.89 | S |
NYBRLSH122 | 0.00 | 68.44 | S |
NYBRLSH123 | 0.00 | 75.56 | HS |
NYBRLSH124 | 0.00 | 56.00 | S |
NYBRLSH125 | 0.00 | 67.56 | S |
NYBRLSH141 | 0.00 | 61.33 | S |
从表1可以看出,所鉴定的23份铁皮石斛种质资源中,1份种质对铁皮石斛茎腐病的抗病级别为抗病(R),3份种质对铁皮石斛茎腐病的抗病级别为中抗(MR),这4份种质可以作为优选抗性资源,15份种质对铁皮石斛茎腐病的抗病级别为感病(S),4份种质对铁皮石斛茎腐病的抗病级别为高感(HS)。
Claims (1)
1.一种铁皮石斛茎腐病组培苗抗性鉴定的方法,其特征在于,包括如下步骤:
(1)病害部位的子实体分离纯化培养,病原菌分子鉴定;
(2)配制组培苗培养基,挑选生长一致的组培苗,转入盛有组培苗培养基的650ml组培瓶中,每瓶接3株,9h光照15h黑暗25℃培养15d;其中,组培苗培养基组分为:MS培养基,5g/L琼脂,pH为5.8-6.0;
(3)配制接种菌液液体培养基,挑取3个菌饼放置于装有接种菌液液体培养基的锥形瓶中,于12h光照12h黑暗,25℃恒温,130r/min振荡培养3d,得到浓度为1×10 6个/mL的孢子悬浮液;其中,接种菌液液体培养基组分为:200g/L马铃薯浸出液,20g/L蔗糖,4g/L酵母浸膏,2g/L MgSO4·7H2O,2g/L K2 HPO4,0.002g/L MnSO4;
(4)采用无菌棉签头蘸取孢子悬浮液,紧贴铁皮石斛组培苗放到培养基上接种;
(5)接种后进行病情调查,依据病情指数评价铁皮石斛种质资源抗病性;其中,组培苗受害分级标准:
按病害严重度将病级分为以下级别,以株为单位
0级:整株无发病症状;
1级:1/4以下叶片变黄,假鳞茎变褐色;
2级:1/4-1/2叶片变黄,假鳞茎轻度腐烂;
3级:1/2-3/4叶片变黄,假鳞茎中度腐烂;
4级:3/4以上叶片变黄,假鳞茎严重腐烂;
5级:假鳞茎完全腐烂,整株倒伏;
病情指数T按公式进行计算:
式中:T—病情指数;XI—各级株数;I—相对级数值;Y—调查总株数;
抗病分级标准:
免疫I:病情指数T=0;
高抗HR:0<病情指数T≤15;
抗病R:15<病情指数T≤30;
中抗MR:30<病情指数T≤45;
感病S:45<病情指数T≤75;
高感HS:病情指数T>75。
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