盐胁迫条件下‘巴麻火麻’内生真菌的分离鉴定与多样性分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0588

摘要/Abstract

摘要：

旨在研究‘巴麻火麻’不同浓度盐处理下植物内生真菌动态变化规律,并筛选到与缓解盐胁迫密切相关的内生真菌。以前期筛选的耐盐工业大麻品种‘巴麻火麻’为研究材料,通过不同浓度（0、100、200和300 mmol/L）NaCl盐处理,利用传统方法对大麻内生真菌进行分离纯化,结合形态学和rDNA-ITS序列分析对菌种进行鉴定。1 400个大麻组织片中共分离到543株真菌,隶属于14个科18个属44个种。研究发现,盐胁迫处理影响大麻内生真菌群落组成,不同浓度NaCl处理下分离到的内生真菌不同,各处理间相似性系数均小于0.5。随着NaCl处理浓度的增加,内生真菌的丰富度、多样性指数、分离率均呈先降后升的趋势。其中,在200 mmol/L NaCl处理下,丰富度和多样性指数最低,而100 mmol/L NaCl处理下,分离率达到最低。在所有的处理中,均分离得到与抗逆性密切相关的毛壳菌（Chaetomium globosum）、哈茨木霉（Trichoderma harzianum）、炭垫菌（Nemania diffusa）、枝孢霉（Cladosporium）、炭团菌（Annulohypoxylon stygium）等。

关键词: 工业大麻, 盐胁迫, 内生真菌

Abstract:

The aim is to study the dynamic changes of plant endophytic fungi under different concentrations of salt treatment in “ Bama hemp ”,and to screen endophytic fungi closely related to alleviating salt stress. The salt tolerant industrial cannabis variety ‘Bama hemp’ screened in the previous period was as study material. It was treated with different concentrations（0,100,200,and 300 mmol/L）of NaCl salt. The traditional method was used to separate and purify endophytic fungi. The strains were identified by rDNA-ITS sequence analysis combined with morphology. A total of 543 fungi were isolated from 1 400 cannabis tissue tablets,belonging to 44 families,18 genera and 44 species. It was found that salt stress treatment affected the composition of endophytic fungi in cannabis. The endophytic fungi isolated under different concentrations of NaCl were different,and the similarity coefficient was < 0.5. With the increase of NaCl concentration,the richness,diversity index and isolation rate of endophytic fungi decreased first and then increased. Among them,the richness and diversity index were the lowest under the treatment of 200 mmol/L NaCl,while the separation rate was the lowest under 100 mmol/L NaCl treatment. In all treatments,Chaetomium globosum,Trichoderma harzianum,Nemania diffusa,Cladosporium,Annulohypoxylon stygium,et al. were isolated,which were closely related to stress resistance.

Key words: industrial hemp, salt stress, endophytic fungi

李娥, 黄勇, 孟园园, 李璇, 杜光辉, 刘飞虎. 盐胁迫条件下‘巴麻火麻’内生真菌的分离鉴定与多样性分析[J]. 生物技术通报, 2021, 37(10): 26-33.

LI E, HUANG Yong, MENG Yuan-yuan, LI Xuan, DU Guang-hui, LIU Fei-hu. Isolation and Identification of the Endophytic Fungi of‘Bama hemp’ Under Salt Stress and Its Diversity Analysis[J]. Biotechnology Bulletin, 2021, 37(10): 26-33.

