松口蘑人工菌塘诱导形成的初步研究

本研究通过土壤筛选找到适合松口蘑菌丝体生长的土壤,并在其土壤中添加适当浓度的橄榄油(0.5%-2.0%),有效地促进了菌丝体在土壤中的生长,菌丝生长状况酷似野外的松口蘑菌塘,且随着添加浓度的增加,菌丝体生物量呈明显的正相关。当土壤中橄榄油浓度为0.5%和1.0%时,松口蘑共生植物赤松苗的生长不但没有受到抑制,而且还能与菌丝体在这种“菌塘”中形成菌根。本研究成功地建立了中国松口蘑人工菌塘诱导体系,同时也为其人工子实体的诱导研究提供了必要的前提条件。     

松口蘑的活性成分及其研究进展

松口蘑是世界上珍稀名贵的食用菌和药用菌,其活性成分(如松茸多糖、多肽、松茸醇、萜类和甾类等)已被证实在抗氧化、抗肿瘤、调节免疫、抗菌和皮肤护理等方面具有良好作用,在膳食营养补充剂、化妆品和药物开发等领域具有巨大潜力。然而,目前松口蘑在国内的消费方式仍以鲜食和初加工产品为主,存在精加工程度低等问题。本文主要综述了松口蘑活性成分及其研究进展,并讨论了其在商业化应用的建议,旨在为松口蘑的国内深加工利用提供理论参考,为带动乡村振兴发展提供科学依据。     

松口蘑与假松口蘑ITS序列测定和分析比较

对松口蘑和假松口蘑进行ITS序列测序,通过DNAStar软件比较分析,发现松口蘑与假松口蘑的5.8SrDNA序列完全一致,ITS1和ITS2呈现不同程度的多态性。松口蘑ITS序列长度为601bp,假松口蘑ITS序列长度为563bp。设计了扩增松口蘑和假松口蘑ITS1的特异性引物,能够快速地区别松口蘑与假松口蘑。     

松口蘑菌丝体的分离和RAPD-PCR分析

针对松口蘑 [Tricholomamatsutake(S .ItoetImai)Sing .]菌丝体分离培养困难和各种相关分离物目前难以用出菇试验鉴定的现实 ,采用 8种培养基配方 ,对 9个不同来源的松口蘑子实体的不同部位及菌根、菌土进行组织分离 ,计接种试管 81 0多支 ,结果从菌褶部位获得 94支慢生型的菌丝体分离菌株 ,从菌柄部位仅获得 1支快生型的菌丝体分离菌株。以马铃薯葡萄糖土壤滤液培养基 (PDAS)、马铃薯葡萄糖麦麸滤液培养基 (PDAW )、BM培养基、马铃薯葡萄糖琼脂培养基 (PDA)对菌褶进行组织分离 ,获慢生型菌丝体的成功率依次为 74.4%、35.5%、156%和 8.9%。以各分离菌株的来源松口蘑子实体和中日两国松口蘑研究者提供的分离菌株作为DNA参照样品 ,对从供试子实体、菌根、菌土进行组织分离获得的各种相关纯培养物进行亲菌鉴定。采用筛选的 1 7个随机引物介导 2 5个供试松口蘑子实体及其分离菌体的RAPD(RandomAmplifiedPolymorphicDNA) PCR反应 ,全部获得了清晰而稳定的DNA指纹图谱 ,结果一致表明 :每个松口蘑子实体的菌盖 (含菌褶…     

