Article(id=1256263562176094258, tenantId=1146029695717560320, journalId=1255847803461844995, issueId=1256263559323967535, articleNumber=null, orderNo=null, doi=10.13346/j.mycosystema.250148, pmid=null, cstr=32115.14.j.mycosystema.250148, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1747238400000, receivedDateStr=2025-05-15, revisedDate=null, revisedDateStr=null, acceptedDate=1751904000000, acceptedDateStr=2025-07-08, onlineDate=1777446173470, onlineDateStr=2026-04-29, pubDate=1771689600000, pubDateStr=2026-02-22, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1777446173470, onlineIssueDateStr=2026-04-29, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1777446173470, creator=13701087609, updateTime=1777446173470, updator=13701087609, issue=Issue{id=1256263559323967535, tenantId=1146029695717560320, journalId=1255847803461844995, year='2026', volume='45', issue='2', pageStart='250058', pageEnd='250280', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1777446172791, creator=13701087609, updateTime=1777447435276, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1256268854674710546, tenantId=1146029695717560320, journalId=1255847803461844995, issueId=1256263559323967535, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1256268854678904851, tenantId=1146029695717560320, journalId=1255847803461844995, issueId=1256263559323967535, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=250148, endPage=, ext={EN=ArticleExt(id=1256263564642345024, articleId=1256263562176094258, tenantId=1146029695717560320, journalId=1255847803461844995, language=EN, title=Promoting effects of Coprinellus micaceus on seed germination of Cremastra appendiculata, columnId=1256263562373226548, journalTitle=Mycosystema, columnName=Research paper, runingTitle=null, highlight=null, articleAbstract=

Coprinellus micaceus, the most common species of coprinoid fungi with extremely abundant resources, having potential in enhancing seed germination of medicinal orchid Cremastra appendiculata was investigated. Fruiting bodies of Co. micaceus were collected from the wild, and the hyphae were isolated from inner side of hollow stipe. Hyphal tip purification method was used to obtain multiple strains, and the two strains first obtained were named as S-1 and S-2 for subsequent experiments. S-1 was co-cultivated with Cr. appendiculata seeds on oat media to observe seed germination. Acid fuchsin staining was used to observe the morphology of protocorms formed on oat media and the hyphal colonization, and ITS sequencing of S-1 was performed. Spawn of S-2 was fragmented and mixed into potting soil, and covered with soil to induce fruiting bodies, while seeds of Cr. appendiculata were sown to simulate field inoculation. Endogenous strain were isolated from the protocorms, yielding S-3, and ITS sequencing of S-3 was performed. Additionally, dried and ground mycelial fragments of Co. micaceus were added to oat media to test whether the fungus could still promote seed germination under sterile conditions. The results showed that the fruiting body morphology and ITS sequences of both S-1 and S-3 matched those of Co. micaceus. Co. micaceus enhanced seed germination in both oat media and soil substrates. Acid fuchsin staining revealed that protocorms could digest invasive hyphae. Moreover, the mycelial powder of Co. micaceus in sterile oat media could also promote the germination of Cr. appendiculata seeds. This study demonstrates that Co. micaceus possesses potential to support the conservation and cultivation of Cr. appendiculata.

, correspAuthors=Xiaojun LI, authorNote=null, correspAuthorsNote=
*E-mail:
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ORCID: SHI Minmin (0009-0001-1197-5941)

, authorsList=Minmin SHI, Shanshan HE, Zhangneng PAN, Xiaoyu JIAN, Wenkai LIU, Xiaojun LI), CN=ArticleExt(id=1256263571642638467, articleId=1256263562176094258, tenantId=1146029695717560320, journalId=1255847803461844995, language=CN, title=晶粒小鬼伞对杜鹃兰种子萌发的促进作用, columnId=1256263563312771301, journalTitle=菌物学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

