CN114847309A - 红果山胡椒叶果混合提取物、野菊花提取物及其混合物在草莓病害防治中的应用 - Google Patents
红果山胡椒叶果混合提取物、野菊花提取物及其混合物在草莓病害防治中的应用 Download PDFInfo
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- CN114847309A CN114847309A CN202210557364.4A CN202210557364A CN114847309A CN 114847309 A CN114847309 A CN 114847309A CN 202210557364 A CN202210557364 A CN 202210557364A CN 114847309 A CN114847309 A CN 114847309A
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- Prior art keywords
- extract
- essential oil
- wild chrysanthemum
- mixed
- rosae
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/24—Lauraceae [Laurel family], e.g. laurel, avocado, sassafras, cinnamon or camphor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
本发明涉及草莓病害Neopestalotiopsis rosae防治方法技术领域，具体涉及红果山胡椒叶果混合提取物和野菊花提取物及其混合物在抑制草莓病害中的应用。将红果山胡椒叶果混合提取物或野菊花提取物按照不同比例添加到PDA培养基中，研究对草莓病害的抑制效果。结果表明，红果山胡椒叶果混合精油在浓度为1μl/ml时的抑菌率大于50%，野菊花精油在浓度为1μl/ml时的抑菌率大于60%，二者的混合物明显高于单一精油的抑菌效果。本发明的原料易得，提取过程简单，对草莓病害有较好的抑制效果，同时植物提取物具有高挥发性、安全、低毒的特点，有利于大棚种植草莓的过程中对病虫害的防治。
Description
技术领域
本发明涉及草莓病害Neopestalotiopsis rosae防治方法技术领域，特别涉及红果山胡椒叶果混合提取物、野菊花提取物及其混合物的新用途，尤其是在抑制Neopestalotiopsis rosae中的新用途。
背景技术
目前，化学药剂在抑制病虫害复制的过程中，起到主要作用，但是由于长期、大量的使用后，造成环境污染严重，同时引发了其他的问题，如杀灭天敌、造成人/畜中毒和产生抗性等问题。因此，减少化学农药的使用量成为急需解决的重要问题之一。植物源药剂具有易分解、安全和抗性低等特点，且其分解产物对人、畜和生态系统不造成危害，来源较广泛。同时，研究发现植物提取物对病虫害具有很强的抑制活性。
草莓(Fragaria ananassa Duch.)属蔷薇科草莓属植物，在我国广为栽培。草莓的营养价值高，含多种营养物质，且有保健功效，同时具有产量高、经济效益好等特点，广受人民群众的喜爱。在草莓的种植过程中，病害对草莓的产量和品质具有很大的影响，而且安全、绿色的防控草莓病虫害，已经成为确保食品安全的主要问题之一。 Neopestalotiopsisrosae能引起草莓的根腐病、冠腐病和叶斑点，其症状为棕色坏死病变，叶上肩瓣黑色，匍匐茎上轻微凹陷的深棕色坏死，果实上凹陷的红褐色坏死。
目前病害的防治仍以化学防治为主，但由于草莓的安全性要求较高，因此，研究新型、安全、高效、低毒、低残留的防治方法成为草莓病虫害防治的热点。在大量的防治技术中，利用植物提取物防治病虫害对环境、生态的危害较小，且清洁无污染、低残留，具有广阔的市场前景。国内外利用植物提取物作为绿色环保抑菌剂应用于植物病害病原菌的防治研究已取得了较大的进展。植物提取物具有很强的挥发性，而且具有安全、低毒等特点，其挥发性有利于大棚种植草莓的过程中对病虫害的防治。
发明内容
针对现有技术中存在的不足，本发明的目的在于提供了防治Neopestalotiopsisrosae 的植物提取物。
