DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.     

DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.     

Genetic control of horizontal virus transmission in the chestnut blight fungus, Cryphonectria parasitica

Vegetative incompatibility in fungi has long been known to reduce the transmission of viruses between individuals, but the barrier to transmission is incomplete. In replicated laboratory assays, we showed conclusively that the transmission of viruses between individuals of the chestnut blight fungus Cryphonectria parasitica is controlled primarily by vegetative incompatibility (vic) genes. By replicating vic genotypes in independent fungal isolates, we quantified the effect of heteroallelism at each of six vic loci on virus transmission. Transmission occurs with 100% frequency when donor and recipient isolates have the same vic genotypes, but heteroallelism at one or more vic loci generally reduces virus transmission. Transmission was variable among single heteroallelic loci. At the extremes, heteroallelism at vic4 had no effect on virus transmission, but transmission occurred in only 21% of pairings that were heteroallelic at vic2. Intermediate frequencies of transmission were observed when vic3 and vic6 were heteroallelic (76 and 32%, respectively). When vic1, vic2, and vic7 were heteroallelic, the frequency of transmission depended on which alleles were present in the donor and the recipient. The effect of heteroallelism at two vic loci was mostly additive, although small but statistically significant interactions (epistasis) were observed in four pairs of vic loci. A logistic regression model was developed to predict the probability of virus transmission between vic genotypes. Heteroallelism at vic loci, asymmetry, and epistasis were the dominant factors controlling transmission, but host genetic background also was statistically significant, indicating that vic genes alone cannot explain all the variation in virus transmission. Predictions from the logistic regression model were highly correlated to independent transmission tests with field isolates. Our model can be used to estimate horizontal transmission rates as a function of host genetics in natural populations of C. parasitica.     

Restriction fragment length polymorphisms in the mushrooms Agaricus brunnescens and Agaricus bitorquis

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species), were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates of both species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in Southern DNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wild type, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species. There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescens strains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA from heterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A. brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplast regenerates from one commercial strain and two strains collected in the wild.     

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.     

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,     

Evolution of Multilocus Genetic Structure in Avena Hirtula and Avena Barbata

Avena barbata, an autotetraploid grass, is much more widely adapted than Avena hirtula, its diploid ancestor. We have determined the 14-locus genotype of 754 diploid and 4751 tetraploid plants from 10 and 50 Spanish sites, respectively. Allelic diversity is much greater in the tetraploid (52 alleles) than in the diploid (38 alleles): the extra alleles of the tetraploid were present in nonsegregating heteroallelic quadriplexes. Seven loci were monomorphic for the same allele (genotypically 11) in all populations of the diploid: five of these loci were also monomorphic for the same allele (genotypically 1111) in all populations of the tetraploid whereas two loci each formed a heteroallelic quadriplex (1122) that was monomorphic or predominant in the tetraploid. Seven of the 14 loci formed one or more highly successful homoallelic and/or heteroallelic quadriplexes in the tetraploid. We attribute much of the greater heterosis and wider adaptedness of the tetraploid to favorable within-locus interactions and interlocus (epistatic) interactions among alleles of the loci that form heteroallelic quadriplexes. It is difficult to account for the observed patterns in which genotypes are distributed ecogeographically except in terms of natural selection favoring particular alleles and genotypes in specific habitats. We conclude that natural selection was the predominant integrating force in shaping the specific genetic structure of different local populations as well as the adaptive landscape of both the diploid and tetraploid.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.     

草菇减数分裂和担孢子形成细胞核行为

本文用苏木精染色和双苯并咪唑(Hoechst 33258)染色法,从草菇子实体“纽期”菌褶分化完开始,每3小时对同一个子实体连续切取菌褶进行染色观察。结果表明草菇子实体“纽期”菌褶形成时,约10％的担子发生了核配;在子实体发育过程中,尤其是子实体成熟期后,不断有少量新的双核担子产生,并发生核配,使草菇减数分裂的同步性不高;草菇从菌褶分化完成(此时已有10％担子发生核配)到子实体完全成熟,菌褶变成深粉红至褐色(此时约70％担子完成减数分裂)需要28—30小时;担子减数分裂的持续时间为18小时,其中细线期和偶线期5.9小时、粗线期6.2小时、双线期和终变期3.4小时、中期10.5小时、后期Ⅰ到四分体2小时;经过对粗线期、双线和终变期以及中期Ⅰ染色体条数的多次反复观察,认为草菇的染色体条数为11(n=11);减数分裂后,4个子核分别进入4个担孢子中,留下无核的担子;绝大部分担孢子是单核的,有约5％的担孢子是双核的。     

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。     

Control of recombination ability of vegetative cells in Saccharomyces cerevisiae

Summary In a diploid strain heteroallelic at the ade3 locus, the mitotic intragenic recombination frequency is enhanced ten fold when the cell population is starved for histidine (Hénaut et Luzzati, 1971). By studying simultaneous recombinational events at two independant loci, it is shown that the effect of histidine starvation is most simply explained in term of an increase in the frequency of cells capable of recombination. In these competent cells, intragenic recombination frequencies during mitosis are equal to those found during meiosis. However, the frequency of recombination between the gene and the centromere appears to be lower during mitosis than during meiosis.We believe that histidine starvation in ade3 strains stimulates chromosome pairing, and that there is no fundamental difference between mitotic and meiotic recombination.     

