CN111869683B - 一种提高油萝卜抗根结线虫效率的方法 - Google Patents
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Abstract
本发明公开一种提高油萝卜抗根结线虫效率的方法,具体包括以下步骤:待油萝卜成熟后采摘油萝卜茎叶,干燥后将油萝卜秸秆用粉碎机粉碎后过150‑300目筛子,所得粉末备用;按90~110g/L的比例将油萝卜秸秆粉末加入蒸馏水中,浸泡45~55h后,离心分离后收集上清液;将上清液浓缩,获得初提物;按100~120g/L的比例在初提物中加入黄腐酸。本发明所述方法工艺简单,实用性强,通过水提取油萝卜秸秆有效成分,可获得较高的富含黄酮的溶液,可以提高油萝卜抗根结线虫效率的方法。
Description
技术领域
本发明涉及一种提高油萝卜抗根结线虫效率的方法,属于液体肥料制备技术领域。
背景技术
根结线虫被认为是最具侵入性的植物寄生线虫。其适应性强,传播途径多样,寄主范围广,可寄生于3000多种植物,对茄科、葫芦科、十字花科植物危害最为严重,且难以根治,严重破坏了全球主要粮食作物和经济作物的农业生产;据不完全统计,全球每年因根结线虫危害所造成的农作物损失多达1000亿美元。
最常见的侵染农作物根结线虫病原种有南方根结线虫(Meloidogyneincognita)、花生根结线虫(M.arenaria)、北方根结线虫(M.hapla)和爪哇根结线虫(M.javanica)4种。它们均属于世界性分布,在因根结线虫引起的农作物损失中所占比例高达90%以上,尤其是南方根结线虫的发生面积大,寄主种类多,在我国危害最大。
油萝卜(Raphanus sativus ssp.Oleiferus,2n=18)是十字花科萝卜属萝卜的野生种,具有抗根瘤病,抗根结线虫和孢囊线虫,及其它抗旱、抗寒、耐酸碱等优良特性。云南省10余个地、州有栽培与分布。油萝卜能作为绿肥熟化,改良土壤,增加土壤孔隙度,提高土壤肥力,提高作物产量。研究表明油萝卜携带抗胞囊线虫和根结线虫的抗性基因。
虽然油萝卜携带抗胞囊线虫和根结线虫的抗性基因,现有关于油萝卜在根结线虫方面的应用主要是将油萝卜秸秆与其他原料、菌种进行发酵制备肥料,由于油萝卜有效成分不高,所以抗根结线虫的效果并不明显。几十年来,防治根结线虫病害主要依靠化学农药,但长期使用造成根结线虫的抗药性,并且其毒性对人类和生态都造成危害而被禁止使用,因此,研发一种新型既可以有效防控根结线虫病害,又可以降低化学肥料的使用具有重要意义。
发明内容
为了解决上述技术问题,本发明的目的在于提供一种提高油萝卜抗根结线虫效率的方法,具体包括以下步骤:
(1)制备油萝卜秸秆粉末:待油萝卜成熟后采摘油萝卜茎叶,干燥后将油萝卜秸秆用粉碎机粉碎后过150-300目筛子,所得粉末备用。
(2)按90~110g/L的比例将油萝卜秸秆粉末加入蒸馏水中,浸泡45~55h后,离心分离后收集上清液。
(3)将步骤(2)的上清液浓缩,获得初提物;按100~120g/L的比例在初提物中加入黄腐酸得到最终的根结线虫抑制剂。
优选的,本发明步骤(1)的干燥过为:油萝卜茎叶在自然条件下晾干或在 60℃-70℃烘箱中烘制20-24h至含水量为12%-15%。
优选的,本发明步骤(3)中浓缩过程为:浓缩至固态含量占比为55~60%。
本发明的有益效果:
(1)本发明油萝卜秸秆粉末用蒸馏水提取且添加黄腐酸,和现有油萝卜秸秆的其他使用方法相比,抗根结线虫的效果明显提升。
(2)黄腐酸溶于水能促进植物生长,能刺激植物产生一些如抗逆蛋白,脯氨酸,可溶性糖等抗逆性物质从而提高植物抗病虫害的能力,本发明将黄腐酸与油萝卜秸秆提取物相配合,可以进一步提高油萝卜秸秆的抗根结线虫的能力,还能起到增产和改善品质作用;主要应用于烟草、瓜果、蔬菜等种植。
附图说明
图1为油萝卜绿色秸秆粉碎及其精粉制备。
图2为油萝卜精粉初提物的光谱扫描部分图片;M:弥勒品系;FY:富源品系; C:XY:祥云品系。
图3油萝卜不同品系精粉分别采用水和乙醇作溶剂初提物抑制根结线虫虫卵孵化M:弥勒品系;FY:富源品系;XY:祥云品系。
