CN100548121C - 治疗微生物引起的植物疾病的方法 - Google Patents

治疗微生物引起的植物疾病的方法 Download PDF

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CN100548121C
CN100548121C CNB2005800125487A CN200580012548A CN100548121C CN 100548121 C CN100548121 C CN 100548121C CN B2005800125487 A CNB2005800125487 A CN B2005800125487A CN 200580012548 A CN200580012548 A CN 200580012548A CN 100548121 C CN100548121 C CN 100548121C
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M·林奇
R·伊什特万
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Abstract

用于治疗和预防植物感染的杀菌方法，所述方法使用包含一种有机酸或有机酸混合物和一种短链阴离子表面活性剂，其具有至少一个大的头基；一个支链烷基链和一个不饱和的烷基链的抗微生物组合物。

Description

治疗微生物引起的植物疾病的方法
发明领域
本发明涉及一种治疗和预防植物感染的杀菌方法。
发明背景
植物受多种微生物引起的常见病的影响。柑橘溃疡病是由革兰氏阴性菌即野油菜黄单胞杆菌引起的一种柑橘树细菌疾病。这种疾病具有高度传染性，会引起叶和果实的过早脱落，从而导致巨大的经济损失。它在染病的树上过冬，并且在多雨天气期间从病斑处渗出而被传播。风吹雨淋是主要的传播模式。此细菌非常适于在高温多雨的适合柑橘生长的地区传播。因为目前没有任何有效的治疗方法，因此根除此疾病的措施是破坏被感染的柑橘树及其周围579米(1,900英尺)半径内的所有其它柑橘树。
野油菜黄单胞杆菌是一种革兰氏阴性菌，属于生物化学上易变的γ变形细菌。溃疡细菌是需氧细菌并且不形成孢子。该细菌在染病的树上过冬，并且在多雨天气期间从病斑处渗出而被传播。风吹雨淋是它的主要传播模式。此细菌非常适于在高温多雨的适合柑橘生长的地区传播。
多种化学组合物的杀菌化合物已经被用于植物以抑制或杀灭对植物有害的微生物。这些微生物包括细菌、酵母和真菌。然而，常规的杀菌剂有几个缺点。
应用大量的生物活性抗微生物化合物也会引起环境危害。许多常规的抗微生物化合物不会迅速降解，将会保留在环境中相当长的一段时间。这些生物活性化合物因为反复应用于植物，当化合物的浓度增加时，可能引起严重的危害。
用于治疗水果、蔬菜和谷物的杀菌剂可能被引入食物链，在食物链中它们最后被人消耗。当这些杀菌化合物在食物链中的浓度增加时，对人和动物的有害效果增加。
目前可用的抗微生物化合物常常是属特异性的。可能需要不同的化学化合物来治疗植物上发现的每一种微生物。每个多重感染的植物因此必须接受一系列的治疗来进行有效的保护或消灭所有的有害微生物。
发明概述
包含一种有机酸或有机酸混合物、一种特异性的具有支链或大的头基的短链阴离子表面活性剂、和任选地一种钙离子清除剂和/或消泡剂的抗微生物组合物在美国专利公布20030235550A1、20040001797A1、和公布的PCT申请WO2004/000016中被描述并受权利要求书保护，其内容被引入本文以供参考。
