黄伞单孢杂交育种的初步研究

以采自山西省五台山和关帝山的WT3 和GD3两个黄伞纯化菌株为材料,显微观察了黄伞担孢子萌发的菌丝及交配特征,在PDA培养基上担孢子首先在其一端萌发出菌丝,宽度约3~4μm,接着产生分枝呈散发状向四周延伸。试验表明:单核菌丝体上可产生类似原基的凸状体,但不具结实性。两个单核菌丝交配后出现锁状联合是形成双核菌丝的典型特征,并具有结实性。     

Induction of Dimorphism in the Basidiomycete Lenzites saepiaria

Sugars can serve as the germinant for basidiospores of the wood-rotting fungus Lenzites saepiaria. Hexoses sterilized by autoclaving were better germinants than hexoses that were sterilized by filtration. The degradation products in heated hexose which were responsible for the stimulation of germination were levulinic and formic acid. Another product of hexose degradation, hydroxymethyl furfural, had a marked effect on outgrowth of L. saepiaria basidiospores and on the development of mycelia. Basidiospores that germinated in the presence of hydroxymethyl furfural yielded large rounded bodies that, in some cases, developed as a chain of yeastlike cells. Addition of hydroxymethyl furfural to developing mycelia resulted in the production of chains of round yeastlike structures. Similar results were obtained by treating basidiospores or mycelia with phenethyl alcohol.     

榆耳有性结构及生活史的研究

本文通过电镜扫描、石腊切片及用苏木精染色法和DAPI荧光染色,对榆耳子实体有性结构进行观察,证实榆耳子实体菌盖结构分三层:上表层为毛层,表面着生有排列较密集顶端游离的菌丝,它们相互粘连呈菌丝束;中间层为髓部,由较疏松而相互交织在一起的薄壁菌丝组成,菌丝间充满胶质物质;下表层为子实层,表面起伏不平,呈不规则的疣状突起,上面着生担子和囊状体,担子无隔膜棍棒形,外表有不规则的网状纹饰,其顶部着生4个瓶梗状小梗,每个小梗上着生1个椭圆形或腊肠形担孢子,大小为2.5—3.0×6.0—6.5μm,担孢子表面有不规则的网状纹饰结构。在担子间的囊状体为长圆柱形或圆锥形,表面有较密的不规则的网状纹饰。 榆耳有性生殖为异宗配合。绝大多数担孢子含一个细胞核,很少数担孢子含两个细胞核。孢子萌发为一端萌发,也有少数为两端萌发。初生菌丝单核,不能形成子实体,当两种不同遗传性的交配型的初生菌丝结合后,形成具有锁状联合结构的双核菌丝,并可发育成子实体。榆耳具有典型减数分裂过程,不具有减数分裂后核分裂行为,四个子核分别进入四个担孢子内。 在初生菌丝或次生菌丝上,均可产生间生的或顶生的厚垣孢子。经过温度、光照和紫外线照射的诱发,均未发现有其它类型的无性孢子产生。因此,榆耳菌的生活史和大多数担子     

Bipolar incompatibility system of an ectomycorrhizal basidiomycete, Rhizopogon rubescens

The mating systems of most ectomycorrhizal fungi have not been elucidated because of two reasons. One is the difficulty of obtaining homokaryotic isolates for mating tests caused by the low germination rate of basidiospores, and another is the difficulty of checking dikaryotization caused by the absence or inconsistent production of clamp connections on heterokaryotic mycelia under laboratory conditions. Basidiospore germination of a few ectomycorrhizal fungi has been induced by living roots of their host plants. Based on this information, we examined methods to obtain homokaryotic isolates of Rhizopogon rubescens using its host plant, Pinus thunbergii. The basidiospores of R. rubescens appeared to germinate well on an agar plate, on which axenic pine seedlings were grown in advance to induce germination, even when the seedlings were removed from the plate at the time of spore inoculation. To enhance the production rate of clamp connections on the heterokaryotic mycelia of R. rubescens, the culture medium composition was modified. The pH of the medium was critical for the production of clamp connections, and the optimal pH was higher for the production of clamp connections than for mycelial growth. These findings made it possible to conduct mating tests, and we found that the mating system of R. rubescens is bipolar with a multiallelic mating type factor.     

