CN111919846B - 一种具有抗真菌活性的氨基羧酸类螯合剂及其与其他杀菌剂的协同应用 - Google Patents

一种具有抗真菌活性的氨基羧酸类螯合剂及其与其他杀菌剂的协同应用 Download PDF

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CN111919846B
CN111919846B CN202010389990.8A CN202010389990A CN111919846B CN 111919846 B CN111919846 B CN 111919846B CN 202010389990 A CN202010389990 A CN 202010389990A CN 111919846 B CN111919846 B CN 111919846B
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edta
dtpa
aminocarboxylic acid
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chelating agent
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CN111919846A (zh
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潘建碧
宋修仕
谷凯鑫
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Nanjing Jixing Biotechnology Development Co ltd
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Abstract

本发明公开了一种具有抗真菌活性的氨基羧酸类螯合剂及其与其他杀菌剂的协同应用，属于农药技术领域。所述氨基羧酸类螯合剂及他们的盐类化合物单用或组合使用，能够有效抑制镰刀菌属、灰葡萄孢属、梨形孢子属、木霉属、青霉属真菌及卵菌的生长发育，抑菌浓度为0.003‑300mM，有效防治小麦赤霉病、锈病、白粉病、水稻稻瘟病、纹枯病和蔬菜灰霉病、菌核病等真菌病害；与有机硫类、抗生素类、苯并咪唑类、三唑类、甲氧基丙烯酸酯类、琥珀酸脱氢酶抑制剂类杀菌剂剂杀卵菌剂组合使用，能够减少农药用量，提高防治效果。

Description

一种具有抗真菌活性的氨基羧酸类螯合剂及其与其他杀菌剂 的协同应用
【技术领域】
本发明属于农药技术领域，具体涉及一种氨基羧酸类螯合剂在真菌病害防治及其与其他杀菌剂的协同应用。
【背景技术】
真菌病害是农业生产中常发性病害，是植物病害中的第一大类病害。随着作物结构调整与耕作制度的变化，加上气候变化等原因，真菌病害的发生在近几年愈发严重，给粮食增产、稳产带来巨大的压力。化学防控作为重要的植保防治措施，在农业生产中发挥着巨大的作用。随着生物技术的发展，单一靶标的选择性杀菌剂因其具有高选择性、安全性等优势，在农业生产中备受青睐。然而，靶标的分化与变异导致了现代杀菌剂极易产生抗药性，造成药剂防治效率降低，病害发生风险升高，农业生产对新药剂的需求增加。
氨基羧酸类螯合剂含羧基和胺(氨基)配位基团，如乙二胺四乙酸(EDTA)及其盐(如EDTANa1-4)，二亚乙基三胺五乙酸(DTPA)及其盐(如DTPANa1-5)等。氨基羧酸类螯合剂用途广泛，可用作漂白定影液，染色助剂，纤维处理助剂，化妆品添加剂，血液抗凝剂，洗涤剂，稳定剂，合成橡胶聚合引发剂，指示剂等。因此，在食品与医药行业EDTA和DTPA广泛用作防腐剂和消毒剂的添加物，增加制剂的稳定性。然而，目前尚未有氨基羧酸类螯合剂及其盐类化合物在植物病害防控、工业防霉和与农用杀菌剂协同应用方面的报道。