本发明提供一种治疗或预防在有此需要的植物中由微生物引起的感染的方法，所述方法包括施用有效量的抗微生物组合物，所述抗微生物组合物包含：a.0.2％至70％的有机酸；和b.0.1％至40％的阴离子表面活性剂混合物，所述阴离子表面活性剂混合物具有i.C4至C12线性烷基链长和至少4埃的总头基大小；和/或ii.C4至C12支链烷基链长；和/或iii.C4至C12支链烷基链长和至少4埃的总头基大小；其中所述组合物的特征在于pH为2.0至4.5。
在一个实施方案中，所述阴离子表面活性剂选自：烷基磺酸甘油、α磺基脂肪酸、烷基膦酸酯、支链烷基磺酸盐和支链烷基苯磺酸盐、仲烷基硫酸盐、磺基琥珀酸单酯、烷基羟乙基磺酸盐、烷基氨基磺酸盐，以及它们的组合。优选的是，所述阴离子表面活性剂用磺酸根、硫酸根或膦酸酯基团取代。
所述有机酸选自：焦谷氨酸、己二酸、葡萄糖酸、内酯型葡萄糖酸、谷氨酸、戊二酸、乙醇酸、酒石酸、抗坏血酸以及它们的组合。
在另一个实施方案中，所述抗微生物组合物还包含非离子剂，其中所述非离子剂优选地选自：1-(2-乙基己基)甘油醚、辛基甘油醚、2-(2-乙基己氧基)丙醇、辛氧基-丙醇、1-(2-乙基己氧基)乙醇、辛氧基乙醇、1，2-己二醇、1，2-环六烷二甲醇、异丙基甘油醚以及它们的组合。所述非离子剂以按组合物的总重量计0.1％至10％的量存在。
在再一个实施方案中，所述微生物感染由至少一种以下生物引起：酵母、霉菌、细菌或病毒，特别是，所述酵母是念珠菌类；所述细菌为革兰氏阴性菌或革兰氏阳性菌。
本发明还涉及一种对农业工具进行消毒的方法，所述方法包括用有效量的抗微生物组合物来处理装置，所述抗微生物组合物包含：a.0.2％至70％的有机酸；和b.0.1％至40％的阴离子表面活性剂混合物，所述阴离子表面活性剂混合物具有i.C4至C12线性烷基链长和至少4埃的总头基大小；和/或ii.C4至C12支链烷基链长；和/或iii.C4至C12支链烷基链长和至少4埃的总头基大小；其中所述组合物的特征在于pH为2.0至4.5。
在一个实施方案中，所述抗微生物组合物以洗涤、喷洒、浸泡或擦拭的方式施用。
发明详述
优选组合物
提供增强的立即的和后遗的抗病毒和抗菌功效的抗微生物组合物，所述病毒和细菌包括鼻病毒、轮状病毒、革兰氏阳性菌、革兰氏阴性菌以及它们的组合，在美国专利公布20030235550A1、20040001797A1、和公布的PCT申请WO2004/000016中被提出，其内容被引入本文以供参考。这些组合物据称具有抗革兰氏阴性菌、革兰氏阳性菌以及病毒的功效。我们相信基于这些短链、大头基表面活性剂的抗微生物组合物的某些制剂对多种发病的农业生物是有效的，包括但不限于柑桔溃疡病。
这些抗微生物组合物包含一种有机酸或有机酸混合物、一种特异性的具有支链或大的头基的短链阴离子表面活性剂、和任选地一种钙离子清除剂和/或消泡剂。为了治疗应用，所述制剂除了表面活性剂之外，还需要一种有机酸和一种非离子剂。优选使用C8-AGS作为表面活性剂，吡咯烷酮羧酸作为有机酸，乙基己基甘油醚(EHOP)作为非离子剂。本领域的技术人员将认识到公开于20030235550A1、20040001797A1、或者WO 2004/000016中的其它试剂可以被取代。优选的组合物具有大于约0.50％的C8AGS，最优选的组合物包含至少1.25％的C8AGS和大于或等于2.0％的吡咯烷酮羧酸。
本领域的技术人员将容易认识到其它活性成分能被加入，以提供不同的特性或改善所述抗特异生物体组合物的效力。当加入其它活性物质时，表面活性剂、酸和/或非离子剂的浓度也将被调节以提供所需的活性度。例如，对氯间二甲苯酚(PCMX)，一种已知的抗微生物剂，可以被加入以提供另一种广谱抗微生物物质。例如当加入PCMX时，将C8AGS的量降低至按重量计1％的浓度之下是可能的。
表1
Figure C20058001254800071
制剂