所述植物提取物为红果山胡椒叶果混合提取物和/或野菊花提取物。
所述红果山胡椒叶果混合提取物是向红果山胡椒的叶和果混合物中加水后，在微波萃取仪中、100℃下提取后分离所得精油；
更具体的，所述红果山胡椒叶果混合提取物的提取方法如下：将红果山胡椒的叶果混合物与水按质量比为1:10混合后，利用微波萃取仪在800W、100℃条件下循环蒸馏3 小时，蒸气经冷凝得到油水混合物，最后经过油水分离得到红果山胡椒叶果混合精油。
所述野菊花提取物是向野菊花中加水后，在微波萃取仪中、100℃下提取后分离所得精油；
所述野菊花提取物的提取方法如下：将干燥的野菊花全株粉末与水按质量比为1:10 混合后，利用微波萃取仪在800W、100℃条件下循环蒸馏3小时，蒸气经冷凝得到油水混合物，最后经过油水分离得到野菊花精油。
为了研究红果山胡椒叶果混合提取物和野菊花提取物对Neopestalotiopsisrosae的防治效果，本发明将红果山胡椒叶果混合精油或野菊花精油按照不同的比例添加到PDA 培养基中，研究对Neopestalotiopsis rosae的抑制效果。本实验分别将不同量的红果山胡椒叶果混合精油或野菊花精油单剂或红果山胡椒叶果混合精油和野菊花精油的混合物加入培养基中混合均匀，配制成不同浓度的培养基，以不加精油的培养基为对照，接上Neopestalotiopsis rosae，在恒温下培养，待对照的Neopestalotiopsis rosae长满后，测量菌落直径，计算抑菌效果。计算方法为抑菌率＝(对照菌落直径-处理菌落直径)*100％/(对照菌落直径-菌饼直径)。
结果表明，野菊花精油在浓度为1μl/ml时对Neopestalotiopsis rosae的抑菌率大于 60％，红果山胡椒叶果混合精油在浓度为1μl/ml对Neopestalotiopsis rosae的抑菌率大于 50％；红果山胡椒叶果混合精油与野菊花精油的混合物按照1:1或1:2比例在浓度为0.5 μl/ml时的抑菌率已大于60％，明显高于单一精油的抑菌效果，0.5μl/ml、1:2的比例已超过单一精油中1μl/ml用量的抑菌效果。
具体实施方式
下面申请人将结合各具体实施例对本发明的新用途做进一步的详细说明。
以下各实施例中，原材料野菊花(Dendranthema indicum)(取全株)为2021年9 月采集于湖北省大悟县，采用微波萃取仪(MKX-H1C1A，青岛迈可威微波创新科技有限公司，下同，不赘述)，提取野菊花精油用于实验；红果山胡椒(Lindera erythrocarpa Makino)为2021年8月采集于湖北省宜昌市，采用微波萃取仪，提取红果山胡椒叶果混合精油用于实验。Neopestalotiopsis rosae均来源于湖北工程学院特色果蔬质量安全控制湖北省重点实验室。植物源农药主要是利用植物中的活性成分包括植物本身和按活性结构合成的化合物及衍生物，使其免受或减轻病害等有害生物危害的植物源制剂，其优点在于：专一性强，不伤害天敌，保护环境等。本研究采用将1)野菊花精油按照0.5 和1μl/ml(精油和培养基体积比)的配比添加到PDA培养基(马铃薯200克，葡萄糖20 克，琼脂18克，蒸馏水1000毫升，自然pH，下同，不赘述)；2)红果山胡椒叶果混合精油按照0.5和1μl/ml(精油和培养基体积比)的配比添加到PDA培养基；3)野菊花精油和红果山胡椒叶果混合精油的混合物按照不同的比例配成总量为0.5和1μl/ml(精油和培养基体积比)的配比添加到PDA培养基；以不加精油的培养基为对照，接上 Neopestalotiopsis rosae，在恒温下培养，待对照的Neopestalotiopsis rosae长满后，测量菌落直径，计算抑菌率，确定提取物对病菌的作用效果。计算方法为抑菌率＝(对照菌落直径-处理菌落直径)*100％/(对照菌落直径-菌饼直径)。
实施例1：野菊花精油的提取及成分含量鉴定
