CN114766499A - 东莨菪碱及其与化学农药联用在防治植物晚疫病害中的新用途 - Google Patents
东莨菪碱及其与化学农药联用在防治植物晚疫病害中的新用途 Download PDFInfo
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- STECJAGHUSJQJN-FWXGHANASA-N scopolamine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-FWXGHANASA-N 0.000 title claims abstract description 121
- 229930000680 A04AD01 - Scopolamine Natural products 0.000 title claims abstract description 120
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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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
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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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
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Abstract
本发明公开了东莨菪碱或其与化学农药联用在抑制致病疫霉(Phytophthorainfestans)或防治植物晚疫病中的应用,所述化学农药为双氧水或银法利。本发明通过实验验证东莨菪碱可抑制致病疫霉的菌丝生长和孢子萌发,减弱其致病力,增强其对逆境的敏感性,还可诱导马铃薯抗病。并且在研究过程中,发现东莨菪碱与双氧水或者银法利联用对致病疫霉产生了更好的抑制效果,东莨菪碱与化学农药的联用能起到协同增效的作用。本发明为植物晚疫病的防治提供了新的生物农药和新的防治思路,有望在植物晚疫病防治中减少化学农药的使用,从而减少化学农药对人和环境的危害。
Description
技术领域
本发明涉及作物病害防治技术领域,具体涉及东莨菪碱及其与化学农药联用在防治植物晚疫病害中的新用途。
背景技术
致病疫霉(Phytophthora infestans)属于卵菌,其引起的马铃薯和番茄晚疫病是世界马铃薯和番茄产区的毁灭性病害之一。
马铃薯(Solanum tuberosum L.)是世界第四大粮食作物。致病疫霉引起的马铃薯晚疫病(PotatoLateBlight)是一种毁灭性的流行性卵菌病害,导致马铃薯茎叶死亡和块茎腐烂,曾引发爱尔兰大饥荒。至今马铃薯晚疫病仍是世界马铃薯产区最具破坏性病害之一,引起巨大经济损失。目前防治该病的主要方法是使用化学杀菌剂。然而,致病疫霉易对化学农药产生耐药性,过量使用化学杀菌剂也会影响人体健康和土壤环境。因此研发安全有效且具有针对性的生防制剂成为了当务之急。
东莨菪碱(Scopolamine)是一种抗胆碱药物,在医疗方面,其对呼吸系统、心血管系统、消化系统、神经系统等疾病治疗有重要作用,也有缓解有机磷农药及乙酰胺中毒等临床应用,同时还有一定的麻醉作用和神经毒性;在植保领域,东莨菪碱可通过抑制孢子萌发和附着胞形成,从而防治稻瘟病等植物病害。然而,有关东莨菪碱防控马铃薯晚疫病的研究尚少。
发明内容
本发明的目的是针对上述问题,一方面提供东莨菪碱在抑制致病疫霉(Phytophthora infestans)中的应用。
本发明的第二方面提供东莨菪碱在防治植物晚疫病中的应用,所述植物为番茄或马铃薯。
本发明的第三方面提供东莨菪碱与化学农药联用在抑制致病疫霉中的应用,所述化学农药为双氧水或银法利。
