块菌属分子系统学及菌根共生机制研究进展

块菌属Tuber是一类珍贵的地下生外生菌根食药用真菌,具有重要的经济和生态价值。分子生物学技术的快速发展尤其是高通量测序技术在块菌属研究领域的应用,极大地推动了块菌属分类、系统学、生态、生化、菌根共生机制及人工种植等研究。本文重点从块菌的分子系统学、群体遗传学及菌根共生机制3个方面对近5年的研究成果进行综述,并对其研究和应用前景进行了展望。     

块菌发育过程中伴生微生物多样性及其生态功能

块菌属Tuber是子囊菌门中一类珍贵的地下生外生菌根真菌,能与松科、壳斗科等多种树木形成菌根共生关系,在森林生态系统中扮演着重要的角色。同时,该属的一些种类因其独特的香味而备受国际食用菌市场推崇,具有极高的经济价值。在块菌属漫长的生活史中,菌丝的生长、菌根的形成、子囊果的产生和发育、特殊芳香化合物及活性物质的产生和积累均与其伴生微生物有着密切的关系。近年来,分子生物学技术的发展尤其是高通量测序技术的应用推动了对块菌属子囊果形成和发育机制的研究,为块菌生态学研究提供了有力的工具,使块菌伴生微生物相关研究取得重要突破。本文综述了近5年来国内外对块菌属生长发育过程中伴生细菌、真菌多样性及其生态功能的相关研究,并探讨了其中尚未清楚的问题及今后可能的研究方向,为块菌属生物学的研究及人工栽培奠定基础。     

夏块菌与青刚栎形成外生菌根形态变化的研究

夏块菌（Tuber aestivum）是一种具有较高经济价值的菌根食用菌。对夏块菌与青刚栎（Cycloba-lanopsis glauca）在形成菌根过程中不同阶段的菌根形态变化进行了研究,结果显示：用夏块菌孢子液接种青刚栎苗后,第14天起开始形成淡乳色的外生菌根,外延菌丝刚毛状;第一至第二个月可形成黄褐色、褐色外生菌根,外延菌丝刚毛或羊毛状。外生菌根为单根,长1～4mm,直径150～250μm。菌套厚12～20μm,平坦或自菌根延伸出刚毛状菌丝,外延菌丝束黄绿色;哈替氏网菌丝直径1～1.5μm。菌根老化后变暗褐或萎缩。外延菌丝束呈黄绿色是夏块菌菌根区别于其它块菌菌根最重要的形态特征。     

内蒙古地区白桦外生菌根形态类型及分子鉴定

以内蒙古不同地区白桦外生菌根为材料,采用形态解剖学方法和分子生物学手段对与白桦共生的外生菌根真菌多样性进行全面的调查,并经对所测序列与GenBank和Database比对。结果显示:在内蒙古地区与白桦共生的外生菌根真菌共13种,其中担子菌7种,子囊菌4种,分别来自于丝膜菌属、丝盖伞属、蜡壳耳属、毛革菌属、滑菇属和空团菌属、块菌属、地怀菌属。其中,菌根类型T₈和T₁₁未能提出其总DNA,根据其外生菌根形态类型并参照Agerer体视显微镜菌根图谱和Haug菌根图谱进行比较,分类鉴定为荷顿氏疣柄牛肝菌和白桦外生菌根真菌一种。结果表明,内蒙古地区白桦外生菌根真菌多样性相对较高,且与利用地上子实体鉴定的外生菌根真菌种类有一定的区别。     

块菌属研究概况和重要进展

块菌属(Tuber F. H. Wigg.)是一类具有重要经济和生态价值的子囊菌门地下生的外生菌根真菌。因能与多种树木共生而在森林生态系统中占据重要地位, 子囊果具独特的香味使其在欧洲市场久负盛名, 一直以来, 该属颇受国内外学者的广泛关注。本文综述了该属在系统分类学、生态学、生物化学及人工栽培等方面的研究概况和进展, 分析了目前研究中存在的问题及尚待解决的主要问题, 并提出了针对性的建议。     

