CN115413655B - 紫苏醛作为农业杀菌剂的用途 - Google Patents
紫苏醛作为农业杀菌剂的用途 Download PDFInfo
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- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
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- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
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- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
- A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
- A01N47/24—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof containing the groups, or; Thio analogues thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
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Abstract
本发明属于农药研发技术领域，具体涉及紫苏醛作为农业杀菌剂的用途。紫苏醛作为抑菌活性成分在制备农业杀菌剂中的用途。本发明的紫苏醛在200mg/L时对茶树炭疽病病原菌山茶刺盘孢菌的抑菌率达到68.37％，具有明显的抑制作用，可以用于防治茶树炭疽病。另外，紫苏醛为植物提取物，为茶树炭疽病天然绿色农药的研制奠定基础。
Description
技术领域
本发明属于农药研发技术领域，具体涉及紫苏醛作为农业杀菌剂的用途。
背景技术
茶树病虫害是茶叶高产优质的重要限制因素，其中炭疽病是茶树叶部的主要真菌病害之一，在我国各茶区均有发生，西湖龙井茶产区温暖湿润的气候更利于其发生和蔓延，主栽品种‘龙井43’易感，近年来发病率逐年上升。受害茶树叶片轻则出现灼烧状病斑，重则造成大面积叶片脱落和植株枯死，严重影响树势和茶叶品质，造成巨大的经济损失。该病还严重为害茶树幼苗及经机采或修剪的茶树。
我国已报到的茶树炭疽菌有16个种及1个未鉴定种，包括异国炭疽菌C.alienum、隐秘炭疽菌C.aenigma、尖孢炭疽菌C.acutatum和博宁炭疽菌C.boninense等。不同种的炭疽菌具有不同的地域分布，对茶树的致病力及对防治药剂的敏感性存在一定的差异，因此明确茶树炭疽菌的种类是开展防治技术研究及抗病品种选育的关键。
目前，防治茶树炭疽病仍以喷施苯并咪唑类和三唑类等化学农药为主，不仅防效难以保障，还易引起茶叶农残超标、抗药性增强及茶园生态环境破坏等一系列问题。近年来，农药残留超标已成为影响我国茶叶出口的主要因素之一。针对茶树病虫害开发绿色高效的生物农药迫在眉睫。
植物提取物因其源于自然植物资源，具有环保低毒、易降解、对非靶标生物相对安全及不易产生抗性等优点，其相关研究越来越引起人们的重视。目前，研究报道较多的天然植物提取物主要有挥发油(精油)、黄酮类、生物碱和多糖类等。很多研究表明，植物提取物具有抑菌、杀虫、杀螨、驱虫和抗氧化等活性。
本发明对采自西湖龙井茶产区“龙井43”茶园的炭疽病病原菌进行分离纯化，通过形态学和分子生物学手段对茶树炭疽病病原菌进行鉴定，同时采用菌丝生长速率法对防治茶树炭疽病的药剂进行筛选，以期为茶树炭疽病天然绿色农药的研制奠定基础。
发明内容
本发明的目的在于提供紫苏醛作为农业杀菌剂的用途，从而克服喷施化学农药不仅防效难以保障，还易引起茶叶农残超标、抗药性增强及茶园生态环境破坏等一系列问题的缺点。
为实现上述目的，本发明提供了如下技术方案：
本发明的第一个目的在于提供紫苏醛作为抑菌活性成分在制备农业杀菌剂中的用途。
作为优选，所述农业杀菌剂用于防治由刺盘孢菌引起的真菌性病害。
作为优选，所述真菌性病害包括茶树炭疽病。
本发明的第二个目的在于提供一种杀菌剂，其活性成分为紫苏醛或由紫苏醛与吡唑醚菌酯复配而成。