本发明的第四方面提供东莨菪碱与化学农药联用在防治植物晚疫病中的应用,所述化学农药为双氧水或银法利,所述植物为番茄或马铃薯。
在上述任意一种技术方案中,东莨菪碱的使用浓度为1-9g/L。
优选地,东莨菪碱的使用浓度为1-7g/L。
进一步优选地,东莨菪碱的使用浓度为2-6g/L或3-6g/L或4.0g/L或4.25g/L或6g/L。
在上述技术方案中,H2O2使用浓度为0.1-0.3mL/L,银法利的使用浓度为5-10μL/L。
优选地,H2O2使用浓度为0.15-0.2mL/L,银法利的使用浓度为6-9μL/L。
东莨菪碱与H2O2联用时,东莨菪碱与6wt%H2O2按照质量体积配比为1-9g:0.1-0.3mL进行混合配制,优选1-7g:0.1-0.3mL或2-6g:0.15-0.2mL。
东莨菪碱与银法利联用时,按照东莨菪碱与银法利质量体积配比为1-9g:5-10μL进行混合配制,优选1-7g:5-10μL或2-6g:6-9μL。银法利采用的是悬浮剂型,含有霜霉威盐酸盐含量625克/升,氟吡菌胺含量62.5克/升。
本发明的有益效果是:
通过平板实验发现东莨菪碱对致病疫霉菌丝生长和孢子萌发的影响;通过离体马铃薯薯块与叶片实验进一步探索出东莨菪碱可以降低致病疫霉的致病力,同时可诱导马铃薯产生抗病性;通过逆境胁迫揭示出东莨菪碱如何影响致病疫霉在逆境下的生长,提高了东莨菪碱防控晚疫病的可行性。本发明通过实验说明东莨菪碱可抑制致病疫霉的菌丝生长和孢子萌发,减弱其致病力,增强其对逆境的敏感性,还可诱导马铃薯抗病。并且在研究过程中,发现东莨菪碱与双氧水或者银法利联用对致病疫霉产生了更好的抑制效果,东莨菪碱与化学农药的联用能起到协同增效的作用。本发明为植物晚疫病的防治提供了新的生物农药和新的防治思路,有望在植物晚疫病防治中减少化学农药的使用,从而减少化学农药对人和环境的危害。
附图说明
图1是东莨菪碱处理对致病疫霉菌丝生长的影响,图(A)、(B)为平板生长情况,图(C)为菌落直径(*P<0.05,**P<0.01,***P<0.001),图(D)为抑制率(*P<0.05,**P<0.01,***P<0.001)。
图2是东莨菪碱对致病疫霉孢子萌发的影响作用,图(A)是用CK或不同浓度的东莨菪碱处理12小时显微镜观察孢子萌发,图(B)致病疫霉孢子萌发率(*P<0.05,**P<0.01,***P<0.001)。
图3是东莨菪碱对致病疫霉逆境胁迫实敏感性影响与抑制率(*P<0.05,**P<0.01,***P<0.001)。
图4是东莨菪碱能降低致病疫霉致病力实验结果,图(A)是不同浓度东莨菪碱处理马铃薯块茎时晚疫病症状;图(B)不同浓度东莨菪碱处理时马铃薯叶片晚疫病症状;图(C)不同浓度东莨菪碱处理时马铃薯块茎病变面积占比(*P<0.05,**P<0.01,***P<0.001);图(D)不同浓度东莨菪碱处理马铃薯叶片时病变面积占比(*P<0.05,**P<0.01,***P<0.001)。
图5是东莨菪碱和化学农药联用对致病疫霉的作用,图中包含致病疫霉在CK、东莨菪碱(IC50)、代森锰锌(7mg/L)、代森锰锌(14mg/L)、代森锰锌(21mg/L)、代森锰锌+东莨菪碱(7mg/L+IC50)、代森锰锌+东莨菪碱(14mg/L+IC50)、代森锰锌+东莨菪碱(21mg/L+IC50)下的生长形态。银法利(3μL/L),银法利(6μL/L),银法利(9μL/L),银法利+东莨菪碱(3μL/L+IC50),银法利+东莨菪碱(6μL/L+IC50),银法利+东莨菪碱(9μL/L+IC50)作用下生长情况,图A-N为平板生长情况,图O-P为用CK、东莨菪碱、银法利、代森锰锌处理的致病疫霉的菌落直径。
图6是东莨菪碱诱导马铃薯块茎产生晚疫病抗性实验结果,图(A)东莨菪碱诱导薯块染病情况,图(B)是病斑面积大小占比(*P<0.05,**P<0.01,***P<0.001)。
具体实施方式
下面结合实施例对本发明作进一步说明,但并不因此而限制本发明。
下述实施例中的实验方法,如无特别说明,均为常规方法;所用生物、化学试剂如无特殊说明,均为本领域常规试剂。
1材料与方法
1.0材料
