CN109328683B - 一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法 - Google Patents

一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法 Download PDF

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CN109328683B
CN109328683B CN201811162355.5A CN201811162355A CN109328683B CN 109328683 B CN109328683 B CN 109328683B CN 201811162355 A CN201811162355 A CN 201811162355A CN 109328683 B CN109328683 B CN 109328683B
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殷辉
赵晓军
周建波
吕红
秦楠
常芳娟
翟世玉
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Abstract

本发明公开了一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法,包括制备扦插生根液、制备扦插穗、扦插与培养、病原物收集、制备吐温80溶液、抗病性鉴定、鉴定结果计算方法。本发明优势在于,对具有优良形状的藜麦品种,种子繁殖会使其性状丧失,而扦插则能使繁殖的后代仍然具有原优良性状,达到准确评价种质资源抗病性的目的。相比种子繁殖苗子繁殖数据准确、重复性高,鉴定效率更高,节约了用种量,在实验室条件下作业,一年可重复多次。本发明采用插穗生根后进行评估鉴定,数据能够准确反映藜麦在田间的抗病能力。

Description

一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法
技术领域
本发明涉及一种植保技术领域,具体是一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法。
背景技术
藜麦又称南美藜、藜谷等,藜科藜属的一年生双子叶植物。藜麦具有良好的适应性用于增加栽培作物的多样性,缓解农业生态系统。藜麦适应性广、耐寒、耐旱、耐瘠薄、耐盐碱等特性特点,在干旱、高海拔地区具有很大的发展前景和空间。藜麦还是一种全营养成分的食品原料,号称“宇航员食品” ,因此,近年来备受关注。随着藜麦产业的不断壮大,未被充分利用的藜麦势必会成为21世纪的重要工业原料和粮食作物。然而,藜麦产业在我国的发展刚刚起步,有很多亟待解决的问题。在植保方面,随着种植面积的扩大及连作栽培,藜麦霜霉病日趋严重,严重影响藜麦产业的发展。
霜霉病属于卵菌病害,是农业生产中的第一大病害。藜麦霜霉病由Peronospora variabilis引起,为活体性寄生菌,其典型症状为叶片有明显粉红色霉层;造成叶片枯黄、脱落、籽粒空秕,严重地块发病率约95%、减产40%左右。选育抗病品种是控制藜麦霜霉病的最有效途径,其中快速、方便、可靠的抗病性鉴定方法是筛选藜麦品种的前提。常规霜霉病抗病性鉴定方法为室内离体叶片接种鉴定法,或采用种子播种待长出植株后鉴定。然而,具体哪种方式更为高效、稳定、准确的能评估藜麦对霜霉病的抗病性,是相关领域关注的焦点问题。
当前,藜麦霜霉病抗病性鉴定中存在几个关键问题:(1)对于一些具有优良形状的藜麦品种,通过种子繁殖进行鉴定会使其性状丧失、导致抗病性鉴定不准确。(2)藜麦霜霉病属于生长中期病害,使用种子繁殖苗子,受环境影响苗子大小、性状不容易控制,致使评价不准确。(3)传统种子繁殖,室外抗病性鉴定周期长,结果易受环境因素影响致使评价不准确。(4)藜麦叶片结构有其特殊性,使用传统叶盘法室内离体接种时,打孔器打取叶盘时很容易破坏叶片结构;加之与黄瓜、葡萄等叶片结构相比极其薄且易失水萎蔫,影响其试验的准确性。
发明内容
针对现有技术存在的问题,本发明的目的在于提供一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法,该方法利用藜麦种子繁殖会使其的性状丧失、而扦插则能使繁殖的后代仍然具有原优良性状的特点,通过扦插繁殖、达到准确评价种质资源抗病性的目的。
本发明是通过下述技术方案实现上述目的的。
一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法,包括如下步骤:
(1)制备扦插生根液 扦插生根液由扦插生根液A和扦插生根液B配置而成。
扦插生根液A:称量1公斤藜麦秸秆加1L水,高压灭菌后,在无菌环境下加1ml浓度为3.5×109cfu/mL枯草芽孢杆菌菌液,置于24~26℃发酵30 d,滤出发酵浸提液。