利用微波萃取仪，在功率800W的条件下，称取15g干燥粉碎的野菊花全株，向其中加入10倍重量的水，然后在100℃条件下循环蒸馏3小时，循环蒸馏产生混合体蒸气，其蒸气导入冷凝器中得到水与精油的液体混合物，在冷凝管处收集浮于水面上的野菊花提取物，即野菊花精油。通过气相色谱－质谱联用仪(7890B-5977A，安捷伦科技有限公司，美国)(GC-MS)对提取出的野菊花精油组成成分进行分析，检测条件：色谱柱为HP-5S毛细管柱(30m×0.25mm×0.25μm)，载气为氦气，进样量为1μl，恒流不分流模式，流速为1ml/min，柱箱升温程序：起始温度50℃，保持1min，以10℃/min升温至110℃后，以5℃/min升温至180℃后，以10℃/min升温至280℃结束，EI离子源，电子能量70eV，质子扫描范围50～550amu，四级杆温度为150℃，离子源温度为230℃，通过与标准谱库(NIST11)的谱图进行比较鉴定化合物，解析图谱后找出匹配度大于 90％的物质作为鉴定标准，鉴定出野菊花精油主要成分为31种，并采用面积归一化法测得各组分的相对百分含量，分析鉴定结果见表1。
表1野菊花精油的成分和含量
实施例2：红果山胡椒叶果混合精油的提取物成分和含量鉴定
利用微波萃取仪，在功率800W的条件下，在鲜重15g的红果山胡椒叶果混合物中加入10倍重量的水，然后在100℃条件下循环蒸馏3小时，循环蒸馏产生混合体蒸气，其蒸气导入冷凝器中得到水与精油的液体混合物，在冷凝管处收集浮于水面上的红果山胡椒叶果提取物，即红果山胡椒叶果混合精油。通过气相色谱－质谱联用仪 (7890B-5977A，安捷伦科技有限公司，美国)(GC-MS)对提取出的红果山胡椒叶果混合精油组成成分进行分析，检测条件：色谱柱为HP-5S毛细管柱(30m×0.25mm×0.25μm)，载气为氦气，进样量为1μl，恒流不分流模式，流速为1ml/min，柱箱升温程序：起始温度50℃，保持1min，以10℃/min升温至110℃后，以5℃/min 升温至290℃结束，EI离子源，电子能量70eV，质子扫描范围50～550amu，四级杆温度为150℃，离子源温度为230℃，通过与标准谱库(NIST11)的谱图进行比较鉴定化合物，解析图谱后找出匹配度大于90％的物质作为鉴定标准，鉴定出红果山胡椒叶果混合精油主要成分为28种，并采用面积归一化法测得各组分的相对百分含量，分析鉴定结果见表2。
表2红果山胡椒叶果混合精油的成分和含量
实施例3：野菊花精油对Neopestalotiopsis rosae的抑菌活性
为了检测实施例1制备的野菊花精油对Neopestalotiopsis rosae的抑菌效果，本发明采取以下实验进行验证：利用不同浓度的野菊花精油对Neopestalotiopsis rosae进行抑制实验。本实验将配置好、已灭菌的PDA培养基，用微波炉加热融化后，待培养基温度降至45-50℃时(下同，不赘述)，分别将野菊花精油按照0.5和1μl/ml(精油和培养基体积比)的配比添加到PDA培养基，倒入培养皿中(直径为90mm)，待其冷却后，将直径为6mm的Neopestalotiopsis rosae菌饼(菌饼是利用内径为6mm的打孔器从25℃培养箱黑暗培养了Neopestalotiopsis rosae菌3天的培养基上获取的，下同，不赘述)接在培养基上，以不加精油的PDA培养基为对照，将接好菌的培养基放置在培养箱中倒置培养，培养条件为25℃，待对照长满后，利用十字交叉法，测量菌落直径，通过计算其抑菌率，确定提取物对病菌的作用效果。结果表明：野菊花精油在浓度为1μl/ml时对 Neopestalotiopsis rosae的抑菌率都处于60％以上。
表3野菊花精油对Neopestalotiopsis rosae的抑菌率
实施例4：红果山胡椒叶果混合精油对Neopestalotiopsis rosae的抑菌活性
为了检测实施例2制备的红果山胡椒叶果混合精油对Neopestalotiopsis rosae的抑菌效果，本发明采取以下实验进行验证：利用不同浓度的红果山胡椒叶果混合精油对Neopestalotiopsis rosae进行抑制实验。本实验分别将红果山胡椒叶果混合精油按照0.5和1μl/ml(精油和培养基体积比)的配比添加到PDA培养基，倒入培养皿中(直径为 90mm)，待其冷却后，将直径为6mm的Neopestalotiopsis rosae菌饼接在培养基上，以不加精油的PDA培养基为对照，将接好菌的培养基放置在培养箱中倒置培养，培养条件为25℃，待对照长满后，利用十字交叉法，测量菌落直径，通过计算其抑菌率，确定提取物对病菌的作用效果。结果表明：红果山胡椒叶果混合精油在浓度为1μl/ml时对 Neopestalotiopsis rosae的抑菌率都处于50％以上。