桦褐孔菌与松口蘑菌粉降血糖作用的研究

为评价桦褐孔菌和松口蘑发酵菌粉的降血糖作用,本文观察了经不同浓度桦褐孔菌和松口蘑菌粉溶液灌胃后,小鼠正常血糖、给予葡萄糖小鼠以及四氧嘧啶糖尿病小鼠血糖的变化.结果表明:桦褐孔菌、松口蘑菌粉溶液(500、1000 mg/kg)对小鼠正常血糖值、正常小鼠糖耐量没有明显影响(P>0.05);对于糖尿病小鼠血糖,桦褐孔菌低剂量组(500 mg/kg)和桦褐孔菌高剂量组(1000 mg/kg)的降糖效果较为明显(P<0.01),用药前与用药后第21 d的血糖值分别为(14.94±1.85)、(10.43±2.22)和(14.89±1.65)、(10.17±2.14) mmol/L,降糖率分别为30.23% 和31.7%.桦褐孔菌和松口蘑菌粉对于小鼠正常血糖、正常小鼠糖耐量无明显影响,对于四氧嘧啶糖尿病小鼠具有一定的降血糖效果.     

长白山松口蘑的驯化研究进展*

对长白山区松口蘑的分布、生态、纯培养菌种、驯化栽培、人工促繁的研究进展进行了专题论述。     

介绍两种野生食用菌

我国幅员辽阔,气候复杂,地形多变,植被种类丰富,为野生食用菌的生长提供了良好环境。现介绍两种: 一、松茸松茸又称松口蘑(Tricholoma matsutake),属伞菌目(Agaricales),口蘑科(Tricholomataceae),口蘑属(Tricholoma),它菌肉肥厚,香气浓郁,风味绝佳,一向被视为菇类中的珍品。     

大白口蘑菌丝的培养优化

从大白口蘑子实体中分离得到菌种,并进行不同营养条件对大白口蘑菌丝生长的影响实验。结果表明,最佳氮源为酵母粉;最佳碳源为果糖;能明显促进菌丝生长的矿质元素为磷酸二氢钾;最佳生长调节因子为吲哚乙酸;最适温度为28－32℃;最适pH4.0－5.0。     

松口蘑复合种形态学及生物地理学研究

松口蘑复合种含有松口蘑Tricholoma matsutake (也称松茸)、北欧口蘑T. nauseosum、喜栎口蘑T. zangii和美洲口蘑T. magnivelare等4个分类单元。在对来自中国、日本、法国、西班牙、瑞典、芬兰和美国的标本进行形态学研究的基础上,认为松口蘑、北欧口蘑、喜栎口蘑形态十分接近,它们的分类地位应该重新考虑。美洲口蘑和它们的差别也不大。对松口蘑复合种的寄主和地理分布也进行了总结,发现松口蘑和美洲口蘑的寄主范围较广,寄主专化性不强,而北欧口蘑和喜栎口蘑的寄主比较专一。在地理分布上,松口蘑和美洲口蘑形成亚洲—北美地理替代种的分布格局;松口蘑和北欧口蘑形成欧亚间断分布的分布格局;喜栎口蘑的地理分布范围完全包含在松口蘑的分布范围中。松口蘑复合种种类的分布区和它们各自寄主的分布不一定完全相同。在松口蘑复合种内,北欧口蘑和松口蘑已被认为是同一个种,而其余两个分类单元的分类地位值得进一步研究。     