【发明内容】
本发明的目的在于提供氨基羧酸类螯合剂及其盐在植物病害防控、工业防霉和农用杀菌剂增效剂方面的应用。所述化合物能够有效防控丝状真菌和卵菌病害，尤其是对镰刀菌、稻瘟病菌、灰葡萄孢、木霉、青霉、疫霉防治效果好，并能与苯并咪唑类(多菌灵)、甲氧基丙烯酸酯类(肟菌酯)、琥珀酸脱氢酶类(啶酰菌胺)药剂协同作用，提高药剂的抑菌效果。发明人测定了氨基羧酸类螯合剂及其盐在一定条件下，对不同真菌的抑菌效果，发现氨基羧酸类螯合剂及其盐对于镰刀菌、稻瘟病菌、灰葡萄孢、木霉、青霉、疫霉具有优异的抑菌效果；活体实验进一步证实了抑菌效果的有效性；大田试验也表现出较好的防治效果；进一步地，对真菌细胞内的离子测定发现氨基羧酸类螯合剂及其盐能螯合真菌细胞内的辅酶金属离子，抑制生命活动重要酶的活性；同时可破坏细胞壁和细胞膜结构，增加真菌细胞的透性，由此发明人发现氨基羧酸类螯合剂及其盐对有机硫类(克菌丹)、抗生素类(多抗霉素)、苯并咪唑类(多菌灵)、三唑类(戊唑醇)、甲氧基丙烯酸酯类(肟菌酯)、琥珀酸脱氢酶抑制剂类(啶酰菌胺)药剂具有增效作用。
【附图说明】
图1为氨基羧酸类螯合剂及其盐对镰刀菌的室内活体防效图。
图2为氨基羧酸类螯合剂及其盐对稻瘟病原菌的室内活体防效图。
图3为氨基羧酸类螯合剂及其盐对灰葡萄孢菌的室内活体防效图。
图4为氨基羧酸类螯合剂及其盐对小麦赤霉病的田间防效图。
图5为氨基羧酸类螯合剂及其盐对真菌细胞内微量元素的影响。
图6为氨基羧酸类螯合剂及其盐对真菌生长关键酶的抑制作用。
【具体实施方式】
以下实施例用于非限制性地解释本发明的技术方案。
在本发明中，如无特殊说明，用于表示浓度的“％”均为重量百分比，“份”均为重量份。
本发明涉及以下培养基，其组成分别为：
PDA培养基：马铃薯200g煮沸15min取浸出液，葡萄糖20g，琼脂15g，蒸馏水定容至1000ml，121℃灭菌20min；
CMC培养基：羧甲基纤维素酯(CMC)7.5g，NH4NO30.5g，KH2PO40.5g，MgSO4·7H2O0.25g，酵母提取物0.5g。用蒸馏水溶解，定容到1000mL，121℃灭菌20min。
SNA培养基：0.1％KH2PO4，0.1％KNO3，0.05％MgSO4·7H2O，0.05％KCl，0.02％葡萄糖和0.02％蔗糖，蒸馏水定容至1000ml。
实施例1：氨基羧酸类螯合剂及其盐对镰刀菌、稻瘟病菌、灰葡萄孢的室内毒理测定。
1.1室内针对镰刀菌毒力测定实验方法及实验结果
镰刀菌室内毒理测定的实验方法及其具体实验步骤：
(1)收集孢子。在25℃培养3d的镰刀菌的边缘均匀上打5个菌碟(5mm)并放于CMC培养基中，25℃ 175rpm条件下摇培5d后使用三层无菌擦镜纸收集滤液，10000rpm/min离心10min，无菌水清洗两次(10000rpm/min，10min/次)，调孢子浓度至1×104ml-1。
(2)药剂浓度梯度设置。利用等比稀释法设置一系列浓度梯度，EDTA及盐(EDTANa，EDTANa2，EDTANa3，EDTANa4)：0，3μM-300mM；DTPA及盐(DTPANa，DTPANa2，DTPANa3，DTPANa4，DTPANa5)：0，3μM-12.7mM；EDTA与DTPA的组合物：3μM-300mM EDTA+3μM-12.7mM DTPA。将药剂与孢子液混匀后吸入细胞培养板中，于25℃恒温培养箱中培养一段时间。
(3)调查结果与统计分析。当对照组孢子萌发率＞90％时，观察统计孢子萌发情况。
孢子萌发率(％)＝萌发孢子数/检查孢子数×100
矫正萌发率(％)＝(对照萌发率-处理萌发率)/(100-对照萌发率)×100
在室内采用孢子萌发法，测定EDTA及盐，DTPA及盐，EDTA与DTPA的组合物抑制镰刀菌生长的EC50值。优选的，EDTA，DTPA抑菌结果如表1所示，
