CN110747130B - 一种哈茨木霉菌新菌株 - Google Patents
一种哈茨木霉菌新菌株 Download PDFInfo
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Abstract
本发明涉及一种哈茨木霉菌(Trichoderma harzianum)新菌株BACC0957,该菌株已作为专利菌种保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC NO:15981。该菌株对小麦赤霉病的主要病原禾谷镰孢菌(Fusarium graminearum)的抑菌率达62.97%。
Description
技术领域
本发明涉及一种哈茨木霉菌新菌株BACC0957,属于微生物领域。
背景技术
木霉菌(Trichoderma spp.)属半知菌类半知菌类的丝孢纲,丛梗孢目,丛梗孢科,广泛存在于土壤等环境中,例如森林腐殖质层,以及农田果园等土壤、植物根围,叶围,种子和球茎等生态环境中大量存在,对多种重要植物病原真菌具有拮抗作用,被认为是有希望的生物防治因子。木霉属中应用最多的是哈茨木霉(Trichoderma harzianum),是多种植物病原菌的寄生菌和拮抗菌之一。1981年在西欧等国家已有用来防治李属果树银叶病的商品化木霉制剂,同时利用其分生孢子制成的产品在防治白绢病、立枯病、疫病等土传病害。被认为是以菌治菌最有希望的生物农药,在英、德、瑞典、俄、美等20余个国家均已走上商品化生产,目前我国尚无商品化木霉制剂。
小麦赤霉病(Fusarium head blight,FHB)是小麦粮食产业链上最具威胁的真菌病害之一,在我国主要集中在长江中下游地区、华南冬麦区及东北春麦区,近年来则逐渐向北扩展蔓延,在黄河流域及周边地区均有发生。另外,随着免耕、秸秆还田种植技术的推广,化肥使用量的增加以及小麦品种抗性的丧失,该病的发生逐年加重。该病害在中国、日本、东南亚和美国南、北部发生较重,能危害小麦的各个生育阶段,引起穗腐、茎腐、苗腐、秆腐,其中以穗腐危害最重。小麦扬花期病原菌最易侵入,造成的经济损失最大。小麦赤霉病在过去十年中,给美国明尼苏达州、伊利诺州、俄亥俄州、印第安纳州造成的经济损失超过10亿美元。我国是世界上小麦赤霉病危害最大的国家,每年受害面积约750万hm2,占全国小麦总面积的1/4,造成产量损失200万~300万t。目前小麦赤霉病发病主要有三大特点,赤霉病危害日益频繁、粮食生产中赤霉毒素污染日益加重、发病区域日益扩大。
目前,控制小麦赤霉病的主要手段仍是使用化学药剂,过去使用最为广泛的是多菌灵、甲基托布津等内吸性杀菌剂,近期研究发现,叶菌唑和丙硫菌唑对小麦赤霉病具有较好的防治效果。但是长期大量使用化学药剂易使土质退化、环境污染、病原菌产生抗药性。急需有新的无污染的生物农药替代。
发明内容
为解决现有技术存在的问题,本发明提供一种哈茨木霉菌新菌株,该菌株对禾谷镰孢菌具有较高抑制活性,由该哈茨木霉菌株培养的孢子粉用于小麦赤霉病的生物防治。
作为本发明的一个方面,本发明提供一种哈茨木霉菌新菌株,其分类命名为Trichoderma harzianum BACC0957,该菌株已作为专利菌种保藏于位于我国北京的中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC NO:15981,保藏日期:2018年6月21日。该菌株对禾谷镰孢菌抑制率为62.97%,明显高于其他筛选的菌株。
通过真菌形态鉴定方法,判定该菌株为哈茨木霉,其培养性状和形态特征如下:
在马铃薯-葡萄糖琼脂(PDA)培养基上25℃培养3天,菌落长满培养皿,圆形,白色,放射状,菌丝稀少,在菌落边缘菌丝较厚;培养5天,产生白色颗粒状产孢簇,在菌落边缘出现绿色产孢簇;培养7天,菌落表面形成放射状、绿色颗粒状产孢簇,边缘产孢簇多,呈垫状,背面白色。菌丝无色,分枝,光滑,具隔膜,宽2.5~5.5μm。在分生孢子梗上单生、对生或轮生产孢细胞。产孢细胞烧瓶形,基部窄,中部宽,向上变细,无色,6.5~9.0×2.5~4.0μm。分生孢子单生,近球形至卵圆形,单胞,绿色,光滑,多个孢子聚集呈头状,2.5~3.5×2.5~3.0μm。无明显气味。厚垣孢子未见。
作为本发明的另一个方面,本发明提供所述哈茨木霉菌新菌株在防治小麦赤霉病中的应用。
作为本发明的另一个方面,本发明提供所述哈茨木霉菌的培养方法,该方法包括如下步骤:
步骤1,菌种通过液体培养进行扩增;
步骤2,将步骤1扩增后的菌种接种到固体培养基中进行固体培养;
步骤3,采收孢子粉。
进一步,步骤2中所述固体培养基为本领域常规固体培养基,如谷物培养基,农作物秸杆培养基,农作物皮壳培养基,经济林木树枝培养基,植物茎杆培养基等;其中优选谷物培养基;所述谷物可选自小麦、玉米、大米、小米、黄豆粉、荞麦、大麦、燕麦、糙米、粳米中的任意一种或几种。