东莨菪碱(CAS登录号51-34-3)购买自杭州泓宇医药有限公司,纯度为98%,用无菌水溶解配置100g/L的母液。马铃薯(品种:青薯9号)由重庆大学植物激素与发育调控重点实验室种植与收获,选取大小相同、健康结实的块茎与无斑点、无黄变的健康叶片。致病疫霉(T30-4、88069、3928A)菌株由本实验室提供,使用固体黑麦培养基在20℃黑暗环境中培养。银法利采用的是拜耳的悬浮剂型,农药登记证号PD20120373,总有效成分含量687.5克/升,霜霉威盐酸盐含量625克/升,氟吡菌胺含量62.5克/升。
1.1东莨菪碱处理对致病疫霉生长的影响
1.1.1东莨菪碱对致病疫霉菌丝生长的影响
配制东莨菪碱浓度为0,0.6,1.2,1.8,2.4,3.0,3.6,4.0,4.2,4.8g/L的培养基。接种直径为6mm的菌块于培养基中央并培养,此后10天内每天采用十字交叉法测量菌落直径。重复3次,计算菌丝生长抑制率及半抑制浓度(IC50)。抑制率定义为:抑制率=(CK菌落直径-东莨菪碱处理后的菌落直径)/(CK菌落直径-初始菌落直径)。IC50通过软件SPSS算出。
1.1.2东莨菪碱对致病疫霉孢子萌发的影响
取致病疫霉菌块在培养基上培养10天,制成孢子悬浮液,加入0.5IC50,IC50浓度东莨菪碱,并设置对照实验,加入96孔板每孔0.2mL,在20℃下恒温培养12h镜检,观察孢子萌发情况。
1.1.3东莨菪碱对致病疫霉逆境胁迫敏感性的影响
配置以下培养基及设置不同实验环境条件:CK,东莨菪碱(IC50),NaCl(0.1mol/L),H2O2(0.2mL/L),东莨菪碱(IC50)+NaCl(0.1mol/L),东莨菪碱(IC50)+H2O2(0.2mL/L),UV(1,350Mw/mm2)10min,4℃24h,37℃2h,东莨菪碱(IC50)+UV(1,350Mw/mm2)10min,东莨菪碱(IC50)+4℃24h,东莨菪碱(IC50)+37℃2h。其中,使用的H2O2溶液的浓度是6wt%。同1.1.1进行接菌培养,9天后测量菌落直径,计算抑制率。
1.1.4东莨菪碱对致病疫霉在马铃薯块茎与叶片上的致病力影响
配制东莨菪碱浓度0,1,2,3,4g/L的培养基,接种致病疫霉培养8天。取经培养后的致病疫霉接种到相同尺寸的分别用75%乙醇和无菌水冲洗,晾干的薯块(4cm×3cm×0.6cm)与叶片上,4组重复。5天后拍照记录病斑面积。
1.1.5东莨菪碱和化学农药对致病疫霉的协同作用
分别配置如下黑麦琼脂平板:CK、东莨菪碱(IC50)、代森锰锌(7mg/L),代森锰锌(14mg/L),代森锰锌(21mg/L),代森锰锌+东莨菪碱(7mg/L+IC50),代森锰锌+东莨菪碱(14mg/L+IC50),代森锰锌+东莨菪碱(21mg/L+IC50),银法利(3μL/L),银法利(6μL/L),银法利(9μL/L),银法利+东莨菪碱(3μL/L+IC50),银法利+东莨菪碱(6μL/L+IC50),银法利+东莨菪碱(9μL/L+IC50);放入20℃培养箱中,在直径为70mm的培养基中心进行培养。培养10天后,采用交叉法测定菌落直径,每个处理重复3次。
1.2东莨菪碱诱导马铃薯产生晚疫病抗性的作用
1.2.1东莨菪碱诱导马铃薯块茎产生晚疫病抗性的作用
另取与1.1.4中薯块相同尺寸的薯块,用0,3,6,9g/L东莨菪碱水溶液对薯块进行浸泡处理1h,取出室温下晾干后接种致病疫霉菌块,设3组重复。培养9天后拍照,用ImageJ求病斑面积,计算所占面积比。病斑所占面积比=(病斑面积)÷(马铃薯面积)×100%。
2.结果与分析
2.1东莨菪碱处理对致病疫霉生长的影响
2.1.1东莨菪碱对致病疫霉菌丝生长的影响
在本实验中,东莨菪碱显著抑制了马铃薯晚疫病菌(T30-4)的菌丝生长。浓度越高,其生长越慢,并表现出DOS依赖性(图1C)。这类似于其他药物抑制T30-4的生长。通过SPSS计算,IC50为4.25g/L。东莨菪碱对其他种类的马铃薯晚疫病菌(包括88069和3928A)也有抑制作用(图1B)。表明东莨菪碱可用于防治不同生理小种马铃薯晚疫病。2.1.2东莨菪碱对致病疫霉孢子萌发的影响