扦插生根液B:量取1L无菌水,溶入2.5mg维生素B1、6.5mg磷酸二氢钾、3.5mg硫酸镁、5.5mg硝酸铵、0.5 mg阿司匹林,配制成培养液,调节培养液的pH值为6.8~7.0。
将扦插生根液A与扦插生根液B按体积比10:1配制成扦插生根液;然后分装于50~100ml的容器中,液面高度3.5~4.0 cm,备用。
(2)制备扦插穗 在藜麦母株上截取长势好、无病虫害、直径为0.8 ~1.2 cm、长30cm的分枝,剪去分枝15 cm以下的叶,切口位于分枝叶柄疤痕下0.5 ~1.0 cm处;切口修剪为与分枝轴线呈约50~60度角的斜面,切面平滑、韧皮部不留毛茬且无损伤,制成扦插穗。
(3)扦插与培养 把(2)制备好的扦插穗的切口浸泡于35%的啤酒水溶液中消毒5~8 min,然后插在(1)制备的扦插生根液中,置于人工气候箱,在18~20℃、光照强度3500lx,光暗交替(光照时间6 h、黑暗18 h)的条件下培养,扦插液蒸发后及时补存至3.5 ~4.0cm高度,当扦插穗切口处明显看到白色细根时,调节气候箱的培养条件为:20~22℃、光照6500 lx(光照时间10h、黑暗14 h)培养,培养10 ~15 d,根系长4.5~5.5 cm时备接种霜霉菌使用。
(4)病原物收集 田间采集具有藜麦霜霉病症状的病叶置于10℃条件下保藏20 ~24 h,用毛刷将新鲜病叶的霜霉菌霉层刷除,无菌水冲洗3次,然后将叶片用湿润的脱脂棉花包裹,置于人工气候箱,培养条件是:18℃、1200 lx、光暗交替(光照时间6 h、黑暗18 h),脱脂棉花处保湿状态,培养36 h后扫刷病叶长出的新鲜霉层(霜霉病菌孢子囊)收集到1.5mL离心管中,置于10℃保藏备用。
(5)制备吐温80溶液 用无菌水配制含1.5%吐温80的溶液。
(6)抗病性鉴定 取(4)收集好的藜麦霜霉病菌孢子囊置于(5)制备的吐温溶液中,调节孢子囊悬浮液浓度为1×108个孢子囊/毫升;用培养(3)的扦插穗进行接种,即用移液器吸取30微升的孢子囊悬浮液涂抹于藜麦的叶片正面,接种后置于人工气候箱,培养条件是:20℃、黑暗培养15 h,然后将气候箱培养条件调为:20~22℃、6500 lx、光暗交替(光照时间10h、黑暗14 h)培养。每插穗接种15片叶子、重复3次,5 d后调查结果。
(7)鉴定结果计算方法 按叶片的感病面积分级记载,计算病情指数进而评价藜麦的抗病性。
病情指数计算公式:
Figure 971731DEST_PATH_IMAGE001
Figure 974191DEST_PATH_IMAGE002
Figure 121139DEST_PATH_IMAGE003
所述的啤酒为酒精度为3%~4% vol、麦汁度10 °P~12 °P的市售啤酒
本发明具有以下有益效果:(1))对具有优良形状的藜麦品种,种子繁殖会使其性状丧失,而扦插则能使繁殖的后代仍然具有原优良性状,达到准确评价种质资源抗病性的目的。相比种子繁殖苗子繁殖数据更科学。(2)特别适合从数量庞大的种质资源中筛选抗病品种,提高鉴定效率;且能减少了不同操作人员间的人为误差。(3))藜麦霜霉病在田间属于藜麦生长中期病害,采用插穗评估鉴定结果能够准确反映,藜麦在田间的抗病能力。(4)采用扦插藜麦穗生根后利用穗上叶片评估,相比传统霜霉病评价的叶盘漂浮法,数据准确、重复性高,接近田间抗病性情况。(5)将藜麦枝剪下进行扦插,可大大节约了用种量;还能经济利用繁殖材料、增加苗子数量。(6)实验室条件下作业,一年可重复多次,比大田生长鉴定节省了大量的时间,提高 了鉴定的工作效率。
实施例1
利用扦插繁殖鉴定藜麦霜霉病抗病性的方法,包括如下步骤:
(1)制备扦插生根液 扦插生根液由扦插生根液A和扦插生根液B配置而成。
制备扦插生根液A:称取10公斤藜麦秸秆,量取10L净水,高压灭菌处理后,在无菌环境下,加10ml浓度为3.5×109cfu/mL枯草芽孢杆菌菌液,置于24~26℃发酵30 d,滤出发酵浸提液,备用。
制备扦插生根液B:量取1L无菌水,溶入2.5mg维生素B1、6.5mg磷酸二氢钾、3.5mg硫酸镁、5.5mg硝酸铵、0.5 mg阿司匹林,配制成培养液,调节培养液的pH值为6.8~7.0,备用。
将扦插生根液A(发酵浸提液)与扦插生根液B(培养液)按体积比10:1配制成扦插生根液,然后分装于50~100ml的容器中,液面高度3.5 ~4.0 cm,备用。
(2)制备扦插穗 六月中下旬,在藜麦母株上截取长势好、无病虫害、直径为0.8 ~1.2 cm、长30 cm的分枝,剪去分枝15 cm以下的叶,切口位于分枝叶柄疤痕下0.5 cm~1.0cm处;切口修剪为与分枝轴线呈约50~60度角的斜面,切面平滑、韧皮部不留毛茬且无损伤,制成扦插穗。
