CN105861334B - 一株能促进台湾相思生物量增长的内生真菌 - Google Patents
一株能促进台湾相思生物量增长的内生真菌 Download PDFInfo
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Abstract
本发明涉及一株能促进台湾相思生物量增长的内生真菌。所述内生真菌为丝黑粉菌属(Filobasidium)B,已于2016年1月27日在中国微生物菌种保藏管理委员会普通微生物中心登记保藏,保藏号为CGMCC No.11910。将台湾相思内生真菌B接种于台湾相思土培苗,测定接种60天后的台湾相思的生物量,台湾相思内生真菌B所处理的植株与对照的生物量差异极显著,生物量比较显示,该菌株较对照组的促生效果良好,其生物量为对照组的1.4倍;根冠比比较显示,该菌株的根冠比为对照的1.5倍,表明该菌株对台湾相思生物量的增长具有明显的促进作用。
Description
技术领域
本发明涉及一株能促进台湾相思生物量增长的内生真菌。
背景技术
植物内生菌的研究始于19世纪末,Vogl从黑麦草Lolium temulentum L.种子中分离出第一株内生菌。但真正开始大量研究植株中的内生菌起始于上世纪八十年代,主要是在温带地区、亚热带地区和热带地区的植被中开展研究。
植物内生真菌的分布广、种类多,前人研究表明从绝大多数植物体内都能分离得到内生真菌,由此可见内生真菌普遍存在于植物体内。在全世界范围内至少在80多个属290多种的禾本科农作物中发现了内生菌。目前从植物中分离的内生菌根据其具有的独特功能可以分为:固氮菌、固钾菌、固磷菌等植物促生菌、具有抗病虫害的生防菌和具有促进植物对不良环境的修复能力的抗性菌。
有关内生真菌对台湾相思生长的影响研究还存在空白。
发明内容
本发明的目的在于提供一株菌株能促进台湾相思生物量增长的内生真菌,该真菌为丝黑粉菌属(Filobasidium)B,已于2016年1月27日在中国微生物菌种保藏管理委员会普通微生物中心登记保藏,地址为北京市朝阳区北辰西路1号院3号,保藏号为CGMCC No.11910。
为实现上述目的,本发明采用如下技术方案:
该菌的分离、纯化包括:
A、材料:将采集得到不同年份的根、枝茎、叶分成三个部分,用自来水清洗干净,分装到自封袋内,于4℃冰箱保存;
B、消毒:75%酒精漂洗30s→无菌水冲洗3-4次→15%的次氯酸钠漂洗(根10min,茎5min,叶2min)→无菌水冲洗4-5次。当样品最后一次浸洗后的水盛于灭菌的小烧杯中,在平板上划线作为对照,以检验样品的消毒是否彻底,若干天后如有菌落生成,则表明样品表面消毒不彻底,需继续调整消毒方法;
C、接种部位的选择:叶片:叶尖部、叶中部和叶基部3个处理;枝茎:上中下分为3部位,其中第3部位为枝茎交接处;根部:分为根尖和根基2个部分;
D 、接种:将消毒后的外植体用接种刀切开,铺放在培养基表面,于28℃恒温培养箱内培养5—7天后观察菌落生长状况。有的菌株繁殖迅速,可先将其产生的菌株进行分离纯化;生长缓慢且数量较少的菌株可延长观察时间,直到其生长稳定后纯化;
固体培养:使用改良马丁固体培养基,配方为蛋白胨 5.0g/L、酵母浸出粉2.0g/L、葡萄糖20.0g/L、磷酸氢二钾1.0g/L、硫酸镁0.5g/L、琼脂14.0g/L、其它为无菌水,pH值6.4±0.2,培养温度为28℃,培养方式为平板培养,培养时间为48h;
E、将分离繁殖出的不同种类的内生真菌接入平板培养基进行纯化,经过2-3次点接纯化、转接后分别得到单一菌种的上述的Filobasidium功能菌株;
上述的一种能促进台湾相思生物量增长的内生真菌应用菌液的制备方法,是将上述的菌株接入液体培养基,摇床振荡培养,培养温度为28℃,培养时间48~72h,利用血球计数板计算菌液浓度,将菌液用超纯水稀释成1.0-9.0×106cfu/ml即为成品备用;液体培养基:为改良马丁培养基,其中蛋白胨 5.0g/L、酵母浸出粉2.0g/L、葡萄糖20.0g/L、磷酸氢二钾1.0g/L、硫酸镁0.5g/L、其它为无菌水、pH值6.4±0.2。
上述的一种能促进台湾相思生物量增长的内生真菌应用菌液的应用方法,是应用该菌株的菌液浇施于台湾相思苗木根际土壤或苗木直接接种。
