CN113373069A - 一株秸秆腐生真菌及其应用 - Google Patents

一株秸秆腐生真菌及其应用 Download PDF

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CN113373069A
CN113373069A CN202110894856.8A CN202110894856A CN113373069A CN 113373069 A CN113373069 A CN 113373069A CN 202110894856 A CN202110894856 A CN 202110894856A CN 113373069 A CN113373069 A CN 113373069A
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saprophytic fungus
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林晓龙
田磊
王新光
田春杰
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Northeast Institute of Geography and Agroecology of CAS
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Abstract

一株秸秆腐生真菌及其应用,本发明秸秆腐生真菌为具疣篮状菌(Talaromycesverruculosus)JG‑542,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号CGMCC No.23042。在冻融条件下具疣篮状菌(Talaromyces verruculosus)JG‑542对水稻秸秆木质素、纤维素、半纤维素均具有优异的降解效果。

Description

一株秸秆腐生真菌及其应用
技术领域
本发明涉及一株秸秆腐生微生物及其应用。
背景技术
秸秆还田是提高秸秆资源综合利用率、平衡农田生态系统和实现农业可持续发展的有效措施。由于水稻秸秆木质纤维素的结晶区域组成,使得还田水稻秸秆难以降解;而我国东北方高寒地区初冬、初春反复冻融等气候因素进一步限制了还田水稻秸秆的有效腐解。因此,在东北地区低温条件下施加外源菌剂加速分解木质纤维素对水稻秸秆资源的开发利用具有重要意义。
发明内容
本发明提供了一株适合高寒地区的秸秆腐生真菌,以及该真菌的应用。
本发明秸秆腐生真菌为具疣篮状菌(Talaromyces verruculosus)JG-542,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号CGMCC No.23042。
上述秸秆腐生真菌在水稻秸秆降解中的应用。
上述秸秆腐生真菌在产β-葡萄糖苷酶中的应用。
上述秸秆腐生真菌在产羧甲基纤维素酶中的应用。
上述秸秆腐生真菌在产漆酶中的应用。
上述秸秆腐生真菌在木质素过氧化物酶中的应用。
上述秸秆腐生真菌在锰过氧化物酶中的应用。
根据秸秆酶解过程以及微生物间的协同关系,直接从自然环境中筛选或组配秸秆高效降解复合菌群,用以提高还田秸秆的降解效率是目前秸秆高效降解的有效途径之一。为了获得水稻秸秆低温高效降解菌以用于北方低温种植区水稻秸秆原位促腐还田,本发明从反复冻融的低温生态环境中采集多年秸秆还田土壤作为水稻秸秆低温降解菌系筛选的菌源材料。
本发明具疣篮状菌(Talaromyces verruculosus)JG-542为具疣篮状菌,属于篮状菌属(Talaromyces);保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期是2021年7月14日,保藏编号CGMCC No.23042。
附图说明
图1是具疣篮状菌(Talaromyces verruculosus)JG-542在孟加拉红培养基上的菌落形态图;
图2是具疣篮状菌(Talaromyces verruculosus)JG-542在纤维素钠-刚果红培养基上降解圈的观察图;
图3是具疣篮状菌(Talaromyces verruculosus)JG-542实验室冻融模拟试验中产β-葡萄糖苷酶的柱状图;
图4是具疣篮状菌(Talaromyces verruculosus)JG-542实验室冻融模拟试验中产羧甲基纤维素酶的柱状图;
图5是具疣篮状菌(Talaromyces verruculosus)JG-542实验室冻融模拟试验中产漆酶的柱状图;