植物菌根块菌分子鉴定及菌丝培养基优化的研究

块菌属(Tuber)是由真菌感染其适宜植物根系形成,部分块菌如法国黑孢块菌(T. melanosporum)和中国印度块菌(T. indicum)等为珍稀药食两用菌,具有很高的经济价值。对块菌的过度挖掘造成野生块菌资源匮乏,块菌人工栽培技术则是实现块菌产业可持续性发展的重要基础。目前国内外人工栽培主要采用块菌子实体匀浆制成的子囊孢子悬浮液,与人工种植苗木根系共生。利用块菌菌丝替代子实体孢子悬浮液进行接种,具有减少对块菌子实体的依赖性和生产成本、保护林下块菌野生资源、促进块菌人工繁育进程的积极意义,但需要成熟高效的菌丝培养技术。该研究从云南、陕西等地林下采取块菌样品,在ITS分子鉴定并纯化得到纯种菌丝的基础上,以菌丝直径为指标,经单因素实验和4因素3水平正交实验L9(34),筛选得出最适宜的菌丝生长培养基。结果表明:(1)形态和ITS分子鉴定得到的13份块菌样品分属印度块菌(T. indicum)和假凹陷块菌(T. pseudoexcavatum)两种。(2)纯化的印度块菌菌丝培养基的最佳碳源为马铃薯浸出液,最佳氮源为酵母膏,VB1对印度块菌菌丝生长并无显著性促进作用。(3)最佳的碳源、氮源、无机盐组合为马铃薯浸出液150 g·L~(-1)、酵母膏3 g·L~(-1)、MgSO_42 g·L~(-1)、KH2PO42 g·L~(-1)。25℃,自然pH下培养8 d菌丝直径可达49.44 mm。     

外生菌根真菌与内生细菌共生互作的研究进展

外生菌根真菌能与很多高等植物共生,广泛存在于自然界,在促进植物生长和养分吸收、增强宿主抗逆性及维持森林生态系统稳定等方面发挥着重要作用。除与寄主植物密切联系外,外生菌根真菌,在其生命周期中与细菌群落进行物理和代谢相互作用常形成共生关系。这些细菌对外生菌根真菌菌丝生长、生物量增加及子实体的形成具有积极影响。本文阐述了外生菌根真菌与内生细菌共生现象的发现、共生关系的建立、内生细菌促进外生菌根真菌生长和发育及宿主与微生物组的研究方法等,以期更好地巩固外生菌根真菌的生物学及生态学等基础性知识,并利用细菌与真菌的相互作用为可食用外生菌根真菌的生物防治、菌肥研究、人工驯化及栽培提供思路。     

外生菌根菌的分子生态学：分子标记基因、遗传个体及生态意义

外生菌根真菌与很多植物形成互利共生关系,在营养物质交换和碳循环等方面起着关键性的作用,是森林生态系统的重要组成部分。近期生物技术的发展使得人们对外生菌根菌的群体遗传学和分子生态学有了更加深入的认识。本文介绍了一些常用的鉴定外生菌根菌的分子标记,并对每种分子标记的特点及其适用范围进行了讨论。文中总结了几种常用的鉴定未知外生菌根菌的方法,指出了一些在研究外生菌根菌过程中需要克服的内在困难,其中之一就是很多外生菌根菌不可以人工培养,所以人们缺少对其地下部分分布规律和动态变化的了解。在寄主专一性、物种多样性和丰富度、遗传个体大小、繁殖方式等方面,近期对外生菌根菌的分子生物学研究已经获得了很多重要的结果。作者讨论了这些研究成果对于今后开展外生菌根菌研究的重要意义以及在森林生态系统保育方面的潜在应用价值。     

攀枝花块菌-华山松菌根根际土壤可培养细菌的多样性研究（英文）

块菌作为可食用的地下外生菌根真菌,有着重要的经济价值和生态学意义。中国白块菌资源虽然被不断的描述和报道,但形成机制尚未为人所知。前人研究表明,块菌的菌根根际土壤微生物群落对块菌的形成有着重要的影响。因此,本研究以攀枝花块菌(Tuber panzhihuanense)-华山松(Pinus armandii)菌根根际土壤为研究对象,用可培养的方法揭示了其根际土壤的细菌多样性。结果显示,在所分离到的细菌中,β-Proteobacteria占了最大的比例(30.98%),以Burkholderia为优势类群,其次是以Pseudomonas为代表类群的γ-Proteobacteria(28.8%),另外,α-Proteobacteria(14.67%)的主要代表类群为Phyllobacterium和根瘤菌Rhizobium;此外,还分离到了分别以Arthrobacter和Bacillus为优势菌群代表的Actinobacteria(12.5%)和Firmicutes(7.6%);Bacteroidetes中只有唯一的代表菌株Chryseobacterium ureilyticum。另外,就目前对块菌属子实体及其根际土壤内的可培养细菌多样性研究进行了比较和探讨。     

