CN113180059A - 一种从菊花杂交后代群体中筛选驱避菊小长管蚜的挥发物的方法 - Google Patents
一种从菊花杂交后代群体中筛选驱避菊小长管蚜的挥发物的方法 Download PDFInfo
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Abstract
本发明涉及一种从菊花杂交后代群体中筛选驱避菊小长管蚜的挥发物的方法。本发明所提供的方法包括：对菊花亲本及杂交后代植株进行抗蚜评价；菊小长管蚜对菊花叶片挥发物的嗅觉行为选择；菊花叶片挥发物的收集和鉴定；对菊花亲本及杂交后代的叶片挥发物进行数据分析，筛选出差异特征挥发物；采用差异特征挥发物的化学标准品对菊小长管蚜进行行为学试验，最终确定具驱避作用的化合物成分。采用本发明所述的方法，首次明确了2‑甲基丙烯醛对菊小长管蚜具有驱避作用。本发明所述方法准确高效地筛选出了对菊小长管蚜具有驱避作用的化合物活性分子，为制备菊小长管蚜驱避剂提供了有力依据。
Description
技术领域
本发明涉及蚜虫驱避剂制备技术领域，特别是对菊小长管蚜具有驱避作用的化合物的测定方法及应用，具体地，本发明涉及一种从菊花中筛选驱避菊小长管蚜的挥发物的方法。
背景技术
菊小长管蚜(Macrosiphoniella sanborni)隶属于同翅目蚜科，是一种主要为害菊科植物的寡食性昆虫。常见寄主如白术，菊属大部分野生种和栽培品种。菊小长管蚜刺吸植物汁液，使叶片褪绿、变黄、萎蔫，甚至干枯，造成菊花生长发育畸形，花期为害更甚，严重影响了观赏菊花和药用菊花的品质。菊小长管蚜分泌的蜜露可导致煤污病发生，也会严重影响观赏价值。此外，菊小长管蚜作为菊花茎坏死病毒、菊花褪绿斑驳类病毒、以及菊花矮化类病毒等病原体的传播媒介，对于栽培菊花的生产具有极其严重的威胁，严重制约了菊花产业的发展。
当前，针对菊小长管蚜的防治研究主要集中在物理防治和化学防治，其中化学防治占据主导地位，但化学防治不是环境友好型途径，无论是对菊花产品本身还是环境都会造成具有较大的负面影响。因此，创制抗蚜菊花栽培品种，研发新型蚜虫驱避剂对于菊小长管蚜的防治具有重要意义。
发明内容
菊花叶片挥发性物质数量大、种类庞杂，本发明的目的是从庞杂的菊花叶片挥发物中筛选对于菊小长管蚜具有抑制作用的成分。
基于上述目的，本发明以构建得到的菊花杂交群体及其亲本作为研究对象，其中，用于构建菊花杂交群体的两个亲本的抗蚜性差异显著，通过对杂交群体及亲本进行群体的代谢组学(即挥发物)分析，再辅以昆虫行为学试验印证，从而在叶片挥发物中筛选出具蚜虫驱避功能的活性成分。
在实际检测中，若直接收集亲本及杂交后代的叶片挥发物，目标性较弱，且挥发物种类繁多，不同杂交后代的挥发物种类及含量差异很大不能准确的判断具抗蚜性的差异挥发物。
本发明首先对亲本及杂交后代进行抗性实验及菊小长管蚜行为学试验，及时从宏观检测鉴别不同菊花杂种后代抗菊小长管蚜水平的高低，以及菊花叶片挥发物成分对菊小长管蚜行为影响的差异。从而在进行叶片挥发物分析收集筛选时，依据后代群体对蚜虫的趋避性结果，缩小差异性叶片挥发物的筛选范围，以两个亲本及杂种后代的挥发物成分进行协方差矩阵下的主成分分析，能够快速(亲本与子代的挥发物有重合，样本量小)、准确地获取特征性挥发物。
基于此，本发明第一方面提供一种筛选驱避菊小长管蚜的挥发物的方法，包括：
