Cytological Analysis of Anastomoses and Vegetative Incompatibility Reactions in <Emphasis Type="Italic">Helicobasidium monpa</Emphasis>

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus. Received: 13 June 2001 / Accepted: 8 August 2001     

Isolation of a homothallic mutant in <Emphasis Type="Italic">Lentinula edodes</Emphasis>

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny. Contribution no. 385 from the Tottori Mycological Institute     

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.     

香菇新菌株选育

采用香菇135双核体秋水仙碱(C22H25NO6)诱变及香菇K303单核体秋水仙碱诱变的菌株388B1与香菇JL-2双核体杂交(单×双)的方法,成功地获得了优良的诱变菌株(编号为K05)和杂交菌株(编号为K48)。经拮抗试验、酯酶同工酶、分子标记等多种方法鉴定和栽培试验证明,诱变、杂交菌株为有别于出发菌株和亲本菌株的新菌株;3年的栽培比较试验结果表明:新菌株比当地主栽香菇135、939高产,K05比135每筒平均增产28.5%,K48比939每筒平均增产10.7%,产量生物学统计显示增产显著;经济性状考评为优质,菌柄短、菇形圆整。该研究成果不仅有望满足菇农对香菇新菌株的需求,而且可为香菇育种提供新的途径。     

以新单倍体化和新单倍体性生物交配为手段鉴定侧耳属的培养

使用牛胆汁琼脂技术对侧耳属的双核体培养物进行新单倍体化(neohaploidization),生成的新单倍性生物(neohaplont)分离物为单核体,并且缺少锁状联合。这些新单倍性生物的分离物在与侧耳属已知单孢子测交菌株交配时,来自每一个亲本双核体的新单倍体性生物只能与一个种的测交菌株亲合。从而表明,该技术对鉴定双核体侧耳培养物有价值,同时对其它一些真菌类群有效。     

Growth and ultrastructural studies on the mitochondrial mutant of Coprinus lagopus

The cytoplasmic acu-10 mutant of Corprinus lagopus has a respiratory deficiency due to an altered cytochrome component and is slower growing than wild type. When growth of wild type and acu-10 monokaryons and dikaryons were compared on solid medium and in liquid culture the mutation was found to restrict growth of the dikaryon more severely than that of the monokaryon. Ultrastructural studies revealed that faster growth of the acu-10 monokaryon occurred at the expense of maintaining the cytoplasmic cell contents and with little increase in the numbers of mitochondria. Cells of the acu-10 dikaryon were comparatively unvacuolated and contained greatly increased numbers of mitochondria. Mitochondria in cells of the mutant had a typical orthodox conformation with clear matrix and well defined cristae. In contrast, mitochondria in wild type cells had a more compact and elongated shape with dense matrix and less obvious cristae. The observed difference in mitochondrial ultrastructure is interpreted as one of conformation rather than structure and is attributed to impaired ability of mutant mitochondria to carry out oxidative phosphorylation. In an old cell of the mutant the mitochondria showed signs of recovering the wild type conformation.     

凤尾菇和裂褶菌原生质体非对称融合研究

具有双重营养缺陷型的裂褶菌单核体突变菌株的原生质体经过灭活处理后作为供体与同样带有营养缺陷型标记的风尾菇单核体原生质体融合,得到大量生长速度和菌落形态差异较大的多核体和双核体融合子．这些融合子主要显示了供体菌株的特性,数次转接后,有的融合子从多核体转变成双核体,有的融合子完全恢复了供体亲本双核体的遗传特性,同时获得了一些有意义值得继续探讨的新的生理特性,如菌丝体在木屑 皮以及废棉培养料上生长速度较快,在平板培养基和木屑培养料上均较早较快地形成原基和子实体等．结果表明原生质体非对称融合是一种效率较高的融合方式,可以作为食用菌育种的一种有效途经．     

Direct microscopic studies of clamp connection formation in growing hyphae of Schizophyllum commune

Summary Photomicroscopic studies of clamp connection formation were collated with microscopic measurements of apical extension and mitosis in rapidly growing dikaryotic hyphae of Schizophyllum commune. Intercalary clamp connection formation was described in sub-terminal regions of the dikaryon. Conventional (i.e., rearward) clamp initiation was compared to forward clamp connection formation. Primary branch emergence was observed from clamp connections in growing hyphae and contrasted to sub-basidial branching in the hymenium of dikaryotic fruit-bodies.     