表1 EDTA，DTPA，EDTA+DTPA对不同镰刀菌的有效中浓度(EC50值)
Figure GSB0000189517580000021
Figure GSB0000189517580000031
1.2室内针对稻瘟病菌毒力测定的试验方法及实验结果
稻瘟病原菌室内毒理测定的实验方法及其具体实验步骤：
(1)收集孢子。利用灭菌水反复冲洗28℃培养7d的稻瘟菌平板，洗涤液使用三层无菌擦镜纸收集滤液，10000rpm/min离心10min，无菌水清洗两次(10000rpm/min，10min/次)，调孢子浓度至1×104ml-1。
(2)药剂浓度梯度设置。按1.1所述设置药剂浓度梯度。将药剂与孢子液混匀后吸入细胞培养板中，于28℃恒温培养箱中培养一段时间。
(3)调查结果与统计分析。
测定EDTA及盐(EDTANa4)，DTPA及盐(DTPANa5)，EDTA与DTPA的组合物抑制稻瘟菌生长的EC50值。优选的，EDTA，EDTANa4，DTPA，DTPANa5，EDTA+DTPA(摩尔比100000∶1和1∶4233)抑菌结果如表2所示，
表2 EDTA，DTPA，EDTA+DTPA对稻瘟病原菌的EC50值
Figure GSB0000189517580000032
1.3室内针对灰葡萄孢毒力测定的试验方法及实验结果
灰葡萄孢菌室内毒理测定的实验方法及其具体实验步骤：
(1)收集孢子：在25℃培养3d的灰葡萄孢菌的边缘均匀上打5个菌碟(5mm)并放于马铃薯培养基中，于25℃培养7d后，利用灭菌水冲洗马铃薯培养基，三层无菌擦镜纸过滤洗涤液并收集滤液，10000rpm/min离心10min，无菌水清洗两次(10000rpm/min，10min/次)，调孢子浓度至1×104ml-1。
(2)药剂浓度梯度设置。按1.1所述设置药剂浓度梯度。将药剂与孢子液混匀后吸入细胞培养板中，于25℃恒温培养箱中培养一段时间。
(3)调查结果与统计分析。
在室内采用孢子萌发法，测定EDTA及盐(EDTANa2)，DTPA及盐(DTPANa3)，EDTA与DTPA的组合物(摩尔比2∶1)抑制灰葡萄孢菌生长的EC50值。结果如表3所示，
表3 EDTA，DTPA，EDTA+DTPA对灰葡萄孢菌的EC50值
Figure GSB0000189517580000041
1.4室内针对青霉、木霉毒力测定的试验方法及实验结果
1)药剂浓度梯度设置。配置一系列浓度梯度的PDA培养基：EDTA及盐(EDTANa2，EDTANa3，EDTANa4)：0，3μM-300mM；DTPA：0，3μM-300mM；EDTA与DTPA的组合物：3μM-300mMEDTA+3μM-12.7mM DTPA。
2)菌丝生长速率法(PDA平板法)测定药剂对青霉和木霉的EC50值
优选结果如表4所示。
表4 EDTA，DTPA，EDTA+DTPA对青霉和木霉的EC50值
Figure GSB0000189517580000042
1.5室内针对疫霉毒力测定的试验方法及实验结果
1)药剂浓度梯度设置。配置一系列浓度梯度的PDA培养基：EDTA及盐(EDTANa2，EDTANa3，EDTANa4)：0，3μM-300mM；DTPA：0，3μM-12.7mM；EDTA与DTPA的组合物：3μM-300mMEDTA+3μM-12.7mM DTPA。
2)菌丝生长速率法(PDA平板法)测定药剂对致病疫霉的EC50值
优选结果如表5所示。
表5 EDTA，DTPA，EDTA+DTPA对致病疫霉的EC50值
Figure GSB0000189517580000051
实施例2 EDTA及盐，DTPA及盐，EDTA与DTPA的组合物对镰刀菌、稻瘟病菌、灰葡萄孢的室内活体药效
(1)EDTA及盐，DTPA及盐，EDTA与DTPA的组合物对镰刀菌的室内活体药效