晶粒小鬼伞Coprinellus micaceus是最常见的鬼伞类真菌,菌种资源极其丰富。为探讨晶粒小鬼伞能否促进杜鹃兰种子萌发,我们从野外收集晶粒小鬼伞,分离菌柄空腔内侧菌丝,以尖端菌丝法纯化得到多个菌株,取最初发现的两株命名为S-1、S-2,进行后续实验,在燕麦培养基上将S-1与杜鹃兰种子共生,观察其萌发情况;酸性品红染色观察燕麦培养基上形成的原球茎形态及菌丝侵染情况,并对S-1的ITS测序;将S-2用菌包扩繁后掰碎放入花盆中,覆土诱导出子实体,并撒入杜鹃兰种子模拟拌菌直播育苗,分离原球茎内生菌,即S-3,并对S-3的ITS测序;将晶粒小鬼伞菌丝体干燥碾碎后加入燕麦培养基中,观察真菌菌丝粉末无菌条件下是否依然能促进杜鹃兰种子萌发。子实体的形态特征,S-1、S-3的ITS序列均与晶粒小鬼伞相符;晶粒小鬼伞在燕麦培养基和土中均能促进杜鹃兰种子萌发;酸性品红染色发现,杜鹃兰原球茎可消化入侵晶粒小鬼伞菌丝;燕麦培养基上,晶粒小鬼伞粉末无菌条件下亦可促进杜鹃兰萌发,说明晶粒小鬼伞可促进杜鹃兰萌发。晶粒小鬼伞资源丰富,可为杜鹃兰保护和种植提供支撑。

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ORCID: SHI Minmin (0009-0001-1197-5941)

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departmentName=null, remark=3 Lichuan Industrial Technology Research Institute, South-Central Minzu University, Lichuan 445400, Hubei, China), AuthorCompanyExt(id=1256263573903368357, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, companyId=1256263573861425313, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3 中南民族大学利川产业技术研究院,湖北 利川 445400)])], figs=[ArticleFig(id=1256263591406199108, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=EN, label=Fig. 1, caption=Habitat and morphology of Coprinellus micaceus.

A: Wild habitat; B: Cheilocystidia; C: Pileipellis; D: Veils composed of yellow-brown globose cells; E: Basidiospores; F: Basidiospores in groups of four on the sterigmata. Bars=10 μm.

, figureFileSmall=mGGMe/ph3V/voVan/uUb/A==, figureFileBig=a4mYRkiXUvV4waxolg1wYA==, tableContent=null), ArticleFig(id=1256263592203116874, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=CN, label=图1, caption=晶粒小鬼伞生境及形态显微特征

A:生长环境;B:褶侧囊状体;C:菌盖皮层;D:剥落的菌幕细胞;E:游离的担孢子;F:孢子梗上4个一组的担孢子;标尺=10 μm

, figureFileSmall=mGGMe/ph3V/voVan/uUb/A==, figureFileBig=a4mYRkiXUvV4waxolg1wYA==, tableContent=null), ArticleFig(id=1256263594262520146, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=EN, label=Fig. 2, caption=Colony of Coprinellus micaceus S-1 on PDA media.

A: Colony morphology; B: Microscopic morphology of hyphae; Yellow arrow indicates the clamp connection; Red arrow indicates an arthrospore. Bars: A=1 cm; B=5 μm.

, figureFileSmall=kw1czMNR4BHepb3mxk5J6A==, figureFileBig=AkDqqS02vZkcmsZXamOFeg==, tableContent=null), ArticleFig(id=1256263594707116371, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=CN, label=图2, caption=菌株S-1菌落形态及菌丝形态

A:S-1菌落形态;B:菌丝显微形态;黄色箭头所指为锁状联合;红色箭头所指为节孢子. 标尺:A=1 cm;B=5 μm

, figureFileSmall=kw1czMNR4BHepb3mxk5J6A==, figureFileBig=AkDqqS02vZkcmsZXamOFeg==, tableContent=null), ArticleFig(id=1256263595101380952, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=EN, label=Fig. 3, caption=Seed germination and growth of Cremastra appendiculata with or without Coprinellus micaceus S-1 inoculation.

A: Growing conditions of 60 days cultivation without S-1; B: Growing conditions of 60 days cultivation with S-1, apical meristem formed in the protocorm; C: Growing conditions of 180 days cultivation with S-1, a seedling with leaves. Bars: A, B=100 μm; C=500 μm.

, figureFileSmall=RqYB3R7Ydt5fFEAJXBPQ3g==, figureFileBig=vzOqDnsd1YJczNIIsmqkBg==, tableContent=null), ArticleFig(id=1256263595483062619, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=CN, label=图3, caption=杜鹃兰种子萌发生长比较

A:无菌培养60 d;B:与菌株S-1共生培养60 d时原球茎形成顶端分生组织;C:与菌株S-1共生培养180 d时带叶片的幼苗;标尺:A, B=100 μm;C=500 μm

, figureFileSmall=RqYB3R7Ydt5fFEAJXBPQ3g==, figureFileBig=vzOqDnsd1YJczNIIsmqkBg==, tableContent=null), ArticleFig(id=1256263595667611997, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=EN, label=Fig. 4, caption=Acid fuchsin staining micrograph of longitudinal section of Cremastra appendiculata protocorm.

