CN109068645B - 包含类芽孢杆菌属菌株或杀镰孢菌素和化学农药的混合物和组合物 - Google Patents
包含类芽孢杆菌属菌株或杀镰孢菌素和化学农药的混合物和组合物 Download PDFInfo
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- CN109068645B CN109068645B CN201780021646.XA CN201780021646A CN109068645B CN 109068645 B CN109068645 B CN 109068645B CN 201780021646 A CN201780021646 A CN 201780021646A CN 109068645 B CN109068645 B CN 109068645B
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
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Abstract
本发明涉及包含作为活性组分的至少一种作为类芽孢杆菌属(Paenibacillus)成员的分离的细菌菌株或其无细胞提取物或其至少一种代谢物,和至少一种化学农药的混合物。本发明还涉及包含至少一种此类细菌菌株、其全培养液或无细胞提取物或级分或其至少一种代谢物和至少一种化学农药的组合物。本发明还涉及通过应用此类组合物防治或抑制植物病原体或防止植物病原体感染的方法。本发明还涉及作为上述菌株产生的农药代谢物的杀镰孢菌素和化学农药的混合物。
Description
描述
发明领域
本发明涉及新的包含作为活性组分的至少一种作为类芽孢杆菌属(Paenibacillus)成员的分离的细菌菌株或其无细胞提取物或其至少一种代谢物,和至少一种化学农药的混合物。本发明还涉及包含至少一种此类细菌菌株、其全培养液或无细胞提取物或级分或其至少一种代谢物和至少一种化学农药的组合物。本发明还涉及通过应用此类组合物防治或抑制植物病原体或防止植物病原体感染的方法。本发明还涉及作为上述菌株产生的农药代谢物的杀镰孢菌素和化学农药的混合物。
发明背景
在防治影响植物或作物的植物病原性真菌的技术领域中,众所周知可以应用生物农药,所述生物农药例如选自对待处理的植物或作物无害的细菌,例如形成孢子的细菌,或真菌,并且所述生物防治剂可以进一步与植物病原体的经典有机化学拮抗剂组合。
已经将生物农药定义为基于微生物(细菌、真菌、病毒、线虫等)或天然产物(来自生物来源的化合物或提取物)的农药形式(U.S.Environmental Protection Agency:http://www.epa.gov/pesticides/biopesticides/)。
生物农药通常通过生长和浓缩天然产生的生物体和/或其代谢物来形成,包括细菌和其它微生物、真菌、病毒、线虫、蛋白质等。它们经常被认为是综合害物治理(IPM)项目的重要组成部分,并且作为合成的化学植物保护产品(PPP)的替代,已经受到了很多实际关注。
生物农药分成两个主要的类别,微生物农药和生物化学农药:
(1)微生物农药由细菌、真菌或病毒(并且通常包括细菌和真菌产生的代谢物)组成。昆虫病原性线虫也归为微生物农药,尽管它们是多细胞的。
(2)生物化学农药是防治害物或提供其它作物保护用途(如以下定义),但对哺乳动物相对无毒的天然物质。
为了防治植物病原性真菌,之前已经描述了几种包含形成孢子的细菌(例如枯草芽孢杆菌(Bacillus subtilis))的微生物农药,参见例如WO1998/050422;WO 2000/029426;WO 1998/50422和WO 2000/58442。
WO 2009/0126473公开了包含细菌或真菌孢子的农业上可接受的含水组合物,所述细菌或真菌孢子包含在水性/有机溶剂中,并且所述组合物可以进一步包含昆虫防治剂、农药、杀真菌剂或其组合。芽孢杆菌(Bacillus)属细菌的孢子是优选的物种。
WO 2006/017361公开了用于防治植物病原体的组合物,并且包含至少一种有益细菌、至少一种有益真菌、至少一种营养物和至少一种延长所述组合物的有效期的化合物。有益细菌组例如包含多粘类芽孢杆菌(Paenibacillus polymyxa)和坚韧类芽孢杆菌(Paenibacillus durum)的细菌。
WO 1999/059412公开了多粘类芽孢杆菌(Paenibacillus polymyxa)菌株PKB1(具有ATCC登记号202127),其具有对抗几种植物病原性真菌的活性。
WO 2011/069227公开了多粘类芽孢杆菌(P.polymyxa)菌株JB05-01-1(具有ATCC登记号PTA-10436),其具有对抗病原性细菌(主要是经食物传播的人病原性细菌)的高度抑制作用。
Raza等人(Brazilian Arch.Biol.Techol.53,1145-1154,2010;Eur.J.PlantPathol.125:471-483,2009)描述了有效对抗尖孢镰刀菌(Fusarium oxysporum)的产生杀镰孢菌素的多粘类芽孢杆菌(Paenibacillus polymyxa)菌株SQR-21。
从Microbial Research 2016(待发表,doi:10.1016/j.micres.2016.01.004)和Green Biotech Co.,Ltd.45-70Yadang-ri,Gyoha-Eup Paju Kyungki-Do,Korea(South)413-830的产品Topseed已知另一个多粘类芽孢杆菌(Paenibacillus polymyxa)菌株,称作AC-1。
又一个假定称作HY96-2的多粘类芽孢杆菌(Paenibacillus polymyxa)菌株从Biocontrol Science and Technology 24(4),426-435(2014)已知并且将按照名称KangDiLeiDe由Shanghai Zeyuan Marine Biotechnology Co.,Ltd销售。
迄今为止,已经公开了几个多粘类芽孢杆菌(Paenibacillus polymyxa)菌株的基因组:特别是菌株M-1(NCBI登记号NC_017542;J.Bacteriol.193(29),5862-63,2011;BMCMicrobiol.13,137,2013)、菌株CR1(GenBank登记号CP006941;Genome Announcements 2(1),1,2014)和菌株SC2(GenBank登记号CP002213和CP002214;NCBI登记号NC_014622;J.Bacteriol.193(1),311-312,2011),关于更多菌株,参见本文中图12的图例。多粘类芽孢杆菌(P.polymyxa)菌株M-1已经以保藏号CGMCC 7581保藏于中国普通微生物菌种保藏管理中心(CGMCC)。
在PCT申请PCT/EP2015/067925(WO 2016/020371)中,已经表征了类芽孢杆菌属(Paenibacillus)的新菌株。已经从德国的作物种植区分离所述细菌菌株Lu16774、Lu17007和Lu17015,并且依据布达佩斯条约于2013年2月20日由BASF SE(德国)保藏于德意志微生物保藏中心(Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ))。
1)以保藏号DSM 26969保藏的类芽孢杆菌属(Paenibacillus)菌株Lu16774;
2)以保藏号DSM 26970保藏的类芽孢杆菌属(Paenibacillus)菌株Lu17007;和
3)以保藏号DSM 26971保藏的类芽孢杆菌属(Paenibacillus)菌株Lu17015。
如本文中使用的术语类芽孢杆菌属(Paenibacillus)菌株与术语类芽孢杆菌属(Paenibacillus)物种菌株相同并且意指从类芽孢杆菌属(Paenibacillus)形成的细菌菌株。类芽孢杆菌属(Paenibacillus)包括类芽孢杆菌属(Paenibacillus)物种的全部物种。
在上述的PCT/EP2015/067925(WO 2016/020371)中,基于以下的形态学和生理学观察(参见PCT/EP2015/067925(WO 2016/020371)中和本文中的实施例2.3),确定了菌株Lu16774、Lu17007和Lu17015属于类芽孢杆菌属(Paenibacillus):
-杆状细胞
-椭圆形孢子
-膨胀的孢子囊
-厌氧生长
-发酵多种糖,包括葡萄糖、阿拉伯糖、木糖、甘露糖醇(mannit)、果糖、棉子糖、海藻糖和甘油,伴随酸形成
-从葡萄糖产生气体
-精氨酸二水解酶阴性
-不利用柠檬酸盐
-在5%或更多氯化钠存在下不生长
-产生降解淀粉、明胶、酪蛋白和七叶苷的胞外水解酶。
此外,还通过16S rDNA分析,依据16S rDNA中具有类芽孢杆菌属(Paenibacillus)-特异性22-碱基序列(5’至3’):
5’-TCGATACCCTTGGTGCCGAAGT-3’
(参见PCT/EP2015/067925(WO 2016/020371)和本文的序列表中的SEQ ID NO:1(核苷酸840-861)、SEQ ID NO:2(840-861)、SEQ ID NO:3(844-865)和SEQ ID NO:4(840-861)),确定这些菌株Lu16774、Lu17007和Lu17015属于类芽孢杆菌属(Paenibacillus)。
此外,与24个不同的类芽孢杆菌属(Paenibacillus)菌株比较,完整16S rDNA的测序导致了菌株Lu16774、Lu17007和Lu17015与模式菌株巴西类芽孢杆菌(Paenibacillusbrasiliensis)、胶冻样类芽孢杆菌(P.kribbensis)、杰米拉类芽孢杆菌(P.jamilae)、皮氏类芽孢杆菌(P.peoriae)和多粘类芽孢杆菌(P.polymyxa),更优选与皮氏类芽孢杆菌(P.peoriae),特别是皮氏类芽孢杆菌(Paenibacillus.peoriae)菌株BD-62的聚簇(参见PCT/EP2015/067925(WO 2016/020371)中和本文中的图1和图2)。已知,多粘类芽孢杆菌(P.polymyxa)和皮氏类芽孢杆菌(P.peoriae)具有99.6%至99.7%的16S rDNA序列同一性值(J.Gen.Appl.Microbiol.48,281-285(2002))。
两个核苷酸序列之间的“同一性百分数”或“相似性百分数”表示,在比对序列的完整长度上残基的同一性百分数,通过在比较窗口(由两个序列之间的局部比对长度定义)中比较两个最佳局部比对的序列来确定,例如,(对于十分相似的序列)借助程序AE2(比对编辑器2)人工比对后计算的同一性。两个序列之间的局部比对只包括根据标准认为足够相似的每个序列的区段,所述标准取决于用于进行比对的算法(例如AE2、BLAST、rRNA分子的二级结构等)。同一性百分数计算如下:确定两个序列中出现相同核酸的位置的数量来产生匹配位置数,匹配位置数除以比较窗口中的总位置数,并且结果乘以100。
为了确定两个核酸序列(例如表1的核苷酸序列之一及其同源物)的序列同一性百分数,为最佳比较目的而比对序列(例如为了与另一核酸最佳比对,可以在一个核酸序列中引入空位)。然后比较位于相应位置的碱基。当一个序列中的一个位置与另一序列中的相应位置被相同碱基占据时,则两个分子在该位置是相同的。可以理解,为确定序列同一性的目的,将DNA序列与RNA序列比较时,胸苷核苷酸等同于尿嘧啶核苷酸。
对于比对,将序列数据输入程序AE2(http://iubio.bio.indiana.edu/soft/ molbio/unix/ae2.readme),根据所得到的rRNA分子的二级结构人工比对,并且与属于厚壁菌门(Firmicutes)的生物的代表性16S rRNA基因序列进行比较(Nucl.Acids Res.27,171-173,1999)。为了获得多个序列的%同一性值,将所有序列彼此比对(多重序列比对)。此外,与多重比对比较,为了获得在比对序列的更长链上两序列之间的%同一性值,按照以上所述,使用AE2,进行了人工成对序列比对(成对序列比对)。
已经使用限制性酶EcoRI,将类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015与皮氏类芽孢杆菌(P.peoriae)BD-62相比,使用QualiconRiboPrintersystem,进行了标准化、自动化核糖体分型(ribotyping),获得了所有三个菌株Lu16774、Lu17007和Lu17015与皮氏类芽孢杆菌(P.peoriae)BD-62的0.24至0.5之间的相似性(参见PCT/EP2015/067925(WO 2016/020371)中和本文中的实施例2.2、图12)。发现类芽孢杆菌属(Paenibacillus)菌株Lu16774和Lu17007属于物种多粘类芽孢杆菌(Paenibacillus polymyxa)。
根据上述PCT/EP2015/067925(WO 2016/020371)(其中和本文中图12至图22)中显示的系统发生分析的结果和德国Borriss教授的未公开结果,物种多粘类芽孢杆菌(Paenibacillus polymyxa)需要新的分类学分类,分成两个亚种:1)多粘类芽孢杆菌多粘亚种(Paenibacillus polymyxa ssp.polymyxa)和2)多粘类芽孢杆菌植物亚种(Paenibacillus polymyxa ssp.plantarum)和3)新物种芽孢杆菌附生新物种(Paenibacillus nov.spec.epiphyticus)。
模式菌株多粘类芽孢杆菌(P.polymyxa)DSM 36,与多粘类芽孢杆菌(P.polymyxa)菌株SQR-21、CF05、CICC 10580、NRRL B-30509和A18一起,在针对五个保守的管家基因(dnaN、gyrB、recA、recN和rpoA)分析的最大可能性树状图的每一个中,形成分开的聚簇(PCT/EP2015/067925(WO 2016/020371)和本文中的图17-21)。
通过测定平均氨基酸同一性(AAI)获得了非常相似的结果,平均氨基酸同一性常常用于确定细菌物种之间的系统发生关系。该方法基于氨基酸水平上核心基因组的平均同一性的计算(Proc.Natl.Acad.USA 102,2567-2572,2005)。根据PCT/EP2015/067925(WO2016/020371)和本文中的图22中得到的AAI-矩阵,多粘类芽孢杆菌(P.polymyxa)DSM 36与多粘类芽孢杆菌(P.polymyxa)SQR-21菌株一起形成亚聚簇,不同于其中所示的另两个亚聚簇。
菌株Lu16674和Lu17007与菌株多粘类芽孢杆菌(P.polymyxa)M-1、1-43、SC2和Sb3-1一起,在针对五个保守的管家基因(dnaN、gyrB、recA、recN和rpoA)分析的最大可能性树状图的每一个中,形成第二亚聚簇(图17-21)。根据PCT/EP2015/067925(WO 2016/020371)和本文中的图22中基于核心基因组分析的AAI-矩阵,这个第二亚聚簇通过其代表性菌株Lu16674和Lu17007与多粘类芽孢杆菌(P.polymyxa)M-1和SC2菌株一起得到确认。
发现两个亚聚簇之间的差异未显著到确立新的物种,但两个聚簇的代表菌株之间的AAI同一性水平约为97.5%,从而说明分成两个分开的亚种是合理的。
因此在PCT/EP2015/067925(WO 2016/020371)中提出,根据模式多粘类芽孢杆菌(P.polymyxa)菌株DSM 36T将第一亚聚簇命名为多粘类芽孢杆菌多粘亚种(Paenibacilluspolymyxa ssp.polymyxa)。除了菌株DSM 36,多粘类芽孢杆菌(P.polymyxa)菌株SQR-21、CF05、CICC 10580、NRRL B-30509和A18应当也属于多粘类芽孢杆菌多粘亚种(Paenibacillus polymyxa ssp.polymyxa)。
此外提出,将第二亚聚簇命名为新的亚种多粘类芽孢杆菌植物亚种(Paenibacillus polymyxa ssp.plantarum)。除了菌株Lu16674和Lu17007,多粘类芽孢杆菌(P.polymyxa)菌株M-1、1-43、SC2和Sb3-1也应当属于多粘类芽孢杆菌植物亚种(Paenibacillus polymyxa ssp.plantarum)。
在核心基因组的基因中,菌株Lu17015与模式菌株多粘类芽孢杆菌(Paenibacillus polymyxa)DSM36=ATCC 842只具有94.9%AAI同一性(PCT/EP2015/067925(WO 2016/020371)和本文中的图22)。因此,菌株Lu17015不能指定为物种多粘类芽孢杆菌(Paenibacillus polymyxa),也不能指定为任何其它已知的类芽孢杆菌属(Paenibacillus)物种。对于菌株E681(94.7%)和CR2(94.9%),发现了相似的值。这三个菌株彼此之间具有至少98.1%同一性(AAI)。根据Konstantinides和Tiedje的物种定义(ProcNatl.Acad.Sci.USA.102,2567-2572,2005),菌株Lu17015以及菌株E681和CR2可以被指定为新的物种。因此,在PCT/EP2015/067925(WO2016/020371)中提出一个新物种类芽孢杆菌附生新物种(Paenibacillus nov.spec.epiphyticus)。因此,菌株Lu17015属于附生类芽孢杆菌(Paenibacillus epiphyticus)。提出了所述菌株应当是模式菌株。同样,基于五个管家基因的序列比较的树状图(PCT/EP2015/067925(WO 2016/020371)中和本文中的图17-21)显示,这个聚簇远离所有其它多粘类芽孢杆菌(P.polymyxa)菌株。除了Lu17015,提出多粘类芽孢杆菌(P.polymyxa)菌株E681、CR2、TD94、DSM 365和WLY78也应当属于类芽孢杆菌附生新物种(Paenibacillus nov.spec.epiphyticus)。
已知类芽孢杆菌属(Paenibacillus)产生许多抗生素代谢物,它们是脂肽例如多粘菌素、八肽菌素、多肽菌素、pelgipeptin和杀镰孢菌素。杀镰孢菌素是从类芽孢杆菌属(Paenibacillus)物种分离的来自环状脂缩酚酸肽(lipodepsipeptides)类别的一组抗生素,其经常共有以下结构特征:由6个氨基酸残基组成的大环,其中三个氨基酸残基是L-Thr、D-allo-Thr和D-Ala,以及通过酰胺键连接N-末端L-Thr残基的15-胍基-3-羟基十五酸尾部(ChemMedChem 7,871-882,2012;J.Microbiol.Meth.85,175-182,2011,其中表1)。这些化合物通过N-末端L-Thr羟基和C-末端D-Ala羰基之间的内酯桥环化。缩酚酸肽(depsipeptide)环内氨基酸残基的位置通常从上述L-Thr(其自身也携带GHPD链)开始编号,并且结束于C-末端D-Ala。从类芽孢杆菌属(Paenibacillus)分离的杀镰孢菌素的非限制性实例称为LI-F03、LI-F04、LI-F05、LI-F07和LI-F08(J.Antibiotics 40(11),1506-1514,1987;Heterocycles 53(7),1533-1549,2000;Peptides 32,1917-1923,2011)以及杀镰孢菌素A(也称为LI-F04a)、杀镰孢菌素B(也称为LI-F04b)、杀镰孢菌素C(也称为LI-F03a)和杀镰孢菌素D(也称为LI-F03b)(J.Antibiotics 49(2),129-135,1996;J.Antibiotics 50(3),220-228,1997)。杀镰孢菌素的氨基酸链不是通过核糖体产生的,而是通过非核糖体肽合成酶产生的。已知的杀镰孢菌素的结构式显示于表1中(BiotechnolLett.34,1327-1334,2012;其中的图1)。称为LI-F03a、LI-F03b直至LI-F08a和LI-F08b的化合物和如本文所述式I和式I.1的杀镰孢菌素,因其在杀镰孢菌素家族内的结构(例如,参见表1),本文中也称为杀镰孢菌素LI-F03a、LI-F03b直至LI-F08a和LI-F08b。
在分离的杀镰孢菌素抗生素中,杀镰孢菌素A已经显示出最有前景的抗微生物活性,对抗多种临床上相关的真菌和革兰氏阳性细菌,例如金黄色葡萄球菌(Staphylococcusaureus)(MIC值范围:0.78-3.12μg/mL)(ChemMedChem 7,871-882,2012)。已经建立了含有替代天然存在的GHPD的12-胍基-十二烷酸(12-GDA)或12-氨基-十二烷酸(12-ADA)的杀镰孢菌素类似物的合成,但是12-ADA替代GHPD导致抗微生物活性的完全失去,而12-GDA替代GHPD保留了抗微生物活性(Tetrahedron Lett.47,8587-8590,2006;ChemMedChem 7,871-882,2012)。
表1:杀镰孢菌素家族的结构
其中箭头定义在GHPD的羰基部分和L-Thr(L-苏氨酸)的氨基之间或者在一个氨基酸的羰基和相邻氨基酸的氨基之间的单个(酰胺)键,其中箭头的尖端表示连接到所述氨基酸L-Thr或所述相邻氨基酸的氨基;并且
其中不带箭头的单线定义D-Ala(D-丙氨酸)的羰基和L-Thr的羟基之间的单个(酯)键;并且其中GHPD是15-胍基-3-羟基十五烷酸。
*在这两种未公开的PCT申请PCT/EP2015/067925(WO 2016/020371)中已知的杀镰孢菌素1A和1B的情况下,尚未阐明环状肽的六个氨基酸的立体构型。
也报道了杀镰孢菌素A、B、C和D抑制植物病原性真菌,例如尖孢镰刀菌(Fusariumoxysporum)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)和托姆青霉(Penicillus thomii)(J.Antibiotics 49(2),129-135,1996;J.Antibiotics 50(3),220-228,1997)。已经发现杀镰孢菌素例如Li-F05、LI-F07和LI-F08具有对抗多种植物病原性真菌例如串珠镰刀菌(Fusarium moniliforme)、尖孢镰刀菌(F.oxysporum)、粉红镰刀菌(F.roseum)、藤仓赤霉(Giberella fujkuroi)、芝麻长蠕孢(Helminthosporium sesamum)和扩展青霉(Penicillium expansum)的一些抗真菌活性(J.Antibiotics 40(11),1506-1514,1987)。杀镰孢菌素还具有对抗革兰氏阳性细菌包括金黄色葡萄球菌(Staphylococcus aureus)的抗细菌活性(J.Antibiotics 49,129-135,1996;J.Antibiotics 50,220-228,1997)。此外,杀镰孢菌素具有对抗斑点小球腔菌(Leptosphaeria maculans)的抗真菌活性(Can.J.Microbiol.48,159-169,2002),斑点小球腔菌(Leptosphaeria maculans)引起油菜(canola)的根黑腐病。此外,发现某些类芽孢杆菌属(Paenibacillus)菌株产生的杀镰孢菌素A和B及其相关的两种化合物(其中D-别-Thr通过其羟基使用酯桥与另外的丙氨酸键合),在培养的欧芹细胞中诱导抗性反应并且抑制尖孢镰刀菌(Fusarium oxysporum)的生长(WO 2006/016558;EP 1 788 074 A1)。
WO 2007/086645描述了从多粘类芽孢杆菌(Paenibacillus polymyxa)菌株E681分离的杀镰孢菌素合成酶及其编码基因,该酶参与杀镰孢菌素A、B、C、D、LI-F03、LI-F04、LI-F05、LI-F07和LI-F08的合成。
在上述的PCT/EP2015/067925(WO 2016/020371)中,发现细菌菌株Lu16774、Lu17007和Lu17015的全培养液、培养物基质和无细胞提取物显示特别对抗链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)和致病疫霉(Phytophthora infestans)的抑制活性。这些菌株的有机提取物的生物活性导向分离导致了两种新的杀镰孢菌素型化合物(本文中称作杀镰孢菌素1A和1B)的分离,其结构通过1D-和2D-NMR谱以及质谱进行了阐明。
已经在多种作物上建立了用于对抗作物疾病的生物农药。例如,生物农药已经在防治霜霉病中起着重要作用。它们的益处包括:0-天安全间隔期(Pre-Harvest Interval)以及能够用于中度至重度疾病压力下。
然而,生物农药在一些条件下也会具有缺陷,例如特异性高(要求害物/病原体的准确识别和使用多重产品)、作用速度慢(因此如果作物受到害物爆发的即时威胁,其是不适合的)、由于多种生物和非生物因素的影响而功效不定(因为生物农药通常是活的生物体,其通过在靶标昆虫害物/病原体内繁殖来实现害物/病原体防治)和抗性产生。
农业实践经验已经显示,防治有害真菌或昆虫或其它害物时反复和排他性施加单个活性组分在许多情况下导致快速选出那些已经对所讨论活性组分形成天然或适应性抵抗力的真菌菌株或害物分离株。随后不再可能用所讨论的活性组分有效防治这些真菌或害物。
为了降低选出抗性真菌菌株或昆虫分离株的风险,如今不同活性组分的混合物常规地用于防治有害真菌或昆虫或其它害物。通过组合有农药活性的化合物和/或具有不同作用机制的生物农药,可以在相对长的时间范围内确保成功防治。
本发明的目的是克服上述缺点并且为了以尽可能低的应用速率有效管理抗性及有效防治植物病原性有害真菌、昆虫或其它害物或有效调节植物生长,提供这样的组合物,其中在减少所应用活性化合物的总量时,所述组合物对有害真菌或害物具有改善的活性或改善的植物生长调节活性(协同性混合物)和拓宽的活性谱,特别对某些适应症而言。
害物防治领域中出现的一个常见问题在于需要减少活性成分的剂量率以减少或避免不利的环境作用或毒理作用,同时仍然允许有效防治害物。就本发明而言,术语害物包括动物害物和有害真菌。
因此,本发明的目的是提供农药混合物,所述农药混合物解决减少剂量率和/或增强活性谱和/或敲减活性与防治延长组合和/或抗性管理和/或促进(增进)植物健康的问题。
发明描述
因此,我们已经发现借助本文中定义的混合物和组合物实现这个目的,所述混合物和组合物包含至少一种类芽孢杆菌属(Paenibacillus)细菌菌株(类芽孢杆菌属(Paenibacillus)菌株)或其无细胞提取物或其至少一种代谢物以及如本文定义的化学农药。
因此,本发明涉及混合物,该混合物包含作为活性组分的
1)至少一种类芽孢杆菌属(Paenibacillus)菌株、其培养物基质或无细胞提取物或其至少一种代谢物;
和
2)至少一种选自组A)至N)的农药II:
A)呼吸抑制剂
-Qo位点的复合体III抑制剂:腈嘧菌酯(azoxystrobin)(A.1.1)、甲香菌酯(coumethoxystrobin)(A.1.2)、丁香菌酯(coumoxystrobin)(A.1.3)、醚菌胺(dimoxystrobin)(A.1.4)、烯肟菌酯(enestroburin)(A.1.5)、烯肟菌胺(fenaminstrobin)(A.1.6)、氟菌螨酯(fenoxystrobin/flufenoxystrobin)(A.1.7)、氟嘧菌酯(fluoxastrobin)(A.1.8)、亚胺菌(kresoxim-methyl)(A.1.9)、甲哌喹(mandestrobin)(A.1.10)、叉氨苯酰胺(metominostrobin)(A.1.11)、肟醚菌胺(orysastrobin)(A.1.12)、啶氧菌酯(picoxystrobin)(A.1.13)、唑菌胺酯(pyraclostrobin)(A.1.14)、唑胺菌酯(pyrametostrobin)(A.1.15)、唑菌酯(pyraoxystrobin)(A.1.16)、肟菌酯(trifloxystrobin)(A.1.17)、2-(2-(3-(2,6-二氯苯基)-1-甲基-亚烯丙基氨基氧基甲基)-苯基)-2-甲氧基亚氨基-N-甲基-乙酰胺(A.1.18)、吡菌苯威(pyribencarb)(A.1.19)、氯啶菌酯(triclopyricarb/chlorodincarb)(A.1.20)、唑酮菌(famoxadone)(A.1.21)、咪唑菌酮(fenamidone)(A.1.21)、甲基-N-[2-[(1,4-二甲基-5-苯基-吡唑-3-基)氧基甲基]苯基]-N-甲氧基-氨基甲酸酯(A.1.22)、1-[3-氯-2-[[1-(4-氯苯基)-1H-吡唑-3-基]氧基甲基]苯基]-4-甲基-四唑-5-酮(A.1.23)、1-[3-溴-2-[[1-(4-氯苯基)吡唑-3-基]氧基甲基]苯基]-4-甲基-四唑-5-酮(A.1.24)、1-[2-[[1-(4-氯苯基)吡唑-3-基]氧基甲基]-3-甲基-苯基]-4-甲基-四唑-5-酮(A.1.25)、1-[2-[[1-(4-氯苯基)吡唑-3-基]氧基甲基]-3-氟-苯基]-4-甲基-四唑-5-酮(A.1.26)、1-[2-[[1-(2,4-二氯苯基)吡唑-3-基]氧基甲基]-3-氟-苯基]-4-甲基-四唑-5-酮(A.1.27)、1-[2-[[4-(4-氯苯基)噻唑-2-基]氧基甲基]-3-甲基-苯基]-4-甲基-四唑-5-酮(A.1.28)、1-[3-氯-2-[[4-(对甲苯基)噻唑-2-基]氧基甲基]苯基]-4-甲基-四唑-5-酮(A.1.29)、1-[3-环丙基-2-[[2-甲基-4-(1-甲基吡唑-3-基)苯氧基]甲基]苯基]-4-甲基-四唑-5-酮(A.1.30)、1-[3-(二氟甲氧基)-2-[[2-甲基-4-(1-甲基吡唑-3-基)苯氧基]甲基]苯基]-4-甲基-四唑-5-酮(A.1.31)、1-甲基-4-[3-甲基-2-[[2-甲基-4-(1-甲基吡唑-3-基)苯氧基]甲基]苯基]四唑-5-酮(A.1.32)、1-甲基-4-[3-甲基-2-[[1-[3-(三氟甲基)苯基]-亚乙基氨基]氧基甲基]苯基]四唑-5-酮(A.1.33)、(Z,2E)-5-[1-(2,4-二氯苯基)吡唑-3-基]-氧基-2-甲氧基亚氨基-N,3-二甲基-戊-3-烯酰胺(A.1.34)、(Z,2E)-5-[1-(4-氯苯基)吡唑-3-基]氧基-2-甲氧基亚氨基-N,3-二甲基-戊-3-烯酰胺(A.1.35)、嘧螨胺(pyriminostrobin)(A.1.36)、吡氟菌酯(bifujunzhi)(A.1.37)、2-(邻((2,5-二甲基苯基-氧基亚甲基)苯基)-3-甲氧基-丙烯酸甲基酯(A.1.38);
-Qi位点的复合体III抑制剂:氰霜唑(cyazofamid)(A.2.1)、吲唑磺菌胺(amisulbrom)(A.2.2)、2-甲基丙酸[(6S,7R,8R)-8-苄基-3-[(3-羟基-4-甲氧基-吡啶-2-羰基)氨基]-6-甲基-4,9-二氧代-1,5-二氧壬环-7-基]酯(A.2.3)、2-甲基丙酸[2-[[(7R,8R,9S)-7-苄基-9-甲基-8-(2-甲基丙酰基氧基)-2,6-二氧代-1,5-二氧壬环-3-基]氨基甲酰基]-4-甲氧基-3-吡啶基]氧基甲基酯(A.2.4)、2-甲基丙酸[(6S,7R,8R)-8-苄基-3-[[4-甲氧基-3-(丙酰基氧基甲氧基)吡啶-2-羰基]氨基]-6-甲基-4,9-二氧代-1,5-二氧壬环-7-基]酯(A.2.5);
-复合体II抑制剂:麦锈灵(benodanil)(A.3.1)、苯并烯氟菌唑(benzovindiflupyr)(A.3.2)、联苯吡菌胺(bixafen)(A.3.3)、啶酰菌胺(boscalid)(A.3.4)、萎锈灵(carboxin)(A.3.5)、呋菌胺(fenfuram)(A.3.6)、氟吡菌酰胺(fluopyram)(A.3.7)、氟酰胺(flutolanil)(A.3.8)、氟唑菌酰胺(fluxapyroxad)(A.3.9)、呋吡唑灵(furametpyr)(A.3.10)、isofetamid(A.3.11)、吡唑萘菌胺(isopyrazam)(A.3.12)、丙氧灭绣胺(mepronil)(A.3.13)、氧化萎锈灵(oxycarboxin)(A.3.14)、氟唑菌苯胺(penflufen)(A.3.15)、吡噻菌胺(penthiopyrad)(A.3.16)、氟唑环菌胺(sedaxane)(A.3.19)、叶枯酞(tecloftalam)(A.3.20)、溴氟唑菌(thifluzamide)(A.3.21)、3-(二氟甲基)-1-甲基-N-(1,1,3-三甲基茚满-4-基)吡唑-4-甲酰胺(A.3.22)、3-(三氟甲基)-1-甲基-N-(1,1,3-三甲基茚满-4-基)吡唑-4-甲酰胺(A.3.23)、1,3-二甲基-N-(1,1,3-三甲基茚满-4-基)吡唑-4-甲酰胺(A.3.24)、3-(三氟甲基)-1,5-二甲基-N-(1,1,3-三甲基茚满-4-基)吡唑-4-甲酰胺(A.3.25)、1,3,5-三甲基-N-(1,1,3-三甲基茚满-4-基)吡唑-4-甲酰胺(A.3.26)、3-(二氟甲基)-1,5-二甲基-N-(1,1,3-三甲基茚满-4-基)吡唑-4-甲酰胺(A.3.27)、3-(二氟甲基)-N-(7-氟-1,1,3-三甲基-茚满-4-基)-1-甲基-吡唑-4-甲酰胺(A.3.28)、(E)-2-[2-[(5-氰基-2-甲基-苯氧基)甲基]苯基]-3-甲氧基-丙-2-烯酸甲基酯(A.3.30);
-其它呼吸抑制剂:氟嘧菌胺(diflumetorim)(A.4.1);硝基苯基衍生物:乐杀螨(binapacryl)(A.4.2)、敌螨通(dinobuton)(A.4.3)、敌螨普(dinocap)(A.4.4)、氟啶胺(fluazinam)(A.4.5);嘧菌腙(ferimzone)(A.4.7);有机金属化合物:三苯基锡盐,例如薯瘟锡(fentin-acetate)(A.4.8)、三苯基锡氯(fentin chloride)(A.4.9)或毒菌锡(fentinhydroxide)(A.4.10);唑嘧菌胺(ametoctradin)(A.4.11);硅噻菌胺(silthiofam)(A.4.12);
B)甾醇生物合成抑制剂(SBI杀真菌剂)
-C14脱甲基酶抑制剂:三唑:戊环唑(azaconazole)(B.1.1)、双苯三唑醇(bitertanol)(B.1.2)、糠菌唑(bromuconazole)(B.1.3)、环唑醇(cyproconazole)(B.1.4)、醚唑(difenoconazole)(B.1.5)、烯唑醇(diniconazole)(B.1.6)、精烯唑醇(diniconazole-M)(B.1.7)、氧唑菌(epoxiconazole)(B.1.8)、腈苯唑(fenbuconazole)(B.1.9)、喹唑菌酮(fluquinconazole)(B.1.10)、氟硅唑(flusilazole)(B.1.11)、粉唑醇(flutriafol)(B.1.12)、己唑醇(hexaconazole)(B.1.13)、酰胺唑(imibenconazole)(B.1.14)、环戊唑醇(ipconazole)(B.1.15)、环戊唑菌(metconazole)(B.1.17)、腈菌唑(myclobutanil)(B.1.18)、咪唑(oxpoconazole)(B.1.19)、多效唑(paclobutrazole)(B.1.20)、戊菌唑(penconazole)(B.1.21)、丙环唑(propiconazole)(B.1.22)、丙硫菌唑(prothioconazole)(B.1.23)、硅氟唑(simeconazole)(B.1.24)、戊唑醇(tebuconazole)(B.1.25)、氟醚唑(tetraconazole)(B.1.26)、三唑酮(triadimefon)(B.1.27)、唑菌醇(triadimenol)(B.1.28)、戊叉唑菌(triticonazole)(B.1.29)、烯效唑(uniconazole)(B.1.30)、1-[rel-(2S;3R)-3-(2-氯苯基)-2-(2,4-二氟苯基)-氧杂环丙烷基甲基]-5-氰硫基-1H-[1,2,4]三唑(B.1.31)、2-[rel-(2S;3R)-3-(2-氯苯基)-2-(2,4-二氟苯基)-氧杂环丙烷基甲基]-2H-[1,2,4]三唑-3-硫醇(B.1.32)、2-[2-氯-4-(4-氯苯氧基)苯基]-1-(1,2,4-三唑-1-基)戊-2-醇(B.1.33)、1-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-环丙基-2-(1,2,4-三唑-1-基)乙醇(B.1.34)、2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1,2,4-三唑-1-基)丁-2-醇(B.1.35)、2-[2-氯-4-(4-氯苯氧基)苯基]-1-(1,2,4-三唑-1-基)丁-2-醇(B.1.36)、2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-3-甲基-1-(1,2,4-三唑-1-基)丁-2-醇(B.1.37)、2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1,2,4-三唑-1-基)丙-2-醇(B.1.38)、2-[2-氯-4-(4-氯苯氧基)苯基]-3-甲基-1-(1,2,4-三唑-1-基)丁-2-醇(B.1.39)、2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1,2,4-三唑-1-基)戊-2-醇(B.1.40)、2-[4-(4-氟苯氧基)-2-(三氟甲基)苯基]-1-(1,2,4-三唑-1-基)丙-2-醇(B.1.41)、2-[2-氯-4-(4-氯苯氧基)苯基]-1-(1,2,4-三唑-1-基)戊-3-炔-2-醇(B.1.42)、2-(氯甲基)-2-甲基-5-(对甲苯基甲基)-1-(1,2,4-三唑-1-基甲基)环戊醇(B.1.43);咪唑:烯菌灵(imazalil)(B.1.44)、稻瘟酯(pefurazoate)(B.1.45)、丙氯灵(prochloraz)(B.1.46)、氟菌唑(triflumizol)(B.1.47);嘧啶、吡啶和哌嗪:异嘧菌醇(fenarimol)(B.1.49)、啶斑肟(pyrifenox)(B.1.50)、嗪氨灵(triforine)(B.1.51)、[3-(4-氯-2-氟-苯基)-5-(2,4-二氟苯基)异唑-4-基]-(3-吡啶基)甲醇(B.1.52);
-δ14-还原酶抑制剂:4-十二烷基-2,6-二甲基吗啉(aldimorph)(B.2.1)、吗菌灵(dodemorph)(B.2.2)、吗菌灵乙酸盐(dodemorph-acetate)(B.2.3)、丁苯吗啉(fenpropimorph)(B.2.4)、克啉菌(tridemorph)(B.2.5)、苯锈啶(fenpropidin)(B.2.6)、粉病灵(piperalin)(B.2.7)、螺茂胺(spiroxamine)(B.2.8);
-3-酮基还原酶抑制剂:环酰菌胺(fenhexamid)(B.3.1);
-其它甾醇生物合成抑制剂:氯苯肟唑(chlorphenomizole)(B.4.1);
C)核酸合成抑制剂
-苯基酰胺或酰基氨基酸杀真菌剂:苯霜灵(benalaxyl)(C.1.1)、精苯霜灵(benalaxyl-M)(C.1.2)、精苯霜灵(kiralaxyl)(C.1.3)、甲霜灵(metalaxyl)(C.1.4)、精甲霜灵(metalaxyl-M)(C.1.5)、甲呋酰胺(ofurace)(C.1.6)、霜灵(oxadixyl)(C.1.7);
-其它核酸合成抑制剂:土菌消(hymexazole)(C.2.1)、异噻菌酮(octhilinone)(C.2.2)、喹菌酮(oxolinic acid)(C.2.3)、磺嘧菌灵(bupirimate)(C.2.4)、5-氟胞嘧啶(C.2.5)、5-氟-2-(对甲苯基甲氧基)嘧啶-4-胺(C.2.6)、5-氟-2-(4-氟苯基甲氧基)嘧啶-4-胺(C.2.7)、5-氟-2-(4-氯苯基甲氧基)嘧啶-4-胺(C.2.8);
D)细胞分裂和细胞骨架抑制剂