印度块菌(Tuber indicum)菌根促生细菌的研究

【目的】筛选对印度块菌菌根量和菌根苗长势有促进作用的菌根促生细菌(Mycorrhization helper bacteria,MHB)。【方法】选择华山松为宿主植物,自块菌菌根根际土壤中分离得到的11种细菌为供试菌株,将印度块菌菌剂与不同浓度的细菌混合于特定基质中后接种于华山松上,并通过对印度块菌与华山松形成的菌根数、华山松的株高和地径三方面的统计与分析,确认MHB。【结果】Pseudomonas sp. JCM 5481 (P143)、Streptomyces sp. EN31 (S191)、Variovorax paradoxus (V633)在浓度为2.4×109 CFU/mL时对印度块菌菌根数、株高和地径均有极显著促进作用(P<0.01);Pseudomonas chlororaphis (P11)、Pseudomonas corrugate (P127)在浓度为0.8×109 CFU/mL时对印度块菌菌根数、株高和地径均有显著促进作用(P<0.05)。4种假单胞菌浓度梯度的设置显示了不同菌株适宜的浓度不同。【结论】实验获得5种MHB,并表明细菌浓度是获得MHB的一个重要影响因素。     

Using common mycorrhizal networks for controlled inoculation of Quercus spp. with Tuber melanosporum: the nurse plant method

The high cost and restricted availability of black truffle spore inoculum for controlled mycorrhiza formation of host trees produced for truffle orchards worldwide encourage the search for more efficient and sustainable inoculation methods that can be applied globally. In this study, we evaluated the potential of the nurse plant method for the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum by mycorrhizal networks in pot cultures. Pine bark compost, adjusted to pH?7.8 by liming, was used as substrate for all assays. Initially, Q. robur seedlings were inoculated with truffle spores and cultured for 12 months. After this period, the plants presenting 74 % mycorrhizal fine roots were transferred to larger containers. Nurse plants were used for two treatments of two different nursling species: five sterilized acorns or five 45-day-old, axenically grown Q. robur or Q. cerris seedlings, planted in containers around the nurse plant. After 6 months, colonized nursling plant root tips showed that mycorrhiza formation by T. melanosporum was higher than 45 % in the seedlings tested, with the most successful nursling combination being Q. cerris seedlings, reaching 81 % colonization. Bulk identification of T. melanosporum mycorrhizae was based on morphological and anatomical features and confirmed by sequencing of the internal transcribed spacer region of the ribosomal DNA of selected root tips. Our results show that the nurse plant method yields attractive rates of mycorrhiza formation by the Périgord black truffle and suggest that establishing and maintaining common mycorrhizal networks in pot cultures enables sustained use of the initial spore inoculum.     

The Socioeconomic Impact of Truffle Cultivation in Rural Spain<Superscript>1</Superscript>

The Socioeconomic Impact of Truffle Cultivation in Rural Spain. Commercial black truffle (Tuber melanosporum) plantations have been promoted in Europe with the intention of benefiting rural economies while conserving biodiversity through the expansion of oak woodlands. In this context, a socioeconomic study was conducted around the town of Sarrión in eastern Spain, where government subsidies have supported oak reforestation and truffle cultivation in unproductive hilly areas since 1987. Currently there are about 4,500 ha of truffle orchards in the surrounding county and 530 members in the local truffle association, which has provided a key forum for truffle cultivators to share technical, financial and administrative experiences. Structured interviews were carried out in 2002 with a number of orchard owners, as well as representatives of financial and governmental institutions. Truffles, which are harvested using trained dogs, typically fetch local cultivators average prices of 220–670 EUR/kg, although retail prices of high-quality specimens may reach twice this amount. In addition to the direct economic impact, an increase in local land prices was also documented, as well as a tendency for continued expansion of truffle orchards, and thus oak reforestation. In conclusion, the promotion of truffle cultivation through autonomous community and provincial government subsidies, in conjunction with support by local banks, a dedicated local truffle association, and growing interest on behalf of local farmers, seems to have achieved the mutual goals of biodiversity conservation and improving the rural economy in this region of Spain.     

