CN114027324B - 地钱在防治根腐病中的应用 - Google Patents
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Abstract
本发明属于生物防治领域,具体涉及地钱在防治根腐病中的应用。技术方案为:地钱在抑制腐皮镰孢菌(Fusarium solani)活性或生长中的应用。本发明通过群落调查分析了花椒园杂草的种类和群落结构,确定出优势伴生杂草,采用生长速率法测定优势伴生杂草对根腐病病原菌腐皮镰孢菌的抑制率,从而筛选出具有抑菌活性的新物种地钱,为进一步开发新型植物源杀菌剂,防治根腐病奠定基础。
Description
技术领域
本发明属于生物防治领域,具体涉及地钱在防治根腐病中的应用。
背景技术
根腐病是一种以腐皮镰孢菌(Fusarium solani)为主要致病菌的毁灭性土传病害,是包括花椒在内的多种经济作物的主要疾病之一。根腐病可导致植物根系腐烂,散发异臭味,根皮与木质部易脱离,地上部分叶片变小且发黄发白,枝条发育不全,果实变小,最终导致整株枯死。
目前多使用化学农药防治根腐病,但这会导致环境污染、生态破坏、病原菌抗性增强等问题。相较于化学农药,植物源杀菌剂具有环境友好、对非靶标生物安全、不易产生抗药性、作用方式特异、能促进作物生长并提高抗病性等特点,可针对性地防治土传病害,是解决根腐病更有效途径。植物源杀菌剂指植物中含有或经诱导产生的某些抗菌物质,包括生物碱类、类黄酮类、蛋白质类、有机酸类和酚类化合物等多种类型。植物源杀菌剂在自然界中广泛存在,是生物农药的重要来源。
从种类繁多的植物资源中筛选出具有抑菌活性的植物,是植物源杀菌剂研究的基础。Rongai等从500种植物筛选出大蒜、牛蒡等15种植物能够抑制番茄枯萎病菌;张燕宁等从56种植物中筛选出黄连、藁本的甲醇提取物能够抑制黄瓜枯萎病菌。孙伟从101种植物中筛选出天明精、苦参和桑白皮的乙醇提取物能够抑制苹果腐烂病菌。然而,关于利用植物源杀菌剂来防治根腐病、尤其是花椒根腐病的研究尚未见报道。
发明内容
本发明的目的是提供地钱在防治根腐病中的应用。
为实现上述发明目的,本发明所采用的技术方案是:地钱在抑制腐皮镰孢菌(Fusarium solani)活性或生长中的应用。
相应的,地钱在防治植物根腐病中的应用。
优选的,所述植物根腐病为花椒根腐病。
相应的,一种可抑制腐皮镰孢菌(Fusarium solani)或防治植物根腐病的抑菌液,所述抑菌液由地钱提取获得。
优选的,所述抑菌液为地钱醇提液。
优选的,所述抑菌液为地钱的甲醇提取液。
优选的,以醇为溶剂浸泡地钱获得抑菌液,溶剂与地钱的料液比为1:5~1:10,g/mL。
相应的,所述抑菌液的制备方法,包括如下步骤:
(1)将地钱洗净、烘干至恒重,粉碎;
(2)以醇为溶剂,按料液比为1:5~1:10,g/mL,浸泡地钱24h以上,离心、过滤,取上清液,过滤除菌,得地钱提取液,即抑菌剂。
优选的,所述溶剂为无水甲醇。
优选的,步骤(2)过滤取上清液后,将滤渣再加入等量甲醇,浸泡滤渣24h以上,离心、过滤,取上清液,合并两次上清液,得地钱提取液。
本发明具有以下有益效果:伴生杂草能够在根腐病爆发的花椒园中稳定生长,其生存机理不可忽视。相较于从庞大的植物资源库中筛选抑菌植物,伴生杂草抑菌活性的筛选将使植物源杀菌剂的开发更具靶向性。因此,发明人课题组通过群落调查分析了花椒园杂草的种类和群落结构,确定出优势伴生杂草,采用生长速率法测定优势伴生杂草对根腐病病原菌腐皮镰孢菌的抑制率,从而筛选出具有抑菌活性的新物种地钱,为进一步开发新型植物源杀菌剂,防治花椒根腐病奠定基础。