可以本领域技术人员已知的任何形式配制使用本发明组合物。局部用、粘膜和气溶胶递送药物的配制方法在Modern Pharmaceutics by Gilbert S.Banker(编者)，Christopher T.Rhodes(编者)Marcel Dekker；第4版(2002年6月15日)ISBN：0824706749中被提出，所有这些文献被引入本文以供参考。也可以参考International Journal of PharmaceuticalCompounding，它能在www.ijpc.com中获取，其内容被引入本文以供参考。这些文献提出并描述了药物配制的基础。本领域的技术人员将知道如何得到本发明的活性成分并配制它们用于递送。这些制剂可以采用洗剂、油膏剂、凝胶、霜膏、滴洗剂、糊剂、栓剂、药糖块、漱口水、含漱剂、冲洗液、泡沫、表面涂层、脂质体、微球体和透皮贴剂的形式。
本领域的技术人员将会知道，能通过选择赋形剂以提供不同的皮肤透过程度或控制释放的方法来影响本组合物的活性。通过在用合适的绷带或包裹物施用后闭合皮肤能增加本制剂的活性。本领域的技术人员也将认识到通过使用延缓活性物质释放的受控释放技术能增加持久的效果。
本文设想的制剂也能被涂敷于或换句话讲掺入医疗装置如擦拭物、海绵、绷带、手术盖布、医用长袍、手术服。制剂能被开发用于合适的消毒医疗装置。这些制剂可以液体形式存在，液体能被用于喷洒表面、浸泡装置、抽吸通过装置或掺入擦拭物中净化表面。
测试
测试上述制剂对白色念珠菌、大肠杆菌、粘质沙雷氏菌、耐甲氧西林金黄色葡萄球菌和粪肠球菌的最低杀菌稀释液和后遗功效，
最低杀菌稀释液
测试表1中的以上组合物的最低杀菌稀释液。受试的生物体生长于斜面上，并且通过划线形成菌苔而被转移到琼脂板上。用无菌接种环从琼脂板上刮掉菌落，悬浮于磷酸缓冲液(PBS)中，稀释至5x106CFU/mL。
表1
Figure C20058001254800081
Figure C20058001254800101
体外杀菌时间
在适宜条件下培养白色念珠菌。(目前的培养生物体的USP程序是合适的。)培养时间持续(典型的120小时，25℃+/-1℃)至密度在1.0E+06至1.0E+07CFUs/mL之间。开始培养时的实际CFUs/mL由连续稀释和平板接种的等分试样决定(典型平板接种10-6、10-5、10-4、10-3、10-2稀释)。酵母培养物的50μL的等分试样被吸移至在无菌闪烁计数瓶中的5.0mL抗微生物溶液中(生物体∶溶液比率＝1∶100)并且涡旋充分混合。测试接种含量为约1.0E+04至1.0E+05CFUs/mL。在预定的时间点，1分钟、5分钟和10分钟，一个0.5mL被接种的抗微生物溶液的等分试样被吸移至4.5mL Dey/Engley(D/E)中和肉汤中(比率＝1∶10)并且涡旋充分混合。包括酵母的中和样本的等分试样使用标准倒平板技术涂于Sabouaud右旋糖琼脂(SDA)上。SDA平板在25℃+/1℃条件下培养5天(约120小时)，计数CFU。计算用于有机体培养的CFUs/mL，并与用于抗微生物溶液的CFUs/mL相比较，测定对数减少量。
后遗功效测试
通过用20μL的活性物质溶液均匀涂敷皮肤贴剂的表面，进行后遗功效测试。移开培养皿的封盖，允许皮肤样本蒸发1分钟、15分钟、60分钟、120分钟、240分钟、360分钟、480分钟和14小时。在合适的时间点，皮肤样本用10μL 1∶10稀释的18-小时微生物悬浮液(约1.0E+08CFUs/mL)接种，均匀的覆盖整个区域，样本恢复并允许保持5分钟。此时皮肤使用无菌钳取出并放入无菌离心管。包含10mL样本溶液并涡旋震荡30秒。包含任何皮肤微生物的提取样本的等分试样使用旋转接种仪(典型的在50μL的指数模式)涂于胰蛋白胨大豆琼脂平板上。此琼脂平板在37℃培养过夜(约18小时)并计数CFU。计算通过基线计数确定的CFUs/mL，并与用于抗微生物/细菌溶液的CFUs/mL相比较以测定对数减少量。基线计数通过在方形皮肤上均匀涂敷10μL稀释细菌悬浮液并依照以上程序处理来完成，只是不加入活性物质溶液。本领域的技术人员将会知道此测试能用任何底物进行重复，包括但不限于树皮、叶、花、果实、根等。