-微管蛋白抑制剂:苯菌灵(benomyl)(D.1.1)、多菌灵(carbendazim)(D.1.2)、麦穗宁(fuberidazole)(D.1.3)、涕必灵(thiabendazole)(D.1.4)、甲基托布津(thiophanate-methyl)(D.1.5)、3-氯-4-(2,6-二氟苯基)-6-甲基-5-苯基-哒嗪(D.1.6)、3-氯-6-甲基-5-苯基-4-(2,4,6-三氟苯基)哒嗪(D.1.7)、N-乙基-2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]丁酰胺(D.1.8)、N-乙基-2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]-2-甲硫基-乙酰胺(D.1.9)、2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]-N-(2-氟乙基)丁酰胺(D.1.10)、2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]-N-(2-氟乙基)-2-甲氧基-乙酰胺(D.1.11)、2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]-N-丙基-丁酰胺(D.1.12)、2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]-2-甲氧基-N-丙基-乙酰胺(D.1.13)、2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]-2-甲硫基-N-丙基-乙酰胺(D.1.14)、2-[(3-乙炔基-8-甲基-6-喹啉基)氧基]-N-(2-氟乙基)-2-甲硫基-乙酰胺(D.1.15)、4-(2-溴-4-氟-苯基)-N-(2-氯-6-氟-苯基)-2,5-二甲基-吡唑-3-胺(D.1.16);
-其它细胞分裂抑制剂:乙霉威(diethofencarb)(D.2.1)、噻唑菌胺(ethaboxam)(D.2.2)、戊菌隆(pencycuron)(D.2.3)、氟吡菌胺(fluopicolide)(D.2.4)、苯酰菌胺(zoxamide)(D.2.5)、苯菌酮(metrafenone)(D.2.6)、焦磷酸烯酮(pyriofenone)(D.2.7);
E)氨基酸和蛋白质合成抑制剂
-甲硫氨酸合成抑制剂:环丙嘧啶(cyprodinil)(E.1.1)、嘧菌胺(mepanipyrim)(E.1.2)、二甲嘧菌胺(pyrimethanil)(E.1.3);
-蛋白质合成抑制剂:灭瘟素(blasticidin-S)(E.2.1)、春雷素(kasugamycin)(E.2.2)、春雷霉素盐酸盐水合物(kasugamycin hydrochloride-hydrate)(E.2.3)、米多霉素(mildiomycin)(E.2.4)、链霉素(streptomycin)(E.2.5)、土霉素(oxytetracyclin)(E.2.6);
F)信号转导抑制剂
-MAP/组氨酸激酶抑制剂:唑呋草(fluoroimid)(F.1.1)、异丙定(iprodione)(F.1.2)、杀菌利(procymidone)(F.1.3)、烯菌酮(vinclozolin)(F.1.4)、咯菌腈(fludioxonil)(F.1.5);
-G蛋白抑制剂:喹氧灵(quinoxyfen)(F.2.1);
G)脂质和膜合成抑制剂
-磷脂生物合成抑制剂:克瘟散(edifenphos)(G.1.1)、异稻瘟净(iprobenfos)(G.1.2)、定菌磷(pyrazophos)(G.1.3)、稻瘟灵(isoprothiolane)(G.1.4);
-脂质过氧化:氯硝胺(dicloran)(G.2.1)、五氯硝基苯(quintozene)(G.2.2)、四氯硝基苯(tecnazene)(G.2.3)、甲基立枯磷(tolclofos-methyl)(G.2.4)、联苯(G.2.5)、地茂散(chloroneb)(G.2.6)、氯唑灵(etridiazole)(G.2.7);
-磷脂生物合成和细胞壁沉积:烯酰吗啉(dimethomorph)(G.3.1)、氟吗啉(flumorph)(G.3.2)、双炔酰菌胺(mandipropamid)(G.3.3)、丁吡吗啉(pyrimorph)(G.3.4)、苯噻菌胺(benthiavalicarb)(G.3.5)、异丙菌胺(iprovalicarb)(G.3.6)、缬氨菌酯(valifenalate)(G.3.7);
-影响细胞膜渗透性的化合物和脂肪酸:百维灵(propamocarb)(G.4.1);
-氧甾醇结合蛋白抑制剂:氟噻唑吡乙酮(oxathiapiprolin)(G.5.1)、2-{3-[2-(1-{[3,5-双(二氟甲基-1H-吡唑-1-基]乙酰基}哌啶-4-基)-1,3-噻唑-4-基]-4,5-二氢-1,2-唑-5-基}苯基甲磺酸酯(G.5.2)、2-{3-[2-(1-{[3,5-双(二氟甲基)-1H-吡唑-1-基]乙酰基}哌啶-4-基)-1,3-噻唑-4-基]-4,5-二氢-1,2-唑-5-基}-3-氯苯基甲磺酸酯(G.5.3);
H)具有多位点作用的抑制剂
-无机活性物质:波尔多液(Bordeaux mixture)(H.1.1)、铜(H.1.2)、乙酸铜(H.1.3)、氢氧化铜(H.1.4)、氯氧化铜(H.1.5)、碱式硫酸铜(H.1.6)、硫(H.1.7);
-硫代-和二硫代氨基甲酸酯:福美铁(ferbam)(H.2.1)、代森锰锌(mancozeb)(H.2.2)、代森锰(maneb)(H.2.3)、威百亩(metam)(H.2.4)、代森联(metiram)(H.2.5)、甲基代森锌(propineb)(H.2.6)、福美双(thiram)(H.2.7)、代森锌(zineb)(H.2.8)、福美锌(ziram)(H.2.9);
-有机氯化合物:敌菌灵(anilazine)(H.3.1)、百菌清(chlorothalonil)(H.3.2)、敌菌丹(captafol)(H.3.3)、克菌丹(captan)(H.3.4)、灭菌丹(folpet)(H.3.5)、抑菌灵(dichlofluanid)(H.3.6)、菌霉净(dichlorophen)(H.3.7)、六氯苯(H.3.8)、五氯酚(H.3.9)及其盐、四氯苯酞(phthalide)(H.3.10)、对甲抑菌灵(tolylfluanid)(H.3.11);
-胍和其它:胍(H.4.1)、多果定(dodine)(H.4.2)、多果定游离碱(dodine freebase)(H.4.3)、双胍盐(guazatine)(H.4.4)、双胍乙酸盐(guazatine-acetate)(H.4.5)、双胍辛胺(iminoctadine)(H.4.6)、双胍辛胺三乙酸盐(iminoctadine-triacetate)(H.4.7)、双胍三辛烷基苯磺酸盐(iminoctadine-tris(albesilate))(H.4.8)、二噻农(dithianon)(H.4.9)、2,6-二甲基-1H,5H-[1,4]二噻二烯并(dithiino)[2,3-c:5,6-c’]二吡咯-1,3,5,7(2H,6H)-四酮(H.4.10);
I)细胞壁合成抑制剂
-葡聚糖合成抑制剂:有效霉素(validamycin)(I.1.1)、多氧霉素(polyoxin)B(I.1.2);
-黑色素合成抑制剂:咯喹酮(pyroquilon)(I.2.1)、三环唑(tricyclazole)(I.2.2)、氯环丙酰胺(carpropamid)(I.2.3)、双氯氰菌胺(dicyclomet)(I.2.4)、氰菌胺(fenoxanil)(I.2.5);
J)植物防御诱导剂
-噻二唑素(acibenzolar-S-methyl)(J.1.1)、噻菌灵(probenazole)(J.1.2)、异噻菌胺(isotianil)(J.1.3)、噻酰菌胺(tiadinil)(J.1.4)、调环酸钙(prohexadione-calcium)(J.1.5);膦酸盐:藻菌磷(fosetyl)(J.1.6)、藻菌磷(fosetyl-aluminum)(J.1.7)、亚磷酸及其盐(J.1.8)、碳酸氢钾或碳酸氢钠(J.1.9);
K)未知作用模式
-溴硝丙二醇(bronopol)(K.1.1)、灭螨猛(chinomethionat)(K.1.2)、环氟菌胺(cyflufenamid)(K.1.3)、清菌脲(cymoxanil)(K.1.4)、棉隆(dazomet)(K.1.5)、双乙氧咪菌威(debacarb)(K.1.6)、双氯氰菌胺(diclocymet)(K.1.7)、哒菌清(diclomezine)(K.1.8)、野燕枯(difenzoquat)(K.1.9)、野燕枯甲基硫酸酯(difenzoquat-methylsulfate)(K.1.10)、二苯胺(K.1.11)、种衣酯(fenitropan)(K.1.12)、胺苯吡菌酮(fenpyrazamine)(K.1.13)、氟联苯菌(flumetover)(K.1.14)、磺菌胺(flusulfamide)(K.1.15)、氟噻菌净(flutianil)(K.1.16)、harpin(K.1.17)、磺菌威(methasulfocarb)(K.1.18)、氯定(nitrapyrin)(K.1.19)、异丙消(nitrothal-isopropyl)(K.1.20)、三氟甲氧威(tolprocarb)(K.1.21)、喹啉铜(oxin-copper)(K.1.22)、丙氧喹啉(proquinazid)(K.1.23)、异丁乙氧喹啉(tebufloquin)(K.1.24)、叶枯酞(tecloftalam)(K.1.25)、唑菌嗪(triazoxide)(K.1.26)、N’-(4-(4-氯-3-三氟甲基-苯氧基)-2,5-二甲基-苯基)-N-乙基-N-甲基甲脒(K.1.27)、N’-(4-(4-氟-3-三氟甲基-苯氧基)-2,5-二甲基-苯基)-N-乙基-N-甲基甲脒(K.1.28)、N’-[4-[[3-[(4-氯苯基)甲基]-1,2,4-噻二唑-5-基]氧基]-2,5-二甲基-苯基]-N-乙基-N-甲基-甲脒(K.1.29)、N’-(5-溴-6-茚满-2-基氧基-2-甲基-3-吡啶基)-N-乙基-N-甲基-甲脒(K.1.30)、N’-[5-溴-6-[1-(3,5-二氟苯基)乙氧基]-2-甲基-3-吡啶基]-N-乙基-N-甲基-甲脒(K.1.31)、N’-[5-溴-6-(4-异丙基环己氧基)-2-甲基-3-吡啶基]-N-乙基-N-甲基-甲脒(K.1.32)、N’-[5-溴-2-甲基-6-(1-苯基乙氧基)-3-吡啶基]-N-乙基-N-甲基-甲脒(K.1.33)、N’-(2-甲基-5-三氟甲基-4-(3-三甲基硅烷基-丙氧基)-苯基)-N-乙基-N-甲基甲脒(K.1.34)、N’-(5-二氟甲基-2-甲基-4-(3-三甲基硅烷基-丙氧基)-苯基)-N-乙基-N-甲基甲脒(K.1.35)、2-(4-氯-苯基)-N-[4-(3,4-二甲氧基-苯基)-异唑-5-基]-2-丙-2-炔基氧基-乙酰胺(K.1.36)、3-[5-(4-氯-苯基)-2,3-二甲基-异唑烷-3-基]-吡啶(氯啶菌酯(pyrisoxazole))(K.1.37)、3-[5-(4-甲基苯基)-2,3-二甲基-异唑烷-3-基]-吡啶(K.1.38)、5-氯-1-(4,6-二甲氧基-嘧啶-2-基)-2-甲基-1H-苯并咪唑(K.1.39)、(Z)-3-氨基-2-氰基-3-苯基-丙-2-烯酸乙酯(K.1.40)、picarbutrazox(K.1.41)、N-[6-[[(Z)-[(1-甲基四唑-5-基)-苯基-亚甲基]氨基]氧基甲基]-2-吡啶基]氨基甲酸戊酯(K.1.42)、2-[2-[(7,8-二氟-2-甲基-3-喹啉基)氧基]-6-氟-苯基]丙-2-醇(K.1.44)、2-[2-氟-6-[(8-氟-2-甲基-3-喹啉基)氧基]苯基]丙-2-醇(K.1.45)、3-(5-氟-3,3,4,4-四甲基-3,4-二氢异喹啉-1-基)喹啉(K.1.46)、3-(4,4-二氟-3,3-二甲基-3,4-二氢异喹啉-1-基)喹啉(K.1.47)、3-(4,4,5-三氟-3,3-二甲基-3,4-二氢异喹啉-1-基)喹啉(K.1.48)、9-氟-2,2-二甲基-5-(3-喹啉基)-3H-1,4-苯并氧杂氮杂(K.1.49)、2-(6-苄基-2-吡啶基)喹唑啉(K.1.50)、2-[6-(3-氟-4-甲氧基-苯基)-5-甲基-2-吡啶基]喹唑啉(K.1.51)、3-[(3,4-二氯异噻唑-5-基)甲氧基]-1,2-苯并噻唑1,1-二氧化物(K.1.52);
L)生长调节剂
脱落酸(abscisic acid)、先甲草胺(amidochlor)、嘧啶醇(ancymidol)、6-苄基氨基嘌呤、芸苔素内酯(brassinolide)、地乐胺(butralin)、矮壮素(chlormequat)、矮壮素(chlormequat chloride)、氯化胆碱(choline chloride)、环丙酸酰胺(cyclanilide)、丁酰肼(daminozide)、敌草克(dikegulac)、噻节因(dimethipin)、2,6-二甲基吡啶(2,6-dimethylpuridine)、乙烯利(ethephon)、氟节胺(flumetralin)、调嘧醇(flurprimidol)、达草氟(fluthiacet)、吡效隆(forchlorfenuron)、赤霉酸(gibberellic acid)、抗倒胺(inabenfide)、吲哚-3-乙酸、抑芽丹(maleic hydrazide)、氟草磺(mefluidide)、助壮素(mepiquat)、氯化助壮素(mepiquat chloride)、萘乙酸、N-6-苄基腺嘌呤、多效唑(paclobutrazol)、调环酸(prohexadione)、调环酸钙(prohexadione-calcium)、茉莉酸诱导体(prohydrojasmon)、赛二唑素(thidiazuron)、抑芽唑(triapenthenol)、脱叶磷(tributyl phosphorotrithioate)、2,3,5-三碘苯甲酸、抗倒酯(trinexapac-ethyl)和烯效唑(uniconazole);
M)选自类别M.1至M.15的除草剂
M.1脂质生物合成抑制剂:枯杀达(alloxydim)、枯杀达(alloxydim-sodium)、丁氧环酮(butroxydim)、烯草酮(clethodim)、炔草酯(clodinafop)、炔草酯(clodinafop-propargyl)、噻草酮(cycloxydim)、氰氟草酯(cyhalofop)、氰氟草酯(cyhalofop-butyl)、氯甲草(diclofop)、禾草灵(diclofop-methyl)、唑禾草灵(fenoxaprop)、唑禾草灵(fenoxaprop-ethyl)、高唑禾草灵(fenoxaprop-P)、高唑禾草灵(fenoxaprop-P-ethyl)、吡氟禾草灵(fluazifop)、吡氟禾草灵(fluazifop-butyl)、精吡氟禾草灵(fluazifop-P)、精吡氟禾草灵(fluazifop-P-butyl)、吡氟氯禾灵(haloxyfop)、吡氟氯禾灵(haloxyfop-methyl)、精吡氟氯禾灵(haloxyfop-P)、精吡氟氯禾灵(haloxyfop-P-methyl)、唑酰草胺(metamifop)、唑啉草酯(pinoxaden)、环苯草酮(profoxydim)、喔草酯(propaquizafop)、喹禾灵(quizalofop)、喹禾灵(quizalofop-ethyl)、喹禾灵(四氢糠基酯)(quizalofop-tefuryl)、精喹禾灵(quizalofop-P)、精喹禾灵(quizalofop-P-ethyl)、精喹禾灵(四氢糠基酯)(quizalofop-P-tefuryl)、稀禾定(sethoxydim)、醌肟草(tepraloxydim)、肟草酮(tralkoxydim)、4-(4’-氯-4-环丙基-2’-氟[1,1’-联苯]-3-基)-5-羟基-2,2,6,6-四甲基-2H-吡喃-3(6H)-酮(CAS 1312337-72-6);4-(2’,4’-二氯-4-环丙基[1,1’-联苯]-3-基)-5-羟基-2,2,6,6-四甲基-2H-吡喃-3(6H)-酮(CAS 1312337-45-3);4-(4’-氯-4-乙基-2’-氟[1,1’-联苯]-3-基)-5-羟基-2,2,6,6-四甲基-2H-吡喃-3(6H)-酮(CAS 1033757-93-5);4-(2’,4’-二氯-4-乙基[1,1’-联苯]-3-基)-2,2,6,6-四甲基-2H-吡喃-3,5(4H,6H)-二酮(CAS 1312340-84-3);5-(乙酰基氧基)-4-(4’-氯-4-环丙基-2’-氟[1,1’-联苯]-3-基)-3,6-二氢-2,2,6,6-四甲基-2H-吡喃-3-酮(CAS 1312337-48-6);5-(乙酰基氧基)-4-(2’,4’-二氯-4-环丙基-[1,1’-联苯]-3-基)-3,6-二氢-2,2,6,6-四甲基-2H-吡喃-3-酮;5-(乙酰基氧基)-4-(4’-氯-4-乙基-2’-氟[1,1’-联苯]-3-基)-3,6-二氢-2,2,6,6-四甲基-2H-吡喃-3-酮(CAS 1312340-82-1);5-(乙酰基氧基)-4-(2’,4’-二氯-4-乙基[1,1’-联苯]-3-基)-3,6-二氢-2,2,6,6-四甲基-2H-吡喃-3-酮(CAS 1033760-55-2);4-(4’-氯-4-环丙基-2’-氟[1,1’-联苯]-3-基)-5,6-二氢-2,2,6,6-四甲基-5-氧代-2H-吡喃-3-基碳酸甲基酯(CAS 1312337-51-1);4-(2’,4’-二氯-4-环丙基-[1,1’-联苯]-3-基)-5,6-二氢-2,2,6,6-四甲基-5-氧代-2H-吡喃-3-基碳酸甲基酯;4-(4’-氯-4-乙基-2’-氟[1,1’-联苯]-3-基)-5,6-二氢-2,2,6,6-四甲基-5-氧代-2H-吡喃-3-基碳酸甲基酯(CAS 1312340-83-2);4-(2’,4’-二氯-4-乙基[1,1’-联苯]-3-基)-5,6-二氢-2,2,6,6-四甲基-5-氧代-2H-吡喃-3-基碳酸甲基酯(CAS1033760-58-5);呋草黄(benfuresate)、苏达灭(butylate)、草灭特(cycloate)、茅草枯(dalapon)、哌草丹(dimepiperate)、EPTC、禾草畏(esprocarb)、乙呋草黄(ethofumesate)、四氟丙酸(flupropanate)、草达灭(molinate)、坪草丹(orbencarb)、克草猛(pebulate)、苄草丹(prosulfocarb)、TCA、杀草丹(thiobencarb)、丁草威(tiocarbazil)、野麦畏(triallate)和灭草猛(vernolate);
M.2ALS抑制剂:磺氨黄隆(amidosulfuron)、四唑黄隆(azimsulfuron)、苄嘧黄隆(bensulfuron)、苄嘧黄隆(bensulfuron-methyl)、氯嘧黄隆(chlorimuron)、氯嘧黄隆(chlorimuron-ethyl)、绿黄隆(chlorsulfuron)、醚黄隆(cinosulfuron)、环丙黄隆(cyclosulfamuron)、胺苯黄隆(ethametsulfuron)、胺苯黄隆(ethametsulfuron-methyl)、乙氧嘧黄隆(ethoxysulfuron)、啶嘧黄隆(flazasulfuron)、氟吡磺隆(flucetosulfuron)、氟定黄隆(flupyrsulfuron)、氟定黄隆(flupyrsulfuron-methyl-sodium)、甲酰氨磺隆(foramsulfuron)、吡氯黄隆(halosulfuron)、吡氯黄隆(halosulfuron-methyl)、啶咪黄隆(imazosulfuron)、碘黄隆(iodosulfuron)、碘甲黄隆钠(iodosulfuron-methyl-sodium)、碘嗪磺隆(iofensulfuron)、碘嗪磺隆钠(iofensulfuron-sodium)、甲基二磺隆(mesosulfuron)、嗪吡嘧磺隆(metazosulfuron)、甲黄隆(metsulfuron)、甲黄隆(metsulfuron-methyl)、烟嘧黄隆(nicosulfuron)、嘧苯胺磺隆(orthosulfamuron)、环丙氧黄隆(oxasulfuron)、氟嘧黄隆(primisulfuron)、氟嘧黄隆(primisulfuron-methyl)、嗪咪唑嘧磺隆(propyrisulfuron)、氟丙黄隆(prosulfuron)、吡嘧黄隆(pyrazosulfuron)、吡嘧黄隆(pyrazosulfuron-ethyl)、玉嘧黄隆(rimsulfuron)、嘧黄隆(sulfometuron)、嘧黄隆(sulfometuron-methyl)、乙黄黄隆(sulfosulfuron)、噻黄隆(thifensulfuron)、噻黄隆(thifensulfuron-methyl)、醚苯黄隆(triasulfuron)、苯黄隆(tribenuron)、苯黄隆(tribenuron-methyl)、三氟啶磺隆(trifloxysulfuron)、氟胺磺隆(triflusulfuron)、氟胺磺隆(triflusulfuron-methyl)、三氟甲磺隆(tritosulfuron)、咪草酯(imazamethabenz)、咪草酯(imazamethabenz-methyl)、咪草啶酸(imazamox)、甲基咪草烟(imazapic)、灭草烟(imazapyr)、灭草喹(imazaquin)、咪草烟(imazethapyr)、唑嘧磺胺酸(cloransulam)、唑嘧磺胺盐(cloransulam-methyl)、唑嘧磺胺(diclosulam)、氟唑啶草(flumetsulam)、双氟磺草胺(florasulam)、唑草磺胺(metosulam)、五氟磺草胺(penoxsulam)、嘧啶硫蕃(pyrimisulfan)和啶磺草胺(pyroxsulam)、双嘧苯甲酸(bispyribac)、双嘧苯甲酸钠(bispyribac-sodium)、嘧苯草肟(pyribenzoxim)、环酯草醚(pyriftalid)、肟啶草(pyriminobac)、肟啶草(pyriminobac-methyl)、嘧硫苯甲酸(pyrithiobac)、嘧硫苯甲酸钠(pyrithiobac-sodium)、4-[[[2-[(4,6-二甲氧基-2-嘧啶基)氧基]苯基]甲基]氨基]苯甲酸-1-甲基乙基酯(CAS 420138-41-6)、4-[[[2-[(4,6-二甲氧基-2-嘧啶基)氧基]苯基]甲基]氨基]苯甲酸丙基酯(CAS 420138-40-5)、N-(4-溴苯基)-2-[(4,6-二甲氧基-2-嘧啶基)氧基]苯甲胺(CAS 420138-01-8)、氟酮磺隆(flucarbazone)、氟酮磺隆钠(flucarbazone-sodium)、丙苯磺隆(propoxycarbazone)、丙苯磺隆(propoxycarbazone-sodium)、噻酮磺隆(thiencarbazone)、噻酮磺隆(thiencarbazone-methyl)、氟酮磺草胺(triafamone);
M.3光合成抑制剂:胺唑草酮(amicarbazone);氯三嗪;莠灭净(ametryn)、莠去津(atrazine)、杀草敏(chloridazone)、草净津(cyanazine)、敌草净(desmetryn)、戊草津(dimethametryn)、六嗪同(hexazinone)、赛克津(metribuzin)、扑灭通(prometon)、扑草净(prometryn)、扑灭津(propazine)、西玛津(simazine)、西草净(simetryn)、甲氧去草净(terbumeton)、特丁津(terbuthylazin)、去草净(terbutryn)、草达津(trietazin);氯溴隆(chlorobromuron)、绿麦隆(chlorotoluron)、枯草隆(chloroxuron)、丁隆(dimefuron)、敌草隆(diuron)、伏草隆(fluometuron)、异丙隆(isoproturon)、异恶隆(isouron)、利谷隆(linuron)、苯嗪草(metamitron)、噻唑隆(methabenzthiazuron)、色满隆(metobenzuron)、甲氧隆(metoxuron)、绿谷隆(monolinuron)、草不隆(neburon)、环草隆(siduron)、丁唑隆(tebuthiuron)、赛二唑素(thidiazuron)、异苯敌草(desmedipham)、卡草灵(karbutilat)、苯敌草(phenmedipham)、乙苯敌草(phenmedipham-ethyl)、杀草全(bromofenoxim)、溴苯腈(bromoxynil)及其盐和酯、碘苯腈(ioxynil)及其盐和酯、除草定(bromacil)、环草定(lenacil)、特草定(terbacil)、噻草平(bentazon)、噻草平(bentazon-sodium)、达草止(pyridatre)、pyridafol、蔬草灭(pentanochlor)、敌稗(propanil);敌草快阳离子(diquat)、敌草快(diquat-dibromide)、对草快阳离子(paraquat)、对草快(paraquat-dichloride)、对草快(paraquat-dimetilsulfate);
M.4原卟啉原-IX氧化酶抑制剂:氟锁草醚(acifluorfen)、氟锁草醚(acifluorfen-sodium)、唑啶炔草(azafenidin)、苯唑磺隆(bencarbazone)、双苯嘧草酮(benzfendizone)、治草醚(bifenox)、氟丙嘧草酯(butafenacil)、氟酮唑草(carfentrazone)、氟酮唑草(carfentrazone-ethyl)、氯硝醚(chlomethoxyfen)、吲哚酮草酯(cinidon-ethyl)、异丙吡草酯(fluazolate)、氟哒嗪草酯(flufenpyr)、氟哒嗪草酯(flufenpyr-ethyl)、酰亚胺苯氧乙酸(flumiclorac)、酰亚胺苯氧乙酸戊酯(flumiclorac-pentyl)、氟嗪酮(flumioxazin)、乙羧氟草醚(fluoroglycofen)、乙羧氟草醚(fluoroglycofen-ethyl)、达草氟(fluthiacet)、达草氟(fluthiacet-methyl)、氟黄胺草醚(fomesafen)、氟硝磺酰胺(halosafen)、乳氟禾草灵(lactofen)、炔丙唑草(oxadiargyl)、恶草灵(oxadiazon)、乙氧氟草醚(oxyfluorfen)、戊唑草(pentoxazone)、氟唑草胺(profluazol)、双唑草腈(pyraclonil)、氟唑草酯(pyraflufen)、氟唑草酯(pyraflufen-ethyl)、嘧啶肟草醚(saflufenacil)、磺胺草唑(sulfentrazone)、噻二唑胺(thidiazimin)、tiafenacil、trifludimoxazin、[3-[2-氯-4-氟-5-(1-甲基-6-三氟甲基-2,4-二氧代-1,2,3,4-四氢嘧啶-3-基)苯氧基]-2-吡啶基氧基]乙酸乙酯(CAS353292-31-6)、N-乙基-3-(2,6-二氯-4-三氟-甲基苯氧基)-5-甲基-1H-吡唑-1-甲酰胺(CAS 452098-92-9)、N-四氢糠基-3-(2,6-二氯-4-三氟甲基苯氧基)-5-甲基-1H-吡唑-1-甲酰胺(CAS915396-43-9)、N-乙基-3-(2-氯-6-氟-4-三氟甲基苯氧基)-5-甲基-1H-吡唑-1-甲酰胺(CAS 452099-05-7)、N-四氢糠基-3-(2-氯-6-氟-4-三氟甲基苯氧基)-5-甲基-1H-吡唑-1-甲酰胺(CAS 452100-03-7)、3-[7-氟-3-氧代-4-(丙-2-炔基)-3,4-二氢-2H-苯并[1,4]嗪-6-基]-1,5-二甲基-6-硫代-[1,3,5]三嗪烷(triazinan)-2,4-二酮(CAS451484-50-7)、2-(2,2,7-三氟-3-氧代-4-丙-2-炔基-3,4-二氢-2H-苯并[1,4]嗪-6-基)-4,5,6,7-四氢-异吲哚-1,3-二酮(CAS1300118-96-0)、1-甲基-6-三氟甲基-3-(2,2,7-三氟-3-氧代-4-丙-2-炔基-3,4-二氢-2H-苯并[1,4]嗪-6-基)-1H-嘧啶-2,4-二酮(CAS1304113-05-0)、(E)-4-[2-氯-5-[4-氯-5-(二氟甲氧基)-1H-甲基-吡唑-3-基]-4-氟-苯氧基]-3-甲氧基-丁-2-烯酸甲基酯(CAS 948893-00-3)、3-[7-氯-5-氟-2-(三氟甲基)-1H-苯并咪唑-4-基]-1-甲基-6-(三氟甲基)-1H-嘧啶-2,4-二酮(CAS 212754-02-4);
M.5漂白剂除草剂:丁酰草胺(beflubutamid)、吡氟草胺(diflufenican)、氟草同(fluridone)、氟咯草酮(flurochloridone)、呋草酮(flurtamone)、达草灭(norflurazon)、氟吡酰草胺(picolinafen)、4-(3-三氟甲基苯氧基)-2-(4-三氟甲基苯基)嘧啶(CAS180608-33-7);苯并双环酮(benzobicyclon)、吡草酮(benzofenap)、氟吡草酮(bicyclopyrone)、异恶草酮(clomazone)、fenquintrione、异氟草(isoxaflutole)、甲基磺草酮(mesotrione)、吡唑氟磺草胺(pyrasulfotole)、吡唑特(pyrazolynate)、苄草唑(pyrazoxyfen)、磺草酮(sulcotrione)、特糠酯酮(tefuryltrione)、环磺酮(tembotrione)、tolpyralate、苯唑草酮(topramezone);苯草醚(aclonifen)、杀草强(amitrole)、氟草隆(flumeturon);
M.6EPSP合成酶抑制剂:草甘膦(glyphosate)、草甘膦异丙胺盐(glyphosate-isopropylammonium)、草甘膦钾(glyphosate-potassium)、草硫膦(glyphosate-trimesium)(sulfosate);
M.7谷氨酰胺合成酶抑制剂:双丙氨酰膦(bilanaphos(bialaphos))、双丙氨酰膦(bilanaphos-sodium)、草铵膦(glufosinate)、草铵膦(glufosinate-P)、草铵膦(glufosinate-ammonium);
M.8DHP合成酶抑制剂:黄草灵(asulam);
M.9有丝分裂抑制剂:氟草胺(benfluralin)、地乐胺(butralin)、敌乐胺(dinitramine)、丁氟消草(ethalfluralin)、氟消草(fluchloralin)、黄草消(oryzalin)、胺硝草(pendimethalin)、氨基丙氟灵(prodiamine)、氟乐灵(trifluralin)、胺草磷(amiprophos)、甲基胺草磷(amiprophos-methyl)、草胺磷(butamiphos)、敌草索(chlorthal)、敌草索(chlorthal-dimethyl)、氟硫草定(dithiopyr)、噻氟啶草(thiazopyr)、拿草特(propyzamide)、丙戊草胺(tebutam);长杀草(carbetamide)、氯苯胺灵(chlorpropham)、氟燕灵(flamprop)、氟燕灵(flamprop-isopropyl)、甲氟燕灵(flamprop-methyl)、强氟燕灵(flamprop-M-isopropyl)、麦草伏(flamprop-M-methyl)、苯胺灵(propham);
M.10VLCFA抑制剂:乙草胺(acetochlor)、甲草胺(alachlor)、丁草胺(butachlor)、克草胺(dimethachlor)、噻吩草胺(dimethanamid)、精噻吩草胺(dimethenamid-P)、吡草胺(metazachlor)、异丙甲草胺(metolachlor)、S-异丙甲草胺(metolachlor-S)、烯草胺(pethoxamid)、丙草胺(pretilachlor)、扑草胺(propachlor)、异丙草胺(propisochlor)、噻醚草胺(thenylchlor)、氟噻草胺(flufenacet)、苯噻草胺(mefenacet)、草乃敌(diphenamid)、萘丙胺(naproanilide)、草萘胺(napropamide)、精草萘胺(napropamide-M)、四唑酰草胺(fentrazamide)、莎稗磷(anilofos)、唑草胺(cafenstrole)、苯磺唑草(fenoxasulfone)、三唑酰草胺(ipfencarbazone)、哌草磷(piperophos)、派罗克杀草砜(pyroxasulfone),式II.1,II.2、II.3、II.4、II.5、II.6、II.7、II.8和II.9的异唑啉化合物:
M.11纤维素生物合成抑制剂:草克乐(chlorthiamid)、敌草腈(dichlobenil)、胺草唑(flupoxam)、茚嗪氟草胺(indaziflam)、异恶草胺(isoxaben)、苯氧丙胺津(triaziflam)、1-环己基-5-五氟苯基氧基-14-[1,2,4,6]硫杂三嗪-3-基胺(CAS 175899-01-1);
M.12去偶剂除草剂:地乐酚(dinoseb)、地乐消酚(dinoterb)、DNOC及其盐;
M.13植物生长素除草剂:2,4-D及其盐和酯、氯酰草膦(clacyfos)、2,4-DB及其盐和酯、氯丙嘧啶酸(aminocyclopyrachlor)及其盐和酯、氨草啶(aminopyralid)及其盐例如氨草啶二甲基铵盐(aminopyralid-dimethylammonium)、氨草啶铵盐(aminopyralid-tris(2-hydroxypropyl)ammonium)及其酯、草除灵(benazolin)、草除灵(benazolin-ethyl)、草灭平(chloramben)及其盐和酯、稗草胺(clomeprop)、二氯皮考啉酸(clopyralid)及其盐和酯、麦草畏(dicamba)及其盐和酯、2,4-滴丙酸(dichlorprop)及其盐和酯、高2,4-滴丙酸(dichlorprop-P)及其盐和酯、氟草烟(fluroxypyr)、氟草烟(fluroxypyr-butometyl)、氟氯胺啶(fluroxypyr-meptyl)、氟氯吡啶酯(halauxifen)及其盐和酯(CAS 943832-60-8)、MCPA及其盐和酯、2甲4氯乙硫酯(MCPA-thioethyl)、MCPB及其盐和酯、2甲4氯丙酸(mecoprop)及其盐和酯、高2甲4氯丙酸(mecoprop-P)及其盐和酯、毒莠定(picloram)及其盐和酯、二氯喹啉酸(quinclorac)、喹草酸(quinmerac)、TBA(2,3,6)及其盐和酯、绿草定(triclopyr)及其盐和酯、4-氨基-3-氯-6-(4-氯-2-氟-3-甲氧基苯基)-5-氟吡啶-2-甲酸、4-氨基-3-氯-6-(4-氯-2-氟-3-甲氧基苯基)-5-氟吡啶-2-甲酸苄基酯(CAS 1390661-72-9);
M.14植物生长素输送抑制剂:二氟吡隆(diflufenzopyr)、二氟吡隆(diflufenzopyr-sodium)、抑草生(naptalam)和抑草生(naptalam-sodium);
M.15其它除草剂:溴丁酰草胺(bromobutide)、氯甲丹(chlorflurenol)、氯甲丹(chlorflurenol-methyl)、环庚草醚(cinmethylin)、苄草隆(cumyluron)、cyclopyrimorate(CAS 499223-49-3)及其盐和酯、茅草枯(dalapon)、棉隆(dazomet)、苯敌快(difenzoquat)、苯敌快(difenzoquat-metilsulfate)、噻节因(dimethipin)、DSMA、香草隆(dymron)、敌草腈(endothal)及其盐、乙苯酰草(etobenzanid)、抑草丁(flurenol)、抑草丁(flurenol-butyl)、调嘧醇(flurprimidol)、膦铵素(fosamine)、膦铵素(fosamine-ammonium)、茚草酮(indanofan)、抑芽丹(maleic hydrazide)、氟草磺(mefluidide)、威百亩(metam)、甲硫唑草啉(methiozolin)(CAS 403640-27-7)、叠氮甲烷(methyl azide)、溴甲烷(methyl bromide)、苯丙隆(methyl-dymron)、碘甲烷(methyl iodide)、MSMA、油酸(oleic acid)、氯嗪草(oxaziclomefone)、壬酸(pelargonic acid)、稗草畏(pyributicarb)、灭藻醌(quinoclamine)、灭草环(tridiphane);
N)选自类别N.1至N.29的杀虫剂
N.1乙酰胆碱酯酶(AChE)抑制剂:涕灭威(aldicarb)、棉铃威(alanycarb)、虫威(bendiocarb)、丙硫克百威(benfuracarb)、丁叉威(butocarboxim)、氧丁叉威(butoxycarboxim)、甲萘威(carbaryl)、虫螨威(carbofuran)、丁硫克百威(carbosulfan)、苯虫威(ethiofencarb)、丁苯威(fenobucarb)、伐虫脒(formetanate)、呋线威(furathiocarb)、异丙威(isoprocarb)、灭虫威(methiocarb)、灭多虫(methomyl)、速灭威(metolcarb)、甲氨叉威(oxamyl)、抗蚜威(pirimicarb)、残杀威(propoxur)、硫双威(thiodicarb)、特氨叉威(thiofanox)、混杀威(trimethacarb)、XMC、灭杀威(xylylcarb)和唑蚜威(triazamate);乙酰甲胺磷(acephate)、唑啶磷(azamethiphos)、乙基谷硫磷(azinphos-ethyl)、谷硫磷(azinphosmethyl)、硫线磷(cadusafos)、壤虫氯磷(chlorethoxyfos)、毒虫畏(chlorfenvinphos)、氯甲磷(chlormephos)、毒死蜱(chlorpyrifos)、甲基毒死蜱(chlorpyrifos-methyl)、蝇毒磷(coumaphos)、杀螟腈(cyanophos)、甲基内吸磷(demeton-S-methyl)、二嗪农(diazinon)、敌敌畏(dichlorvos)/DDVP、百治磷(dicrotophos)、乐果(dimethoate)、甲基毒虫畏(dimethylvinphos)、乙拌磷(disulfoton)、EPN、乙硫磷(ethion)、灭克磷(ethoprophos)、氨磺磷(famphur)、克线磷(fenamiphos)、杀螟松(fenitrothion)、倍硫磷(fenthion)、噻唑酮磷(fosthiazate)、庚虫磷(heptenophos)、imicyafos、丙胺磷(isofenphos)、O-(甲氧基氨基硫代-磷酰基)水杨酸异丙酯、异唑磷(isoxathion)、马拉硫磷(malathion)、灭蚜磷(mecarbam)、甲胺磷(methamidophos)、杀扑磷(methidathion)、速灭磷(mevinphos)、久效磷(monocrotophos)、二溴磷(naled)、氧化乐果(omethoate)、砜吸磷(oxydemeton-methyl)、一六零五(parathion)、甲基一六零五(parathion-methyl)、稻丰散(phenthoate)、甲拌磷(phorate)、伏杀磷(phosalone)、亚胺硫磷(phosmet)、磷胺(phosphamidon)、辛硫磷(phoxim)、虫螨磷(pirimiphos-methyl)、丙溴磷(profenofos)、烯虫磷(propetamphos)、丙硫磷(prothiofos)、吡唑硫磷(pyraclofos)、打杀磷(pyridaphenthion)、喹恶磷(quinalphos)、硫特普(sulfotep)、嘧丙磷(tebupirimfos)、硫甲双磷(temephos)、特丁磷(terbufos)、杀虫畏(tetrachlorvinphos)、甲基乙拌磷(thiometon)、三唑磷(triazophos)、敌百虫(trichlorfon)、蚜灭多(vamidothion);
N.2GABA门控氯离子通道拮抗剂:硫丹(endosulfan)、氯丹(chlordane);乙虫腈(ethiprole)、氟虫腈(fipronil)、丁虫腈(flufiprole)、吡嗪氟虫腈(pyrafluprole)、吡啶氟虫腈(pyriprole);
N.3钠通道调节剂:氟丙菊酯(acrinathrin)、丙烯除虫菊(allethrin)、右旋丙烯菊酯(d-cis-trans allethrin)、右旋反式丙烯菊酯(d-trans allethrin)、联苯菊酯(bifenthrin)、生物烯丙菊酯(bioallethrin)、2-环戊烯基生物烯丙菊酯(bioallethrinS-cyclopentenyl)、生物苄呋菊酯(bioresmethrin)、乙氰菊酯(cycloprothrin)、氟氯氰菊酯(cyfluthrin)、高效氟氯氰菊酯(beta-cyfluthrin)、(RS)氯氟氰菊酯(cyhalothrin)、高效氯氟氰菊酯(lambda-cyhalothrin)、精高效氯氟氰菊酯(gamma-cyhalothrin)、氯氰菊酯(cypermethrin)、甲体氯氰菊酯(alpha-cypermethrin)、乙体氯氰菊酯(beta-cypermethrin)、高效反式氯氰菊酯(theta-cypermethrin)、己体氯氰菊酯(zeta-cypermethrin)、苯醚氰菊酯(cyphenothrin)、溴氰菊酯(deltamethrin)、烯炔菊酯(empenthrin)、顺式氰戊菊酯(esfenvalerate)、醚菊酯(etofenprox)、甲氰菊酯(fenpropathrin)、杀灭菊酯(fenvalerate)、氟氰戊菊酯(flucythrinate)、氟氯苯菊酯(flumethrin)、氟胺氰菊酯(tau-fluvalinate)、溴氟醚菊酯(halfenprox)、heptafluthrin、咪炔菊酯(imiprothrin)、氯氟醚菊酯(meperfluthrin)、甲氧苄氟菊酯(metofluthrin)、莫氟杀林(momfluorothrin)、氯菊酯(permethrin)、苯醚菊酯(phenothrin)、炔酮菊酯(prallethrin)、丙氟菊酯(profluthrin)、除虫菊酯(pyrethrin(除虫菊(pyrethrum)))、灭虫菊(resmethrin)、灭虫硅醚(silafluofen)、七氟菊酯(tefluthrin)、四氟醚菊酯(tetramethylfluthrin)、胺菊酯(tetramethrin)、四溴菊酯(tralomethrin)和四氟菊酯(transfluthrin);DDT、甲氧滴滴涕(methoxychlor);