Changes in isozyme patterns between monokaryons and dikaryons of a bipolar Coprinus

Proteins and isozymes of several different classes of enzymes in partially purified protein extracts of monokaryons, dikaryons, and monokaryon mixtures of a bipolar Coprinus sp. were separated on polyacrylamide gels by slab electrophoresis. Differences in protein and isozyme spectra were correlated with the operation of the incompatibility factors and with the results of Wang and Raper on Schizophyllum. It was concluded that the shift from monokaryon to dikaryon mediated a major change in the nature, quantity, or distribution of the proteins of this Coprinus sp.     

Sexual selection in mushroom-forming basidiomycetes

We expect that sexual selection may play an important role in the evolution of mushroom-forming basidiomycete fungi. Although these fungi do not have separate sexes, they do play female and male roles: the acceptance and the donation of a nucleus, respectively. The primary mycelium (monokaryon) of basidiomycete fungi, growing from a germinating sexual spore, is hermaphroditic, but it loses female function upon the acceptance of a second nucleus. The resulting dikaryon with two different nuclei in each cell retains a male potential as both nuclei can fertilize receptive mycelia. We tested the occurrence of sexual selection in the model species of mushroom-forming basidiomycetes, Schizophyllum commune, by pairing monokaryons with fully compatible dikaryons. In most pairings, we found a strong bias for one of the two nuclei although both were compatible with the monokaryon when paired alone. This shows that sexual selection can occur in mushroom-forming basidiomycetes. Since the winning nucleus of a dikaryon occasionally varied depending on the receiving monokaryon, we infer that sexual selection can operate through choosiness of the receiving individual (analogous to female choice). However, in other cases the same nucleus won, irrespective of the receiving monokaryon, suggesting that competition between the two nuclei of the donating mycelium (analogous to male–male competition) might also play a role.     

The thn mutation of Schizophyllum commune, which suppresses formation of aerial hyphae, affects expression of the Sc3 hydrophobin gene.

The spontaneous and recessive mutation thn in the basidiomycete Schizophyllum commune suppresses the formation of aerial hyphae in the monokaryon and, if present as a double dose, the formation of both aerial hyphae and fruit-bodies in the dikaryon. In the monokaryon, the mutation prevents accumulation of mRNA of the Sc3 gene, and in the dikaryon it also prevents the accumulation of fruiting-specific mRNAs, including mRNAs of the Sc1 and Sc4 genes, which are homologous to the Sc3 gene. These three genes code for hydrophobins, a family of small hydrophobic cysteine-rich proteins. In the thn monokaryon, the only detectable change in synthesized proteins is the disappearance of an abundant protein of apparent Mr = 28 K from the culture medium and from the cell walls. Protein sequencing shows that this is the product of the Sc3 gene. The Sc3 hydrophobin is present in the walls of aerial hyphae as a hot-SDS-insoluble complex. Submerged hyphae excrete large amounts of the hydrophobin into the medium.     

SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune

Two monokaryons of Schizophyllum commune can form a fertile dikaryon when the mating-type genes differ. Monokaryons form sterile aerial hyphae, while dikaryons also form fruiting bodies that function in sexual reproduction. The SC3 hydrophobin gene is expressed both in monokaryons and in dikaryons. The SC4 hydrophobin is dikaryon specific. In the monokaryon, SC3 lowers the water surface tension, coats aerial hyphae with a hydrophobic layer and mediates attachment of hyphae to hydrophobic surfaces. The SC4 protein lines gas channels within fruiting bodies with a hydrophobic membrane. Using gene disruptions, in this study, we show that in dikaryons SC3 fulfils the same roles as in monokaryons. SC4, on the other hand, has a role within fruiting bodies. In contrast to gas channels in fruiting bodies of the wild type, those of a DeltaSC4 strain easily filled with water. Thus, SC4 prevents gas channels filling with water under wet conditions, probably serving uninterrupted gas exchange. Other dikaryon-specific hydrophobin genes, SC1 and SC6, apparently do not substitute for the SC4 gene. In addition, by expressing the SC4 gene behind the SC3 promoter in a DeltaSC3 monokaryon, it was shown that SC4 cannot fully substitute for SC3, indicating that both hydrophobins evolved to fulfil specific functions.     