(3)扦插与培养 量取2 L的净水,加入0.7 L市售普通啤酒,制成扦插穗消毒液。把(2)制备好的扦插穗的切口浸泡该消毒液中5 ~8 min,然后插在(1)制备的扦插生根液中,每个容器插一个扦插穗,置于人工气候箱,在18~20℃、光照强度3500 lx,光暗交替(光照时间6 h、黑暗18 h)的条件下培养,扦插液蒸发后及时补存至3.5~4.0 cm高度,当扦插穗切口处明显看到白色细根时,调节气候箱的培养条件为:20~22℃、光照6500 lx(光照时间10h、黑暗14 h)培养,培养10 ~15 d,根系长4.5~5.5 cm时备接种霜霉菌使用。
(4)病原物收集 六月上中旬,在田间采集具有藜麦霜霉病症状的病叶置于10℃条件下保藏20 ~24 h,用毛刷将新鲜病叶的霜霉菌霉层刷除,无菌水冲洗3次,然后将叶片用湿润的脱脂棉花包裹,置于人工气候箱,培养条件是:18℃、1200 lx、光暗交替(光照时间6h、黑暗18 h),脱脂棉花处保湿状态,培养36 h后扫刷病叶长出的新鲜霉层(霜霉病菌孢子囊)收集到1.5mL离心管中,置于10℃保藏备用。
(5)制备吐温80溶液 用无菌水配制含1.5%吐温80的溶液。
(6)抗病性鉴定 取(4)收集好的藜麦霜霉病菌孢子囊置于(5)制备的吐温溶液中,调节孢子囊悬浮液浓度为1×108个孢子囊/毫升;用培养(3)的扦插穗进行接种,即用移液器吸取30微升的孢子囊悬浮液涂抹于藜麦的叶片正面,接种后置于人工气候箱,培养条件是:20℃、黑暗培养15 h,然后将气候箱培养条件调为:20~22℃、6500 lx、光暗交替(光照时间10h、黑暗14 h)培养。每株接种15片叶子、重复3次,5 d后调查结果。
(7)鉴定结果计算方法 按叶片的感病面积分级记载,计算病情指数进而评价藜麦的抗病性。
病情指数计算公式:
Figure 617979DEST_PATH_IMAGE001
Figure 268403DEST_PATH_IMAGE002
Figure 926918DEST_PATH_IMAGE003
本发明实施例1制备的扦插生根液与常用的生根粉对藜麦插穗生根效果的比较
1、试验方法
将市售的生根粉稀释溶液为浓度分别为:2mg/L、4mg/L、8mg/L三个梯度溶液;按实施例1方法制备扦插生根液,所有生根液均分装于50~100ml的容器中,液面高度3.5 ~4.0cm。扦插穗的制备、扦插与培养方法完全同实施例1方法。每个处理重复3次,培养10 d时,分别测量并统计生根率、平均根长、平均生根数量,统计分析本发明扦插生根液和常规生根粉对藜麦生根的影响。
2、试验结果
试验结果见表1。
Figure 544850DEST_PATH_IMAGE004
试验结果表明,本发明扦插生根液对藜麦扦插穗的生根率、平均根长、平均根数显著高于普通生根粉各浓度梯度的培养液,与最好的生根粉浓度培养液的效果相比,生根率提高了60.3%,平均根长提高了98.5%,平均根数提高了116.8%。
本发明与传统叶盘漂浮法鉴定藜麦霜霉病抗病性对比试验
病原物收集与制备接种孢子囊悬浮液方法相同。
本发明方法采用扦插生根后在插穗的叶片上接种,用移液器吸取30微升的孢子囊悬浮液涂抹于插穗株5~10位的叶片正面,接种后置于人工气候箱,培养条件是:20℃、黑暗培养15 h,然后将气候箱培养条件调为:20~22℃、6500 lx、光暗交替(光照时间10h、黑暗14 h)培养。每株接种15片叶子、重复3次,5 d后调查结果。
叶盘漂浮法 取供试藜麦茎上的5~10位健康叶片,质量百分浓度为0.5%的次氯酸钠溶液消毒10 s,无菌水冲洗2次,打孔器打取直径1.5 cm的叶盘,放入铺有3层湿润滤纸的培养皿中进行保湿。每个平皿均匀摆放8个叶盘,叶正面朝上。在每个叶盘中央用移液器接种30微升上述孢子囊悬浮液。接种后20℃黑暗培养15 h,然后再将气候箱调为温度20℃~22℃、光照6500 lx(光照时间10h、黑暗14 h)培养。每处理8个叶盘,3次重复,3 d后调查结果。
上述2种方法都按照按叶片的感病面积分级记载,统计病情指数(DI);最后结合病情指数和抗病性分级表进行抗病性评价。
试验结果见表2。
Figure 579802DEST_PATH_IMAGE005
本发明方法为插穗上整叶接种,发病速度适中,接近田间发病进程,不同品种间发病梯度差异明显。然而,采用叶盘漂浮法接种时发病较快、较重,不同品种间的病情指数差异不明显,不能充分揭示不同品种间抗病性差异情况、鉴定结果不准确。此外,由于叶盘漂浮法属于离体叶片接种,接种期间叶片容易腐烂、滋生杂菌,为整个实验带来很多不确定因素。本发明方法属于活体接种,发病速度接近田间发病进程,能充分揭示不同品种间抗病性差异情况,数据准确、重复性高,鉴定结果接近田间抗病性情况。
此外,藜麦叶片结构有其特殊性,使用传统叶盘漂浮法室内离体接种时,打孔器打取叶盘时很容易破坏叶片结构;加之与霜霉病常规寄主的黄瓜、葡萄等叶片结构相比极其薄且易失水萎蔫,利用叶盘漂浮法鉴定影响其试验的准确性。