The left image shows the staining of the longitudinal section of the protocorm formed after 60 days of symbiosis; The right image is an enlarged view of the surface tissue from the left image, showing loose hyphae within the cells and deeply stained hyphae in the center of the rhizoid. Bars=1 mm.

, figureFileSmall=KK5gPI6wCNTsxxMJ0jr7cA==, figureFileBig=SIEpKyT88e5Y1+r5GjoMPg==, tableContent=null), ArticleFig(id=1256263596049293666, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=CN, label=图4, caption=杜鹃兰原球茎纵切片酸性品红染色图

左图为共生60 d形成的原球茎纵切面染色照片,右图为左图表层组织局部放大,可见细胞内松散的菌丝,假根毛中央可见深染的菌丝. 标尺=1 mm

, figureFileSmall=KK5gPI6wCNTsxxMJ0jr7cA==, figureFileBig=SIEpKyT88e5Y1+r5GjoMPg==, tableContent=null), ArticleFig(id=1256263596204482918, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=EN, label=Fig. 5, caption=Coprinellus micaceus symbiotic with Cremastra appendiculata in a flowerpot.

A: Fruiting bodies of Coprinellus micaceus, with slender white Cremastra appendiculata seeds attached to brown mycelial bundles at the base; B: Germinated seeds developing into protocorms of different sizes. Bars=100 μm.

, figureFileSmall=pdD+n7qY7yoXwfsmRemfIA==, figureFileBig=YpvBEdtd8InCgZ5aveUybg==, tableContent=null), ArticleFig(id=1256263596317729129, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=CN, label=图5, caption=晶粒小鬼伞菌株在花盆内与杜鹃兰共生

A:菌包生出的子实体,基部褐色菌丝束上可见白色细长的杜鹃兰种子;B:种子萌发形成大小不等的原球茎. 标尺=100 μm

, figureFileSmall=pdD+n7qY7yoXwfsmRemfIA==, figureFileBig=YpvBEdtd8InCgZ5aveUybg==, tableContent=null), ArticleFig(id=1256263598070948205, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=EN, label=Fig. 6, caption=The promoting effect of the mycelial powder of Coprinellus micaceus on the seed germination of Cremastra appendiculata.

A: 0 g/L; B: 0.2 g/L; C: 1 g/L; D: 5 g/L. Bars=1 mm.

, figureFileSmall=l8axIxbUUcTa4qalMyT/YA==, figureFileBig=4FW2c5u38trRw9pKQosUag==, tableContent=null), ArticleFig(id=1256263598305829234, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=CN, label=图6, caption=晶粒小鬼伞菌丝粉末对杜鹃兰种子萌发的促进作用, figureFileSmall=l8axIxbUUcTa4qalMyT/YA==, figureFileBig=4FW2c5u38trRw9pKQosUag==, tableContent=null), ArticleFig(id=1256263598461018484, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=EN, label=Fig. 7, caption=NJ phylogenetic analysis constructed based on ITS sequences.

The values on the branches indicate the statistic support (based on 1 000 bootstrap replicates). S-1: Coprinellus micaceus strain isolated from the wild. S-3: Endophytic Co. micaceus strain isolated from the protocorm of Cremastra appendiculata in a flowerpot. Coprinus comatus is the outgroup.

, figureFileSmall=7USkSJZc13Gx6L50Pl86Cw==, figureFileBig=DNDoqK0cMpHsgOM0VLbjkA==, tableContent=null), ArticleFig(id=1256263598528127351, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263562176094258, language=CN, label=图7, caption=基于ITS序列构建的NJ系统发育分析

分支上数值表示支持率(基于1 000次自举重复);分支后为菌种拉丁名和NCBI登录号;S-1为野外采集的菌株、S-3为花盆内杜鹃兰原球茎分离的内生菌,毛头鬼伞Coprinus comatus为外类群