进一步,步骤2接种量为15%-25%;更进一步,接种量为18-23%;更进一步优选为20%。
进一步,步骤2固体培养过程中,在菌丝体生长阶段采用遮光培养,培养温度22-25℃;待菌丝体长满后转为光照培养,培养温度为20-25℃;培养过程中相对湿度为60-95%。
本发明哈茨木霉菌株培养的孢子粉可用于世界性病害小麦赤霉病的生物防治。
附图说明
图1为菌株BACC0957对禾谷镰孢菌的抑制作用(PDA平板);
其中,图1-A为平板正面图;图1-B为平板背面图。
生物材料保藏信息
哈茨木霉菌(Trichoderma harzianum)BACC0957,已作为专利菌种保藏于位于我国北京的中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC NO:15981,保藏日期:2018年6月21日。
具体实施方式
实施例1哈茨木霉菌株的分离
采用土壤中真菌的检测和分离方法,对多种样本的土壤取样进行分离,分离方法参考“方中达编著《植病研究方法》P132页(7)土壤中真菌的检测和分离”方法。分离培养基为:马丁氏(Martin)培养基:葡萄糖10g,蛋白胨5g,KH2PO4 1g,MgSO4 7H2O 0.5g,琼脂20g,链霉素30.0mg,1/30000孟加拉红水溶液100ml,水900ml。各种成分溶解在水中,加热,开始沸腾时加孟加拉红,然后分装灭菌,在倒平板前加链霉素。用于从土壤中分离真菌。
实施例2哈茨木霉菌对禾谷镰孢菌的抑制率测定
对从土壤中分离获得的200多株植物病原真菌,测定其对对禾谷镰孢菌的抑制率,采用菌落对峙法测定哈茨木霉对禾谷镰孢菌的抑菌能力,具体方法如下:
①配制PDA平板,分别培养待测的菌种和禾谷镰孢菌,待基本长满皿后,然后用灭菌的4mm的打孔器,分别在两种培养基生长一致的部位打孔(距皿边缘等距离)。
②在PDA空平板上,分别用灭菌的小镊子挑取打孔的培养基块,反转接种两种真菌,距离基本一致,然后每12小时度量一次抑菌圈大小(十字交叉法)。依据以下公式计算抑菌率。
抑菌率=[(对照菌落直径-4)-(处理菌落直径-4)]÷(对照菌落直径-4)×100%
注:“4”为打孔器所取菌落的直径。
不同菌株对禾谷镰孢菌的抑制率见表1(表中只记载了抑制率高于20%的菌株,其余菌株的抑制率低于20%)。
表1不同菌株对禾谷镰孢菌的抑制作用
选用菌种(株) | 对禾谷镰孢菌的抑制率% |
哈茨木霉(BACC0957) | 62.97 |
47号木霉 | 55.81 |
84号木霉 | 44.76 |
22号木霉 | 31.00 |
19号木霉 | 36.91 |
76号木霉 | 24.70 |
绿粘帚霉 | 27.20 |
蝉棒束孢 | 0 |
由表1可见,菌株BACC0957对禾谷镰孢菌的抑菌率最高,为62.97%。该菌株的PDA平板见图1,平板上半部分为菌株BACC0957,下半部分为禾谷镰孢菌。由图1可见,哈茨木霉对赤霉病的病原-禾谷镰孢菌表现出明显的抑制作用。
通过真菌形态鉴定方法,判定该菌株为哈茨木霉,其培养性状和形态特征如下:
在马铃薯-葡萄糖琼脂(PDA)培养基上25℃培养3天,菌落长满培养皿,圆形,白色,放射状,菌丝稀少,在菌落边缘菌丝较厚;培养5天,产生白色颗粒状产孢簇,在菌落边缘出现绿色产孢簇;培养7天,菌落表面形成放射状、绿色颗粒状产孢簇,边缘产孢簇多,呈垫状,背面白色。菌丝无色,分枝,光滑,具隔膜,宽2.5~5.5μm。在分生孢子梗上单生、对生或轮生产孢细胞。产孢细胞烧瓶形,基部窄,中部宽,向上变细,无色,6.5~9.0×2.5~4.0μm。分生孢子单生,近球形至卵圆形,单胞,绿色,光滑,多个孢子聚集呈头状,2.5~3.5×2.5~3.0μm。无明显气味。厚垣孢子未见。
实施例3哈茨木霉孢子粉的培养
所用菌种为BACC0957,将斜面上的孢子接入摇瓶中进行培养摇瓶培养,培养液为土豆200g煮汁、加2%葡萄糖,补水至1L,温度25℃,转速180rpm,培养7天。待摇瓶中的培养液呈粘稠状,绿色至墨绿色时,即可中止培养。
然后将种子液接到固体培养基上发酵7d,发酵条件:温度25℃,湿度95%;固态发酵培养基为:麸皮4.6%、稻壳13.9%、玉米粉13.9%、木屑4.6%、水63.0%。接种量20%,接种后先进行遮光暗培养24℃2-3天,待菌丝生长满盒时,转光培养25℃5-7天,孢子丝长满,且全盘墨绿时,即可收获,固体培养物55℃干燥箱内烘干,用双层筛子筛分,得到孢子粉。可用于小麦赤霉病的生物防治。
Claims (2)
1.一株哈茨木霉(Trichoderma harzianum)新菌株BACC0957,该菌株已作为专利菌种保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC NO:15981。
2.如权利要求1所述的菌株在防治小麦赤霉病中的应用。
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