金针菇担孢子核相及遗传属性的研究

以3个不同的金针菇菌株为材料,研究了其担孢子的核相及遗传属性。荧光染色观察显示,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80.2%、7.5%和12.3%。源于单孢分离物的菌丝为有隔膜、无锁状联合的多核菌丝。在交配试验中,源于不同菌株单孢分离物的菌丝原生质体的配对形成具锁状联合的菌落,而源于同一单孢分离物的菌丝原生质体的配对则形成无锁状联合的菌落,暗示担孢子中的两个核具有相同的交配型。RAPD分析显示,源于同一单孢分离物的菌丝原生质体为10个随机引物所扩增的图谱彼此完全相同,印证了担孢子中的双核是同质的。此外,观察表明,一个担子上着生有4个担孢子。因此,金针菇是一种具4个含同质双核担孢子的四极性蕈菌。     

银耳二型态细胞差异性的初步研究

以银耳的担孢子、节孢子和菌丝体为材料,通过显微观察和电泳分析揭示银耳二型态细胞间的差异性。显微形态及核相观察表明:节孢子直径略大于担孢子,担孢子为单核细胞,节孢子绝大多数为单核细胞,少数为双核细胞,菌丝体为双核细胞并具有典型的锁状联合结构。总蛋白及同工酶电泳分析表明:菌丝相总蛋白谱带多于酵母相,过氧化物及酯酶与酵母相存在一定差异,而多酚氧化酶基本相同。因此,伴随银耳二型态细胞两相形态的转变,细胞代谢水平发生了相应变化。     

长根小奥德蘑双孢菌株与四孢菌株核相变化的比较

用双苯并咪唑（Hoechst 33258）染色法分别对长根小奥德蘑Oudemansiella radicata双孢菌株和四孢菌株的菌丝、子实体、担孢子进行染色观察,结果表明：双孢长根小奥德蘑菌丝细胞多为单核,无锁状联合;原担子中单核进行一次有丝分裂形成两个横向或纵向排列的子核,这2个子核分别进入2个担孢子中,留下无核的空担子;成熟担孢子具有一个核。四孢长根小奥德蘑菌丝细胞大多数为双核,具有锁状联合;进入原担子中的两个单倍性细胞核先发生核配,形成一个二倍性的核,再经过减数分裂形成四个染色体减半的单倍性子核,     

桦纤孔菌有性生殖相关特征及交配型位点分析

对桦纤孔菌菌株MDJCBS88的显微形态、菌丝及担孢子核相进行了观察。采用棉籽壳培养基对担孢子萌发形成的菌株进行栽培试验,筛选出不形成子实体或子实体发育不完整的菌株,将这些菌株在平板上进行了亲和试验,分析桦纤孔菌的有性生殖方式;并基于基因组序列进行交配型基因克隆验证,分析桦纤孔菌的交配型位点结构。显微观察发现,桦纤孔菌菌丝没有锁状联合结构,菌丝细胞无核到多核;子实层担孢子可含0-4个不等的细胞核,不同时期弹射的担孢子含有的细胞核数量不同。桦纤孔菌担孢子萌发率极低,能萌发的担孢子多为早期弹射的担孢子;培养基也影响担孢子的萌发率,与PDA培养基和CYM培养基相比,桦木屑培养基最适合桦纤孔菌担孢子萌发,萌发率为4.55%。从担孢子萌发的96个菌株中获得了2个不结实菌株和9个结实不产孢菌株,占11.5%,这些菌株间亲和试验出现不同的表现特征,包括形成产孢子实体,产生菌丝纽结,相互融合和相互拮抗等现象,认为桦纤孔菌的有性生殖以次级同宗结合为主,并受交配型基因控制。交配型位点克隆测序后分析发现,桦纤孔菌交配型A位点共14 034 bp,含有一个MIP基因和两组HD1和HD2基因;交配型B位点包含3个疑似信息素受体基因和1个信息素前体编码基因。     

Quest to elucidate the life cycle of Puccinia psidii sensu lato

There is controversy surrounding the described life cycle of the rust fungus Puccinia psidii sensu lato, which causes disease on several plant species in the family Myrtaceae. The objective of this study was to determine whether P. psidii s.l. is autoecious by performing basidiospore inoculations, and microscopically examining the fate of basidiospores on the leaf surface and nuclear condition at different stages of rust development. No spermogonia developed on leaves of Agonis flexuosa inoculated either with a teliospore suspension or basidiospores naturally discharged from telia. Uredinial sori that developed in all three inoculations with teliospore suspensions and in one of the five inoculations with naturally-discharged basidiospores from telia were most likely the result of urediniospore infections. Microsatellite analysis revealed that isolates made from these uredinial sori had the same multilocus genotype as that of the original isolate. No signs of penetration of plant cells by basidiospores were observed on A. flexuosa and Syzygium jambos. The nuclear condition of mycelia of uredinial sori, urediniospores, teliospores, and four-celled metabasidia was typical of that in many rust fungi. Our study could not provide unequivocal proof that P. psidii s.l. is autoecious. While it is possible that it could be heteroecious, with an unknown alternate aecial host, it is also possible that basidiospores have lost the ability to infect Myrtaceae or are infrequently operational.     

