CN114391554B - 一种紫罗兰在抑制葡萄霜霉病中的应用 - Google Patents
一种紫罗兰在抑制葡萄霜霉病中的应用 Download PDFInfo
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- CN114391554B CN114391554B CN202210210648.6A CN202210210648A CN114391554B CN 114391554 B CN114391554 B CN 114391554B CN 202210210648 A CN202210210648 A CN 202210210648A CN 114391554 B CN114391554 B CN 114391554B
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Abstract
本发明提供了一种紫罗兰在抑制葡萄霜霉病中的应用，所述应用为通过使用紫罗兰与葡萄间作的种植方式，利用紫罗兰植物在生长过程中释放的混合挥发性组分来抑制葡萄霜霉病致病菌Plasmopara viticola的生长，来减轻葡萄园葡萄霜霉病的发生。实现葡萄霜霉病的绿色防治的同时还能增加单位土地经济价值，具有广阔的应用前景。
Description
技术领域
本发明涉及生物防治领域，更为具体的，本发明涉及一种紫罗兰在抑制葡萄霜霉病中的应用。
背景技术
紫罗兰(Matthiola incana(L.)R.Br.)十字花科植物，是二年生或多年生芳香观赏植物，具有丰富的挥发性物质。但对于其挥发物的鉴定及其挥发物对病原菌的作用目前还没有相关研究报道。
葡萄霜霉病是世界范围内最严重的葡萄病害之一。它的致病菌是卵菌Plasmoparaviticola，其侵染循环是从成熟游动孢子囊释放游动孢子开始，游动孢子通过风或雨滴传播，通过气孔侵入葡萄叶片和幼果。霜霉病不仅会影响葡萄植株的生长，还会降低葡萄果实的品质和产量。目前，葡萄霜霉病的防治主要以化学防治为主，但化学农药的大量和高频率不科学使用不仅会使病原菌产生耐药性，还会带来农药残留、环境污染等问题(Vezzulli，S.，Malacarne，G.，Masuero，D.，Vecchione，A.，Dolzani，C.，Goremykin，V.et al.(2019)The Rpv3-3haplotype and stilbenoid induction mediate downy mildew resistancein a grapevine interspecific population.Front Plant Sci.10：234.)。因此寻找替代的环保型杀菌剂或防治策略势在必行。
发明内容
为了填补现有技术的空白，实现葡萄病害的绿色防控，本发明提供如下的技术方案：
本发明的第一个方面，提供一种紫罗兰在抑制葡萄霜霉病中的应用。
在一种实施方式中，所述应用为将紫罗兰与葡萄在非开放性大棚中以间作的方式种植。
在另一种实施方式中，所述应用为利用紫罗兰地面部分挥发物抑制葡萄霜霉病菌。优选的，所述紫罗兰地面部分挥发物选自丁香酚、苯乙醇、十三烷、柠檬烯、2-甲基丁酸乙酯、1，8-桉树脑、2，6-二叔丁基-4-甲基苯酚、乙醛二乙缩醛、苯乙酸甲酯、惕各酸乙酯、苯甲酸乙酯、乙酸戊酯、β-石竹烯、α-蒎烯、β-蒎烯或β-金合欢烯中的一种或几种。
本发明的第二个方面，提供一种紫罗兰用于制备葡萄霜霉病抑制剂的应用。
在一种实施方式中，所述抑制剂包含紫罗兰地上部分的挥发物单体中的一种或多种。优选的，所述挥发物单体为丁香酚、苯乙醇、十三烷、柠檬烯、2-甲基丁酸乙酯、1，8-桉树脑、2，6-二叔丁基-4-甲基苯酚、乙醛二乙缩醛、苯乙酸甲酯、惕各酸乙酯、苯甲酸乙酯、乙酸戊酯、β-石竹烯、α-蒎烯、β-蒎烯或β-金合欢烯。