N.4烟碱乙酰胆碱受体激动剂(nAChR):啶虫脒(acetamiprid)、噻虫胺(clothianidin)、环氧虫啶(cycloxaprid)、呋虫胺(dinotefuran)、吡虫啉(imidacloprid)、烯啶虫胺(nitenpyram)、噻虫啉(thiacloprid)、噻虫嗪(thiamethoxam);(2E-)-1-[(6-氯吡啶-3-基)甲基]-N’-硝基-2-亚戊基氨基胍(hydrazinecarboximidamide);1-[(6-氯吡啶-3-基)甲基]-7-甲基-8-硝基-5-丙氧基-1,2,3,5,6,7-六氢咪唑并[1,2-a]吡啶;烟碱;
N.5烟碱乙酰胆碱受体别构活化剂:艾克敌105(spinosad)、乙基多杀菌素(spinetoram);
N.6氯离子通道活化剂:齐墩螨素(abamectin)、甲氨基阿维菌素苯甲酸盐(emamectin benzoate)、伊维菌素(ivermectin)、雷皮菌素(lepimectin)、米尔螨素(milbemectin);
N.7保幼激素模拟物:蒙五一二(hydroprene)、蒙七七七(kinoprene)、蒙五一五(methoprene);双氧威(fenoxycarb)、蚊蝇醚(pyriproxyfen);
N.8混杂非特异性(多位点)抑制剂:甲基溴和其它烷基卤化物;氯化苦(chloropicrin)、硫酰氟、硼砂、吐酒石(tartar emetic);
N.9选择性同翅目进食阻断剂:拒嗪酮(pymetrozine)、氟啶虫酰胺(flonicamid);
N.11昆虫中肠膜的微生物干扰物:苏云金芽孢杆菌(Baccillus thuringiensis)、球形芽孢杆菌(Bacillus sphaericus)及其产生的杀虫蛋白质:苏云金芽孢杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis)、球形芽孢杆菌(Bacillussphaericus)、苏云金芽孢杆菌鲇泽亚种(Bacillus thuringiensis subsp.aizawai)、苏云金芽孢杆菌库斯塔亚种(Bacillus thuringiensis subsp.kurstaki)、苏云金芽孢杆菌拟步行甲亚种(Bacillus thuringiensis subsp.tenebrionis)、Bt作物蛋白质:Cry1Ab、Cry1Ac、Cry1Fa、Cry2Ab、mCry3A、Cry3Ab、Cry3Bb、Cry34/35Ab1;
N.12线粒体ATP合成酶抑制剂:杀螨硫隆(diafenthiuron);唑环锡(azocyclotin)、三环锡(cyhexatin)、杀螨锡(fenbutatin oxide)、克螨特(propargite)、三氯杀螨砜(tetradifon);
N.13通过质子梯度干扰的氧化磷酸化去偶剂:氟唑虫清(chlorfenapyr)、DNOC、氟虫胺(sulfluramid);
N.14烟碱乙酰胆碱受体(nAChR)通道阻断剂:杀虫磺(bensultap)、巴丹(cartaphydrochloride)、硫环杀(thiocyclam)、杀虫单(thiosultap sodium);
N.150型壳多糖生物合成抑制剂:双三氟虫脲(bistrifluron)、定虫隆(chlorfluazuron)、氟脲杀(diflubenzuron)、氟螨脲(flucycloxuron)、氟虫脲(flufenoxuron)、氟铃脲(hexaflumuron)、氟丙氧脲(lufenuron)、双苯氟脲(novaluron)、多氟脲(noviflumuron)、伏虫隆(teflubenzuron)、杀虫隆(triflumuron);
N.161型壳多糖生物合成抑制剂:噻嗪酮(buprofezin);
N.17蜕皮干扰剂:灭蝇胺(cyromazine);
N.18蜕皮激素受体激动剂:甲氧苯酰肼(methoxyfenozide)、双苯酰肼(tebufenozide)、特丁苯酰肼(halofenozide)、呋喃虫酰肼(fufenozide)、环虫酰肼(chromafenozide);
N.19章鱼胺受体激动剂:虫螨脒(amitraz);
N.20线粒体复合体III电子传输抑制剂:灭蚁腙(hydramethylnon)、灭螨醌(acequinocyl)、嘧螨酯(fluacrypyrim);
N.21线粒体复合体I电子传输抑制剂:喹螨醚(fenazaquin)、唑螨酯(fenpyroximate)、嘧胺苯醚(pyrimidifen)、哒螨酮(pyridaben)、吡螨胺(tebufenpyrad)、唑虫酰胺(tolfenpyrad);鱼藤酮(rotenone);
N.22电压依赖性钠通道阻断剂:二唑虫(indoxacarb)、氰氟虫胺(metaflumizone)、2-[2-(4-氰基苯基)-1-[3-(三氟甲基)苯基]亚乙基]-N-[4-(二氟甲氧基)苯基]氨基脲、N-(3-氯-2-甲基苯基)-2-[(4-氯苯基)-[4-[甲基(甲基磺酰基)氨基]苯基]亚甲基]氨基脲;
N.23乙酰CoA羧化酶抑制剂:螺螨酯(spirodiclofen)、螺甲螨酯(spiromesifen)、螺虫乙酯(spirotetramat);
N.24线粒体复合体IV电子传输抑制剂:磷化铝、二磷化三钙、膦、磷化锌;氰化物;
N.25线粒体复合体II电子传输抑制剂:腈吡螨酯(cyenopyrafen)、丁氟螨酯(cyflumetofen);
N.28鱼尼汀(Ryanodine)受体调节剂:氟虫双酰胺(flubendiamide)、氯虫酰胺(chlorantraniliprole)、氰虫酰胺(cyantraniliprole)、环溴虫酰胺(cyclaniliprole)、氟氰虫酰胺(tetraniliprole);(R)-3-氯-N1-{2-甲基-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]苯基}-N2-(1-甲基-2-甲基磺酰基乙基)邻苯二甲酰胺、(S)-3-氯-N1-{2-甲基-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]苯基}-N2-(1-甲基-2-甲基磺酰基乙基)邻苯二甲酰胺、甲基-2-[3,5-二溴-2-({[3-溴-1-(3-氯吡啶-2-基)-1H-吡唑-5-基]羰基}氨基)苯甲酰基]-1,2-二甲基肼甲酸酯;N-[4,6-二氯-2-[(二乙基-λ-4-亚硫烷基(sulfanylidene))氨基甲酰基]-苯基]-2-(3-氯-2-吡啶基)-5-(三氟甲基)吡唑-3-甲酰胺;N-[4-氯-2-[(二乙基-λ-4-亚硫烷基)氨基甲酰基]-6-甲基-苯基]-2-(3-氯-2-吡啶基)-5-(三氟甲基)吡唑-3-甲酰胺;N-[4-氯-2-[(二-2-丙基-λ-4-亚硫烷基)氨基甲酰基]-6-甲基-苯基]-2-(3-氯-2-吡啶基)-5-(三氟甲基)吡唑-3-甲酰胺;N-[4,6-二氯-2-[(二-2-丙基-λ-4-亚硫烷基)氨基甲酰基]-苯基]-2-(3-氯-2-吡啶基)-5-(三氟甲基)吡唑-3-甲酰胺;N-[4,6-二溴-2-[(二乙基-λ-4-亚硫烷基)氨基甲酰基]-苯基]-2-(3-氯-2-吡啶基)-5-(三氟甲基)吡唑-3-甲酰胺;N-[2-(5-氨基-1,3,4-噻二唑-2-基)-4-氯-6-甲基苯基]-3-溴-1-(3-氯-2-吡啶基)-1H-吡唑-5-甲酰胺;3-氯-1-(3-氯-2-吡啶基)-N-[2,4-二氯-6-[[(1-氰基-1-甲基乙基)氨基]羰基]苯基]-1H-吡唑-5-甲酰胺;3-溴-N-[2,4-二氯-6-(甲基氨基甲酰基)苯基]-1-(3,5-二氯-2-吡啶基)-1H-吡唑-5-甲酰胺;N-[4-氯-2-[[(1,1-二甲基乙基)氨基]羰基]-6-甲基苯基]-1-(3-氯-2-吡啶基)-3-(氟甲氧基)-1H-吡唑-5-甲酰胺;氯氟氰虫酰胺(cyhalodiamide);
N.29.未知或不确定作用模式的杀虫活性化合物:啶喃环丙虫酯(afidopyropen)、阿福拉纳(afoxolaner)、印楝素(azadirachtin)、磺胺螨酯(amidoflumet)、苯螨特(benzoximate)、联苯肼酯(bifenazate)、溴氟酰胺(broflanilide)、溴螨酯(bromopropylate)、喹菌酮(chinomethionat)、冰晶石(cryolite)、环胺普罗(dicloromezotiaz)、三氯杀螨醇(dicofol)、嘧虫胺(flufenerim)、氟美托奎(flometoquin)、氟噻虫砜(fluensulfone)、氟己芬(fluhexafon)、氟吡菌酰胺(fluopyram)、氟吡呋喃酮(flupyradifurone)、氟拉蓝那(fluralaner)、虫酮(metoxadiazone)、增效醚(piperonyl butoxide)、派氟丁胺(pyflubumide)、啶虫丙醚(pyridalyl)、氟虫吡喹(pyrifluquinazon)、氟啶虫胺腈(sulfoxaflor)、替恶杀芬(tioxazafen)、三氟苯嘧啶(triflumezopyrim)、11-(4-氯-2,6-二甲基苯基)-12-羟基-1,4-二氧杂-9-氮杂双螺[4.2.4.2]-十四碳-11-烯-10-酮、3-(4’-氟-2,4-二甲基联苯-3-基)-4-羟基-8-氧杂-1-氮杂螺[4.5]癸-3-烯-2-酮、1-[2-氟-4-甲基-5-[(2,2,2-三氟乙基)亚磺酰基]苯基]-3-(三氟甲基)-1H-1,2,4-三唑-5-胺、坚硬芽胞杆菌(Bacillusfirmus)、(E/Z)-N-[1-[(6-氯-3-吡啶基)甲基]-2-亚吡啶基]-2,2,2-三氟-乙酰胺;(E/Z)-N-[1-[(6-氯-5-氟-3-吡啶基)甲基]-2-亚吡啶基]-2,2,2-三氟-乙酰胺;(E/Z)-2,2,2-三氟-N-[1-[(6-氟-3-吡啶基)甲基]-2-亚吡啶基]乙酰胺;(E/Z)-N-[1-[(6-溴-3-吡啶基)甲基]-2-亚吡啶基]-2,2,2-三氟-乙酰胺;(E/Z)-N-[1-[1-(6-氯-3-吡啶基)乙基]-2-亚吡啶基]-2,2,2-三氟-乙酰胺;(E/Z)-N-[1-[(6-氯-3-吡啶基)甲基]-2-亚吡啶基]-2,2-二氟-乙酰胺;(E/Z)-2-氯-N-[1-[(6-氯-3-吡啶基)甲基]-2-亚吡啶基]-2,2-二氟-乙酰胺;(E/Z)-N-[1-[(2-氯嘧啶-5-基)甲基]-2-亚吡啶基]-2,2,2-三氟-乙酰胺;(E/Z)-N-[1-[(6-氯-3-吡啶基)甲基]-2-亚吡啶基]-2,2,3,3,3-五氟-丙酰胺);N-[1-[(6-氯-3-吡啶基)甲基]-2-亚吡啶基]-2,2,2-三氟-硫代乙酰胺;N-[1-[(6-氯-3-吡啶基)甲基]-2-亚吡啶基]-2,2,2-三氟-N’-异丙基-乙脒;三氟咪啶酰胺(fluazaindolizine);4-[5-(3,5-二氯苯基)-5-(三氟甲基)-4H-异唑-3-基]-2-甲基-N-(1-噻烷-3-基)苯甲酰胺;fluxametamide;5-[3-[2,6-二氯-4-(3,3-二氯烯丙基氧基)苯氧基]丙氧基]-1H-吡唑;3-(苯甲酰基甲基氨基)-N-[2-溴-4-[1,2,2,3,3,3-六氟-1-(三氟甲基)丙基]-6-(三氟甲基)苯基]-2-氟-苯甲酰胺;3-(苯甲酰基甲基氨基)-2-氟-N-[2-碘-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]-6-(三氟甲基)苯基]-苯甲酰胺;N-[3-[[[2-碘-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]-6-(三氟甲基)苯基]氨基]羰基]苯基]-N-甲基-苯甲酰胺;N-[3-[[[2-溴-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]-6-(三氟甲基)苯基]氨基]羰基]-2-氟苯基]-4-氟-N-甲基-苯甲酰胺;4-氟-N-[2-氟-3-[[[2-碘-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]-6-(三氟甲基)苯基]氨基]羰基]苯基]-N-甲基-苯甲酰胺;3-氟-N-[2-氟-3-[[[2-碘-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]-6-(三氟甲基)苯基]氨基]羰基]苯基]-N-甲基-苯甲酰胺;2-氯-N-[3-[[[2-碘-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]-6-(三氟甲基)苯基]氨基]羰基]苯基]-3-吡啶甲酰胺;4-氰基-N-[2-氰基-5-[[2,6-二溴-4-[1,2,2,3,3,3-六氟-1-(三氟甲基)丙基]苯基]氨基甲酰基]苯基]-2-甲基-苯甲酰胺;4-氰基-3-[(4-氰基-2-甲基-苯甲酰基)氨基]-N-[2,6-二氯-4-[1,2,2,3,3,3-六氟-1-(三氟甲基)丙基]苯基]-2-氟-苯甲酰胺;N-[5-[[2-氯-6-氰基-4-[1,2,2,3,3,3-六氟-1-(三氟甲基)丙基]苯基]氨基甲酰基]-2-氰基-苯基]-4-氰基-2-甲基-苯甲酰胺;N-[5-[[2-溴-6-氯-4-[2,2,2-三氟-1-羟基-1-(三氟甲基)乙基]苯基]氨基甲酰基]-2-氰基-苯基]-4-氰基-2-甲基-苯甲酰胺;N-[5-[[2-溴-6-氯-4-[1,2,2,3,3,3-六氟-1-(三氟甲基)丙基]苯基]氨基甲酰基]-2-氰基-苯基]-4-氰基-2-甲基-苯甲酰胺;4-氰基-N-[2-氰基-5-[[2,6-二氯-4-[1,2,2,3,3,3-六氟-1-(三氟甲基)丙基]苯基]氨基甲酰基]苯基]-2-甲基-苯甲酰胺;4-氰基-N-[2-氰基-5-[[2,6-二氯-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]苯基]氨基甲酰基]苯基]-2-甲基-苯甲酰胺;N-[5-[[2-溴-6-氯-4-[1,2,2,2-四氟-1-(三氟甲基)乙基]苯基]氨基甲酰基]-2-氰基-苯基]-4-氰基-2-甲基-苯甲酰胺;2-(1,3-二烷-2-基)-6-[2-(3-吡啶基)-5-噻唑基]-吡啶;2-[6-[2-(5-氟-3-吡啶基)-5-噻唑基]-2-吡啶基]-嘧啶;2-[6-[2-(3-吡啶基)-5-噻唑基]-2-吡啶基]-嘧啶;N-甲基磺酰基-6-[2-(3-吡啶基)噻唑-5-基]吡啶-2-甲酰胺;N-甲基磺酰基-6-[2-(3-吡啶基)噻唑-5-基]吡啶-2-甲酰胺;N-乙基-N-[4-甲基-2-(3-吡啶基)噻唑-5-基]-3-甲硫基-丙酰胺;N-甲基-N-[4-甲基-2-(3-吡啶基)噻唑-5-基]-3-甲硫基-丙酰胺;N,2-二甲基-N-[4-甲基-2-(3-吡啶基)噻唑-5-基]-3-甲硫基-丙酰胺;N-乙基-2-甲基-N-[4-甲基-2-(3-吡啶基)噻唑-5-基]-3-甲硫基-丙酰胺;N-[4-氯-2-(3-吡啶基)噻唑-5-基]-N-乙基-2-甲基-3-甲硫基-丙酰胺;N-[4-氯-2-(3-吡啶基)噻唑-5-基]-N,2-二甲基-3-甲硫基-丙酰胺;N-[4-氯-2-(3-吡啶基)噻唑-5-基]-N-甲基-3-甲硫基-丙酰胺;N-[4-氯-2-(3-吡啶基)噻唑-5-基]-N-乙基-3-甲硫基-丙酰胺;1-[(6-氯-3-吡啶基)甲基]-1,2,3,5,6,7-六氢-5-甲氧基-7-甲基-8-硝基-咪唑并[1,2-a]吡啶;1-[(6-氯吡啶-3-基)甲基]-7-甲基-8-硝基-1,2,3,5,6,7-六氢咪唑并[1,2-a]吡啶-5-醇;1-异丙基-N,5-二甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;1-(1,2-二甲基丙基)-N-乙基-5-甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;N,5-二甲基-N-哒嗪-4-基-1-(2,2,2-三氟-1-甲基-乙基)吡唑-4-甲酰胺;1-[1-(1-氰基环丙基)乙基]-N-乙基-5-甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;N-乙基-1-(2-氟-1-甲基-丙基)-5-甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;1-(1,2-二甲基丙基)-N,5-二甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;1-[1-(1-氰基环丙基)乙基]-N,5-二甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;N-甲基-1-(2-氟-1-甲基-丙基]-5-甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;1-(4,4-二氟环己基)-N-乙基-5-甲基-N-哒嗪-4-基-吡唑-4-甲酰胺;1-(4,4-二氟环己基)-N,5-二甲基-N-哒嗪-4-基-吡唑-4-甲酰胺、N-(1-甲基乙基)-2-(3-吡啶基)-2H-吲唑-4-甲酰胺;N-环丙基-2-(3-吡啶基)-2H-吲唑-4-甲酰胺;N-环己基-2-(3-吡啶基)-2H-吲唑-4-甲酰胺;2-(3-吡啶基)-N-(2,2,2-三氟乙基)-2H-吲唑-4-甲酰胺;2-(3-吡啶基)-N-[(四氢-2-呋喃基)甲基]-2H-吲唑-5-甲酰胺;2-[[2-(3-吡啶基)-2H-吲唑-5-基]羰基]肼甲酸甲基酯;N-[(2,2-二氟环丙基)甲基]-2-(3-吡啶基)-2H-吲唑-5-甲酰胺;N-(2,2-二氟丙基)-2-(3-吡啶基)-2H-吲唑-5-甲酰胺;2-(3-吡啶基)-N-(2-嘧啶基甲基)-2H-吲唑-5-甲酰胺;N-[(5-甲基-2-吡嗪基)甲基]-2-(3-吡啶基)-2H-吲唑-5-甲酰胺、N-[3-氯-1-(3-吡啶基)吡唑-4-基]-N-乙基-3-(3,3,3-三氟丙基硫烷基)丙酰胺;N-[3-氯-1-(3-吡啶基)吡唑-4-基]-N-乙基-3-(3,3,3-三氟丙基亚磺酰基)丙酰胺;N-[3-氯-1-(3-吡啶基)吡唑-4-基]-3-[(2,2-二氟环丙基)甲基硫烷基]-N-乙基-丙酰胺;N-[3-氯-1-(3-吡啶基)吡唑-4-基]-3-[(2,2-二氟环丙基)甲基亚磺酰基]-N-乙基-丙酰胺;sarolaner、lotilaner。
称作组分2)的活性物质、其制备及其农药活性是已知的(参见:http://www.alanwood.net/pesticides/);这些物质是可商购获得的。依据IUPAC命名法描述的化合物、其制备及其农药活性也是已知的(例如Can.J.Plant Sci.48(6),587-94,1968;EP-A141 317;EP-A 152 031;EP-A 226 917;EP-A 243 970;EP-A 256 503;EP-A 428 941;EP-A532 022;EP-A 1 028125;EP-A 1 035 122;EP-A 1 201 648;EP-A 1 122 244、JP2002316902;DE 19650197;DE 10021412;DE 102005009458;US 3,296,272;US 3,325,503;WO 98/46608;WO 99/14187;WO 99/24413;WO 99/27783;WO 00/29404;WO 00/46148;WO00/65913;WO 01/54501;WO 01/56358;WO 02/22583;WO 02/40431;WO 03/10149;WO 03/11853;WO 03/14103;WO 03/16286;WO 03/53145;WO 03/61388;WO 03/66609;WO 03/74491;WO 04/49804;WO 04/83193;WO 05/120234;WO 05/123689;WO 05/123690;WO 05/63721;WO 05/87772;WO 05/87773;WO 06/15866;WO 06/87325;WO 06/87343;WO 07/82098;WO 07/90624、WO 10/139271、WO 11/028657、WO2012/168188、WO 2007/006670、WO2011/77514;WO13/047749、WO 10/069882、WO 13/047441、WO 03/16303、WO 09/90181、WO13/007767、WO 13/010862、WO 13/127704、WO 13/024009、WO 13/024010和WO 13/047441、WO 13/162072、WO 13/092224、WO 11/135833、CN 1907024、CN 1456054)。
优选的是,混合物包含彼此独立选自A)、B)、C)、D、E)、F)、G)、H)、I)、J)和K)的杀真菌化合物作为农药II。
根据本发明的另一个实施方案,混合物包含选自组L)的植物生长调节剂化合物作为农药II。
根据本发明的另一个实施方案,混合物包含选自组M)的除草剂化合物作为农药II。
根据进一步的实施方案,混合物包含选自组N)的杀虫化合物作为农药II。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组A)中Qo位点的复合体III抑制剂,更优选选自化合物(A.1.1)、(A.1.4)、(A.1.8)、(A.1.9)、(A.1.10)、(A.1.12)、(A.1.13)、(A.1.14)、(A.1.17)、(A.1.21)、(A.1.24)、(A.1.25)、(A.1.26)、(A.1.27)、(A.1.28)、(A.1.29)、(A.1.30)、(A.1.31)、(A.1.32)、(A.1.33)、(A.1.34)和(A.1.35);特别选自(A.1.1)、(A.1.4)、(A.1.8)、(A.1.9)、(A.1.13)、(A.1.14)、(A.1.17)、(A.1.24)、(A.1.25)、(A.1.26)、(A.1.27)、(A.1.28)、(A.1.29)、(A.1.30)、(A.1.31)、(A.1.32)、(A.1.33)、(A.1.34)和(A.1.35)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组A)中Qi位点的复合体III抑制剂,更优选选自化合物(A.2.1)、(A.2.3)、(A.2.4)和(A.2.5);特别选自(A.2.3)、(A.2.4)和(A.2.5)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组A)中复合体II抑制剂,更优选选自化合物(A.3.2)、(A.3.3)、(A.3.4)、(A.3.7)、(A.3.9)、(A.3.11)、(A.3.12)、(A.3.14)、(A.3.16)、(A.3.19)、(A.3.20)、(A.3.21)、(A.3.22)、(A.3.23)、(A.3.24)、(A.3.25)、(A.3.27)和(A.3.28);特别选自(A.3.2)、(A.3.3)、(A.3.4)、(A.3.7)、(A.3.9)、(A.3.12)、(A.3.14)、(A.3.17)、(A.3.19)、(A.3.22)、(A.3.23)、(A.3.24)、(A.3.25)、(A.3.27)、(A.3.28)和(A.3.29)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组A)中其它呼吸抑制剂,更优选选自化合物(A.4.4)和(A.4.11);特别是(A.4.11)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组B)中C14脱甲基酶抑制剂,更优选选自化合物(B.1.4)、(B.1.5)、(B.1.8)、(B.1.10)、(B.1.11)、(B.1.12)、(B.1.13)、(B.1.17)、(B.1.18)、(B.1.21)、(B.1.22)、(B.1.23)、(B.1.25)、(B.1.26)、(B.1.29)、(B.1.31)、(B.1.32)、(B.1.33)、(B.1.34)、(B.1.35)、(B.1.36)、(B.1.37)、(B.1.38)、(B.1.39)、(B.1.40)、(B.1.41)、(B.1.42)、(B.1.43)和(B.1.46);特别选自(B.1.5)、(B.1.8)、(B.1.10)、(B.1.17)、(B.1.23)、(B.1.25)、(B.1.31)、(B.1.32)、(B.1.33)、(B.1.34)、(B.1.35)、(B.1.36)、(B.1.37)、(B.138)、(B.1.39)、(B.1.40)、(B.1.41)、(B.1.42)、(B.1.43)和(B.1.46)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组B)中δ14-还原酶抑制剂,更优选选自化合物(B.2.4)、(B.2.5)、(B.2.6)和(B.2.8);特别是(B.2.4)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组C)中苯基酰胺和酰基氨基酸杀真菌剂,更优选选自化合物(C.1.1)、(C.1.2)、(C.1.4)和(C.1.5);特别选自(C.1.1)和(C.1.4)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组C)中其它核酸合成抑制剂,更优选选自化合物(C.2.6)和(C.2.7)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组D),更优选选自化合物(D.1.1)、(D.1.2)、(D.1.5)、(D.2.4)和(D.2.6);特别选自(D.1.2)、(D.1.5)和(D.2.6)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组E),更优选选自化合物(E.1.1)、(E.1.3)、(E.2.2)和(E.2.3);特别是(E.1.3)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组F)、更优选选自化合物(F.1.2)、(F.1.4)和(F.1.5)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组G),更优选选自化合物(G.3.1)、(G.3.3)、(G.3.6)、(G.5.1)、(G.5.2)和(G.5.3);特别选自(G.3.1)、(G.5.1)、(G.5.2)和(G.5.3)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组H),更优选选自化合物(H.2.2)、(H.2.3)、(H.2.5)、(H.2.7)、(H.2.8)、(H.3.2)、(H.3.4)、(H.3.5)、(H.4.9)和(H.4.10);特别选自(H.2.2)、(H.2.5)、(H.3.2)、(H.4.9)和(H.4.10)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组I),更优选选自化合物(I.2.2)和(I.2.5)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组J),更优选选自化合物(J.1.2)、(J.1.5)和(J.1.8);特别是(J.1.5)。
还优选混合物,所述混合物包含作为组分2)的至少一种农药II,所述农药II选自组K),更优选选自化合物(K.1.41)、(K.1.42)、(K.1.44)、(K.1.45)、(K.1.46)、(K.1.47)、(K.1.48)和(K.1.49);特别选自(K.1.41)、(K.1.44)、(K.1.45)、(K.1.46)、(K.1.47)、(K.1.48)和(K.1.49)。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的如上定义的至少一种微生物的全培养液、培养物基质或无细胞提取物或级分或至少一种代谢物,其优选显示出对抗至少一种植物病原体的拮抗活性。
如本文中使用的,“全培养液”是指含有悬浮在培养基中的营养细胞和/或孢子和任选由相应微生物产生的代谢物的微生物液体培养物。
如本文中使用的,“培养物基质”是指可通过如下方法获得的培养基:在所述培养基(优选液体肉汤)中培养微生物,并且保留除去生长在培养基中的细胞后的剩余物,例如通过离心、过滤、沉淀或本领域公知的其它方式除去生长在液体肉汤中的细胞后剩余的上清液;包含例如由相应微生物产生并且分泌至培养基中的代谢物。可以例如通过以约5,000至20,000×g(更优选约15,000×g),在约2至30℃的温度(更优选在4至20℃的温度),离心约10至60分钟(更优选约15至30分钟),获得有时候也称为“上清液”的“培养物基质”。
如本文中使用的,“无细胞提取物”是指微生物的营养细胞、孢子和/或全培养液的提取物,其包含可以通过本领域已知的细胞破坏方法获得的相应微生物产生的细胞代谢物,所述细胞破坏方法例如基于溶剂的(例如,有机溶剂,例如醇,有时候与适合的盐组合)、基于温度的、应用剪切力、使用超声发生器进行的细胞破坏。可以通过常规浓缩技术,例如干燥、蒸发、离心等来浓缩所需的提取物。优选在使用前,也可以使用基于有机溶剂和/或水的介质,对粗提物进行一些洗涤步骤。
如本文中使用的,术语“代谢物”是指由微生物(例如真菌和细菌,特别是本发明的菌株)产生的具有本文中所述任何有益作用的任何组分、化合物、物质或副产物(包括但不限于小分子次生代谢物、聚酮化合物、脂肪酸合成酶产物、非核糖体肽、核糖体肽、蛋白质和酶),所述有益作用例如农药活性或改善植物生长、植物的水利用效率、植物健康、植物外观或与本文植物活性有关的土壤中的有益微生物群体。
如本文中使用的,“分离物”是指从其天然来源分离的纯微生物培养物,例如通过培养微生物单菌落获得的分离物。分离物是源自异质的野生微生物群体的纯培养物。
如本文中使用的,“菌株”是指显示出属于相同谱系的,而不同于相同物种的其它分离物或菌株的表型和/或遗传性状的细菌分离物或一组分离物。
如本文所用,“其培养物基质”是指如紧邻术语“其培养物基质”之前限定的细菌菌株的培养物基质,如以下情况中:“菌株Lu16774、Lu17007和Lu17015及其培养物基质”意指菌株Lu16774、Lu17007和Lu17015和菌株Lu16774、Lu17007和Lu17015各自的培养物基质。同样地,相同逻辑适用于相似术语例如“其无细胞提取物”、“其全培养液”和“其代谢物”以及如这类术语的组合,例如“其无细胞提取物或至少一种代谢物”。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一中类芽孢杆菌属(Paenibacillus)菌株,所述菌株具有以下特征:
-革兰氏阳性结构的杆状细胞,
-革兰氏染色弱反应,常常甚至是阴性染色的,
-分化成椭圆形内生孢子,其明显胀大了孢子囊(母细胞),
-兼性厌氧生长,在不存在空气的情况下强烈生长,与是否存在硝酸盐无关,
-发酵多种糖,
-从多种糖(包括葡萄糖)形成酸和气体,
-从核糖醇和山梨醇不产生酸,
-脲酶阴性(强壮类芽孢杆菌(P.validus)除外),
-精氨酸二水解酶(dihydrolase)阴性,
-不利用柠檬酸盐,
-在10%氯化钠存在下不生长,
-分泌多种降解DNA、蛋白质、淀粉的胞外水解酶;和/或
-40%至54%的DNA的G+C含量;
或其培养物基质、无细胞提取物或至少一种代谢物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种类芽孢杆菌属(Paenibacillus)菌株,所述的菌株选自物种多粘类芽孢杆菌(Paenibacilluspolymyxa)、附生类芽孢杆菌(Paenibacillus epiphyticus)、皮氏类芽孢杆菌(Paenibacillus peoriae)、土地类芽孢杆菌(Paenibacillus terrae)、杰米拉类芽孢杆菌(Paenibacillus jamilae)、胶冻样类芽孢杆菌(Paenibacillus kribbensis);解淀粉类芽孢杆菌(Paenibacillus amylolyticu)、巴塞罗那类芽孢杆菌(Paenibacillusbarcinonensis)、苔原类芽孢杆菌(Paenibacillus tundra)、伊利诺伊类芽孢杆菌(Paenibacillus illinoisensis)、马阔里类芽孢杆菌(Paenibacillus macquariensis)、台中类芽孢杆菌(Paenibacillus taichungensis)、解聚糖类芽孢杆菌(Paenibacillusglycanilyticus)和Paenibacillus odorifer;或其培养物基质、无细胞提取物或至少一种代谢物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种类芽孢杆菌属(Paenibacillus)菌株,所述的菌株选自物种多粘类芽孢杆菌(Paenibacilluspolymyxa)、附生类芽孢杆菌(Paenibacillus epiphyticus)、皮氏类芽孢杆菌(Paenibacillus peoriae)、土地类芽孢杆菌(Paenibacillus terrae)、杰米拉类芽孢杆菌(Paenibacillus jamilae)和胶冻样类芽孢杆菌(Paenibacillus kribbensis);或其培养物基质、无细胞提取物或至少一种代谢物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种细菌菌株,所述的菌株选自物种多粘类芽孢杆菌(Paenibacillus polymyxa)、附生类芽孢杆菌(Paenibacillus epiphyticus)、皮氏类芽孢杆菌(Paenibacillus peoriae)和杰米拉类芽孢杆菌(Paenibacillus jamilae);或其培养物基质、无细胞提取物或至少一种代谢物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种细菌菌株,所述的菌株选自物种多粘类芽孢杆菌(Paenibacillus polymyxa)、附生类芽孢杆菌(Paenibacillus epiphyticus)和皮氏类芽孢杆菌(Paenibacillus peoriae);或其培养物基质、无细胞提取物或至少一种代谢物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种细菌菌株,所述的菌株选自物种多粘类芽孢杆菌(Paenibacillus polymyxa)和附生类芽孢杆菌(Paenibacillus epiphyticus);或其培养物基质、无细胞提取物或至少一种代谢物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种类芽孢杆菌属(Paenibacillus)菌株,所述菌株选自:
1)以保藏号DSM 26969保藏于DSMZ的Lu16774;
2)以保藏号DSM 26970保藏于DSMZ的Lu17007;和
3)以保藏号DSM 26971保藏于DSMZ的Lu17015;
或其培养物基质、无细胞提取物或至少一种代谢物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种类芽孢杆菌属(Paenibacillus)菌株,所述菌株选自:
1)以保藏号DSM 26969保藏于DSMZ的多粘类芽孢杆菌植物亚种(Paenibacilluspolymyxa ssp.plantarum)菌株Lu16774,
2)以保藏号DSM 26970保藏于DSMZ的多粘类芽孢杆菌植物亚种(Paenibacilluspolymyxa ssp.plantarum)菌株Lu17007,
3)以保藏号DSM 26971保藏于DSMZ的附生类芽孢杆菌(Paenibacillusepiphyticus)菌株Lu17015。
或其培养物基质、无细胞提取物或至少一种代谢物。
除了包含作为组分1)的至少一种菌株Lu16774、Lu17007和Lu17015的混合物之外,本发明还涉及包含作为组分1)的任何类芽孢杆菌属(Paenibacillus)菌株的混合物,不管其是物理上源自任何菌株Lu16774、Lu17007和Lu17015的原始保藏物的或是独立分离的,只要它们保留保藏的类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015的至少一个鉴别性特征。此类本发明的类芽孢杆菌属(Paenibacillus)菌株包括任何菌株Lu16774、Lu17007和Lu17015的任何后代,包括所述菌株的突变株。
术语“突变株”是指通过直接突变选择获得的微生物,但也包括已经进行了进一步诱变或操纵(例如通过引入质粒)的微生物。因此,实施方案包括代表性微生物的突变株、变体和或衍生物,天然产生的和人工诱导的突变株。例如,可以通过使微生物接受已知的诱变剂(例如X-射线、UV照射或N-甲基-亚硝基胍),使用常规方法,来诱导突变株。所述处理后,可以进行显示出所需特征的突变菌株的筛选。
可以通过本领域已知的任何方法来获得突变菌株,例如直接突变选择、化学诱变或基因操纵(例如,通过引入质粒)。例如,可以通过应用已知的诱变剂(例如X-射线、UV照射或N-甲基-亚硝基胍)来获得此类突变株。所述处理后,可以进行显示出所需特征的突变菌株的筛选。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含DNA序列,所述DNA序列与菌株Lu16774、Lu17007和Lu17015的16S rDNA序列的任何一个(即序列表中SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中所示的那些核苷酸序列的任何一个)显示至少99.6%、优选至少99.8%、甚至更优选至少99.9%并且特别是100.0%核苷酸序列同一性。
根据进一步的实施方案,本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含DNA序列,所述DNA序列与菌株Lu16774、Lu17007和Lu17015的16S rDNA序列的任何一个(即序列表中SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中所示那些核苷酸序列的任何一个)显示100%核苷酸序列同一性。
根据进一步的实施方案,本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株的完整16S rDNA序列,在比对序列窗口内最佳比对后,与序列SEQ ID NO:1和SEQ ID NO:2中的至少一个具有至少99.6%同一性或与SEQ ID NO:3具有至少99.8%同一性;优选与序列SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中的至少一个具有至少99.8%同一性;更优选与序列SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中的至少一个具有至少99.9%同一性;甚至更优选与序列SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3中的至少一个具有高于99.9%同一性;特别是与序列SEQ ID NO:1、SEQ ID NO:2和SEQ IDNO:3中的至少一个具有100%同一性。
根据进一步的实施方案,本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株选自:
a)以保藏号DSM 26969保藏于DSMZ的Lu16774;
b)以保藏号DSM 26970保藏于DSMZ的Lu17007;
c)以保藏号DSM 26971保藏于DSMZ的Lu17015;和
