CN1234277C - 一种高效生物除草菌剂的制备方法及用途 - Google Patents
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Abstract
本发明公开的高效生物除草菌剂的制备方法,其菌种筛选工艺中采用从反枝苋、马唐、藜、千金子、决明等杂草根际5-15cm土壤、地上茎、叶片取样,分离时用NPC培养基、蛋白核小球藻(Chlorella pyrenoidosa)生长抑制试验和谷氨酰胺合成酶抑制剂筛选模型,选择除草生物活性高、杀草谱广的菌株。经测定,其菌剂对目前田地内大部分阔叶杂草和部分主要禾本科杂草有较好的防除效果,茎叶喷雾处理对反枝苋、藜、决明、蓼的防效达90%以上,对早熟禾、马唐、千金子的防效分别达75%、85%、80%。并对大部分草坪草和苗木安全。
Description
技术领域
本发明涉及一种生物除草菌株野油菜黄单胞菌反枝苋致病变种(Xanthomonas campestris pv.retroflexus)菌剂的制备方法及用途。
背景技术
化学除草剂能有效控制许多恶性杂草,但其大量使用也引发了一系列环境问题,而且新化学除草剂的开发难度大、成功率低、耗资高。相比之下,微生物除草剂低毒、易降解、环境相容性好,具有巨大的研制潜力。
微生物除草剂的研究始于上世纪六十年代,先后推出一批商品化的真菌除草剂,我国山东农科院开发了“鲁保一号”菟丝子盘长孢状刺盘孢的培养物防治大豆田菟丝子;这些真菌除草剂均是孢子制剂,作用条件要求严格,在批量生产、配方、储藏等技术上要求过高,未被广泛接受,没有产生显著的社会效益和经济效益。九十年代初人们将研究目标转向细菌,从杂草根系土壤的微生物菌群中筛选出具有抑制作用的细菌,通过培养获得使宿主致病的胞外毒素。由于细菌较真菌生长周期短、发酵工艺简单、生产流程易控制,其胞外毒素对环境条件要求不象真菌孢子严格、易被土壤降解,具有良好的应用开发前景,成为微生物除草剂研制的一大热点。
具有除草作用的根细菌主要是革兰氏阴性菌的假单胞菌属(Pseudomonas)、欧文氏菌属(Erwinia)、黄单胞菌属(Xanthomonas),其中研究得最多的是假单胞菌属。Pseudomonas syringae pv.phaseolicola所产生的植物毒素phaseolotoxin能使野葛(Kudzu)的叶片出现萎黄病的病症,产生局部坏死;这种毒素一旦进入植物根部将向枝端感染,导致植株的矮化、失绿,严重的导致植物叶片坏死。Gurusiddaiah.S将导致病害的Pseudomonas fluorescensstrainD7菌株发酵液初步纯化后的粗毒素用以控制早雀麦等等。Johnson B.J.最先研究黄单胞菌用作生物除草剂,他筛选出多株有除草活性的野菜黄单胞菌(Xanthomonas Campestris pv.poannua)用于控制百慕大草坪中的一年生早熟禾(Poa annua L.)。主要适用于草坪、花生地、土豆田、道路两旁、高尔夫球场等旱绿地。
发明内容
本发明的目的在于以植物生态学原理为基础,从根际土壤或植物组织上分离得到产细菌素的植物活性菌;并提供这种对田间主要杂草有防除作用的生物活性菌剂的制备方法。因此,本发明的着眼点在于筛选对田间主要杂草有防除作用并对大部分草坪草和苗木安全的菌株,其能防治多种田间杂草,其除草活性强、杀草谱广、选择性好。经大量筛选工作,从根际细菌中筛选出具有较高除草活性的菌株野油菜黄单胞菌反枝苋致病变种,明确了其目标杂草、杀草谱、使用剂量和应用范围。
本发明生物除草菌剂的制备方法,具体步骤如下:
1)从反枝苋、马唐、藜、千金子、决明等杂草根际5-15cm土壤、地上茎、叶片取样,在200ml无菌水中漂洗1-3次,投入100ml 0.01-2.0%吐温-80溶液中400-600rpm摇瓶10min,用无菌磷酸盐缓冲液(5-20Mm K3PO4-KH2PO4,0.05-2MNaCl,PH6.4-8.2)作10-20倍系列稀释,取0.1-0.3ml涂布于NPC培养基平板,20-32℃培养20-30h观察结果,挑选有色的菌落,保存于营养琼脂上,对初筛出的单菌落作进一步分离纯化:①抑菌试验,用接种针接一环待测菌,穿刺接种到大肠杆菌平板,20-32℃培养箱培养20-30h,观察抑菌斑;②蛋白核小球藻(Chlorella pyrenoidosa)生长抑制实验,将藻液接种到锥形瓶中,初始浓度为8×103-8×106个细胞/ml;处理加入待测液,对照不加,20-30℃、3000-60001ux光照度持续光照和100-200rpm旋转振荡培养2-7d,以培养液为参比,在最大吸收波长680nm下测定吸光值(光程1cm),测抑制率;③谷氨酰胺合成酶抑制剂模型筛选,分别在没有添加谷氨酰胺和添加了浓度为0.05-10%的谷氨酰胺的常规培养基上涂枯草芽孢杆菌细菌悬液平板,观察抑菌效果,从中选出菌落生长快,抑菌效果好,生物活性高的菌株。
2)菌株在常规固体培养基中进行斜面培养,复壮,25-30℃培养9-24h;然后进行种子培养,在400ml三角瓶中盛2/3体积的液体培养基,液体培养基的配方为:牛肉膏0.5-2.2%,蛋白胨0.2-4%,葡萄糖0.2-4%,NH4H2PO40.05-0.5%,NaCl 0.1-2.0%,以水补足体积;调初始PH6.0-7.4,在121-125℃下灭菌20-30mi n,冷却后以1%的接种量接种,25-30℃培养1 2-32h。
3)发酵罐中盛2/3体积的液体培养基,液体培养基的配方同种子液体培养基的配方,以6-12%的接种量接种,通气量为1∶0.6-1.5(V∶V),25-30℃培养54-120h;菌发酵液中含菌量在105-9cfu/ml;添加助剂,助剂为0.5-5%吐温80或氨硅,得到除草菌剂;直接包装成产品。
上述第一步所选出的具有高效生物除草活性菌株野油菜黄单胞菌反枝苋致病变种对大肠杆菌的抑菌圈是15-30mm,对谷氨酰胺合成酶抑制能力强,对蛋白核小球藻的抑制在发酵液稀释10-30倍下有60-90%。
野油菜黄单胞菌反枝苋致病变种的群体特征:菌落在固体平板上呈圆形,全缘,表面光滑湿润,半透明,黄色。菌落直径为5-6mm/24h;在液体培养基中静止培养时无菌膜形成,细胞稳定,菌液变混浊并有沉淀产生。
个体形态:细胞为短杆状,1.5-2.0×0.5-1.0 um,无芽孢,极生鞭毛,运动,革氏染色阴性,有类脂粒。
理化特征:过氧化氢酶反应产气,好氧,不固定氮素,能利用一碳化合物作为唯一的碳源。生长温度为8-50℃,最适生长温度为25-35℃;PH5-9都能生长,最适PH6-7.5。能产生非水溶性的类胡萝卜素,氧化酶反应阴性,不能还原硝酸盐。能水解葡萄糖、明胶,不能水解淀粉。
根据上述特征,该菌属于第八版“伯杰氏鉴定细菌学手册”中的黄单胞菌属(Xanthomonas),该菌在中国专利局指定的保藏单位中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为:CGMCC NO.0902。该菌株的培养特征、个体特征与生理生化特征见表1、2。
本发明高效生物除草菌剂的应用
本发明的高效生物除草菌剂用于草坪草及木本苗木和草本苗木中防除阔叶杂草和禾本科杂草。
所说的草坪草为高羊茅、狗牙根、马蹄金、黑麦草、结缕草等。苗木为香樟、雪松、广玉兰、桂花、柳杉、国槐、黄杨、女贞、红花木莲、金叶含笑、紫藤、凌霄等。阔叶杂草和禾本科杂草主要为反枝苋、马齿苋、刺苋、荠菜、稗草、狗尾草、牛筋草、马唐、早熟禾、看麦娘、双穗省稗、藜、苍耳、荠菜、猪殃殃、婆婆纳、田旋花、空心莲子草、黄花篙、棉毛酸模叶廖、碎米荠、香附子等。
使用方法、剂量和时期:采用喷雾的方法施药,使用剂量为500-1000L/ha;使用期在杂草萌发至五叶期为最佳,一般上半年在3、4月份,下半年在9、10月份;菌剂作一次也可作二次施用,第二次施药在第一次施药后一至四个星期。
本发明的生物除草菌剂经温度、湿度和光照条件试验表明,该菌剂对环境的要求较为宽松,温度在10-35,湿度在20%以上,对目标杂草的致病性均良好;一般在施药后1星期表现出致病性,可持续到4星期以上。没有残留,没有致病性,对环境安全,菌剂还可起到富集土壤微生物的作用。同时该菌剂还适用于大规模生产,有效菌株单纯,工艺简单,成本低廉,使用方便。