作物驯化的遗传学研究及其在大豆育种中的应用

驯化遗传学是进化生物学研究的一个重要分支,对作物驯化历程进行深入探索,可以更好地认识人类早期农耕文明的产生和发展,了解物种的形成乃至生命的起源和进化,对指导人们利用野生资源来改良现有的栽培作物和培育新的有价值的品种也具有重要的意义。本文综述了作物驯化的遗传学研究内容和进展,以及重要作物大豆驯化起源的研究概况,探讨了目前作物驯化研究对大豆驯化遗传学及野生大豆资源综合利用的指导意义。     

作物驯化的遗传学研究及其在大豆育种中的应用

驯化遗传学是进化生物学研究的一个重要分支, 对作物驯化历程进行深入探索, 可以更好地认识人类早期农耕文明的产生和发展, 了解物种的形成乃至生命的起源和进化, 对指导人们利用野生资源来改良现有的栽培作物和培育新的有价值的品种也具有重要的意义。本文综述了作物驯化的遗传学研究内容和进展, 以及重要作物大豆驯化起源的研究概况, 探讨了目前作物驯化研究对大豆驯化遗传学及野生大豆资源综合利用的指导意义。     

长江流域野生猕猴桃遗传资源的潜在价值、现状分析与保护策略

长江流域是猕猴桃属(Actinidia)植物起源和演化的关键分布区, 富集了全世界重要的猕猴桃属野生物种资源和中华-美味猕猴桃物种复合体(Actinidia chinensis-A. deliciosa species complex)的种群遗传资源。作为水果作物, 猕猴桃在100多年前从长江流域经上海到新西兰, 通过栽培、驯化, 逐渐发展成新兴果树产业。目前, 猕猴桃植物的研究已在二倍体“红阳”中华猕猴桃(Actinidia chinensis cv. Hongyang)的基因组测序、种间关系的重测序分析和基础的分子系统发育、种群遗传结构等方面取得长足进步, 但基于最新研究成果的基本资源评价还相当匮乏, 对猕猴桃野生资源的保护与可持续利用亟待加强。本文回顾了栽培猕猴桃的驯化简史与猕猴桃属植物系统分类的研究进展, 通过与其他流域的比较, 对长江流域野生猕猴桃资源的潜在价值和现状进行分析, 阐述了该流域猕猴桃属植物的分布特点和受威胁的状况, 并针对目前存在的问题提出建立长效的保护机制、加强遗传资源的基础科研调查和系统评价, 以及健全种质资源保存规范和促进可持续利用等相应保护的策略。     

红花深山含笑形态变异与繁殖应用

红花深山含笑是2007年发现并快速繁殖应用的木兰科种类。本文在野外调查的基础上,结合文献资料,对红花深山含笑的发现历程、形态性状变异、繁殖推广应用现状进行分析。结果表明,红花深山含笑是优良的乡土树种,发现时野外数量极少;实生苗间存在一定的性状差异,是资源开发的优良种质资源;目前红花深山含笑繁殖主要以嫁接为主,通过正确的嫁接方法及良好的后期管理可有效繁殖苗木。     

Synthesis and establishment of <Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt. ectomycorrhizae on two <Emphasis Type="Italic">Nothofagus</Emphasis> species in Chile

Axenically germinated seedlings of two species of Southern beech (Nothofagus obliqua, N. glauca) from Chile were inoculated with spores of the Périgord black truffle (Tuber melanosporum). Ectomycorrhizal development was monitored for 6 months in the greenhouse and compared to the performance of the natural host species Quercus ilex and Quercus robur. Seedling survival and mycorrhization showed major differences in both Nothofagus species: T. melanosporum readily formed ectomycorrhizae with seedlings of N. obliqua, although at a lower rate than with Q. ilex but at a proportion very similar to Q. robur; survival and colonization rates were high, and seedling growth was not visibly affected by the high soil pH required by T. melanosporum. In contrast, more than 50% of N. glauca seedlings died after inoculation, and mycorrhiza formation was very sparse. In both species, no colonization by adventive ectomycorrhizal fungi could be observed, whereas both species of Quercus showed minor colonization by another fungus, probably Inocybe or Hebeloma. Our results show that it is possible to infect N. obliqua with the Périgord black truffle under greenhouse conditions, which opens up the possibility of cultivating this truffle as a secondary crop during reforestation with N. obliqua in Chile.     