具体实施方式
下面结合具体实施例本发明作进一步的详细说明,但本发明的保护范围并不限于所述内容。
实施例1
一种提高油萝卜抗根结线虫效率的方法,具体包括以下步骤:
(1)制备油萝卜秸秆粉末:待油萝卜成熟后采摘油萝卜茎叶,干燥后将油萝卜茎在65℃烘箱中烘制20h至含水量为13%,然后用粉碎机粉碎后过200目筛子,所得粉末备用。
(2)按100g/L的比例将油萝卜秸秆粉末加入蒸馏水中,浸泡45h后, 12000rpm离心10min后收集上清液。
(3)将步骤(2)的上清液浓缩至固态含量占比为55%,获得初提物;按 120g/L的比例在初提物中加入黄腐酸得到最终的根结线虫抑制剂。
实施例2
一种提高油萝卜抗根结线虫效率的方法,具体包括以下步骤:
(1)制备油萝卜秸秆粉末:待油萝卜成熟后采摘油萝卜茎叶,干燥后将油萝卜茎叶在自然条件下晾干15%,然后用粉碎机粉碎后过150目筛子,所得粉末备用。
(2)按110g/L的比例将油萝卜秸秆粉末加入蒸馏水中,浸泡55h后, 12000rpm离心10min后收集上清液。
(3)将步骤(2)的上清液浓缩至固态含量占比为60%,获得初提物;按 110g/L的比例在初提物中加入黄腐酸得到最终的根结线虫抑制剂。
实施例3
一种提高油萝卜抗根结线虫效率的方法,具体包括以下步骤:
(1)制备油萝卜秸秆粉末:待油萝卜成熟后采摘油萝卜茎叶,干燥后将油萝卜茎叶在自然条件下晾干至含水量为12%,然后用粉碎机粉碎后过300目筛子,所得粉末备用。
(2)按90g/L的比例将油萝卜秸秆粉末加入蒸馏水中,浸泡50h后,12000rpm 离心10min后收集上清液。
(3)将步骤(2)的上清液浓缩至固态含量占比为56%,获得初提物;按 100g/L的比例在初提物中加入黄腐酸得到最终的根结线虫抑制剂。
取本发明实施例1~3中提取物,研究其对根结线虫的抑制效果,使用方法:在云南玉溪的15亩烤烟种植试验区,种植烤烟前,按根结线虫抑制剂与水质量比为1:50的比例进行稀释,喷洒于烟塘中,与烟塘中的土壤混合均匀,每亩80kg;使用后实施例1的根结线虫防治效率达99.8%,实施例2中根结线虫防治效率达 99.2%,实施例3中中根结线虫防治效率达99.1%,(结线虫防治效率=(没有感染的烤烟数/烤烟总数)×100%)。
实施例4
进一步研究本发明所述方法抑制根结线虫的机理
(1)取3个油萝卜品系各取绿色秸秆1kg,分别放入植物秸秆捣碎仪中,捣碎成均匀细粉,经60目过筛,收集过筛的细粉。
(2)取每种细粉20g,于离心管中,分别加入蒸馏水、乙醇200mL,混匀,浸泡48h后,12000rpm离心10min,分别收集上清液。(3)将收集的上清液在滤纸上过滤,收集滤液。
(4)分别取收集的滤液1ml,以对应的溶剂为空白对照,通过DU730型分光光度计,在全波长范围检测吸收光波长。
(5)利用旋转真空蒸发仪浓缩,获得初提物,备用。
(6)初提物的主要化学成分检测。
一、提取有效成分的检测:
本实施例共制备了油萝卜3个品系秸秆粉,参见图1。对制备的3种绿色秸秆粉样品进行初提取后,经光谱扫描,确认初提物中,提液均在特定波长范围含有极高吸收光谱的物质,参见图2。初步分析,在290-450nm波长范围内,光谱吸收OD值较高,说明提取物中含有较多的成分在此波长范围吸收光波;并且,从图2中光波吸收曲线可以看出,290-450nm波长范围内,蒸馏水作为溶剂的提取物中,3个油萝卜品系初提物中吸收光谱的物质含量更高,显示出此类物质适于以水作为溶剂进行提取。
将油萝卜绿色秸秆发酵肥(将油萝卜秸秆粉碎;加入地衣芽孢杆菌、枯草芽孢杆菌、绿色木霉和黑曲霉,几个菌种的比例为1:1:1:1;在密闭容器中发酵6-7 周,每周一次翻拌均匀)和秸秆粉分别用水和乙醇进行提取,送至云南省农业科学院质量标准与测试分析研究所进行主要化学成分(总酸、总糖、总黄酮和蛋白质)的初步检测,结果参见表1,黄酮类和总酸的含量较高,并且,以水作为溶剂时,这两类成分的含量比以乙醇作为溶剂高出约2倍。据报道,黄酮类化合物的吸收光谱分为带Ⅰ(240-280nm)和带Ⅱ(240-280nm),酸类化合物在253nm、300-500nm均有吸收峰。