图/表 7

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盐浓度Salt concentration /（mmol·L^-1）	科Families	属Genera	种Species
0	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Pleosporaceae多孢菌科,Cladosporiaceae枝孢菌科,Trichosphaeriaceae毛球藻科,Hypocreales incertae sedis,Diaporthaceae,Nectriaceae从赤壳科,Glomerellaceae小从赤壳科,Hypocreaceae竹盖菌科（9）	Annulohypoxylon炭团菌属,Nigrospora 黑孢霉属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Nemania炭垫菌属,Alternaria链格孢属,Aphanocladium丝枝霉属,Colletotrichum炭疽病属,Diaporth茎点霉属,Fusarium镰孢菌属,Neocosmospora炭疽病属,Pleosporales格孢腔菌,Hypoxylon炭团菌属,Trichoderma木霉菌属（14）	Diaporthe novem,Nemania diffusa②,Alternaria sp. R60.1,Annulohypoxylon bovei,Cladosporium sp②.,Annulohypoxylon stygium,Aphanocladium album,Pleosporales sp. 9 MSP-2018,Colletotrichum kahawae,Neocosmospora rubicola,Hypoxylon fragiforme,Nigrospora oryzae,Trichoderma harzianum,Nigrospora sphaerica,Annulohypoxylon atroroseum,Fusarium oxysporum②,Chaetomium globosum③（22）
100	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Pleosporaceae多孢菌科,Cladosporiaceae枝孢菌科,Trichosphaeriaceae毛球藻科,Hypocreales incertae sedis,Nectriaceae从赤壳科,Hypocreaceae竹盖菌科,Xylariales incertae sedis（9）	Annulohypoxylon炭团菌属,Nigrospora 黑孢霉属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Nemania炭垫菌属,Alternaria链格孢属,Aphanocladium丝枝霉属,Fusarium镰孢菌属,Neocosmospora炭疽病属,Hypoxylon炭团菌属,Trichoderma木霉菌属,Hansfordia汉斯霉属,Kretzschmaria炭墩属（13）	Nigrospora sphaerica,Nemania diffusa,Nigrospora oryzae,Annulohypoxylon stygium,Aphanocladium album,Hansfordia sp.,Kretzschmaria pavimentosa,Alternaria sp.,Fusarium equiseti②,Neocosmospora rubicola,Trichoderma harzianum②,Fusarium solani,Chaetomium globosum②,Hypoxylon pilgerianum,Alternaria macrospora,Cladosporium sp.,Hypoxylon trugodes（20）
200	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Cladosporiaceae枝孢菌科,Nectriaceae从赤壳科,Hypocreaceae竹盖菌科,Xylariales incertae sedis（6）	Annulohypoxylon炭团菌属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Nemania炭垫菌属,Fusarium镰孢菌属,Trichoderma木霉菌属,Hansfordia汉斯霉属,Diaporth茎点霉属（8）	Nemania sp. AX40,Diaporthe novem,Nemania diffusa,Annulohypoxylon stygium,Fusarium sp.,Hansfordia sp. JP38-6,Chaetomium globosum,Trichoderma harzianum,Cladosporium sp.,Fusarium equiseti,Fusarium oxysporum（11）
300	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Pleosporaceae多孢菌科,Cladosporiaceae枝孢菌科,Trichosphaeriaceae毛球藻科,Diaporthaceae,Glomerellaceae小从赤壳科,Hypocreaceae竹盖菌科,Leptosphaeriaceae小球腔菌科,Ceratobasidiaceae（9）	Annulohypoxylon炭团菌属,Nigrospora 黑孢霉属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Alternaria链格孢属,Colletotrichum炭疽病属,Diaporth茎点霉属,Fusarium镰孢菌属,Pleosporales格孢腔菌,Hypoxylon炭团菌属,Trichoderma木霉菌属,Leptosphaeria小球腔菌属（13）	Diaporthe novem②,Nemania diffusa,Leptosphaeria microscopica,Annulohypoxylon stygium,Nigrospora oryzae,Fusarium sp.,Pleosporales sp. 9 MSP-2018,Alternaria sp,Annulohypoxylon atroroseum,Hypoxylon fragiforme,Colletotrichum sp.,Trichoderma harzianum,Chaetomium globosum②,Cladosporium sp.,Fusarium equiseti,Fusarium oxysporum②,Thanatephorus cucumeris（20）

