Screening and biological characteristics of cellulose degrading Trichoderma strains

doi:10.3969/j.issn.1000-1565.2021.06.010

Abstract

Abstract: We selected 7 strains of Trichoderma spp.from the rhizosphericsoil of medicinal plants in Anguo, Hebei Province, China. The cellulolytic Trichoderma strains were screened by sodium carboxymethyl cellulose medium and solid state fermentation(SSF)medium of corn straw and liquorice residues.Morphological and molecular biological methods were used to identify the strains. And its biological characteristics were investigated.The results showed that after the preliminary screening of sodium carboxymethyl cellulose medium, four Trichoderma strains had cellulose degradation ability, which were HQ1, BB1, DS3 and DS1. The results of SSF showed that BB1 and HQ1 display the highest FPase activity when using corn straw and liquorice residues as fermentation substrate,respectively.Combining colony morphology, microscopic structure and DNA molecular identification,HQ1 and BB1 were- DOI:10.3969/j.issn.1000-1565.2021.06.010纤维素降解木霉菌株的筛选及其生物学特性探究李婉云,杨静雅,赵爽,李敏,赵丽莉,贺学礼(河北大学生命科学学院,河北保定 071002)摘要:选取分离自河北安国中药材种植基地不同药用植物根区土壤的7株木霉菌,通过羧甲基纤维素钠培养基初筛及玉米秸秆、甘草药渣固态发酵培养基复筛,联合筛选纤维素降解木霉菌株,采用形态学和分子生物学相结合方法进行菌株鉴定,并对其生物学特性进行探究.结果表明,经羧甲基纤维素钠培养基初筛,有4株菌表现出纤维素降解能力,分别为HQ1、BB1、DS3和DS1.经2种木质纤维素底物固态发酵培养基复筛发现,BB1以玉米秸秆为发酵基质时滤纸纤维素酶(filter paper cellulase,FPase)活性最高,HQ1以甘草药渣为基质时FPase活性最高.结合菌落形态、显微结构和DNA分子鉴定,HQ1被鉴定为长枝木霉(Trichoderma longibrachiatum),BB1为非洲哈茨木霉(Trichoderma afroharzianum).2株菌生物学特性存在趋同性,适宜生长产孢pH为5~6,适宜培养温度为28~33 ℃,且都表现出抵御干旱胁迫的能力.变差分解表明,不同培养条件对木霉菌生长速率及产孢有重要影响.本实验可为后续进一步优化HQ1和BB1降解不同木质纤维素底物固态发酵培养条件提供依据.关键词:药用植物;纤维素降解菌;非洲哈茨木霉;长枝木霉;生物学特性中图分类号:Q939.9 文献标志码:A 文章编号:1000-1565(2021)06-0698-11Screening and biological characteristics of cellulose degrading Trichoderma strainsLI Wanyun, YANG Jingya, ZHAO Shuang, LI Min, ZHAO Lili, HE Xueli(College of Life Sciences, Hebei University, Baoding 071002, China)Abstract: We selected 7 strains of Trichoderma spp.from the rhizosphericsoil of medicinal plants in Anguo, Hebei Province, China. The cellulolytic Trichoderma strains were screened by sodium carboxymethyl cellulose medium and solid state fermentation(SSF)medium of corn straw and liquorice residues.Morphological and molecular biological methods were used to identify the strains. And its biological characteristics were investigated.The results showed that after the preliminary screening of sodium carboxymethyl cellulose medium, four Trichoderma strains had cellulose degradation ability, which were HQ1, BB1, DS3 and DS1. The results of SSF showed that BB1 and HQ1 display the highest FPase activity when using corn straw and liquorice residues as fermentation substrate,respectively.Combining colony morphology, microscopic structure and DNA molecular identification,HQ1 and BB1 were- 收稿日期:2020-09-17 基金项目:国家自然科学基金资助项目(31470533);河北省研究生创新资助项目(CXZZBS2020021) 第一作者:李婉云(1995—),女,河北石家庄人,河北大学在读博士研究生,主要从事药用植物与相关微生物互作等领域研究.E-mail:1964910366@qq.com 通信作者:贺学礼(1963—),男,陕西蒲城人,河北大学教授,博士生导师,主要从事菌根生物学、药用植物学和生态学等领域研究. E-mail: xlh3615@126.com第6期李婉云等:纤维素降解木霉菌株的筛选及其生物学特性探究identified as Trichoderma longibrachiatum and Trichoderma afroharzianum,respectively.The biological characteristics of the two Trichoderma strains were similar. The suitable culture pH was 5~6, and the suitable culture temperature was 28~33 ℃. Both Trichoderma strains showed the ability to resist mild drought stress.The variation partitioning indicated that different culture conditions had important influence on the growth rate and sporulation of Trichoderma strains.This experiment can provide a basis for further optimization of fermentation conditions for HQ1 and BB1 degraded different lignocellulosic substrates using SSF.