d)包含DNA序列的菌株,所述DNA序列显示
d1)与DNA序列SEQ ID NO:4或SEQ ID NO:9具有至少99.6%核苷酸序列同一性;或
d2)与DNA序列SEQ ID NO:14具有至少99.8%核苷酸序列同一性;或
d3)与DNA序列SEQ ID NO:5或SEQ ID NO:10具有至少99.9%核苷酸序列同一性;或
d4)与DNA序列SEQ ID NO:15具有至少99.2%核苷酸序列同一性;或
d5)与DNA序列SEQ ID NO:6或SEQ ID NO:11具有至少99.2%核苷酸序列同一性;或
d6)与DNA序列SEQ ID NO:16具有至少99.8%核苷酸序列同一性;或
d7)与DNA序列SEQ ID NO:7或SEQ ID NO:12具有至少99.8%核苷酸序列同一性;或
d8)与DNA序列SEQ ID NO:17具有至少99.3%核苷酸序列同一性;或
d9)与DNA序列SEQ ID NO:8或SEQ ID NO:13具有100.0%核苷酸序列同一性;或
d10)与DNA序列SEQ ID NO:18具有至少99.9%核苷酸序列同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含与DNA序列SEQ ID NO:4或SEQ ID NO:9显示至少99.6%核苷酸序列同一性的dnaN DNA序列或包含与DNA序列SEQ ID NO:14显示至少99.8%核苷酸序列同一性的DNA序列。
根据进一步的实施方案,本发明的混合物是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株的完整dnaN DNA序列,在比对序列窗口内最佳比对后,与DNA序列SEQ ID NO:4和SEQ ID NO:9中的至少一个具有至少99.6%同一性或与SEQ ID NO:14具有至少99.8%同一性;优选与SEQ ID NO:14具有至少99.9%同一性;特别是与SEQ IDNO:14具有100%同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含DNA序列,所述DNA序列与菌株Lu16774、Lu17007和Lu17015的dnaN DNA序列的任何一个(即DNA序列SEQ ID NO:4、SEQ ID NO:9和SEQ ID NO:14中的任何一个)显示至少99.8%,特别是100.0%核苷酸序列同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含与DNA序列SEQ ID NO:5或SEQ ID NO:10显示至少99.9%核苷酸序列同一性的gyrB DNA序列或包含与DNA序列SEQ ID NO:15显示至少99.2%核苷酸序列同一性的DNA序列。
根据进一步的实施方案,本发明的混合物是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株的完整gyrB DNA序列,在比对序列窗口内最佳比对后,与DNA序列SEQ ID NO:5和SEQ ID NO:10中的至少一个显示至少99.9%同一性或与SEQ ID NO:15具有至少99.9%同一性;优选与SEQ ID NO:15具有至少99.9%同一性;特别是与SEQ IDNO:15具有100%同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含DNA序列,所述DNA序列与菌株Lu16774、Lu17007和Lu17015的gyrB DNA序列的任何一个(即那些DNA序列SEQ ID NO:5、SEQ ID NO:10和SEQ ID NO:15中的任何一个)显示100.0%核苷酸序列同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含与DNA序列SEQ ID NO:6或SEQ ID NO:11显示至少99.2%核苷酸序列同一性的recF DNA序列或包含与DNA序列SEQ ID NO:16显示至少99.8%核苷酸序列同一性的DNA序列。
根据进一步的实施方案,本发明的混合物是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株的完整recF DNA序列,在比对序列窗口内最佳比对后,与DNA序列SEQ ID NO:6和SEQ ID NO:11中的至少一个具有至少99.2%同一性或与SEQ ID NO:16具有至少99.8%同一性;优选与SEQ ID NO:16具有至少99.9%同一性;特别是与SEQ IDNO:16具有100%同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含DNA序列,所述DNA序列与菌株Lu16774、Lu17007和Lu17015的recF DNA序列的任何一个(即DNA序列SEQ ID NO:6、SEQ ID NO:11和SEQ ID NO:16中的任何一个)显示至少99.8%,特别是100.0%核苷酸序列同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含与DNA序列SEQ ID NO:7或SEQ ID NO:12显示至少99.8%核苷酸序列同一性的recN DNA序列或包含与DNA序列SEQ ID NO:17显示至少99.3%核苷酸序列同一性的DNA序列。
根据进一步的实施方案,本发明的混合物是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株的完整recN DNA序列,在比对序列窗口内最佳比对后,与DNA序列SEQ ID NO:7和SEQ ID NO:12中的至少一个具有至少99.8%同一性或与SEQ ID NO:17具有至少99.3%同一性;优选与SEQ ID NO:17具有至少99.6%同一性;特别是与SEQ IDNO:17具有100%同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含DNA序列,所述DNA序列与菌株Lu16774、Lu17007和Lu17015的recN DNA序列的任何一个(即DNA序列SEQ ID NO:7、SEQ ID NO:12和SEQ ID NO:17中的任何一个)显示至少99.8%,特别是100.0%核苷酸序列同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含与DNA序列SEQ ID NO:8或SEQ ID NO:13显示100.0%核苷酸序列同一性的rpoA DNA序列或包含与DNA序列SEQ ID NO:18显示至少99.9%核苷酸序列同一性的DNA序列。
根据进一步的实施方案,本发明的混合物是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株的完整rpoA DNA序列,在比对序列窗口内最佳比对后,与DNA序列SEQ ID NO:8和SEQ ID NO:13中的至少一个具有100.0%同一性或与SEQ ID NO:18具有至少99.9%同一性;优选与SEQ ID NO:17具有至少99.9%同一性;特别是与SEQ ID NO:18具有100%同一性。
本发明的混合物特别是一种包含作为组分1)的至少一种细菌菌株的混合物,所述细菌菌株包含DNA序列,所述DNA序列与菌株Lu16774、Lu17007和Lu17015的rpoA DNA序列的任何一个(即DNA序列SEQ ID NO:8、SEQ ID NO:13和SEQ ID NO:18中的任何一个)显示100.0%核苷酸序列同一性。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种分离的微生物,所述分离的微生物是类芽孢杆菌属(Paenibacillus)成员,具有以下菌株之一的至少一个鉴别性特征:
1)以保藏号DSM 26969保藏于DSMZ的类芽孢杆菌菌株Lu16774;
2)以保藏号DSM 26970保藏于DSMZ的类芽孢杆菌菌株Lu17007;或
3)以保藏号DSM 26971保藏于DSMZ的类芽孢杆菌菌株Lu17015。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一个类芽孢杆菌属(Paenibacillus)菌株,其选自:
1)以保藏号DSM 26969保藏于DSMZ的菌株Lu16774;
2)以保藏号DSM 26970保藏于DSMZ的菌株Lu17007;
3)以保藏号DSM 26971保藏于DSMZ的菌株Lu17015;和
4)具有所述菌株Lu16774、Lu17007和Lu17015之一的至少一个鉴别性特征的菌株。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种分离的微生物,其选自以下菌株:
1)以保藏号DSM 26969保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu16774;
2)以保藏号DSM 26970保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17007;和
3)以保藏号DSM 26971保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17015。
其显示出对抗至少一种植物病原体的拮抗活性,并且能够产生至少一种杀镰孢菌素;或其保留所述能力的突变菌株,即保留所述对抗至少一种植物病原体的拮抗活性和保留所述产生至少一种杀镰孢菌素的能力。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种微生物,其选自:
1)以保藏号DSM 26969保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu16774;
2)以保藏号DSM 26970保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17007;
3)以保藏号DSM 26971保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17015;
或具有所述菌株之一的全部鉴别性特征的突变菌株。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的如以上任意一个优选实施方案中所定义的至少一种类芽孢杆菌属(Paenibacillus)菌株的培养物基质。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的如以上任意一个优选实施方案中所定义的至少一种类芽孢杆菌属(Paenibacillus)菌株的无细胞提取物。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的如以上任意一个优选实施方案中所定义的至少一种类芽孢杆菌属(Paenibacillus)菌株的至少一种代谢物;优选所述至少一种代谢物是脂肽并且甚至更优选选自多粘菌素、八肽菌素、多肽菌素、pelgipeptin和杀镰孢菌素。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种杀镰孢菌素。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种式I的杀镰孢菌素
其中
R选自15-胍基-3-羟基十五烷酸(GHPD)和12-胍基十二烷酸(12-GDA);
X1是苏氨酸;
X2选自异亮氨酸和缬氨酸;
X3选自酪氨酸、缬氨酸、异亮氨酸和苯丙氨酸;
X4是苏氨酸;
X5选自谷氨酰胺和天冬酰胺;
X6是丙氨酸;并且
其中箭头定义R的羰基部分和氨基酸X1的氨基之间或一个氨基酸的羰基和相邻氨基酸的氨基之间的单个(酰胺)键,其中箭头的尖端表示连接到所述氨基酸X1或所述相邻氨基酸的氨基;并且
其中无箭头头部的单线定义X6的羰基和X1的羟基之间的单个(酯)键。
进一步的实施方案涉及混合物,所述混合物包含作为组分1)的至少一种式I.1的杀镰孢菌素
其中
R选自15-胍基-3-羟基十五烷酸(GHPD)和12-胍基十二烷酸(12-GDA);
X1是苏氨酸;
X2是异亮氨酸;
X3是酪氨酸;
X4是苏氨酸;
X5选自谷氨酰胺和天冬酰胺;
X6是丙氨酸;并且
其中箭头定义R的羰基部分和氨基酸X1的氨基之间或一个氨基酸的羰基和相邻氨基酸的氨基之间的单个(酰胺)键,其中箭头的尖端表示连接到所述氨基酸X1或所述相邻氨基酸的氨基;并且
其中无箭头头部的单线定义X6的羰基和X1的羟基之间的单个(酯)键。
根据进一步的实施方案,式I中的X1优选是L-苏氨酸。根据进一步的实施方案,式I中的X2优选是D-异亮氨酸或D-别-异亮氨酸。根据进一步的实施方案,式I中的X3优选是L-酪氨酸。根据进一步的实施方案,式I中的X4优选是D-别-苏氨酸。根据进一步的实施方案,式I中的X5优选是D-谷氨酰胺或D-天冬酰胺。根据进一步的实施方案,式I中的R优选是GHPD。
对于式I.1的杀镰孢菌素,式I.1也可以如下描绘:
其中
X选自-NH-(C=O)-CH2-CH(OH)-(CH2)12-NH-C(=NH)NH2和-NH-(C=O)-(CH2)11-NH-C(=NH)NH2;
R1是1-羟基乙基;
R2是1-甲基丙基(仲丁基);
R3是4-羟基苄基;
R4是1-羟基乙基;
R5选自氨基甲酰基乙基和氨基甲酰基甲基;
R6是甲基。
类似地,基于上述可选择的式I.1描述的优选实施方案如下:
该式I中的R1优选是(1S,2R)-1-羟基乙基。
该式I中的R2优选是(1R,2R)-1-甲基丙基或(1R,2S)-1-甲基丙基。
该式I中的R3优选是(S)-4-羟基-苄基。
该式I中的R4优选是(1S,2R)-1-羟基乙基。
该式I中的R5优选是(R)-氨基甲酰基乙基和(R)-氨基甲酰基甲基。
该式I中的X优选是-NH-(C=O)-CH2-CH(OH)-(CH2)12-NH-C(=NH)NH2。
根据进一步的实施方案,本发明进一步涉及混合物,所述混合物包含作为组分1的至少一种选自杀镰孢菌素1A和1B的杀镰孢菌素,其中所述杀镰孢菌素具有式I,其中R是GHPD,并且在杀镰孢菌素1A的情况中,X5是天冬酰胺,在杀镰孢菌素1B的情况中,X5是谷氨酰胺。
此外,可以以类似本领域已知的方法(Biopolymers 80(4),541,2005;J.PeptideSci.12S,219,2006;Tetrahedron Lett.47(48),8587-90,2006;Biopolymers 88(4),568,2007;ChemMedChem 7,871-882,2012)来合成式I的杀镰孢菌素,包括其中R是GHTD的那些杀镰孢菌素。
本发明还涉及包含本发明混合物的组合物,所述混合物包含作为组分1)的本发明的菌株、全培养液、无细胞提取物、培养物基质或式I的杀镰孢菌素及其盐,以及涉及所述组合物用于防治或抑制植物病原体或预防植物病原体感染或用于保护材料以对抗有害微生物的侵染破坏的用途;并且涉及相应的方法,包括用有效量的本发明的组合物、菌株、全培养液、无细胞提取物、培养物基质或式I的杀镰孢菌素及其盐,处理病原体、其栖息地或待接受抗病原体攻击保护的材料或植物,或土壤或繁殖材料。
以下的发明详述、权利要求和附图中公开了本发明的进一步的实施方案。
保藏的类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015的一个鉴别性特征是,它们能够产生至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别是产生杀镰孢菌素1A和1B(其是相应菌株的代谢物)。
因此,根据本发明的一个方面,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株能够产生至少一种式I的杀镰孢菌素,更优选产生杀镰孢菌素1A或1B,特别是产生杀镰孢菌素1A和1B;更优选在包含至少一种碳源和一种氮源(如本文中定义)的生长培养基中产生。
因此,根据本发明的另一个方面,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株在包含至少一种碳源和一种氮源(如本文中定义)的生长培养基中产生至少一种式I的杀镰孢菌素,更优选产生杀镰孢菌素1A或1B,特别是产生杀镰孢菌素1A和1B。
本发明的另一个实施方案涉及混合物,所述混合物包含作为组分1)的至少一种分离的微生物,其选自
1)以保藏号DSM 26969保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu16774;
2)以保藏号DSM 26970保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17007;和
3)以保藏号DSM 26971保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17015。
其显示出对抗至少一种植物病原体的拮抗活性,并且能够产生至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别是产生杀镰孢菌素1A和1B;或其保留所述能力的突变菌株,即保留所述对抗至少一种植物病原体的拮抗活性和保留所述产生至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别是产生杀镰孢菌素1A和1B的能力。
类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015或其突变菌株的进一步的鉴别性特征是,除了能够产生至少一种式I的杀镰孢菌素(优选选自杀镰孢菌素1A和1B,特别是产生杀镰孢菌素1A和1B)以外,它们还能够产生至少一种选自杀镰孢菌素A、杀镰孢菌素B、杀镰孢菌素C、杀镰孢菌素D、LI-F06a、LI-F06b和LI-F08b的杀镰孢菌素。
因此,根据本发明的进一步的方面,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株能够产生至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别是产生杀镰孢菌素1A和1B(如本文中公开的)并且能够产生至少一种选自杀镰孢菌素A、杀镰孢菌素B、杀镰孢菌素C、杀镰孢菌素D、LI-F06a、LI-F06b和LI-F08b的化合物。
根据本发明的进一步的方面,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株能够产生至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别是产生杀镰孢菌素1A和1B(如本文中公开的),并且能够产生至少三种选自杀镰孢菌素A、杀镰孢菌素B、杀镰孢菌素C、杀镰孢菌素D、LI-F06a、LI-F06b和LI-F08b的化合物。
类芽孢杆菌属(Paenibacillus)菌株的进一步的鉴别性特征是它们的抗真菌活性。特别地,发现这些菌株能有效对抗植物病原体感染,包括链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotinia sclerotiorum),其中链格孢属(Alternaria)物种优选选自茄链格孢(A.solani)和链格孢(A.alternata),特别是茄链格孢(A.solani)。
因此,根据本发明的进一步的方面,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株具有抗真菌活性,特别是对抗选自链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotiniasclerotiorum)的植物病原体,其中链格孢属(Alternaria)物种优选选自茄链格孢(A.solani)和链格孢(A.alternata),特别是茄链格孢(A.solani)。更特别地,本发明的类芽孢杆菌属(Paenibacillus)菌株具有对抗所述病原体中的至少两种或对抗全部四种的抗真菌活性。
根据本发明的进一步的方面,本发明的类芽孢杆菌属(Paenibacillus)菌株具有对抗植物病原体茄链格孢(Alternaria.solani)、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotinia sclerotiorum)的抗真菌活性。
可以使用所需的植物病原性真菌,例如链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotiniasclerotiorum),在体外对抗测试中,证实类芽孢杆菌属(Paenibacillus)菌株对抗植物病原体的拮抗活性,其中链格孢属(Alternaria)物种优选选自茄链格孢(A.solani)和链格孢(A.alternata),特别是茄链格孢(A.solani)。
作为这些植物病原性真菌的生长培养基,使用每升包含以下物质的ISP2培养基:10g麦芽提取物(Sigma Aldrich,70167);4g细菌培养用酵母提取物(Becton Dickinson,212750);4g葡萄糖一水合物(Sigma Aldrich,16301);20g琼脂(Becton Dickinson,214510),pH约7,重蒸水。作为PHYTIN的生长培养基,使用每升包含以下物质的V8培养基:200mL蔬菜汁,3g碳酸钙(Merck Millipore,1020660250);30g琼脂(Becton Dickinson,214510),pH 6.8,重蒸水。
将类芽孢杆菌属(Paenibacillus)菌株点种在琼脂平板的一侧。将含有一种活跃生长的植物病原体的琼脂块(约0.3cm2)放在平板中央。在约25℃温育7-14天后,检查植物病原体的生长,特别是针对抑制区。可以评价以下拮抗作用:通过评价无真菌区(抑制区)的直径,进行抗生作用的评分。通过将带有细菌菌株的平板上真菌病原体的生长直径与对照平板比较,对竞争作用评分。在细菌生长超过真菌病原体以及真菌寄生物生长超过病原体的情况中,可以记录真菌寄生作用。这可以通过显微镜来观察。
更特别的是,本发明涉及混合物,所述混合物包含作为组分1)的至少一种细菌菌株,所述菌株选自类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015以及任何具有保藏菌株的一个、二个、三个或更多个鉴别性特征的类芽孢杆菌属(Paenibacillus)菌株,其中鉴别性特征选自:
(a)如本文中公开的,对抗选自链格孢属(Alternaria)物种、灰葡萄孢(Botrytiscinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotinia sclerotiorum)的植物病原体的抗真菌活性,其中链格孢属(Alternaria)物种优选选自茄链格孢(A.solani)和链格孢(A.alternata),特别是茄链格孢(A.solani);
(b)如本文中公开的,产生至少一种式I的杀镰孢菌素,特别是杀镰孢菌素1A和/或1B的能力;
(c)如本文中公开的,产生至少一种选自杀镰孢菌素A、B、C、D、LI-F06a、LI-F06b和LI-F08b的杀镰孢菌素的能力;
(d)如本文中公开的,产生和分泌至少一种水解酶的能力,所述酶选自壳多糖酶、纤维素酶和淀粉酶。
更优选的是,所述类芽孢杆菌属(Paenibacillus)菌株在如本文中定义的包含至少一种碳源和一种氮源的生长培养基中具有(b)、(c)和(d)所述及的能力。
特别的是,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株具有保藏菌株的两个或更多个鉴别性特征,特别优选所述菌株具有至少特征(a)和(b)。例如,根据优选实施方案,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株(a)具有对抗选自链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthorainfestans)和核盘菌(Sclerotinia sclerotiorum)的植物病原体的抗真菌活性,其中链格孢属(Alternaria)物种优选选自茄链格孢(A.solani)和链格孢(A.alternata),特别是茄链格孢(A.solani);和(b)能够产生至少一种式I的杀镰孢菌素,并且特别是杀镰孢菌素1B。根据进一步优选的实施方案,本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株(a)具有对抗选自链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotinia sclerotiorum)的三种或全部植物病原体的抗真菌活性,其中链格孢属(Alternaria)物种优选选自茄链格孢(A.solani)和链格孢(A.alternata),特别是茄链格孢(A.solani);和(b)能够产生至少一种式I的杀镰孢菌素,更优选产生杀镰孢菌素1A或1B,特别是产生杀镰孢菌素1A和1B。
根据本发明的实施方案,以分离或基本上纯化的形式提供本发明混合物的组分1)的类芽孢杆菌属(Paenibacillus)菌株。
术语“分离的”或“基本上纯化的”用来表示本发明的菌株已经从天然环境取出并且已经进行分离或分开,并且至少60%不含,优选至少75%不含,并且更优选至少90%不含,甚至更优选至少95%不含并且最优选至少99%不含与其天然相关的其它组分。通过培养微生物单菌落获得的分离物是本发明的分离菌株的实例。
本发明的混合物包含作为组分1)的至少一种处于任何生理(例如活性或休眠)状态的类芽孢杆菌属(Paenibacillus)菌株。例如可以以冷冻、干燥或冻干或部分脱水(WO2008/002371中给出了产生部分脱水的生物体的程序)或孢子的形式提供休眠的类芽孢杆菌属(Paenibacillus)菌株。
根据本发明的实施方案,混合物包含作为组分1)的孢子形式的至少一种类芽孢杆菌属(Paenibacillus)菌株。
根据本发明的实施方案,混合物包含作为组分1)的全培养液形式的至少一种类芽孢杆菌属(Paenibacillus)菌株。
培养物优选是分离的或基本上纯化的培养物。
术语“分离的培养物”或“基本上纯化的培养物”是指本发明菌株的培养物,其不包含显著含量的通常在菌株生长和/或菌株通常可获自的天然栖息地中发现的其它物质。因此,此类“分离的培养物”或“基本上纯化的培养物”至少60%不含,优选至少75%不含,并且更优选至少90%不含,甚至更优选至少95%不含并且最优选至少99%不含通常在菌株生长的和/或通常菌株可获自的天然栖息地中发现的其它物质。此类“分离的培养物”或“基本上纯化的培养物”通常不包含含量足以干扰本发明菌株复制的任何其它微生物。
如本发明混合物中所使用的类芽孢杆菌属(Paenibacillus)菌株可以连续培养,或在分批工艺中或在补料分批或重复的补料分批工艺中不连续培养。已知的培养方法的综述参见Chmiel的教科书(Bioprozesstechnik 1.Einführung in dieBioverfahrenstechnik(Gustav Fischer Verlag,Stuttgart,1991))或Storhas的教科书(Bioreaktoren und periphere Einrichtungen(Vieweg Verlag,Braunschweig/Wiesbaden,1994))。
用于微生物培养的培养基必须以适合的方式满足特定菌株的需求。用于多种微生物的培养基描述在美国细菌学协会(American Society for Bacteriology)的手册“普通细菌学方法手册(Manual of Methods for General Bacteriology)”(Washington D.C.,USA,1981)中。
根据本发明可以使用的这些培养基通常包含一种或多种碳源、氮源、无机盐、维生素和/或微量元素。优选的碳源是糖,例如单糖、双糖或多糖。非常好的碳源例如是葡萄糖、果糖、甘露糖、半乳糖、核糖、山梨糖、核酮糖、乳糖、麦芽糖、蔗糖、棉子糖、淀粉或纤维素。糖也可以通过复杂化合物加入培养基中,例如糖蜜或其它来自糖精制的副产物。添加多种碳源的混合物也可以是有利的。其它可能的碳源是油和脂肪,例如大豆油、葵花油、花生油和椰子油,脂肪酸例如棕榈酸、硬脂酸或亚油酸,醇例如甘油、甲醇或乙醇,以及有机酸例如乙酸或乳酸。氮源通常是有机或无机氮化合物或含有这些化合物的物质。氮源的实例包括氨气或铵盐(例如硫酸铵、氯化铵、磷酸铵、碳酸铵或硝酸铵)、硝酸盐、尿素、氨基酸或复杂氮源,例如玉米浆、大豆粉、大豆蛋白质、酵母提取物、肉膏等。氮源可以分开使用或作为混合物使用。可以存在于培养基中的无机盐化合物包括钙、镁、钠、钴、钼、钾、锰、锌、铜和铁的氯化物、磷酸盐或硫酸盐。无机含硫化合物,例如硫酸盐、亚硫酸盐、连二亚硫酸盐、连四硫酸盐、硫代硫酸盐、硫化物,以及有机硫化合物,例如硫醇(mercaptan)和硫醇(thiol),可以用作硫源。磷酸、磷酸二氢钾或磷酸氢二钾或相应的含钠盐可以用作磷源。可以将螯合剂加入培养基中,以将金属离子保持在溶液中。特别适合的螯合剂包括二羟基酚,例如儿茶酚或原儿茶酸,或有机酸,例如柠檬酸。根据本发明使用的发酵培养基还可以含有其它生长因子,例如维生素或生长促进剂,其包括例如生物素、核黄素、硫胺素、叶酸、烟酸、泛酸和吡哆醇。生长因子和盐常常来自培养基的复杂成分,例如酵母提取物、糖蜜、玉米浆等。此外,可以将适合的前体加入培养基中。培养基中化合物的确切组成强烈取决于特定的试验并且必须针对每种特定的情况单独确定。关于培养基优化的信息可以在教科书“应用微生物学、生理学、实用方法(Applied Microbiol.Physiology,A Practical Approach)”(Publ.P.M.Rhodes,P.F.Stanbury,IRL Press(1997),第53-73页,ISBN 0 19 9635773)中找到。生长培养基也可以获自商业供应商,例如Standard 1(Merk)或BHI(脑心浸液,DIFCO)等。
根据本发明可以使用的优选生长培养基包含一种或多种碳源,选自L-阿拉伯糖、N-乙酰基-D-葡糖胺、D-半乳糖、L-天冬氨酸、D-海藻糖、D-甘露糖、甘油、D-葡糖酸、D-木糖、D-甘露醇、D-核糖、D-果糖、α-D-葡萄糖、麦芽糖、D-蜜二糖、胸苷、α-甲基-D-半乳糖苷、α-D-乳糖、乳果糖、蔗糖、尿苷、α-羟基戊二酸-γ-内酯、β-甲基-D-葡糖苷、核糖醇、麦芽三糖、2-脱氧腺苷、腺苷、柠檬酸、粘酸、D-纤维二糖、肌苷、L-丝氨酸、L-丙氨酰基-甘氨酸、D-半乳糖醛酸、α-环糊精、β-环糊精、糊精、菊粉、果胶、苦杏仁苷、龙胆二糖、乳糖醇、D-松三糖、α-甲基-D-葡糖苷、β-甲基-D-半乳糖苷、β-甲基-D-木糖苷、帕拉金糖、D-棉子糖、水苏糖、松二糖、γ-氨基丁酸、D-葡糖胺、D-乳酸、L-赖氨酸、3-羟基2-丁酮;和一种或多种氮源,选自氨、亚硝酸盐、硝酸盐、L-丙氨酸、L-天冬酰胺、L-天冬氨酸、L-谷氨酸、L-谷氨酰胺、甘氨酸、氨基酸二聚物:Ala-Asp、AlaGln、Ala-Glu、Ala-His、Gly-Gln、Gly-Glu、Gly-Met和Met-Ala;特别是硝酸盐。这些培养基可以补充无机盐和维生素和/或微量元素。所述菌株能够在这些生长培养基中产生杀镰孢菌素1A和1B。
通过加热(在2.0巴和121℃20分钟)或通过无菌过滤,将培养基的所有组分灭菌。组分可以一起灭菌,或如果需要,分开灭菌。培养基的所有组分可以在培养开始时就存在,或任选可以连续或通过分批补料来添加。
培养温度通常为15℃至36℃,优选25℃至33℃,并且在试验过程中可以保持恒定或可以改变。培养基的pH值范围应当为5至8.5,优选约7.0。可以通过添加碱性化合物,例如氢氧化钠、氢氧化钾、氨或氨水,或酸性化合物,例如磷酸或硫酸,控制生长过程中用于生长的pH值。消泡剂,例如脂肪酸多元醇酯,可以用于控制泡沫。为了维持质粒的稳定性,具有选择性作用的适合物质,例如抗生素,可以加入培养基。将氧或含氧气体混合物,例如环境空气,加入培养物中,以维持需氧条件。培养温度通常为20℃至45℃。持续培养直至形成最大所需产物。这通常在10小时至160小时内实现。
特别地,本发明的菌株可以在多种标准微生物学培养基,例如Luria-Bertani肉汤(LB)、胰胨豆胨肉汤(TSB)、酵母提取物/麦芽提取物/葡萄糖肉汤(YMG,ISP2)中,在液体培养基或在皮氏培养皿上琼脂固化的培养基中,在15℃至36℃,培养18至360小时。通气可能是需要的。可以将细菌细胞(营养细胞和孢子)洗涤并且浓缩(例如通过在7,000×g,在约15至30℃温度,离心约15分钟)。
本发明还涉及包含作为组分1)的培养物基质的混合物,所述培养物基质通过以下方式可获得:在培养基中培养至少一种如以上任意一个优选实施方案中所定义的类芽孢杆菌属(Paenibacillus)菌株并且从培养液中分离出培养基(因此,从全培养液中除去生长在培养基中的细胞后的剩余物),例如,全培养液的上清液,即通过离心、过滤、沉淀或本领域公知的其它方式,除去生长在培养液中的细胞和其它碎片时剩余的液体培养液。例如,通过在约5,000至20,000×g(更优选在约15,000×g),在约2至30℃的温度(更优选在4至20℃的温度),离心约10至60分钟(更优选约15至30分钟),可以获得上清液。
这样的培养物基质含有培养菌株产生的农药代谢物。
本发明还涉及混合物,所述混合物包含作为组分1)的至少从如以上任意一个优选实施方案中所定义的类芽孢杆菌属(Paenibacillus)菌株可获得的无细胞提取物。为了产生无细胞提取物,可以如以上所述培养菌株。还可以通过高频超声波,通过高压(例如,在弗氏压碎器中),通过渗压裂解(osmolysis),通过去污剂、水解酶或有机溶剂的作用,通过均质机或通过所列方法中的几种的组合,来破坏细胞。提取优选使用有机溶剂或溶剂混合物来进行,更优选使用醇(例如甲醇、乙醇、正丙醇、2-丙醇等),甚至更优选使用2-丙醇(例如以与培养物体积1:1的比例)。通过添加盐,例如NaCl,可以增强相分离。可以收集有机相,并且可以通过常规蒸馏和/或干燥,除去溶剂或溶剂混合物,接着重悬浮于甲醇中,并且过滤。
此类提取物含有培养菌株产生的农药代谢物。
可以根据常规方法从此类培养基或提取物回收本发明菌株特异性的农药代谢物,特别是在已经按照以上所述培养了本发明的菌株后。
本发明的方法可以进一步包括回收单个农药代谢物的步骤。
术语“回收”包括从培养物基质或无细胞提取物提取、收获、分离或纯化化合物。可以根据本领域已知的任何常规分离或纯化方法来进行化合物的回收,所述方法包括但不限于,使用常规树脂(例如阴离子或阳离子交换树脂、非离子型吸附树脂等)处理、使用常规吸附剂(例如活性炭、硅酸、硅胶、纤维素、矾土等)处理、改变pH、溶剂提取(例如使用常规溶剂,例如醇、乙酸乙酯、己烷等)、蒸馏、透析、过滤、浓缩、结晶、重结晶、pH调节、冻干等。例如可以通过首先除去微生物,从培养物基质回收代谢物。然后将剩余的培养液通过阳离子交换树脂,以除去不需要的阳离子,然后通过阴离子交换树脂,以除去不需要的无机阴离子和有机酸。
因此,本发明还涉及混合物,所述混合物包含作为组分1)的微生物的全培养液,所述全培养液包含至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别的是所述全培养液包含杀镰孢菌素1A和1B。
根据进一步的实施方案,本发明还涉及混合物,所述混合物包含作为组分1)的微生物类芽孢杆菌属(Paenibacillus)菌株的全培养液,所述全培养液包含至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别的是所述全培养液包含杀镰孢菌素1A和1B。
所述杀镰孢菌素型代谢物分泌入能够产生的相应微生物的培养物基质。
因此,本发明还涉及混合物,所述混合物包含作为组分1)的微生物的培养物基质和/或无细胞提取物,所述培养物基质和/或无细胞提取物包含至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别的是所述培养物基质和/或无细胞提取物包含杀镰孢菌素1A和1B。
根据进一步的实施方案,本发明还涉及混合物,所述混合物包含作为组分1)的类芽孢杆菌属(Paenibacillus)微生物的培养物基质和/或无细胞提取物,所述培养物基质和/或无细胞提取物包含至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别的是所述培养物基质和/或无细胞提取物包含杀镰孢菌素1A和1B。
根据进一步的实施方案,本发明还涉及混合物,所述混合物包含作为组分1)的如以上任意一个优选实施方案中所定义的至少一种本发明类芽孢杆菌属(Paenibacillus)菌株的培养物基质和/或无细胞提取物,所述培养物基质和/或无细胞提取物包含至少一种式I的杀镰孢菌素,优选选自杀镰孢菌素1A和1B,特别的是所述培养物基质和/或无细胞提取物包含杀镰孢菌素1A和1B。
本发明进一步涉及农用化学组合物,其包含如下定义的助剂和本发明的混合物,所述混合物包含至少一种如以上任意一个优选实施方案中所定义的细菌菌株、全培养液、无细胞提取物、培养物基质和/或式I的杀镰孢菌素。
如本文中使用的,涉及本发明产品(微生物菌株、试剂或制剂)的“组合物”是指成分的组合,其中“配制”是,针对待加入形成制剂的成分的组合,使用配方(formula)(例如制法(recipe))的过程。此类组合物在本文中也称为制剂。
混合物包含作为组分1)的如以上任意一个优选实施方案中所定义的细菌菌株、全培养液、无细胞提取物、培养物基质、式I的杀镰孢菌素,和作为组分2)的如以上任意一个优选实施方案中所定义的至少一种化学农药,并且本发明的组合物适合作为抗真菌剂或杀真菌剂。它们的出众之处在于:突出的对抗广谱植物病原性真菌的有效性,所述真菌包括土传真菌,特别是源自根肿菌纲(Plasmodiophoromycetes)、Peronosporomycetes(与卵菌纲(Oomycetes)同义)、壶菌纲(Chytridiomycetes)、接合菌纲(Zygomycetes)、子囊菌纲(Ascomycetes)、担子菌纲(Basidiomycetes)和半知菌纲(Deuteromycetes)(与半知真菌(Fungi imperfecti)同义)的真菌。一些是系统有效的并且它们可以作为叶杀真菌剂、拌种杀真菌剂和土壤杀真菌剂用于作物保护中。此外,它们适用于防治有害真菌,特别是出现在植物的木材或根部的那些真菌。
本发明的混合物和组合物,对于多种栽培植物上以及在这些植物的植物繁殖材料(例如种子)和作物材料上的多种植物病原性真菌防治,各自特别重要,所述植物例如谷物,例如小麦、黑麦、大麦、黑小麦、燕麦或稻;甜菜,例如糖用甜菜或饲料甜菜;水果,例如仁果、核果或无核小果(soft fruit),例如苹果、梨、李、桃、杏、樱桃、草莓、树莓、黑莓或醋栗;豆科植物,例如小扁豆、豌豆、苜蓿或大豆;油料植物,例如油菜、芥菜、橄榄、向日葵、椰子、可可豆、蓖麻油植物、油棕、花生或大豆;葫芦科植物,例如南瓜、黄瓜或甜瓜;纤维植物,例如棉花、亚麻、大麻或黄麻;柑桔水果,例如橙、柠檬、葡萄柚或柑橘;蔬菜,例如菠菜、莴苣、芦笋、卷心菜、胡萝卜、洋葱、番茄、马铃薯、葫芦或红辣椒;月桂科植物,例如鳄梨、肉桂或樟脑;能量和原料植物,例如玉米、大豆、油菜、甘蔗或油棕;玉米;烟草;坚果;咖啡;茶;香蕉;藤本植物(食用葡萄(table grapes)和葡萄汁葡萄);蛇麻草;坪草;甜叶(也称为甜叶菊);天然橡胶植物或观赏植物和森林植物,例如花、灌木、阔叶树或常绿树,例如针叶树。
优选本发明的混合物和组合物用于防治田间作物(例如马铃薯、糖用甜菜、烟草、小麦、黑麦、大麦、燕麦、稻、玉米、棉花、大豆、油菜、豆、向日葵、咖啡或甘蔗);水果;藤本植物;观赏植物;或蔬菜(例如黄瓜、番茄、菜豆或南瓜)上的多种真菌。
术语“植物繁殖材料”应当理解为表示植物的所有生殖部分例如种子和可以用于繁殖植物的营养性植物材料,例如插枝和块茎(例如马铃薯)。这包括植物的种子、根、果实、块茎、球茎、根茎、枝、芽和其它部分,包括秧苗和幼苗,其可以在萌发后或从土壤萌出后移栽。也可以在移栽前通过浸没或倾倒进行整体或部分处理来保护这些幼苗。
优选用本发明的菌株、全培养液、无细胞提取物、培养物基质、式I的杀镰孢菌素和组合物处理植物繁殖材料,用于防治谷物,例如小麦、黑麦、大麦和燕麦;稻、玉米、棉花和大豆上的多种真菌。
术语“栽培的植物”应当理解为包括已经通过育种、诱变或遗传工程修饰的植物,包括但不限于销售的或研发中的农业生物技术产品(参考http://cera-gmc.org/,参见其中的GM作物数据库)。遗传修饰的植物是其遗传物质已经通过使用重组DNA技术修饰的植物,该修饰在自然环境下通过杂交育种、突变或自然重组不能容易获得。通常,将一个或多个基因整合至遗传修饰植物的遗传物质中,以改善植物的某些特性。此类遗传修饰还包括但不限于蛋白质、寡肽或多肽的靶向翻译后修饰,例如,通过糖基化或聚合物添加(例如异戊二烯化、乙酰化或法尼基化部分或PEG部分)。
