生物技术通报 ›› 2021, Vol. 37 ›› Issue (10): 26-33.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0588
李娥1(), 黄勇1, 孟园园1, 李璇2, 杜光辉2(), 刘飞虎1,2()
收稿日期:
2020-05-14
出版日期:
2021-10-26
发布日期:
2021-11-12
作者简介:
李娥,女,硕士研究生,研究方向:植物耐盐性及植物内生真菌;E-mail: 基金资助:
LI E1(), HUANG Yong1, MENG Yuan-yuan1, LI Xuan2, DU Guang-hui2(), LIU Fei-hu1,2()
Received:
2020-05-14
Published:
2021-10-26
Online:
2021-11-12
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。
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1 内生真菌灭菌效果检测图 A、B、C分别代表组织压片法检验、涂布检验、超净工作台检验
Fig. 1 Sterilization effect of endophytic fungi A, B and C respectively refers to tissue compression test,coating test and ultra clean bench test
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) |
表1 不同浓度盐胁迫下“巴麻火麻”内生真菌分离种类比较
Table 1 Comparison of species of endophytic fungi isolated from “Bama hemp” under different concentrations of salt stress
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 |
表2 不同浓度盐胁迫下“巴麻火麻”始终存在的内生真菌及其ITS序列分析
Table 2 Endophytic fungi and its sequence analysis of ‘Bama hemp’ under different concentrations of salt stress
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 | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
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 | ||
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 |
表3 不同浓度盐胁迫下“巴麻火麻”始终存在的内生真菌分离数目
Table 3 Number of endophytic fungi isolated from ‘Bama hemp’ under different concentrations of salt stress
共有菌名称 Name of common endophytic fungi | CK | 100 mmol/L | 200 mmol/L | 300 mmol/L | 总计 Total | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
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 | ||
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 |
图3 不同浓度盐处理下大麻内生真菌的多样性分析 小写字母a、b、c表示差异显著(P<0. 05),下同
Fig. 3 Diversity analysis of endophytic fungi in hemp under different concentrations of salt treatment Small letters a,b and c indicate significant difference(P < 0.05). The same below
盐浓度Salt concentr ation/(mmol·L-1) | 0 | 100 | 200 | 300 |
---|---|---|---|---|
0 | ||||
100 | 0.36±0.12a | |||
200 | 0.30±0.08a | 0.20±0.13a | ||
300 | 0.33±0.06a | 0.17±0.17a | 0.35±0.21a |
表4 不同盐浓度间大麻内生真菌相似性系数比较
Table 4 Comparison of similarity coefficient of hemp end-ophytic fungi among different salt concentrations
盐浓度Salt concentr ation/(mmol·L-1) | 0 | 100 | 200 | 300 |
---|---|---|---|---|
0 | ||||
100 | 0.36±0.12a | |||
200 | 0.30±0.08a | 0.20±0.13a | ||
300 | 0.33±0.06a | 0.17±0.17a | 0.35±0.21a |
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