草业学报 ›› 2021, Vol. 30 ›› Issue (7): 179-189.DOI: 10.11686/cyxb2020267
收稿日期:
2020-06-08
修回日期:
2020-07-16
出版日期:
2021-07-20
发布日期:
2021-06-03
通讯作者:
李春杰
作者简介:
李春杰(1968-),男,甘肃镇原人,教授,博士。E-mail: chunjie@lzu.edu.cn基金资助:
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
摘要:
内生真菌侵染禾草既可提高宿主植物在群落中的竞争力,又可引起家畜中毒, 因其特殊的双重特性而受到国内外学术界的广泛关注。由于禾草内生真菌具有宿主特异性使其资源的利用开发受到限制。随着分子生物学和基因组学的发展与应用,越来越多优良的内生真菌被发现,禾草组织培养再生体系也已逐步建立,对禾草内生真菌人工接种技术的传统方法有一定程度的改进。筛选具有优良特性的内生真菌菌株,通过人工接种的方法建立既对家畜无毒又能提高宿主品质及抗性的共生体是当前和今后研究的方向与热点。对现有的禾草内生真菌人工接种技术进行了综述,发现目前主要的接种方法有:无菌苗接种法、注射接种法、愈伤组织接种法、种子接种法和成株接种法。其中无菌苗接种法技术趋于成熟而应用广泛,但该方法与注射接种法均对植物有所伤害,增加了幼苗死亡率;愈伤组织接种法潜在成功率较高,但目前成熟的组织培养再生体系建立不完全而应用较少;种子切口接种法是新的接种方法的尝试,该方法操作简便,但目前并无成功接入的范例;种子浸菌接种法对植物组织造成的物理伤害小,幼苗存活率高,能够有效提高接种成功率。通过对现有人工接种技术的研究与探讨,以期借鉴开发其他微生物人工接种方法,同时探索新的接种成功率更高、相容性更好的接种手段,拓展内生真菌在禾草的共生范围,获得更多具有优良性状的共生体。
李春杰, 郎鸣晓, 陈振江, 王正凤, 陈泰祥. 禾草-内生真菌人工接种技术研究进展[J]. 草业学报, 2021, 30(7): 179-189.
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 |
表1 不同禾草-内生真菌组合人工接种结果
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 | [ |
表2 不同接种方法操作步骤及接种成功率
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 | [ |
表3 不同植物及外植体诱导愈伤组织所需激素浓度
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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