(1)在菊花亲本及杂交后代植株上，接种菊小长管蚜成虫，8-12天后统计蚜虫数量；
(2)对菊花杂交后代及亲本进行菊小长管蚜的行为选择试验；
(3)利用固相顶空微萃取结合气相色谱质谱连用仪收集和鉴定菊花杂交后代及亲本的叶片挥发物；
(4)将步骤(1)和步骤(2)的蚜虫趋避性结果与挥发物的差异性相关联，对步骤(3)中收集得到的两个亲本及杂种后代的叶片挥发物成分进行协方差矩阵下的主成分分析，根据载荷值筛选出候选活性挥发物；
(5)使用所述候选挥发物的化学标准品，进行菊小长管蚜的行为学试验，确定驱避化合物成分。
具体地，在本发明步骤(1)中，接种的菊小长管蚜成虫为4龄若虫蜕皮后24h内的无翅成虫；接种后，采用透气的PC盒罩住植株。
本发明步骤(2)使用直径为19公分，高度为20公分的Y型嗅觉仪样品瓶进行行为选择试验，将植物连盆放入样品瓶中，盆土使用锡箔纸严密包裹。
在本发明步骤(3)中，以癸酸乙酯标准品为内标；所用萃取头型号为50/30μm DVB/CAR/PDMS；气相色谱以氦气为载气；质谱采用电子离子源。
在本发明步骤(4)中，利用GCMS Solution 4.11软件参考NIST.11谱库检索结果进行定性分析；通过内标峰面积进行定量分析；利用Origin2019软件对叶片挥发物进行主成分分析，根据载荷值筛选出驱蚜植株的显著差异成分，为候选活性挥发物。
在本发明步骤(5)中，以正己烷标准品为对照，候选活性挥发物标准品以正己烷为溶剂配制成不同浓度。
具体地，一种筛选驱避菊小长管蚜的挥发物的方法，包括：
(1)在菊花亲本及杂交后代植株上，接种菊小长管蚜成虫，8-12天后统计蚜虫数量；在接种蚜虫时，菊花植株长至8-12枚叶，接种的菊小长管蚜统一为4龄若虫蜕皮后24h内的无翅成虫。接种完成后，植株被规格为7×7×12cm，带有透气孔的PC盒罩住。
(2)对菊花亲本及杂交后代进行菊小长管蚜的行为选择试验；所用Y型嗅觉仪样品瓶是经过特殊改造的直径为19公分，高度为20公分的气密PC瓶，植株连盆放入样品瓶中，盆土用锡箔纸严密包裹。
(3)利用固相顶空微萃取结合气相色谱质谱连用仪，收集和鉴定菊花亲本及杂交后代菊花植株的叶片挥发物；
以癸酸乙酯标准品为内标；所用萃取头型号为50/30μm DVB/CAR/PDMS，萃取条件为50℃，20分钟；热脱附进样条件为250℃，5分钟；所用气相色谱质谱平台为岛津CGMS-QP2010，色谱型号为DB-5MS capillary column(30m×0.25mm×0.25μm)；气相色谱以氦气为载气，流速为1mL/min；运行程序为：起始温度40℃，维持2分钟，随后以5℃/min的速率增加到200℃，保持5分钟；电离电压为70eV，质谱扫描范围为40–500m/z，离子源温度为200℃，接口温度为250℃。
(4)将步骤(1)和步骤(2)的蚜虫趋避性结果与挥发物的差异性相关联，对步骤(3)中收集得到的两个亲本及杂种后代的叶片挥发物成分进行协方差矩阵下的主成分分析，根据载荷值筛选出候选活性挥发物；
利用GCMS Solution 4.11软件参考NIST.11谱库检索结果进行定性分析，并通过内标峰面积进行定量分析；利用Origin2019软件对杂交后代挥发物进行主成分分析，根据载荷值筛选出驱蚜植株的显著差异成分，为候选活性挥发物。
(5)使用所述候选挥发物的化学标准品，进行菊小长管蚜的行为学试验，确定驱避化合物成分；以正己烷标准品为对照，候选活性挥发物标准品以正己烷为溶剂配制成不同浓度。