附图说明
图1是将分离得到的野油菜黄单胞菌反枝苋致病变种制备菌剂的工艺流程图。
具体实施方式
以下通过实例进一步说明生物除草菌剂的制备方法,具体步骤如下:
1)用从反枝苋杂草根际10cm土壤、地上茎、叶片取样,去掉杂草根部多余的土壤,在200ml无菌水中漂洗2次,投入100ml 1.0%吐温-80溶液中500rpm摇瓶10min,用无菌磷酸盐缓冲液(6MmK3PO4-KH2PO4,0.2MNaCl,PH7.2)作10倍系列稀释,取0.1ml涂布于NPC培养基平板,NPC培养基平板指在Davis氏最小培养基中添加青霉素、新生霉素、放线菌酮,24℃培养22h观察结果,挑选有色的菌落,保存于营养琼脂上。对初筛出的单菌落作进一步分离纯化:①抑菌试验,用接种针接一环待测菌,穿刺接种到大肠杆菌平板,24℃培养箱培养22h,观察抑菌斑,所用到的培养基均为常规培养基。②蛋白核小球藻(Chlorellapyrenoidosa)生长抑制实验,将藻液接种到锥形瓶中,初始浓度为8×103个细胞/ml。处理加入待测菌发酵液,对照不加,22℃、3000lux光照度持续光照和100rpm旋转振荡培养3d,以培养液为参比,在最大吸收波长680nm下测定吸光值(光程1cm),测抑制率。③谷氨酰胺合成酶抑制剂模型筛选,分别在没有添加谷氨酰胺和添加了浓度为2%的谷氨酰胺的常规培养基上涂枯草芽孢杆菌细菌悬液平板,观察抑菌效果。从中选出菌落生长快,抑菌效果好,生物活性高的菌株。所选出的菌株野油菜黄单胞菌反枝苋致病变种对大肠杆菌的抑菌圈是23mm,对谷氨酰胺合成酶抑制能力强,对蛋白核小球藻的抑制在发酵液稀释30倍下有70%以上。
2)菌株在常规固体培养基中进行斜面培养,复壮,26℃培养22h;然后进行种子培养,在400ml三角瓶中盛2/3体积的液体培养基,液体培养基的配方为;牛肉膏0.5%,蛋白胨3%,葡萄糖2%,NH4H2PO4 0.05%,NaCl 1.5%,以水补足体积;调初始PH7.4,在121-125℃下灭菌20min,冷却后以1%的接种量接种,26℃培养28h。
3)发酵罐中盛2/3体积的液体培养基,液体培养基的配方同种子液体培养基的配方,以6%的接种量接种,通气量为1∶0.7(V:V),26℃培养96h;菌发酵液中含菌量在107cfu/ml;添加助剂,助剂为4%吐温80或氨硅,得到除草菌剂;直接包装成产品。
高效生物除草菌剂的应用实例如下:
实施例1:反枝苋
在反枝苋种子露白出芽至4真叶期喷施菌剂,剂量在500 L/ha,表现出较好的防除效果(见表3)。1d后新叶萎蔫,2d后老叶开始萎蔫,4d后叶子基本枯萎,防效达90%以上。3星期后防效略有下降,反枝苋的部分茎叶开始恢复,但长势差,已对受保护植物不构成影响。
实施例2:决明
在决明种子露白出芽至第4轮真叶期喷施菌剂,剂量在500 L/ha,表现出较好的防除效果。2d后新叶开始萎蔫,3d后部分老叶开始萎蔫,7d后叶子基本枯萎,防效达85%以上。3星期后防效有所下降,决明的少部分茎叶开始恢复,但长势极差,对受保护植物不构成影响。
实施例3:小藜
在小藜种子露白出芽至4真叶期喷施菌剂,剂量在500 L/ha,防除效果良好。5d后叶子基本枯萎,防效达85%以上,3星期后防效略有下降,但已被控制在经济阈植以下。
实施例4:早熟禾
在早熟禾种子露白出芽至3叶期喷施菌剂,剂量在500 L/ha,防除效果较好。4d后大部分叶子枯萎,防效达75%。2星期后防效有所下降。
实施例5:马唐
在马唐种子露白出芽至4真叶期喷施菌剂,剂量在500 L/ha,表现出较好的防除效果。1d后新叶萎蔫,2d后老叶萎蔫,4d后叶子基本枯萎,防效达85%以上,3星期后防效开始下降。
实施例6:高羊茅
在高羊茅出芽2叶期以后喷施菌剂,剂量在900 L/ha,表现出较好的安全性(见表4)。3叶期以上无药害,2叶期植株略有矮化,1星期后既可恢复,低剂量下更为安全。故田间在高羊茅长至2叶期后施药较安全。