Nuclear and Non-nuclear Factors influencing Clamp Connection Formation in Coprinus disseminatus

Matings between sister single spore lines of Coprinus disseminatusshowed a cryptic tetrapolar pattern. The two groups of matingsthat resulted in formation of mycelia with clamp connections(apparent dikaryons) differed in rate of nuclear penetrationduring mating. In one group penetration occurred at rates comparablewith nuclear migration in other species and in the other groupit was often extensive but at a rate similar to, or less than,the dikaryon growth-rate. No differences were detected betweenthese two groups in stability, colony extension rate, frequencyof clamp connections, proportions of true clamp connectionsand pseudoclamps, or number of nuclei per hyphal tip cell. Cytological studies and the isolation of hyphal tips showedthat both groups of apparent dikaryons were heterokaryotic di-or trikaryons. The di- and trikaryotic conditions co-existedin the same mycelium, but adjacent cells of individual branchingsystems usually contained equal numbers of nuclei. Within apparentdikaryons the number and kinds of nuclei per cell were similarin hyphae with clamp connections and those with simple septa.Treatments that prevented clamp connection formation did notalter the nuclear status of most of the hyphae. Irregularities in nuclear distribution were infrequent and mostwere associated with pseudoclamps. Forty per cent of nodes withprobable pseudoclamps yielded homokaryotic branches, which wereof either constituent mating type. There was some indicationthat irregularities in nuclear distribution could also occurduring divisions associated with simple septa.     

Fungal fidelity: Nuclear divorce from a dikaryon by mating or monokaryon regeneration

Basidiomycete fungi perform fertilizations by incorporation of nuclei into a monokaryotic mycelium to establish a dikaryon. The dikaryon cannot incorporate another type of nucleus, but can still act as a nucleus donor in a dikaryon–monokaryon (di–mon) mating, known as the Buller phenomenon. Previously, it has been observed that: (1) in a particular di–mon mating, one of the nuclear types of the dikaryon generally performs better as a donor than the other, and (2) when nuclei from a dikaryon are separated to form monokaryons again (dedikaryotisation), recovery of monokaryons of the two nuclear types is usually unequal. In this study, we investigated if these two observations of asymmetry are functionally related. We tested this hypothesis by performing both di–mon matings and dedikaryotisation of dikaryons derived from five different monokaryons. When a single mechanism controls both processes, the nucleus better at fertilizing a monokaryon in a Buller pairing should also be recovered upon dedikaryotisation with a higher frequency. The results showed a hierarchical structure for recovery among nuclei in dedikaryotisation, but this hierarchy did not correspond to the fertilization success during di–mon mating. These findings thus show that the mechanism causing asymmetric regeneration of nuclei, is most likely not the same as the mechanism responsible for increased chance of fertilization in di–mon matings. We discuss the complexity of the interactions that occur during di–mon matings with regards to the mating type loci.     

Regulation of the pattern of protein synthesis in Schizophyllum commune by the incompatibility genes

The pattern of protein synthesis in various coisogenic mycelial types of Schizophyllum commune, viz. monokaryon, dikaryon, and homokaryons carrying primary mutations in the A and the B factors, was studied by two-dimensional gel electrophoresis. After pulse-labeling with ³⁵S-methionine, approximately 650 of 710 proteins analyzed were common to all mycelial types. Coisogenic monokaryons differed by only 2%, whereas the largest difference was found between these monokaryons and the dikaryon derived from them (6.6 and 7.7%). The majority of these differences fell into two about equally sized categories, i.e., proteins which were either specifically absent (“switched-off” proteins) or present (“switched-on” proteins) in the dikaryon. “Switched-on” proteins were on the average larger and slightly more acidic than “switched-off” proteins. The double factor mutant which best mimicked the dikaryon in morphology also best resembled the dikaryon in types of proteins synthesized. Unexpected, however, was the large overlap in proteins apparently controlled by each of the two incompatibility factors individually, despite the distinct morphological sequences directed by each of them.     