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晶粒小鬼伞对杜鹃兰种子萌发的促进作用
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石敏敏 1 , 何姗姗 1 , 潘长能 1 , 简晓宇 1 , 刘文凯 2 , 李小军 1, 3, *
菌物学报 | 研究论文 2026,45(2): 250148
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菌物学报 | 研究论文 2026, 45(2): 250148
晶粒小鬼伞对杜鹃兰种子萌发的促进作用
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石敏敏1, 何姗姗1, 潘长能1, 简晓宇1, 刘文凯2, 李小军1, 3, *
作者信息
  • 1 中南民族大学药学院,湖北 武汉 430074
  • 2 湖北鹤峰县林源林业发展有限公司,湖北 鹤峰 445800
  • 3 中南民族大学利川产业技术研究院,湖北 利川 445400
Promoting effects of Coprinellus micaceus on seed germination of Cremastra appendiculata
Minmin SHI1, Shanshan HE1, Zhangneng PAN1, Xiaoyu JIAN1, Wenkai LIU2, Xiaojun LI1, 3, *
Affiliations
  • 1 School of Pharmaceutical Sciences, South-Central Minzu University, Wuhan 430074, Hubei, China
  • 2 Hubei Linyuan Forestry Development Co., Ltd., Hefeng 445800, Hubei, China
  • 3 Lichuan Industrial Technology Research Institute, South-Central Minzu University, Lichuan 445400, Hubei, China
  • ORCID: SHI Minmin (0009-0001-1197-5941)

出版时间: 2026-02-22 doi: 10.13346/j.mycosystema.250148
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晶粒小鬼伞Coprinellus micaceus是最常见的鬼伞类真菌,菌种资源极其丰富。为探讨晶粒小鬼伞能否促进杜鹃兰种子萌发,我们从野外收集晶粒小鬼伞,分离菌柄空腔内侧菌丝,以尖端菌丝法纯化得到多个菌株,取最初发现的两株命名为S-1、S-2,进行后续实验,在燕麦培养基上将S-1与杜鹃兰种子共生,观察其萌发情况;酸性品红染色观察燕麦培养基上形成的原球茎形态及菌丝侵染情况,并对S-1的ITS测序;将S-2用菌包扩繁后掰碎放入花盆中,覆土诱导出子实体,并撒入杜鹃兰种子模拟拌菌直播育苗,分离原球茎内生菌,即S-3,并对S-3的ITS测序;将晶粒小鬼伞菌丝体干燥碾碎后加入燕麦培养基中,观察真菌菌丝粉末无菌条件下是否依然能促进杜鹃兰种子萌发。子实体的形态特征,S-1、S-3的ITS序列均与晶粒小鬼伞相符;晶粒小鬼伞在燕麦培养基和土中均能促进杜鹃兰种子萌发;酸性品红染色发现,杜鹃兰原球茎可消化入侵晶粒小鬼伞菌丝;燕麦培养基上,晶粒小鬼伞粉末无菌条件下亦可促进杜鹃兰萌发,说明晶粒小鬼伞可促进杜鹃兰萌发。晶粒小鬼伞资源丰富,可为杜鹃兰保护和种植提供支撑。

小脆柄菇科  /  小鬼伞属  /  山慈菇  /  共生真菌

Coprinellus micaceus, the most common species of coprinoid fungi with extremely abundant resources, having potential in enhancing seed germination of medicinal orchid Cremastra appendiculata was investigated. Fruiting bodies of Co. micaceus were collected from the wild, and the hyphae were isolated from inner side of hollow stipe. Hyphal tip purification method was used to obtain multiple strains, and the two strains first obtained were named as S-1 and S-2 for subsequent experiments. S-1 was co-cultivated with Cr. appendiculata seeds on oat media to observe seed germination. Acid fuchsin staining was used to observe the morphology of protocorms formed on oat media and the hyphal colonization, and ITS sequencing of S-1 was performed. Spawn of S-2 was fragmented and mixed into potting soil, and covered with soil to induce fruiting bodies, while seeds of Cr. appendiculata were sown to simulate field inoculation. Endogenous strain were isolated from the protocorms, yielding S-3, and ITS sequencing of S-3 was performed. Additionally, dried and ground mycelial fragments of Co. micaceus were added to oat media to test whether the fungus could still promote seed germination under sterile conditions. The results showed that the fruiting body morphology and ITS sequences of both S-1 and S-3 matched those of Co. micaceus. Co. micaceus enhanced seed germination in both oat media and soil substrates. Acid fuchsin staining revealed that protocorms could digest invasive hyphae. Moreover, the mycelial powder of Co. micaceus in sterile oat media could also promote the germination of Cr. appendiculata seeds. This study demonstrates that Co. micaceus possesses potential to support the conservation and cultivation of Cr. appendiculata.