在一种实施方式中，上述十三烷指的是正十三烷，是指直链无任何取代基的。分子式为C13H28，英文名：tridecane或n-tridecane，其结构式如下：
本发明获得了如下突出的技术效果：
1.现有技术主要使用微生物作为挥发物的生产源，而本发明创新性的使用紫罗兰这一芳香花卉植物，该植物种植广泛，挥发物丰富，而且本身存在经济价值，与葡萄间种在提高葡萄产量和品质的同时，还能增加额外的经济收益；
2.本发明对紫罗兰挥发物中的23个物质(包括萜烯类、酯类、醇类、酚类、醛类、酮类和烷烃类)进行了抑菌活性测定，明确了每种单体对葡萄霜霉病菌的抑菌能力，从小分子物质化感作用的层面探讨了紫罗兰与葡萄间作控病的机理，为田间的试验现象做出了理论解释。同时也探讨了单一物质开发成新型植物源杀菌剂的潜力。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1为田间红提葡萄单作和红提葡萄与紫罗兰间作模式图；
图2为紫罗兰与葡萄间作对葡萄霜霉病防治效果图(不同的小写字母代表处理间的差异显著性，one-way ANOVA，Duncan’s multiple range test，P＜0.05)；
图3为紫罗兰地上部分挥发物对葡萄霜霉病菌抑制效果图(不同的小写字母代表处理间的差异显著性，one-way ANOVA，Duncan’s multiple range test，P＜0.05)；
图4为紫罗兰挥发物收集装置图；
图5为23个紫罗兰挥发物单体对葡萄霜霉病菌抑制活性图(不同的小写字母代表处理间的差异显著性，one-way ANOVA，Duncan’s multiple range test，P＜0.05)；
图6为正十三烷对葡萄霜霉病菌的抑制率变化图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
实施例1紫罗兰地上部分挥发物对葡萄霜霉病抑制作用
于2017和2018年在云南寻甸大河桥农场(N25.56°，E103.25°；海拔1885米，年均气温为14.9℃，年均降雨量为1022.4mm，年均日照时数为2061.6h)约2000m2的葡萄园内设置试验。田间试验设置是在葡萄园开放性空间中进行的。葡萄株高一般是150-160cm，紫罗兰株高一般是60cm左右，所以葡萄对间作其中的紫罗兰形成了一个相对的屏障。
试验包括红提葡萄单作、以及紫罗兰与红提葡萄间作两种种植模式(具体如图1所示)。其中，红提葡萄也叫红地球(Red Globe)；紫罗兰的品种为“Stock Yume no Uta”。试验所用葡萄在园中已生长了9年；紫罗兰是先用种子在温室育苗，当长到8-10片叶时，于葡萄开花期(3-4月)移栽到葡萄园中，当葡萄成熟期(7-8月)紫罗兰可开花。每种种植模式设置3个重复，每个重复地块长22.5m宽2.2m，其间种植2行葡萄约有50株(行间距是2.2m×1.0m)，在间作的地块中，2行葡萄之间种植约180株紫罗兰，葡萄与紫罗兰之间的距离是58cm，紫罗兰之间的行间距是35cm×35cm。为避免紫罗兰挥发物对单作葡萄的影响，在2种种植模式之间有2个重复地块(种植红提葡萄)作为间隔。每隔7天调查一次田间葡萄霜霉病发病情况，直到葡萄果实采收结束。根据葡萄霜霉病的分级标准，计算出病情指数和防治效果。霜霉病的病害严重度分级参照臧超群等(2014)的分级方法，具体如下：
0级，叶片无病斑；
1级，病斑面积占整个叶面积的5％以下(即S叶＜5％)；
3级，病斑面积占整个叶面积的5％～25％(即5％≤S叶≤25％)；
5级，病斑面积占整个叶面积的26％～50％(即25％＜S叶≤50％)；
7级，病斑面积占整个叶面积的51％～75％(即50％＜S叶≤75％)；
9级，病斑面积占整个叶面积的75％以上(即S叶＞75％)。
(参考文献：臧超群，赵奎华，刘长远，梁春浩，于舒怡，刘丽.生防细菌SY286的筛选及其对葡萄霜霉病的防治效果研究[J].中国生物防治学报，2014，30(03)：402-407.)