上述的一种能促进台湾相思生物量增长的内生真菌应用菌液的应用方法,是用5.5×106cfu/ml菌液,按每株100ml在林地浇于每株台湾相思的根际。
本发明涉及的菌株是从台湾相思植株中分离纯化得到的,经接种于台湾相思土培苗,根据各项指标综合评判,进一步证实其对植株生长具有促进台湾相思生物量增长的作用,寻找到对促进植株生物量增长的有益的功能内生真菌可应用于生产。
通过菌液浇施的方法接种于台湾相思幼苗,测定初接种4月时和接种60天6月时的台湾相思的生物量、根冠比。台湾相思内生真菌B所处理的植株与对照的生物量、根冠比差异显著。生物量比较显示,菌株B处理较对照组的促生效果良好,其生物量为对照组的1.4倍。根冠比比较显示,菌株B的根冠比为对照的1.5倍。表明菌株B对台湾相思生物量的增长具有明显的促进作用。
附图说明
图1为不同处理对台湾相思幼苗生物量的影响;
图2为不同处理对台湾相思幼苗根冠比的影响。
具体实施方式
根际土壤浇施与苗木直接接种应用
本试验采用土培盆栽试验,试验所用材料为福建省漳州市市***提供的台湾相思一年生苗。选择长势一致的台湾相思苗木定植于直径15cm,高10cm的塑料盆中。所用土壤是严格经过熏蒸消毒的黄心土。经过混匀称量,每盆放入等量的3kg土壤。经过一个月的恢复性生长后,连续三天于台湾相思根际施入浓度为5.5×106cfu/ml的100mL菌液,用蒸馏水溶液作为空白对照。
菌液制备:将供试菌株接入40mL的液体培养基,培养基为改良马丁培养基,其中蛋白胨 5.0g/L、酵母浸出粉2.0g/L、葡萄糖20.0g/L、磷酸氢二钾1.0g/L、硫酸镁0.5g/L、其它为无菌水、pH值6.4±0.2,在恒温振荡培养箱中经过72h的培养。用无菌生理盐水按十倍稀释法将菌液稀释,利用血球计数板计算菌液浓度配制成5.5×106cfu/mL。接种60天后进行苗高、地径等指标的测定,检测方法如下:
生物量的测定
将植株的不同部位分别放入信封中并标上记号,放入烘箱105℃烘干半小时(钝化酶),在60℃下烘干,经过12h后,称重后再放入烘箱中,重复测定直至恒重为止。称得样品的重量(W),即为样品干重,以此表示植株的生物量,精确到0.01g。
结果与分析
不同处理对台湾相思生物量和根冠比的影响
生物量是反映苗木生产力水平的重要指标,它与苗木的生长发育存在着非常密切的关系。如图1所示,接种菌株B的幼苗的生物量比对照组大37.12%,且与对照组间呈极显著的差异性(sig=0.008,显著水平为0.01),由此可得,菌株对台湾相思幼苗的生物量的增长具有极显著的促进作用。图2所示,接种菌株B的幼苗的根冠比对照组大46.89%,且与对照组间极显著的差异性(sig=0.005,显著水平为0.01),说明菌株B对台湾相思幼苗根冠比的增大具有极显著的促进作用。
本发明发现两株混合菌株能促进台湾相思苗生物量增长的内生真菌,将该株台湾相思内生真菌菌株采用浇根的方法接种于台湾相思幼苗。通过对植株的生物量、根冠比的测定,得出接种该菌株的处理的生物量、根冠比增长均较大程度高于对照,表明其对于促进植株生物量的增长具有很大功用,从而进一步证实得到该株内生真菌能促进台湾相思生物量的增长。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (3)
1.一株能促进台湾相思生物量增长的内生真菌,其特征在于:该真菌为丝黑粉菌属(Filobasidium)B,已于2016年1月27日在中国微生物菌种保藏管理委员会普通微生物中心登记保藏,保藏号为CGMCC No. 11910。
2.一种如权利要求1所述的一株能促进台湾相思生物量增长的内生真菌的应用菌液,其特征在于:所述应用菌液的制备方法,将所述的丝黑粉菌属(Filobasidium)B菌株接入液体培养基,摇床振荡培养,培养温度为28℃,培养时间48~72h,利用血球计数板计算菌液浓度,将菌液用超纯水稀释成1.0×106-9.0×106cfu/ml,备用;液体培养基:为改良马丁培养基,其中蛋白胨 5.0g/L、酵母浸出粉2.0g/L、葡萄糖20.0g/L、磷酸氢二钾1.0g/L、硫酸镁0.5g/L、其它为无菌水、pH值6.4±0.2。
3.一种如权利要求2所述的应用菌液的用途,其特征在于:所述应用菌液用于台湾相思苗木根际土壤浇施或苗木直接接种。
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