用0.1％升汞消毒豫麦35种子5min，用无菌水冲洗3遍后浸泡2h；将种子均匀置于铺有双层无菌滤纸的塑料盒中，每盒放置30粒种子，25℃，90％湿度，12h光照，12h黑暗的光照培养箱中培养2d至胚芽鞘长至2.5cm时分别喷施EDTA及盐(3μM-300mM)，DTPA及盐(3μM-12.7mM)，EDTA与DTPA的组合物溶液，12h后剪去胚芽鞘尖端，向伤口分别接种5μL 5×105个/mL Fg2021孢子液，于光照培养箱中培养，7d后调查病斑长度，优选的结果如图1所示：7.5mM EDTA及盐(EDTANa4)，15μM DTPA及盐(DTPANa5)，EDTA与DTPA的组合物(摩尔比100000∶1；3000∶1；1∶200；1∶2000)处理后明显抑制镰刀菌Fg2021的侵染能力，处理后麦苗的病斑显著短于对照麦苗。
(2)EDTA及盐，DTPA及盐，EDTA与DTPA的组合物对稻瘟病原菌的室内活体药效
大麦幼苗30℃，12h光照，12h黑暗培养6.5d，喷施EDTA及盐(3μM-300mM)，DTPA及盐(3μM-12.7mM)，EDTA与DTPA的组合物于大麦叶片上，12h后用灭菌的剪刀剪取尖端7～8cm的叶片放于滤纸保湿的培养皿中，接种5μL，2×104个/mL的稻瘟病菌Guy11孢子液于叶片上，相同条件下培养6d后统计分析病斑长度。
分析表明，EDTA及盐，DTPA及盐，EDTA与DTPA的组合物处理后能显著降低稻瘟病菌致病力。优选的，30mM EDTANa2，10mM DTPANa3，EDTA+DTPA(摩尔比2∶1；1∶10)处理后明显抑制稻瘟病菌Guy11的侵染能力，表现为病斑显著减小(图2)。
(3)EDTA及盐，DTPA及盐，EDTA与DTPA的组合物对灰葡萄孢菌的室内活体药效
黄瓜幼苗在30℃，12h光照，12h黑暗的温室中培养14d，喷施(3μM-300mM)，DTPA及盐(3μM-12.7mM)，EDTA与DTPA的组合物于黄瓜叶片上，12h后用灭菌的剪刀剪取黄瓜叶片放于的培养皿中叶柄处使用棉球保湿，将10μL 1×105个/mL的灰葡萄孢菌的孢子液接于叶片上，相同条件下培养4d后统计分析病斑大小。
分析表明，EDTA及盐，DTPA及盐，EDTA与DTPA的组合物可抑制灰葡萄孢侵染。优选的，25mM EDTA，30mM EDTAMg2，10mM DTPA，EDTA+DTPA(摩尔比2∶1)处理后黄瓜叶片的病斑显著短于对照叶片(图3)。
实施例3田间药效实验
试验时间：2018年、2019年
试验地点：江苏南京
供试作物：小麦
防治对象：小麦赤霉病
试验方法：小麦扬花期分别喷施EDTA及盐(EDTANa2，EDTANa3，EDTANa4)，DTPA及盐(DTPACa2Na)：0，7，70，100g ha-1；EDTA与DTPA的组合物(摩尔比200∶1；20∶1；2∶1；1∶20；1∶200；1∶2000)，以140gha-1的多菌灵作为药剂对照，24h后在麦穗喷施1×104个/mL的Fg2021孢子悬浮液1ml。每个处理100株小麦。
实验结果：分别于14d或21d观察并统计发病小穗数。优选的，图4展示了140g ha-1的多菌灵，7g ha-1EDTA，7g ha-1DTPA，EDTA与DTPA的组合物(摩尔比2∶1)溶液处理后的小穗发病情况，可从图片中直观的看出，药剂处理后小麦穗发病情况明显弱于对照组，田间不同小区的防效为37.6％～88.4％。
实施例4氨基羧酸类螯合物及其盐在真菌中的主要靶标与作用机制
(1)氨基羧酸类螯合物及其盐影响细胞壁的形成及细胞透性
以在25℃条件下将镰刀菌孢子分别于SNA和含氨基羧酸类螯合物及其盐的SNA培养基中培养48h后收集菌丝，测定处理后菌丝的细胞壁透性。以0.15mM的EDTA为例，氨基羧酸类螯合物及其盐处理菌丝后，细胞透性显著降低。