盐浓度Salt concentration /（mmol·L^-1）	科Families	属Genera	种Species
0	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Pleosporaceae多孢菌科,Cladosporiaceae枝孢菌科,Trichosphaeriaceae毛球藻科,Hypocreales incertae sedis,Diaporthaceae,Nectriaceae从赤壳科,Glomerellaceae小从赤壳科,Hypocreaceae竹盖菌科（9）	Annulohypoxylon炭团菌属,Nigrospora 黑孢霉属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Nemania炭垫菌属,Alternaria链格孢属,Aphanocladium丝枝霉属,Colletotrichum炭疽病属,Diaporth茎点霉属,Fusarium镰孢菌属,Neocosmospora炭疽病属,Pleosporales格孢腔菌,Hypoxylon炭团菌属,Trichoderma木霉菌属（14）	Diaporthe novem,Nemania diffusa②,Alternaria sp. R60.1,Annulohypoxylon bovei,Cladosporium sp②.,Annulohypoxylon stygium,Aphanocladium album,Pleosporales sp. 9 MSP-2018,Colletotrichum kahawae,Neocosmospora rubicola,Hypoxylon fragiforme,Nigrospora oryzae,Trichoderma harzianum,Nigrospora sphaerica,Annulohypoxylon atroroseum,Fusarium oxysporum②,Chaetomium globosum③（22）
100	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Pleosporaceae多孢菌科,Cladosporiaceae枝孢菌科,Trichosphaeriaceae毛球藻科,Hypocreales incertae sedis,Nectriaceae从赤壳科,Hypocreaceae竹盖菌科,Xylariales incertae sedis（9）	Annulohypoxylon炭团菌属,Nigrospora 黑孢霉属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Nemania炭垫菌属,Alternaria链格孢属,Aphanocladium丝枝霉属,Fusarium镰孢菌属,Neocosmospora炭疽病属,Hypoxylon炭团菌属,Trichoderma木霉菌属,Hansfordia汉斯霉属,Kretzschmaria炭墩属（13）	Nigrospora sphaerica,Nemania diffusa,Nigrospora oryzae,Annulohypoxylon stygium,Aphanocladium album,Hansfordia sp.,Kretzschmaria pavimentosa,Alternaria sp.,Fusarium equiseti②,Neocosmospora rubicola,Trichoderma harzianum②,Fusarium solani,Chaetomium globosum②,Hypoxylon pilgerianum,Alternaria macrospora,Cladosporium sp.,Hypoxylon trugodes（20）
200	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Cladosporiaceae枝孢菌科,Nectriaceae从赤壳科,Hypocreaceae竹盖菌科,Xylariales incertae sedis（6）	Annulohypoxylon炭团菌属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Nemania炭垫菌属,Fusarium镰孢菌属,Trichoderma木霉菌属,Hansfordia汉斯霉属,Diaporth茎点霉属（8）	Nemania sp. AX40,Diaporthe novem,Nemania diffusa,Annulohypoxylon stygium,Fusarium sp.,Hansfordia sp. JP38-6,Chaetomium globosum,Trichoderma harzianum,Cladosporium sp.,Fusarium equiseti,Fusarium oxysporum（11）
300	Xylariaceae炭角科,Chaetomiaceae 毛壳科,Pleosporaceae多孢菌科,Cladosporiaceae枝孢菌科,Trichosphaeriaceae毛球藻科,Diaporthaceae,Glomerellaceae小从赤壳科,Hypocreaceae竹盖菌科,Leptosphaeriaceae小球腔菌科,Ceratobasidiaceae（9）	Annulohypoxylon炭团菌属,Nigrospora 黑孢霉属,Chaetomium毛壳菌属,Cladosporium 枝孢霉属,Alternaria链格孢属,Colletotrichum炭疽病属,Diaporth茎点霉属,Fusarium镰孢菌属,Pleosporales格孢腔菌,Hypoxylon炭团菌属,Trichoderma木霉菌属,Leptosphaeria小球腔菌属（13）	Diaporthe novem②,Nemania diffusa,Leptosphaeria microscopica,Annulohypoxylon stygium,Nigrospora oryzae,Fusarium sp.,Pleosporales sp. 9 MSP-2018,Alternaria sp,Annulohypoxylon atroroseum,Hypoxylon fragiforme,Colletotrichum sp.,Trichoderma harzianum,Chaetomium globosum②,Cladosporium sp.,Fusarium equiseti,Fusarium oxysporum②,Thanatephorus cucumeris（20）

菌株编号 Strain code	鉴定结果 Identification results	中文名 Chinese name	登录号 Login number	相似度 Similarity/%
B17,B51,B58	Nemania diffusa	炭垫菌	MK247796.1	100
B26,B19,B33,B52,B23,B60	Annulohypoxylon stygium	炭团菌	FJ848865.1	100
B44,B37,B56,B62,B16,B18	Chaetomium globosum	球毛壳菌	KF156299.1	100
B10,B70,B67,B65	Cladosporium sp.	枝孢霉	MK268136.1	99.80
B96,B101,B91,B86,B95,B90,B76,B93	Trichoderma harzianum	哈茨木霉	KT323178.2	100

菌株编号 Strain code	鉴定结果 Identification results	中文名 Chinese name	登录号 Login number	相似度 Similarity/%
B17,B51,B58	Nemania diffusa	炭垫菌	MK247796.1	100
B26,B19,B33,B52,B23,B60	Annulohypoxylon stygium	炭团菌	FJ848865.1	100
B44,B37,B56,B62,B16,B18	Chaetomium globosum	球毛壳菌	KF156299.1	100
B10,B70,B67,B65	Cladosporium sp.	枝孢霉	MK268136.1	99.80
B96,B101,B91,B86,B95,B90,B76,B93	Trichoderma harzianum	哈茨木霉	KT323178.2	100

共有菌名称 Name of common endophytic fungi	CK							100 mmol/L							200 mmol/L							300 mmol/L							总计 Total
共有菌名称 Name of common endophytic fungi	上叶	中叶	下叶	上茎	中茎	下茎	根	上叶	中叶	下叶	上茎	中茎	下茎	根	上叶	中叶	下叶	上茎	中茎	下茎	根	上叶	中叶	下叶	上茎	中茎	下茎	根	总计 Total
Nemania diffusa	—	1	—	—	—	—	—	—	1	2	—	—	—	—	—		1	—	—	—	—	—	—	1	—	—	—	—	6
Annulohypoxylon stygium	—	—	4	—	—	—	—	—	1	1	—	—	—	—	—	15	1	—	—	—	—	—	—	1	—	—	—	—	23
Chaetomium globosum	—	1	4	—	—	—	—	—	2	5	—	—	—	—	—	—	4	—	—	—	—	—	—	1	—	—	—	—	17
Cladosporium sp.	—	2	8	6	—	1	—	—	5	5	2	6	1	—	2	—	2	4	6	3	0	3	2	5	—	5	4	—	112
Trichoderma harzianum	—	—	—	—	—	—	50	—	—	—	—	—	8	18	—	—	—	—	—	2	30	—	—	—	—	—	7	46	161