在实际抑菌应用中,因地钱喜湿润环境,且其体内的抑菌活性物质在自然条件下无法发挥作用,因此直接将地钱种植到需要抑菌的区域难以起效。故发明人课题组对地钱进行醇提,获得具有可直接使用的抑菌液。本领域技术人员也在该抑菌液的基础上,进一步利用活性追踪法、各种色谱技术等,对抑菌液内各种可能的抑菌活性成分进行分离纯化。
附图说明
图1为空白对照组抑菌效果示意图;
图2为抑菌浓度为0.08g/mL下培养5d后地钱抑菌示意图;
图3为抑菌浓度为0.08g/mL下培养5d后菊叶香藜抑菌示意图;
图4为抑菌浓度为0.08g/mL下培养5d后老鹳草抑菌示意图。
具体实施方式
本发明提供了一种利用地钱防治包括根腐病在内的由腐皮镰孢菌引发的疾病的应用。优选的方案为:用醇或其他溶剂浸泡地钱,获得地钱提取液。因甲醇极性大于乙醇,对大部分亲脂性成分的溶解性也优于乙醇,且甲醇易挥发,便于后续进一步试验时蒸发去除溶剂,因此,更优选的方案为:用甲醇浸泡地钱。所述地钱提取液即为腐皮镰孢菌的抑菌剂,可采用业内常规技术手段,直接喷施或稀释后喷施到待治理区域,或干燥成粉/制粒保存和使用,或作为肥料等的添加剂使用。提取液的保存温度≤4℃。
更优选的方案为:将地钱洗净、烘干至恒重,粉碎过40目筛,以无水甲醇为溶剂,按料液比(g/mL)为1:5~1:10浸泡地钱24h以上,离心、过滤,取上清液。以料液比1:5为例,表示1g植物干样加入5mL无水甲醇。滤渣可再加入等量甲醇,重复浸泡、离心、过滤,取上清液。合并滤液,用0.22μm滤膜过滤除菌,得地钱提取液,即抑菌剂。所述地钱提取液优选在4℃下冷藏保存。使用时,将地钱提取液直接喷施到待处理区域,或根据需要将地钱提取液稀释后使用。所述地钱提取液的使用浓度优选为≥0.01g/mL。所述浓度指将地钱干样充分提取后在最终使用溶液中的溶度,以0.01g/mL为例,指0.5g地钱干样充分提取后获得的提取液稀释到50mL进行使用。
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。所获得的数据均为进行至少3次重复后获得的平均值,且各重复获得的均为有效数据。
实施例一:花椒园伴生杂草组成及抑菌效果展示
1、为提高数据准确性,本发明采集的致病菌、伴生杂草来自多个花椒园的不同位置,涉及花椒园分别位于四川省阿坝藏族羌族自治州茂县的黑虎镇苏家坪村、沟口镇鸡公寨村、沟口镇水草坪村和叠溪镇较场村。
从花椒园内根腐病发病花椒的根系中分离、筛选出致病菌,经基因鉴定为腐皮镰孢菌(Fusarium solani)。根据文献查阅及花椒种植试验验证,发现该微生物对花椒具有强致病性。将所述腐皮镰孢菌在PDA培养基上充分活化,备用。
2、从花椒园地共发现、采集伴生杂草45种,分属于23科42属,具体如表1所示。
表1花椒伴生杂草种类一览
3、从表1中选择重要值>0.05的杂草及经前期试验证明可能有一定抑菌效果的杂草共计20种,洗净、65℃烘箱烘干至恒重,粉碎过40目筛。再用水或无水甲醇对各杂草分别浸泡24h,杂草与水的料液比(g/mL)为1:10,杂草与甲醇的料液比(g/mL)为1:10。浸泡过程中摇床振荡(220r/min),振荡结束后再超声(300w)浸提30min,离心(4000r/min,5min),滤纸过滤后收集上清液。将滤渣再加入等量相同溶剂(水或无水甲醇),重复上述步骤,合并滤液。将合并后的滤液于旋转蒸发仪中蒸发浓缩后经超声15min充分混合,再用相同溶剂稀释至不同浓度的提取液,再用0.22μm滤膜过滤除菌,即得各杂草浸提液。