本发明提供的方法，用于从扦插繁殖得到的菊花亲本杂交后代中筛选驱避菊小长管蚜的挥发物。
在从菊花亲本杂交后代中筛选驱避菊小长管蚜的挥发物时，选取生长状态良好无病虫害的芙蓉菊与甘菊杂种后代的脚芽，截取长6-8公分插穗，扦插至含草炭、珍珠岩和蛭石的基质中，待杂种后代无性系长至8-12枚叶时，接种菊小长管蚜成虫；其中，选取的是杂交后代母株的健壮脚芽进行扦插，插穗长6-8公分，扦插基质为草炭：珍珠岩：蛭石＝2：1：1。
根据本领域技术人员的理解，本发明还要求保护，上述制备方法在培育抗蚜菊花栽培品种，或制备菊小长管蚜驱避剂中的应用。
本发明还要求保护2-甲基丙烯醛用于制备菊小长管蚜驱避剂的新用途。
本发明的有益效果至少在于：
(1)本发明首次发现2-甲基丙烯醛具有驱避菊小长管蚜的作用，可用于制备菊小长管蚜驱避剂；
(2)本发明所提供方法中，通过蚜虫抗性试验和行为选择试验筛选出显著差异的杂交后代株系，有效地缩小了样本量；
(3)本发明利用固相顶空微萃取结合气相色谱质谱联用，对差异杂交后代的挥发物成分进行了鉴定分析，筛选出了它们的特征挥发物成分，又利用挥发物的标准化学品进行菊小长管蚜的行为学选择试验，从而成功地从众多地挥发物成分中，筛选出了具有驱避活性功能的成分；
(4)本发明所提供方法，从两个抗性差异亲本的杂交后代中，筛选出抗性个体，鉴定出活性挥发物成分，为其他植物的抗蚜虫鉴定提供了准确依据。
附图说明
图1为本发明实施例1中接种蚜虫10天后芙蓉菊(Cr)、甘菊(Ch)以及杂种后代上菊小长管蚜的虫情图。
图2为本发明实施例1中接种蚜虫10天后芙蓉菊(Cr)、甘菊(Ch)以及杂种后代上菊小长管蚜的数量统计图。
图3为本发明实施例1中菊小长管蚜对亲本及杂种后代挥发物行为选择的试验装置图。
图4为本发明实施例1中菊小长管蚜对亲本及杂种后代挥发物的行为选择结果统计图。
图5为本发明实施例1中对亲本及子代挥发物主成分分析的得分图。
图6为本发明实施例1中对亲本及子代挥发物主成分分析的载荷图。
图7为本发明实施例1中菊小长管蚜对2-甲基丙烯醛行为选择的结果图。
具体实施方式
以下实例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的保护范围。
若未特别指明，本发明实例中所用的实验材料、试剂、仪器等均可市售获得；若未具体指明，本发明实例中所有的技术手段均为本领域技术人员所熟知的常规手段。
实施例1
本实施例提供一种从芙蓉菊与甘菊杂交后代中筛选驱避菊小长管蚜的挥发物的方法，步骤如下：
1、杂种后代无性系获得：选取生长状态良好无病虫害的芙蓉菊与甘菊杂种后代的脚芽，截取长6-8公分插穗，扦插至草炭：珍珠岩：蛭石＝2：1：1的基质中。每株系扩繁5株。
2、杂种后代对菊小长管蚜抗性测定：待步骤1杂种后代无性系长至8-12枚叶时，人工接种菊小长管蚜4龄若虫蜕皮后24h内的无翅成虫。接种完成后，利用规格为7×7×12cm的带有透气孔的PC盒罩住植株，底部基质用直径为10cm的滤纸覆盖。接种后第10天统计PC盒内存活蚜虫数量(图1)。
统计结果发现，接种蚜虫十天后母本芙蓉菊、杂种后代H1、H2、H6、H8、H9、H11上的蚜虫数量显著低于父本甘菊以及杂种后代H3、H5、H10。说明芙蓉菊、杂种后代H1、H2、H6、H8、H9、H11对于蚜虫有较好的抗性(图2)。
