Acta Prataculturae Sinica ›› 2021, Vol. 30 ›› Issue (7): 179-189.DOI: 10.11686/cyxb2020267
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Chun-jie LI(), Ming-xiao LANG, Zhen-jiang CHEN, Zheng-feng WANG, Tai-xiang CHEN
Received:
2020-06-08
Revised:
2020-07-16
Online:
2021-07-20
Published:
2021-06-03
Contact:
Chun-jie LI
Chun-jie LI, Ming-xiao LANG, Zhen-jiang CHEN, Zheng-feng WANG, Tai-xiang CHEN. Advances in artificial inoculation technology for grass-endophytic fungi[J]. Acta Prataculturae Sinica, 2021, 30(7): 179-189.
物种 Species | 接种材料 Inoculation material type | 内生真菌 Endophytic fungi | 结果 Result | 原宿主 Original host | 参考文献 References | 备注 Remarks |
---|---|---|---|---|---|---|
黑麦草 Lolium perenne | 无菌苗 Sterile seedling | Acremonium loliae | 9% | 黑麦草L. perenne | [ | 最高侵染率Highest infection rate |
Gliocladium-like sp. | 80% | 黑麦草L. perenne | ||||
Acremonium coenophialum | 未侵染Non infection | 高羊茅Festuca arundinacea | ||||
Phialophora-like sp. | 3% | 高羊茅F. arundinacea | ||||
Epichloё typhina | 44% | 紫羊茅Festuca rubra | ||||
Neotyphodium typhinum | 0.7% | 鹅观草Roegneria kamoji、鹅观草属Roegneria sp. | [ | |||
Neotyphodium starrii | 1.6% | 小糠草Agrostis alba、鹅观草属Roegneria sp. | ||||
Neotyphodium coenophialum | 未侵染Non infection | 高羊茅F. arundinacea | ||||
Epichloё sp. | 2.5% | 鹅观草R. kamoji | ||||
愈伤组织 Callus | N. typhinum | 3.3% | 小糠草A. alba、鹅观草属Roegneria sp. | [ | ||
N. starrii | 2.5% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 愈伤组织褐化 Callus browning | 高羊茅F. arundinacea | ||||
Epichloё sp. | 20.0% | 鹅观草R. kamoji | ||||
高羊茅 F. arundinacea | 无菌苗 Sterile seedlings | A. loliae | 11% | 黑麦草L. perenne | [ | 最高侵染率Highest infection rate |
A. coenophialum | 10% | 高羊茅F. arundinacea | ||||
Gliocladium-like sp. | 9% | 黑麦草L. perenne | ||||
Phialophora-like sp. | 32% | 高羊茅F. arundinacea | ||||
E. typhina | 12% | 紫羊茅F. rubra | ||||
N. typhinum | 3.3% | 鹅观草R. kamoji、鹅观草属Roegneria sp. | [ | |||
N. starrii | 5.0% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 3.3% | 高羊茅F. arundinacea | ||||
Epichloё sp. | 再生植株死亡Death | 鹅观草R. kamoji | ||||
Neotyphodium sp. | 2.5% | 羊茅属Festuca spp. | [ | |||
种子Seeds | Neotyphodium sp. | 2.1% | 羊茅属Festuca spp. | [ | ||
愈伤组织 Callus | N. typhinum | 3.3% | 鹅观草R. kamoji、鹅观草属Roegneria sp. | [ | ||
N. starrii | 5.0% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 3.3% | 高羊茅F. arundinacea | ||||
Epichloё sp. | 再生植株死亡Death | 鹅观草R. kamoji | ||||
成株Mature plant | N. starrii | 未侵染Non infection | 羊茅Festuca ovina | [ | ||
紫羊茅 F. rubra | 无菌苗 Sterile seedling | A. coenophialum | 未侵染Non infection | 高羊茅F. arundinacea | [ | 最高侵染率Highest infection rate |
Phialophora-like sp. | 未侵染Non infection | 高羊茅F. arundinacea | ||||
E. typhina | 50% | 紫羊茅F. rubra | ||||
醉马草Achnatherum inebrians | 无菌苗 Sterile seedlings | Epichloё gansuensis | 21% | 醉马草A. inebrians | [ | |
