CN1165536A - 壳二孢属(Ascochyta)的微生物及其用途 - Google Patents
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Abstract
本发明的采用壳二孢属的一种微生物的除草剂和本发明的运用这一除草剂的控制方法,可以选择性地杀死莎草科莎草属的杂草或抑制它们的生长而不污染或破坏环境。
Description
本发明涉及壳二孢属的一个新菌株以及利用壳二孢属微生物的一种除草剂和一种杂草控制方法。
作为一种控制生长在稻田和田地的主要杂草莎草科(Cyperaceae)莎草属(Cyperus)杂草的方法,使用化学除草剂是主要采用的方法。最近,考虑到大量使用农药所引起的环境问题,人们希望发展不依赖于农药的除草剂和其利用方法。特别是,人们迫切希望研制出利用植物致病性真菌的微生物除草剂。到目前为止,DeVine(美国;目标杂草:马利筋科的Stranglervine)、Collego(美国;目标杂草:豆科的Northern jointvetch)、BioMal(加拿大;目标杂草:锦葵科的圆叶锦葵)作为除草剂已经注册并成为商品。然而,尚未研制出针对莎草科莎草属的微生物除草剂。
本发明的一个目的是提供一种用微生物控制杂草(如莎草科植物)的方法。
通过对解决上述问题的方法的广泛深入研究,本发明的发明者发现了一种壳二孢属的微生物,它能极好地控制象莎草科植物那样的杂草。因而完成了本发明。
本发明涉及壳二孢属的JTKA-644菌株,它对莎草属的植物有致病性。
本发明也涉及一种除草剂,它包括作为活性成份的壳二孢属的一种微生物,该微生物对杂草有致病性。
本发明还涉及一种用上述除草剂控制杂草的方法。
以下将对本发明进行详细描述。
首先,要对本发明的新菌株-壳二孢属的JTKA-644菌株进行描述。通过收集莎草属染病杂草,分离对上述杂草有致病性的菌株,纯培养菌株,并测定它们对上述杂草的控制效果,同时采用一种筛选方法以测定对其他作物(特别是稻谷)的致病性,获得了这一菌株。把本发明新菌株的主要真菌学特征列举如下:
该菌株为需氧真菌,其能够生长的pH值范围为3-13,优选为5-7。生长温度为15-30℃,最适温度为25-30℃,能在马铃薯葡萄糖培养基上活跃生长。菌落产生大量向各方向辐射排列的黑色细颗粒,随后逐渐呈白色菌丝体弯曲,分生菌丝数量少。分生孢子无色,分裂成两个细胞,呈卵形,大小约为13-18μm×约4-6μm。
通过对上述真菌学特征用“图解植物真菌”(Kobayashi等,1992,2-52页,384页,农村教育协会)和“图解半知菌的属”第四版(Barnett,H.L.和Hunter,B.B.,1987,6-39页,178页,麦克米伦出版公司)进行检索,发明者将该菌株鉴定为壳二孢属的微生物。而且,虽然已知壳二孢属的一些微生物是黄瓜、番茄的致病性真菌,但由于尚未了解到壳二孢属的微生物能对莎草科莎草属植物产生致病性,因此该菌株被认为是一个新菌株并且被定名壳二孢属的JTKA-644菌株。该菌株保藏于工业科学和技术署国立生命科学和人体技术研究所(1-3,Higashi 1-chome,Tsukuba City,Ibaraki Pref,日本),入藏登记号FERM BP-5176(最初保藏日期为1995年7月21日)。
本菌株的发酵无需特殊方法。可采用与用于发酵已知壳二孢属菌株的方法类似的方法。无论是合成培养基还是天然培养基均可用作为培养基,只要含有适宜的可利用的碳源、氮源、矿物质和必要的生长促进物质。可列举为特殊培养基的例子是马铃薯葡萄糖琼脂培养基(PDA)、察氏琼脂培养基等等。在发酵过程中,最好是将温度维持在15-30℃下,优选为25-30℃,pH值维持在3-13,优选为5-7。当该菌株在上述条件下培养7-14天后,在培养基表面会形成足够量的分生孢子。该菌株能将生长在稻田和田地中的莎草科莎草属的杂草杀死。另一方面,该菌株不影响禾本科作物,如稻谷、小麦、大麦、玉米等,也不影响其他作物如茄子、大豆、甘蓝、黄瓜等等。该菌株能进行大量培养,并且同时很容易获得大量的孢子。
现将本发明的除草剂和控制方法描述如下。