Key words: medicinal plants, cellulose degrading fungi, Trichoderma afroharzianum, Trichoderma longibrachiatum, biological characteristics

CLC Number:

Q939.9

LI Wanyun, YANG Jingya, ZHAO Shuang, LI Min, ZHAO Lili, HE Xueli. Screening and biological characteristics of cellulose degrading Trichoderma strains[J]. Journal of Hebei University(Natural Science Edition), 2021, 41(6): 698-708.

References

[1] SIQUEIRA J G W,RODRIGUES C,VANDENBERGHE L P D S,et al. Current advances in on-site cellulase production and application on lignocellulosic biomass conversion to biofuels:A review[J]. Biomass Bioenerg,2020,132:105419.DOI:10.1016/j.biombioe.2019.105419.
[2] ANWAR Z,GULFRAZ M,IRSHAD M. Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy:A brief review[J]. J Radiat Res Appl Sc,2014,7(2):163-173.DOI:10.1016/j.jrras.2014.02.003.
[3] 贺超,王文全,侯俊玲. 中药药渣生物有机肥的研究进展[J]. 中草药,2017,48(24):5286-5292.DOI: 10.7501/j.issn.0253-2670.2017.24.035.
[4] ZHAO X,WANG F,FANG Y,et al. High-potency white-rot fungal strains and duration of fermentation to optimize corn straw as ruminant feed[J]. Bioresource Technol,2020,312:123512. DOI:10.1016/j.biortech.2020.123512.
[5] 高亮,王延龙,翟奎林,等. 中药渣生物有机肥在人参上的应用技术[J]. 人参研究,2017,29(5):11-12.DOI:10.19403/j.cnki.1671-1521.2017.05.004.
[6] UDEIGWE T K,TEBOH J M,EZE P N,et al. Implications of leading crop production practices on environmental quality and human health[J]. J Environ Manage,2015,151:267-279.DOI:10.1016/j.jenvman.2014.11.024.
[7] MEDINA J,MONREAL C M,ORELLANA L,et al. Influence of saprophytic fungi and inorganic additives on enzyme activities and chemical properties of the biodegradation process of wheat straw for the production of organo-mineral amendments[J]. J Environ Manage,2020,255:109922.DOI:10.1016/j.jenvman.2019.109922.
[8] 季陆鹰,葛胜,郭静,等. 作物秸秆还田的存在问题及对策[J]. 江苏农业科学,2012,40(6):342-344. DOI:10.15889/j.issn.1002-1302.2012.06.078.
[9] 王士超,闫志浩,王瑾瑜,等. 秸秆还田配施氮肥对稻田土壤活性碳氮动态变化的影响[J].中国农业科学,2020,53(4):782-794.DOI:10.3864/j.issn.0578-1752.2020.04.010.
[10] HAN W,HE M. The application of exogenous cellulase to improve soil fertility and plant growth due to acceleration of straw decomposition[J]. Bioresource Technol,2010,101(10):3724-3731. DOI:10.1016/j.biortech.2009.12.104.
[11] 张野,王吉平,苏天明,等. 筛选微生物降解木质纤维素的研究进展[J].中国生物工程杂志,2020,40(6):100-105.DOI:10.13523/j.cb.2001053
[12] SHEN F,ZHONG B,WANG Y,et al. Cellulolytic microflora pretreatment increases the efficiency of anaerobic co-digestion of rice straw and pig manure[J].Bioenerg Res,2019,12(3):703-713.DOI:10.1007/s12155-019-10013-w.
[13] 王恒震,李化强,吴菲菲,等. 木霉菌降解果渣的研究进展[J]. 食品安全质量检测学报,2019,10(24):8289-8295.DOI:10.19812/j.cnki.jfsq11-5956/ts.2019.24.014.
[14] 袁扬,王胤晨,韩玉竹,等. 木霉菌在农业中的应用研究进展[J]. 江苏农业科学,2018,46(3):10-14. DOI:10.15889/j.issn.1002-1302.2018.03.003.
[15] BIZOS G,PAPATHEODOROU E M,CHATZISTATHIS T,et al. The role of microbial inoculants on plant protection, growth stimulation, and crop productivity of the olive tree(Olea europea L.)[J]. Plants,2020,9(6):743.DOI:10.3390/plants9060743.