(2)氨基羧酸类螯合物及其盐对真菌的抑菌活性主要通过螯合辅因子发挥作用
分别添加一系列不同浓度的MgCl2，CaCl2或MnCl2(0，0.3，0.6，0.9，1.2，2.4mM)至含氨基羧酸类螯合物及其盐的SNA溶液中，取50μl混合液并将其与50μl 2×103个/ml-1镰刀菌孢子悬浮液均匀混合并置于96孔细胞培养板中于25℃培养52h后，使用分光光度计测定其OD290的值。同时，接种104个/ml-1孢子液至SNA、含氨基羧酸类螯合物及其盐的SNA培养基中，25℃培养7d，收集菌丝，用于测定菌丝内的微量元素。
结果如图5显示，氨基羧酸类螯合剂可显著降低真菌细胞内关键酶的活性辅助因子含量，造成真菌生长迟缓。
(3)氨基羧酸类螯合物及其盐对真菌关键酶的抑制作用
以真菌生长关键酶(细胞壁合成相关酶)为例，利用液体闪烁仪测定不同辅因子存在时细胞壁合成相关酶的活性，结果发现辅因子存在时细胞壁合成相关酶活性最高，添加氨基羧酸类螯合物及其盐类化合物，抑制细胞壁合成相关酶活性可达85％以上(图6)。
实施例5氨基羧酸类螯合物及其盐对有机硫类(代森锰锌和克菌丹)、抗生素类(多抗霉素)、三唑类(戊唑醇)、苯并咪唑类(多菌灵)、甲氧基丙烯酸酯类(肟菌酯)、琥珀酸脱氢酶类(啶酰菌胺)、杀卵菌剂(霜脲氰)药剂协同增效作用
组分A是指EDTA及盐，DTPA及盐，EDTA与DTPA的组合物中的一种，组分B指有机硫类(以代森锰锌和克菌丹为例)、抗生素类(以多抗霉素为例)、三唑类(以戊唑醇为例)、苯并咪唑类(以多菌灵为例)、甲氧基丙烯酸酯类(以肟菌酯为例)、琥珀酸脱氢酶抑制剂类(以啶酰菌胺为例)、杀卵菌剂(以霜脲氰为例)中的一种。按照有效成分(A)∶(B)质量配比为：0.5∶50，1∶30，1∶10，1∶1，10∶1，50∶1的复配比进行试验，测定其对真菌的共毒系数或SR值。
以灰葡萄孢为例，结果如表6所示，不同复配比增效效果不同，氨基羧酸类螯合物及其盐与不同杀菌剂组合处理时效果也不同(＞1.5表示增效效果较强)，优选复配比为0.5-30∶1-50。
表6氨基羧酸类螯合物及其盐对有机硫类(代森锰锌和克菌丹)、抗生素类(多抗霉素)、三唑类(戊唑醇)、苯并咪唑类(多菌灵)、甲氧基丙烯酸酯类(肟菌酯)、琥珀酸脱氢酶类(啶酰菌胺)、杀卵菌剂(霜脲氰)药剂混用协同增效作用
Figure GSB0000189517580000071
Figure GSB0000189517580000081
以上所揭露的仅为本发明的较佳实例而已，不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，均属于本发明所涵盖的范围。

Claims (3)

1.一种氨基羧酸类螯合剂的用途，所述用途为氨基羧酸类螯合剂防治稻瘟病菌、木霉、青霉中的应用；所述氨基羧酸类螯合剂为单剂或组合物，所述单剂为EDTA、DTPA、EDTA的盐类化合物EDTA Me+/++(n)、DTPA的盐类化合物DTPAMe+/++(n)中的一种，所述组合物由EDTA和DTPA组成；所述Me+/++为钠、钙、镁、锰、锌、铁离子，n为1-5。
2.根据权利要求1所述的氨基羧酸类螯合剂的用途，其特征在于，所述单剂使用浓度为0.003-300mM/L；所述组合物EDTA与DTPA的摩尔比为1-100000∶4233-1。
3.根据权利要求1所述的氨基羧酸类螯合剂的用途，其特征在于，EDTA Me+/++(n)和DTPAMe+/++(n)中一种或者几种混合与硫代氨基甲酸酯类、抗生素类、三唑类、甲氧基丙烯酸酯类、琥珀酸脱氢酶抑制剂类和杀卵菌剂中的一种或几种组合杀菌剂的组合应用，螯合剂与杀菌剂组合摩尔比为1-50∶50-1。
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