已经通过育种、诱变或遗传工程修饰的植物,例如作为常规育种或遗传工程方法的结果,已被赋予对特定类别的除草剂施用的耐受性,所述除草剂例如植物生长素除草剂,例如麦草畏(dicamba)或2,4-D;漂白剂除草剂,例如羟基苯基丙酮酸双加氧酶(HPPD)抑制剂或八氢番茄红素去饱和酶(PDS)抑制剂;乙酰乳酸合酶(ALS)抑制剂,例如磺酰脲或咪唑啉酮;烯醇丙酮莽草酸-3-磷酸合酶(EPSPS)抑制剂,例如草甘膦(glyphosate);谷氨酰胺合成酶(GS)抑制剂,例如草铵膦(glufosinate);原卟啉原-IX氧化酶抑制剂;脂质生物合成抑制剂,例如乙酰CoA羧化酶(ACCase)抑制剂;或苯腈(即溴苯腈或碘苯腈)除草剂。此外,通过多重遗传修饰,可以使得植物抵抗多种类别的除草剂,例如抵抗草甘膦和草铵膦两者或抵抗草甘膦和来自另一种类别的除草剂,例如ALS抑制剂、HPPD抑制剂、生长素除草剂或ACCase抑制剂。这些除草剂抗性技术例如描述于Pest Managem.Sci.61,2005,246;61,2005,258;61,2005,277;61,2005,269;61,2005,286;64,2008,326;64,2008,332;WeedSci.57,2009,108;Austral.J.Agricult.Res.58,2007,708;Science 316,2007,1185;以及其中引用的参考文献。已经通过常规育种(诱变)方法赋予了几种栽培植物对除草剂的耐受性,例如,对咪唑啉酮(例如咪草啶酸(imazamox))耐受的夏油菜(Canola,BASF SE,德国),或对磺酰脲(例如苯磺隆)耐受的向日葵(DuPont,USA)。遗传工程方法已经用于赋予栽培植物(例如大豆、棉花、玉米、甜菜和油菜)对除草剂(例如草甘膦和草铵膦)的耐受性,其中一些可以以商品名(草甘膦-耐受,Monsanto,U.S.A.)、(咪唑啉酮耐受,BASF SE,德国)和(草铵膦-耐受,Bayer CropScience,德国)商购可获得。
此外,还涵盖这样的植物,所述植物通过使用重组DNA技术而能够合成一种或多种杀虫蛋白质,特别是:已知来自细菌芽孢杆菌属(Bacillus),特别是来自苏云金芽孢杆菌(Bacillus thuringiensis)的杀虫蛋白质,例如δ-内毒素,例如CryIA(b)、CryIA(c)、CryIF、CryIF(a2)、CryIIA(b)、CryIIIA、CryIIIB(b1)或Cry9c;植物杀虫蛋白质(VIP),例如VIP1、VIP2、VIP3或VIP3A;定殖线虫的细菌(例如光杆状菌属(Photorhabdus)物种或致病杆菌属(Xenorhabdus)物种)的杀虫蛋白质;动物产生的毒素,例如蝎子毒素、蜘蛛毒素、黄蜂毒素,或其它昆虫特异性神经毒素;真菌产生的毒素,例如链霉菌纲(Streptomycetes)毒素;植物凝集素(lectin),例如豌豆或大麦凝集素;凝集素(agglutinin);蛋白酶抑制剂,例如胰蛋白酶抑制剂、丝氨酸蛋白酶抑制剂、马铃薯块茎储藏蛋白(patatin)、半胱氨酸蛋白酶抑制剂(cystatin)或木瓜蛋白酶抑制剂;核糖体-失活蛋白(RIP),例如蓖麻毒素、玉米-RIP、相思豆毒素、软瓜蛋白、皂草毒蛋白或异株泻根毒蛋白;类固醇代谢酶,例如3-羟基类固醇氧化酶、蜕皮类固醇-IDP-糖基-转移酶、胆固醇氧化酶、蜕皮素抑制剂或HMG-CoA-还原酶;离子通道阻断剂,例如钠或钙通道的阻断剂;保幼激素酯酶;利尿激素受体(棉铃虫激肽(helicokinin)受体);芪合酶、联苄合酶、壳多糖酶或葡聚糖酶。在本发明的上下文中,这些杀虫蛋白质或毒素也应当明确地被理解为前毒素、杂合蛋白质、截短的或其它修饰的蛋白质。杂合蛋白质的特征在于蛋白质结构域的新组合(参见,例如WO 02/015701)。此类毒素或能够合成此类毒素的遗传修饰植物的进一步的实例公开于例如EP A 374753、WO 93/007278、WO 95/34656、EP-A 427 529、EP-A 451 878、WO 03/18810和WO 03/52073中。用于产生此类遗传修饰植物的方法通常是本领域技术人员已知的并且描述于例如上述出版物中。遗传修饰植物中含有的这些杀虫蛋白质赋予产生这些蛋白质的植物对来自所有节肢动物分类学群组的有害害物的耐受性,特别是甲虫(鞘翅目(Coeloptera))、双翅昆虫(双翅目(Diptera))和蛾(鳞翅目(Lepidoptera))以及线虫(线虫钢(Nematoda))。能够合成一种或多种杀虫蛋白质的遗传修饰植物例如描述于上述出版物中,并且其中一些是商购可获得的,例如(产生Cry1Ab毒素的玉米栽培品种)、Plus(产生Cry1Ab和Cry3Bb1毒素的玉米栽培品种)、(产生Cry9c毒素的玉米栽培品种)、RW(产生Cry34Ab1、Cry35Ab1和酶膦丝菌素-N-乙酰转移酶[PAT]的玉米栽培品种);33B(产生Cry1Ac毒素的棉花栽培品种)、I(产生Cry1Ac毒素的棉花栽培品种)、II(产生Cry1Ac和Cry2Ab2毒素的棉花栽培品种);(产生VIP-毒素的棉花栽培品种);(产生Cry3A毒素的马铃薯栽培品种); Bt11(例如CB)和来自Syngent Seeds SAS法国的Bt176(产生Cry1Ab毒素和PAT酶的玉米栽培品种)、来自Syngenta Seeds SAS法国的MIR604(产生修饰形式的Cry3A毒素的玉米栽培品种,参考WO 03/018810)、来自Monsanto Europe S.A.比利时的MON 863(产生Cry3Bb1毒素的玉米栽培品种)、来自Monsanto Europe S.A.比利时的IPC 531(产生修饰形式的Cry1Ac毒素的棉花栽培品种)和来自Pioneer Overseas Corporation比利时的1507(产生Cry1F毒素和PAT酶的玉米栽培品种)。
此外,还涵盖这样的植物,所述植物通过使用重组DNA技术而能够合成一种或多种蛋白质来提高这些植物对细菌、病毒或真菌病原体的抗性或耐受性。此类蛋白质的实例是所谓的“病原相关蛋白质”(PR蛋白质,参见,例如EP-A 392 225)、植物疾病抗性基因(例如马铃薯栽培品种,其表达对抗源自墨西哥野生马铃薯Solanum bulbocastanum的致病疫霉(Phytophthora infestans)的抗性基因)或T4-溶菌酶(例如能够合成这些蛋白质的马铃薯栽培品种,具有提高的对抗细菌例如解淀粉欧文氏菌(Erwinia amylvora)的抗性)。用于产生此类遗传修饰植物的方法通常是本领域技术人员已知的并且描述于例如上述出版物中。
此外,还涵盖这样的植物,所述植物通过使用重组DNA技术而能够合成一种或多种蛋白质以提高生产力(例如生物量生产、粮食产量、淀粉含量、油含量或蛋白质含量),对干旱、盐度或其它生长限制环境因素的耐受性、或对这些植物的害物和真菌、细菌或病毒病原体的耐受性。
此外,还涵盖这样的植物,所述植物通过使用重组DNA技术而含有改变含量的内含物质或新的内含物质,特别是以改善人或动物营养,例如产生促进健康的长链ω-3脂肪酸或不饱和ω-9脂肪酸的油料作物(例如油菜,DOW Agro Sciences,加拿大)。
本发明的混合物和组合物特别适用于防治以下植物疾病:
观赏植物、蔬菜(例如,白锈菌(A.candida))和向日葵(例如婆罗门参白锈(A.tragopogonis))上的白锈菌属(Albugo)物种(白锈病);蔬菜,油菜(芸苔链格孢(A.brassicola或A.brassicae))、糖用甜菜(细链格孢(A.tenuis))、水果、稻、大豆、马铃薯(例如茄链格孢(A.solani)或链格孢(A.alternata))、番茄(例如茄链格孢(A.solani)或链格孢(A.alternata))和小麦上的链格孢属(Alternaria)物种(链格孢叶斑病);糖用甜菜和蔬菜上的丝囊霉属(Aphanomyces)物种;谷物和蔬菜上的壳二孢属(Ascochyta)物种,例如小麦上的小麦壳二孢(A.tritici)(炭疽病)和大麦上的大麦壳二孢(A.hordei);平脐蠕孢属(Bipolaris)物种和内脐蠕孢属(Drechslera)物种(有性型:旋孢腔菌属(Cochliobolus)物种),例如玉米上的小斑病(玉蜀黍内脐蠕孢(D.maydis))或大斑病(玉米平脐蠕孢(B.zeicola)),例如谷物上的斑枯病(麦根腐平脐蠕孢(B.sorokiniana))以及例如稻和坪草上的稻平脐蠕孢(B.oryzae);谷物上(例如小麦或大麦上)的禾本科布氏白粉菌(Blumeria graminis)(前称禾生白粉菌(Erysiphe graminis)(白粉病);水果和无核小果(例如草莓)、蔬菜(例如莴苣、胡萝卜、芹菜和卷心菜)、油菜、花、藤本植物、森林植物和小麦上的灰葡萄孢(Botrytis cinerea)(有性型:富氏葡萄核盘菌(Botryotinia fuckeliana):灰霉病);莴苣上的莴苣盘梗霉(Bremia lactucae)(霜霉病);阔叶树和常绿树上的长喙壳属(Ceratocystis)物种(同义词:Ophiostoma)(腐病或枯萎),例如榆树上的榆长喙壳(C.ulmi)(荷兰榆树病(Dutch elm disease));玉米(例如灰斑病:玉蜀黍尾孢(C.zeae-maydis))、稻、糖用甜菜(例如甜菜生尾孢(C.beticola))、甘蔗、蔬菜、咖啡、大豆(例如大豆尾孢(C.sojina)或菊池尾孢(C.kikuchii))和稻上的尾孢属(Cercospora)物种(尾孢菌叶斑(Cercospora leaf spot));番茄(例如黄枝孢(C.fulvum):叶霉病)和谷物上的枝孢属(Cladosporium)物种,例如小麦上的多主枝孢(C.herbarum)(黑穗病(black ear));谷物上的麦角菌(Claviceps purpurea)(麦角病(ergot));玉米(炭色旋孢腔菌(C.carbonum))、谷物(例如禾旋孢腔菌(C.sativus),无性型:麦根腐平脐蠕孢(B.sorokiniana))和稻(例如宫部旋孢腔菌(C.miyabeanus),无性型:稻长蠕孢(H.oryzae))上的旋孢腔菌属(Cochliobolus)(无性型:平脐蠕孢属(Bipolaris)的长蠕孢属(Helminthosporium))物种(叶斑病);棉花(例如棉刺盘孢(C.gossypii))、玉米(例如禾生刺盘孢(C.graminicola):炭疽病茎腐)、无核小果、马铃薯(例如C.coccodes:黑斑病)、菜豆(例如豆刺盘孢(C.lindemuthianum))和大豆(例如平头刺盘孢(C.truncatum)或盘长孢状刺盘孢(C.gloeosporioides))上的刺盘孢属(Colletotrichum)(有性型:小丛壳属(Glomerella))物种(炭疽病);伏革菌属(Corticium)物种,例如稻上的笹木伏革菌(C.sasakii)(纹枯病(sheath blight));大豆和观赏植物上的山扁豆生棒孢菌(Corynespora cassiicola)(叶斑病);孔雀斑病菌属(Cycloconium)物种,例如橄榄树上的油橄榄孔雀斑病菌(C.oleaginum);果树上、藤本植物上(例如鹅掌楸柱孢(C.liriodendri),有性型:鹅掌楸新丛赤壳(Neonectria liriodendri):黑脚病(Black Foot Disease))和观赏植物上的柱孢属(Cylindrocarpon)物种(例如果树溃疡病或幼藤衰败,有性型:丛赤壳属(Nectria)或新丛赤壳属(Neonectria)物种);大豆上的Dematophora necatrix(有性型:褐座坚壳(Rosellinia necatrix)(根和茎腐病);间座壳属(Diaporthe)物种,例如大豆上的菜豆间座壳(D.phaseolorum)(猝倒);玉米、谷物例如大麦(例如大麦网斑内脐蠕孢(D.teres),网斑病(net blotch))和小麦(例如偃麦草内脐蠕孢(D.tritici-repentis):黄褐斑病(tanspot))、稻和坪草上的内脐蠕孢属(Drechslera,同义词:长蠕孢属(Helminthosporium),有性型:核腔菌属(Pyrenophora))物种;藤本植物上的Esca(梢枯病(dieback)、干枯病(apoplexy)),由层卧孔菌(Fomitiporia punctata)(同义词:斑孔木层孔菌(Phellinuspunctata))、地中海层卧孔菌(F.mediterranea)、厚孢小褐球壳(Phaeomoniellachlamydospora)(之前Phaeoacremonium chlamydosporum)、褐枝顶孢霉(Phaeoacremoniumaleophilum)和/或Botryosphaeria obtusa引起;仁果(梨痂囊腔菌(E.pyri))、无核小果(E.veneta:炭疽病)和藤本植物(痂囊腔菌(E.ampelina):炭疽病)上的痂囊腔菌属(Elsinoe)物种;稻上的稻叶黑粉菌(Entyloma oryzae)(叶黑粉病(leaf smut));小麦上的附球菌属(Epicoccum)物种(黑霉病(black mold));糖用甜菜(甜菜白粉菌(E.betae))、蔬菜(例如豌豆白粉菌(E.pisi)),例如葫芦(例如二孢白粉菌(E.cichoracearum))、卷心菜、油菜(例如十字花科白粉菌(E.cruciferarum))上的白粉菌属(Erysiphe)物种(白粉病);果树、藤本植物和观赏木上的Eutypa lata(弯孢壳(Eutypa)溃疡病或梢枯病,无性型:Cytosporina lata,同义词:Libertella blepharis);玉米(例如大斑凸脐蠕孢(E.turcicum))上的凸脐蠕孢属(Exserohilum)(同义词:长蠕孢属(Helminthosporium))物种;多种植物上的镰孢属(Fusarium)(有性型:赤霉属(Gibberella))物种(萎蔫、根或茎腐病),例如谷物(例如小麦或大麦)上的禾本科镰孢(F.graminearum)或大刀镰孢(F.culmorum)(根腐病、疮痂病(scab)或赤霉病(head blight))、番茄上的尖镰孢(F.oxysporum)、大豆上的腐皮镰孢(F.solani)(大豆专化型(f.sp.glycines),同义词:F.virguliforme)和F.tucumaniae及F.brasiliense(均引起猝死症)和玉米上的轮枝镰孢(F.verticillioides);谷物(例如小麦或大麦)和玉米上的禾顶囊壳(Gaeumannomycesgraminis)(全蚀病(take-all));谷物(例如玉蜀黍赤霉(G.zeae)和稻(例如藤仓赤霉(G.fujikuroi):恶苗病(Bakanae disease))上的赤霉属(Gibberella)物种;藤本植物、仁果和其它植物上的围小丛壳(Glomerella cingulata)和棉花上的棉小丛壳(G.gossypii);稻上的颗粒染色复合体(Grainstaining complex);藤本植物上的葡萄球座菌(Guignardiabidwellii)(黑腐病(black rot));蔷薇科植物和杜松上的胶锈菌属(Gymnosporangium)物种,例如梨上的G.sabinae(锈病);玉米、谷物和稻上的长蠕孢属(Helminthosporium)物种(同义词:内脐蠕孢属(Drechslera),有性型:旋孢腔菌属(Cochliobolus));驼孢锈菌属(Hemileia)物种,例如咖啡上的咖啡驼孢锈菌(H.vastatrix)(咖啡叶锈病);藤本植物上的褐斑拟棒束孢(Isariopsis clavispora)(同义词:Cladosporium vitis);大豆和棉花上的菜豆壳球孢(Macrophomina phaseolina)(同义词:Macrophomina phaseoli)(根茎腐病);谷物(例如小麦或大麦)上的Microdochium nivale(同义词:雪腐镰孢(Fusarium nivale))(粉雪霉病(pink snow mold));大豆上的扩散叉丝壳(Microsphaera diffusa)(白粉病);核果和其它蔷薇科植物上的链核盘菌属(Monilinia)物种,例如核果链核盘菌(M.laxa)、M.fructicola和果生链核盘菌(M.fructigena)(花腐枝枯病、褐腐病);谷物、香蕉、无核小果和落花生上的球腔菌属(Mycosphaerella)物种,例如小麦上的禾生球腔菌(M.graminicola)(无性型:小麦壳针孢(Septoria tritici),壳针孢斑枯病)或香蕉上的斐济球腔菌(M.fijiensis)(黑叶斑病(black Sigatoka disease));卷心菜(例如芸苔霜霉(P.brassicae))、油菜(例如寄生霜霉(P.parasitica))、洋葱(例如葱霜霉(P.destructor))、烟草(烟草霜霉(P.tabacina))和大豆(例如东北霜霉(P.manshurica))上的霜霉属(Peronospora)物种(霜霉病);大豆上的豆薯层锈菌(Phakopsora pachyrhizi)和山马蝗层锈菌(P.meibomiae)(大豆锈病);瓶霉属(Phialophora)物种,例如藤本植物(例如P.tracheiphila和P.tetraspora)和大豆(例如P.gregata:茎腐病)上;油菜和卷心菜上的黑胫茎点霉(Phoma lingam)(根茎腐病)和糖用甜菜上的甜菜茎点霉(P.betae)(根腐病、叶斑病和猝倒);向日葵、藤本植物(例如葡萄生拟茎点霉(P.viticola):蔓和叶斑病)和大豆(例如茎腐病:P.phaseoli,有性型:菜豆间座壳(Diaporthe phaseolorum))上的拟茎点霉(Phomopsis)物种;玉米上的玉蜀黍节壶菌(Physoderma maydis)(褐斑病);在多种植物上的疫霉属(Phytophthora)物种(萎蔫、根、叶、果实和茎腐病),例如红辣椒和葫芦(例如辣椒疫霉(P.capsici))、大豆(例如大雄疫霉(P.megasperma),同义词:大豆疫霉(P.sojae))、马铃薯和番茄(例如致病疫霉(P.infestans):晚疫病)和阔叶树(例如P.ramorum:栎树猝死);卷心菜、油菜、萝卜和其它植物上的芸苔根肿菌(Plasmodiophora brassicae)(根肿病(club root));单轴霉属(Plasmopara)物种,例如藤本植物上的葡萄生单轴霉(P.viticola)(葡萄霜霉病)和向日葵上的霍尔斯单轴霉(P.halstedii);蔷薇科植物、蛇麻、仁果和无核小果上的叉丝单囊壳属(Podosphaera)物种(白粉病),例如苹果上的白叉丝单囊壳(P.leucotricha);多粘霉属(Polymyxa)物种,例如谷物例如大麦和小麦(禾谷多粘霉(P.graminis))和糖用甜菜(甜菜多粘霉(P.betae))上的,和由此传播的病毒病;谷物例如小麦或大麦上的Pseudocercosporella herpotrichoides(眼斑病,有性型:Tapesiayallundae);多种植物上的假霜霉属(Pseudoperonospora)(霜霉病),例如葫芦上的古巴假霜霉(P.cubensis)或蛇麻上的葎草假霜霉(P.humili);藤本植物上的八孢假无柄盘菌(Pseudopezicula tracheiphila)(红火病(red fire disease)或rotbrenner,无性型:瓶霉属(Phialophora));多种植物上的柄锈菌属(Puccinia)物种(锈病),例如谷物例如小麦、大麦或黑麦上的小麦柄锈菌(P.triticina)(褐锈病或叶锈病)、条形柄锈菌(P.striiformis)(条锈病或黄锈病)、大麦柄锈菌(P.hordei)(矮锈病)、禾柄锈菌(P.graminis)(秆锈病或黑锈病)或隐匿柄锈菌(P.recondita)(棕锈病或叶锈病),甘蔗上的屈恩柄锈菌(P.kuehnii)(柑橘锈病)和芦笋上的天门冬属柄锈菌(P.asparagi);小麦上的偃麦草核腔菌(Pyrenophora tritici-repentis)(无性型:偃麦草内脐蠕孢(Drechsleratritici-repentis)(褐斑病)或大麦上的圆核腔菌(P.teres)(网斑病);梨孢属(Pyricularia)物种,例如稻上的稻梨孢(P.oryzae)(有性型:Magnaporthe grisea,稻瘟病)和坪草和谷物上的灰梨孢(P.grisea);坪草、稻、玉米、小麦、棉花、油菜、向日葵、大豆、糖用甜菜、蔬菜和多种其它植物上的腐霉属(Pythium)物种(猝倒)(例如终极腐霉(P.ultimum)或瓜果腐霉(P.aphanidermatum));柱隔孢属(Ramularia)物种,例如大麦上的R.collo-cygni(柱隔孢叶斑病、生理叶斑病)和糖用甜菜上的甜菜生柱隔孢(R.beticola);棉花、稻、马铃薯、坪草、玉米、油菜、马铃薯、糖用甜菜、蔬菜和多种其它植物上的丝核菌属(Rhizoctonia)物种,例如大豆上的立枯丝核菌(R.solani)(根茎腐病)、稻上的立枯丝核菌(R.solani)(纹枯病)或者小麦或大麦上的禾谷丝核菌(R.cerealis)(丝核菌春叶枯病);草莓、胡萝卜、卷心菜、藤本植物和番茄上的匐枝根霉(Rhizopus stolonifer)(黑霉病、软腐病);大麦、黑麦和黑小麦上的黑麦喙孢(Rhynchosporium secalis)(灼枯病(scald));稻上的稻帚枝杆孢菌(Sarocladium oryzae)和S.Attenuatum(鞘腐病);蔬菜和大田作物,例如油菜、向日葵(例如核盘菌(S.sclerotiorum))和大豆(例如,S.rolfsii或核盘菌(S.sclerotiorum))上的核盘菌属(Sclerotinia)物种(茎腐病或白霉);多种植物上的壳针孢属(Septoria)物种,例如大豆上的大豆壳针孢(S.glycines)(褐斑病)、小麦上的小麦壳针孢(S.tritici)(壳针孢叶枯病)和谷物上的颖枯壳针孢(S.nodorum)(同义词:颖枯壳多孢(Stagonospora nodorum)(壳多孢叶枯病);藤本植物上的葡萄钩丝壳(Uncinulanecator)(同义词:葡萄白粉菌(Erysiphe necator)(白粉病,无性型:葡萄粉孢(Oidiumtuckeri);玉米(例如S.turcica,同义词:大斑病长蠕孢(Helminthosporium turcicum))和坪草上的大斑病菌属(Setospaeria)物种(叶枯病);玉米、高粱和甘蔗上的轴黑粉菌属(Sphacelotheca)物种(黑穗病)(例如,丝轴黑粉菌(S.reiliana):丝黑穗病);葫芦上的单丝壳(Sphaerotheca fuliginea)(白粉病);马铃薯上的马铃薯粉痂菌(Spongosporasubterranea)(粉痂病)和由此传播的病毒病;谷物上的壳多孢属(Stagonospora)物种,例如小麦上的颖枯壳多孢(S.nodorum)(壳多孢黑斑病,有性型:颖枯小球腔菌(Leptosphaeria nodorum))[同义词:颖枯暗球腔菌(Phaeosphaeria nodorum)];马铃薯上的内生集壶菌(Synchytrium endobioticum)(马铃薯癌肿病);外囊菌属(Taphrina)物种,例如桃上的畸形外囊菌(T.deformans)(缩叶病)和李树上的李外囊菌(T.pruni)(李囊果病(plum pocket));烟草、仁果、蔬菜、大豆和棉花上的根串珠霉属(Thielaviopsis)物种(黑根腐病),例如根串珠霉(T.basicola)(同义词:Chalara elegans);谷物上的腥黑粉菌属(Tilletia)物种(腥黑穗病(common bunt或stinking smut)),例如小麦上的小麦腥黑粉菌(T.tritici)(同义词:小麦网腥黑粉菌(T.carie),小麦腥黑穗病)和小麦矮腥黑粉菌(T.controversa)(矮腥黑穗病);大麦或小麦上的肉孢核瑚菌(Typhula incarnate)(灰雪腐霉病(grey snow mold));条黑粉菌属(Urocystis)物种,例如黑麦上的隐条黑粉菌(U.occulta)(秆黑穗病(stem smut));蔬菜例如菜豆(例如疣顶单胞锈菌(U.appendiculatu),同义词:菜豆单胞锈菌(U.phaseoli))和糖用甜菜(例如甜菜单胞锈菌(U.betae))上的单胞锈菌属(Uromyces)物种(锈病);谷物(例如麦散黑粉菌(U.nuda)和燕麦散黑粉菌(U.avaenae))、玉米(例如玉蜀黍黑粉菌(U.maydis):玉米黑粉病)和甘蔗上的黑粉菌属(Ustilago)物种(散黑穗病(loose smut));苹果(例如苹果黑星菌(V.inaequalis))和梨上的黑星菌属(Venturia)物种(疮痂病(scab));和多种植物例如水果和观赏植物、藤本植物、无核小果、蔬菜和大田作物上的轮枝孢属(Verticillium)物种(萎蔫),例如草莓、油菜、马铃薯和番茄上的大丽花轮枝孢(V.dahliae)。
本发明的混合物和组合物还适用于储存品或收获物的保护中和用于材料的保护中防治有害病原体,特别是真菌。术语“材料的保护”应当被理解为表示,向技术性无生命材料提供对抗有害微生物(例如真菌和细菌)的侵染和破坏的保护作用,所述材料例如粘合剂、胶、木材、纸张和纸板、纺织品、皮革、颜料分散体、塑料、冷却润滑剂、纤维或织物。关于木材和其它材料的保护,特别关注以下有害真菌:子囊菌纲(Ascomycetes),例如蛇口壳(Ophiostoma)物种、长喙壳属(Ceratocystis)物种、出芽短梗霉菌(Aureobasidiumpullulans)、核茎点属(Sclerophoma)物种、毛壳菌属(Chaetomium)物种、腐质霉属(Humicola)物种、彼得壳属(Petriella)物种、毛束霉属(Trichurus)物种;担子菌纲(Basidiomycetes),例如粉孢革菌属(Coniophora)物种、革盖菌属(Coriolus)物种、粘褶菌属(Gloeophyllum)物种、香菇属(Lentinus)物种、侧耳属(Pleurotus)物种、卧孔属(Poria)物种、龙介属(Serpula)物种和干酪菌属(Tyromyces)物种;半知菌纲(Deuteromycetes),例如曲霉属(Aspergillus)物种、枝孢属(Cladosporium)物种、青霉属(Penicillium)物种、木霉属(Trichorma)物种、链格孢属(Alternaria)物种、拟青霉属(Paecilomyces)物种;和接合菌纲(Zygomycetes),例如毛霉属(Mucor)物种;并且此外,在储存品和收获物的保护中,以下的酵母真菌值得关注:假丝酵母属(Candida)物种和酿酒酵母(Saccharomycescerevisiae)。根据本发明的处理方法也可以用于保护储存品或收获物以对抗真菌和微生物攻击的领域中。根据本发明,术语“储存品”应当被理解为表示,植物或动物来源的天然物质及其经加工的形式,其已经取自天然生命周期并且对其需要进行长期保护。作物来源的储存品,例如植物或其部分,例如茎秆、叶、块茎、种子、果实或谷粒,可以以新鲜采收状态或以加工形式来保护,所述加工形式例如预先干燥的、润湿的、切碎的、研磨的、压制的或烘烤的,该过程也称为采收后处理。木材也落入储存品的定义中,不管是粗制木材形式,例如建筑木材、电缆塔和障碍物,或成品的形式,例如家具或从木材制得的物品。动物来源的储存品是兽皮、皮革、毛皮、毛发等。根据本发明的组合物可以防止不利影响,例如腐烂、脱色或发霉。优选,“储存品”应当被理解为植物来源的天然物质和其加工形式,更优选果实和其加工形式,例如仁果、核果、无核小果和柑桔类水果及其加工形式。
本发明的混合物和组合物对防治多种栽培植物上以及这些植物的植物繁殖材料(例如种子)和作物材料上的多种植物病原性昆虫或其它害物(例如鳞翅类、甲虫、双翅类、蓟马类、异翅类、半翅目、同翅目、白蚁类、直翅类、蛛形动物和线虫)特别重要,所述栽培植物例如谷物,例如小麦、黑麦、大麦、黑小麦、燕麦或稻;甜菜,例如糖用甜菜或饲料甜菜;水果,例如仁果、核果或无核小果,例如苹果、梨、李、桃、杏、樱桃、草莓、树莓、黑莓或醋栗;豆科植物,例如小扁豆、豌豆、苜蓿或大豆;油料植物,例如油菜、芥菜、橄榄、向日葵、椰子、可可豆、蓖麻油植物、油棕、花生或大豆;葫芦科植物,例如南瓜、黄瓜或甜瓜;纤维植物,例如棉花、亚麻、大麻或黄麻;柑桔类水果,例如橙、柠檬、葡萄柚或柑橘;蔬菜,例如菠菜、莴苣、芦笋、卷心菜、胡萝卜、洋葱、番茄、马铃薯、葫芦或红辣椒;月桂科植物,例如鳄梨、肉桂或樟脑;能量和原料植物,例如玉米、大豆、油菜、甘蔗或油棕;玉米;烟草;坚果;咖啡;茶;香蕉;藤本植物(食用葡萄(table grapes)和葡萄汁葡萄);蛇麻草;坪草;天然橡胶植物或观赏植物和森林植物,例如花、灌木、阔叶树或常绿树,例如针叶树。
优选,本发明的混合物和组合物用于防治田间作物,例如马铃薯、糖用甜菜、烟草、小麦、黑麦、大麦、燕麦、稻、玉米、棉花、大豆、油菜、菜豆、向日葵、咖啡或甘蔗;水果;藤本植物;观赏植物;或蔬菜,例如黄瓜、番茄、菜豆或南瓜上的多种害物。
本发明的混合物及其组合物特别适用于防治来自以下类目的有害昆虫
鳞翅类(鳞翅目(Lepidoptera)),例如小地老虎(Agrotis ypsilon)、黄地老虎(Agrotis segetum)、棉叶波纹夜蛾(Alabama argillacea)、大豆夜蛾(Anticarsiagemmatalis)、苹实巢蛾(Argyresthia conjugella)、丫纹夜蛾(Autographa gamma)、松尺蠖(Bupalus piniarius)、枞卷叶蛾(Cacoecia murinana)、烟卷蛾(Capua reticulana)、冬尺蠖(Cheimatobia brumata)、云杉卷叶蛾(Choristoneura fumiferana)、西方云杉色卷蛾(Choristoneura occidentalis)、一星粘虫(Cirphis unipuncta)、苹果蠹蛾(Cydiapomonella)、欧洲松毛虫(Dendrolimus pini)、黄瓜绢野螟(Diaphania nitidalis)、西南玉米杆草螟(Diatraea grandiosella)、埃及钻夜蛾(Earias insulana)、南美玉米苗斑螟(Elasmopalpus lignosellus)、环针单纹卷蛾(Eupoecilia ambiguella)、欧洲松梢小卷蛾(Evetria bouliana)、粒肤地老虎(Feltia subterranea)、大蜡螟(Galleriamellonella)、李小食心虫(Grapholitha funebrana)、梨小食心虫(Grapholithamolesta)、棉铃虫(Heliothis armigera)、烟芽夜蛾(Heliothis virescens)、玉米夜蛾(Heliothis zea)、菜螟(Hellula undalis)、Hibernia defoliaria、美国白蛾(Hyphantriacunea)、苹果巢蛾(Hyponomeuta malinellus)、番茄蠹蛾(Keiferia lycopersicella)、东方铁杉尺蠖(Lambdina fiscellaria)、甜菜夜蛾(Laphygma exigua)、咖啡潜叶蛾(Leucoptera coffeella)、旋纹潜叶蛾(Leucoptera scitella)、潜叶细蛾(Lithocolletisblancardella)、葡萄花翅小卷蛾(Lobesia botrana)、草地螟(Loxostege sticticalis)、舞毒娥(Lymantria dispar)、模毒蛾(Lymantria monacha)、桃潜蛾(Lyonetiaclerkella)、天幕毛虫(Malacosoma neustria)、甘蓝夜蛾(Mamestra brassicae)、黄杉毒蛾(Orgyia pseudotsugata)、欧洲玉米螟(Ostrinia nubilalis)、小眼夜蛾(Panolisflammea)、棉红铃虫(Pectinophora gossypiella)、疆夜蛾(Peridroma saucia)、圆掌舟蛾(Phalera bucephala)、马铃薯块茎蛾(Phthorimaea operculella)、柑桔潜叶蛾(Phyllocnistis citrella)、大菜粉蝶(Pieris brassicae)、苜蓿绿夜蛾(Plathypenascabra)、小菜蛾(Plutella xylostella)、大豆尺蠖(Pseudoplusia includens)、松梢卷叶蛾(Rhyacionia frustrana)、Scrobipalpula absoluta、麦蛾(Sitotroga cerealella)、葡萄长须卷蛾(Sparganothis pilleriana)、草地贪夜蛾(Spodoptera frugiperda)、海灰翅夜蛾(Spodoptera littoralis)、斜纹夜蛾(Spodoptera litura)、松异舟蛾(Thaumatopoeapityocampa)、栎绿卷蛾(Tortrix viridana)、粉纹夜蛾(Trichoplusia ni)和云杉线小卷蛾(Zeiraphera canadensis),
甲虫(鞘翅目(Coleoptera)),例如梨窄吉丁(Agrilus sinuatus)、直条叩甲(Agriotes lineatus)、暗色叩甲(Agriotes obscurus)、六月金龟(Amphimallussolstitialis)、北方材小蠹(Anisandrus dispar)、墨西哥棉铃象甲(Anthonomusgrandis)、苹花象(Anthonomus pomorum)、甜菜隐食甲(Atomaria linearis)、纵坑切梢小蠹(Blastophagus piniperda)、Blitophaga undata、蚕豆象(Bruchus rufimanus)、豌豆象(Bruchus pisorum)、欧洲兵豆象(Bruchus lentis)、梨卷叶象(Byctiscus betulae)、甜菜大龟甲(Cassida nebulosa)、菜豆莹叶甲(Cerotoma trifurcata)、白菜籽龟象(Ceutorhynchus assimilis)、芫菁龟象(Ceuthorrhynchus napi)、甜菜胫跳甲(Chaetocnema tibialis)、烟草金针虫(Conoderus vespertinus)、石刁柏叶甲(Criocerisasparagi)、长角叶甲(Diabrotica longicornis)、南美叶甲(Diabrotica speciosa)、十二斑叶甲(Diabrotica 12-punctata)、玉米根萤叶甲(Diabrotica virgifera)、阿根廷兜虫(Diloboderus abderus)、墨西哥豆瓢虫(Epilachna varivestis)、烟草跳甲(Epitrixhirtipennis)、棉灰蒙象变种(Eutinobothrus brasiliensis)、松树皮象(Hylobiusabietis)、埃及苜蓿叶象(Hypera brunneipennis)、紫苜蓿叶象(Hypera postica)、云杉八齿小蠹(Ips typographus)、烟草负泥虫(Lema bilineata)、黑角负泥虫(Lemamelanopus)、马铃薯叶甲(Leptinotarsa decemlineata)、甜菜叩甲(Limoniuscalifornicus)、稻水象甲(Lissorhoptrus oryzophilus)、玉米金针虫(Melanotuscommunis)、油菜花露尾甲(Meligethes aeneus)、大栗鳃金龟(Melolonthahippocastani)、五月鳃金龟(Melolontha melolontha)、稻负泥虫(Oulema oryzae)、葡萄黑耳喙象(Ortiorrhynchus sulcatus)、Oryazophagus oryzae、草莓根象甲(Otiorrhynchus ovatus)、辣根猿叶甲(Phaedon cochleariae)、Phyllotretachrysocephala、鳃金龟属(Phyllophaga)物种、Phyllophaga cuyabana、Phyllophagatriticophaga、庭园发丽金龟(Phyllopertha horticola)、芫菁淡足跳甲(Phyllotretanemorum)、黄曲条跳甲(Phyllotreta striolata)、日本弧丽金龟(Popillia japonica)、条纹根瘤象甲(Sitona lineatus)和谷象(Sitophilus granaria),
双翅类(双翅目(Diptera)),例如埃及伊蚊(Aedes aegypti)、刺扰伊蚊(Aedesvexans)、墨西哥按实蝇(Anastrepha ludens)、五斑按蚊(Anopheles maculipennis)、地中海实蝇(Ceratitis capitata)、蛆症金蝇(Chrysomya bezziana)、Chrysomyahominivorax、腐败金蝇(Chrysomya macellaria)、高粱瘿蚊(Contarinia sorghicola)、噬人瘤蝇(Cordylobia anthropophaga)、淡色库蚊(Culex pipiens)、瓜实蝇(Dacuscucurbitae)、橄榄实蝇(Dacus oleae)、芸薹荚瘿蚊(Dasineura brassicae)、夏厕蝇(Fannia canicularis)、马胃蝇(Gasterophilus intestinalis)、刺舌蝇(Glossinamorsitans)、扰血蝇(Haematobia irritans)、Haplodiplosis equestris、种蝇(Hylemyiaplatura)、纹皮蝇(Hypoderma lineata)、美洲斑潜蝇(Liriomyza sativae)、三叶草斑潜蝇(Liriomyza trifolii)、Lucilia caprina、铜绿蝇(Lucilia cuprina)、丝光绿蝇(Luciliasericata)、Lycoria pectoralis、小麦瘿蚊(Mayetiola destructor)、家蝇(Muscadomestica)、厩腐蝇(Muscina stabulans)、羊狂蝇(Oestrus ovis)、黑麦秆蝇(Oscinellafrit)、甜菜潜叶蝇(Pegomya hysocyami)、葱蝇(Phorbia antiqua)、甘蓝草种蝇(Phorbiabrassicae)、Phorbia coarctata、樱桃绕实蝇(Rhagoletis cerasi)、苹果绕实蝇(Rhagoletis pomonella)、牛虻(Tabanus bovinus)、甘蓝大蚊(Tipula oleracea)和欧洲大蚊(Tipula paludosa)、
蓟马类(缨翅目(Thysanoptera)),例如烟蓟马(Frankliniella fusca)、西花蓟马(Frankliniella occidentalis)、美东花蓟马(Frankliniella tritici)、柑橘蓟马(Scirtothrips citri)、稻蓟马(Thrips oryzae)、棕榈蓟马(Thrips palmi)和葱蓟马(Thrips tabaci),
膜翅类(膜翅目(Hymenoptera)),例如Acromyrmex ambuguus、Acromyrmexcrassispinus、Acromyrmex heiery、Acromyrmex landolti、Acromyrmex subterraneus、新疆菜叶蜂(Athalia rosae)、Atta capiguara、大头南美切叶蚁(Atta cephalotes)、Attalaevigata、Atta robusta、塞氏美切叶蚁(Atta sexdens)、德州切叶蚁(Atta texana)、李实叶蜂(Hoplocampa minuta)、苹实叶蜂(Hoplocampa testudinea)、小黄家蚁(Monomoriumpharaonis)、火蚁(Solenopsis geminata)和红火蚁(Solenopsis invicta),
异翅类(异翅亚目(Heteroptera)),例如拟绿蝽(Acrosternum hilare)、玉米长蝽(Blissus leucopterus)、黑斑烟盲蝽(Cyrtopeltis notatus)、Dichelops furcatus、棉二点红蝽(Dysdercus cingulatus)、棉红蝽(Dysdercus intermedius)、Euchistos heros、麦扁盾蝽(Eurygaster integriceps)、棉褐蝽(Euschistus impictiventris)、叶足缘蝽(Leptoglossus phyllopus)、美洲牧草盲蝽(Lygus lineolaris)、牧草盲蝽(Lyguspratensis)、稻绿蝽(Nezara viridula)、方背皮蝽(Piesma quadrata)、璧蝽(Piezodorusguildini)、Solubea insularis和Thyanta perditor,