设置不同倍数的IC50浓度实验组和空白对照组,按顺序处理T30-4菌株的孢子,每组重复三次,观察东莨菪碱对疫霉孢子萌发的影响。对照组中孢子萌发率组为61.43%,而用0.5IC50,IC50浓度的东莨菪碱处理后的孢子萌发率仅为16.16%,3.99%,表明东莨菪碱(IC50)很大程度上抑制了致病疫霉的孢子萌发(图2)。
2.1.3东莨菪碱对致病疫霉逆境胁迫敏感性的影响
用NaCl、H2O2、低温、高温和UV等不同的逆境条件处理T30-4,结果显示T30-4的生长受到抑制(图3C、E、G、I、K、M),表明致病疫霉对这些压力敏感。当这些逆境胁迫与东莨菪碱处理相结合时,致病疫霉对低温、H2O2、NaCl的敏感性增加,抑制效果均好于单逆境处理,但UV和高温组无明显影响(图3D、F、H、J、L、M)。这说明东莨菪碱在逆境环境下仍具有抑菌作用,此外可以增强致病疫霉对环境的敏感性,并可能在一定程度上减少晚疫病的传播。
2.1.4东莨菪碱对致病疫霉在马铃薯块茎与叶片上致病力的影响
随着东莨菪碱浓度升高,病斑面积变小,腐烂程度降低(图4A,B),当东莨菪碱的添加量达到4g/L时,致病疫霉对薯块与叶片几乎没有侵染能力(图4C,D),表明东莨菪碱能够降低致病疫霉致病力。
2.1.5东莨菪碱和化学农药对致病疫霉的协同作用
银法利(infinito)和代森锰锌(mancozeb)是控制马铃薯晚疫病的两种最常用的化学农药。化学农药银法利对马铃薯晚疫病有很好的抑制作用(图5C、G、K、O),当用该化学农药与东莨菪碱(IC50)联合处理时,与单独用该化学农药处理相比,能更好地抑制致病疫霉的菌丝生长(图5D,H,L,O)。化学杀菌剂代森锰锌也能抑制菌丝生长,但无论是单独使用还是与东莨菪碱联合使用,都不如单独使用东莨菪碱有效(图5F,I,J,K)。因此,东莨菪碱可以作为杀菌剂的添加剂,以减少杀菌剂的使用。
2.2东莨菪碱诱导马铃薯块茎产生晚疫病抗性的作用
随东莨菪碱浓度升高,病斑面积变小,颜色变浅(图6A),表明东莨菪碱可诱导马铃薯产生晚疫病抗性,当东莨菪碱浓度达到6g/L,继续提高东莨菪碱浓度并不会使薯块的抗病能力增加(图6B),说明6g/L东莨菪碱处理下其抗病能力较好。
3结论
东莨菪碱能直接抑制致病疫霉菌丝生长,还能够抑制致病疫霉孢子萌发,减弱致病疫霉致病性,且在逆境条件下对致病疫霉有抑制作用,进而有防治马铃薯晚疫病的效果。此外,东莨菪碱还能诱导马铃薯块茎与叶片抗病。因此,东莨菪碱可以作为生防治剂,以防治马铃薯晚疫病。
Claims (10)
1.东莨菪碱在抑制致病疫霉(Phytophthora infestans)中的应用。
2.东莨菪碱在防治植物晚疫病中的应用,其特征在于:所述植物为番茄或马铃薯。
3.东莨菪碱与化学农药联用在抑制致病疫霉中的应用,其特征在于:所述化学农药为双氧水或银法利。
4.东莨菪碱与化学农药联用在防治植物晚疫病中的应用,其特征在于:所述化学农药为双氧水或银法利,所述植物为番茄或马铃薯。
5.如权利要求1至4任一项所述的应用,其特征在于:东莨菪碱的使用浓度为1-9g/L。
6.如权利要求5所述的应用,其特征在于:东莨菪碱的使用浓度为1-7g/L。
7.如权利要求5所述的应用,其特征在于:东莨菪碱的使用浓度为2-6g/L或3-6g/L或4.0g/L或4.25g/L或6g/L。
8.如权利要求5至7任一项所述的应用,其特征在于:H2O2使用浓度为0.1-0.3mL/L,银法利的使用浓度为5-10μL/L。
9.如权利要求8所述的应用,其特征在于:H2O2使用浓度为0.15-0.2mL/L,银法利的使用浓度为6-9μL/L。
10.如权利要求3或4所述的应用,其特征在于:东莨菪碱与H2O2联用时,东莨菪碱与6wt%H2O2按照质量体积配比为1-9g:0.1-0.3mL进行混合配制,优选1-7g:0.1-0.3mL或2-6g:0.15-0.2mL;
东莨菪碱与银法利联用时,按照东莨菪碱与银法利质量体积配比为1-9g:5-10μL进行混合配制,优选1-7g:5-10μL或2-6g:6-9μL。
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