[16] CORONADO-RUIZ C,AVENDANÑO R,ESCUDERO-LEYVA E,et al. Two new cellulolytic fungal species isolated from a 19^th-century art collection[J]. Sci Rep-UK,2018,8(1):7492.DOI:10.1038/s41598-018-24934-7.
[17] GAUTAM S P,BUNDELA P S,PANDEY A K,et al. Diversity of cellulolytic microbes and the biodegradation of municipal solid waste by a potential strain[J].Int J Microbiol,2012,2012:325907.DOI:10.1155/2012/325907.
[18] ABD EL ATY A A,SALEH S A A,EID B M,et al. The rmodynamics characterization and potential textile applications of Trichoderma longibrachiatum KT693225 xylanase[J]. Biocatal Agric Biotechnol,2018,14:129-137. DOI:10.1016/j.bcab.2018.02.011.
[19] BAGEWADI Z K,MULLA S I,NINNEKAR H Z. Optimization of endoglucanase production from Trichoderma harzianum strain HZN11 by central composite design under response surface methodology[J]. Biomass Convers Bior,2018,8(2): 305-316. DOI:10.1007/s13399-017-0285-3.
[20] LIBARDI N,SOCCOL C R,GÓES-NETO A,et al. Domestic wastewater as substrate for cellulase production by Trichoderma harzianum[J]. Process Biochem,2017,57:190-199.DOI:10.1016/j.procbio.2017.03.006.
[21] 黄亚丽,黄媛媛,马慧媛,等. 低温秸秆降解真菌的筛选及在秸秆还田中的应用[J]. 中国农学通报,2020,36(21):53-60.
[22] LEGODI L M,LA GRANGE D,JANSEN VAN RENSBURG E L,et al. Isolation of cellulose degrading fungi from decaying banana pseudostem and Strelitzia alba[J]. Enzyme Res,2019,2019:1-10.DOI:10.1155/2019/1390890.
[23] 张家麟,柯涛,王曦,等. 哈茨木霉产纤维素酶菌株筛选及培养基优化[J]. 生物技术,2019,29(6):566-572. DOI:10.16519/j.cnki.1004-311x.2019.06.0094.
[24] BARELLI L,WALLER A S,BEHIE S W,et al. Plant microbiome analysis after Metarhizium amendment reveals increases in abundance of plant growth-promoting organisms and maintenance of disease-suppressive soil[J]. PLoS One,2020,15(4):e0231150.DOI:10.1371/journal.pone.0231150.
[25] 肖艳红,李菁,刘祝祥,等. 药用植物根际微生物研究进展[J]. 中草药,2013,44(4):497-504.DOI: 10.7501/j.issn.0253-2670.2013.04.024.
[26] HE C,WANG W,HOU J. Characterization of dark septate endophytic fungi and improve the performance of liquorice under organic residue treatment[J]. Front Microbiol,2019,10:1364. DOI:10.3389/fmicb.2019.01364.
[27] 周德庆,徐德强. 微生物学实验教程[M]. 北京:高等教育出版社,2013.
[28] WONOPUTRI V,SUBIANTORO,KRESNOWATI M T A P,et al. Solid state fermentation parameters effect on cellulase production from empty fruit bunch[J]. Bull Chem React Eng,2018,13(3):553-559. DOI:10.9767/bcrec.13.3.1964.553-559.
[29] 赵爽,李婉云,杨静雅,等. 两种不同来源的绿色木霉降解木质纤维素研究[J]. 菌物学报,2020,39(5): 856-869.DOI:10.13346/j.mycosystema.190398.
[30] IRFAN M,NADEEM M,SYED Q. One-factor-at-a-time(OFAT)optimization of xylanase production from Trichoderma viride-IR05 in solid-state fermentation[J]. J Radiat Res Appl Sc,2014,7(3):317-326.DOI:10.1016/j.jrras.2014.04.004.
[31] 杨合同. 木霉分类与鉴定[M]. 北京:中国大地出版社,2009.
