对92株茶树菇菌株的遗传多样性分析和农艺性状的评价

为了解茶树菇（Agrocybe aegerita）种质资源的遗传多样性和筛选优良的茶树菇新品种,采用菌株拮抗试验方法观察了92株茶树菇菌株间拮抗反应及其类型,ISSR-PCR（inter-simple sequence repeat-PCR）分子标记方法对92株茶树菇菌株的遗传多样性进行了综合分析。拮抗试验将92株茶树菇菌株分为27组;筛选出的20条ISSR引物共扩增出317条清晰条带,多态性条带平均比率为82.60%;在遗传相似系数为0.742时,ISSR分子标记分析可将92株茶树菇划分为6大类群,拮抗试验和ISSR分子标记分析的结果基本一致。通过对比农艺性状分析,初步筛选出滇农5、滇农14、茶5-800、白茶、闽农5及滇农8作为工厂化生产茶树菇菌种。结果表明茶树菇的遗传多样性丰富,结合栽培出菇试验可为茶树菇品种选育和杂交育种的亲本选择提供参考。     

利用地域野生资源创制地方适栽的优质香菇新品种

香菇是我国深受欢迎的食药用菌,产量居食用菌产业之首。为拓宽栽培香菇菌株的遗传背景,解决现有主栽香菇菌株种性退化的问题,本研究以香菇‘808’菌株与3个香菇野生资源、3个栽培菌株（辽抚4号、BY1和荷香）开展单-双杂交育种,开发适合当地栽培环境的主栽品种。研究结果显示：在270对单-双杂交组合中,通过锁状联合观察和与亲本的拮抗试验,鉴定出89个杂交子,进一步通过栽培试验筛选出10个性状相对优良的候选菌株;最终通过农艺性状对比试验,筛选出ZJXG 5和ZJXG 8两个优良杂交菌株,这两个菌株均以当地长白山野生资源S1和本地主栽菌株BY1为双核杂交亲本选育而来,表现出超亲显著、菇形圆整、颜色呈浅褐色的特性,前4潮生物学效率在86%-88%之间。本研究结果揭示,在香菇杂交育种中,当亲本菌株来源地域与杂交后代菌株筛选地域相近时,单双杂交亲和率、杂交菌株的出菇率和丰产性指标较高,这提示我们,利用当地野生香菇资源更有利于创制适合当地自然环境的地方品种。     

茯苓基本生物学特性研究

以11个不同来源的茯苓菌株为材料,研究了茯苓菌丝体、子实体和担孢子的形态特征及适宜的生长、发育条件。结果表明,茯苓菌丝体为少分枝、有隔膜、无锁状联合的多核菌丝,茯苓担孢子核相以双核为主,双核孢子,单核孢子和无核孢子分别占87.2%,4.7%和8.1%。配对试验结果表明,同一菌株及不同菌株原生质体分离株间的配对均能融洽生长,同一菌株担孢子间的配对均产生拮抗线,但其中有少数配对在交接区形成扇形区域,拮抗线随后消失,而不同菌株担孢子间的配对则全部形成稳定的栅栏型菌落,暗示茯苓担孢子中的两个细胞核是具遗传互补性,能形成独立个体的异双核,茯苓可能是一种次级同宗结合菌。     

有性杂交选育银耳优良菌株的初步研究

为了解决银耳栽培生产中存在菌株优良性状单一,生产效率较低等问题,采用银科1#和Tr21两个不同品种菌株为亲本菌株,通过单孢子分离选取50个有性孢子单核体、两两配对有性杂交试验,显微观察镜检鉴定,统计杂交成功率为36%、杂交菌株与亲本的拮抗试验选育出7株杂交株,栽培对比农艺性状评价选育出1株(GT3)比亲本菌株生长快,生物转化率较高的菌株。试验结果表明有性杂交育种可作为一种行之有效的筛选银耳优良菌株的方法。     

黑龙江黑木耳优良菌株筛选研究

在2012年春季初筛和秋季复筛基础上,对选出的4个野生黑木耳菌株和5个栽培品种,以及在多个木耳基地分离获得的7个菌株进行了进一步栽培比较试验。基于菌丝生长速度、长势、拮抗试验、抗菌试验、栽培农艺性状和产量等因素,最终从16个菌株中筛选出适合黑龙江栽培的优良菌株6个,其中野生菌株2个：A14和A15;栽培菌株4个：M23、M43、M44和M46。     