半翅目(Hemiptera)和同翅目(Homoptera),例如拟绿蝽(Acrosternum hilare)、玉米长蝽(Blissus leucopterus)、黑斑烟盲蝽(Cyrtopeltis notatus)、柑橘木虱(Diaphorina citri)、棉二点红蝽(Dysdercus cingulatus)、棉红蝽(Dysdercusintermedius)、麦扁盾蝽(Eurygaster integriceps)、棉褐蝽(Euschistusimpictiventris)、叶足缘蝽(Leptoglossus phyllopus)、美洲牧草盲蝽(Lyguslineolaris)、牧草盲蝽(Lygus pratensis)、稻绿蝽(Nezara viridula)、方背皮蝽(Piesmaquadrata)、Solubea insularis、Thyanta perditor、无网长管蚜(Acyrthosiphononobrychis)、落叶松球蚜(Adelges laricis)、Aphidula nasturtii、黑豆蚜(Aphisfabae)、草莓根蚜(Aphis forbesi)、苹果蚜(Aphis pomi)、棉蚜(Aphis gossypii)、北美茶簏子蚜(Aphis grossulariae)、施奈德蚜(Aphis schneideri)、绣线菊蚜(Aphisspiraecola)、接骨木蚜(Aphis sambuci)、豇豆无网长管蚜(Acyrthosiphon pisum)、茄粗额蚜(Aulacorthum solani)、飞廉短尾蚜(Brachycaudus cardui)、李短尾蚜(Brachycaudus helichrysi)、桃短尾蚜(Brachycaudus persicae)、Brachycaudusprunicola、甘蓝蚜(Brevicoryne brassicae)、Capitophorus horni、Cerosiphagossypii、草莓钉蚜(Chaetosiphon fragaefolii)、茶藨隐瘤蚜(Cryptomyzus ribis)、高加索冷杉椎球蚜(Dreyfusia nordmannianae)、云杉椎球蚜(Dreyfusia piceae)、居根西圆尾蚜(Dysaphis radicola)、Dysaulacorthum pseudosolani、车前圆尾蚜(Dysaphisplantaginea)、梨西圆尾蚜(Dysaphis pyri)、蚕豆微叶蝉(Empoasca fabae)、桃大尾蚜(Hyalopterus pruni)、莴苣超瘤蚜(Hyperomyzus lactucae)、麦长管蚜(Macrosiphumavenae)、马铃薯长管蚜(Macrosiphum euphorbiae)、蔷薇管蚜(Macrosiphon rosae)、巢菜修尾蚜(Megoura viciae)、Melanaphis pyrarius、麦无网长管蚜(Metopolophiumdirhodum)、烟蚜(Myzodes persicae)、冬葱瘤额蚜(Myzus ascalonicus)、Myzus cerasi、李瘤蚜(Myzus varians)、莴苣衲长管蚜(Nasonovia ribis-nigri)、褐飞虱(Nilaparvatalugens)、囊柄瘿绵蚜(Pemphigus bursarius)、甘蔗扁角飞虱(Perkinsiellasaccharicida)、蛇麻疣额蚜(Phorodon humuli)、苹果木虱(Psylla mali)、梨木虱(Psyllapiri)、冬葱缢瘤蚜(Rhopalomyzus ascalonicus)、玉米缢管蚜(Rhopalosiphum maidis)、禾谷缢管蚜(Rhopalosiphum padi)、莲缢管蚜(Rhopalosiphum insertum)、Sappaphismala、苹果圆尾蚜(Sappaphis mali)、麦二叉蚜(Schizaphis graminum)、Schizoneuralanuginosa、麦长管蚜(Sitobion avenae)、温室粉虱(Trialeurodes vaporariorum)、Toxoptera aurantiiand、葡萄根瘤蚜(Viteus vitifolii)、温带臭虫(Cimexlectularius)、热带臭虫(Cimex hemipterus)、Reduvius senilis、锥蝽属(Triatoma)物种和Arilus critatus,
白蚁类(等翅目(Isoptera)),例如木白蚁(Calotermes flavicollis)、堆角白蚁(Cornitermes cumulans)、细异白蚁(Heterotermes tenuis)、Leucotermes flavipes、欧氏新域扭白蚁(Neocapritemes opacus)、Procornitermes triacifer;欧洲散白蚁(Reticulitermes lucifugus)、蔗稻共白蚁(Syntermes molestus)和白蚁(Termesnatalensis)、
直翅类(直翅目(Orthoptera)),例如家蟋蟀(Acheta domestica)、东方蜚蠊(Blatta orientalis)、德国小蠊(Blattella germanica)、普通蠼螋(Forficulaauricularia)、欧洲蝼蛄(Gryllotalpa gryllotalpa)、飞蝗(Locusta migratoria)、双带黑蝗(Melanoplus bivittatus)、赤胫黑蝗(Melanoplus femur-rubrum)、墨西哥黑蝗(Melanoplus mexicanus)、血黑蝗(Melanoplus sanguinipes)、石栖黑蝗(Melanoplusspretus)、红翅蝗(Nomadacris septemfasciata)、美洲大蠊(Periplaneta americana)、美洲沙漠蝗(Schistocerca americana)、Schistocerca peregrina、Stauronotusmaroccanus和庭疾灶螽(Tachycines asynamorus)、蛛形纲(Arachnoidea),例如以下科的蛛形动物:软蜱科(Argasidae)、硬蜱科(Ixodidae)和疥螨科(Sarcoptidae),例如美洲钝眼蜱(Amblyomma americanum)、彩饰花蜱(Amblyomma variegatum)、波斯锐缘蜱(Argaspersicus)、具环牛蜱(Boophilus annulatus)、消色牛蜱(Boophilus decoloratus)、微小牛蜱(Boophilus microplus)、森林革蜱(Dermacentor silvarum)、长喙璃眼蜱(Hyalommatruncatum)、篦子硬蜱(Ixodes ricinus)、浅红硬蜱(Ixodes rubicundus)、非洲钝缘蜱(Ornithodorus moubata)、耳刺残喙蜱(Otobius megnini)、鸡皮刺螨(Dermanyssusgallinae)、绵羊痒螨(Psoroptes ovis)、具尾扇头蜱(Rhipicephalus appendiculatus)、埃氏扇头蜱(Rhipicephalus evertsi)、人疥螨(Sarcoptes scabiei),和瘿螨科(Eriophyidae)物种,例如斯氏针剌瘿螨(Aculus schlechtendali)、桔皱叶刺瘿螨(Phyllocoptrata oleivora)和柑橘瘿螨(Eriophyes sheldoni);跗线螨科(Tarsonemidae)物种,例如樱草植食螨(Phytonemus pallidus)和侧多食跗线螨(Polyphagotarsonemus latus);细须螨科(Tenuipalpidae)物种,例如紫红短须螨(Brevipalpus phoenicis);叶螨科(Tetranychidae)物种,例如朱砂叶螨(Tetranychuscinnabarinus)、神泽叶螨(Tetranychus kanzawai)、太平洋叶螨(Tetranychuspacificus)、棉叶螨(Tetranychus telarius)和二斑叶螨(Tetranychus urticae)、榆全爪螨(Panonychus ulmi)、柑桔全爪螨(Panonychus citri)和草地小爪螨(Oligonychuspratensis)。
特别的是,本发明混合物适合用于对抗鞘翅目(Coleoptera)、鳞翅目(Lepidoptera)、缨翅目(Thysanoptera)、同翅目(Homoptera)、等翅目(Isoptera)和直翅目(Orthoptera)的害物。
它们还适合用于防治以下植物寄生线虫,例如根结线虫、花生根结线虫(Meloidogyne arenaria)、哥伦比亚根结线虫(Meloidogyne chitwoodi)、短小根结线虫(Meloidogyne exigua)、北方根结线虫(Meloidogyne hapla)、南方根结线虫(Meloidogyneincognita)、爪哇根结线虫(Meloidogyne javanica)和其它根结线虫属(Meloidogyne)物种;囊胞线虫,马铃薯金线虫(Globodera rostochiensis)、马铃薯白线虫(Globoderapallida)、烟草球形胞囊线虫(Globodera tabacum)和其它球形胞囊属(Globodera)物种、禾谷胞囊线虫(Heterodera avenae)、大豆孢囊线虫(Heterodera glycines)、甜菜胞囊线虫(Heterodera schachtii)、三叶草胞囊线虫(Heterodera trifolii)和其它孢囊线虫属(Heterodera)物种;种子瘿线虫,剪股颖粒线虫(Anguina funesta)、小麦粒线虫(Anguinatritici)和其它粒线虫属(Anguina)物种;柄和叶线虫,贝西拟滑刃线虫(Aphelenchoidesbesseyi)、草莓拟滑刃线虫(Aphelenchoides fragariae)、菊花拟滑刃线虫(Aphelenchoides ritzemabosi)和其它拟滑刃线虫属(Aphelenchoides)物种;刺线虫,长尾刺线虫(Belonolaimus longicaudatus)和其它刺线虫属(Belonolaimus)物种;松树线虫,松材线虫(Bursaphelenchus xylophilus)和其它伞滑刃线虫属(Bursaphelenchus)物种;环线虫,环线虫属(Criconema)物种、小环线虫属(Criconemella)物种、拟环线虫属(Criconemoides)物种和中环线虫属(Mesocriconema)物种;柄和鳞茎线虫,腐烂茎线虫(Ditylenchus destructor)、鳞球茎茎线虫(Ditylenchus dipsaci)、食菌茎线虫(Ditylenchus myceliophagus)和其它茎线虫属(Ditylenchus)物种;锥线虫,锥线虫属(Dolichodorus)物种;螺旋线虫、双宫螺旋线虫(Helicotylenchus dihystera)、多带螺旋线虫(Helicotylenchus multicinctus)和其它螺旋属(Helicotylenchus)物种、强壮盘旋线虫(Rotylenchus robustus)和其它盘旋属(Rotylenchus)物种;鞘线虫,鞘线虫属(Hemicycliophora)物种和半轮线虫属(Hemicriconemoides)物种;潜根线虫属(Hirshmanniella)物种;矛线虫、哥伦比亚纽带线虫(Hoplolaimus columbus)、帽状纽带线虫(Hoplolaimus galeatus)和其它纽带线虫属(Hoplolaimus)物种;假根结线虫、异常珍珠线虫(Nacobbus aberrans)和其它珍珠线虫属(Nacobbus)物种;长针线虫(needlenematode)、横带长针线虫(Longidorus elongatus)和其它长针属(Longidorus)物种;针线虫(pin nematode)、针线虫属(Paratylenchus)物种;根腐线虫、最短尾短体线虫(Pratylenchus brachyurus)、咖啡短体线虫(Pratylenchus coffeae)、Pratylenchuscurvitatus、全体短体线虫(Pratylenchus goodeyi)、落选短体线虫(Pratylencusneglectus)、穿刺短体线虫(Pratylenchus penetrans)、斯克里布纳短体线虫(Pratylenchus scribneri)、伤残短体线虫(Pratylenchus vulnus)、玉米短体线虫(Pratylenchus zeae)和其它短体属(Pratylenchus)物种;棕榈细杆滑刃线虫(Radinaphelenchus cocophilus)和其它细杆滑刃属(Radinaphelenchus)物种;穿孔线虫、香蕉穿孔线虫(Radopholus similis)和其它穿孔属(Radopholus)物种;肾形线虫、肾形线虫(Rotylenchulus reniformis)和其它肾状线虫属(Rotylenchulus)物种;盾线虫属(Scutellonema)物种;残根线虫(stubby root nematode)、原始毛刺线虫(Trichodorusprimitivus)和其它毛刺属(Trichodorus)物种;较小拟毛刺线虫(aratrichodorus minor)和其它拟毛刺属(Paratrichodorus)物种;矮化线虫、克莱顿矮化线虫(Tylenchorhynchusclaytoni)、不定矮化线虫(Tylenchorhynchus dubius)和其它矮化属(Tylenchorhynchus)物种和默林属(Merlinius)物种;柑橘线虫、半穿刺线虫(Tylenchulus semipenetrans)和其它半穿刺属(Tylenchulus)物种;剑线虫、美洲剑线虫(Xiphinema americanum)、标准剑线虫(Xiphinema index)、异尾剑线虫(Xiphinema diversicaudatum)和其它剑线虫属(Xiphinema)物种;和其它植物寄生线虫物种。
在同等优选的实施方案中,本发明涉及防治动物害物(昆虫、螨虫或线虫)的方法,其中用有效量的包含如上文定义的类芽孢杆菌属(Paenibacillus)菌株和农药II的本发明混合物处理动物害物(昆虫、螨虫或线虫)、其栖息地、繁殖地、其场所或待保护对抗动物害物(昆虫、螨虫或线虫)攻击的植物。
通常而言,“农药用有效量”意指为了对生长实现可观测作用所需要的本发明混合物或包含混合物的组合物的量,可观测作用包括坏死、死亡、阻滞、阻止和移除、破坏作用或另外削弱目标生物的出现和活动。对于本发明中使用的多种混合物/组合物,农药用有效量可以不同。混合物/组合物的农药用有效量也将根据优势条件而不同,例如所需的农药作用和持续时间、天气、目标物种、部位、应用模式等。
可以用本发明的混合物和组合物在种植或移栽时或之前预防性地处理植物繁殖材料。
特别地,本发明涉及用于保护植物繁殖材料免受害物的方法,其中用有效量的本发明混合物处理植物繁殖材料。
在同等优选的实施方案中,本发明涉及用于保护植物繁殖材料免受有害真菌的方法,其中用有效量的本发明混合物处理植物繁殖材料。
在同等优选的实施方案中,本发明涉及用于改善植物健康的方法,其中用有效量的本发明混合物处理植物。
术语“植物健康有效量”指足以实现如下文定义的植物健康作用的本发明混合物的量。下文给出更多关于待使用的量、应用方式和适合比例的示例性信息。无论如何,技术人员是非常清楚以下事实:此类量可以在宽范围内不同并且取决于多种因素,例如处理的栽培植物或材料和气候条件。
更健康的植物是合乎需要的,因为它们可以导致例如植物或作物的更高产量和/或更高品质,特别是采收的植物部分的更高品质。更健康的植物也能更好地抵抗生物和/或非生物胁迫。对抗生物胁迫的高抗性又可以允许本领域技术人员降低使用的农药量并且因此减缓对抗相应农药的抗性的产生。
因此,本发明的目的是提供农药组合物,其解决了如上文概述的问题并且特别应当改善植物的健康,特别是植物的产量。
术语“植物的健康”或“植物健康”被定义为植物和/或其产物的状态,所述状态由单独或彼此组合的几个方面例如增加的产量、植物活力、已收获植物部分的品质和非生物胁迫和/或生物胁迫耐受性决定。
必须强调,在植物没有处于生物胁迫下时,并且特别是植物没有处于害物压力下时,本发明混合物的以上所述作用,即增强的植物健康,也是存在的。
对于种子处理例如接种和/或叶面应用形式,显而易见,与已经针对致病真菌或任何其它相关害物接受治愈性或预防性处理并且可以无生物胁迫因素所致损伤的情况下生长的植物相比,遭受真菌攻击或杀虫攻击的植物产生更少的生物量并且导致产量减少。然而,根据本发明的方法导致增强的植物健康,即使在不存在任何生物胁迫的情况下。这意味着,本发明混合物的积极影响不能仅依据组分1)的细菌菌株和农药II的农药活性解释,而是还基于其它的活性特征。因此,本发明混合物的应用也可以在不存在害物压力的情况下进行。
以下所列的每个植物健康指标(选自产量、植物活力、品质以及植物对非生物和/或生物胁迫的耐受性),将被理解为各自单独地或优选彼此组合地构成本发明的优选实施方案。
根据本发明,植物“产量增加”意指将这种植物的产物的产量增加可度量的量,超过在相同条件下但不应用本发明混合物时所产生的该植物的相同产物的产量。
对于种子处理,例如作为接种和/或叶面应用形式,产量增加可以通过例如以下改善的植物特性来表征:植物重量增加;和/或植物高度提高;和/或生物量增加,例如更高的整体鲜重(FW);和/或每株植物的花数量增加;和/或谷粒和/或果实产量更高;和/或分蘖或侧芽(分枝)更多;和/或叶子更大;和/或芽生长增加;和/或蛋白质含量增加;和/或含油量增加;和/或淀粉含量增加;和/或色素含量增加;和/或叶绿素含量增加(叶绿素含量与植物的光合作用速率具有正相关,并且因此叶绿素含量越高,植物产量越高)和/或植物品质提高。
“谷粒”和“果实”应当被理解为由植物产生的具有任何经济价值的任何植物产物,其在采收后进一步被利用,例如本来意义上的果实、蔬菜、坚果、谷粒、种子、木材(例如在造林植物的情况中)、花卉(例如在园林植物、观赏植物的情况中)等。
根据本发明,产量提高至少4%。通常,产量提高甚至可以更高,例如5至10%,更优选10至20%,或者甚至20至30%。
根据本发明,如果在不存在害物压力的情况下,产量提高至少2%。通常,产量提高甚至可以更高,例如直至4%至5%,或甚至更高。
用于植物状况的另一个指标是植物活力。植物活力显示在几个方面中,例如一般视觉外观。
对于叶面应用,改善的植物活力可以例如通过以下改善的植物特性来表征:改善的植物活力;和/或改善的植物生长;和/或改善的植物发育;和/或改善的视觉外观;和/或改善的植株成活(较少植物歪倒(verse)/植物倒伏和/或较大的叶片;和/或较大的大小;和/或提高的植物高度;和/或提高的分蘖数量;和/或提高的侧芽数量;和/或提高的每株植物的花数量;和/或提高的芽生长;和/或增强的光合活性(例如基于提高的气孔导度和/或提高的CO2同化速率));和/或较早的开花;和/或较早的结果;和/或较早的谷粒成熟;和/或较少的无效分蘖;和/或较少的死基生叶;和/或较少的输入需求(例如肥料或水);和/或更绿的叶子;和/或在缩短的营养期下的完全成熟;和/或更容易的采收;和/或更快和更均匀的成熟;和/或较长的货架期;和/或较长的穗(panicle);和/或衰老的延迟;和/或更强壮和/或更有效分蘖;和/或更好的成分可提取性;和/或改善的种子品质(对于用于种子生产的下一季中的播种);和/或降低的乙烯产生和/或抑制其被植物吸收。
用于植物状况的另一个指标是植物和/或其产物的“品质”。根据本发明,增强的品质表示某些植物特征,例如某些成分的含量或组成,与相同条件下但没有应用本发明混合物而产生的植物的相同因子相比,增加或改善了可测量的或显著的量。增强的品质可以例如通过以下改善的植物或其产物的品质来表征:提高的营养素含量;和/或提高的蛋白质含量;和/或提高的含油量;和/或提高的淀粉含量;和/或提高的脂肪酸含量;和/或提高的代谢物含量;和/或提高的类胡萝卜素含量;和/或提高的糖含量;和/或提高的必需氨基酸含量;和/或改善的营养素组成;和/或改善的蛋白质组成;和/或改善的脂肪酸组成;和/或改善的代谢物组成;和/或改善的类胡萝卜素组成;和/或改善的糖组成;和/或改善的氨基酸组成;和/或改善的或优化的果实颜色;和/或改善的叶色;和/或较高的存储力;和/或采收产物更好的加工性。
植物状况的另一个指标是植物对生物和/或非生物胁迫因素的耐受性或抵抗力。生物和非生物胁迫,特别是长时间的,可以对植物具有有害影响。
生物胁迫是由活的生物体引起的,而非生物胁迫是例如由环境极端情况引起的。根据本发明,“对生物和/或非生物胁迫因素增强的耐受性或抵抗力”表示:(1.)与暴露于相同条件但没有用本发明混合物处理的植物相比,由生物和/或非生物胁迫引起的某些负面因素可以以可测量的或显著的量被减少;和(2.)通过本发明混合物对胁迫因素的直接作用,例如通过其直接破坏微生物或害物的杀真菌或杀虫作用,负面影响不能被减少,但通过刺激植物自身对抗所述胁迫因素的防御反应则可以。
由生物胁迫(例如病原体和害物)引起的负面因素是公知的并且是由活的生物体引起的,例如竞争性植物(例如杂草)、微生物(例如植物病原性真菌和/或细菌)和/或病毒。
由非生物胁迫引起的负面因素也是公知的并且常常可以作为降低的植物活力(参见上文)观察到,例如:
较低产量和/或较低活力,对于这两种影响,实例可以是例如焦枯的叶子、较少的花、过早的成熟、延后的作物成熟、降低的营养价值。
非生物胁迫可以例如通过以下引起:温度的极端情况,例如热或冷(热胁迫/冷胁迫);和/或温度的强烈变化;和/或对于特定季节不寻常的温度;和/或干旱(干旱胁迫);和/或极端湿度;和/或高盐度(盐胁迫);和/或辐射(例如由于减少的臭氧层引起的提高的UV辐射);和/或提高的臭氧水平(臭氧胁迫);和/或有机污染(例如农药的植物毒性量);和/或无机污染(例如重金属污染)。
作为生物和/或非生物胁迫因素的结果,受胁迫植物的数量和品质下降。就品质(如上文定义的)而言,生殖发育通常受到严重影响,在作物上造成对果实或种子重要的后果。蛋白质的合成、累积和储存主要受到温度的影响;几乎所有类型的胁迫会减缓生长;多糖合成,结构和存储都降低或改变:这些影响导致生物量(产量)降低和植物营养价值的改变。
如上文指出的,以上针对植物的健康状况定义的指标可以相互依赖并且可以互为因果。例如,提高的对生物和/或非生物胁迫的抵抗力可以导致更好的植物活力,例如更好和更大的作物,并且因此导致提高的产量。相反,发育更好的根系可以导致提高的对生物和/或非生物胁迫的抵抗力。然而,这些相互依赖性和相互作用不是全部都是已知的,也没有全面了解,因此分开描述这些不同的指标。
在一个实施方案中,本发明的混合物实现了植物或其产物提高的产量。在另一个实施方案中,本发明的混合物实现了植物或其产物提高的活力。在另一个实施方案中,本发明的混合物实现了植物或其产物提高的品质。在另一个实施方案中,本发明的混合物实现了植物或其产物提高的对抗生物胁迫的耐受性和/或抵抗力。在另一个实施方案中,本发明的混合物实现了植物或其产物提高的对抗非生物胁迫的耐受性和/或抵抗力。
本发明还涉及农用化学组合物,其包含助剂和至少一种如本文定义的类芽孢杆菌属(Paenibacillus)菌株或其无细胞提取物或至其少一种代谢物和至少一种根据本发明的农药II。
农用化学组合物包含杀真菌有效量或杀虫有效量的至少一种如本文定义的类芽孢杆菌属(Paenibacillus)菌株或其无细胞提取物或其至少一种代谢物和至少一种农药II。术语“有效量”指组合物的量或至少一种如本文定义的类芽孢杆菌属(Paenibacillus)菌株或其无细胞提取物或其至少一种代谢物和至少一种农药II的量,所述量足以促进植物健康,足以防治栽培植物上的有害真菌或有害害物或足以保护材料并且对处理的植物或材料不产生实质损害。这样的量可以在宽范围中不同并且取决于多种因素,例如待防治的真菌或害物物种,处理的栽培植物或材料,气候条件。
至少一种如本文定义的类芽孢杆菌属(Paenibacillus)菌株或其无细胞提取物或其至少一种代谢物和至少一种农药II可以转化成惯用类型的农用化学组合物,例如溶液、乳液、悬浮液、散粉剂、粉末、糊状物、颗粒、压制物、胶囊及其混合物。组合物类型的实例是悬浮液(例如SC、OD、FS)、可乳化浓缩物(例如EC)、乳液(例如EW、EO、ES、ME)、胶囊(例如CS、ZC)、糊状物、锭剂、可润湿的粉末或撒粉剂(例如WP、SP、WS、DP、DS)、压制物(例如BR、TB、DT)、颗粒(例如WG、SG、GR、FG、GG、MG)、杀虫物品(例如LN),以及用于处理植物繁殖材料(例如种子)的凝胶制剂(例如GF)。这些和进一步组合物类型定义于“Catalogue of pesticideformulation types and international coding system”,Technical Monograph No.2,第6版,2008年5月,CropLife International。
本发明的混合物可以配制成用于植物的接种物。术语“接种物”表示包括本发明的分离菌株和任选的载体(其可以包含生物学上可接受的介质)的组合物。
此类接种物和其它适合的组合物可以通过常规方式(参见,例如H.D.Burges:Formulation of Microbial Biopesticides,Springer,1998)制成组合物,所述组合物除了活性成分以外,还包含至少一种助剂(惰性成分)。
为了制备干制剂,可以将细菌细胞优选孢子悬浮于适合的干载体(例如粘土)中。为了制备液体制剂,可以将细胞优选孢子悬浮于适合的液体载体(例如基于水的)至所需的孢子密度。可以通过在琼脂培养基(例如马铃薯右旋糖琼脂)上在约20至约30℃的温度温育几天后鉴定集落形成单位(CFU)的数量来测定每mL的孢子密度数。
根据一个实施方案,本发明的组合物的各单独组分,例如试剂盒的部件或二元或三元混合物的部分,可以通过使用者自身在喷雾罐或任何其它种类的用于施用的容器(例如种子处理器鼓、种子造粒机、背负式喷雾器)中混合,并且如果适合,可以添加其它助剂。当活的生物体,例如本发明的类芽孢杆菌属(Paenibacillus)菌株,形成此类试剂盒的一部分时,必须小心,试剂盒的其它部分(例如化学农药)和其它助剂的选择和含量应当不影响微生物农药在使用者混合的组合物中的活力。特别是对于杀细菌剂和溶剂,必须考虑与相应微生物农药的相容性。
如本文定义的类芽孢杆菌属(Paenibacillus)菌株、全培养液、无细胞提取物、培养物基质和/或式I的杀镰孢菌素,与至少一种农药一起,可以转化成惯用类型的农用化学组合物,例如溶液、乳液、悬浮液、散粉剂、粉末、糊状物、颗粒、压制物、胶囊及其混合物。组合物类型的实例是悬浮液(例如SC、OD、FS)、可乳化浓缩物(例如EC)、乳液(例如EW、EO、ES、ME)、胶囊(例如CS、ZC)、糊状物、锭剂、可润湿的粉末或撒粉剂(例如WP、SP、WS、DP、DS)、压制物(例如BR、TB、DT)、颗粒(例如WG、SG、GR、FG、GG、MG)、杀虫物品(例如LN),以及用于处理植物繁殖材料(例如种子)的凝胶制剂(例如GF)。这些和进一步的组合物类型定义于“Catalogue of pesticide formulation types and international coding system”,Technical Monograph No.2,第6版,2008年5月,CropLife International。
可以以已知方式制备组合物,例如Mollet and Grubemann,Formulationtechnology,Wiley VCH,Weinheim,2001;或Knowles,New developments in cropprotection product formulation,Agrow Reports DS243,T&F Informa,London,2005中所述。
适合的助剂是溶剂、液体载体、固体载体或填充剂、表面活性剂、分散剂、乳化剂、润湿剂、佐剂、增溶剂、渗透增强剂、保护性胶体、粘附剂、增稠剂、湿润剂、驱避剂、引诱剂、摄食刺激剂、配伍剂、杀细菌剂、抗冻剂、消泡剂、着色剂、增粘剂和粘合剂。
适合的溶剂和液体载体是水和有机溶剂,例如中至高沸点的矿物油级分,例如煤油、柴油;植物或动物来源的油;脂肪族、环状或芳香族烃,例如甲苯、石蜡、四氢化萘、烷基化萘;醇,例如乙醇、丙醇、丁醇、苄醇、环己醇;二元醇;DMSO;酮,例如环己酮;酯,例如乳酸酯、碳酸酯、脂肪酸酯、γ-丁内酯;脂肪酸;膦酸酯;胺;酰胺,例如N-甲基吡咯烷酮、脂肪酸二甲基酰胺;及其混合物。
适合的固体载体或填充剂是矿物土,例如硅酸盐、硅胶、滑石、高岭土、石灰石、石灰、粘土、白云石、硅藻土、膨润土、硫酸钙、硫酸镁、氧化镁;多糖,例如纤维素、淀粉;肥料,例如硫酸铵、磷酸铵、硝酸钠、尿素;植物来源的产物,例如谷粉、树皮粉、木粉、坚果壳粉,及其混合物。
适合的表面活性剂是表面活性化合物,例如阴离子型、阳离子型、非离子型和两性表面活性剂、嵌段聚合物、聚电解质,及其混合物。此类表面活性剂可以用作乳化剂、分散剂、增溶剂、润湿剂、渗透增强剂、保护性胶体或助剂。表面活性剂的实例列于McCutcheon’s,Vol.1:Emulsifiers&Detergents,McCutcheon’s Directories,Glen Rock,USA,2008(国际版或北美版)。
适合的阴离子表面活性剂是磺酸、硫酸、磷酸、羧酸的碱、碱土或铵盐,及其混合物。磺酸盐的实例是烷基芳基磺酸盐、二苯基磺酸盐、α-烯烃磺酸盐、木质素磺酸盐、脂肪酸和油的磺酸盐、乙氧基化烷基酚的磺酸盐、烷氧基化芳基酚的磺酸盐、缩合萘的磺酸盐、十二烷基-和十三烷基苯的磺酸盐、萘和烷基萘的磺酸盐、磺基琥珀酸盐或磺基琥珀酰胺酸盐。硫酸盐的实例是脂肪酸和油的硫酸盐、乙氧基化烷基酚的硫酸盐、醇的硫酸盐、乙氧基化醇的硫酸盐,或脂肪酸酯的硫酸盐。磷酸盐的实例是磷酸盐酯。羧酸盐的实例是烷基羧酸盐和羧化醇或烷基酚乙氧基化物。
适合的非离子型表面活性剂是烷氧基化物、N-取代的脂肪酸酰胺、氧化胺、酯、基于糖的表面活性剂、聚合表面活性剂,及其混合物。烷氧基化物的实例是例如醇、烷基酚、胺、酰胺、芳基酚、脂肪酸或脂肪酸酯的化合物,其已经进行了1至50当量的烷氧基化。环氧乙烷和/或环氧丙烷可以用于烷氧基化,优选环氧乙烷。N-取代的脂肪酸酰胺的实例是脂肪酸葡糖胺或脂肪酸链烷醇酰胺。酯的实例是脂肪酸酯、甘油酯或单甘油酯。基于糖的表面活性剂的实例是山梨聚糖、乙氧基化山梨聚糖、蔗糖和葡萄糖酯或烷基聚葡糖苷。聚合表面活性剂的实例是乙烯吡咯烷酮、乙烯醇或乙酸乙烯酯的同聚物或共聚物。
适合的阳离子型表面活性剂是季盐型表面活性剂,例如具有一个或两个疏水性基团的季铵化合物,或长链伯胺的盐。适合的两性表面活性剂是烷基甜菜碱和咪唑啉。适合的嵌段聚合物是包含聚氧乙烯和聚氧丙烯嵌段的A-B或A-B-A型的嵌段聚合物,或包含烷醇、聚氧乙烯和聚氧丙烯的A-B-C型的嵌段聚合物。适合的聚电解质是聚酸或聚碱。聚酸的实例是聚丙烯酸或聚酸梳形聚合物的碱盐。聚碱的实例是聚乙烯基胺或聚乙撑胺。
适合的助剂是其自身具有可忽略的或甚至不具有农药活性并且其改善无细胞提取物、培养物基质或代谢物对靶标的生物学性能的化合物。实例是表面活性剂、矿物油或植物油,和其它助剂。更多的实例由Knowles,Adjuvants and additives(助剂和添加剂),Agrow Reports DS256,T&F Informa UK,2006,第5章列出。
适合的增稠剂是多糖(例如黄原胶、羧甲基纤维素)、无机粘土(有机修饰或未修饰的)、聚羧酸盐和硅酸盐。
适合的杀细菌剂是溴硝醇和异噻唑啉酮衍生物,例如烷基异噻唑啉酮和苯并异噻唑啉酮。适合的抗冻剂是乙二醇、丙二醇、尿素和甘油。适合的消泡剂是硅酮、长链醇和脂肪酸的盐。适合的着色剂(例如红色、蓝色或绿色)是低水溶性的色素和水溶性染料。实例是无机着色剂(例如氧化铁、氧化钛、六氰基高铁酸盐)和有机着色剂(例如茜素-、偶氮-和酞菁着色剂)。适合的增粘剂或粘合剂是聚乙烯吡咯烷酮、聚乙烯乙酸酯、聚乙烯醇、聚丙烯酸酯、生物或合成蜡和纤维素醚。
当活的微生物(例如类芽孢杆菌属(Paenibacillus)的细菌菌株)以细胞或孢子的形式形成组合物的一部分时,此类组合物可以通过常规方式(参见例如H.D.Burges:Formulation of Microbial Biopesticides,Springer,1998)制成除了活性成分外还包含至少一种助剂(惰性成分)的组合物。适合的惯用类型的此类组合物是悬浮液、散粉剂、粉末、糊状物、颗粒、压制物、胶囊,及其混合物。组合物类型的实例是悬浮液(例如SC、OD、FS)、胶囊(例如CS、ZC)、糊状物、锭剂、可润湿的粉末或散粉剂(例如WP、SP、WS、DP、DS)、压制物(例如BR、TB、DT)、颗粒(例如WG、SG、GR、FG、GG、MG)、杀虫物品(例如LN),以及用于处理植物繁殖材料(例如种子)的凝胶制剂(例如GF)。在此,必须考虑,每种制剂类型或助剂的选择不应当影响组合物储存过程中以及最终施用于土壤、植物或植物繁殖材料时微生物的活力。适合的制剂例如在WO 2008/002371、US 6,955,912、US 5,422,107中有提及。
适合助剂的实例是本文中前文提及的那些,其中必须考虑此类助剂的选择和含量不应当影响组合物中的微生物农药的活力。特别对于杀细菌剂和溶剂,必须考虑与相应微生物农药中的相应微生物的相容性。此外,含有微生物农药的组合物可以进一步含有稳定剂或营养素和UV防护剂。适合的稳定剂或营养素例如为α-生育酚、海藻糖、谷氨酸盐、山梨酸钾、多种糖,例如葡萄糖、蔗糖、乳糖和麦芽糖糊精(H.D.Burges:Formulation ofMicrobial Biopesticides,Springer,1998)。适合的UV防护剂例如是无机化合物,例如二氧化钛、氧化锌和氧化铁色素,或有机化合物,例如二苯酮、苯并三唑和苯基三嗪。除了针对本文组合物提及的助剂,组合物还可以任选包含0.1-80%稳定剂或营养素和0.1-10%UV防护剂。
农用化学组合物通常包含0.01至95%,优选0.1至90%,并且特别是0.5至75%重量的活性物质。活性物质可以以90%至100%的纯度,优选95%至100%的纯度(根据NMR谱)来使用。
组合物类型及其制备的实例为:
i)水溶性浓缩物(SL、LS)
将10-60wt%本发明混合物和5-15wt%润湿剂(例如醇烷氧基化物)溶解于水和/或水溶性溶剂(例如醇)中,补足100wt%。用水稀释后,活性物质溶解。
ii)可分散浓缩物(DC)
将5-25wt%本发明混合物和1-10wt%分散剂(例如聚乙烯吡咯烷酮)溶解于有机溶剂(例如环己酮)中,补足100wt%。用水稀释获得分散体。
iii)可乳化浓缩物(EC)
将15-70wt%本发明混合物和5-10wt%乳化剂(例如十二烷基苯磺酸钙和蓖麻油乙氧基化物)溶解于水不溶性有机溶剂(例如芳烃)中,补足100wt%。用水稀释获得乳液。
iv)乳液(EW、EO、ES)
将5-40wt%本发明混合物和1-10wt%乳化剂(例如十二烷基苯磺酸钙和蓖麻油乙氧基化物)溶解于20-40wt%水不溶性有机溶剂(例如芳烃)中。通过乳化机,将该混合物引入水中,补足100wt%,并且制成均质乳液。用水稀释获得乳液。
v)悬浮液(SC、OD、FS)
在搅拌球磨机中,将20-60wt%本发明混合物粉碎,并且添加2-10wt%分散剂和润湿剂(例如木质素磺酸钠和醇乙氧基化物)、0.1-2wt%增稠剂(例如黄原胶)和水,补足100wt%,获得细的活性物质悬浮液。用水稀释获得稳定的活性物质的悬浮液。对于FS型组合物,加入高达40wt%的粘合剂(例如聚乙烯基醇)。
vi)水分散颗粒和水溶性颗粒(WG、SG)
将50-80wt%本发明混合物细磨,并且添加分散剂和润湿剂(例如木质素磺酸钠和醇乙氧基化物),补足100wt%,并且通过技术设备(例如挤出、喷雾塔、流化床)制成水分散或水溶性颗粒。用水稀释获得稳定的活性物质的分散液或溶液。
vii)水分散性粉末和水溶性粉末(WP、SP、WS)
将50-80wt%本发明混合物在转子-定子研磨机中研磨,并且添加1-5wt%分散剂(例如木质素磺酸钠)、1-3wt%润湿剂(例如醇乙氧基化物)和固体载体(例如硅胶),补足100wt%。用水稀释获得稳定的活性物质的分散液或溶液。
viii)凝胶(GW、GF)
在搅拌球磨机中,将5-25wt%本发明混合物粉碎,并且添加3-10wt%分散剂(例如木质素磺酸钠)、1-5wt%增稠剂(例如羧甲基纤维素)和水,补足100wt%,获得活性物质的细悬浮液。用水稀释获得稳定的活性物质的悬浮液。
ix)微乳液(ME)
将5-20wt%本发明混合物加入至5-30wt%有机溶剂混合物(例如脂肪酸二甲基酰胺和环己酮)、10-25wt%表面活性剂混合物(例如醇乙氧基化物和芳基苯酚乙氧基化物)和水,补足100%。将该混合物搅拌1小时,以自发地产生热力学稳定的微乳液。
x)微胶囊(CS)
将包含5-50wt%本发明的混合物、0-40wt%水不溶性有机溶剂(例如芳烃)、2-15wt%丙烯酸单体(例如甲基丙烯酸甲酯、甲基丙烯酸和二-或三丙烯酸酯)的油相,分散于保护性胶体(例如聚乙烯基醇)的水溶液中。通过自由基引发剂启动的自由基聚合导致聚(甲基)丙烯酸酯微胶囊的形成。
可选择的是,将包含5-50wt%本发明的全培养液、无细胞提取物、培养物基质或代谢物、0-40wt%水不溶性有机溶剂(例如芳烃)和异氰酸酯单体(例如二苯基亚甲基-4,4’-二异氰酸酯)的油相,分散于保护性胶体(例如聚乙烯基醇)的水溶液中。添加聚胺(例如六亚甲基二胺)导致聚脲微胶囊的形成。单体占1-10wt%。wt%相对于整个CS组合物。
xi)粉剂(DP、DS)
将1-10wt%本发明的全培养液、无细胞提取物、培养物基质或代谢物细磨并且与固体载体(例如细碎的高岭土)密切混合,补足100wt%。
xii)颗粒(GR、FG)
将0.5-30wt%本发明的全培养液、无细胞提取物、培养物基质或代谢物细磨并且与固体载体(例如硅酸盐)组合,补足100wt%。通过挤出、喷雾干燥或流化床来造粒。
xiii)超低容量液体(UL)
将1-50wt%本发明的全培养液、无细胞提取物、培养物基质或代谢物溶解于有机溶剂(例如芳烃)中,补足100wt%。
组合物类型i)至xiii)可以任选包含其它助剂,例如0.1-1wt%杀细菌剂、5-15wt%抗冻剂、0.1-1wt%消泡剂和0.1-1wt%着色剂。
用于种子处理的溶液(LS)、悬乳液(SE)、可流动浓缩物(FS)、用于干处理的粉末(DS)、用于浆液处理的水分散粉末(WS)、水溶性粉末(SS)、乳液(ES)、可乳化浓缩物(EC)和凝胶(GF)通常用于植物繁殖材料(特别是种子)的处理目的。
用于预混组合物的种子处理制剂类型或土壤施用的优选实例是WS、LS、ES、FS、WG或CS-类型。
通常,用于种子处理应用的预混制剂包含0.5至99.9%,特别是1至95%所需成分,和99.5至0.1%,特别是99至5%固体或液体助剂(包括例如溶剂,例如水),其中基于预混制剂,助剂可以是0至50%,特别是0.5至40%含量的表面活性剂。而商业产品将优选配制成浓缩物(例如预混组合物(制剂)),最终使用者将正常地使用稀释制剂(例如罐混组合物)。
用于将本发明的菌株、全培养液、无细胞提取物、培养物基质、式I的杀镰孢菌素和组合物应用于或处理植物繁殖材料(特别是种子)的种子处理方法是本领域已知的,并且包括繁殖材料的拌药、包衣、薄膜包衣、丸粒化和浸没应用方法。此类方法也适用于根据本发明的组合。在优选的实施方案中,本发明的混合物和组合物通过萌发不受到负面影响的方法,应用于或处理植物繁殖材料。因此,用于应用于(或处理)植物繁殖材料(例如种子)的适合方法的实例是种子拌种、种子包衣或种子丸粒化等。
优选植物繁殖材料是种子、插条(即茎秆)或种球(seed bulb)。
尽管认为本发明的方法可以应用于任何生理状态的种子,但优选种子处于足够坚固的状态,使得在处理过程中不发生损伤。通常,种子将是已经从田间采收的种子;从植物取下的种子;以及从任何穗轴、茎秆、外壳和周围果肉或其它非种子植物材料分离的种子。种子优选还将是一定程度生物稳定的,从而处理不会对种子引起生物损伤。认为可以在种子采收与种子播种之间的任何时间或在播种过程中的任何时间,对种子施以处理(种子指向的应用)。还可以在处理之前或之后引发(prime)种子。
在繁殖材料处理过程中,希望本发明的混合物和组合物中的成分均匀分布并且均匀粘附于种子上。处理可以不同,从植物繁殖材料(例如种子)上的含有组合(例如活性成分的混合物)的制剂的薄膜(拌药(dressing))(其中初始大小和/或形状可识别),至中间状态(liru包衣(coating)),然后至较厚的膜(例如用许多层不同材料(例如载体,例如粘土;不同制剂,例如其它活性成分的制剂;聚合物;和着色剂)丸粒化(pelleting)),其中种子的初始形状和/或大小不再可识别。
本发明的一个方面包括将本发明的混合物和组合物以靶向方式应用于植物繁殖材料,包括将组合中成分放置在整个植物繁殖材料上或仅放置在其一部分上,包括只放置在单侧或单侧的一部分上。从EP954213B1和WO06/112700提供的描述中,本领域普通技术人员将理解这些应用方法。