[32] BISSETT J. A revision of the genus Trichoderma. I. Section Longibrachiatum sect. nov[J]. Can J Bot,1984,62:924-931.
[33] 孙军,段玉玺,吕国忠. 辽宁木霉属(Trichoderma)真菌的形态分类研究[J]. 菌物研究,2006,4(2):38 -44. DOI:10.13341/j.jfr.2006.02.009.
[34] 张广志,张新建,陈泉,等. 哈茨木霉复合种内3个中国新记录种的分离和鉴定[J]. 山东科学,2015, 28(6):43-46.DOI:10.3976/j.issn.1002-4026.2015.06.007.
[35] CHAVERRI P,BRANCO-ROCHA F,JAKLITSCH W,et al. Systematics of the Trichoderma harzianum species complex and the re-identification of commercial biocontrol strains[J]. Mycologia,2015,107(3):558-590. DOI:10.3852/14-147.
[36] HARMAN G E,HOWELL C R,VITERBO A,et al. Trichoderma species opportunistic,avirulent plant symbionts[J]. Nat Rev Microbiol,2004,2(1):43-56.DOI:10.1038/nrmicro797.
[37] HEWEDY O A,ABDEL-LATEIF K S,BAKR R A. Genetic diversity and biocontrol efficacy of indigenous Trichoderma isolates against Fusarium wilt of pepper[J]. J Basic Microb,2020,60(2):126-135.DOI:10.1002/jobm.201900493.
[38] 李作森,何月秋,夏贤仁. 5个木霉菌株的抑菌谱及部分生物学特性[J]. 云南农业大学学报,2004(3):267-271.DOI:10.16211/j.issn.1004-390x(n).2004.03.007.
[39] LI J X,ZHANG F,JIANG D D,et al. Diversity of cellulase-producing filamentous fungi from tibet and transcriptomic analysis of a superior cellulase producer Trichoderma harzianum LZ117[J]. Front Microbiol,2020,11:1617. DOI:10.3389/fmicb.2020.01617.
[40] DELABONA P D S,FARINAS C S,SILVA M R,et al. Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production[J]. Bioresource Technol,2012,107:517-521. DOI:10.1016/j.biortech.2011.12.048.
[41] AN Q,WU X J,HAN M L,et al. Sequential solid-state and submerged cultivation of the white rot fungus pleurotusostreatus on biomass and the activity of lignocellulolytic enzymes[J]. Bioresources,2016,11(4):8791-8805.DOI:10.15376/biores.11.4.8791-8805.
[42] HAWROT-PAW M,IZWIKOW M. Cellulolytic activity of Trichoderma viride with regard to selected lignocellulosic waste materials[J]. J Ecol Eng,2016,17(1):119-122.DOI:10.12911/22998993/61199.
[43] CHANDRA M,KALRA A,SHARMA P K,et al. Cellulase production by six Trichoderma spp. fermented on medicinal plant processings[J]. J Ind Microbiol Biot,2009,36(4):605-609.DOI:10.1007/s10295-009-0544-9.
[44] TAHERZADEH-GHAHFAROKHI M,PANAHI R,MOKHTARANI B. Optimizing the combination of conventional carbonaceous additives of culture media to produce lignocellulose-degrading enzymes by Trichoderma reesei in solid state fermentation of agricultural residues[J].Renew Energ,2019,131:946-955.DOI:10.1016/j.renene.2018.07.130.
[45] 李琳,张雅梅,张祥辉,等. 生防棘孢木霉T31菌株的分离筛选及其生物学特性[J]. 植物保护学报,2014,41(1):54-60.DOI:10.13802/j.cnki.zwbhxb.2014.01.009.
[46] 郭成,张小杰,徐生军,等. 长枝木霉菌株GAAS L3-1-0.8对玉米形态学指标影响及其生物学特性[J]. 玉米科学,2018,26(3):153-159.DOI:10.13597/j.cnki.maize.science.20180325.
[47] 柯仿钢,黄思良,付岗,等. 西贡蕉枯萎病生防木霉菌株gz-2的鉴定及生物学特性研究[J]. 西南农业学报,2010,23(5):1533-1539.DOI:10.16213/j.cnki.scjas.2010.05.054.
[48] 尹大川,邓勋,Ilan Chet,等. 干旱和NaCl胁迫下哈茨木霉的响应[J]. 安徽农业科学,2013,41(30):11997-12000. DOI:10.13989/j.cnki.0517-6611.2013.30.048. (