酿酒酵母异源表达纤维二糖转运蛋白LacY与纤维二糖利用菌株的构建北大核心

【目的】在酿酒酵母体内设计代谢通路,使酿酒酵母能利用纤维素水解产物纤维二糖生产乙醇。【方法】首先,用大肠杆菌DH5α总DNA为模板克隆编码大肠杆菌乳糖透过酶的LacY基因。为过表达LacY基因,以质粒YEplac181作为载体,将酿酒酵母PGK1p强启动子加到LacY基因之前,CYC1t终止子加到LacY基因之后,构建质粒YEplac181-PGK1p-LacY-CYC1t。之后,将纤维二糖转运蛋白LacY表达质粒和β-葡萄糖苷酶(β-glucosidase,BGL)表达质粒pRS316-PGK1p-gh1-1-CYC1t依次转入野生型酿酒酵母W303-1A中,使野生型酿酒酵母W303-1A异源表达可转运纤维二糖的LacY蛋白和β-葡萄糖苷酶GH1-1,构建可利用纤维二糖的酿酒酵母工程菌W303-1A GL。最后,通过发酵测定酿酒酵母工程菌W303-1A GL的纤维二糖利用情况和乙醇产量,并对纤维二糖代谢通路中纤维二糖酶活力进行测定。【结果】本研究构建了纤维二糖转运蛋白LacY和β-葡萄糖苷酶GH1-1协同表达的酿酒酵母工程菌W303-1AGL。W303-1AGL可以有效利用纤维二糖发酵生产乙醇,W303-1A GL发酵24 h时乙醇产量达到3.25 g/L,得率为0.325 g乙醇/g纤维二糖,利用葡萄糖产乙醇理论得率为0.511 g乙醇/g纤维二糖,达到葡萄糖产乙醇理论得率的64%,细胞密度最高在第54 h达到OD600=10.84,胞内β-葡萄糖苷酶的酶活在72 h最高,可达到0.51 U/mg。【结论】本研究成功构建了能有效利用纤维二糖的重组酿酒酵母工程菌W303-1A GL,为提高纤维素乙醇生产效率、降低纤维素乙醇生产成本提供了新思路。     

The ectomycorrhizal fungus Tricholoma matsutake is a facultative saprotroph in vitro

Tricholoma matsutake is an economically important ectomycorrhizal fungus of coniferous woodlands. Mycologists suspect that this fungus is also capable of saprotrophic feeding. In order to evaluate this hypothesis, enzyme and chemical assays were performed in the field and laboratory. From a natural population of T. matsutake in southern Finland, samples of soil-mycelium aggregate (shiro) were taken from sites of sporocarp formation and nearby control (PCR-negative) spots. Soil organic carbon and activity rates of hemicellulolytic enzymes were measured. The productivity of T. matsutake was related to the amount of utilizable organic carbon in the shiro, where the activity of xylosidase was significantly higher than in the control sample. In the laboratory, sterile pieces of bark from the roots of Scots pine were inoculated with T. matsutake and the activity rates of two hemicellulolytic enzymes (xylosidase and glucuronidase) were assayed. Furthermore, a liquid culture system showed how T. matsutake can utilize hemicellulose as its sole carbon source. Results linked and quantified the general relationship between enzymes secreted by T. matsutake and the degradation of hemicellulose. Our findings suggest that T. matsutake lives mainly as an ectomycorrhizal symbiont but can also feed as a saprotroph. A flexible trophic ecology confers T. matsutake with a clear advantage in a heterogeneous environment and during sporocarp formation.     

四川省雅江松茸菌的分离与系统发育

对外生菌根真菌-松茸的纯培养条件进行了探讨,并从采集自四川省雅江县的松茸子实体中获得了10株松茸菌的纯培养物;分别以NS1和NS6 ,NS1和NS8,ITS4和ITS5为引物,对分离获得的松茸菌进行了18S r DNA PCR- RFL P和ITS PCR-RFL P分析,结果显示,用Alu I,H ae III,H inf I和Msp I四种限制性内切酶,这些松茸菌株的18S r DNA、ITS片段的酶切图谱完全相同;代表菌E7的ITS序列分析结果表明,本研究分离的松茸菌与Tricholoma matsutake的菌株在系统发育上高度同源,在分类上应属于同一个种     

Tricholoma matsutake dominates diverse microbial communities in different forest soils