实施例7:金叶含笑
在金叶含笑出芽后长至4cm以上喷施菌剂,剂量在900 L/ha,表现出较好的安全性(见表5)。在3-4cm喷施生长有所受抑制,但很快恢复。故田间在金叶含笑长至4.5cm高后施药安全。
实施例8:红花木莲
在红花木莲出芽后长至5cm以上喷施菌剂,剂量在900 L/ha,安全性较好。
表1.野油菜黄单胞菌反枝苋致病变种培养特征和个体特征
固体平板菌落特征 | 大小 | 5-6mm |
形状 | 圆形 | |
表面结构 | 光滑湿润 | |
边缘 | 全缘 | |
光学特征 | 半透明 | |
菌苔颜色 | 黄色 | |
产生色素 | 有 | |
液养体培 | 菌膜 | 无 |
混浊 | 有 | |
沉淀 | 有 | |
个体特征 | 宽(um) | 0.5-1.0 |
长(um) | 1.5-2.0 | |
革氏染色 | 阴性 | |
运动性 | 有 | |
鞭毛 | 极生>1根 | |
类脂粒染色 | 有 |
表2.野油菜黄单胞菌反枝苋致病变种生理生化特征
过氧化氢酶 | + |
氧化酶 | - |
葡萄糖氧化发酵试验 | + |
明胶水解 | + |
淀粉水解 | - |
L-缬氨酸 | + |
β-丙氨酸 | + |
DL-精氨酸 | + |
硝酸盐还原 | - |
41℃生长 | + |
精氨酸双水解酶的测定 | - |
荧光色素的产生 | - |
表3 菌剂对不同叶龄期杂草防效(鲜重抑制率%)
杂草名称 | 叶龄 | ||||
1 | 2 | 3 | 4 | 5 | |
反枝苋 | 98.1 | 90.5 | 86.6 | 82.4 | 45.2 |
决明 | 100.0 | 93.4 | 84.1 | 81.3 | 50.7 |
小藜 | 100.0 | 94.3 | 88.6 | 84.2 | 67.8 |
蓼 | 95.6 | 86.4 | 80.1 | 71.8 | 39.6 |
车前 | 98.2 | 91.4 | 86.3 | 69.4 | 41.8 |
酸模 | 94.0 | 88.7 | 79.4 | 71.4 | 52.6 |
早熟禾 | 95.0 | 87.7 | 78.5 | 63.4 | 41.7 |
马唐 | 100.0 | 96.3 | 90.5 | 86.2 | 66.4 |
千金子 | 94.7 | 86.7 | 74.5 | 65.7 | 44.9 |
牛筋草 | 90.4 | 80.0 | 67.5 | 53.3 | 37.7 |
表4菌剂对不同叶龄期草坪草的药害情况(鲜重抑制率%)
草坪草名称 | 叶龄 | |||
1 | 2 | 3 | 4 | |
高羊茅 | 45.7 | 15.6 | 6.3 | 3.4 |
狗牙根 | 51.3 | 20.4 | 5.5 | 0 |
马蹄金 | 66.7 | 17.3 | 9.1 | 3.6 |
黑麦草 | 53.4 | 19.3 | -1.3 | 2.7 |
表5 菌剂对不同株高苗木的药害情况(鲜重抑制率%)
草坪草名称 | 株高(cm) | |||
2 | 3 | 4 | 5 | |
金叶含笑 | 55.7 | 31.6 | 7.8 | 4.4 |
峨嵋含笑 | 56.3 | 40.4 | 22.5 | 4.9 |
红花木莲 | 62.7 | 47.6 | 27.6 | 6.6 |
Claims (5)
1.一种生物除草菌剂的制备方法,其特征是包括以下步骤:
1)从反枝苋、马唐、藜、干金子、决明等杂草根际5-15cm土壤、地上茎、叶片取样,在200ml无菌水中漂洗1-3次,投入100ml 0.01-2.0%吐温-80溶液中400-600rpm摇瓶10min,用PH6.4-8.2的无菌磷酸盐缓冲液,成分为5-20MmK3PO4-KH2PO4,0.05-2M NaCl,作10-20倍系列稀释,取0.1-0.3ml涂布于NPC培养基平板,20-32℃培养20-30h观察结果,挑选有色的菌落,保存于营养琼脂上,对初筛出的单菌落作进一步分离纯化:①抑菌试验,用接种针接一环待测菌,穿刺接种到大肠杆菌平板,20-32℃培养箱培养20-30h,观察抑菌斑,对大肠杆菌的抑菌圈是15-30mm;②蛋白核小球藻(Chlorella