结果显示，2017年7月初至7月中旬，红提与紫罗兰间作后，霜霉病病情指数略低于对照(图2a)，但差异不显著性(P＞0.05)，从7月下旬至8月初，病情指数显著低于对照(P＜0.05)，平均防效为26.51±3.79％，尤其在紫罗兰开花盛期(7月21日-7月26日)防效最好；2018年病情指数变化规律与2017年基本一致(图2b)，平均防效为26.56±3.68％。在本试验中，对照(葡萄单作)和间作(葡萄和紫罗兰间作)是处于相同的一片葡萄园中，气候条件一致，农事处理一致，所以可以认为对照和间作葡萄园病情指数的差异主要是由间作了紫罗兰引起的。
进一步采用离体叶盘接种法(图3a)分析紫罗兰地上部分挥发物对葡萄霜霉病的抑制作用。2017年，选长势相当且无霜霉病症状的新鲜红提葡萄叶片，打取叶盘(D＝2.5cm)。在培养皿(D＝15cm)内铺上润湿的滤纸，叶盘背面朝上放置，一皿20片。在每片叶盘背面接种20μL浓度为105个/mL的霜霉病菌游动孢子悬浮液。将1.0，3.0，5.0g的紫罗兰(花+叶片)放入锡箔纸碗中，放入培养皿，用Parafilm封口膜密封。培养皿置于培养箱培养(20℃，12h光照/12h黑暗)。以不放置紫罗兰的处理为对照，每个处理重复三次。发病后测量病斑面积，计算防治效果。2018年，重复该试验，在2018年的实验中将紫罗兰质量改为0.1，0.5，1.0，2.0，3.0，4.0，5.0g。
结果显示，紫罗兰地上部分挥发物对葡萄霜霉病起关键抑制作用。随着紫罗兰质量从1.0g增大到5.0g，对叶盘霜霉病的抑制率从29.93±2.06％增大到41.65±7.17％(2017)，紫罗兰质量从0.1g增大到5.0g，对霜霉病抑制率从10.32±5.31％增大到47.68±8.43％(2018)(图3b)。
实施例2：用GC-MS对紫罗兰挥发物成分进行分析
基于常温常压吹气法收集紫罗兰挥发物成分(装置如图4所示)，具体的方法如下：
(1)用锡箔纸对开花的紫罗兰根部进行密封包裹，按50株一缸至于挥发物收集缸中；
(2)使用大气采样仪(QC-1B，Beijing Municipal Institute of LabourProtection，Beijing，China)，将空气经由活性炭和硅胶除去杂质和水分后抽入收集缸中；
(3)使用大气采样仪抽出含有紫罗兰挥发物的气体至装有0.5g干净的吸附树脂(PorapakTM Q，80-100mesh，Waters，USA)的吸附柱中；
(4)整个收集过程持续48h，每个紫罗兰样品收集3个重复。
采用GC-MS对收集所得的挥发物进行检测，具体操作方法如下：
(1)吸附柱吸附48h后，用5mL正己烷洗脱吸附柱内的挥发物成分，氮吹浓缩至2mL后通过0.22um微孔滤膜过滤后装入样品瓶中，上机检测。
(2)待测样品的进样量为1μL，气象色谱仪的升温程序为：初始温度40℃，以3℃/min的升温速度升至80℃，再以5℃/min的速度升至260℃，保持10min。质谱条件如下：扫描间隔为0.3sec，质谱扫描范围35-500。
(3)数据下机后使用GCMS solution软件(Shimadzu，Kyoto，Japan)比对NIST14、NIST14s图谱库，排除相似度低于80％且保留时间>35min的物质。并采用ChemStationIntegrator积分方式进行相对含量分析。
结果显示(见下表1)，紫罗兰挥发物中主要含有36种成分(相似度≥80％，相对含量≥0.05％)，其中酯类物质17种(按相对含量从高到低排序：2-甲基丁酸乙酯、丁酸乙酯、己酸乙酯、苯乙酸甲酯、反-3-己稀酸乙酯、辛酸乙酯、苯甲酸乙酯、惕各酸乙酯、反-2-己烯酸乙酯、乙酸戊酯、水杨酸甲酯、丁二酸二乙酯、乙酸己酯、丙酸乙酯、丙二酸二乙酯、乙酸异戊酯、戊酸乙酯)，萜烯类物质8种(α-金合欢烯、β-金合欢烯、α-蒎烯、β-石竹烯、茴香脑、柠檬烯、1，8-桉树脑、β-蒎烯)，醇类物质3种(苯乙醇、1-辛烯-3-醇、3-己醇)，烷烃类物质3种(正十三烷、3-甲基十三烷、2，6-二甲基十一烷)、酮类物质2种(6-甲基-5-庚烯-2-酮、2-己酮)和酚类物质各2种(丁香酚、2，6-二叔丁基-4-甲基苯酚)、醛类1种(乙醛二乙缩醛)。