图6是具疣篮状菌(Talaromyces verruculosus)JG-542实验室冻融模拟试验中产木质素过氧化物酶的柱状图;
图7是具疣篮状菌(Talaromyces verruculosus)JG-542实验室冻融模拟试验中产锰过氧化物酶的柱状图;
图8是具疣篮状菌(Talaromyces verruculosus)JG-542系统发育树;
图9是实施例1试验重复组中的一组水稻秸秆和CK组水稻秸秆经过56天反复冻融后的降解对比观察图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。
具体实施方式一:本实施方式秸秆腐生真菌为具疣篮状菌(Talaromycesverruculosus)JG-542,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号CGMCC No.23042。
本发明2019年4月从吉林省松原市水稻土中分离获得具疣篮状菌(Talaromycesverruculosus)JG-542。
分离及纯化方法:取土壤样品1.0g,风干研磨分散均匀,以1∶10的质量比例加入无菌水中,过滤取滤液作为10-1初始液,再依次获得10-2和10-3土壤滤液梯度稀释液;分别在孟加拉红培养基上进行稀释涂布,28℃培养直至有单个菌落长出,挑取单菌落在培养基上纯化,至最终获得单一菌株。
筛选:采用纤维素钠-刚果红固体培养基(纤维素钠-刚果红固体培养基中硝酸钠1.0g/L;磷酸氢二钠1.2g/L;磷酸二氢钾0.9g/L;硫酸镁0.5g/L;氯化钾0.5g/L;酵母浸出粉0.5g/L;酸水解酪蛋白0.5g/L;刚果红0.2g/L;纤维素粉5.0g/L;琼脂15.0g/L;pH值7.0±0.2,121℃高压灭菌15分钟。),先将分离及纯化获得的单一菌株分别接种在孟加拉红培养基上进行活化。在无菌操作台上,用1cm打孔器取菌饼放置在纤维素钠-刚果红平板中央。每组3个重复,置于28℃恒温培养箱中培养,观察菌落长势,并在第5d记录菌的最小半径以及降解圈的半径。计算公式=降解圈的半径/菌落半径。其中,菌株JG-542对纤维素的降解率为3.24±0.02,最为优异。
菌株JG-542的ITS SrRNA序列及系统发育分析:获得菌株ITS S rRNA基因序列与GenBank数据库中的已知序列进行BLAST比对分析,与(MN429191.1)Talaromycesverruculosus isolate F8138相似度到达99.82%,与(MW958046.1)Talaromycesverruculosus isolate 18Crab相似度到达99.64%,采用MEGA 5.1构建系统进化树,菌株JG-542与Talaromyces verruculosus属于同一分支,其相似度达到100%。并结合形态特征学鉴定结果,命名为具疣篮状菌(Talaromyces verruculosus)JG-542。
本实施例共分离获得了21株真菌,其中具疣篮状菌(Talaromyces verruculosus)JG-542对纤维素降解效果最佳。
实验室冻融模拟试验:
将称取20g水稻秸秆固体培养基装于底面直径为70mm、容积为200mL的圆柱形组培瓶中,121℃高压灭菌40min;待组培瓶冷却至室温,将孢子悬浮液浓度为1×108CFU/mL的具疣篮状菌(Talaromyces verruculosus)JG-542按0.5%的接种量接种到上述组培瓶中摇匀,放置于-10℃~10℃冻融条件下培养8周(-10℃、10℃各12h,交替进行)中培养,每7天称取1g固体培养基进行酶学检测,具疣篮状菌(Talaromyces verruculosus)JG-542产β-葡萄糖苷酶的能力最高为123.28±0.16U/min(试验第6周),产羧甲基纤维素酶的能力最高为89.44±1.84U/min(试验第6周),产漆酶的能力最高为22.48±0.48nmol/min/mL(试验第7周),产木质素过氧化物酶的能力最高为19.10±2.89nmol/min/mL(试验第6周),产锰过氧化物酶的能力最高为12.36±0.50nmol/min/mL(试验第5周),如图3~7所示。
实验结果说明在冻融条件下具疣篮状菌(Talaromyces verruculosus)JG-542对水稻秸秆木质素、纤维素、半纤维素均具有优异的降解效果。
其中,水稻秸秆固体培养基由96.9%(w/v)的秸秆粉(水稻秸秆剪成3-5cm小段)、1%(w/v)的蔗糖、0.3%(w/v)的MgSO4·7H2O、1.5%(w/v)的(NH4)2SO4、0.3%(w/v)的KH2PO4组成,调节pH值至7.0±0.2,121℃灭菌40min。