可以把壳二孢属的JTKA-644菌株用作为用于本发明的除草剂的微生物。除了这一菌株外,任何属于壳二孢属并且对莎草属的植物有致病性的微生物也可以被利用。本发明的除草剂可以通过把孢子悬浮于水中来配制。这些孢子是大量培养壳二孢属的微生物而获得的,但配制方法不限于这一种。在本方法中,孢子的浓度约为106-107个孢子/ml,但不限于这一范围。在水中悬浮孢子时,可加入佐剂,如表面活性剂和展布剂。作为主要物质的微生物可以是刚经过发酵的新鲜微生物,或者可以将曾经贮藏过的微生物经过水或其它物质复原。可采用熟知的如超低温冷藏(-80℃)和真空冷冻干燥等等作为贮藏方法。
本发明的控制方法的实施是通过对田地施上述除草剂来进行的。所施的量可根据杂草生长的旺盛程度决定。通常最好是以每10英亩地中用1011-1012的孢子量的比率来施该除草剂。
对本发明的除草剂和控制方法的目的植物没有特别限制,但本发明的除草剂和控制方法对莎草属的植物特别有效。同样,也没有特别限定目的植物的生长阶段。本发明的除草剂和控制方法可应用于从刚发芽植株到长成植株的很宽的植物范围。
实施例1)本发明新菌株的产生方法
收集生长在稻田或田地中并且有患病症状的莎草科莎草属的杂草,切下患病部分。切下的叶片在70%乙醇溶液中浸泡30-60秒,然后悬浮在通过向1%次氯酸钠溶液加入等体积0.1%吐温80溶液制成的溶液中,浸泡1-3分钟进行表面消毒。随后叶片用0.1%的吐温80溶液洗一次,用无菌水洗两次。然后将叶片置于含有抗菌素的PDA培养基上,25℃下温育2-5天。将延伸菌落的边缘部分转至新鲜PDA培养基上,使之形成分生孢子。分生孢子再划线于新鲜PDA培养基上并培养,以此获得单菌落。这样获得这上微生物的纯分离。
对分离的各菌茱,再测定其对莎草科莎草属杂草的致病性,并且也测定它们对稻谷、小麦、玉米、蕃茄和黄瓜的影响。结果分离到壳二孢属JTKA-644新菌株,它显示出很好的杀草效果而不影响稻谷、小麦、玉米、蕃茄和黄瓜。2)所获菌株的鉴定
本发明菌株的鉴定主要是通过观察分生孢子的形态来进行。结果如上所述将这一菌株鉴定为壳二孢属的微生物。3)大量培养和配制的方法
通过向生长在PDA培养基上的壳二孢属微生物加入无菌水并且搅动可以制得高浓度的孢子悬浮液,再将约100μl的这种悬浮液滴入V-8计琼脂培养基上,然后用灭菌的L型玻璃棒分散悬浮液,有可能一次性地在培养皿(直径9cm)中接种大量的微生物。结果孢子产量约为每一培养皿6×107个孢子。
如上所述,通过平板培养法很容易从本发明的微生物获得大量的孢子。随后,可通过悬浮于10%脱脂牛奶和真空干燥的方法把所得的孢子制成可湿性粉剂。这样所得的可湿性粉剂通过悬浮于水中并且加入一种表面活性剂(如吐温80)后可施于杂草。4)对具芒碎米莎草(Cyperus microiria)的致病性试验。
在一个商品盆中种植具芒碎米莎草,用2-5叶时期的植株作为试验样品。
本发明的微生物培养在马铃薯琼脂培养基上,所得的孢子悬浮于0.1%吐温80溶液中使具有104-107个孢子/ml的浓度。所得的悬浮液以空中喷雾方式接种于具芒碎米莎草。样品立即置于一个保湿室中,放置48小时。然后转至25℃的温室中。二个星期之后。测定具芒碎米莎草中的发病率和控制百分比。在107个孢子/ml浓度下,本发明的微生物对具芒碎米莎草的发病率为100%;被本发明微生物所杀死的具芒碎米莎草的百分比为100%;本发明微生物对具芒碎米莎草的控制百分比为100%。
表1 JTKA-644菌株对具芒碎米莎草的控制效果
孢子浓度 | 发病率 | 杀死百分比(%) | 控制百分比(%) |
0 | 0 | 0 | 0 |
104 | 0 | 0 | 0 |
105 | 100 | 0 | 5 |
106 | 100 | 0 | 70 |
107 | 100 | 100 | 100 |
*发病率:得病植株的数目/接种植株的数目×100
*杀死百分比:杀死植株的数目/接种植株的数目×100
*控制百分比:总体上植株减少的比率(远看的景象)
从表1中可以清楚地看出,本发明的微生物在107个孢子/ml浓度下对具芒碎米莎草有很好的控制效果。5)对莎草属杂草的至病性试验
对莎草属杂草的致病性试验采用与在具芒碎米莎草致病性试验中所用的方法相似的方法进行。接种物的孢子浓度为107个孢子/ml。