3、菊小长管蚜对亲本及杂种后代挥发物行为反应测定：利用直径为2.8cm，主臂长21cm,支臂长17cm的玻璃Y型管进行行为选择试验。支臂连接两个样品瓶，样品瓶为一个自制的密闭性良好的PC瓶(直径＝19cm，高度＝20cm)。一个样品瓶内放入用锡箔纸严密包裹的空花盆，作为对照(图3)。另一个样品瓶内放入用锡箔纸严密包裹花盆的杂种后代植株。将菊小长管蚜的无翅成蚜放在主臂末端，利用气泵将经过木炭净化后的空气，以0.5L/min的流速输入，将杂种后代气味和对照组气味随机带入两个支臂内。8分钟后记录超过每个支臂至少2cm的蚜虫数量，每组试验重复5次，每50只无翅成虫为一个重复。
结果表明，母本芙蓉菊的挥发物对于菊小长管蚜有显著的驱避作用，而父本甘菊的挥发物对于菊小长管蚜有显著的吸引作用。杂种后代挥发物相对于对照组，对于菊小长管蚜都有一定的驱避性，其中H1,H2和H11的挥发物驱避作用较为明显(图4)。
通过本实施例的步骤2的菊小长管蚜抗性测定，可以确定杂交后代中具有蚜虫抗性的菊花植株；通过步骤3的蚜虫对杂种后代挥发物的行为选择试验，明确对蚜虫具有驱避作用或者吸引作用的菊花杂交后代；在后续鉴定具蚜虫驱避作用的叶片挥发物时，可进一步缩小样本量。
4、挥发物的收集与鉴定：从健壮的植株上采集0.1克样品，放入样品瓶内，利用型号为50/30μm DVB/CAR/PDMS的萃取头，在50℃条件下萃取20min。然后将萃取头插入气相色谱质谱联用仪CGMS-QP2010的色谱进样口中，在250℃条件下热脱附5min。气相色谱型号为DB-5MS capillary column(30m×0.25mm×0.25μm)，以氦气为载气，流速为1mL/min，运行程序为：起始温度40℃，维持2分钟，随后以5℃/min的速率增加到200℃，保持5分钟。质谱采用电子离子源，电离电压为70eV，质谱扫描范围为40–500m/z，离子源温度为200℃，接口温度为250℃。利用GCMS Solution 4.11软件对色谱图和质谱图进行解析，通过比对NIST11谱库，从芙蓉菊、甘菊及其杂种后代叶片中，一共鉴定到挥发物成分47种(表1)。
表1芙蓉菊、甘菊及其杂种后代叶片中挥发物及其含量
5、挥发物分析筛选：利用SPSS20.0软件对挥发物成分的定量结果进行分析。先利用单因素ANOVAs分析检测方差齐性，若方差齐则使用Tukey HSD检验进行多重比较；若方差不齐，则使用Bonferroni’s检验进行多重比较。随后，利用Origin2019对两个亲本及杂种后代的挥发物成分进行协方差矩阵下的主成分分析，发现主成分1和主成分2各能解释31.6％和22.9％的变异，并且在两个主成分下，亲本与子代均有明显的分离(图5)。其中，杂种后代株系H1,H2和H11和其它株系有显著的分离。在主成分1下，通过筛选载荷值>0.15的变量，筛选出了15种代表了H1,H2和H11的特征性挥发物(图6)。
6、菊小长管蚜对挥发物标品行为反应测定：以正己烷标准品为溶剂，将候选挥发物标准品配置成4个不同浓度。在如步骤3嗅觉仪中，将5×40mm的滤纸放入梨形玻璃样品瓶中，分别连接嗅觉仪两个支臂，两个样品瓶中分别滴入10μL的正己烷和配置好的候选挥发物溶液。其它操作程序与步骤3一致，统计蚜虫选择反应。结果发现，100ng/μL的2-甲基丙烯醛对菊小长管蚜有显著的驱避效果(图7)。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (10)