表1.不同原料中总酸、总糖、总黄酮和蛋白质的含量
检测成分类别 | 秸秆粉-水 | 秸秆粉-乙醇 | 发酵肥-水 | 发酵肥-乙醇 |
总酸(%) | 0.46 | 0.27 | 0.05 | 0.02 |
总糖(%) | 0.027 | 0.014 | 0.003 | 0.003 |
总黄酮(mg/100g) | 8.1 | 4.4 | 6.9 | 2.5 |
蛋白质(%) | 0.28 | 0.04 | 0.02 | 0.04 |
从表1可以看出,本发明所述方法通过水提取秸秆粉中的有效成分明显高于乙醇提取的含量,在提取溶剂相同的情况下,用秸秆粉提取的成分明显高于发酵肥料。
二、虫卵孵化抑制试验
①将发病的烟草根冲洗干净,在解剖镜下用镊子挑取新鲜卵块,放入直径 9cm的培养皿内,用无菌水清洗3次后收集备用。
②将试验分为空白对照(蒸馏水)、油萝卜M绿色秸秆粉-水的提取物、油萝卜M绿色秸秆粉-乙醇的提取物、油萝卜FY绿色秸秆粉-水的提取物、油萝卜FY 绿色秸秆粉-乙醇的提取物、油萝卜XY绿色秸秆粉-水的提取物、油萝卜X绿色秸秆粉-乙醇的提取物,油萝卜绿色秸秆发酵肥-水的提取物,油萝卜绿色秸秆发酵肥-乙醇的提取物,共8个处理,每个处理设3个重复。
③将上述制备的南方根结线虫卵块,分别加入12孔培养板中,每孔分别加入大小以及发育一致的卵块3枚,再加入蒸馏水及3个油萝卜品系的初提物,以只加蒸馏水处理为空白对照,3次重复,于25℃恒温培养箱中。
④线虫卵块经孵化3天、7天、14天和21天,依次在解剖镜下检测培养板每孔中J2的数量。
结果参见图3,采用从烟株根系挑取的卵块(3卵块/孔),分别加入蒸馏水及 3个油萝卜品系绿色秸秆粉的提取物及发酵肥的水提取物,以只加蒸馏水处理作为空白对照,25℃培养箱中孵化21天的结果表明,加有初提物的处理,极大程度的抑制了虫卵孵化成二龄幼虫(J2),处理间差异达到极显著水平 (F=15.70>F0.01=4.29),但用不同油萝卜品系、分别以水与乙醇作为溶剂的提取物处理间,差异不明显。油萝卜绿色秸秆发酵肥的水提取物的抑制效果(85.8%)也有所减弱,推测与堆捂发酵过程中持续高温和微生物的活动有关。
通过综合分析上述检测和抑制虫卵孵化试验的结果,初步归纳为如下结果: 3个油萝卜品系的光谱吸收极为相似;抑制虫卵孵化试验结果表明:3个油萝卜品系及采用水或乙醇作为溶剂的初提物,对根结线虫虫卵孵化为J2均具有极显著的抑制作用,不同品系或不同溶剂的初提物处理间,抑制效果的差异不显著,但是,以水作为溶剂的提取中,含有更高浓度的总酸、总糖、总黄酮和蛋白质,抑制效果更好。油萝卜绿色秸秆发酵肥中,同样具有这4类化合物。各处理对虫卵孵化的抑制率达到81.5-100%。因此,分别以水和乙醇作为溶剂,3个油萝卜品系的绿色秸秆粉及其发酵肥分别在不同溶剂中浸泡和抽提,均含有抑制根结线虫虫卵孵化成J2的成分,所以,推测油萝卜绿色秸秆发酵肥防控根结线虫病害的作用机理,主要是抑制虫卵孵化成J2的作用。
Claims (3)
1.一种提高油萝卜抗根结线虫效率的方法,其特征在于,具体包括以下步骤:
(1)制备油萝卜秸秆粉末:待油萝卜成熟后采摘油萝卜茎叶,干燥后将其用粉碎机粉碎后过150-300目筛子,所得粉末备用;
(2)按90~110g/L的比例将油萝卜秸秆粉末加入蒸馏水中,浸泡45~55h后,离心分离后收集上清液;
(3)将步骤(2)的上清液浓缩,获得初提物;按100~120g/L的比例在初提物中加入黄腐酸得到最终的根结线虫抑制剂。
2.根据权利要求1所述提高油萝卜抗根结线虫效率的方法,其特征在于:步骤(1)的干燥过为:油萝卜茎叶在自然条件下晾干或在60℃-70℃烘箱中烘制20-24h至含水量为12%-15%。
3.根据权利要求1所述提高油萝卜抗根结线虫效率的方法,其特征在于:浓缩至固态含量占比为55~60%。
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