Psathyrellaceae  /  Coprinellus  /  Cremastra appendiculata  /  symbiotic fungi
石敏敏, 何姗姗, 潘长能, 简晓宇, 刘文凯, 李小军. 晶粒小鬼伞对杜鹃兰种子萌发的促进作用. 菌物学报, 2026 , 45 (2) : 250148 - . DOI: 10.13346/j.mycosystema.250148
Minmin SHI, Shanshan HE, Zhangneng PAN, Xiaoyu JIAN, Wenkai LIU, Xiaojun LI. Promoting effects of Coprinellus micaceus on seed germination of Cremastra appendiculata[J]. Mycosystema, 2026 , 45 (2) : 250148 - . DOI: 10.13346/j.mycosystema.250148
杜鹃兰Cremastra appendiculata (D. Don) Makino为兰科Orchidaceae杜鹃兰属Cremastra多年生植物。其干燥假鳞茎为名贵中药材—山慈菇的基原之一(国家药典委员会 2020),具有清热解毒、活血止痛、抗肿瘤等功效(Liu et al. 2021)。杜鹃兰已被列入《国家重点保护野生植物名录》,紫金锭、周氏回生丸、慈丹胶囊等多种中成药均含有山慈菇,广州肺炎一号方等新药的开发,使近年来市场对杜鹃兰的需求量大增,过度采挖导致杜鹃兰野生资源进一步枯竭。与大多数兰科植物一样,杜鹃兰种子细小如尘,缺少胚乳(Shao et al. 2024),在自然条件下无法依靠自身储存的营养物质萌发(Dearnaley & Cameron 2017),必须依赖共生真菌提供的营养物质才能萌发并形成幼苗(Kauth et al. 2008;赵泽宇等 2021)。在需求刺激下,许多组培工厂培育杜鹃兰,但此法成本极高,种苗移栽成活率低,只是权宜之计,无法保障临床用药。因此,找到特异的真菌,开发适宜的种子拌菌直播技术,是平衡杜鹃兰资源保护和利用的可行举措。
已知能促进杜鹃兰萌发的真菌集中在小脆柄菇科小鬼伞属和堪多脆柄菇属,如辐毛小鬼伞Co. radians (王静等 2023)、家园鬼伞Co. domesticus (Yagame et al. 2013)、庭院小鬼伞Co. xanthothrix及辛格小脆柄菇Psathyrella singer (Pan et al. 2024)等。晶粒小鬼伞Co. micaceus (Bull.) Vilgalys, Hopple & Jacq. Johnson 2001是我国分布最广泛、最常见的鬼伞类真菌(冶晓燕和朱学泰 2020),极易辨认且有着非常丰富的菌种资源,其能否促进杜鹃兰种子萌发,尚未见报道。本研究旨在探讨晶粒小鬼伞对杜鹃兰种子萌发的促进作用,为杜鹃兰野生资源的保护和人工育苗提供技术支撑。
杜鹃兰种子由湖北省巴东一零八药材种植专业合作社提供,经中南民族大学药学院李小军副教授鉴定,确认其为杜鹃兰Cr. appendiculata。4-5月经人工授粉,当年10月份待其蒴果近成熟、未开裂前采收,室温下自然阴干,置于4-10 ℃保存备用。
马铃薯葡萄糖琼脂(potato dextrose agar, PDA)培养基:称取马铃薯200 g切碎,加水500 mL煮30 min后过滤,取其提取液加入琼脂粉15 g,补水至1 000 mL,高压蒸汽灭菌锅121 ℃灭菌25 min,备用。燕麦琼脂(oatmeal agar, OMA)培养基:称取燕麦3 g,加水500 mL煮 30 min后过滤,取其提取液加入琼脂粉15 g,加水至1 000 mL,灭菌备用。马铃薯液体培养基:称取马铃薯200 g切碎,加水500 mL煮 30 min后过滤,稀释后,加入葡萄糖20 g、KH2PO4 3.0 g、MgSO4·7H2O 1.5 g、维生素B1 10 mg,将pH调至6.0,补水至1 000 mL,灭菌备用。水琼脂培养基:称取15 g琼脂粉,加入锥形瓶中,补水至1 000 mL,灭菌备用。
菌包:棉籽壳800 g,麸皮160 g,加水适量至握之成团、松之即散的状态,装入菌袋,密封,高压蒸汽灭菌锅121 ℃灭菌3 h,备用。