4、将所述腐皮镰孢菌接种至PDA培养基上,30℃黑暗培养48h,获得菌丝块。再将步骤3制备的各提取液1mL与49mL熔融状态(40~60℃)的PDA培养基混合均匀后制成PDA平板,以加入等量溶剂替代提取液作为空白对照(CK)。用直径为6.5mm的打孔器在活化病原菌的培养基内,按同心圆周打孔,将菌丝块接种至各个PDA平板中心,每个处理设置3次重复,每个重复包含3块平板,将所有平板置于30℃恒温培养箱中黑暗倒置培养。待菌落直径长至培养皿2/3时采用十字交叉法测量菌丝直径,抑制率计算方法为:抑制率=(空白对照菌落平均直径-各处理菌落平均直径)/(空白对照菌落平均直径-菌饼直径)×100%。测试各杂草浸提液浓度为0.02g/mL时的抑菌效果,结果如表2所示。
表2各杂草抑制腐皮镰孢菌的效果展示
从表2可以看出,当提取物浓度为0.02g/mL时,伴生杂草水提取液与甲醇提取液对腐皮镰孢菌菌丝生长的作用存在明显差异。在水提取液中,除老鹳草外,其余供试杂草均能极显著促进腐皮镰孢菌菌丝生长。在甲醇提取液中,有14种供试杂草对腐皮镰孢菌菌丝生长存在促进作用,6种供试杂草对腐皮镰孢菌的菌丝生长存在显著抑制作用,其中地钱、黄花蒿、菊叶香藜、老鹳草的抑制率分别为34.01%、20.09%、17.41%和11.40%,与CK差异达到极显著水平,覆盆子、羽叶蓼的抑制率分别为7.77%、5.58%,与CK差异达到显著水平。
实施例二:花椒园伴生杂草提取液浓度及抑菌效果关系展示1、选择醇提取液对腐皮镰孢菌有抑制作用的6种伴生杂草(地钱、黄花蒿、菊叶香藜、老鹳草、覆盆子、羽叶蓼),以实施例一相同的方法分别制备0.01g/mL、0.02g/mL、0.04g/mL和0.08g/mL的4个梯度浓度的甲醇提取液。
2、按照实施例一测试腐皮镰孢菌抑制率的方法,使用6种伴生杂草分别测试在培养3d、4d、5d后的抑制率,并参照实施例一的方法同样设置空白对照,结果如表3所示。表3中,浓度单位为g/mL,菌落直径单位为mm,抑制率单位为%。
表3各杂草醇提液在不同浓度下抑制腐皮镰孢菌的效果展示
从表3可以看出:在浓度为0.01g/mL时,仅地钱、老鹳草表现出极显著的抑菌效果,在培养第5d时抑菌效果均达到最大,抑制率分别为28.65%、11.18%。在浓度为0.02g/mL时,地钱、黄花蒿、老鹳草和菊叶香藜均表现出极显著的抑菌作用,抑制强度为地钱>老鹳草>菊叶香藜>黄花蒿。在浓度为0.04g/mL时,除羽叶蓼外,其余5种杂草都表现出极显著抑菌作用,抑制强度表现为地钱>老鹳草>黄花蒿>覆盆子>菊叶香藜。在浓度为0.08g/mL时,6种杂草提取物抑菌作用均达到极显著水平,抑制强度表现为地钱>黄花蒿>菊叶香藜>老鹳草>覆盆子>羽叶蓼。在0.08g/mL下培养5d后,空白对照示意图如图1所示,地钱抑菌示意图如图2所示,菊叶香藜抑菌示意图如图3所示,老鹳草抑菌示意图如图4所示。因组别过多,只展示了抑菌效果最明显的几个组别,其余抑菌效果图未一一展示。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形、变型、修改、替换,均应落入本发明权利要求书确定的保护范围内。
Claims (2)
1.地钱的甲醇提取液在抑制腐皮镰孢菌(Fusarium solani)的活性或生长或杀灭腐皮镰孢菌中的应用。
2.地钱的甲醇提取液在防治植物根腐病中的应用,其特征在于:所述植物根腐病为花椒根腐病,所述花椒根腐病由腐皮镰孢菌引起。
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