食用外共生菌根真菌兰茂牛肝菌Lanmaoa asiatica纯培养条件下不同发育时期的核相

食用外共生菌根菌是一类可食用并与植物共生的大型真菌,其在纯培养条件下菌丝生长缓慢,不会扭结发育成原基,不能完成生活史。因此关于食用菌根菌纯培养条件下生活史的研究报道极少。本研究的兰茂牛肝菌Lanmaoa asiatica已能在纯培养条件下诱导出原基,这使生活史的研究得以完成。利用扫描电子显微镜、透射电子显微镜及荧光显微镜对兰茂牛肝菌子实体担子、担孢子、纯培养菌丝和原基的核相进行了观察。结果表明,子实体菌丝细胞为双核,担子经过减数分裂形成4个单核担孢子;担孢子萌发时长轴端长出芽状突起伸长为菌丝,原孢子中的细胞核不分裂,直接进入菌丝,形成单核初生菌丝;初生菌丝5d后变为双核菌丝;纯培养菌丝及原基细胞均为双核,其菌丝表面光滑,未观察到锁状联合。     

Genetische Untersuchungen zur Fruchtkörper- und Artbildung bei Basidiomyceten: Genetische Kontrolle der Fruchtkörperbildung beiPolyporus ciliatus

Using mainly the basidiomycetePolyporus ciliatus, we were able to produce fully developed fruitbodies not only in the normal meiotic cycle (teleomorphic = perfect) but in some stocks also in the mitotic cycle (anamorphic = imperfect) of exclusively haploid mycelia. These haploid and mitotic fruitbodies can be distinguished from dikaryotic ones only by microscopic examination, i.e. by their lack of clamp connections, karyogamy and meiosis, the production of two-spored basidia with few, but larger basidiospores. Using three other stocks of isogenic mycelial strains that did not form any fruiting structures from either haploid or dikaryotic mycelia with clamp connections, we could not find any evidence for a “fruiting initiation gene” as was postulated byEsser & al. (1977). No fruitbodies occurred in compatible combinations within one isogenic stock, but normal fruitbodies are produced abundantly in crossings of different isogenic stocks. We were able to confirm these results with the help of the non-fruiting strains originally used byStahl & Esser (1976).—Our experimental results are compatible with the hypothesis suggested byKniep (1930): The genetic control of the sexual cycle by the mating type factors A and B on the one hand, and of the fruitbody formation on the other, are essentially independent. For the latter, a multitude of different genes has been proposed. Fruitbody formation thus is a typical polygenic character. The results of experiments on two other morphological characters i.e. primordia and coralloid structures, also correspond with this genetic concept.     

Identification and concentration of airborne basidiospores

Although clinical investigations have shown that basidiospores are allergenic, very little information exists on the airborne concentration or identification of these spores. The atmosphere in Tulsa, Oklahoma has been monitored for the presence of basidiospores using Burkard Volumetric Spore Traps. The concentration of total basidiospores shows that they are a significant component of the Tulsa atmosphere during certain periods. Overall spores of 18 genera have been identified from the atmosphere; the morphology of these spores is described. Field studies have shown that fruiting bodies of all these genera are common within the city. In addition other basidiospores present in the atmosphere include those identifiable only to the family level as well as many morphologically indistinct hyaline basidiospores.     

Stereum sanguinolentum: Is it an amphithallic basidiomycete?

Monobasidiospore isolates were prepared from basidiocarps of Stereum sanguinolentum. Five isolates per basidiome were paired with each other and with isolates from the trama. Interbasidiome pairings of the trama isolates and of a selection of single-spore isolates also were performed. Thin sections of the hymenium were stained with DAPI and examined by fluorescence microscopy to study the nuclei in the basidia. Spore prints were stained with DAPI to count the number of nuclei per spore. SEM was used to determine the number of basidiospores per basidium. All intrabasidiome pairings were compatible. In contrast, interbasidiome pairings, except one, were incompatible, independent of whether single-spore or trama isolates were paired. Fertile basidiomes were formed in single-basidiospore cultures. Basidia were regularly four-spored. On average, 5% of the basidiospores possessed one nucleus, 82% two, 2% three and 1% four nuclei. Ten percent of the spores appeared to be empty. Karyogamy, meiosis and postmeiotic mitosis were observed in the basidia. Nuclei resulting directly from meiosis, i.e., without having undergone postmeiotic mitosis, sometimes were observed in the sterigmata or spore primordia. The high number of vegetative compatibility groups (VCG) of S. sanguinolentum observed in this study and earlier studies is difficult to explain without sexual or parasexual recombination. We suppose that the majority of spores with ≥2 nuclei are amphithallic, possessing at least one nucleus of each mating type. Recombination could occur by exchange of nuclei among VCGs via anastomoses between homothallic compartments. Transfer of nuclei from heterothallic to homothallic mycelia or matings between homothallic mycelia, which originate from monokaryotic spores, might be other paths for gene exchange.     