ZSA0850连续变倍体视显微镜(重庆重光实业有限公司),EX33生物显微镜(宁波舜宇仪器有限公司),苏净安泰/SW-CJ-1FD-Ⅱ洁净工作台、DHP-9162恒温培养箱(太仓市科教器材厂),2720 thermal cycler PCR仪(Applied Biosystems)。
早春至初冬,晶粒小鬼伞常密集簇生于树木基部湿润土上;子实体偏小,菌柄呈污白色,中空易碎;孢子黑色;菌褶随子实体成熟逐渐变成墨汁状并萎缩,即出现自溶;菌盖表面小块菌幕残留(朱力扬等 2022)。2023年于武汉市内采集到多株具有上述特征的真菌子实体,并带回实验室纯化菌株。
1.2中真菌子实体,用消毒后的刀片切开菌柄,挑取少量组织,接种至PDA培养基上,于室温下培养3-5 d诱导菌丝生长。选择长势良好且无污染的菌种,利用菌丝尖端纯化法纯化3次(冯连荣等 2022)。采用插片培养法,在显微镜下观察菌丝形态。纯化后接种至PDA斜面试管,室温培养至2/3,4 ℃保存,最初分离的两株命名为S-1、S-2,均采自中南民族大学校园内。
从蒴果中取适量种子,以自来水冲洗表面杂质,75%乙醇浸泡30 s,蒸馏水冲洗3次除去乙醇;0.1%升汞溶液浸泡7 min,蒸馏水冲洗3次除去升汞;转移至超净工作台,以1%次氯酸钠溶液浸泡1 min,无菌水冲洗5次后,将种子悬浮于0.1%水琼脂中。
用无菌胶头滴管吸取约2 mL杜鹃兰种子悬浮液(含约100粒种子)滴在OMA培养皿上,接种S-1菌株,放入人工气候培养箱中,(23±2) ℃、60%-70%湿度,光照强度20%、12 h,黑暗12 h,定期观察记录菌株的生长和杜鹃兰种子的萌发情况。相同条件下无菌培养的杜鹃兰种子作为空白对照,每组3个平行皿。
突破种皮形成原球茎视为萌发,未突破种皮的杜鹃兰种子视为未萌发。根据公式计算萌发率(Yang et al. 2020)。
萌发率%=原球茎数/(原球茎数+未萌发杜鹃兰种子数)×100%。
1.4中培养皿内共生培养形成的杜鹃兰原球茎进行观察,首先在体视显微镜下观察S-1菌与杜鹃兰共生结构;然后取原球茎徒手纵切,酸性品红染色(冯加平等 2020),光学显微镜观察真菌定殖情况。
将晶粒小鬼伞S-2菌株接种至马铃薯液体培养基中,置于摇床,25 ℃、150 r/min培养7 d,得到菌液。将菌液接种至菌包中,阴暗通风环境下放置15 d,待菌丝基本长满后,将菌包掰碎置于花盆里,覆土,在其上撒上杜鹃兰种子,喷水保湿,诱导子实体产生并观察杜鹃兰种子是否能萌发。
挑取1.6中的杜鹃兰原球茎进行表面消毒,用无菌刀片切片后置于PDA培养基上,将培养出的菌株命名为S-3。
对S-1、S-3进行分子学鉴定,具体操作如下。DNA提取:按Ezup柱式真菌基因组DNA抽提试剂盒(B518259)操作。PCR扩增采用通用引物,ITS1:5′-TCCGTAGGTGAACCTGCGG-3′,ITS4:5′-TCCTCCGC TTATTGATATGC-3′。PCR反应体系:0.5 μL DNA、2.5 μL 10×Buffer (with Mg2+)、1 μL dNTPs、0.2 μL Taq Plus DNA Polymerase、上、下游引物各0.5 μL (至终浓度为0.2 μmol/L),加双蒸水至25 μL。程序设定为:94 ℃预变性30 s;94 ℃变性45 s,55 ℃退火45 s,72 ℃延伸1 min,30个循环;72 ℃再延伸10 min,4 ℃下终止反应。琼脂糖凝胶电泳:1%琼脂糖凝胶电泳,150 V、40 min电泳观察。将电泳产物进行切胶回收,并按SanPrep柱式DNA胶回收试剂盒(B518131)方法纯化DNA片段。将纯化产物送至生工生物工程(上海)股份有限公司进行测序。将测序结果提交至NCBI进行BLAST,从NCBI下载相关真菌ITS序列,依托MEGA软件,使用Neighbor-Joining法构建系统发育分析,重复抽样1 000次分析系统树各分支的置信度(杨琴等 2020)。
将晶粒小鬼伞菌种活化后接种至含200 mL马铃薯葡萄糖培养液的500 mL锥形瓶中,在25 °C、160 r/min条件下振荡10 d。纱布过滤发酵液获得菌丝体,干燥后研磨成粉末状。
分别称取菌丝粉末0、0.02、0.1、0.5 g,加入100 mL OMA培养基,配制成含晶粒小鬼伞 0、0.2、1、5 g/L的培养基,121 °C灭菌20 min后取出,摇匀,将冷却至40 ℃左右的培养基倒入无菌培养皿中,凝固后使用。