测试结果
杀真菌/酵母活性
测试配方R、M、H、KS和KSM抗白色念珠菌的功效并与Chloraprep比较活性。这些制剂的杀真菌特性以前是未经测试的和未知的。体外杀菌时间测试按上述方法进行。测试结果见表2。
表2
对白色念珠菌的后遗皮肤功效测试按上述方法进行。数据见下表3。
表3白色念珠菌(白色念珠菌10231)后遗功效
对数减少量   Chlora-prep   RID   M   H   KS   KSM
  1分钟   3.6   0.8   2.3   3.4   3.6   3.6
  15分钟   2.2   1.1   2.7   3.9   3.6   3.6
  60分钟   2.5   0.9   2.9   3.9   3.6   3.6
  120分钟   1.8   2.1   3.2   3.9   3.6   3.6
  240分钟   1.1   0.2   2.7   3.9   3.6   3.6
  完全杀菌＝Log 4
以上数据显示RID杀灭念珠菌的强度医学上可以接受。通过加入已知的广谱抗微生物剂(PCMX)能获得更快的杀菌速度，但是不能获得更强的后遗活性。
大肠杆菌
测试配方R、M、H、KS和KSM大肠杆菌的功效并与Chloraprep作比较。对大肠杆菌的后遗皮肤功效的测试按上述方法进行。数据见下表4。
表4
大肠杆菌(大肠杆菌11229)后遗功效测试。
对数减少量   Chlora-prep   RID   M   H   KS   KSM
  1分钟   4.6   0.6   4.6   4.6   4.6   4.6
  15分钟   4.5   2.7   4.6   4.6   3.7   4.5
  60分钟   4.1   4.5   4.6   4.6   4.6   4.6
  120分钟   3.3   3.7   4.6   4.2   4.6   4.6
  240分钟   3.9   4.0   4.6   4.1   4.6   4.6
粘质沙雷氏菌(粘质沙雷氏菌14756)
测试配方R、M、H、KS和KSM抗粘质沙雷氏菌的功效并与Chloraprep作比较。对粘质沙雷氏菌的后遗皮肤功效的测试按上述方法进行。数据见下表5。
表5粘质沙雷氏菌14756后遗功效结果
对数减少量   Chlora-prep   RID   M   H   KS   KSM
  1分钟   1.8   0.1   3.6   6.0   5.1   5.1
  15分钟   0.7   0.1   3.6   6.0   5.1   5.1
  60分钟   2.0   0.9   2.0   6.0   4.5   5.1
  120分钟   1.1   0.0   3.7   6.0   5.1   5.1
  240分钟   1.0   0.2   2.8   6.0   5.1   5.1
  完全杀菌＝Log 6
耐甲氧西林金黄色葡萄球菌(MRSA)
测试配方R、M、H、KS和KSM抗MRSA的功效并与Chloraprep作比较。对MRSA的后遗皮肤功效的测试按上述方法进行。数据见下表6。
表6MRSA(葡萄球菌33591)后遗功效结果
对数减少量   Chlora-prep   R   M   H   KS   KSM