大麦 Hordeum vulgare | 无菌苗 Sterile seedling | Epichloё bromicola | 2% | 野大麦Hordeum brevisubulatum | [ | |
针茅属 Stipa | 幼苗 Seedlings | Atkinsonella hypoxylon | 35% | 针茅属Stipa sp. | [ | |
A. hypoxylon | 未侵染Non infection | 扁芒草Danthonia spicata | ||||
香附子 Cyperus rotundus | 无菌苗 Sterile seedling | Balansia cyperi | 19% | 香附子C. rotundus | [ | |
A. hypoxylon | 未侵染Non infection | 扁芒草D. spicata | ||||
扁芒草 D. spicata | 无菌苗 Sterile seedling | A. hypoxylon | 100% | 扁芒草D. spicata | [ | |
小麦 Triticum aestivum | 无菌苗 Sterile seedling | Epichloё sp. | 植株矮化Dwarf | 狭颖披碱草Elymus mutabilis | [ | |
翦股颖 Agrostis sp. | 愈伤组织 Callus | N. typhinum | 3.3% | 鹅观草R. kamoji、鹅观草属Roegneria sp. | [ | |
N. starrii | 12.5% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 未侵染Non infection | 高羊茅F. arundinacea | ||||
Epichloё sp. | 33.3% | 鹅观草R. kamoji |
Table 1 Artificial inoculation results of different grass-endophytic fungi combinations
物种 Species | 接种材料 Inoculation material type | 内生真菌 Endophytic fungi | 结果 Result | 原宿主 Original host | 参考文献 References | 备注 Remarks |
---|---|---|---|---|---|---|
黑麦草 Lolium perenne | 无菌苗 Sterile seedling | Acremonium loliae | 9% | 黑麦草L. perenne | [ | 最高侵染率Highest infection rate |
Gliocladium-like sp. | 80% | 黑麦草L. perenne | ||||
Acremonium coenophialum | 未侵染Non infection | 高羊茅Festuca arundinacea | ||||
Phialophora-like sp. | 3% | 高羊茅F. arundinacea | ||||
Epichloё typhina | 44% | 紫羊茅Festuca rubra | ||||
Neotyphodium typhinum | 0.7% | 鹅观草Roegneria kamoji、鹅观草属Roegneria sp. | [ | |||
Neotyphodium starrii | 1.6% | 小糠草Agrostis alba、鹅观草属Roegneria sp. | ||||
Neotyphodium coenophialum | 未侵染Non infection | 高羊茅F. arundinacea | ||||
Epichloё sp. | 2.5% | 鹅观草R. kamoji | ||||
愈伤组织 Callus | N. typhinum | 3.3% | 小糠草A. alba、鹅观草属Roegneria sp. | [ | ||
N. starrii | 2.5% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 愈伤组织褐化 Callus browning | 高羊茅F. arundinacea | ||||
Epichloё sp. | 20.0% | 鹅观草R. kamoji | ||||
高羊茅 F. arundinacea | 无菌苗 Sterile seedlings | A. loliae | 11% | 黑麦草L. perenne | [ | 最高侵染率Highest infection rate |
A. coenophialum | 10% | 高羊茅F. arundinacea | ||||
Gliocladium-like sp. | 9% | 黑麦草L. perenne | ||||
Phialophora-like sp. | 32% | 高羊茅F. arundinacea | ||||
E. typhina | 12% | 紫羊茅F. rubra | ||||
N. typhinum | 3.3% | 鹅观草R. kamoji、鹅观草属Roegneria sp. | [ | |||
N. starrii | 5.0% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 3.3% | 高羊茅F. arundinacea | ||||
Epichloё sp. | 再生植株死亡Death | 鹅观草R. kamoji | ||||
Neotyphodium sp. | 2.5% | 羊茅属Festuca spp. | [ | |||
种子Seeds | Neotyphodium sp. | 2.1% | 羊茅属Festuca spp. | [ | ||
愈伤组织 Callus | N. typhinum | 3.3% | 鹅观草R. kamoji、鹅观草属Roegneria sp. | [ | ||
N. starrii | 5.0% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 3.3% | 高羊茅F. arundinacea | ||||