吸取2 mL消毒的杜鹃兰种子悬浮液,均匀分散于培养基表面,放入人工气候培养箱中,培养条件:(23±2) ℃、60%-70%湿度,光照强度20%、12 h,黑暗12 h。定期观察记录杜鹃兰种子的萌发情况。设置3个平行皿,相同条件下不添加菌丝粉末的培养皿作为空白对照。
收集的真菌子实体簇生在柳树根部周边地面上(图1)。菌盖直径2-4 cm,卵圆形或钟形,具沟状条纹,表面黄色,有白色颗粒状晶体;菌褶黑色、不等长,成熟后自溶为墨汁状,留下黑色印迹;菌柄长2-11 cm,宽0.3-0.5 cm,中空易碎,污白色。每个担子周围被2-4个担孢子,担孢子7-10 × 5-5.5 μm,水溶液中呈现黄褐色,卵圆形至椭圆形,光滑,有萌发孔;菌盖表面有发光的鳞片,菌盖菌幕中有球形或近球形细胞;盖皮细胞“Paraderm”型(朱力扬等 2022),近球形或椭圆形;褶侧囊状体无色,透明,短圆柱形;菌柄基部有菌丝束。
PDA培养基上,菌落呈圆形,灰白色,菌丝生长速度为(0.764±0.02) cm/d,气生菌丝蓬松且茂密(图 2A)。光学显微镜下观察菌丝较粗,隔膜明显;呈分枝状,分枝几近直角;菌丝细胞壁出现喙状突起,突起向下弯曲形成锁状联合,属于典型的担子菌菌丝结构;菌丝还会产生节孢子,呈短棒状(图2B)。
将纯化的S-1菌株与杜鹃兰种子在OMA培养基上进行共生培养,成熟杜鹃兰种子呈细长形,淡黄色,种胚较小、种子较长,约1 mm,位于中央的椭圆形种胚被致密的种皮包裹。共生培养60 d,空白组在光学显微镜下并未观察到种胚吸水膨胀(图 3A),处理组杜鹃兰种胚发育成肉眼可见的、顶端具分生组织细胞的梨形原球茎,其表面可见假根毛,萌发率为42.96% (图3B);共生培养6个月时,种子成功发育成带叶片的幼苗(图3C)。
以共生培养60 d的杜鹃兰原球茎为原材料,观察S-1菌与杜鹃兰的共生结构。体视镜下观察发现,菌丝附着并侵入杜鹃兰原球茎(图 3B)。徒手切原球茎,酸性品红染色观察纵切面,原球茎中央已形成细长的维管组织,菌丝侵染主要在原球茎中下部,顶端1/5无侵染迹象,表皮假根毛中亦有菌丝,大量菌丝在浅部皮层细胞中定殖,放大后可见大量菌丝疏松盘绕在细胞内,菌丝结构完整,可见横隔;随着菌丝的深入,菌丝团在深部皮层细胞中逐渐被消化,菌丝紧密缠绕逐渐形成更小的菌丝团块,难以看清其中的菌丝结构,直至消亡,维管柱周围无菌丝侵染(图4)。
晶粒小鬼伞菌株制作成的菌包在花盆内培养得到子实体(图 5A),子实体表面有白色颗粒状晶体、菌盖具有明显沟纹,成熟后变黑自溶,孢子黑色,与晶粒小鬼伞菌株特征一致。撒入的杜鹃兰种子3个月后萌发形成原球茎(图5B)。
将不同浓度的晶粒小鬼伞菌丝粉末添加至OMA培养基中,能够促进杜鹃兰种子萌发,且呈现剂量依赖性(图6)。无添加组杜鹃兰种子未能萌发,在0.2 g/L和1 g/L处理组萌发效果较差,萌发率仅为1.9%和4.35%;而5 g/L的处理组萌发率最高,为11.49%。
为了确定S-1、S-3菌株的物种身份,对其分别进行分子生物学鉴定。S-1、S-3基于ITS1/ITS4引物PCR扩增产物长度分别为679 bp和683 bp;序列已上传至GenBank,获得序列号分别为PV067750、PV761652。
将ITS序列提交至NCBI进行BLAST序列比对,S-1、S-3菌株序列与晶粒小鬼伞Co. micaceus (MK120529.1)序列同源性分别为97.78%、99.40%。从NCBI下载相关参考序列,运用NJ法构建系统发育分析,结果表明,S-1、S-3菌株与晶粒小鬼伞聚类于同一分支,支持上述鉴定结果(图7)。结合形态学特征,鉴定S-1、S-3菌株均为晶粒小鬼伞Co. micaceus