pyrenoidosa)生长抑制实验,将藻液接种到锥形瓶中,初始浓度为8×103-8×106个细胞/ml;处理加入待测液,对照不加,20-30℃、3000-6000lux光照度持续光照和100-200rpm旋转振荡培养2-7d,以培养液为参比,在最大吸收波长680nm下测定吸光值,光程为1cm,测抑制率,对蛋白核小球藻的抑制在发酵液稀释10-30倍下有60-90%抑制效果;③谷氨酰胺合成酶抑制剂模型筛选,分别在没有添加谷氨酰胺和添加了浓度为0.05-10%的谷氨酰胺的常规培养基上涂枯草芽孢杆菌细菌悬液平板,观察抑菌效果,从中选出菌落生长快,抑菌效果好,生物活性高的菌株。
2)菌株在常规固体培养基中进行斜面培养,复壮,25-30℃培养9-24h;然后进行种子培养,在400ml三角瓶中盛2/3体积的液体培养基,液体培养基的配方为:牛肉膏0.5-2.2%,蛋白胨0.2-4%,葡萄糖0.2-4%,NH4H2PO40.05-0.5%,NaCl 0.1-2.0%,以水补足体积;调初始PH6.0-7.4,在121-125℃下灭菌20-30min,冷却后以1%的接种量接种,25-30℃培养12-32h。
3)发酵罐中盛2/3体积的液体培养基,液体培养基的配方同种子液体培养基的配方,以6-12%的接种量接种,通气量为1∶0.6-1.5(V∶V),25-30℃培养54-120h;菌发酵液中含菌量在105-9cfu/ml;添加助剂,助剂为0.5-5%吐温80或氨硅,得到除草菌剂;
2.权利要求1所述的生物除草菌剂,在草坪草及木本苗木和草本苗木中防除阔叶杂草和禾本科杂草的应用。
3.权利要求1所述的生物除草菌剂,在高羊茅、狗牙根、马蹄金、黑麦草或结缕草防除阔叶杂草和禾本科杂草的应用。
4.权利要求1所述的生物除草菌剂,在香樟、雪松、广玉兰、桂花、柳杉、国槐、黄杨、女贞、红花木莲、金叶含笑、紫藤或凌霄防除阔叶杂草和禾本科杂草的应用。
5.权利要求1所述的生物除草菌剂,在防除反枝苋、马齿苋、刺苋、荠菜、稗草、狗尾草、牛筋草、马唐、早熟禾、看麦娘、双穗省稗、藜、苍耳、荠菜、猪殃殃、婆婆纳、田旋花、空心莲子草、黄花篙、棉毛酸模叶廖、碎米荠或香附子的应用。
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CN100565187C (zh) * | 2007-09-06 | 2009-12-02 | 浙江工业大学 | 一种用于除草剂高通量的96孔板筛选方法 |
CN101611707B (zh) * | 2009-08-12 | 2014-07-16 | 海南利蒙特生物农药有限公司 | 一种枯草芽孢杆菌油悬浮剂及其制备方法 |
CN102206604A (zh) * | 2011-04-28 | 2011-10-05 | 南京大学 | 一株肠杆菌菌株及其在除草中的应用 |
CN109090144A (zh) * | 2018-10-17 | 2018-12-28 | 马书文 | 一种新型环保生物除草剂 |
CN110129062A (zh) * | 2019-06-14 | 2019-08-16 | 阿尔格生命科学(江苏)有限公司 | 一种藻类活性细胞土壤调理剂生产工艺 |
CN110590593B (zh) * | 2019-09-23 | 2022-10-18 | 青岛农业大学 | 一种酚酸酰胺衍生物及其制备方法和应用 |
CN114441491B (zh) * | 2022-01-25 | 2023-04-04 | 河北科技大学 | 一种利用蛋白核小球藻荧光检测阿特拉津生物毒性的方法 |
CN114657068A (zh) * | 2022-04-11 | 2022-06-24 | 广西地源之本肥业有限公司 | 一种康土抑草菌的制备方法 |
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