Epichloё sp. | 再生植株死亡Death | 鹅观草R. kamoji | ||||
成株Mature plant | N. starrii | 未侵染Non infection | 羊茅Festuca ovina | [ | ||
紫羊茅 F. rubra | 无菌苗 Sterile seedling | A. coenophialum | 未侵染Non infection | 高羊茅F. arundinacea | [ | 最高侵染率Highest infection rate |
Phialophora-like sp. | 未侵染Non infection | 高羊茅F. arundinacea | ||||
E. typhina | 50% | 紫羊茅F. rubra | ||||
醉马草Achnatherum inebrians | 无菌苗 Sterile seedlings | Epichloё gansuensis | 21% | 醉马草A. inebrians | [ | |
大麦 Hordeum vulgare | 无菌苗 Sterile seedling | Epichloё bromicola | 2% | 野大麦Hordeum brevisubulatum | [ | |
针茅属 Stipa | 幼苗 Seedlings | Atkinsonella hypoxylon | 35% | 针茅属Stipa sp. | [ | |
A. hypoxylon | 未侵染Non infection | 扁芒草Danthonia spicata | ||||
香附子 Cyperus rotundus | 无菌苗 Sterile seedling | Balansia cyperi | 19% | 香附子C. rotundus | [ | |
A. hypoxylon | 未侵染Non infection | 扁芒草D. spicata | ||||
扁芒草 D. spicata | 无菌苗 Sterile seedling | A. hypoxylon | 100% | 扁芒草D. spicata | [ | |
小麦 Triticum aestivum | 无菌苗 Sterile seedling | Epichloё sp. | 植株矮化Dwarf | 狭颖披碱草Elymus mutabilis | [ | |
翦股颖 Agrostis sp. | 愈伤组织 Callus | N. typhinum | 3.3% | 鹅观草R. kamoji、鹅观草属Roegneria sp. | [ | |
N. starrii | 12.5% | 小糠草A. alba、鹅观草属Roegneria sp. | ||||
N. coenophialum | 未侵染Non infection | 高羊茅F. arundinacea | ||||
Epichloё sp. | 33.3% | 鹅观草R. kamoji |
接种方法 Inoculation method | 接种过程 Inoculation process | 接种材料 Inoculation material | 接种成功率Infected rate (%) | 参考文献 References |
---|---|---|---|---|
无菌苗接种Sterile seedling inoculation | 1) 50%硫酸和次氯酸钠溶液消毒。Sterilized in the solution of 50% sulfuric acid and sodium hypochlorite. 2) 无菌水冲洗。Washed with sterile water. 3) 种子于20~23 °C的黑暗条件下孵育5~16 d。Seeds are incubated for 5-16 days in the darkness at 20-23 ℃. 4) a.菌丝体置于幼芽中胚轴和胚芽鞘的交界处。The mycelium is placed at the junction of the mesocotyl and coleoptile. b.菌丝体放入除去切口组织的中胚轴和胚芽鞘交界处3~5 mm切口。The mycelia placed in a 3-5 mm long section of cut at the junction of the mesocotyl and coleoptile with removed cut tissue. c.菌丝体放入中胚轴和胚芽鞘交界处3~5 mm的纵向切口。The mycelium is placed in a 3-5 mm longitudinal slit at the junction of the mesocotyl and coleoptile. | 多年生黑麦草L. perenne, 高羊茅F. arundinacea, 紫羊茅F. rubra | 10.80 | [ |
注射接种法 Injection inocu lation | 1)95%的乙醇和clorox(5.25%的次氯酸钠)消毒。Sterilized in the 95% ethanol and clorox (5.25% sodium hypochlorite). 2)转移回95%乙醇中30 s,无菌水冲洗。Transfer back to 95% ethanol for 30 seconds and rinsed with sterile water. 3)于12 h·d-1光照保存至种子发芽。Stored at 12 h·d-1until seed germinated. 4)将真菌菌丝体和分生孢子用针头挑起同无菌水注入幼苗分生组织部。Fungal mycelia and conidia picked up with the tip of needle, and introduced into the meristem of seedlings with a drop of sterile water. | 针茅Stipa capillata, 香附子C. rotundus | 0~100 | [ |