兰科植物与真菌存在较强专一性(唐燕静 2021),兰科共生真菌主要类群为胶膜菌属Tulasnella、角担菌属Ceratobasidium、蜡壳菌属Sebacina (Zhao et al. 2013;Rasmussen et al. 2015;Rafter et al. 2016;Chen et al. 2025)。杜鹃兰较为特殊,目前为止,发现的萌发菌主要在小鬼伞属,少数属于堪多小脆柄菇属(Pan et al. 2024),据此推测,晶粒小鬼伞可能也会促进其萌发,本研究通过OMA培养基和模拟野外环境共生培养证实了该推测。真菌促萌发的机制,主要涉及3个层面(Feldman et al. 2020):一是侵入,真菌分泌细胞壁降解酶消化宿主种皮,进入宿主种胚细胞;二是被消化,宿主细胞消化入侵的菌丝,从共生关系中获益;三是识别,真菌“致病因子”和宿主抗病性的相互识别并达到平衡共生关系。白假小鬼伞Co. disseminatus与杜鹃兰种子共培养时,前者纤维素酶基因上调,分解后者种皮木质纤维素,为菌丝入侵创造条件(吉军 2023)。本研究发现,晶粒小鬼伞菌丝能侵入杜鹃兰种子,提示晶粒小鬼伞也可能分泌了降解酶助其侵入。无菌培养时,晶粒小鬼伞菌丝粉末促进杜鹃兰种子萌发;共生培养时晶粒小鬼伞菌丝被杜鹃兰皮层细胞消化,说明晶粒小鬼伞可能为杜鹃兰萌发提供了营养。本实验室还进行了其他一些探索,晶粒小鬼伞的发酵液中无法检测到常见的植物激素。共生萌发杜鹃兰的根茎叶中没有发现晶粒小鬼伞菌丝侵入的证据,即使将杜鹃兰根切伤,菌丝依然无法侵入。说明晶粒小鬼伞菌丝可以入侵杜鹃兰种胚和原球茎的中下部,但是对于顶端分生组织及由其分化产生的根茎叶组织没有入侵能力,两者的共生关系是短暂而非终生式的,母株并不携带促进萌发的真菌,无法为种子萌发提供条件,这也许是杜鹃兰在野外散生而几乎没有成片生长的原因。
晶粒小鬼伞是野外极为常见的鬼伞类真菌,广泛分布于北温带地区,它在春季最早现身,直至冬季最晚一批隐退,菌种资源极其丰富。以武汉为例,2024年12月5日,8-15 ℃,在中南民族大学校园最后一次出菇,2025年3月16日,2-12 ℃,在沙湖公园发现出菇。杜鹃兰分布在海拔800 m以上的山区,其生长环境亦有晶粒小鬼伞分布。在杜鹃兰的种植过程中,可采取一种生态化种植方法,即在长有晶粒小鬼伞的树桩下或林地上撒播杜鹃兰种子,并轻轻覆土,这种仿野生的方法操作简便,有助于保障杜鹃兰供应,且有利于恢复杜鹃兰种群,保护生物多样性。
石敏敏:采集实验材料、实验及数据分析、论文撰写;何姗姗:采集实验材料、实验;潘长能:实验指导、论文修改;简晓宇:实验、数据处理;刘文凯:提供实验材料和思路;李小军:论文指导及审核、实验思路指导。
该研究不存在任何潜在利益冲突的商业或财务关系。
  • 湖北省中医药管理局中医药科研项目(ZY2025L082)
  • 中南民族大学大学生创新训练项目(XCX2024123)
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doi: 10.13346/j.mycosystema.250148
  • 接收时间:2025-05-15
  • 首发时间:2026-04-29
  • 出版时间:2026-02-22
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  • 收稿日期:2025-05-15
  • 录用日期:2025-07-08
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Scientific Research Project of Hubei Provincial Administration of Traditional Chinese Medicine(ZY2025L082)
湖北省中医药管理局中医药科研项目(ZY2025L082)
College Students' Innovative Training Program of South-Central Minzu University(XCX2024123)
中南民族大学大学生创新训练项目(XCX2024123)
作者信息
    1 中南民族大学药学院,湖北 武汉 430074
    2 湖北鹤峰县林源林业发展有限公司,湖北 鹤峰 445800
    3 中南民族大学利川产业技术研究院,湖北 利川 445400

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https://castjournals.cast.org.cn/joweb/jwxb/CN/10.13346/j.mycosystema.250148
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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