本发明的菌株、全培养液、无细胞提取物、培养物基质、式I的杀镰孢菌素和组合物还可以以“丸剂”或“丸粒”或适合基质的形式来使用,将处理过的丸剂或基质放置或播种在植物繁殖材料旁边。这样的技术是本领域已知的,特别是在EP1124414、WO 07/67042和WO07/67044中。将本文中所述菌株、全培养液、无细胞提取物、培养物基质、式I的杀镰孢菌素和组合物应用于植物繁殖材料上还包括通过将一种或多种含有农药的颗粒放置在经农药处理过的种子旁边,以保护用本发明的组合处理过的植物繁殖材料,其中农药的含量将使得经农药处理过的种子和含有农药的颗粒一起包含有效剂量的农药,并且农药处理过的种子中含有的农药剂量低于或等于农药的最大无植物毒性剂量。此类技术是本领域已知的,特别是在WO2005/120226中。
将本发明的菌株、全培养液、无细胞提取物、培养物基质、式I的杀镰孢菌素和组合物应用于种子上还包括种子上控制释放包衣,其中组合中的成分掺入可以随着时间释放所述成分的材料中。控制释放种子处理技术的实例是本领域公知的并且包括聚合物膜、蜡或其它种子包衣,其中所述成分可以掺入控制释放材料中或应用在材料层之间,或两者。
可以以任何所需顺序或同时,将本发明的混合物和组合物施用于种子来处理种子。
种子处理可以针对未播种的种子进行,并且术语“未播种的种子”意在包括处于种子采收与为了植物发芽和生长的目的在田间播种种子之间的任何时间段的种子。
对未播种的种子的处理不旨在包括其中将活性物质应用于土壤的那些实践,但包括种植过程中靶向种子的任何应用实践。
优选,处理在种子播种前进行,使得播种的种子已经用本发明的混合物和组合物预处理过。特别地,优选种子包衣或种子丸粒化。作为处理的结果,成分粘附于种子上并且因此可用于害物防治。
处理过的种子可以按照与任何其它活性成分处理过的种子一样的方式储存、操作、播种和耕作。
特别的是,本发明涉及用于保护植物繁殖材料免受害物、有害真菌和/或改善从所述植物繁殖材料生长的植物的健康的方法,其中用有效量的本发明混合物或组合物,处理播种植物繁殖材料的土壤。
使用者通常从预定剂量装置、背负式喷雾器、喷雾罐、喷洒飞机或灌溉系统来应用本发明的组合物。通常,用水、缓冲液和/或其它助剂将农用化学组合物制成所需的应用浓度,并且因此获得根据本发明的即用的喷雾液体或农用化学组合物。通常,每公顷农业有用面积应用20至2000升,优选50至400升即用的喷雾液体。
处理植物繁殖材料(特别是种子)时,本文中公开的组合物在二至十倍稀释后,在即用的制备物中提供0.01至60%重量,优选0.1至40%的活性组分浓度。应用可以在播种前或播种过程中进行。用于将本发明的菌株、无细胞提取物、培养物基质、代谢物或组合物施用于植物繁殖材料(特别是种子)上的方法包括植物繁殖材料的拌药、包衣、丸粒化、撒粉、浸泡和犁沟内(in-furrow)应用方法。优选,通过使得不会诱导发芽的方法,例如通过种子拌药、丸粒化、包衣或撒粉,将本发明的菌株、全培养液、无细胞提取物、培养物基质、式I的杀镰孢菌素或组合物应用于植物繁殖材料上。
将细菌菌株用于作物保护中时,其中菌株作为叶面处理应用或应用至土壤,应用率范围通常为约1×106至5×1016(或更高)CFU/ha,优选约1×107至约1×1016CFU/ha,甚至更优选1×1012至5×1015CFU/ha。
将本发明的菌株用于种子处理中时,关于植物繁殖材料的应用率范围通常为约1×101至1×1012(或更高)CFU/种子,优选约1×103至约1×1010CFU/种子,并且甚至更优选约1×103至约1×106CFU/种子。可选择的是,关于植物繁殖材料的应用率范围优选为约1×107至1×1016(或更高)CFU/100kg种子、优选1×109至约1×1015CFU/100kg种子,甚至更优选1×1011至约1×1015CFU/100kg种子。
使用无细胞提取物、培养物基质和/或代谢物例如式I的杀镰孢菌素时,固体材料(干物质)被认为是活性组分,例如在液体制剂的情况中,在干燥或蒸发提取介质或悬浮介质后获得的。用于植物保护中时,取决于所需的作用类型,应用的活性组分量为0.001至2kg/ha,优选0.005至2kg/ha,更优选0.05至0.9kg/ha,并且特别是0.1至0.75kg/ha。在植物繁殖材料(例如种子)的处理中,例如通过给种子撒粉、包衣或浇灌,通常需要每100公斤植物繁殖材料(优选种子)0.1至1000g,优选1至1000g,更优选1至100g并且最优选5至100g活性组分的量。用于材料或储存产品的保护中时,应用的活性组分的量取决于应用区域的种类和所需的效果。通常用于材料保护中的量为每立方米处理材料0.001g至2kg,优选0.005g至1kg活性组分。
根据一个实施方案,本发明组合物的各组分,例如试剂盒的部分或者二元或三元混合物的部分,可以通过使用者自己在喷雾罐或任何其它种类的用于施用的容器(例如种子处理器鼓、种子粒化机、背负式喷雾器)中混合,并且如果适合,可以加入其它助剂。
如果活的生物体,例如类芽孢杆菌属(Paenibacillus)细菌菌株,形成此类试剂盒的一部分,必须小心,组分(例如化学农药)和其它助剂的选择和量不应当影响微生物在使用者混合的组合物中的活力。特别是对于杀细菌剂和溶剂,必须考虑与相应微生物的相容性。
因此,本发明的一个实施方案是用于制备可用农药组合物的试剂盒,所述试剂盒包含a)组合物,其包含如本文定义的组分1)和至少一种助剂;和b)组合物,其包含如本文定义的组分2)和至少一种助剂;和任选的c)组合物,其包含至少一种助剂和任选的如本文定义的其它活性组分3)。
多种类型的油、润湿剂、助剂、肥料或微量营养素和其它农药(例如除草剂、杀虫剂、杀真菌剂、生长调节剂、安全剂、生物农药)可以加入本发明的菌株、无细胞提取物、培养物基质、代谢物、式I的杀镰孢菌素和组合物,作为预混物,或如果适合,直到临施用前加入(罐混合)。这些试剂可以与本发明的组合物以1:100至100:1的重量比混合,优选1:10至10:1。优选本发明的组合物包含其它生物农药。甚至更优选,本发明的组合物除了助剂和至少一种式I的杀镰孢菌素外,还包含微生物农药。
农药通常是通过其作用来阻止害物、使害物失能、杀灭害物或妨碍害物的化学或生物学试剂(例如病毒、细菌、抗微生物剂或消毒剂)。靶害物可以包括破坏财产、造成滋扰、传播疾病或构成疾病载体的昆虫、植物病原体、杂草、软体动物、鸟类、哺乳动物、鱼、线虫(蛔虫)和微生物。术语农药还包括改变预期的植物生长、开花或繁殖速率的植物生长调节剂;引起叶或其它树叶从植物掉落的落叶剂,通常以有助于采收;促进活组织(例如不需要的植物顶端)干燥的干燥剂;激活植物用于对抗某些害物的防御生理学的植物激活剂;降低农药在作物上的不合需要的除草作用的安全剂;和影响植物生理学以提高植物生长、作物的可采收物的生物量、产量或任何其它品质参数的植物生长促进剂。
通过连同至少一种来自组A)至组N)的农药II一起应用至少一种如以上任意一个优选实施方案中所定义的类芽孢杆菌属(Paenibacillus)菌株或其培养物基质或无细胞提取物或其至少一种代谢物,可以获得协同作用,即获得大于各自作用简单相加(协同性混合物)。
根据一个实施方案,混合物以协同有效量包含组分1)和组分2)。
术语“协同作用”应当被理解为特别意指Colby’s式定义的作用(Colby,S.R.,“Calculating synergistic and antagonistic responses of herbicidecombinations”,Weeds,15,pp.20-22,1967)。
术语“协同作用”还应当被理解为意指Tammes方法定义的作用(Tammes,P.M.L.,“Isoboles,a graphic representation of synergism in pesticides”,Netherl.J.Plant Pathol.70,1964)。
这可以通过以下方式获得:将至少一种如以上任意一个优选实施方案中所定义的类芽孢杆菌属(Paenibacillus)菌株或其培养物基质或无细胞提取物或其至少一种代谢物和至少一种农药II同时(联合(作为罐混物)或分别)或依次应用,其中选择各个应用之间的时间间隔以确保首先应用的活性物质在应用其它活性物质时仍以足够量出现在作用部位处。应用顺序对于实施本发明并不重要。
当依次应用如以上任意一个优选实施方案中所定义的类芽孢杆菌属(Paenibacillus)菌株或其培养物基质或无细胞提取物或其代谢物和农药II时,两次应用之间的时间可以不同,例如在2小时至7天之间不同。更宽的范围也还可能,范围从0.25小时至30天、优选0.5小时至14天、特别是1小时至7天或1.5小时至5天,甚至更优选2小时至1天。优选农药II作为首次处理应用。
可以使用相应微生物的CFU量,按以下等式计算相应活性组分的总重量:1×1010CFU等于一克相应活性组分的总重量,确定以活微生物细胞形式(包括休眠形式)包含至少一种微生物农药的组合物的总重量比例。菌落形成单位是活的微生物细胞、特别是真菌细胞和细菌细胞的量度。
在本发明的混合物和组合物中,组分1)和组分2)的重量比通常取决于使用的活性组分的性质,通常它的范围为1:10,000至10,000:1,经常它的范围为1:100至100:1,常规地范围为1:50至50:1,优选范围为1:20至20:1,更优选范围为1:10至10:1,甚至更优选范围为1:4至4:1并且特别是范围为1:2至2:1。
根据混合物和组合物的进一步的实施方案,组分1)和组分2)的重量比通常范围为1000:1至1:1,经常范围为100:1至1:1,常规地范围为50:1至1:1,优选范围为20:1至1:1,更优选范围为10:1至1:1,甚至更优选范围为4:1至1:1并且特别是范围为2:1至1:1。
根据混合物和组合物的进一步的实施方案,组分1)和组分2)的重量比通常范围为20,000:1至1:10,经常范围为10,000:1至1:1,常规地范围为5,000:1至5:1,优选范围为5,000:1至10:1,更优选范围为2,000:1至30:1,甚至更优选范围为2,000:1至100:1并且特别是范围为1,000:1至100:1。
根据混合物和组合物的进一步的实施方案,组分1)和组分2)的重量比通常范围为1:1至1:1000,经常范围为1:1至1:100,常规地范围为1:1至1:50,优选范围为1:1至1:20,更优选范围为1:1至1:10,甚至更优选范围为1:1至1:4并且特别是范围为1:1至1:2。
根据混合物和组合物的进一步的实施方案,组分1)和组分2)的重量比通常范围为10:1至1:20,000,经常范围为1:1至1:10,000,常规地范围为1:5至1:5,000,优选范围为1:10至1:5,000,更优选范围为1:30至1:2,000,甚至更优选范围为1:100至1:2,000并且特别是范围为1:100至1:1,000。
作为杀真菌剂和/或杀虫剂的使用形式时,本发明的混合物和组合物还可以与其它活性物质共存,例如与除草剂、杀虫剂、生长调节剂、杀真菌剂或者还与肥料共存在,作为预混物,或如果适合,至临用前(罐混物)才共存。
将本发明的二元混合物或包含它们的组合物与其它杀真菌剂混合在许多情况下导致杀真菌活性谱扩展或导致阻止杀真菌剂抗性形成。另外,在许多情况下,获得协同作用。
将本发明的二元混合物或包含它们的组合物与其它杀虫剂混合在许多情况下导致杀虫活性谱扩展或导致阻止杀虫剂抗性形成。另外,在许多情况下,获得协同作用。
根据本发明,可以优选混合物和包含它们的组合物,除至少一种如以上任意一个优选实施方案中所定义的类芽孢杆菌属(Paenibacillus)菌株或其培养物基质或无细胞提取物或其至少一种代谢物(组分1)、和农药II(组分2)之外还包含作为组分3)的其它农药,优选以协同有效量包含。另一个实施方案涉及其中组分3)是选自如下文定义的组SF)和组SI)中农药III的混合物,条件是这些三元混合物每者中,农药III与选择的农药II不同。这些三元混合物特别适于处理植物繁殖材料(即处理种子)。
可以结合本发明二元混合物使用以下列表的农药III,该列表意在显示可能的组合,而非限制它们:
SF)杀真菌剂
-Qo位点的复合体III抑制剂,其选自:唑菌胺酯(pyraclostrobin)、腈嘧菌酯(azoxystrobin)、啶氧菌酯(picoxystrobin)、肟菌酯(trifloxystrobin)、醚菌胺(dimoxystrobin)、烯肟菌酯(enestroburin)、烯肟菌胺(fenaminstrobin)、氟嘧菌酯(fluoxastrobin)、亚胺菌(kresoxim-methyl)、甲哌喹(mandestrobine)、叉氨苯酰胺(metominostrobin)、肟醚菌胺(orysastrobin)、唑胺菌酯(pyrametostrobin)、唑菌酯(pyraoxystrobin);
-复合体II的广谱吡啶和吡唑抑制剂,其选自:氟唑菌酰胺(fluxapyroxad)、啶酰菌胺(boscalid)、苯并烯氟菌唑(benzovindiflupyr)、氟唑菌苯胺(penflufen)、吡噻菌胺(penthiopyrad)、氟唑环菌胺(sedaxane)、氟吡菌酰胺(fluopyram)、联苯吡菌胺(bixafen)、吡唑萘菌胺(isopyrazam);
-担子菌纲(Basidiomycetes)特异性复合体II抑制剂,其选自:萎锈灵(carboxin)、麦锈灵(benodanil)、呋菌胺(fenfuram)、氟酰胺(flutolanil)、呋吡唑灵(furametpyr)、丙氧灭锈胺(mepronil)、氧化萎锈灵(oxycarboxin)、溴氟唑菌(thifluzamide);
ATP产生抑制剂:硅噻菌胺(silthiofam);
-杀真菌性吡咯(azole)化合物,其选自:环戊唑醇(ipconazole)、醚唑(difenoconazole)、丙硫菌唑(prothioconazole)、丙氯灵(prochloraz)、戊叉唑菌(triticonazole)、粉唑醇(flutriafol)、环唑醇(cyproconazole)、烯唑醇(diniconazole)、精烯唑醇(diniconazole-M)、喹唑菌酮(fluquinconazole)、氟硅唑(flusilazole)、己唑醇(hexaconazole)、烯菌灵(imazalil)、酰胺唑(imibenconazole)、环戊唑菌(metconazole)、腈菌唑(myclobutanil)、硅氟唑(simeconazole)、戊唑醇(tebuconazole)、唑菌醇(triadimenol)、烯效唑(uniconazole)、涕必灵(thiabendazole);
-卵菌纲(Oomycetes)杀真菌剂,其选自:氟噻唑吡乙酮(oxathiapiprolin)、缬氨菌酯(valifenalate)、甲霜灵(metalaxyl)、精甲霜灵(metalaxyl-M)、噻唑菌胺(ethaboxam)、烯酰吗啉(dimethomorph)、苯酰菌胺(zoxamide)、氟吗啉(flumorph)、双炔酰菌胺(mandipropamid)、丁吡吗啉(pyrimorph)、苯噻菌胺(benthiavalicarb)、异丙菌胺(iprovalicarb);
-MAP/组氨酸激酶抑制剂:咯菌腈(fludioxonil);
-苯并咪唑化合物,其选自:甲基托布津(thiophanate-methyl)、多菌灵(carbendazim);
-二硫代氨基甲酸酯化合物,其选自:福美双(thiram)、福美锌(ziram);
SI)杀虫剂
-GABA拮抗剂化合物,其选自:氟虫腈(fipronil)、乙虫腈(ethiprole)、氟吡唑虫(vaniliprole)、吡嗪氟虫腈(pyrafluprole)、吡啶氟虫腈(pyriprole)、5-氨基-1-(2,6-二氯-4-甲基-苯基)-4-氨亚磺酰基-1H-吡唑-3-硫代甲酰胺;
-鳞翅类(lepidopteran)-特异性兰尼碱受体抑制剂,其选自:氯虫酰胺(chlorantraniliprole)和氟虫双酰胺(flubendiamide);
-交叉谱兰尼碱受体抑制剂:氰虫酰胺(cyantraniliprole);
-拟除虫菊酯钠通道调节剂,其选自:七氟菊酯(tefluthrin)、联苯菊酯(bifenthrin)、氯氰菊酯(cypermethrin)、甲体氯氰菊酯(alpha-cypermethrin)、氟氯氰菊酯(cyfluthrin)、高效氟氯氰菊酯(beta-cyfluthrin)、高效氟氯氰菊酯(lambda-cyhalothrin)、溴氰菊酯(deltamethrin)、顺式氰戊菊酯(esfenvalerate)、醚菊酯(etofenprox)、氰戊菊酯(fenvalerate)、氟氰戊菊酯(flucythrinate)、氯菊酯(permethrin);
-全身活性新烟碱(neonicotinoid)化合物:噻虫胺(clothianidin)、吡虫啉(imidacloprid)、噻虫嗪(thiamethoxam)、呋虫胺(dinotefuran)、啶虫脒(acetamiprid)、氟吡呋喃酮(flupyradifurone)、噻虫啉(thiacloprid)、三氟苯嘧啶(triflumezopyrim)、烯啶虫胺(nitenpyram);
-乙酰胆碱酯酶抑制剂、氯通道激活剂和亚砜亚胺(sulfoximine):氟啶虫胺腈(sulfoxaflor)、乙酰甲胺磷(acephate)、毒死蜱(chlorpyrifos)、硫双威(thiodicarb)、齐墩螨素(abamectin)、艾克敌105(spinosad);
-其它杀虫剂:替恶杀芬(tioxazafen)。
更优选地,农药III选自以下组SF)和SI):
SF)杀真菌剂
腈嘧菌酯(azoxystrobin)、肟菌酯(trifloxystrobin)、啶氧菌酯(picoxystrobin)、唑菌胺酯(pyraclostrobin)、氟唑环菌胺(sedaxane)、吡噻菌胺(penthiopyrad)、氟唑菌苯胺(penflufen)、氟吡菌酰胺(fluopyram)、氟唑菌酰胺(fluxapyroxad)、啶酰菌胺(boscalid)、氟噻唑吡乙酮(oxathiapiprolin)、甲霜灵(metalaxyl)、精甲霜灵(metalaxyl-M)、噻唑菌胺(ethaboxam)、烯酰吗啉(dimethomorph)、缬氨菌酯(valifenalate)、环唑醇(cyproconazole)、苯醚甲环唑(cifenoconazole)、丙硫菌唑(prothioconazole)、粉唑醇(flutriafol)、涕必灵(thiabendazole)、环戊唑醇(ipconazole)、戊唑醇(tebuconazole)、唑菌醇(triadimenol)、丙氯灵(prochloraz)、喹唑菌酮(fluquinconazole)、戊叉唑菌(triticonazole)、咯菌腈(fludioxinil)、萎锈灵(carboxin)、硅噻菌胺(silthiofam)、福美锌(ziram)、福美双(thiram)、多菌灵(carbendazim)、甲基托布津(thiophanate-methyl);
SI)杀虫剂
氟虫腈(fipronil)、噻虫胺(clothianidin)、噻虫嗪(thiamethoxam)、啶虫脒(acetamiprid)、呋虫胺(dinotefuran),、吡虫啉(imidacloprid)、噻虫啉(thiacloprid)、氟啶虫胺腈(sulfoxaflor)、灭虫威(methiocarb)、七氟菊酯(tefluthrin)、联苯菊酯(bifenthrin)、氯氰菊酯(cypermethrin)、甲体氯氰菊酯(alpha-cypermethrin)、艾克敌105(spinosad)、氯虫酰胺(chlorantraniliprole)、氰虫酰胺(cyantraniliprole)、硫双威(thiodicarb)、三氟苯嘧啶(triflumezopyrim)、乙酰甲胺磷(acephate)、毒死蜱(chlorpyriphos)、氟吡呋喃酮(flupyradifurone)、齐墩螨素(abamectin)、替恶杀芬(tioxazafen)。
实施例
将通过以下实施例更详细地描述本发明。以下实施例是为了举例说明的目的,并且不是用来限制本发明的范围。
涉及类芽孢杆菌属(Paenibacillus)菌株及其代谢物的实施例
实施例1:类芽孢杆菌属(Paenibacillus)菌株的分离
从多个欧洲位置(包括德国)收集了土壤样品。通过对这些土壤应用公知的微生物分离程序,发明人获得了多种细菌,将其进一步使用常规分离技术以提供本文中所述纯分离物。
按照标准微生物富集技术(C.A.Reddy,T.J.Beveridge,J.A.Breznak,G.A.Marzluf,T.M.Schmidt和L.R.Snyder(编辑).Methods for General and MolecularMicrobiology,Am.Soc.Microbiol.,Washington,District of Columbia),分离每种类型的细菌。
以下菌株已经分离并且于2013年2月20日依据布达佩斯条约保藏于德意志微生物和培养物保藏中心(Deutsche Sammlung von Mikroorganismen und Zellkulturen,DSMZ):
a)以保藏号DSM 26969保藏于DSMZ的Lu16774
b)以保藏号DSM 26970保藏于DSMZ的Lu17007,
c)以保藏号DSM 26971保藏于DSMZ的Lu17015。
实施例2-类芽孢杆菌属(Paenibacillus)菌株的表征
实施例2.1:16S-rDNA测序
在DSMZ,Braunschweig,德国,通过PCR扩增的16S rDNA的直接测序,测定了类芽孢杆菌属(Paenibacillus)菌株的16S rRNA基因序列。
根据制造商的说明,使用来自Epicentre Biotechnologies的MasterPureTM革兰氏阳性DNA纯化试剂盒,进行了基因组DNA提取。按照之前所述(Int.J.Syst.Bacteriol.46,1088-1092,1996),进行了16S rDNA的PCR-介导的扩增和PCR产物的纯化。按照制造商试验方案的指导,使用Terminator v1.1环测序试剂盒(Applied Biosystems),对纯化的PCR产物测序。使用来自Applied Biosystems的3500xL遗传分析仪,将序列反应物电泳。序列的不定性可能是由于单个基因组内存在几种具有不同序列的编码16S rRNA的顺反子(J.Bacteriol.178(19),5636-5643,1996)。
将从菌株得到的序列数据输入比对编辑器AE2(http://iubio.bio.indiana.edu/soft/molbio/unix/ae2.readme)中,根据所得到的rRNA分子的二级结构进行人工比对,并且与属于厚壁菌门(Firmicutes)的生物体的代表性16S rRNA基因序列进行比较(Nucl.Acids Res.27,171-173,1999)。为了比较,从EMBL和RDP数据库获得16S rRNA序列。
本发明菌株的16S rRNA序列列于序列表中,如表2中所示。
表2:类芽孢杆菌属(Paenibacillus)菌株的16S rRNA的序列表参考
菌株 | SEQ ID NO |
Lu16774 | 1 |
Lu17007 | 2 |
Lu17015 | 3 |
通过在所比较序列的比对中进行序列的成对比较,计算了以%计的16S rRNA基因同一性值。
基于成对序列比对进行的仅两个序列的比较,在本文中表示为二元值(binaryvalue)。其它值所基于的是比较中全部序列的多重序列比对。来自多重序列比较的更高同一性值是由所比较序列的序列数据具有不同长度引起的,导致了较短的比对。
来自三种菌株Lu16774、Lu17007和Lu17015的完整rDNA序列的成对比较的%同一性为99.5%至99.9%(表3,二元值)。
表3:三种类芽孢杆菌属(Paenibacillus)菌株的完整16S rRNA序列的%同一性(括号中为二元值)。
三种菌株Lu16774、Lu17007和Lu17015的完整16S rRNA序列与相关分类群的比较(参见图9),揭示了与皮氏类芽孢杆菌(Paenibacillus peoriae)(模式菌株DSM 8320)具有99.8%的高同一性百分数。对于皮氏类芽孢杆菌(P.peoriae)与菌株Lu16774、Lu17007和Lu17015的成对序列比对,二元值分别如下:Lu16774:99.5%,Lu17007:99.5%;和Lu17015,99.7%同一性。
基于16S rRNA序列数据,不可能最终评价类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015所属物种。
针对皮氏类芽孢杆菌(Paenibacillus peoriae)NRRL BD-62的完整rDNA的测序获得与皮氏类芽孢杆菌(P.peoriae)(模式菌株DSM 8320)的100.0%同一性,证实针对这种菌株BD-62的物种命名皮氏类芽孢杆菌(P.peoriae)(参见图9)。
通过与皮氏类芽孢杆菌(P.peoriae)菌株BD-62的16S rRNA序列的比较,获得了99.8%的同一性值,证实了所有三种类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015与皮氏类芽孢杆菌(P.peoriae)的密切关系(参见图9)。
为了构建系统发生树,使用了ARB包的操作(Nucl.Acids Res.35,7188-7196,2007):基于进化距离值,使用Jukes和Cantor的校正(Mol.Biol.Evol.4,406-425,1987),通过邻接法(Jukes,T.H.和Cantor C.R.(1969).Evolution of protein molecules.InMammalian protein metabolism,第21-132页.H.N.Munro.编辑New York:Academicpress),构建了系统发生树。通过将耐热科恩氏菌(Cohnella thermotolerans)的16S rRNA基因序列包括在分析中来确定树根。进化系统树下方的比例尺表示每100个核苷酸1个核苷酸置换。图10中给出了结果。
这些序列的系统发生树(图10)显示出三种菌株Lu16774、Lu17007和Lu17015彼此最相关,并且对于它们之每一个,已知的最相关亲属是皮氏类芽孢杆菌(Paenibacilluspeoriae)菌株NRRL BD-62。
实施例2.2:RiboPrint-分析
使用Qualicon RiboPrintersystem进行了标准化的、自动化的核糖体分型。RiboPrinter系统将核糖体分型的分子处理步骤组合在单机自动化仪器中。该程序包括细胞裂解、用限制性酶EcoRI消化染色体DNA、通过电泳分离片段、DNA片段转移至尼龙膜、与自大肠杆菌(E.coli)的rrnB操纵子产生的探针杂交、化学发光检测探针与含有rrn操纵子序列的片段的杂交、图像检测和RiboPrint模式的计算机化分析(Food Technology 50(1),77-81,1996;Proc.Natl.Acad.Sci.USA 92,5229-5233,1995;Int.Journ.Syst.Bact.44(3),454-460,1994)。
使用限制性酶EcoRI,通过类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015与皮氏类芽孢杆菌(P.peoriae)菌株BD-62比较,由德国DSMZ执行核糖体分型。使用RiboPrinter系统的软件、集成DuPont识别文库以及BioNumerics软件(Applied Maths,比利时),比较了所得到的模式。
所有三种菌株Lu16774、Lu17007和Lu17015与BD-62的相似性在0.24至0.5之间(图11)。三种菌株归入两个组,第一组包含Lu17015,而第二组包含菌株Lu16774和Lu17007。没有一个菌株与DuPont识别文库内的任何菌株具有高于0.84的相似性,并且因此没有得到自动识别。
基于DuPont识别文库的条目DUP-13142(基于皮氏类芽孢杆菌(Paenibacilluspeoriae)DSM 8320的条目),菌株BD-62已经被鉴定为皮氏类芽孢杆菌(Paenibacilluspeoriae)。
实施例2.3:形态学和生理学表征
在DSMZ,以类似于Gordon,R.E.,Haynes,W.C.和Pang.C.H.-N.(1973):The GenusBacillus,Agriculture Handbook no.427.Washington DC:US Department ofAgriculture中所述方法,表征了菌株。结果在表4中给出。
表4:本发明的类芽孢杆菌属(Paenibacillus)菌株的表征数据以及与已知的皮氏类芽孢杆菌(Paenibacillus peoriae)菌株NRRL BD-62的比较。
*n.g.=无生长
在DSMZ进行了细胞脂肪酸分析,所有菌株显示出类芽孢杆菌属(Paenibacillus)物种的典型特征。
使用可得的遗传、生理和生物化学数据,显示菌株Lu16774、Lu17007和Lu17015属于类芽孢杆菌属(Paenibacillus)。因为菌株Lu16774、Lu17007和Lu17015以及BD-62从葡萄糖产生气体,所以它们均不属于杰米拉类芽孢杆菌(Paenibacillus jamilae)。
使用从一些底物产酸的特征(Int.J.Syst.Bacteriol.43(2),388-390,1993;In.J.Syst.Bacteriol.46(6),988-1003,1996),皮氏类芽孢杆菌(Paenibacilluspeoriae)和多粘类芽孢杆菌(Paenibacillus polymyxa)之间的表型区分基本上可行。菌株Lu16774、Lu17007和Lu17015无一与表4中针对这两个物种的任何一个完全列出的特征完全匹配,但可得的遗传、生理和生物化学数据总体上最可能指向物种皮氏类芽孢杆菌(Paenibacillus peoriae)和多粘类芽孢杆菌(P.polymyxa)或至少与皮氏类芽孢杆菌(Paenibacillus peoriae)和多粘类芽孢杆菌(P.polymyxa)非常密切相关的另一个物种。
由于迄今为止描述的类芽孢杆菌属(Paenibacillus)物种的众多性,故不可能基于表4的生理学和形态学标准确定所测试的三种分离物的正确分类学物种(RainerBorriss,Humboldt University Berlin,未公开的结果)。
然而,完全确定这个属内的物种是不可能的。基于16S-rDNA分析,发现最密切相关的物种和菌株是皮氏类芽孢杆菌(Paenibacillus peoriae)BD-62(参见例如图11)。
实施例2.4:基于编码DnaN、GyrB、RecF、RecN和RpoA的基因的系统发生分析
已经从完整基因组序列或从公共数据库中提取了编码DnaN、GyrB、RecF、RecN和RpoA的基因的核苷酸序列(如表28中列出的序列表)。
用全对全的方法(all against all approach)产生了同一性表(图12至16),在所述方法中,将每个序列与每另一个序列比对。用程序针(EMBOSS package 6.6.0;Trends inGenetics 16(6);276-277)进行了序列比对。使用了标准参数(空位形成10.0;空位延伸0.5)。基于比对计算了同一性评分,没有考虑任何空位。
对于系统发生树(图17至21),使用Clustal Omega(版本1.2.0;MolecularSystems Biology 7:539,doi:10.1038/msb.2011.75)进行了多序列比对。通过最大似然法,使用软件Dnaml(在Phylip 3.696包中执行;Felsenstein 1981,http://evolution.genetics.washington.edu/phylip.html),计算了系统发生树。使用F84距离模型,同时使用二(2)的转换/颠换率(transition/transversion ratio),建立了系统树图。用工具Dendroscope(http://dendroscope.org/)绘制树。
表28:类芽孢杆菌属(Paenibacillus)菌株的dnaN、gyrB、recF、recN和rpoA DNA序列的序列表参考
菌株 | 基因 | SEQ ID NO |
Lu16774 | dnaN | 4 |
Lu16774 | gyrB | 5 |
Lu16774 | recF | 6 |
Lu16774 | recN | 7 |
Lu16774 | rpoA | 8 |
Lu17007 | dnaN | 9 |
Lu17007 | gyrB | 10 |
Lu17007 | recF | 11 |
Lu17007 | recN | 12 |
Lu17007 | rpoA | 13 |
Lu17015 | dnaN | 14 |
Lu17015 | gyrB | 15 |
Lu17015 | recF | 16 |
Lu17015 | recN | 17 |
Lu17015 | rpoA | 18 |
实施例2.5:核心基因组比较和AAI矩阵
使用Gieβen大学的软件包EDGAR(BMC Bioinformatics 10,154,2009;(https://edgar.computational.bio.uni-giessen.de/cgi-bin/edgar.cgi))进行了基因组比较。使用软件包EDGAR,基于完整基因组序列和AAI矩阵值,进行了核心基因组的确定和系统发生树。结果显示于图22。
实施例3:用于体内试验的菌株的生长(可发酵性)
对于温室和田间试验,首先将类芽孢杆菌属(Paenibacillus)菌株生长于ISP2平板上(来自BD[USA]的即用型琼脂,目录编号277010)。此后,在含有液体ISP2培养基的带挡板的摇瓶中接种来自琼脂平板的菌落并且在150rpm和25℃温育5-7天。取决于该试验,将全培养液或离心且H2O洗后的细胞沉淀物或上清液应用于植物。可以扩大规模至10L发酵罐。
将类芽孢杆菌属(Paenibacillus)菌株在ISP2液体培养基(10g/L麦芽提取物、4g/L细菌培养用酵母提取物、4g/L葡萄糖一水合物)在22℃和150rpm生长6天。在不同时间点测量指示细菌生长的OD600nm。
表5:液体ISP2培养基中类芽孢杆菌属(Paenibacillus)菌株的细菌生长
实施例4-针对抗真菌活性的体外对抗测试
在体外对抗测试中显示了类芽孢杆菌属(Paenibacillus)菌株对抗植物病原体的拮抗活性。使用的植物病原性真菌是核盘菌(Sclerotina sclerotiorum)(SCLSCL)、灰葡萄孢(Botrytis cinerea)(BOTRCI)、链格孢属(Alternaria)物种(ALTESP)和致病疫霉(Phytophthora infestans)(PHYTIN)。
作为用于BOTRCI、ALTESP、SCLSCL的生长培养基,使用了ISP2培养基,其每升包含:10g麦芽提取物(Sigma Aldrich,70167);4g细菌培养用酵母提取物(Becton Dickinson,212750);4g葡萄糖一水合物(Sigma Aldrich,16301);20g琼脂(Becton Dickinson,214510),pH约7,重蒸水。作为用于PHYTIN的生长培养基,使用了V8培养基,其每升包含:200mL蔬菜汁,3g碳酸钙(Merck Millipore,1020660250);30g琼脂(Becton Dicknson,214510),pH 6.8,重蒸水。
将类芽孢杆菌属(Paenibacillus)菌株点接种于琼脂平板的一侧。将含有一种活跃生长的植物病原体的琼脂块(大约0.3cm2)放在平板中央。在25℃温育7-14天后,检查植物病原体的生长,特别是抑制区。
此后,在评价前,将琼脂平板在℃温育约7-14天。通过评价无真菌区(抑制区)的直径,对抗生作用进行评分。通过将带有细菌菌株的平板上真菌病原体的生长直径与对照平板比较,对竞争作用评分。在细菌生长超过真菌病原体并且还寄生于病原体的情况中,可以记录真菌寄生作用。这可以通过显微镜来观察。
这些类芽孢杆菌属(Paenibacillus)菌株显示出对抗所有试验的植物病原体的抗真菌活性。
表6:体外对抗测试结果
实施例5-温室试验抗植物病原性真菌活性
用途实施例5.1:使用保护性应用,对抗番茄上致病疫霉(Phytophthorainfestans)所致晚疫病的活性
将商业上可得的番茄幼苗(“Goldene”)用于所述温室试验。每个处理使用2个重复(每盆各1株植物)。将植物在温室中,在商业上可得的基质(Universal,Floragard)中,在约22℃生长。使用专用设备控制湿度(~90%湿度)。使用喷雾箱,用相应类芽孢杆菌属(Paenibacillus)菌株6日龄培养物的粗/全培养液喷洒植物(取决于设置)至滚落。用于菌株的培养条件描述于实施例3中。应用一天后,给处理过的植物接种致病疫霉(Phytophthora infestans)(PHYTIN)的孢子囊悬浮液。接种后,将试验植物立即转移至潮湿的房间中。在接种后5-7天,目视评定叶子上真菌攻击的程度。未处理的对照中,真菌攻击在80-100%之间,并且出于比较原因,将其设为100%。
表7:
用途实施例5.2:使用保护性应用,对抗辣椒上灰葡萄孢(Botrytis cinerea)所致灰霉病的活性
将商业上可得的辣椒幼苗(“Neusiedler Ideal”)用于所述温室试验。每个处理使用2个重复(每盆各1株植物)。将植物在温室中,在商业上可得的基质(Universal,Floragard)中,在约22℃生长。使用专用设备控制湿度(~90%湿度)。使用喷雾箱,用相应类芽孢杆菌属(Paenibacillus)菌株6日龄培养物的粗养液喷洒植物(取决于设置)至滚落。用于菌株的培养条件描述于实施例3中。应用一天后,给处理过的植物接种灰葡萄孢(Botrytis cinerea)(BOTRCI)的孢子悬浮液。接种后,将试验植物立即转移至潮湿的房间中。在接种后5-7天,目视评定叶子上真菌攻击的程度。未处理的对照中,真菌攻击在80-100%之间,并且出于比较原因,将其设为100%。
表8:
用途实施例5.3:使用保护性应用,对抗番茄上茄链格孢(Alternaria solani)所致早疫病的活性
将商业上可得的番茄幼苗(“Goldene”)用于所述温室试验。每个处理使用2个重复(每盆各1株植物)。将植物在温室中,在商业上可得的基质(Universal,Floragard)中,在约22℃生长。使用专用设备控制湿度(~90%湿度)。使用喷雾箱,用相应类芽孢杆菌属(Paenibacillus)菌株6日龄培养物的粗/全培养液喷洒植物(取决于设置)至滚落。用于菌株的培养条件描述于实施例3中。应用一天后,给处理过的植物接种茄链格孢(Alternaria solani)(ALTESO)的孢子悬浮液。接种后,将试验植物立即转移至潮湿的房间中。在接种后5-7天,目视评定叶子上真菌攻击的程度。未处理的对照中,真菌攻击在80-100%之间,并且出于比较原因,将其设为100%。
表9:
用途实施例5.4:使用保护性应用,对抗大豆上豆薯层锈菌(Phakopsorapachyrhizi)所致大豆锈病的活性
将商业上可得的大豆幼苗(“Mentor”)用于所述温室试验。每个处理使用2个重复(每盆各1株植物)。将植物在温室中,在商业上可得的基质(Universal,Floragard)中,在约22℃生长。使用专用设备控制湿度(~90%湿度)。使用喷雾箱,用类芽孢杆菌属(Paenibacillus)物种2-6日龄培养物的粗养液喷洒植物(取决于设置)至滚落。应用一天后,给处理过的植物接种豆薯层锈菌(Phakopsora pachyrhizi)(PHAKPA)的孢子悬浮液。接种后,将试验植物立即转移至潮湿的房间中。在接种后5-7天,目视评定叶子上真菌攻击的程度。
用途实施例5.5:使用保护性应用,对抗小麦上禾本科镰孢(Fusariumgraminearum)所致赤霉病(Fusarium Head Blight)的活性
将商业上可得的小麦幼苗用于所述温室试验。每个处理使用2个重复(每盆各1株植物)。将植物在温室中,在商业上可得的基质(Universal,Floragard)中,在约22℃生长。使用专用设备控制湿度(~90%湿度)。使用喷雾箱,用类芽孢杆菌属(Paenibacillus)物种2-6日龄培养物的粗养液喷洒植物(取决于设置)至滚落。培养条件描述于实施例3中。应用一天后,给处理过的植物接种禾本科镰孢(Fusarium graminearum)(GIBBZE)的孢子悬浮液。接种后,将试验植物立即转移至潮湿的房间中。在接种后5-7天,目视评定叶子上真菌攻击的程度。
用途实施例5.6:使用保护性应用,对抗小麦上小麦壳针孢(Septoria tritici)所致叶枯病(speckled leaf blotch)的活性
将商业上可得的小麦幼苗用于所述温室试验。每个处理使用2个重复(每盆各1株植物)。将植物在温室中,在商业上可得的基质(Universal,Floragard)中,在约22℃生长。使用专用设备控制湿度(~90%湿度)。使用喷雾箱,用类芽孢杆菌属(Paenibacillus)物种2-6日龄培养物的粗养液喷洒植物(取决于设置)至滚落。培养条件描述于实施例3中。应用一天后,给处理过的植物接种小麦壳针孢(Septoria tritici)(SEPTTR)的孢子悬浮液。接种后,将试验植物立即转移至潮湿的房间中。在接种后21-28天,目视评定叶子上真菌攻击的程度。
用途实施例5.7:使用保护性应用,类芽孢杆菌属(Paenibacillus)细胞和上清液对抗多种病原体的活性