Claims (3)

1.一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法,包括如下步骤:
(1)制备扦插生根液 扦插生根液由扦插生根液A和扦插生根液B配置而成:扦插生根液A:称量1公斤藜麦秸秆加1L水,高压灭菌后,在无菌环境下加1ml浓度为3.5 ×109 cfu/mL枯草芽孢杆菌菌液,置于24~26℃发酵30 d,滤出发酵浸提液,备用;扦插生根液B:量取1L无菌水,溶入2.5mg维生素B1、6.5mg磷酸二氢钾、3.5mg硫酸镁、5.5mg硝酸铵、0.5 mg阿司匹林,配制成培养液,调节培养液的pH值为6.8~7.0,备用; 将扦插生根液A与扦插生根液B按体积比10:1配制成扦插生根液,然后分装于50~100ml的容器中,液面高度3.5~4.0cm,备用;
(2)制备扦插穗 在藜麦母株上截取长势好、无病虫害、直径为0.8~1.2 cm、长30cm的分枝,剪去分枝15cm以下的叶片,切口位于分枝叶柄疤痕下0.5~1.0 cm处;切口修剪为与分枝轴线呈约50-60度角的斜面,切面平滑、韧皮部不留毛茬且无损伤,制成扦插穗;
(3)扦插与培养 把(2)制备好的扦插穗的切口浸泡于35%的啤酒水溶液中消毒5~8min,然后插在(1)制备的扦插生根液中,置于人工气候箱,在18~20℃、光照强度3500lx,光照时间6h、黑暗18h的条件下培养,扦插液蒸发后及时补存至3.5~4.0cm高度,当扦插穗切口处明显看到白色细根时,调节气候箱的培养条件为:20~22℃、光照 6500 lx,光照时间10h、黑暗14h培养,培养10~15d,根系长4.5~5.5cm时备接种霜霉菌使用;
(4)病原物收集 田间采集具有藜麦霜霉病症状的病叶置于10℃条件下保藏20~24h,用毛刷将新鲜病叶的霜霉菌霉层刷除,无菌水冲洗3次,然后将叶片用湿润的脱脂棉花包裹,置于人工气候箱,培养条件是:18℃、1200lx、光照时间6h、黑暗18h,脱脂棉花处保湿状态,培养36 h后扫刷病叶长出的新鲜霉层,新鲜霉层即为霜霉病菌孢子囊,收集到1.5mL离心管中,置于10℃保藏,备用;
(5)制备吐温80溶液 用无菌水配制含1.5%吐温80的溶液;
(6)抗病性鉴定 取(4)收集好的藜麦霜霉病菌孢子囊置于(5)制备的吐温溶液中,调节孢子囊悬浮液浓度为1×108个孢子囊/毫升;用培养(3)的扦插穗进行接种,即用移液器吸取30微升的孢子囊悬浮液涂抹于藜麦的叶片正面,接种后置于人工气候箱,培养条件是:20℃、黑暗培养15h,然后将气候箱培养条件调为:20~22℃、6500 lx、光暗交替,光照时间10h、黑暗14 h培养,每插穗接种15片叶子、重复3次,5d后调查结果;
(7)鉴定结果计算方法 按叶片的感病面积分级记载,计算病情指数进而评价藜麦的抗病性。
2.根据权利要求1所述的一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法,其特征在于,所述的啤酒为酒精度为3%~4% vol、麦汁度10 °P~12 °P的市售啤酒。
3.根据权利要求1所述的一种利用扦插繁殖鉴定藜麦霜霉病抗病性的方法,其特征在于,所述的病情指数的计算公式为:
Figure DEST_PATH_IMAGE001
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1966669A (zh) * 2006-11-24 2007-05-23 东北农业大学 黄瓜霜霉菌孢子囊离体保存方法
CN102643892A (zh) * 2012-03-23 2012-08-22 云南农业大学 一种用于霜霉病菌抗药性频率监测的方法
CN103173365A (zh) * 2013-03-11 2013-06-26 河北省农林科学院植物保护研究所 一种用于室内培养繁殖葡萄霜霉病菌的方法
CN104313117A (zh) * 2014-10-14 2015-01-28 西北农林科技大学 利用葡萄白粉菌接种葡萄离体叶片快速鉴定抗病性的方法
WO2015127185A1 (en) * 2014-02-21 2015-08-27 Two Blades Foundation Methods for identifying plant pathogen resistance genes
CN105760871A (zh) * 2014-12-14 2016-07-13 仲恺农业工程学院 一种植物叶斑类病害抗性鉴定新方法
CN106399455A (zh) * 2016-08-30 2017-02-15 中国烟草总公司广东省公司 烟草抗赤星病的抗病性强弱温室苗期接种鉴定方法
CN206061778U (zh) * 2016-10-09 2017-04-05 黑龙江省农业科学院耕作栽培研究所 一种植物抗病性离体鉴定培养观察箱