  1分钟   5.5   1.3   0.9   4.9   5.1   5.1
  15分钟   1.4   0.2   0.6   3.9   5.0   4.9
  60分钟   2.7   0.1   0.6   2.9   5.8   4.9
  120分钟   3.9   0.3   1.3   4.0   4.8   4.5
  240分钟   3.7   0.4   1.7   4.6   5.0   4.8
  完全杀菌＝Log 6
万古霉素抗药性肠球菌(VRE)
测试配方R、M、H、KS和KSM抗粪肠球菌的功效并与Chloraprep作比较。对粪肠球菌的后遗皮肤功效的测试按上述方法进行。数据见下表7。
表7VRE(51299)后遗功效结果
对数减少量   Chlora-prep   RID   GMP/M   GMP/H   GMP/KS   GMP/KSM
  1分钟   4.3   0.5   4.7   4.7   4.8   4.8
  15分钟   1.1   1.3   4.5   4.7   4.8   4.4
  60分钟   1.5   2.0   4.7   4.7   4.8   4.8
  120分钟   1.7   2.8   4.7   4.7   4.6   4.8
  240分钟   3.2   0.6   4.7   4.7   4.8   4.8
本发明的植物杀菌组合物被期望对所有已知的植物病原体有效，包括但不限于柑橘溃疡病细菌即野油菜黄单孢杆菌柑桔致病变种。本发明的植物杀菌化合物将以含水混合物的形式通过任何常规方法例如喷洒来施用于植物上。在此化合物被施用于柑橘树之后，此树木被保护不受野油菜黄单胞杆菌的感染。
本发明的杀菌剂优选以水溶液的形式施用。本发明的植物杀菌化合物将能通过常规技术例如通过喷洒或喷雾来施用于植物上。本发明的植物杀菌化合物也能被涂敷于植物上。
以下实施例用于进一步说明本发明，但同时不对其构成任何限制。
实施例I
表1中的植物杀菌化合物的溶液将被喷洒于柑橘树苗的幼嫩的和成熟的叶片上，并观察其危害植物的毒性。每日观察叶、芽、枝、树干或根的变化。
实施例II
为了测试人工接种的测试表面上的柑橘溃疡病细菌(野油菜黄单孢杆菌柑桔致病变种)的根除效果，实施例I中的杀菌化合物将被施用到几个表面上。被提议的表面包括牛皮纸，它代表一种多孔的无生命表面，以及未经洗涤的柑橘类果实以测试在柑橘类果实表面的杀菌能力。接种浓度为水中106个细胞/毫升，这大约是从天然损害中渗出的柑橘溃疡病细菌的期望最高浓度。
接种到纸上10分钟后或果实上2分钟后，测试表面用无菌棉签擦拭并在营养琼脂上划线。未处理的对照物也进行测试。在每个测试中，测试表面被重复测试三次。施用无菌水淋洗果实表面，并通过无菌棉签转移小量到营养琼脂上检查未死的野油菜黄单孢杆菌细胞。
实施例III
红冬小麦种子(Arrowhead Mills，Inc.Hereford Texas)被浸泡在本发明的植物杀菌化合物的水溶液中15分钟或三十分钟。所述种子被风干、种植于泥炭罐中并每日暴露于人造光下16小时。每天进行观察以测定萌芽种子的百分比、平均萌芽时间和平均生长率。
当然，应当理解前述内容仅仅涉及本发明的优选实施方案，在不背离附加的权利要求书中阐明的精神和范围的条件下，可以对其进行许多修改或更改。
在发明详述中引用的所有文献的相关部分均引入本文以供参考；任何文献的引用不可解释为是对其作为本发明的现有技术的认可。在本书面文献中术语的任何意义或定义与引入本文以供参考的文献中术语的任何意义或定义冲突时，将以赋予本书面文献中术语的意义或定义为准。