1.一种筛选驱避菊小长管蚜的挥发物的方法，其特征在于，包括：
(1)在菊花亲本及杂交后代植株上，接种菊小长管蚜成虫，8-12天后统计蚜虫数量；
(2)对菊花杂交后代及亲本进行菊小长管蚜的行为选择试验；
(3)利用固相顶空微萃取结合气相色谱质谱连用仪收集和鉴定菊花杂交后代及亲本的叶片挥发物；
(4)将步骤(1)和步骤(2)的蚜虫趋避性结果与挥发物的差异性相关联，对步骤(3)中收集得到的两个亲本及杂种后代的叶片挥发物成分进行协方差矩阵下的主成分分析，根据载荷值筛选出候选活性挥发物；
(5)使用所述候选挥发物的化学标准品，进行菊小长管蚜的行为学试验，确定驱避化合物成分。
2.根据权利要求1所述的方法，其特征在于，步骤(1)接种的菊小长管蚜成虫为4龄若虫蜕皮后24h内的无翅成虫；接种后，采用透气的PC盒罩住植株。
3.根据权利要求1所述的方法，其特征在于，步骤(2)使用Y型嗅觉仪样品瓶进行行为选择试验，将菊花植株连盆放入样品瓶中，盆土使用锡箔纸严密包裹。
4.根据权利要求1所述的方法，其特征在于，步骤(3)中，以癸酸乙酯标准品为内标；所用萃取头型号为50/30μmDVB/CAR/PDMS；气相色谱以氦气为载气；质谱采用电子离子源。
5.根据权利要求1所述的方法，其特征在于，步骤(4)中，利用GCMS Solution 4.11软件参考NIST.11谱库检索结果进行定性分析；通过内标峰面积进行定量分析；利用Origin2019软件对叶片挥发物进行主成分分析，根据载荷值筛选出驱蚜植株的显著差异成分，得到候选活性挥发物。
6.根据权利要求1所述的方法，其特征在于，步骤(5)中，以正己烷标准品为对照，候选活性挥发物标准品以正己烷为溶剂配制成不同浓度。
7.根据权利要求1-6任一项所述的方法，其特征在于，从扦插繁殖得到的菊花亲本杂交后代中筛选驱避菊小长管蚜的挥发物。
8.根据权利要求7所述的方法，其特征在于，选取生长状态良好无病虫害的芙蓉菊与甘菊杂种后代的脚芽，截取长6-8公分插穗，扦插至含草炭、珍珠岩和蛭石的基质中，待杂种后代无性系长至8-12枚叶时，接种菊小长管蚜成虫。
9.权利要求1-8任一项所述方法在培育抗蚜菊花栽培品种，或制备菊小长管蚜驱避剂中的应用。
10.2-甲基丙烯醛用于制备菊小长管蚜驱避剂的新用途。
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| CN110771603A (zh) * | 2019-11-08 | 2020-02-11 | 西北农林科技大学 | 对柑橘大实蝇成虫具有行为反应的柑橘幼果挥发物的测试方法 |
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| US5792467A (en) * | 1995-06-07 | 1998-08-11 | Proguard, Inc. | Repellent compositions containing aromatic aldehydes |
| CN110771603A (zh) * | 2019-11-08 | 2020-02-11 | 西北农林科技大学 | 对柑橘大实蝇成虫具有行为反应的柑橘幼果挥发物的测试方法 |
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