实施例1
将本发明具疣篮状菌(Talaromyces verruculosus)JG-542接种于高氏一号液体培养基中28℃培养3d,然后12000rpm转速下离心10min获得菌体,加等体积PBS液体重悬并离心2次后,再将获得的JG-542菌体用PBS配制成1×108cfu/mL的菌液,分4组处理,其中3组作为试验重复组(加入具疣篮状菌JG-542菌液),另一组没有任何处理的CK组,试验重复组将具疣篮状菌(Talaromyces verruculosus)JG-542按0.5%的接种量(1×108CFU/mL的孢子悬浮液)接种到组培瓶中摇匀;将冻结温度设置为-10℃,融化温度为10℃,冻结阶段和融化阶段的持续时间都为12h,即24h为一个冻融循环,试验共计56天。结果显示:加入具疣篮状菌(Talaromyces verruculosus)JG-542的处理,可以显著降解水稻秸秆,而未加拮抗菌JG-542的CK组水稻秸秆没有腐烂(如图9所示),说明具疣篮状菌(Talaromycesverruculosus)JG-542对水稻秸秆具有降解作用。
实施例2
具疣篮状菌(Talaromyces verruculosus)JG-542的ITS S rRNA基因序列及系统发育分析
获得具疣篮状菌(Talaromyces verruculosus)JG-542的ITS S rRNA基因序列,DNA序列如SEQ ID NO:1所示,然后与GenBank数据库中的已知序列进行BLAST比对分析,采用MEGA 5.1构建系统进化树JG-542菌株与Talaromyces verruculosus属于同一分支,其相似度达到100%。通过形态特征和分子生物学鉴定结果,JG-542菌株为具疣篮状菌(Talaromyces verruculosus)(如图8所示)。
序列表
<110> 中国科学院东北地理与农业生态研究所
<120> 一株秸秆腐生真菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 556
<212> DNA
<213> 具疣篮状菌(Talaromyces verruculosus)
<400> 1
ggggttcggg gtgcgggtct cgcggcccac ctcccaccct tgtctctcta cacctgttgc 60
tttggcgggc ccactggggc tccctggtcg ccgggggaca cccgtccccg ggcccgcgcc 120
cgccgaagcg cttcgtgaac cctgatgaag aagggctgtc tgagtactat gaaaattgtc 180
aaaactttca acaatggatc tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga 240
taagtaatgt gaattgcaga attccgtgaa tcatcgaatc tttgaacgca cattgcgccc 300
cctggcattc cggggggcat gcctgtccga gcgtcatttc tgccctcaag cacggcttgt 360
gtgttgggtg tggtcccccc ggggacctgc ccgaaaggca gcggcgacgt ccgtctggtc 420
ctcgagcgta tggggctctg tcactcgctc gggaaggacc tgcgggggtt ggtcaccacc 480
acattttcca ttatggttga cctcggatca ggtaggagtt acccgctgaa cttaagcata 540
tcaataagcg gaggaa 556

Claims (7)

1.一株秸秆腐生真菌,其为具疣篮状菌(Talaromyces verruculosus)JG-542,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号CGMCC No.23042。
2.如权利要求1所述的秸秆腐生真菌在水稻秸秆降解中的应用。
3.如权利要求1所述的秸秆腐生真菌在产β-葡萄糖苷酶中的应用。
4.如权利要求1所述的秸秆腐生真菌在产羧甲基纤维素酶中的应用。
5.如权利要求1所述的秸秆腐生真菌在产漆酶中的应用。
6.如权利要求1所述的秸秆腐生真菌在木质素过氧化物酶中的应用。
7.如权利要求1所述的秸秆腐生真菌在锰过氧化物酶中的应用。
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