Mycorrhization of Pecan trees (Carya illinoinensis) with commercial truffle species: Tuber aestivum Vittad. and Tuber borchii Vittad

Pecan (Carya illinoinensis) is an economically important nut tree native to the Mississippi basin and cultivated worldwide. In North America, species of truffles are regularly found fruiting in productive pecan orchards and the truffle genus Tuber appears to be abundant in pecan ectomycorrhizal (EM) communities. As an initial step to determine the feasibility of co-cropping European truffle species with pecan, we evaluated whether mycorrhizae of highly esteemed European truffle species (Tuber aestivum Vittad. T. borchii and T. macrosporum) could be formed on pecan seedlings. Seedlings were inoculated with truffle spores and were grown in a greenhouse for 10?months. Levels of EM colonization were estimated visually and quantified by counting EM tips. Ectomycorrhizae were identified both morphologically and molecularly with species-specific amplification and by sequencing of the ITS region of the nuclear ribosomal DNA (nrDNA). Both T. borchii and T. aestivum spores produced well-formed ectomycorrhizae on pecan seedlings with average root colonization levels of about 62% and 42%, respectively, whereas no ectomycorrhizae of T. macrosporum were formed. The anatomy and morphology of these truffle ectomycorrhizae on pecan was characterized. The co-cropping of T. aestivum and T. borchii may hold promise as an additional stream of revenue to pecan growers, although, further studies are needed to assess whether this symbiosis is maintained after planting in the field and whether truffle production can be supported by this host species.     

Can NPK fertilizers enhance seedling growth and mycorrhizal status of Tuber melanosporum-inoculated Quercus ilex seedlings?

Although successful cultivation of the black truffle (Tuber melanosporum) has inspired the establishment of widespread truffle orchards in agricultural lands throughout the world, there are many unknowns involved in proper management of orchards during the 6–10 years prior to truffle production, and there are conflicting results reported for fertilizer treatments. Here, we systematically evaluate the combined effects of nitrogen, phosphorous, and potassium with different doses of each element, applied to either foliage or roots, on plant growth parameters and the mycorrhizal status of outplanted 3-year-old seedlings in five experimental Quercus ilex–T. melanosporum orchards. Fertilization did not significantly improve seedling aboveground growth, but the plants treated with the fertilizer 12-7-7 applied to the roots (HNr) displayed longer field-developed roots. Only the fertilizer with the highest dose of K (10-6-28) applied to the foliage (HKf) increased the probability of fine root tip colonization by T. melanosporum in field-developed roots. However, the plants treated with the same fertilizer applied to the soil (HKr) presented the highest probability for colonization by other competing mycorrhizal soil fungi. Potassium seems to have an important role in mycorrhizal development in these soils. Apart from T. melanosporum, we found 14 ectomycorrhizal morphotypes, from which seven were identified to species level, three to genus, two to family, and two remained unidentified by their morphological characteristics and DNA analyses.     

The combined effects of Pseudomonas fluorescens and Tuber melanosporum on the quality of Pinus halepensis seedlings

The ecological, economic and social values of the ectomycorrhizal fungi of the black truffle found in the rural Mediterranean are well known. The inoculation of Pinus halepensis seedlings with mycorrhizal fungi and rhizobacteria can improve the morphology and physiology of the seedlings and benefit the regeneration of arid regions and the reintroduction of inocula of mycorrhizal fungi into these areas. Some rhizobacteria can improve the establishment and functioning of ectomycorrhizal symbiosis. In this study, seedlings of P. halepensis were inoculated with the mycorrhizal fungus Tuber melanosporum and the rhizobacteria Pseudomonas fluorescens CECT 844 under non-limiting greenhouse conditions. Five months after inoculation, we analysed the growth, water parameters (osmotic potential at saturation, osmotic potential at turgor loss and modulus of elasticity), concentrations of mycorrhizal colonies, nutrient concentration and nutrient contents (N, P, K, Ca, Mg and Fe) in roots and aerial parts of the seedlings. Subsequently, tests were performed to estimate the root growth potentials. None of the treatments changed the water parameters or growth potentials of the roots. The inoculations improved the growth and nutrient uptake of the seedlings, although the combination of P. fluorescens CECT 844 and T. melanosporum did not generally lead to a significant improvement over the positive effects of a simple inoculation of T. melanosporum; however, the addition of P. fluorescens CECT 844 did double the rate of the mycorrhization of T. melanosporum. These results may be promising for enhancing the cultivation of truffles.