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。     

糙皮侧耳染色体计数和担孢子形成过程中细胞核行为的研究

糙皮侧耳(Pleurotus ostreatus)菌褶经有丝分裂阻断剂预处理、原生质体铺片和Gie-msa 染色并结合苏木精染色后,经过多次反复实验观察,证明糙皮侧耳的染色体条数为 9(n=9);糙皮侧耳从菌褶分化完成到子实体完全成熟的过程中,不断有少量新的双核担子产生,发生核配直到释放担孢子。其减数分裂同步性不高。减数分裂后,4个子核分别进入4个担孢子中,留下中空的担子。     

Biochemical aspects of basidiospore maturation in <Emphasis Type="Italic">Agaricus bisporus</Emphasis> at various temperatures

Phosphatidylethanolamine is the main phospholipid of Agaricus bisporus basidiospores obtained under sterile conditions from young basidiomes with closed partial veils. Storing the basidiospores for five months at room temperature resulted in a complete loss of their germinating capacity. Conversely, storing them at a low temperature increased their germination rate by 15–20%. At both temperature levels, the phosphatidyl-choline ratio significantly increased during storage to the level found in mature basidiospores. In addition, a drastic (8–10-fold) decrease in trehalose content occurred after two months of storage at room temperature. The trehalose content decreased only 1.5-fold at low temperatures. The involvement of trehalose and lipids in the retention of spore viability is discussed.     

Genetic analysis of Phytophthora infestans populations in the Nordic European countries reveals high genetic variability

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). The pathogen is highly adaptable and to get an overview of the genetic variation in the Nordic countries, Denmark, Finland, Norway and Sweden we have analyzed 200 isolates from different fields using nine simple-sequence repeat (SSR) markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all four Nordic countries shared the most common alleles across the loci. In total 169 multilocus genotypes (based on seven loci) were identified among 191 isolates. The genotypic diversities, quantified by a normalized Shannon's diversity index (H(s)), were 0.95 for the four Nordic countries. The low F(ST) value of 0.04 indicates that the majority of variation is found within the four Nordic countries. The large number of genotypes and the frequency distribution of mating types (60% A1) support the hypothesis that sexual reproduction is contributing notably to the genetic variation of P. infestans in the Nordic countries.     

Genotypic diversity of Armillaria gallica from mixed oak forests in Massachusetts

The population structure of Armillaria gallica, an important pathogen of Quercus spp., was investigated from mixed oak forests in central Massachusetts, encompassing a sampling area over 500 km(2). From 16 plots at four sites a total of 153 isolates (34-40 isolates per site) was analyzed with amplified fragment length polymorphisms (AFLPs). Analyses of 204 polymorphic loci detected 38 AFLP genotypes from a sample area of 4.51 hectares (ha). Genets ranged in distribution from five to 33 genets per hectare (GPH), with a mean of eight GPH and the average A. gallica genet occupying 0.13 ha. Allele frequencies produced an unbiased expected heterozygosity (H(E)) value of 0.112 (SE = 0.006) and a Nei's expected heterozygosity (H(J)) value of 0.190 (SE = 0.009), indicating low genetic diversity within the population. Analysis of molecular variation (Φ(PT) = 0.301; P < 0.001) indicates high genetic differentiation, with 70% of the molecular variation explained at the site-level within A. gallica subpopulations. However, results of the Mantel test, used to assess the isolation-by-distance hypothesis, were inconclusive in determining whether the subpopulations were truly isolated by distance. A neighbor-joining tree constructed from a genetic distance matrix grouped genotypes from the same site (subpopulation) together, but from three of four sites genotypes were randomly clustered at the plot level. The results suggest that basidiospore dispersal is an important means of new genet formation at linear distances up to 2000 m.     

Formation of atypical fruiting structures inGanoderma lucidum isolates on a nutrient agar medium

Effects of light and ventilation on the formation of atypical fruiting structures (AFSs) and fruit body primordia (FBPs) ofGanoderma lucidum on nutrient agar media were investigated. Although the mycelial growth was inhibited by illumination and ventilation, brown AFSs appeared on the white mycelial colony, and basidia and basidiospores were produced on the AFSs. On the other hand, FBPs were induced by illumination alone, regardless of ventilation. However, the primordia could not develop to mature fruit bodies. In the dark, only vegetative growth of the fungus progressed. Twenty-three isolates ofG. lucidum collected from four countries were tested for the formation of AFSs and FBPs under light and ventilation. Thirteen isolates formed AFSs, and another five isolates produced FBPs. Of the remaining five isolates, one formed callus-like structures without elaborating basidiospores, and the other four did not induce AFSs or FBPs. Microscopical observation showed that the basidia were formed directly from generative hyphae on the surface of AFSs. Basidiospores formed on the basidia were brown and ellipsoid with an eccentric hilar appendix on the rounded spore base. They had a double wall and mostly contained one or two large vacuoles. The surface of basidiospores was smooth or wrinkled and had shallow holes. The spore size was (4.5–)6.4–9.6(–10.3) × (2.6–)3.2–5.1(–6.4), 7.3 × 4.2 µm on average.     

Biochemical aspects of basidiospore maturation in Agaricus bisporus at various temperatures

Phosphatidylethanolamine is the main phospholipid of Agaricus bisporus basidiospores obtained under sterile conditions from young basidiomes with closed partial veils. Storing the basidiospores for five months at room temperature resulted in a complete loss of their germinating capacity. Conversely, storing them at a low temperature increased their germination rate by 15-20%. At both temperature levels, the phosphatidylcholine ratio significantly increased during storage to the level found in mature basidiospores. In addition, a drastic (8-10-fold) decrease in trehalose content occurred after two months of storage at room temperature. The trehalose content decreased only 1.5-fold at low temperatures. The involvement of trehalose and lipids in the retention of spore viability is discussed.