Fungal and actinobacterial communities were analyzed together with soil chemistry and enzyme activities in order to profile the microbial diversity associated with the economically important mushroom Tricholoma matsutake. Samples of mycelium-soil aggregation (shiro) were collected from three experimental sites where sporocarps naturally formed. PCR was used to confirm the presence and absence of matsutake in soil samples. PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and direct sequencing were used to identify fungi and actinobacteria in the mineral and organic soil layers separately. Soil enzyme activities and hemicellulotic carbohydrates were analyzed in a productive experimental site. Soil chemistry was investigated in both organic and mineral soil layers at all three experimental sites. Matsutake dominated in the shiro but also coexisted with a high diversity of fungi and actinobacteria. Tomentollopsis sp. in the organic layer above the shiro and Piloderma sp. in the shiro correlated positively with the presence of T. matsutake in all experimental sites. A Thermomonosporaceae bacterium and Nocardia sp. correlated positively with the presence of T. matsutake, and Streptomyces sp. was a common cohabitant in the shiro, although these operational taxonomic units (OTUs) did not occur at all sites. Significantly higher enzyme activity levels were detected in shiro soil. These enzymes are involved in the mobilization of carbon from organic matter decomposition. Matsutake was not associated with a particular soil chemistry compared to that of nearby sites where the fungus does not occur. The presence of a significant hemicellulose pool and the enzymes to degrade it indicates the potential for obtaining carbon from the soil rather than tree roots.     

Artificial shiro formation of Tricholoma matsutake

One type of soil collected from Maoer-shan in Heilongjiang Province,China was selected to induce hyphal growth of Tricholoma matsutake by a soil screening experiment.It was confirmed that hyphal growth of all the tested T.matsutake isolates was significantly stimulated in soil by supplemented with 0.5%～2.0% olive oil.The aggregation of hyphae and soil resembled natural Shiro.The biomass of hyphae in the soil increases with increasing olive oil concentrations.Moreover,seedlings of Pinus densiflora grew well in the soil containing 0.5%～1.0% olive oil and were also successfully infected by T.matsutake isolate A in the soil containing 1.0% olive oil.This study established a culture system of artificial Shiro formation and also provided a premise for formulation of culture substratum for fruit body formation of T.matsutake.     

四川松茸内生细菌群落结构与多样性

为了解松茸的内生细菌群落结构与多样性,以采自四川7个松茸主产区的14个松茸样品为材料,通过变性梯度凝胶电泳(DGGE)分析其内生细菌的群落结构与多样性差异.结果表明： 各个产区松茸样品的内生细菌群落结构与多样性存在明显差异.但采自相近地点、相似环境的松茸样品内生细菌群落结构相似性较高;样品的丰度范围为15～25,多样性指数依次为盐源＞冕宁＞会东＞木里＞雅江＞盐边＞小金;条带回收测序构建的系统发育树显示,松茸内生细菌种群丰富,各个产区松茸优势种群存在一定的差异,但假单胞菌属、爱文菌属、芽孢杆菌属在所有样品中均有分布,而且都占据一定的优势地位.产碱杆菌属与鞘氨醇杆菌属在大部分样品中为优势种群;而杜擀氏菌属、赖氨酸芽孢杆菌属在特定样品中为优势种群.表明松茸内生细菌具有丰富的多样性,研究结果可为筛选促进松茸生长的内生菌提供依据.     

Saprobic potential of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>: growth over pine bark treated with surfactants

Saprotrophic growth of Tricholoma matsutake isolates was investigated over Pinus densiflora bark fragments either on soil or on agar media. Preferential colonization of pine bark fragments by hyphae, in glucose-deprived environments suggested that Matsutake was able to extract some nutrients to sustain its growth. This was confirmed in glucose-free liquid nutrient medium, where bark as sole carbon source significantly stimulated (up to two-fold) growth of T. matsutake isolates. The addition of surfactants (Tween 80 and Tween 40) in liquid medium further stimulated mycelium growth over pine bark by up to 55%. Such growth stimulation was associated with a sharp increase in protein and beta-glucosidase excretion by hyphae in culture filtrates. As T. matsutake has some saprotrophic ability, the initiation and extension of Matsutake Shiro in forest soil might require simultaneously nutrients derived from the host plant and from soil organic compounds. Data reported here may contribute to the formulation of new culture substrates adapted to the co-culture of T. matsutake and its host plant under controlled conditions.