作为优选，所述紫苏醛与吡唑醚菌酯的质量比1-9：12-1。
作为优选，所述紫苏醛与苯醚甲环唑的质量比1-20：20-1。
与现有技术相比，本发明具有如下有益效果：
(1)本发明的紫苏醛在200mg/L时对茶树炭疽病病原菌山茶刺盘孢菌的抑菌率达到68.37％，具有明显的抑制作用，可以用于防治茶树炭疽病。另外，紫苏醛为植物提取物，为茶树炭疽病天然绿色农药的研制奠定基础。
(2)本发明紫苏醛与吡唑醚菌酯或苯醚甲环唑复配时表现出增效作用，与单一有效成分相比，可以提高对茶树炭疽病的防治效果，基于此可以减少农药施用剂量，降低茶叶中的农药残留，避免超标。
附图说明
图1为茶园发生炭疽病病斑的位置及叶片症状；
图2为菌株QG5在PDA平板上培养7d后形态特征(a：正面；b：反面)；
图3为有伤接种后茶树叶片上病斑；
图4为病原菌rDNA-ITS区PCR扩增产物的电泳结果；
图5为菌株QG5的系统发育树。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：茶树炭疽病病原菌鉴定
1.1试验材料与方法
1.1.1试剂
Ezup柱式真菌基因组DNA抽提试剂盒，购自上海生工；
2×Taq PCRMsaterMix，购自天根生化科技(北京)有限公司。
1.1.2培养基
PDA培养基：去皮马铃薯200g、蔗糖20g、琼脂粉16g，蒸馏水定容至1000mL。
1.2试验方法
1.2.1病菌分离纯化
2021年6月，从西湖龙井茶产区‘龙井43’茶园采集茶树炭疽病典型病叶，用组织分离法进行病原菌分离。将新鲜采摘的茶树病叶冲洗30min，晾干后在病健交界处剪取0.5cm×0.5cm组织块，分别用75％乙醇和5％次氯酸钠消毒后用无菌水冲洗3次，在无菌滤纸上晾干后置于PDA固体培养基上，置于25℃培养3d长出菌丝后取菌落边缘的菌丝块转接到新鲜的PDA平板中央，重复此操作数次直至获得纯培养物。
1.2.2病原菌的形态学观察
在纯化后的菌落平板边缘打取0.5cm×0.5cm菌丝块接种于PDA平板中央，置于25℃下培养7d后，定期观察病原菌菌落特征及孢子形态，计算菌落生长速度，并通过柯赫氏法则将分离病原菌进行初步回接侵染验证。
1.2.3病原菌分子鉴定
采用Ezup柱式真菌基因组DNA抽提试剂盒提取病原菌基因组DNA，选用真菌通用引物ITS1和ITS4对病原菌进行PCR扩增，其中引物序列如表1所示，PCR反应体系如表2所示。
表1 ITS rRNA引物
表2 PCR反应体系
PCR程序为：94℃，3min；94℃45s，55℃45s，72℃1min，循环30次；72℃，10min。
扩增产物经1％琼脂琼脂糖电泳(150V100mA，20min)检测后进行测序。将所述的序列在NCBI的GenBank数据库中进行BLAST对比，利用MEGA7.0软件对与其同源性较高的序列构建系统发育树。
1.2.4致病性鉴定
取健康的‘龙井43’新稍从上往下数的第1-5叶位叶片作为一组处理，经无菌水冲洗后自然晾干，用75％乙醇进行擦拭，将叶片表面划伤后，用直径5mm打孔器打取菌饼。将长有菌丝的一面放于伤口处进行接种，以接种无菌PDA培养基块及无伤接种病原菌作为对照，将接种后的叶片置于25℃恒温箱中进行培养，定期观察不同叶位叶片的发病情况。对接种发病的叶片，再次按照1.2.1的方法分离纯化致病菌，观察其与原接种分离物是否一致。
1.3.试验结果
1.3.1茶园茶树炭疽病症状
茶园茶树炭疽病的症状见图1。
从图1可知，西湖龙井茶区茶树炭疽病新发病斑主要出现在成叶上，以芽下3、4叶为主(图1a)。初为暗绿色水渍状病斑，后逐渐扩展为黄褐色病斑，最后变为灰白色枯斑，其上生有粒状黑色小颗粒，为病原菌的分生孢子盘，个别大型枯斑有时可跨过中央叶脉蔓延至整叶(图1b)。
1.3.2茶树炭疽病致病菌的分离纯化及形态观察
在西湖龙井茶产区茶园采集新近发病的茶树叶片并立即进行病原菌分离，纯化4-5次得到该病原菌的纯培养物。根据分离获得的真菌培养性状和菌落形态，在茶树病叶上共分离到了8个真菌菌株，经初步回接验证其中QG3和QG5两株菌株可使茶树叶片发病，QG5的致病性最强，其在PDA平板上培养7d后形态特征见图2。