根据用途实施例3,获得来自类芽孢杆菌属(Paenibacillus)菌株Lu17007的6天培养物的全培养液,并且用于用途实施例5.1至5.3的试验设置中。可选择的是,将该全培养液通过具有0.2μm孔径的滤器进行过滤,以获得培养物基质和粗细胞级分。可以用初始体积的磷酸盐缓冲盐水,将粗细胞级分进一步洗涤三次,以获得洗涤过的细胞。
按照以上针对各病原体致病疫霉(Phytophthora infestans)、灰葡萄孢(Botrytis cinerea)和茄链格孢(Alternaria solani)的用途实施例5.1、5.2和5.3所述,进行了温室试验。在接种后5-7天,目视评定叶子上真菌攻击的程度。未处理的对照中,真菌攻击在80-100%之间,并且出于比较原因,将其设为100%。
表10:
实施例6-酶试验
用途实施例6.1:壳多糖酶
壳多糖酶试验固体培养基:
2g/L NaNO3、1g/L K2HPO4、0.5g/L MgSO4、0.5g/L KCl、0.2g/L蛋白胨、15g/L琼脂、10g/L来自蟹壳的壳多糖(Sigma-Aldrich C7170)。
将试验固体培养基高压灭菌并且装入9cm皮氏培养皿中。将类芽孢杆菌属(Paenibacillus)菌株接种在平板中心并且在27℃温育两天。此后,将平板用1:3稀释的Lugol溶液(Carl Roth N052.2)染色5至10分钟。将Lugol溶液倒出并且将平板拍照并且评价。不同菌株的生长不超过5-10mm。未染色区(用壳多糖酶活性校正)从0mm(无活性,表11中的“-”)至几cm(表11中的“+”)不同。
用途实施例6.2:纤维素酶
纤维素酶试验固体培养基:
2g/L NaNO3、1g/L K2HPO4、0.5g/L MgSO4、0.5g/L KCl、0.2g/L蛋白胨、15g/L琼脂、羧甲基纤维素,钠盐(Sigma-Aldrich 419273)。
将培养基高压灭菌,倾入9cm皮氏培养皿中。将类芽孢杆菌属(Paenibacillus)菌株接种在平板中心并且在27℃温育两天。温育后,将平板用1:3稀释的Lugol溶液(CarlRoth N052.2)染色5至10分钟。将Lugol溶液倒出并且将平板拍照。
用途实施例6.3:淀粉酶
淀粉酶试验固体培养基:
2g/L NaNO3、1g/L K2HPO4、0.5g/L MgSO4、0.5g/L KCl、0.2g/L蛋白胨、15g/L琼脂、10g/L可溶性淀粉(Merck 1.01252)。
将培养基高压灭菌,倾入9cm皮氏培养皿中。将类芽孢杆菌属(Paenibacillus)菌株接种在平板中心并且在27℃温育两天。温育后,将平板用1:3稀释的Lugol溶液(CarlRoth N052.2)染色5至10分钟。将Lugol溶液倒出并且将平板拍照。
表11:类芽孢杆菌属(Paenibacillus)菌株的壳多糖酶、纤维素酶和淀粉酶活性。
菌株 | 壳多糖酶 | 纤维素酶 | 淀粉酶 |
Lu16774 | + | + | - |
Lu17007 | ++ | + | + |
Lu17015 | + | + | + |
BD-62 | - | - | - |
-,无活性;(+),低活性;+,常规活性;++,高活性。
实施例7-从类芽孢杆菌属(Paenibacillus)菌株获得的杀镰孢菌素型代谢物
实施例7.1:大规模培养细菌分离物和提取杀镰孢菌素型代谢物
a)培养
将类芽孢杆菌属(Paenibacillus)菌株在含有GYM培养基(10g/L葡萄糖、4g/L酵母提取物、10g/L麦芽提取物;pH 5.5,在高压灭菌前调节)和20g/L琼脂的琼脂平板上培养。培养在室温进行10至20天。对于维持,使用具有相同培养基的琼脂斜面,并且储存在4℃。
给小规模液体培养物(500mL烧瓶中250mL GYM培养基)接种4-5块生长良好的琼脂培养物并且在室温(20-23℃)在120rpm下在轨道摇床中培养。
大规模发酵在20L发酵罐中进行,使用15L GYM培养基(由于形成泡沫,没有使用发酵罐的总容量),接种了250mL生长良好的液体培养物并且在室温(20-23℃)使用搅拌(120rpm)和通气(3L/分钟)进行发酵5至8天。
b)提取
将一份等体积的异丙醇加入全培养液中(没有自液体培养物分离生物质)。搅拌并且温育2至16小时后,将普通食盐(氯化钠-100至200g/L)加入混合物中,直至可见有机相和水相的相分离。
将异丙醇相真空浓缩。将所得到的提取物,仍然含有大量盐,溶解于甲醇中,离心以更好地沉淀盐残余物,并且将有机相再次浓缩。重复这个步骤,直至不存在任何盐沉淀物。
c)纯化
i)硅胶色谱
将30克提取物溶解于甲醇中并且结合于50g硅胶(Merck,K60,70-230目),在40℃干燥,并且在1kg硅胶上(柱直径约10cm,高30cm)分层。
如下进行四步洗脱:
步骤1-4L乙酸乙酯
步骤2-4L乙酸乙酯:甲醇(3:1,v/v)
步骤3-7L乙酸乙酯:甲醇(1:1,v/v)
步骤4-4L甲醇。
将含有活性化合物的第三级分(中间体1)真空干燥并且溶解于在0.1%甲酸(FA)中的40%甲醇(MeOH)中(浓度:100mg/mL)。丢弃其它级分。
ii)Chromabond HR-X分级
将20mL中间体1上样至之前(用在0.1%FA中的40%MeOH)平衡的Chromabond HR-X柱(Macherey-Nagel,1000mg,ref 730941)上。用100mL在0.1%FA中的40%MeOH洗涤柱并且用60mL在0.1%FA中的70%MeOH洗脱。然后将该中间体1-1真空干燥。
iii)Sunfire C18柱上制备性HPLC
将中间体1-1溶解于DMSO中(浓度:200mg/mL)并且将300μL的中间体1-1在SunfireC18柱(19×250mm,5μm,Waters)上按如下进行色谱:
以10mL/分钟,16分钟,等度70%0.2FA;30%乙腈(ACN),
以14mL/分钟,1分钟,梯度至65%0.2%FA;35%ACN,
以14mL/分钟,5分钟,等度65%0.2%FA;35%ACN。
可以检测到五个级分。将所有五个所得到的级分真空干燥并且溶解于DMSO中(浓度:125mg/mL)。使用相同柱进行进一步纯化,并且针对每个级分,调节等度条件(流速:10.5mL/分钟)(12.5mg/运行):
级分1:69%0.2FA;31%ACN;检测到两个峰(1-1和1-2)
级分2:69%0.2FA;31%ACN;检测到两个峰(2-1和2-2)
级分3:69%0.2FA;31%ACN;检测到三个峰(3-1、3-2和3-3)
级分4/5:67%0.2FA;33%ACN;检测到一个峰(4/5)
级分6:65%0.2FA;35%ACN;检测到两个峰(6-1和6-2)
以下样品的纯度和品质足以用于NMR分析和结构解析:峰1-2、2-1、3-2、4/5和6-1。
实施例7.2:化合物1A和1B的结构解析
对于级分2的峰2-1,获得了作为棕色油状物的化合物1A和1B(比例约3:7)的混合物([α]D 25=+20.9(c=0.6,DMSO-d6))。
从HR-ESI-MS谱推断出主要组分化合物1B的分子式C47H78N10O12,其在m/z 975.5863[M+H]+产生峰;ESI-MS:975.6(100%,[M+H]+),488.4(51%,[M+2H]2+)。
此外,混合物还含有作为主要组分的较轻的同系物1A,并且两种化合物之间的质量差为14amu。在ESI-MS谱中在m/z 961.6处观察到的第二个峰支持了这种观察结果。
除了δ6.83和8.58之间可交换质子的信号,NMR谱(表12)还包括δ166.0-174.5范围的羰基共振以及δ47.8和δ60.4之间的次甲基信号(指示肽)。
化合物1B的1D-和2D-NMR数据的广泛分析揭示了六个氨基酸的存在,包括酪氨酸(Tyr)、谷氨酰胺(Gln)、丙氨酸(Ala)、两个苏氨酸(Thr1和Thr2)和异亮氨酸(Ile)。使用跨酰胺官能的两个或三个键联系,发现了它们的序列。因此,COSY、NOESY(图2)和HMBC(图3)谱描绘了从δ8.58的Thr2氮质子到δ3.84的Thr2的次甲基质子信号和δ166.7的Tyr的羰基的联系,同时在δ8.52的Tyr的氮质子以及δ2.60的Tyr的亚甲基质子信号和δ170.4的Ile的羰基之间,注意到了相同关系。此外,δ4.16的Ile的次甲基氢与δ170.4的Ile的羰基信号具有强烈联系,并且与δ168.6的Thr1的羰基信号具有弱接触;δ5.30的Thr1的β-次甲基质子信号与δ170.4的Ala的羰基信号关联。除了上述关联,从δ7.27的Ala的N-质子到δ4.20的相同氨基酸的次甲基质子展示了其它关联,而这后一质子与其氨基的羰基和Gln的羰基具有相同的相互作用。此外,从δ8.20的Gln的可交换质子到δ3.87的Gln的次甲基氢和δ170.6的Thr2的羰基,揭示了交叉峰;这些上述的数据表明了化合物1B的环酯肽结构。
这种环酯肽1B含有连接至Thr1的末端胍β-羟基脂肪酸,因为在δ4.39的α-次甲基质子信号和δ171.9的羰基共振之间观察到了关键的联系;进一步观察到HMBC接触,从δ171.9的羰基至δ2.35的α-亚甲基质子和δ3.77的β-次甲基质子,以及在δ3.03的亚甲基质子和δ157.2的胍碳之间。基于在m/z 256.2的母体[M+H]+离子的APCI-MS-MS谱中观察到的碎片离子,推断侧链在β-羟基和胍基之间含有十二个亚甲基。同样,这个谱提供了信息(图4b),其证实了氨基酸的连接序列并且导致阐明如图1中所示的化合物1B的结构。
在1D-和2D-谱中在2.80、2.52/36.3的CH2基团信号推测对应于化合物1A中的天冬酰胺(Asn)的β-CH2基团。这种结论得到已报道的数据(Heterocycles 53,1533-1549,2000)和获自m/z 961.6的母峰的MS/MS的片段的支持(图4a)。同样,后面的分析提供了信息(图4a、4b),其证实了两种化合物中的氨基酸连接序列并且导致阐明如图1中所示的化合物1A和1B的结构。
实施例7.3:作为杀镰孢菌素C和D的化合物2A和2B的结构鉴定
从级分1的峰1-2,获得了作为棕色油状物的化合物2A和2B的混合物(比例约为1:1)。基于低分辨率质谱,较重组分化合物2B的分子式测定为C46H76N10O12。NMR数据的分析(表13)允许鉴定化合物2B为杀镰孢菌素D。混合物的较轻组分化合物2A,同样鉴定为杀镰孢菌素C,其中杀镰孢菌素C的Gln残基被Asn替代。
分别针对化合物2B和2A的m/z 961.6和947.6的母体离子的质谱碎裂方式(图5a、5b)证实了取代的脂肪酸侧链的长度与化合物1B中的相同。杀镰孢菌素C和D之前已经被Kajimura等报道过(J.Antibiot.50,220-228,1997)。
实施例7.4:作为LI-F08b的化合物3的结构鉴定
从级分6的峰6-1,分离了作为棕色油状物的化合物3,并且其低分辨率显示了m/z925.6[M+H]+的峰,这结合NMR数据(表14),形成分子式C44H80N10O11。化合物3在NMR谱中显示出与化合物1B和2B(杀镰孢菌素D)相似的特征,除了芳香族信号的存在(表14)。因此,观察到了肽的特征性共振,即δ6.89和8.49之间连接氮的质子的十个信号,范围在δ168.1和174.3之间的羰基的八个共振,以及δ48.0和59.5之间包括的N-次甲基的六个信号。HMQC、COSY和TOCSY谱的详细分析揭示了存在六个氨基酸,包括Gln,两个Thr单元,两个Ile单元和Ala。此外,这些谱显示出归因于与化合物1A、1B以及杀镰孢菌素C(2A)和D(2B)相同的具有末端胍的β-羟基脂肪酸的化学位移。基于δ4.44的Thr1的N-次甲基的质子信号和δ172.1的脂肪酸的羰基信号之间的HMBC谱发现的长范围关联,确定了这个侧链的位置。从NOESY相互作用和碎裂方式推断氨基酸的序列(图6)。
NMR数据(表14)和质谱的组合导致将代谢物化合物3鉴定为LI-F08b,在本文中也称为杀镰孢菌素LI-F08b,首次由Kuroda等报道(Heterocycles 53,1533-1549,2000)。
实施例7.5:分别作为LI-F06a和LI-F06b的化合物4A和4B以及作为杀镰孢菌素A和B的化合物5A和5B的结构鉴定
从级分4/5的峰4/5,获得了两种另外的代谢物化合物4A和4B的混合物(比例约为1:3),其在ESI-MS谱中给出了m/z 897.5(4A)和911.6(4B)的两个峰,表明两种另外的同系环酯肽。在其NMR谱(表15)中观察到了表示肽的共振以及末端为胍基团的β-羟基脂肪酸的那些。针对化合物4A和4B(图7a、7b)发现的两种母体离子的碎裂方式允许确定氨基酸的序列以及将混合物的组分分别鉴定为LI-F06a(4A)和LI-F06b(4B)。
以相同方式分析了从级分3的峰3-2获得的化合物5A和5B的混合物(比例约为1:3)。混合物的ESI质谱显示出m/z 883.6(5A)和897.5(5B)的两个峰,并且这些母体离子的碎裂方式(图8a,8b)结合NMR数据(表16)允许将组分鉴定为杀镰孢菌素A(5A)和杀镰孢菌素B(5B)。针对4A、4B、5A和5B发现的数据与之前报道的那些匹配。(J.Antibiot.50,220-228,1997;Heterocycles 53,1533-1549,2000)。
表12.化合物1A和1B的1H(DMSO-d6,600MHz)和13C-NMR(DMSO-d6,150MHz)数据。
*Pos.=位置;FA=脂肪酸;Gu=胍;nf=未发现。图例也适用于表13至16。
表13.化合物2A和2B的1H(DMSO-d6,600MHz)和13C-NMR(DMSO-d6,150MHz)数据
表14.作为LI-F08b的化合物3的1H(DMSO-d6,600MHz)和13C-NMR(DMSO-d6,150MHz)数据
表15.化合物4A和4B的1H(DMSO-d6,600MHz)和13C-NMR(DMSO-d6,150MHz)数据
表16.化合物5A和5B的1H(DMSO-d6,600MHz)和13C-NMR(DMSO-d6,150MHz)数据
没有进行水解试验来测定组成氨基酸的构型。
实施例8-类芽孢杆菌属(Paenibacillus)菌株产生的代谢物
实施例8.1:类芽孢杆菌属(Paenibacillus)菌株的代谢物产生
遵循上文实施例7.1中所述程序步骤,对类芽孢杆菌属(Paenibacillus)菌株测定总体的杀镰孢菌素并且特别是杀镰孢菌素A、B、C、D、LI-F06a、LI-F06b、LI-F08b、1A和1B的存在。
表17:类芽孢杆菌属(Paenibacillus)菌株的杀镰孢菌素型代谢物产生。
图例:-,化合物不可检出;+,化合物可检出;++,与等级+相比,化合物以更高含量可检出。
所有类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015的全培养液都含有至少一种在实施例7中鉴定的杀镰孢菌素(表17)。这些杀镰孢菌素无一在皮氏类芽孢杆菌(P.peoriae)菌株BD-62的全培养液中检出。
类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015的全培养液全部含有杀镰孢菌素1A和1B。此外,类芽孢杆菌属(Paenibacillus)菌株Lu16774、Lu17007和Lu17015的全培养液全部含有杀镰孢菌素A、B、C和D以及LI-F08b。此外,类芽孢杆菌属(Paenibacillus)菌株Lu17007和Lu17015的全培养液含有杀镰孢菌素LI-F06a和LI-F06b。
在密切相关的皮氏类芽孢杆菌(P.peoriae)菌株BD-62的全培养液中没有检测到杀镰孢菌素1A和1B。在皮氏类芽孢杆菌(P.peoriae)菌株BD-62的全培养液中也没有杀镰孢菌素A、B、C和D、LI-F06a、LI-F06b和LI-F08b。
实施例9:类芽孢杆菌属(Paenibacillus)菌株的代谢物对抗多种真菌病原体的活性
将获得的杀镰孢菌素A、B、D、1A和1B用于以下试验中。
在96孔平板中进行真菌生长测试,使用病原体灰葡萄孢(Botrytis cinerea)(BOTRCI,在YBA中[10g细菌培养用蛋白胨(Becton Dickinson211677),10g酵母提取物(Becton Dickinson 212750),20g乙酸钠,用重蒸水补足1000mL])或茄链格孢(Alternariasolani)(ALTESO,在YBG中[10g细菌培养用蛋白胨(Becton Dickinson 211677),10g酵母提取物(Becton Dickinson 212750),20g甘油99%,用重蒸水补足1000mL])的孢子悬浮液。将杀镰孢菌素以及化合物1A和1B在DMSO中溶解并且稀释。将范围从60μM降至0.3μM的不同浓度吸移至微滴定平板中。加入104个孢子/mL的水性悬浮液。将平板在约18℃温育。通过在接种孢子后3天和7天在微平板读数仪中在600nm下测量光密度来测定真菌生长并且与未处理对照(DMSO)进行比较。此后测定了IC50(真菌生长抑制50%所需要的各代谢物的浓度[μM])。
值得注意的是,化合物1A和1B显示出最高的抗真菌功效,具有0.4-0.6μM的IC50值(表18)。
表18.类芽孢杆菌属(Paenibacillus)代谢物的抗真菌生长抑制IC50值
此外,按照以上针对各自的病原体灰葡萄孢(Botrytis cinerea)(BOTRCI)、茄链格孢(Alternaria solani)(ALTESO)、致病疫霉(Phytophthora infestans)(PHYTIN)、豆薯层锈菌(Phakopsora pachyrhizi)(PHAKPA)和禾本科镰孢(Fusarium graminearum)(GIBBZE)的用途实施例5.1至5.5所述,用杀镰孢菌素1A和1B进行了温室测试。在接种后5-7天,目视评定叶子上真菌攻击的程度。
值得注意的是,化合物1A和1B已经在低如7.2ppm的剂量水平在防治作物上的重要真菌疾病中是有效的并且显示出高于杀镰孢菌素A、B和D的抗真菌功效(表19至21)。
表19.在植物中原位测定的代谢物的抗真菌功效。
表20.使用保护性应用时,代谢物对抗番茄上致病疫霉(Phytophthorainfestans)所致晚疫病(late blight)的功效
表21.使用保护性应用时,代谢物对抗小麦上禾本科镰孢(Fusariumgraminearum)所致赤霉病(head blight)的功效
表22.使用保护性应用时,代谢物对抗小麦上小麦壳针孢(Septoria tritici)所致赤霉病的功效
实施例10:温室试验中比较多粘类芽孢杆菌植物新亚种(Paenibacilluspolymyxa nov.ssp.plantarum)菌株Lu16674和Lu17007与多粘类芽孢杆菌植物新亚种(Paenibacillus polymyxa nov.ssp.plantarum)M-1对抗多种病原体的活性
根据用途实施例3获得了类芽孢杆菌属(Paenibacillus)菌株Lu17007、Lu16674和M1的6天培养物的全培养液并且按照用途实施例5.1至5.5的试验设置来使用。按照以上针对各自病原体的用途实施例5.1至5.5中所述来进行温室测试。在接种后5-7天,目视评定叶子上真菌攻击的程度。
值得注意的是,类芽孢杆菌属(Paenibacillus)菌株Lu16774和Lu17007对防治作物上的重要真菌疾病是有效的,即使在高稀释因子下,并且显示出高于密切相关的菌株M-1的抗真菌功效(表22至表27)。
表22.
表23.
表24.
表25.
表26.
表27.
实施例11:涉及本发明混合物和组合物的实施例
活性化合物分别在二甲基亚砜中配制为具有浓度10000ppm的储备液。将类芽孢杆菌属(Paenibacillus)LU17007作为试验制剂使用并且用水稀释至活性化合物的所述浓度。将产品picarbutrazox作为商用成品制剂使用并且用水稀释至活性化合物的所述浓度。
1.微量滴定板试验中对抗灰霉灰葡萄孢(Botrytis cinerea)(Botrci)活性
将储备液根据比例混合,吸到微量滴定平板(MTP)上并且用水稀释至所述浓度。随后添加在含水biomalt或酵母-细菌用蛋白胨-乙酸钠溶液中的灰葡萄孢(Botrytiscinerea)孢子悬浮液。将平板置于18℃温度的水蒸气饱和的培养室内。使用吸收光度计,接种7天后在405nm测量MTP。表28中描述结果。
2.微量滴定板试验中对抗稻瘟病稻梨孢(Pyricularia oryzae)(Pyrior)活性
将储备液根据比例混合,吸到微量滴定平板(MTP)上并且用水稀释至所述浓度。随后添加在含水biomalt或酵母-细菌用蛋白胨-甘油溶液中的稻梨孢(Pyricularia oryzae)孢子悬浮液。将平板置于18℃温度的水蒸气饱和的培养室内。使用吸收光度计,接种7天后在405nm测量MTP。表29中描述结果。
3.对抗小麦上小麦壳针孢(Septoria tritici)(Septtr)所致叶斑病(leafblotch)的活性
将储备液根据比例混合,吸到微量滴定平板(MTP)上并且用水稀释至所述浓度。随后添加在含水biomalt或酵母-细菌用蛋白胨-甘油溶液中的小麦壳针孢(Septoriatritici)孢子悬浮液。将平板置于18℃温度的水蒸气饱和的培养室内。使用吸收光度计,接种7天后在405nm测量MTP。表30中描述结果。
4.对抗茄链格孢(Alternaria solani)(Alteso)所致早疫病的活性
将储备液根据比例混合,吸到微量滴定平板(MTP)上并且用水稀释至所述浓度。随后添加在含水biomalt或酵母-细菌用蛋白胨-甘油溶液中的茄链格孢(Alternariasolani)孢子悬浮液。将平板置于18℃温度的水蒸气饱和的培养室内。使用吸收光度计,接种7天后在405nm测量MTP。表31中描述结果。
5.对抗颖枯小球腔菌(Leptosphaeria nodorum)(Leptno)所致小麦叶斑病的活性
将储备液根据比例混合,吸到微量滴定平板(MTP)上并且用水稀释至所述浓度。随后添加在含水biomalt或酵母-细菌用蛋白胨-甘油溶液中的颖枯小球腔菌(Leptosphaerianodorum)孢子悬浮液。将平板置于18℃温度的水蒸气饱和的培养室内。使用吸收光度计,接种7天后在405nm测量MTP。表32中描述结果。
测量参数与无活性化合物的对照变体的生长(100%)和无真菌和无活性化合物空白值比较,以确定在相应的活性化合物中病原体的以%计的相对生长。
将这些百分数换算成功效。
使用Colby公式计算预期功效(EColby)
使用Colby公式(Colby,S.R.,计算除草剂组合的协同性和拮抗性应答(Calculating synergistic and antagonistic responses of herbicidecombinations),Weeds,15,第20-22页,1967年),测定活性化合物组合的预期功效并且与观察的功效比较。
Colby公式:EColby=PA+PB-PA*PB/100
EColby使用处于浓度a和b的活性化合物A和B的混合物时的预期功效,以未处理的对照的%表示。
PA使用处于浓度a的活性化合物A时的功效,以未处理的对照的%表示。
PB使用处于浓度b的活性化合物B时的功效,以未处理的对照的%表示。
协同因子(SF)的计算
为了确定协同作用,将混合物观察的试验功效E测量和混合物预期的功效Ecolby之间的协同因子(SF)计算为
SF=E测量/EColby
大于或小于1的协同因子表示距离独立作用假设的偏差,其意指生物学上二种组分共同发挥作用或彼此对抗作用。如果SF>1,观察到协同作用;如果SF<1,观察到拮抗作用。
表28.Botrci
Mefentrifluconazol是2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1H-1,2,4-三唑-1-基)丙-2-醇。
表29.Pyrior
表30.Septtr
Mefentrifluconazol是2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1H-1,2,4-三唑-1-基)丙-2-醇。
表31.Alteso
Mefentrifluconazol是2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1H-1,2,4-三唑-1-基)丙-2-醇。
表32.Leptno
Mefentrifluconazol是2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1H-1,2,4-三唑-1-基)丙-2-醇。
本文中引用的文献通过引用并入作为参考。
附图简述
图1.化合物1A、1B、2A、2B、3、4A、4B、5A和5B。
图2.化合物1B的主要NOESY和COSY关联。
图3.化合物1B的HMBC关联。
图4.化合物1A的碎裂方式a)和化合物1B的碎裂方式b)。
图5.化合物2A(杀镰孢菌素C)的碎裂方式a)和化合物2B(杀镰孢菌素D)的碎裂方式b)。
图6.化合物3(LI-F08b)的碎裂方式。
图7.化合物4A(LI-F06a)的碎裂方式a)和化合物4B(LI-F06b)的碎裂方式b)。
图8.化合物5A(杀镰孢菌素A)的碎裂方式a)和化合物5B(杀镰孢菌素B)的碎裂方式b)。
图9显示了多个序列比对后本发明的类芽孢杆菌属(Paenibacillus)菌株与相关分类群的完整16S rDNA序列的同一性百分数。
图例:*菌株编号:1=类芽孢杆菌属(Paenibacillus)菌株Lu16774;2=类芽孢杆菌属(Paenibacillus)菌株Lu17015;3=类芽孢杆菌属(Paenibacillus)菌株Lu17007;4=皮氏类芽孢杆菌(Paenibacillus peoriae)NRRL BD-62;5=安艾瑞克类芽孢杆菌(Paenibacillus anaericanus)MH21;6=巴西类芽孢杆菌(Paenibacillus brasiliensis)PB172;7=坎皮纳斯类芽孢杆菌(Paenibacillus campinasensis)324;8=奇本类芽孢杆菌(Paenibacillus chibensis)JCM 9905;9=解葡糖类芽孢杆菌(Paenibacillusglucanolyticus)DSM 5162;10=湖南类芽孢杆菌(Paenibacillus hunanensis)FeL05;11=杰米拉类芽孢杆菌(Paenibacillus jamilae)CECT 5266;12=胶冻样类芽孢杆菌(Paenibacillus kribbensis)AM49;13=乳类芽孢杆菌(Paenibacillus lactis)MB 1871;14=灿烂类芽孢杆菌(Paenibacillus lautus)JCM 9073;15=浸麻类芽孢杆菌(Paenibacillus macerans)IAM 12467;16=玛斯里类芽孢杆菌(Paenibacillusmassiliensis)2301065;17=饲料类芽孢杆菌(Paenibacillus pabuli)HSCC 492;18=皮氏类芽孢杆菌(Paenibacillus peoriae)DSM 8320(BD-57);19=皮尼类芽孢杆菌(Paenibacillus pini)S22;20=多粘类芽孢杆菌(Paenibacillus polymyxa)IAM 13419;21=普里斯帕蒂类芽孢杆菌(Paenibacillus purispatii)ES_MS17;22=沉积物类芽孢杆菌(Paenibacillus sediminis)GT-H3;23=土地类芽孢杆菌(Paenibacillus terrae)AM141;24=土壤类芽孢杆菌(Paenibacillus terrigena)A35;25=提摩类芽孢杆菌(Paenibacillus timonensis)2301032;26=图列茨类芽孢杆菌(Paenibacillusturicensis)MOL722;27=潮湿类芽孢杆菌(Paenibacillus uliginis)N3/975;28=耐热科恩氏菌(Cohnella thermotolerans)CCUG 47242。菌株6至28是用于各自物种的模式菌株。
菌株Lu16774、Lu17007和Lu17015与皮氏类芽孢杆菌(Paenibacillus peoriae)(NRRL BD-62和DSM 8320)的相似性已经以粗体标注。
图10显示了从本发明的类芽孢杆菌属(Paenibacillus)菌株与其它分类群的16S-rDNA序列的%同一性(图9)计算的系统发育树。通过将耐热科恩氏菌(Cohnellathermotolerans)的16S rRNA基因序列包括在分析中来确定树根。进化系统树下方的比例尺表示每100个核苷酸1个核苷酸置换。
图11显示了使用RiboPrinter微生物表征系统和从其得到的系统发育树,从本发明的类芽孢杆菌属(Paenibacillus)菌株的样品与密切相关的皮氏类芽孢杆菌(P.peoriae)BD-62的样品相比获得的RiboPrint模式。
图12显示了多序列比对后本发明的类芽孢杆菌属(Paenibacillus)菌株的dnaN基因的DNA序列与相关类芽孢杆菌属(Paenibacillus)菌株的同一性百分数。
图例:*菌株编号:1=类芽孢杆菌属(Paenibacillus)菌株Lu16774;2=类芽孢杆菌属(Paenibacillus)菌株Lu17007;3=类芽孢杆菌属(Paenibacillus)菌株Lu17015;4=皮氏类芽孢杆菌(P.peoriae)DSM 8320T=KCTC 3763T(GenBank登记号AGFX00000000;J.Bacteriol.194,1237-1238,2012);5=多粘类芽孢杆菌(P.polymyxa)1-43(GenBank登记号ASRZ01000000;保藏号GCMCC 4965;CN 102352332B);6=多粘类芽孢杆菌(P.polymyxa)A18(GenBank登记号JWJJ00000000.1;NCBI Project ID 225496);7=多粘类芽孢杆菌(P.polymyxa)ATCC 842T=DSM36T=KCTC 3858T(GenBank登记号AFOX00000000;J.Bacteriol.193(18),5026-5027,2011);8=多粘类芽孢杆菌(P.polymyxa)CF05(GenBank登记号CP009909;Genome Announc 3(2):e00198-15.Doi:10.1128/genomeA.00198-15);9=多粘类芽孢杆菌(P.polymyxa)CICC10580(GenBank登记号JNCB00000000;GenomeAnnounc.2(4):e00854-14.doi:10.1128/genomeA.00854-14);10=多粘类芽孢杆菌(P.polymyxa)DSM365(GenBank登记号JMIQ00000000;J.Biotechnol.195,72-73,2015);11=多粘类芽孢杆菌(P.polymyxa)E681(GenBank登记号CP000154;GenomeNet Ref Seq NC_014483.2;J.Bacteriol.192(22),6103-6104,2010);12=多粘类芽孢杆菌(P.polymyxa)M-1(GenBank登记号HE577054.1;GenomeNet Ref Seq NC_017542.1);13=多粘类芽孢杆菌(P.polymyxa)NRRL B-30509(GenBank登记号JTHO00000000;Genome Announc.2015Mar-Apr;3(2):e00372-15);14=多粘类芽孢杆菌(P.polymyxa)SC2(GenBank登记号CP002213;J.Bacteriol.193(1),311-312,2011);15=多粘类芽孢杆菌(P.polymyxa)SQR-21(GenBank登记号CP006872;GenomeNet Ref Seq NZ_CP006872.1;Genome Announc.2014Mar-Apr;2(2):e00281-14);16=多粘类芽孢杆菌(P.polymyxa)Sb3-1(GenBank登记号CP010268;Genome Announc.2015Mar-Apr;3(2):e00052-15);17=多粘类芽孢杆菌(P.polymyxa)TD94(GenBank登记号ASSA00000000);17=多粘类芽孢杆菌(P.polymyxa)WLY78(GenBank登记号ALJV00000000);土地类芽孢杆菌(P.terrae)HPL 003(GenBank登记号CP003107;NCBI RefSeq NC_016641.1);多粘类芽孢杆菌(P.polymyxa)CR1(GenBank登记号CP006941;GenomeAnnounc.2014Jan-Feb;2(1):e01218-13)。
图13显示了多序列比对后本发明的类芽孢杆菌属(Paenibacillus)菌株的完整gyrB基因的DNA序列与相关类芽孢杆菌属(Paenibacillus)菌株的同一性百分数。菌株编号如图12中图例所述。
图14显示了多序列比对后本发明的类芽孢杆菌属(Paenibacillus)菌株的完整recF基因的DNA序列与相关类芽孢杆菌属(Paenibacillus)菌株的同一性百分数。菌株编号如图12中图例所述。
图15显示了多序列比对后本发明的类芽孢杆菌属(Paenibacillus)菌株的完整recN基因的DNA序列与相关类芽孢杆菌属(Paenibacillus)菌株的同一性百分数。菌株编号如图12中图例所述。
图16显示了多序列比对后本发明的类芽孢杆菌属(Paenibacillus)菌株的完整rpoA基因的DNA序列与相关类芽孢杆菌属(Paenibacillus)菌株的同一性百分数。菌株编号如图12中图例所述。
图17显示了基于多粘类芽孢杆菌(P.polymyxa)复合物菌株的完整dnaN基因序列的最大似然聚类图。所示的0.1刻度对应于1%核苷酸交换。
图18显示了基于多粘类芽孢杆菌(P.polymyxa)复合物菌株的完整gyrB基因序列的最大似然聚类图。所示的0.1刻度对应于1%核苷酸交换。
图19显示了基于多粘类芽孢杆菌(P.polymyxa)复合物菌株的完整recF基因序列的最大似然聚类图。所示的0.1刻度对应于1%核苷酸交换。
图20显示了基于多粘类芽孢杆菌(P.polymyxa)复合物菌株的完整recN基因序列的最大似然聚类图。所示的0.1刻度对应于1%核苷酸交换。
图21显示了基于多粘类芽孢杆菌(P.polymyxa)复合物菌株的完整rpoA基因序列的最大似然聚类图。所示的0.1刻度对应于1%核苷酸交换。
图22显示了根据实施例2.5进行的多粘类芽孢杆菌(P.polymyxa)复合物的代表性基因组的氨基酸指数(AAI)矩阵。菌株编号如图12中图例所述。
Claims (7)
1.混合物,包含作为仅有的活性组分的
1)至少一种类芽孢杆菌属(Paenibacillus)菌株,其选自:
a)以保藏号DSM 26969保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu16774;
b)以保藏号DSM 26970保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17007;和
c)以保藏号DSM 26971保藏于DSMZ的类芽孢杆菌属(Paenibacillus)菌株Lu17015;
其中所述1)中的类芽孢杆菌属(Paenibacillus)菌株能够在包含至少一种碳源和一种氮源的生长培养基中产生以下两种化合物:
和
2)至少一种选自组A)、B)、C)、E)、F)和K)的农药:
A)呼吸抑制剂
-Qo位点的复合体III抑制剂:腈嘧菌酯(azoxystrobin)、醚菌胺(dimoxystrobin)、唑菌胺酯(pyraclostrobin);
-复合体II抑制剂:苯并烯氟菌唑(benzovindiflupyr)、氟吡菌酰胺(fluopyram)、氟唑菌酰胺(fluxapyroxad);
B)甾醇生物合成抑制剂(SBI杀真菌剂)
-C14脱甲基酶抑制剂:醚唑(difenoconazole)、环戊唑菌(metconazole)、丙硫菌唑(prothioconazole)、戊叉唑菌(triticonazole)、2-[4-(4-氯苯氧基)-2-(三氟甲基)苯基]-1-(1,2,4-三唑-1-基)丙-2-醇;
C)核酸合成抑制剂
-苯基酰胺或酰基氨基酸杀真菌剂:甲霜灵(metalaxyl)、精甲霜灵(metalaxyl-M);
E)氨基酸和蛋白质合成抑制剂
-甲硫氨酸合成抑制剂:二甲嘧菌胺(pyrimethanil);
F)信号转导抑制剂
-MAP/组氨酸激酶抑制剂:咯菌腈(fludioxonil);
K)未知作用模式
-picarbutrazox。
2.权利要求1的混合物,其中所述至少一种类芽孢杆菌属(Paenibacillus)菌株具有对抗至少两种植物病原体的抗真菌活性,所述植物病原体选自链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotinia sclerotiorum)。
3.权利要求1或2的混合物,其中组分1)包含至少一种代谢物,所述代谢物选自多粘菌素、八肽菌素、多肽菌素、pelgipeptin和杀镰孢菌素。
4.如权利要求1至3中任一项定义的混合物的用途,用于防治或抑制植物病原性真菌或防止植物被植物病原性真菌感染或用于保护材料对抗植物病原性真菌的侵袭破坏。
5.防治、抑制植物病原性真菌或防止植物病原性真菌感染的方法,其中用有效量的如权利要求1至3中任一项定义的混合物处理植物病原性真菌、其栖息地或待对抗植物病原性真菌攻击而受到保护的材料或植物,或土壤或繁殖材料。
6.权利要求4的用途,其中植物病原性真菌选自链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotiniasclerotiorum)。
7.权利要求5的方法,其中植物病原性真菌选自链格孢属(Alternaria)物种、灰葡萄孢(Botrytis cinerea)、致病疫霉(Phytophthora infestans)和核盘菌(Sclerotiniasclerotiorum)。
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PCT/EP2017/052532 WO2017137351A1 (en) | 2016-02-09 | 2017-02-06 | Mixtures and compositions comprising paenibacillus strains or fusaricidins and chemical pesticides |
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BR112018015535B1 (pt) | 2023-03-07 |
UA127242C2 (uk) | 2023-06-21 |
US20190110480A1 (en) | 2019-04-18 |
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CN115005215A (zh) | 2022-09-06 |
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EP3413714B1 (en) | 2023-07-26 |
BR112018015535A2 (pt) | 2018-12-26 |
CL2023000924A1 (es) | 2023-09-15 |
EP3205208A1 (en) | 2017-08-16 |
JP6875409B2 (ja) | 2021-05-26 |
KR20180104165A (ko) | 2018-09-19 |
CA3012437A1 (en) | 2017-08-17 |
WO2017137351A1 (en) | 2017-08-17 |
EP4292433A3 (en) | 2024-03-06 |
EP4292433A2 (en) | 2023-12-20 |
ES2959636T3 (es) | 2024-02-27 |
AR107562A1 (es) | 2018-05-09 |
AU2022200847B2 (en) | 2024-03-07 |
PL3413714T3 (pl) | 2024-01-22 |
RU2018131441A3 (zh) | 2020-07-02 |
CN109068645A (zh) | 2018-12-21 |
MX2018009719A (es) | 2018-09-10 |
CL2018002197A1 (es) | 2018-09-14 |
MX2023010171A (es) | 2023-09-14 |
JP2019509991A (ja) | 2019-04-11 |
EP3413714A1 (en) | 2018-12-19 |
BR112018015535B8 (pt) | 2023-03-28 |
AU2022200847A1 (en) | 2022-03-03 |
RU2018131441A (ru) | 2020-03-11 |
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