钴60诱变选育平菇新菌株的研究

【目的】利用~(60)Co-γ射线诱变平菇原生质体创制平菇新种质。【方法】通过3个剂量的~(60)Co-γ射线辐照处理平菇原生质体悬浮液,统计不同剂量的致死率,利用拮抗试验、SSR分子标记筛选突变菌株,通过出菇试验筛选优良的平菇新菌株。【结果】平菇原生质体在0.9 KGy的诱变处理下致死率为0%,在1.2KGy的诱变处理下菌株致死率63.33%,在1.5KGy的诱变处理下菌株致死率为76.67%。经拮抗试验和SSR分子标记筛选,获得了8株突变菌株,出菇试验结果表明7个突变菌株产量和生物学效率均有不同程度的降低,菌盖颜色也略有变化,其中突变菌株1.5-P4的产量较对照降低了41.92%;而突变菌株1.2-P1为高产菌株,其产量达437.95±12.22 g/袋,较对照菌株提高了9.76%,生物学效率较CK提高了10.38%。【结论】利用~(60)Co-γ射线诱变育种技术得到了1个性状较为优良的平菇新菌株,这为其他食药用菌诱变育种研究提供了参考。     

茯苓种质资源的RAPD分析

为探讨国内茯苓人工栽培的主要品种和贵州野生茯苓菌株亲缘关系,利用RAPD技术对供试菌株的基因组DNA进行了分析。从41个随机引物中筛选出17个有效引物,共检测到101个RAPD标记位点,其中39个位点(38.6%)具有多态性,通过PAUP软件进行数据处理,采用最大简约法(MP法)进行聚类分析,并建立系统树。结果表明:不同来源的茯苓菌株亲缘关系非常相近,仅在某些引物的扩增物上存在较小的差异。     

16种茯苓菌株液体发酵产胞内多糖与三萜的比较研究

筛选茯苓高产胞内多糖和胞内三萜的优良液体发酵出发菌株。采用PDA富集固体平板培养与液体发酵培养测定菌丝体生长速率;采用液体发酵策略分析16种茯苓菌株产胞内多糖与胞内三萜的潜能。实验结果表明菌株生长于固体培养基与种子培养基的生长速率之间没有关联性;降低一级种子培养基初始pH值到4.0时能有效缓解茯苓菌株培养物褐化现象;AS5.137胞内多糖含量最高,达377.60±0.10 mg/g,而DB菌株显示出最高的胞内多糖产量,达1.01±0.13 g/L;Y1菌株胞内三萜含量最高,达83.89±4.28 mg/g,而Jingzhou28菌株胞内三萜产量最高,达136.63±26.66 mg/L。就生产茯苓胞内多糖与胞内三萜而言,AS5.137与DB菌株适合作为液体发酵产胞内多糖的出发菌株;Y1,Jingzhou28,Z(z)与Xingpinzhong菌株均较适合作为液体发酵产胞内三萜的出发菌株。     

常压室温等离子体诱变选育花脸香蘑新菌株

花脸香蘑作为一种营养丰富、菇型美观的珍稀食用菌,具有良好的开发利用价值。利用常压室温等离子体（ARTP）技术对野生花脸香蘑菌株610双核菌丝片段进行了诱变处理。利用拮抗试验进行了诱变菌株初筛。通过连续传代培养,自然淘汰劣势菌株并获得诱变效应稳定的48个菌株。综合平板培养和基于ISSR和SSR联合遗传多样性分析的结果,筛选出7个诱变菌株与出发菌株共同进行出菇比较试验。出菇试验及子实体蛋白和氨基酸营养分析的结果显示：诱变菌株Ls2和Ls3为筛选出的花脸香蘑优质高产新菌株。诱变菌株较出发菌株生物学效率分别提高10.27%和14.75%,总氨基酸含量、必需氨基酸含量以及氨基酸评分等指标也均显著高于出发菌株,品质提升效果极显著。     

茯苓原生质体单核化及同核体杂交

茯苓Wolfiporia hoelen是一种食药兼用的大型真菌,在我国栽培历史悠久,但由于长期无性繁殖,导致菌种退化,栽培产量下降,严重影响产业发展。为解决茯苓育种的难题,我们前期建立了茯苓同核体鉴定方法,明确了其交配系统,建立了单孢同核体杂交育种体系,但是部分茯苓菌株子实体形成困难或子实体贴生,导致担孢子收集困难,因此原生质体单核化的研究具有重要意义。本研究以两株存在较大遗传差异且同核体类型不同的菌株776和775为材料,核荧光染色表明超过60%的原生质体具有细胞核,原生质体再生率分别为11.0%和7.6%,不易生长的再生菌株中既有同核也有异核菌株。原生质体单核化获得了两个菌株的同核体菌株,比例可达到10%以上,菌株776获得了两种交配型的同核体菌株(5:3),不存在偏分离(χ²=0.5),但菌株775原生质体单核化仅获得了一种交配型的同核体菌株,表现出严重的交配型偏分离。通过杂交菌株与两亲本对峙试验及基于rpb2的杂合位点验证,证实获得了原生质体同核体杂交菌株25株,原生质体同核体与单孢同核体杂交菌株50株,表明茯苓原生质体同核体杂交育种的可行性。     

