CN108395995A - 丛枝菌根真菌高效菌种快速筛选与鉴定方法 - Google Patents
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Abstract
本发明公开了一种丛枝菌根真菌高效菌种快速筛选与鉴定方法,通过在岩溶干旱环境中,采集植物根围土壤样品,通过高通量测序先进技术及群落多样性指标筛选当地土著丛枝菌根真菌优势菌种,然后将所筛选优势菌种经扩繁后接种于原植物,通过干旱胁迫处理观测植物生长、生理等性状,鉴定是否属于高效菌种。本发明可以快速筛选获得当地土著优势菌种,并通过进一步验证得到高效菌种,从而增强岩溶区植物抗旱性。
Description
技术领域
本发明涉及微生物的筛选与鉴定领域,具体涉及一种丛枝菌根真菌高效菌种快速筛选与鉴定方法。
背景技术
我国南方岩溶地区是全球三大岩溶集中连片区域中分布面积最大、发育类型最全的一个区域,岩石出露面积达5.5×105km2。由于长期存在不合理的土地利用,水土流失严重,地表干旱缺水、土被不连续、土壤富钙偏碱缺磷等造成植被生境严酷、植被覆盖率锐减,现已成为制约该地区可持续发展的重要生态环境问题。近年来,选用适应性强,兼具生态与经济效益且区域性突出的先锋树种进行植被恢复。然而,在年降水量丰沛的西南喀斯特区域,因受季风气候影响季节性分配不均,加上土壤蓄水能力低、岩石渗透性强等原因,地表和地下水土流失相互叠加,土壤水分亏缺仍然是岩溶区植被恢复重建的主要障碍因素。水分胁迫通过抑制植物光合作用使植物生长减缓、繁殖力下降乃至死亡,通过菌根技术可以有效缓解干旱胁迫对植物造成的伤害,并缩短岩溶区生态修复周期并保证修复效果的稳定性。
丛枝菌根真菌是一类能与大多数高等植物形成共生关系的功能真菌。在岩溶干旱环境中,丛枝菌根真菌的菌丝比植物根系窄的多,能够深入岩石裂缝或裂隙中吸收水分并将水分运输到植物根内,从而促进植物生长并增强植物抗岩溶干旱能力。但是,不同丛枝菌根真菌具有不同的功能,并且喀斯特石漠化地区的生态环境不同于其它地区,可能蕴藏着具有特殊功能尤其是抗逆性强的丛枝菌根真菌种类。因此,针对岩溶干旱环境亟需建立优良、高效丛枝菌根真菌的快速筛选与鉴定方法。
目前的优良、高效丛枝菌根真菌筛选与鉴定方法大多采用以下方法:
随机购买一些丛枝菌根真菌菌种,通过设计一些接种与不接种处理,以及土壤水分处理等,将植物移栽或撒播于不同处理土壤中,观测植物生长性状、生理特性后,将显著促进植物生长发育的菌种筛选鉴定为丛枝菌根真菌优良菌种。但此方法忽视了两个关键问题:
1.随机购买的菌种缺乏一定的科学依据。
2.直接筛选购买的菌种不一定完全适宜当地土壤环境。
发明内容
基于以上两个核心问题,本发明提供了一种通过先进技术直接从岩溶干旱区植物根围快速筛选丛枝菌根真菌优势菌种,并将其接种至原植物以鉴定是否属于增强植物抗旱性的高效菌种的方法,从而促进丛枝菌根真菌在当地土壤环境中的生态应用。
为实现上述目的,本发明采取的技术方案为:
丛枝菌根真菌高效菌种快速筛选与鉴定方法,包括如下步骤:
S1、在夏季炎热干旱时,采集岩溶区不同类型植物根围土壤样品,并迅速冷冻;
S2、利用土壤基因组DNA提取试剂盒快速提取样品DNA,随后用丛枝菌根真菌两对引物AML1-AML2和NS31-AM1进行巢式PCR扩增,并用琼脂糖凝胶电泳和荧光定量系统检测扩增产物;
S3、通过第二代高通量测序手段对PCR扩增产物进行测序,获得土壤中不同科属丛枝菌根真菌;
S4、利用生物群落相关指数筛选获得土著丛枝菌根真菌优势菌种;
S5、通过显微镜分离出上述土著丛枝菌根真菌优势菌种的孢子,并进一步利用盆栽试验将孢子放入适量的河沙中并借助三叶草和玉米作为寄主扩繁出各优势菌种;
S6、将上述扩繁的优势菌种接种于灭菌土壤,并撒播或移栽所需植物,正常培养数月后,做干旱胁迫处理及胁迫后复水处理,在不同处理阶段注意观测植物生长状况;
S7、根据上述所观测的植物生长状况结果,将干旱胁迫与复水处理后,植物长势表现依旧较好的菌种鉴定为高效菌种。
本发明具有以下有益效果:
本发明可以快速筛选获得当地土著优势菌种,并通过进一步验证得到高效菌种,从而增强岩溶区植物抗旱性。
具体实施方式