所试的植株为具芒碎米莎草、水莎草(Cyperus serotinus)、Cyperusbrevifolia的变种leiolepis和异型莎草(Cyperus difformis)。试验结果如表2所示。
表2 JTKA-644菌株对莎草属杂草的控制效果
植株 | 效果的有或无 |
具芒碎米莎草 | + |
水莎草 | + |
Cyperus brevefolia变种leiolepis | + |
异型莎草 | + |
+:杀死或抑制生长
-:没有影响
从表2清楚可见,本发明的微生物对莎草属的杂草有很好的控制效果。6)对作物的影响
对作物的致病性试验采用与在具芒碎米莎草的致病性试验中所用的方法相似的方法进行。接种物的孢子浓度为107个孢子/ml。
用于试验的植物有稻谷、小麦、玉米、蕃茄和黄瓜。试验结果见表3。
表3对作物的影响
植株 有或无 |
稻谷 -小麦 -玉米 -茄子 -大豆 -甘蓝 -黄瓜 -具芒碎米莎草 + |
+:杀死或抑制生长
-:没有影响
从表3可清楚看出,本发明的微生物对稻谷、小麦、玉米、茄子、黄瓜、甘蓝和大豆没有任何影响。【制剂例1】(液剂)
把从壳二孢属FTKA-644菌株获得的分生孢子(1010个分生孢子)和吐温80(1g)加入1升水中,混合而制成液剂。【制剂例2】(可湿性粉剂)
在每1ml含有麦芽糖(9%)、粘土(1%)和水(90%)的混合液中悬浮108JTKA-644菌株的分生孢子。风干悬浮液,然后将干燥的物质混合并压碎而制成可湿性粉剂。【制剂例3】(可湿性粉剂)
在每1ml含有脱脂牛奶(10%)和水(90%)的混合液中悬浮108JTKA-644菌株的分生孢子。将悬浮液真空干燥,然后混合干物质并且压碎而制成可湿性粉剂。【制剂例4】(粉剂)
在每克含有羟丙基-β-环糊精(14%)、白碳(12%)和粘土(74%)的混合物中混入108个JTKA-644菌株的分生孢子。所得的混合物进行干燥并均匀地压碎而制成粉剂。【制剂例5】(颗粒)
在每克含有β-环糊精(15%)、淀粉(12%)、皂土(18%)、碳酸钙(36%)和水(29%)的混合物中加入108个JTKA-644菌株的分生孢子,并且进行捍和。然后将捍和后的物质用成粒机制成颗粒,再进行干燥而制成颗粒。【制剂6】(乳油)
在每克含有聚氧乙烯壬基苯基醚磷酸铵(18%)、聚氧乙烯壬基苯基醚(6%)、磷酸三乙酯(29%)和磷酸三丁酯(47%)的混合物中加入108JTKA-644菌株的分生孢子,均匀悬浮而得到乳油。【制剂例7】(油剂)
在lml含有锭子油(95%)、蓖麻油(4%)和硅油(1%)的混合液中悬浮108JTKA-644菌株的分生孢子而制成油剂。【制剂例8】(干流动剂型)(Dry Flowable)
在1ml含有烷基苯磺酸钠(12%)和聚乙二醇醚(88%)的组合物中,悬浮108JTKA-644菌株的分生孢子而制成干流动剂型。【制剂例9】(胶囊)
在1ml由藻酸钠(0.7%)、高岭土(5%)、甘油(15%)和水(79.3%)组成的混合液中悬浮108JTKA-644菌株的分生孢子。将悬浮液滴入0.2M乙酸钙溶液中而得到胶囊样产物。将这一产物切成小块、过筛并风干而制成胶囊。
本发明的除草剂有选择地杀死莎草科莎草属的杂草或抑制其生长,因此控制那些杂草的生长而不影响作物(如稻谷)。而且,与农药不同,本发明的除草剂不污染也不破坏环境。
Claims (6)
1.壳二孢属(Ascochyta)的JTKA-644菌株,它对莎草属(Cyperus)的植物具有致病性。
2.一种除草剂,它含有作为活性成份的壳二孢属的一种微生物,它对杂草具致病性,但对有用作物没有致病性。
3.权利要求2的除草剂,其中该壳二孢属的微生物是壳二孢属的JTKA-644菌株。
4.权利要求2或3的除草剂,其中的杂草是莎草属的植物。
5.权利要求4的除草剂,其中莎草属的植物是具芒碎米莎草(Cyperusmicroiria)、水莎草(Cyperus serotinus)、异型莎草(Cyperus difformis)或Cyperusbrevifolia变种leiolepis中的任一种植物。
6.一种控制杂草的方法,它使用权利要求2-5中任意一项的除草剂。
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