表1.紫罗兰挥发物中36种主要成分
(注：表中带*的23物质在每类物质中其相对含量覆盖了高、中、低三个范围，用于后面的单一挥发物成分对葡萄霜霉病菌抑菌活性测定)
实施例3：紫罗兰挥发物单体对葡萄霜霉病抑制效果
采用离体叶盘接种法，选取表1中23个紫罗兰挥发物用于测定其对葡萄霜霉病菌的抑菌活性，其中，23个挥发物的选取标准为：
(1)能购买到纯品；
(2)曾在文献中报道有抑菌或杀菌活性的物质：如萜烯类物质被报道具有多种生态功能，可直接抑菌或杀菌(Kanchiswamy et al.，2015)；含苯环类化合物大多有抑菌作用，如丁香酚对子囊菌、卵菌、丝核菌和半知菌等均有抑制作用(Schiestl，2015)；脂肪酸衍生物如醇、醛类的物质也可直接抑菌(Baldwin et al.，2006)；
(3)根据相对含量的高低，在表1的每组类型的物质中，选择的物质其相对含量覆盖高(相对含量≥1)、中(0.1≤相对含量＜1)、低(相对含量＜0.1)三个范围。
具体实验步骤如下：选长势相当且无霜霉病症状的新鲜红提葡萄叶片，打取叶盘(D＝2.5cm)。在培养皿(D＝15cm)内铺上润湿的滤纸，叶盘背面朝上放置，一皿20片。在每片叶盘背面接种20μL浓度为105个/mL的霜霉病菌游动孢子悬浮液。将23个单一挥发物分别放入离心管盖中，在培养皿中形成0.5，1.0，2.0，4.0，6.0，8.0，10.0，15.0，20.0mg.L-1不同浓度。以等量无菌水处理为对照。每个处理3个重复。培养皿密封后放于培养箱(20±1℃，12h光照/12h黑暗)培养。发病后测量病斑面积，计算防治效果和EC50值。
结果显示(图5)，在23个紫罗兰挥发物中，有17个紫罗兰挥发物单体表现出对葡萄 霜霉病菌的直接抑菌活性。按EC50值的大小可分为4组，第一EC50≤1mg·L-1，包括丁香酚、苯乙醇和正十三烷，其中，正十三烷对葡萄霜霉病菌的抑制率变化趋势如图6所示，随着十三烷浓度从0.5增大到20.00mg/L，平均抑制率从57.18％上升到100％。本试验设计的最低浓度是0.5mg/L，此时它对霜霉病菌的平均抑制率已达到57.18％，3次重复试验的EC50值分别为0.77mg/L、0.42mg/L、0.42mg/L，平均EC50值为0.54mg/L。
第二组1mg·L-1＜EC50≤5mg·L-1，由9个物质构成(柠檬烯、2-甲基丁酸乙酯、1，8-桉树脑、2，6-二叔丁基-4-甲基苯酚、乙醛二乙缩醛、苯乙酸甲酯、惕各酸乙酯、苯甲酸乙酯)，其中柠檬烯抑菌活性最高；第三组包括乙酸戊酯、β-石竹烯、α-蒎烯和β-蒎烯，其5mg·L-1＜EC50≤20mg·L-1；第四组仅有β-金合欢烯，其EC50＝126.92mg·L-1，显著高于其他所有单一化合物(P＜0.05)。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (3)
1.紫罗兰地面部分挥发物在抑制葡萄霜霉病中的应用，其特征在于，所述紫罗兰地面部分挥发物为正十三烷。
2.如权利要求1所述的应用，其特征在于，所述正十三烷的使用剂量为20.00 mg/L。
3.紫罗兰地面部分挥发物在抑制葡萄霜霉病中的应用，其特征在于，所述紫罗兰地面部分挥发物为丁香酚、苯乙醇和正十三烷。
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