Mechanisms for the development of genetically variable mycorrhizal mycelia in the ectomycorrhizal fungus Laccaria bicolor.

An in vitro study investigated mechanisms for the development of genetically variable mycorrhizal mycelia for Laccaria bicolor. Seedlings of jack pine (Pinus banksiana) grown nonaseptically in an autoclaved soil substrate were given different L. bicolor inoculum treatments. These included (i) a dikaryotic mycelium genotype (D); (ii) D and basidiospores collected from one group of five sporophores (T1); (iii) D and basidiospores collected from 10 sporophores, two from each of five different groups (T5); (iv) T1 alone; (v) T5 alone; and (vi) a noninoculated control. Dikaryotic mycelial inoculum was provided at the time of sowing, while basidiospore inoculum was added at 10 weeks after seed germination. Sporophore formation was induced after 20 weeks of growth, and dikaryotic cultures were isolated from their tissue. Seedlings were harvested, and growth and mycorrhization were assessed. Levels of both were generally lower for T1-treated seedlings, compared with seedlings receiving D, while levels for T5-treated seedlings were intermediate. Sporophore genotype variability was assessed for inoculum treatments by using the isoenzymatic marker leucine aminopeptidase. The greatest genetic variability was seen with the basidiospore treatments T1 and T5, with up to four leucine aminopeptidase patterns per seedling. The mixed treatments D plus T1 and D plus T5 produced most frequently, but not exclusively, the inoculated dikaryon genotype. After isoenzyme results were assessed, variable sporophore isolates of mixed treatments were analyzed with randomly amplified polymorphic DNA and PCR mitochondrial DNA markers to determine if they were formed by dikaryon-monokaryon crosses between the inoculated dikaryon and monosporous mycelia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological variation among Tricholoma matsutake isolates generated from basidiospores obtained from one basidioma

Matsutake (Tricholoma matsutake) is a commercially valuable edible ectomycorrhizal mushroom. The physiological traits of T. matsutake have been previously assessed using mycelial isolates isolated from basidiomata; however, few studies have focused on basidiospores. Here, we report that sibling T. matsutake isolates generated from basidiospores on a single basidioma show distinct physiological variation. We first established 145 isolates of T. matsutake on modified Norkrans' C (MNC) agar medium and found that their radial growth varied significantly. The mycelial biomasses of nine isolates with different growth rates were reduced on low-carbon and low-nitrogen MNC media. However, the colony diam of one isolate was significantly elevated on low-carbon medium, and the colony diam of two isolates were significantly elevated on low-nitrogen medium. In co-cultures of two or three isolates, commensal and amensal interactions were observed. The physiological variation induced by low carbon and nitrogen levels and the mycelial interactions between sibling isolates imply mechanisms for the genetic and functional characteristics of mycelia of T. matsutake.     

中国香菇交配型和基因型的分析

自1987—1990,在长江以南九省(区)采集52个香菇品系,43株制作了孢子印用以检测交配型,又以检测A.B因子位点的等位基因方法测定了担孢子的基因型,保藏了以A_xB_x(或A_xB_y……)标记的标准单核菌丝体,这是建立中国香菇种质库的第一步,将为香菇杂交育种提供有效亲本。结果表明:每一菌株的四个交配型呈随机分配。具有四个交配型的菌株百分率是79%,有二个交配型的香菇菌株百分率是21%。有9个菌株的单核菌丝的基因型从A_1B_1……A_(18)B_(18)不同,A.B因子等位基因差异明显,这些菌株显示了A.B因子的很低的地区性重复频率。本项研究中建立了区分同宗A同宗B以及四个交配型的试验方法,遵循着核移动的规律,这个方法能够重复。     

Battling the biotypes of balsam: the biological control of Impatiens glandulifera using the rust fungus Puccinia komarovii var. glanduliferae in GB