茯苓原生质体制备与再生条件的研究

研究了酶、酶解时间、菌龄、稳渗剂等对茯苓原生质体制备与再生的影响。茯苓原生质体制备的最佳条件为:纤维素酶(1．5%)和蜗牛酶(1．5%)的等量混合酶解系统,酶解时间3h,7d菌龄菌丝,产量可达1．77×10~7个/mL。以甘露醇为稳渗剂,采用CYM再生培养基,酶解时间3h,7d菌龄菌丝,其原生质体再生率最高,为0．164%。这一结果为茯苓通过原生质体技术进行菌种改良提供了重要技术参数。     

Molecular phylogeny of Laetiporus and other brown rot polypore genera in North America

Phylogenetic relationships were investigated among North American species of Laetiporus, Leptoporus, Phaeolus, Pycnoporellus and Wolfiporia using ITS, nuclear large subunit and mitochondrial small subunit rDNA sequences. Members of these genera have poroid hymenophores, simple septate hyphae and cause brown rots in a variety of substrates. Analyses indicate that Laetiporus and Wolfiporia are not monophyletic. All North American Laetiporus species formed a well supported monophyletic group (the "core Laetiporus clade" or Laetiporus s.s.) with the exception of L. persicinus, which showed little affinity for any genus for which sequence data are available. Based on data from GenBank, the southern hemisphere species L. portentosus also fell well outside the core Laetiporus clade. Wolfiporia dilatohypha was found to represent a sister group to the core Laetiporus clade. Isolates of Phaeolus, Pycnoporellus and members of the core Laetiporus clade all fell within the Antrodia clade of polypores, while Leptoporus mollis and Laetiporus portentosus fell within the phlebioid clade of polypores. Wolfiporia cocos isolates also fell in the Antrodia clade, in contrast to previous studies that placed W. cocos in the core polyporoid clade. ITS analyses resolved eight clades within Laetiporus s.s., three of which might represent undescribed species. A combined analysis using the three DNA regions resolved five major clades within Laetiporus s.s.: a clade containing conifer-inhabiting species ("Conifericola clade"), a clade containing L. cincinnatus ("Cincinnatus clade"), a clade containing L. sulphureus s.s. isolates with yellow pores ("Sulphureus clade I"), a clade containing L. sulphureus s.s. isolates with white pores ("Sulphureus clade II") and a clade containing L. gilbertsonii and unidentified isolates from the Caribbean ("Gilbertsonii clade"). Although there is strong support for groups within the core Laetiporus clade, relationships among these groups remain poorly resolved.     

茯苓健脾作用活性部位的研究

通过建立饮食失节、过食肥甘所致的小鼠脾虚模型,以体重、饮食量、外部表现特征及脾指数为指标,从茯苓总三萜类、茯苓总多糖及茯苓水煎剂中确定具有健脾作用的有效活性部位。结果表明:茯苓总三萜类对脾虚小鼠有明显的恢复作用,茯苓水煎剂也有一定作用,而茯苓总多糖作用不明显。说明茯苓总三萜类是茯苓治疗过食肥甘所致小鼠脾虚证的活性部位。     

茯苓中三萜类和多糖类成分的研究进展

主要对中药茯苓中的三萜类和多糖类成分进行了综述。到目前为止已从茯苓的菌核和菌丝中分离到三萜类物质39个,其中羊毛甾-8-烯型三萜12个,羊毛甾-7,9(11)-二烯型三萜16个,3,4-开环-羊毛甾-7,9(11)-二烯型三萜7个,3,4-开环-羊毛甾-8-烯型三萜2个,三环二萜类1个,齐墩果烷型三萜1个;分离到多糖类物质23个。     

Evaluating the basidiomycetes Poria medula-panis and Wolfiporia cocos for xylanase production

Xylanase, oxidative enzymes and iron-binding compounds were detected in the filtrates of Wolfiporia cocos and Poria medula-panis grown in wheat bran liquid medium. Xylanase and iron-binding compounds were produced at high levels by the brown-rot fungus (BR) W. cocos and at low levels by the white-rot fungus (WR) P. medula-panis. Phenoloxidase was produced only by P. medula-panis. Polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) showed a wide variety of bands for extracellular proteins produced by W.cocos, with low molecular weight (<30 kDa) and minor bands with molecular weight above 45 kDa. Two bands with xylanase activity derived from W. cocos extracts were detected in the gels, whereas many different bands with xylanase activity were found in the extracts from P. medula-panis. P. medula-panis is a selective lignin degrader, whereas W. cocos preferentially removes cellulose from wood.     

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production

Aims:  The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability.
Methods and Results:  W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe³⁺-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe³⁺-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible.
Conclusions:  W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators.
Significance and Impact of the Study:  This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.     

发酵茯苓菌丝体和天然茯苓多糖的研究

本文对发酵茯苓菌丝体和天然茯苓中多糖的提取分离进行了研究,并分别测定了二者总多糖的提取率及总糖的平均含量。发酵茯苓菌丝体和天然茯苓中总多糖的提取率分别为24.47%和82.93%;发酵茯苓菌丝体和天然茯苓总糖的平均含量分别为45.17%和87.24%。