经形态学观察发现，QG5在PDA培养基上培养7d，其生长速率为11.9-12.9mm/d，菌落呈圆形，边缘整齐，气生菌丝致密，绒毛状，初为白色后为灰白色，背面白色至灰白色。
1.3.3致病性测定
采用有伤和无伤接种的方法对菌株QG5的致病性进行测定，接种2d后的病斑见图3。
由图3可知，有伤接种的茶树叶片上产生了红褐色病斑，边缘有水渍状晕圈。其中第1-3叶位叶片上的病斑更为明显。通过观察和测量病斑面积，发现幼嫩叶片中病斑面积更大，扩展更快，而第4-5叶位相对成熟的叶片病斑面积小。
将病斑组织用PDA培养基重新进行分离培养，所获得的菌落形态和原接种菌株QG5一致，结果符合柯赫氏法则。无伤接种QG5及有伤接种PDA培养基的对照没有出现病斑。
1.3.4病原菌序列系统发育分析
提取病原菌QG5的基因组DNA，利用真菌通用引物ITS1/ITS4进行PCR扩增，经1％琼脂糖电泳检测，PCR产物电泳检测如图4所示。
由图4可知，PCR产物条带出现在600bp左右，条带清晰，说明成功PCR产物扩增成功。
将所测QG5的序列及与其同源性较高的序列用MEGA7软件的NJ法构建系统发育树，结果见图5。
由图5可知，菌株QG5与Colletotrichum camelliae(登录号ON528180.1)聚类在同一分支上，支持率为67％，亲缘关系最近。因此，结合病原菌的形态特征和分子生物学鉴定结果，确定所分离获得的菌株QG5为日本山茶刺盘孢。
实施例2：植物提取物对茶树炭疽病病原菌的抑制作用
2.1试验药剂
百里香精油、柠檬精油、肉桂精油、牛至精油、柑橘精油、香茅精油、薰衣草精油、薄荷油、紫苏醛、香荆芥酚、柠檬醛和肉桂醛，以上药剂均购自上海源叶生物科技有限公司。
2.2.抑菌活性测定方法
以分离所得的茶树炭疽病菌为研究对象，采用菌丝生长速率法测定12种植物提取物对菌丝生长的抑制作用。将精油与等体积的乳化剂Tween 80混合后加灭菌水配成20g/L的母液，使用时用灭菌水稀释到所需浓度。
将2ml药液加入到刻度试管中，再用灭菌后冷却至50℃左右的PDA培养基定容至20mL，充分混匀后倒入90mm培养皿中配成培养基平板，并设置不含植物提取物的对照，每个处理重复3次，所有制剂现用现配。将黑暗培养5d的供试病原菌用0.5cm打孔器打取长势相同的菌饼，带有菌丝的一面朝下接入带药培养基中央，置于25℃恒温培养7d后用十字交叉法测量各处理的菌落直径，计算不同处理对菌丝生长抑制率。
2.3.抑菌活性测定结果
12种植物提取物对QG5菌株的抑菌活性测定结果见下表3。
表3 12种植物提取物对QG5菌株的抑菌活性测定结果
注：表中数据为平均值±标准误差，同列中不同小写字母表示差异显著(P＜0.05)，同列中不同大写字母表示差异极显著(P＜0.01)。
由表3可知，在质量浓度为200mg/L时，香荆芥酚、牛至精油对茶树炭疽病菌菌丝生长的抑制率最高，达到了100％，紫苏醛也具有较强的抑菌活性，在浓度为200mg/L时，对病原菌的抑菌率达到68.37％，与其他处理差异极显著。
实施例3：茶树炭疽病病原菌复配药剂筛选
3.1.供试菌株：菌株QG5
3.2供试药剂
95％紫苏醛原药(上海源叶生物科技有限公司)，98％吡唑醚菌酯原药(宁夏格瑞精细化工有限公司)、95％苯醚甲环唑原药(江苏澄扬作物科技有限公司)
将供试药剂用0.1％吐温-80水溶液稀释(其中，吡唑醚菌酯和苯醚甲环唑原药先用二甲基亚砜溶解再用0.1％吐温-80水溶液稀释)，配制成单剂母液，设置多组配比，各单剂和配比混剂均按等比方法设置5个质量浓度梯度。
3.3试验方法
采用菌丝生长速率法，将预先融化的PDA培养基9mL加入无菌锥形瓶中，从低浓度到高浓度依次定量吸取药液1mL，分别加入上述锥形瓶中，充分摇匀后等量倒入3个直径为9cm的培养皿中，制成相应浓度的含药平板；并设不含药剂的处理作空白对照，每个处理设置3个重复，所有制剂现用现配。
将黑暗培养5d的供试病原菌用0.5cm打孔器打取长势相同的菌饼，带有菌丝的一面朝下接入带药培养基中央，置于25℃恒温培养7d后用十字交叉法测量各处理的菌落直径，计算不同处理对菌丝生长抑制率。
3.4数据分析：采用DPS软件进行数据统计分析，以杀菌剂浓度对数值为x，对应的菌丝生长抑制率几率值为y进行线性回归，得出毒力回归方程及药剂对靶标病菌的毒力EC50值，并根据孙云沛法计算共毒系数(CTC)。
3.5.测定结果