尽管已用具体实施方案来说明和描述了本发明，但对于本领域的技术人员显而易见的是，在不背离本发明的精神和保护范围的情况下可作出许多其它的变化和修改。因此，有意识地在附加的权利要求书中包括属于本发明范围内的所有这些变化和修改。

Claims (12)

1.一种治疗或预防在有此需要的植物中由微生物引起的感染的方法，所述方法包括施用有效量的抗微生物组合物，所述抗微生物组合物包含：a.0.2％至70％的有机酸；和b.0.1％至40％的阴离子表面活性剂混合物，所述阴离子表面活性剂混合物具有i.C4至C12线性烷基链长和至少4埃的总头基大小；和/或ii.C4至C12支链烷基链长；和/或iii.C4至C12支链烷基链长和至少4埃的总头基大小；其中所述组合物的特征在于pH为2.0至4.5。
2.如权利要求1所述的方法，其中所述阴离子表面活性剂选自：烷基磺酸甘油、α磺基脂肪酸、烷基膦酸酯、支链烷基磺酸盐和支链烷基苯磺酸盐、仲烷基硫酸盐、磺基琥珀酸单酯、烷基羟乙基磺酸盐、烷基氨基磺酸盐，以及它们的组合。
3.如权利要求1所述的方法，其中所述阴离子表面活性剂用磺酸根、硫酸根或膦酸酯基团取代。
4.如权利要求1所述的方法，其中所述有机酸选自：焦谷氨酸、己二酸、葡萄糖酸、内酯型葡萄糖酸、谷氨酸、戊二酸、乙醇酸、酒石酸、抗坏血酸以及它们的组合。
5.如权利要求1所述的方法，其中所述抗微生物组合物还包含非离子剂。
6.如权利要求5所述的方法，其中所述非离子剂选自：1-(2-乙基己基)甘油醚、辛基甘油醚、2-(2-乙基己氧基)丙醇、辛氧基-丙醇、1-(2-乙基己氧基)乙醇、辛氧基乙醇、1，2-己二醇、1，2-环六烷二甲醇、异丙基甘油醚以及它们的组合。
7.如权利要求5所述的方法，其中所述非离子剂以按组合物的总重量计0.1％至10％的量存在。
8.如权利要求1所述的方法，其中所述微生物感染由至少一种以下生物引起：酵母、霉菌、细菌或病毒。
9.如权利要求8所述的方法，其中所述酵母是念珠菌类。
10.如权利要求8所述的方法，其中所述细菌为革兰氏阴性菌或革兰氏阳性菌。
11.一种对农业工具进行消毒的方法，所述方法包括用有效量的抗微生物组合物来处理装置，所述抗微生物组合物包含：a.0.2％至70％的有机酸；和b.0.1％至40％的阴离子表面活性剂混合物，所述阴离子表面活性剂混合物具有i.C4至C12线性烷基链长和至少4埃的总头基大小；和/或ii.C4至C12支链烷基链长；和/或iii.C4至C12支链烷基链长和至少4埃的总头基大小；其中所述组合物的特征在于pH为2.0至4.5。
12.如权利要求11所述的方法，其中所述抗微生物组合物以洗涤、喷洒、浸泡或擦拭的方式施用。
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HK1105771A1 (en) 2008-02-22
BRPI0510326A (pt) 2007-10-23
WO2005104843A2 (en) 2005-11-10
CN1946291A (zh) 2007-04-11
AU2005237547A1 (en) 2005-11-10
WO2005104843A3 (en) 2006-03-09
NO20065459L (no) 2006-11-27
JP2007534765A (ja) 2007-11-29
AR052398A1 (es) 2007-03-21
AU2009201512A1 (en) 2009-05-14
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US20050260243A1 (en) 2005-11-24
CA2584366C (en) 2010-10-26

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