为了使本发明的目的及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例
第一步:在夏季炎热干旱时,采集贵州省毕节市岩溶区桑树根围土壤样品,并放入便携式低温箱,迅速带回实验室用液氮速冻,随后放入-80℃超低温冰箱保存备用。
第二步:利用土壤基因组DNA提取试剂盒(MPSpin Kit for Soil)快速提取样品DNA,随后用丛枝菌根真菌两对引物AML1-AML2和NS31-AM1进行巢式PCR扩增,并用2%琼脂糖凝胶电泳和QuantiFluor-ST蓝色荧光定量系统(Promega公司)检测扩增产物。
第三步:通过第二代高通量测序平台(Roche Genome Sequencer FLX+)上机测序,获得土壤中不同科属丛枝菌根真菌。
第四步:综合利用Shannon-Winner多样性、Simpson优势度和Pielou均匀度等生物群落指数筛选获得土著丛枝菌根真菌优势菌种2个,摩西斗管囊霉和根内根孢囊霉。
第五步:通过显微镜从原土壤中分离出上述土著丛枝菌根真菌优势菌种Fm和Ri的孢子,进一步利用盆栽试验将孢子放入适量的河沙中并借助三叶草和玉米作为寄主扩繁出各优势菌种。
第六步:将上述扩繁的优势菌种接种于灭菌土壤,移栽灭菌基质中培养出的桑树幼苗,正常培养2个月后,观测此时桑树幼苗的株高、茎粗、叶面积、叶片数、叶绿素、光合等生长、生理指标,以及桑树根系菌根侵染率。随后对其他剩余桑树接种与未接种盆栽进行干旱胁迫和复水处理,同时观测桑树生长与生理指标,以及菌根侵染率。
第七步:根据上述所观测的桑树生长数据,将干旱胁迫与复水处理后,综合考虑桑树菌根侵染率及长势状况,将桑树根系菌根侵染率高,同时桑树长势表现依旧较好的菌种鉴定为高效菌种,本实施例中将优势菌种Fm鉴定为强化桑树抗旱性的土著丛枝菌根真菌高效菌种。。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (1)
1.丛枝菌根真菌高效菌种快速筛选与鉴定方法,其特征在于,包括如下步骤:
S1、在夏季炎热干旱时,采集岩溶区不同类型植物根围土壤样品,并迅速冷冻;
S2、利用土壤基因组DNA提取试剂盒快速提取样品DNA,随后用丛枝菌根真菌两对引物AML1-AML2和NS31-AM1进行巢式PCR扩增,并用琼脂糖凝胶电泳和荧光定量系统检测扩增产物;
S3、通过第二代高通量测序手段对PCR扩增产物进行测序,获得土壤中不同科属丛枝菌根真菌;
S4、利用生物群落相关指数筛选获得土著丛枝菌根真菌优势菌种;
S5、通过显微镜分离出上述土著丛枝菌根真菌优势菌种的孢子,并进一步利用盆栽试验将孢子放入适量的河沙中并借助三叶草和玉米作为寄主扩繁出各优势菌种;
S6、将上述扩繁的优势菌种接种于灭菌土壤,并撒播或移栽所需植物,正常培养数月后,做干旱胁迫处理及胁迫后复水处理,在不同处理阶段注意观测植物生长状况;
S7、根据上述所观测的植物生长状况结果,将干旱胁迫与复水处理后,植物长势表现依旧较好的菌种鉴定为高效菌种。
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CN111808936A (zh) * | 2020-08-07 | 2020-10-23 | 中国农业科学院植物保护研究所 | 基于高通量测序技术检测玉米叶部病原真菌的方法及应用 |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2020103359A1 (zh) * | 2018-11-20 | 2020-05-28 | 上海派森诺生物科技股份有限公司 | 一种基于sanger测序快速鉴定人体真菌的方法 |
CN111793680A (zh) * | 2020-07-30 | 2020-10-20 | 华南农业大学 | 一种基于高通量测序的促生菌株筛选方法及其应用 |
CN111808936A (zh) * | 2020-08-07 | 2020-10-23 | 中国农业科学院植物保护研究所 | 基于高通量测序技术检测玉米叶部病原真菌的方法及应用 |
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