根据计算的共毒系数(CTC)评价药剂的增效作用，CTC≤80为拮抗作用，80＜CTC＜120为相加作用，CTC≥120为增效作用，结果见表4-5。
表4紫苏醛与吡唑醚菌酯复配对茶树炭疽病菌的室内生物活性测定
| 药剂名称及配比 | EC50(mg/L) | ATI | TTI | CTC |
| 紫苏醛 | 109.5054 | 100.0000 | -- | -- |
| 吡唑醚菌酯 | 4.2012 | 2606.5267 | -- | -- |
| 紫苏醛1：吡唑醚菌酯12 | 3.5475 | 3086.8330 | 2413.7170 | 127.8871 |
| 紫苏醛1：吡唑醚菌酯9 | 3.2321 | 3388.0573 | 2355.8740 | 143.8132 |
| 紫苏醛1：吡唑醚菌酯6 | 4.0159 | 2726.7960 | 2248.4515 | 121.2744 |
| 紫苏醛1：吡唑醚菌酯3 | 2.6832 | 4081.1494 | 1979.8950 | 206.1296 |
| 紫苏醛1：吡唑醚菌酯1 | 6.3380 | 1727.7595 | 1353.2634 | 127.6736 |
| 紫苏醛3：吡唑醚菌酯1 | 7.9842 | 1371.5263 | 726.6317 | 188.7512 |
| 紫苏醛6：吡唑醚菌酯1 | 16.6469 | 657.8126 | 458.0752 | 143.6036 |
| 紫苏醛9：吡唑醚菌酯1 | 24.3814 | 449.1350 | 350.6527 | 128.0854 |
由表4可知，有效成分紫苏醛与吡唑醚菌酯复配后，在质量比为1-9：12-1内时对茶树炭疽病菌的共毒系数均大于120，表现为增效作用。
表5紫苏醛与苯醚甲环唑复配对茶树炭疽病菌的室内生物活性测定
| 药剂名称及配比 | EC50(mg/L) | ATI | TTI | CTC |
| 紫苏醛 | 109.5054 | 100.0000 | -- | -- |
| 苯醚甲环唑 | 7.2532 | 1509.7529 | -- | -- |
| 紫苏醛1：苯醚甲环唑20 | 6.2683 | 1746.9713 | 1442.6218 | 121.0970 |
| 紫苏醛1：苯醚甲环唑10 | 5.1689 | 2118.5436 | 1381.5936 | 153.3406 |
| 紫苏醛1：苯醚甲环唑5 | 6.1618 | 1777.1658 | 1274.7941 | 139.4081 |
| 紫苏醛1：苯醚甲环唑3 | 7.6681 | 1428.0643 | 1157.3147 | 123.3946 |
| 紫苏醛1：苯醚甲环唑1 | 9.3966 | 1165.3726 | 804.8765 | 144.7890 |
| 紫苏醛3：苯醚甲环唑1 | 13.8388 | 791.2926 | 452.4382 | 174.8952 |
| 紫苏醛5：苯醚甲环唑1 | 15.1305 | 723.7395 | 334.9588 | 216.0682 |
| 紫苏醛10：苯醚甲环唑1 | 32.3964 | 338.0172 | 228.1594 | 148.1496 |
| 紫苏醛20：苯醚甲环唑1 | 19.6812 | 556.3960 | 167.1311 | 332.9099 |
由表5可知，有效成分紫苏醛与苯醚甲环唑复配后，在质量比为1-20：20-1内时对茶树炭疽病菌的共毒系数均大于120，表现为增效作用。
综上所述，紫苏醛与吡唑醚菌酯或苯醚甲环唑复配后具有很好的协同增效作用，与单一有效成分相比，可以提高茶树炭疽病的防治效果，基于此可以减少农药施用剂量，降低茶叶中的农药残留，避免超标。
上所述仅是本发明的优选实施方式，对于本领域技术人员来说，在不脱离本发明原理的前提下，可以做适当的改进，这些改进也在本发明的保护范围之内。
Claims (1)
1.一种杀菌剂，其特征在于，其活性成分由紫苏醛与吡唑醚菌酯或苯醚甲环唑复配而成，所述紫苏醛与吡唑醚菌酯的质量比1-9：12-1；所述紫苏醛与苯醚甲环唑的质量比1-20：20-1。
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