灵芝尿嘧啶营养缺陷型菌株的筛选和分子鉴定

【背景】营养缺陷型是一种应用广泛的分子标记,但是目前在灵芝中还未有研究和应用报道。【目的】为灵芝遗传转化研究、杂交育种和菌种鉴别提供亲本材料和技术支持。【方法】采用紫外光诱变、单单杂交、孢子单核化的方法从灵芝单核体菌株出发得到尿嘧啶营养缺陷型双核体菌株。【结果】获得8株稳定的尿嘧啶营养缺陷型单核体突变菌株和7株尿嘧啶营养缺陷型双核体菌株。【结论】灵芝尿嘧啶营养缺陷型菌株在添加外源营养物的基础上可恢复正常生长,可以为灵芝遗传转化体系的构建和灵芝育种提供材料。     

Development of the Amut Bmut strain of Schizophyllum commune

Summary In search for genetically uniform system for the cytological and biochemical analysis of sexual morphogenesis in Schizophyllum, a dikaryon mimic, the Amut Bmut strain, was studied. The course of nuclear distribution and clamp formation was followed in germlings of the double mutant. It was found that this mutant mimics the interaction between two compatible strains. The developmental sequence leading to dikaryon formation can be precisely timed. Thus, it is suggested that this strain be used for determination of the sequential induction of the enzymatic systems operating in sexual morphogenesis.     

Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune.

Complementary DNA was synthesized on polyadenylated RNA from a dikaryotic mycelium of the basidiomycete Schizophyllum commune bearing fruiting body initials. The complementary DNA was cloned into the PstI site of pBR327 by the deoxyguanidylate-deoxycytidylate tailing approach. After transformation into Escherichia coli cells, a differential screening was performed by colony hybridization with complementary [32P]DNA made on the RNAs of the monokaryon and dikaryon strains. Two clones were selected for further analysis by Northern blotting and hybrid release translation. Clone 1D10 hybridized with an mRNA of 775 nucleotides, coding for a polypeptide with an Mr of 15,000. Although this RNA was present in both monokaryotic and dikaryotic mycelia, its concentration appeared to change considerably over time and with different cultivation conditions. This mRNA is probably the most abundantly expressed sequence in S. commune. Clone 1G2 and its homologs hybridized with an mRNA of 650 nucleotides, coding for a polypeptide with an Mr of 13,000. This gene was exclusively expressed in the dikaryon strain. In liquid-grown cultures, the concentration of this mRNA was low but increased ca. 20-fold during the establishment of fruiting body primordia. A chromosomal fragment of 9 kilobase pairs which contained the 1G2 gene was cloned into pBR327 and used as a probe in Northern blot hybridization. It was found that surrounding sequences were not expressed at the same time or to the same extent as the 1G2 gene.     

Isolation and characterization of a sporeless mutant in Pleurotus eryngii

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.     

双单杂交技术选育亮盖灵芝与白肉灵芝优良性状杂交子

以亮盖灵芝Ganoderma lucidum和白肉灵芝G. leucocontextum为亲本,采用亮盖灵芝异核体双核菌丝与通过原生质体单核化获得的白肉灵芝单核体进行双单杂交,最终获得34株杂交子。采用ISSR技术对34株杂交子及亲本进行DNA多态性聚类分析,杂交子主要分为两个类群。对杂交子进行生长速度和灵芝酸含量对比,筛选到4株优质杂交子。进一步进行杂交子及亲本间HPLC指纹图谱对比,发现4株杂交子中有3株HPLC指纹图谱在具备两个亲本特征灵芝酸的同时,还有低极性灵芝酸的产生。