表4红果山胡椒叶果混合精油对Neopestalotiopsis rosae的抑菌率
实施例5：野菊花精油和红果山胡椒叶果混合精油的混合物对Neopestalotiopsisrosae的抑菌活性
为了检测实施例1制备的野菊花精油和实施例2制备的红果山胡椒叶果混合精油的混合物对Neopestalotiopsis rosae的抑菌效果，本发明采取以下实验进行验证：利用不同比例的混合物对Neopestalotiopsis rosae进行抑制实验。本实验分别将野菊花精油和红果山胡椒叶果混合精油的混合物按照0.5和1μl/ml(精油和培养基体积比)的配比添加到PDA培养基，倒入培养皿中(直径为90mm)，待其冷却后，将直径为6mm的 Neopestalotiopsisrosae菌饼接在培养基上，以不加精油的PDA培养基为对照，将接好菌的培养基放置在培养箱中倒置培养，培养条件为25℃，待对照长满后，利用十字交叉法，测量菌落直径，通过计算其抑菌率，确定提取物对病菌的作用效果。结果表明：野菊花精油与红果山胡椒叶果混合精油体积比1:1和2:1的混合物在混合物浓度为0.5 μl/ml时对Neopestalotiopsis rosae的抑菌率都处于60％以上，明显高于相同用量时单一精油的抑菌效果，0.5μl/ml、2:1的比例已超过实施例3-4中1μl/ml用量的抑菌效果。
表5野菊花精油和红果山胡椒叶果混合精油混合物对Neopestalotiopsis rosae的抑菌率
Claims (9)
1.红果山胡椒叶果混合提取物和/或野菊花提取物在抑制Neopestalotiopsis rosae中的应用。
2.红果山胡椒叶果混合提取物和野菊花提取物的混合物在抑制Neopestalotiopsis rosae中的应用。
3.红果山胡椒叶果混合提取物和/或野菊花提取物在制备植物用Neopestalotiopsis rosae抑制剂中的应用。
4.红果山胡椒叶果混合提取物和野菊花提取物的混合物在制备植物用Neopestalotiopsis rosae抑制剂中的应用。
5.红果山胡椒叶果混合提取物和/或野菊花提取物在制备草莓用Neopestalotiopsis rosae抑制剂中的应用。
6.红果山胡椒叶果混合提取物和野菊花提取物的混合物在制备草莓用Neopestalotiopsis rosae抑制剂中的应用。
7.根据权利要求1-6中任一所述的应用，其特征在于：
所述红果山胡椒叶果混合提取物是向红果山胡椒的叶和果混合物中加水后，在微波萃取仪中、100℃下提取后分离所得精油；
所述野菊花提取物是向野菊花中加水后，在微波萃取仪中、100℃下提取后分离所得精油。
8.根据权利要求7所述的应用，其特征在于：所述红果山胡椒叶果混合提取物包括如下成分：莰烯、3-蒈烯、邻-异丙基苯、桉油精、β-罗勒烯、4-萜烯醇、乙酸龙脑酯、Alpha-荜澄茄油烯、a-蒎烯、正癸酸(2-乙基己酸)、β-榄香烯、(Z)-9-十六碳烯醛、石竹烯、布藜烯、香橙烯、新异长叶烯、律草烯、丙位依兰油烯、香树烯、2,3,4,4a,5,6-六氢-1, 4a -二甲基-7-(1-甲基乙基)萘、(1S,8aR)-1-异丙基-4,7-二甲基-1,2,3,5,6,8a-六氢萘、丁位愈创烯、橙花叔醇、Tau.-杜松醇、异香橙烯环氧化物、长叶松萜烯、3,7,11-三甲基-2,6,10-十二烷三烯-1-醇、n-十六酸。
9.根据权利要求7所述的应用，其特征在于：所述野菊花提取物包括如下成分：莰烯、β-蒎烯、对伞花烃、桉叶油醇、崖柏酮、樟脑、2-莰醇、α-松油醇、反式香芹醇、乙酸冰片酯、3-叔丁基苯酚、丁香酚、β-榄香烯、石竹烯、β-倍半萜-水芹烯、律草烯、1-(1,5-二甲基-4-己烯基)-4-甲基苯、丙位依兰油烯、1,2,4a,5,6,8a-六氢-4,7-二甲基-1-(1-甲基乙基)萘、异香橙烯环氧化物、Tau.-杜松醇、β-人参烯、别香树烯氧化物、母菊薁、十四烷酸、十五烷酸、正十七烷酸、叶绿醇、亚麻酸、十八烷、二十四烷。
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