种子接种法 Seed inoculation method | 种子浸菌接种法Seed soaking inoculation: 1)9%次氯酸钠溶液消毒。Sterilized in 9% sodium hypochlorite solution. 2)剪破种子。Cut the seeds. 3)种子置于装有菌悬液的培养皿并封口。Seeds are placed in petri dishes with fungal suspension and sealed. | 高羊茅 F. arundinacea | 2.10 | [ |
种子切口接种法Seed incision inoculation: 1)酒精和次氯酸钠消毒。Sterilized in alcohol and sodium hypochlorite. 2)种子胚附近切一小口。Cut a small slit near the seed embryo. 3)取菌丝塞入伤口中。Inserted mycelia into the slit. | 高羊茅 F. arundinacea | 0 | [ | |
愈伤组织接菌法 Callus inoculation | 1)种子于50%硫酸溶液中脱皮30 min。Seeds are peeled in 50% sulfuric acid solution for 30 min. 2)酒精和次氯酸钠消毒1~2 min,无菌水冲洗。Sterilized with alcohol and sodium hypochlorite for 1-2 min, rinsed with sterile water. 3)取种子/胚为外植体,置于诱导培养基,于20~25 ℃暗培养。Seeds/embryos are picked as explants, placed in inducement culture medium, and cultured in the darkness at 20-25 ℃. 4)将愈伤组织转移到继代培养基暗培养2~3次。Transfer the callus to the subculture medium and 2-3 times in the darkness. 5)选择优质的愈伤组织,转移到分化培养基,于20~25 ℃ 16 h·d-1光照,获得分化苗。Select high-quality callus, transfer to differential medium, at 20-25 ℃ for 16 h·d-1, obtaining differentiation seedlings. 6)分化芽切小口,将菌悬液滴入伤口。Differentiated buds are cut into small slit, drop the fungal suspension into the slit. | 高羊茅 F. arundinacea | 17.00 | [ |
成株接种法 Adult plant inoculation | 1)70%酒精消毒植株基部。Sterilized the plant base with 70% alcohol. 2)无菌注射器尖在生长点划小口。Needle of the tip cut an incision at growth point. 3)镊子取少量菌丝塞入小口。Take a small amount of mycelium into the wound. 4)无菌封口膜包好伤口。Cover the wound with a sterile parafilm. | 高羊茅 F. arundinacea | 0 | [ |
Table 2 Different inoculation methods and inoculation rate
接种方法 Inoculation method | 接种过程 Inoculation process | 接种材料 Inoculation material | 接种成功率Infected rate (%) | 参考文献 References |
---|---|---|---|---|
无菌苗接种Sterile seedling inoculation | 1) 50%硫酸和次氯酸钠溶液消毒。Sterilized in the solution of 50% sulfuric acid and sodium hypochlorite. 2) 无菌水冲洗。Washed with sterile water. 3) 种子于20~23 °C的黑暗条件下孵育5~16 d。Seeds are incubated for 5-16 days in the darkness at 20-23 ℃. 4) a.菌丝体置于幼芽中胚轴和胚芽鞘的交界处。The mycelium is placed at the junction of the mesocotyl and coleoptile. b.菌丝体放入除去切口组织的中胚轴和胚芽鞘交界处3~5 mm切口。The mycelia placed in a 3-5 mm long section of cut at the junction of the mesocotyl and coleoptile with removed cut tissue. c.菌丝体放入中胚轴和胚芽鞘交界处3~5 mm的纵向切口。The mycelium is placed in a 3-5 mm longitudinal slit at the junction of the mesocotyl and coleoptile. | 多年生黑麦草L. perenne, 高羊茅F. arundinacea, 紫羊茅F. rubra | 10.80 | [ |
注射接种法 Injection inocu lation | 1)95%的乙醇和clorox(5.25%的次氯酸钠)消毒。Sterilized in the 95% ethanol and clorox (5.25% sodium hypochlorite). 2)转移回95%乙醇中30 s,无菌水冲洗。Transfer back to 95% ethanol for 30 seconds and rinsed with sterile water. 3)于12 h·d-1光照保存至种子发芽。Stored at 12 h·d-1until seed germinated. 4)将真菌菌丝体和分生孢子用针头挑起同无菌水注入幼苗分生组织部。Fungal mycelia and conidia picked up with the tip of needle, and introduced into the meristem of seedlings with a drop of sterile water. | 针茅Stipa capillata, 香附子C. rotundus | 0~100 | [ |