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1966669A (zh) * 2006-11-24 2007-05-23 东北农业大学 黄瓜霜霉菌孢子囊离体保存方法
CN102643892A (zh) * 2012-03-23 2012-08-22 云南农业大学 一种用于霜霉病菌抗药性频率监测的方法
CN103173365A (zh) * 2013-03-11 2013-06-26 河北省农林科学院植物保护研究所 一种用于室内培养繁殖葡萄霜霉病菌的方法
WO2015127185A1 (en) * 2014-02-21 2015-08-27 Two Blades Foundation Methods for identifying plant pathogen resistance genes
CN104313117A (zh) * 2014-10-14 2015-01-28 西北农林科技大学 利用葡萄白粉菌接种葡萄离体叶片快速鉴定抗病性的方法
CN105760871A (zh) * 2014-12-14 2016-07-13 仲恺农业工程学院 一种植物叶斑类病害抗性鉴定新方法
CN106399455A (zh) * 2016-08-30 2017-02-15 中国烟草总公司广东省公司 烟草抗赤星病的抗病性强弱温室苗期接种鉴定方法
CN206061778U (zh) * 2016-10-09 2017-04-05 黑龙江省农业科学院耕作栽培研究所 一种植物抗病性离体鉴定培养观察箱

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
36个苹果树品种对腐烂病菌的抗病性评价;殷辉 等;《山西农业科学》;20171231;第45卷(第6期);第998-1001页 *
应用相对抗病性指数评价胡萝卜种质资源黑腐病抗性;周建波 等;《中国农学通报》;20151231;第31卷(第19期);第154-158页 *
茶树种质资源抗病性鉴定;曾莉 等;《生态科学》;19971231;第16卷(第2期);第61-64页 *

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