Impatiens glandulifera, or Himalayan balsam, is a prolific invader of riverine habitats. Introduced from the Himalayas for ornamental purposes in 1839, this annual species has naturalised across Great Britain (GB) forming dense monocultures with negative affects across whole ecosystems. In 2006 a programme exploring biocontrol as an alternative control method was initiated and to date, two strains of the rust fungus Puccinia komarovii var. glanduliferae have been released. To better understand the observed differences in susceptibility of GB Himalayan balsam stands to the two rust strains, inoculation studies were conducted using urediniospores and basidiospores. Experiments revealed large variation in the susceptibility of stands to urediniospores of the two rust strains, with some resistant to both. Furthermore, the infectivity of basidiospores was found to differ, with some stands fully susceptible to the urediniospore stage, being immune to basidiospore infection. Therefore, before further rust releases at new sites, it is necessary to ensure complete compatibility of the invasive stands with both urediniospores and basidiospores. However, for successful control across GB it is essential that plant biotypes are matched to the most virulent rust strains. This will involve additional strains from the native range to tackle those biotypes resistant to the strains currently released.     

Basidiome formation of an edible wild, putatively ectomycorrhizal fungus, Phlebopus portentosus without host plant

Phlebopus portentosus is a popular wild edible ectomycorrhizal fungus in northern Thailand. In general ectomycorrhizal fungi produce basidiomes when associated with a host plant. In this paper mycelium growth and basidiome production of P. portentosus were examined in pure culture both in vitro and in pot-culture experiments. Five mycelial strains of P. portentosus were isolated from basidiomes and used in the experiments. The mycelia grew fastest on sorghum grains supplemented with fungal-host solution. The mycelia produced sclerotia-like structures after 3 wk incubation in darkness at 30 C. All strains of P. portentosus had the ability to form primordia. The primordia were formed under lowered temperature, high humidity and a 12 h photo-period. They developed to mature basidiomes after 8-12 d in in vitro. In the pot-culture primordia were found after 28-35 d incubation in the greenhouse and mature basidiomes released basidiospores within 6-8 d. Basidiospores were germinated on fungal-host medium and formed mycelial colonies. This fungus showed an ability to produce basidiomes even 2 y after the original isolation from tissues. This research provides valuable information concerning the techniques and protocols for the large scale commercial production of P. portentosus basidiomes in the absence of a host plant.     

Frequent diploidisation of haploid Armillaria ostoyae strains in an outdoor inoculation experiment

Very little is known about the biology and ecology of haploid Armillaria strains in nature. In this outdoor inoculation experiment, we assessed the virulence of six haploid Armillariaostoyae strains along with their diploid parent towards 2-year-old seedlings and 4-year-old saplings of Norway spruce (Picea abies), and determined their ability to colonise freshly cut stumps. As inoculum source an Armillaria-colonised hazelnut (Corylus avellana) stem segment was inserted into the soil substrate. Re-isolations from mycelial fans at the root collar of infected trees or stumps were made. Surprisingly, not a single haploid re-isolate could be recovered. Microsatellite genotyping of 133 re-isolates suggests that the inoculated haploid strains were diploidised either by mating propagules (basidiospores or haploid mycelia) already present in the soil substrate or naturally disseminated in the course of the experiment from nearby forests. Consequently, no conclusion about the infectious ability of haploid Armillaria mycelia under natural conditions can be drawn. Nonetheless, the diploid half-sib families resulting from the diploidisation showed varying degrees of virulence, with a high correlation between the experiment with 2-year-old seedlings and 4-year-old saplings. Despite extensive genotyping of re-isolates, no evidence for somatic recombination between haploid mating propagules and diploidised mycelia was detected, suggesting that this is an uncommon phenomenon in A. ostoyae.     

Mating System and Basidiospore Formation in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Colonies arising from single conidia of various wild-type strains were all capable of producing fruit bodies and basidiospores. In addition, single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from these fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when allowed to form a heterokaryon, implying that these isolates do not represent mating types. All this evidence indicates that P. chrysosporium has a primary homothallic mating system. In addition to fruiting and nonfruiting phenotypes, basidiospores from strain OGC101, a derivative of ME-446, gave rise to colonies which did not grow on cellulose (Cel⁻). The fruiting, nonfruiting, and Cel⁻ phenotypes differed from each other and from the parental wild-type strain in a variety of characteristics, including growth, conidiation, and evolution of ¹⁴CO₂ from ¹⁴C-side chain-labeled lignin, indicating that strain OCG101 is a heterokaryon.