种子接种法 Seed inoculation method | 种子浸菌接种法Seed soaking inoculation: 1)9%次氯酸钠溶液消毒。Sterilized in 9% sodium hypochlorite solution. 2)剪破种子。Cut the seeds. 3)种子置于装有菌悬液的培养皿并封口。Seeds are placed in petri dishes with fungal suspension and sealed. | 高羊茅 F. arundinacea | 2.10 | [ |
种子切口接种法Seed incision inoculation: 1)酒精和次氯酸钠消毒。Sterilized in alcohol and sodium hypochlorite. 2)种子胚附近切一小口。Cut a small slit near the seed embryo. 3)取菌丝塞入伤口中。Inserted mycelia into the slit. | 高羊茅 F. arundinacea | 0 | [ | |
愈伤组织接菌法 Callus inoculation | 1)种子于50%硫酸溶液中脱皮30 min。Seeds are peeled in 50% sulfuric acid solution for 30 min. 2)酒精和次氯酸钠消毒1~2 min,无菌水冲洗。Sterilized with alcohol and sodium hypochlorite for 1-2 min, rinsed with sterile water. 3)取种子/胚为外植体,置于诱导培养基,于20~25 ℃暗培养。Seeds/embryos are picked as explants, placed in inducement culture medium, and cultured in the darkness at 20-25 ℃. 4)将愈伤组织转移到继代培养基暗培养2~3次。Transfer the callus to the subculture medium and 2-3 times in the darkness. 5)选择优质的愈伤组织,转移到分化培养基,于20~25 ℃ 16 h·d-1光照,获得分化苗。Select high-quality callus, transfer to differential medium, at 20-25 ℃ for 16 h·d-1, obtaining differentiation seedlings. 6)分化芽切小口,将菌悬液滴入伤口。Differentiated buds are cut into small slit, drop the fungal suspension into the slit. | 高羊茅 F. arundinacea | 17.00 | [ |
成株接种法 Adult plant inoculation | 1)70%酒精消毒植株基部。Sterilized the plant base with 70% alcohol. 2)无菌注射器尖在生长点划小口。Needle of the tip cut an incision at growth point. 3)镊子取少量菌丝塞入小口。Take a small amount of mycelium into the wound. 4)无菌封口膜包好伤口。Cover the wound with a sterile parafilm. | 高羊茅 F. arundinacea | 0 | [ |
植物Plant | 外植体Explant | 激素Hormone | 浓度Concentration (mg·L-1) | 参考文献References |
---|---|---|---|---|
多年生黑麦草L. perenne | 成熟胚Mature embryo | 2,4-D | 4 | |
种子Seed | 7 | [ | ||
高羊茅F. arundinacea | 离体成熟胚Isolated mature embryo | 2,4-D | 2.5 | [ |
种子Seed | 5.5 | [ | ||
西伯利亚翦股颖Agrostis stolonifera | 种子Seed | 2,4-D | 3 | [ |
胚根生长点Radicle growing point | 3 | |||
胚芽生长点Apical growing point | 3 | |||
冰草Agropyron cristatum | 茎尖Shoot tip | 2,4-D | 1.5 | [ |
幼胚Immature embryos | 2 | [ |
Table 3 Hormone concentration required for callus induction in different plant species and explants
植物Plant | 外植体Explant | 激素Hormone | 浓度Concentration (mg·L-1) | 参考文献References |
---|---|---|---|---|
多年生黑麦草L. perenne | 成熟胚Mature embryo | 2,4-D | 4 | |
种子Seed | 7 | [ | ||
高羊茅F. arundinacea | 离体成熟胚Isolated mature embryo | 2,4-D | 2.5 | [ |
种子Seed | 5.5 | [ | ||
西伯利亚翦股颖Agrostis stolonifera | 种子Seed | 2,4-D | 3 | [ |
胚根生长点Radicle growing point | 3 | |||
胚芽生长点Apical growing point | 3 | |||
冰草Agropyron cristatum | 茎尖Shoot tip | 2,4-D | 1.5 | [ |
幼胚Immature embryos | 2 | [ |
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