CN105861335A - 一株在低磷环境下促进台湾相思营养元素吸收的内生真菌 - Google Patents
一株在低磷环境下促进台湾相思营养元素吸收的内生真菌 Download PDFInfo
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Abstract
本发明涉及一株在低磷环境下促进台湾相思营养元素吸收的内生真菌。所述内生真菌为丝黑粉菌属(Filobasidium)F,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.12102。将台湾相思内生真菌F接种于台湾相思土培苗,通过对植株碳含量和氮含量的测定,得出接种该菌株在低磷环境下的处理植株的碳含量和氮含量均基本较大程度高于对照,表明其在低磷环境下对促进植株营养元素吸收具有很大功用,从而进一步证实得到该株内生真菌能在低磷环境下促进台湾相思植株营养元素的吸收。
Description
技术领域
本发明涉及一株在低磷环境下促进台湾相思营养元素吸收的内生真菌。
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在低磷环境下促进台湾相思营养元素吸收的内生真菌,该真菌为丝黑粉菌属(Filobasidium)F,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. 12102。
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;
上述的一种在低磷环境下促进台湾相思营养元素吸收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。
上述的一种在低磷环境下促进台湾相思营养元素吸收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。
上述的一种在低磷环境下促进台湾相思营养元素吸收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,进一步证实其在低磷环境下对植株生长具有促进台湾相思营养元素吸收的作用,寻找到对促进植株在低磷环境下营养元素吸收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,并在接种15天后进行低磷胁迫试验,在胁迫90d时测定各营养元素。根据测得的各项指标综合评判,进一步证实其对植株生长具有缓解低磷胁迫的作用,寻找到对促进植株营养元素吸收有益的功能内生真菌可应用于生产。通过对植株碳和氮的测定,得出接种该菌株的处理的碳和氮均基本较大程度高于对照,表明其在低磷环境下对促进植株碳和氮吸收具有很大功用,从而进一步证实得到该株内生真菌能在低磷环境下促进台湾相思植株碳和氮的吸收。
附图说明
图1为不同处理对台湾相思幼苗碳含量的影响;
图2为不同处理对台湾相思幼苗氮含量的影响。
具体实施方式
一、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林业局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。
二、低磷胁迫试验设计
盆栽试验土壤的基质为黄心土。经测定该土壤中各养分物质见表1。接种15天后进行低磷胁迫试验处理,这段时间主要是让接种菌株侵入植株。
表1 基质土壤养分情况
单位:mg/g
本试验设计了4个磷处理水平,每个水平3个重复(表2)。磷肥采用KH2PO4,定期施氮肥、钾肥及其他微量元素,直至收获。低磷胁迫于2015年5月4日进行。根据天气状况及时为台湾相思苗补充水分。
表2 低磷胁迫试验设计
单位:mg/kg
在胁迫90d时测定植株的碳含量和氮含量。检测方法如下:
(1)植株碳含量采用重铬酸钾氧化—外加热法测定植株全碳(即有机质)含量进行测定,每个样品做三个重复。
计算公式:
式中:为有机碳含量,g/kg;0.8000为重铬酸钾标准溶液的浓度,mol/L;5.0为重铬酸钾标准溶液的体积,mL;为空白标定用去硫酸亚铁溶液体积,mL;V为测定土样用去硫酸亚铁溶液体积,mL;0.003为1/4碳原子的摩尔质量,g/mmol;1.1为氧化校正系数;m1为风干土样质量,g;K2为风干土换算到烘干的水分换算系数。
(2)植株氮含量采用凯氏定氮法测定进行测定,每个样品做三个重复。
计算公式:
式中:WN为氮含量,g/kg;V为测定样品用去盐酸标准溶液体积,mL;V0为滴定试剂空白试验用去盐酸标准溶液体积,mL;c为盐酸标准溶液的浓度,mol/L;0.014为氮原子的摩尔质量,g/mmol;m为烘干样品质量,g。
(3)数据处理
数据整理与作图采用Excel2003,数据的统计学分析采用SPSS20.0。
三、结果与分析
(1)不同低磷胁迫下不同处理对台湾相思碳含量的影响
四个不同低磷胁迫条件分别为:重度胁迫、中度胁迫、轻度胁迫、正常条件。如图1所示,在四个不同低磷胁迫程度下,接种菌株I的幼苗的地上部分碳含量均较明显大于对照,分别为对照的1.27倍、1.14倍、1.38倍、1.10倍,地下部分碳含量均大于对照,分别为对照的1.44倍、1.14倍、1.22倍、1.15倍。由此可知,菌株F有利于促进植株对碳元素的吸收。
(2)不同低磷胁迫下不同处理对台湾相思氮含量的影响
如图2所示,在四个不同低磷胁迫程度下,接种菌株I的幼苗的地上部分氮含量均明显大于对照,分别为对照的1.35倍、1.95倍、1.93倍、1.23倍,地下部分碳含量在中度胁迫和轻度胁迫下均大于对照,分别为对照的1.50倍、1.31倍。由此可知,菌株F有利于促进植株对氮元素的吸收。
本发明发现一株在低磷环境下能促进台湾相思苗对碳氮等营养元素吸收的内生真菌,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。通过对植株碳含量和氮含量的测定,得出接种该菌株的处理的碳含量和氮含量均较大程度高于对照,表明其对于促进植株营养元素的吸收具有很大功用,从而进一步证实得到该株内生真菌在低磷环境下能促进台湾相思对营养元素的吸收。
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)
1.一株在低磷环境下促进台湾相思营养元素吸收的内生真菌,其特征在于:该真菌为丝黑粉菌属(Filobasidium)F,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. 12102。
2.一种如权利要求1所述的一株在低磷环境下促进台湾相思营养元素吸收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)F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,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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