Nuclear selection in oidium formation from dikaryotic mycelia ofFlammulina velutipes

The effect of nuclear dominance in monokaryotic oidium formation from dikaryotic mycelia in a tetrapolar basidiomycete,Flammulina velutipes, was examined. A total of 46 monokaryotic stocks were used to produce 194 hybrid dikaryotic stocks by crossing. The proportion of homokaryons among the oidium isolates from dikaryotic mycelia was over 95%. The staining of nuclei of oidia with propidium iodide showed that over 90% of oidia were monokaryotic and suggested that these oidia had single haploid nuclei at the G1 stage. The monokaryotic oidium isolates from hybrid dikaryons were backrossed to parental monokaryotic stocks. Although most of the monokaryotic oidium isolates (except for those from 17 hybrid dikaryons from a total of 194 test stocks) showed nuclear types similar to only one of the parental stocks, the process seems to produce essentially the split nuclear type composition. Therefore, the monokaryotization in oidium formation from dikaryotic mycelia essentially involves the process of nuclear selection. The two separate results of hierarchies of relative dominance among two nuclei of the parental dikaryons in the monokaryotic oidium formation by grouping with incompatibility factor compositions were determined. Only a few discrepancies were found in the hierarchies between the two specific nuclear compositions of hybrid dikaryons.     

MUTATIONS AFFECTING HETEROKARYOSIS IN SCHIZOPHYLLUM COMMUNE

Mutations that affect the basic characteristics of heterokaryons of S. commune occur spontaneously and are preferentially selected in the common-A heterokaryon and in its homokaryotic mimics, strains carrying a mutated B factor or strains disomic for heteroallelic B factors. Nine independent mutations were compared: all segregate independently of A and B incompatibility factors, and at least 3 distinct loci, of which 2 are linked, are involved. None of the mutations is phenotypically expressed in the homokaryon or in the common-AB heterokaryon. All 9 mutations increase vegetative vigor of the common-A the effects of all the mutations are additive in both heteroallelic and homoallelic combinations. At least 1 type-II mutation also affects nuclear distribution in common-B heterokaryons. Type-II mutations appear to reduce common-A, common-B, and compatible heterokaryons to a single type unlike any of the normal heterokaryons. Pseudoclamping often persists for extended periods in modified homokaryons isolated from modified heterokaryons. Several cases of somatic recombination have been observed among components of modified heterokaryons.     

Adaptive Significance of Vegetative Incompatibility in NEUROSPORA CRASSA

Certain features reminiscent of sexuality occur in the vegetative life cycle of some filamentous fungi such as Neurospora crassa. Hyphal fusions can occur between genetically different individuals, thereby endowing the new composite mycelium, a heterokaryon, with some of the advantages of heterozygosity usually associated with diploid organisms. In N. crassa, however, there are a number of incompatibility loci which prevent formation of heterokaryons unless the alleles at the incompatibility loci are identical in the two mycelia. The selection pressures that maintain incompatibility polymorphisms are not known. We suggest here that they are maintained because they prevent a kind of exploitation of heterokaryons by nuclei that are nonadaptive in homokaryons but that enjoy a proliferative advantage over other nuclei in heterokaryons. A mathematical model that abstracts the major features of the vegetative life cycle of Neurosopra crassa has been developed, and the action of selection in this model and various extensions of it is such as to maintain polymorphisms of vegetative incompatibility factors.     

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.     

Isolation of the <Emphasis Type="Italic">B</Emphasis> incompatibility factor mutants in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes. Received: August 3, 2001 / Accepted: January 18, 2002     

Genetics and QTL mapping of somatic incompatibility and intraspecific interactions in the basidiomycete Heterobasidion annosum s.l

Somatic incompatibility (SI) is a system by which filamentous fungi can distinguish self from non-self by delimiting the own mycelia from that of other individuals of the same species. In this study, we show that SI in the basidiomycete Heterobasidion annosum sensu lato is controlled by four loci by observing the frequency of somatically compatible pairings in two experiments where isolates were paired in all possible combination. The first experiment utilized 63 heterokaryons each with one unique nucleus chosen from an array of sibling homokaryons paired with one unrelated nucleus of homokaryotic isolate TC-39-7. The second experiment used 39 heterokaryons each with one unique nucleus from the array of sibling homokaryons backcrossed with one of the parental strains (TC-122-12). We observed that SI allelic differences in a pairing alone are not enough to determine the degree of somatic incompatibility. In the first experiment, we also observed other interactions such as hyphal walls in interaction zones, increased exudation of dark-coloured metabolites and increased production of aerial hyphae. QTLs for the respective traits were positioned to a genetic linkage map of the H. annosum genome. Map-based cloning of the corresponding loci will shed much new light on intraspecific interactions in basidiomycetes.     

BIPOLAR INCOMPATIBILITY IN THE HYMENOMYCETE POLYPORUS PALUSTRIS

The basic features of bipolar incompatibility in the hymenomycete, Polyporus palustris (Berk. and Curt.), have been investigated by a comparison of compatible and incompatible interactions leading to the formation of dikaryons and common-factor heterokaryons, respectively. Nuclear migration is severely restricted and nuclear pairing and conjugate nuclear division fail to occur in the interaction of incompatible homokaryons. It is suggested that the single incompatibility factor of P. palustris combines the functions of the two incompatibility factors of tetrapolar species.     

Protein mapping and genome expression variations in the basidiomycete Agrocybe aegerita

Summary A procedure suitable for the extraction and mapping of total proteins from the basidiomycete, Agrocybe aegerita, was developed. A. aegerita mycelia were fragmented either with a Dangoumeau grinder, an X-press bomb or a sonicator and the efficiency of these three disruption methods were compared. The extraction buffer composition was optimized to avoid proteolytic activities. 2D-SDS-PAGE analysis of protein extracts showed that the rate of reproducibility depending on extractions and electrophoretic separations was always greater than 96% for all strains. The differences in efficiency observed between the breaking procedures indicate that the A. aegerita cell wall is more mechanically resistant than that of other basidiomycetes. The efficient action of protease inhibitors (PMSF and SDS) showed that A. aegerita mycelia contains numerous and/or highly active proteases. Reproducibility of protein extraction and separation methods allowed the establishment and the comparison of standard maps. Qualitative and quantitative variations in gene products between a wild dikaryotic strain and 11 homokaryotic strains from its progeny were examined. The genetic diversity, determined by comparing the distribution of proteic variations in 11 homokaryons from the same progeny, was comparable to that observed between co-isogenic homokaryons of another basidiomycete.     

Investigation of Mitochondrial Transmission in Selected Matings between Homokaryons from Commercial and Wild-Collected Isolates of Agaricus bisporus (= Agaricus brunnescens)

Ten heterokaryons of Agaricus bisporus (= Agaricus brunnescens) were shown to carry four different mitochondrial (mt) genotypes by analysis of mt restriction fragment length polymorphisms (RFLPs). Fifteen homokaryons derived from these strains were used to investigate mt inheritance in A. bisporus. One hundred eighty-nine pairings were performed in 25 different combinations. Pairings in 15 different combinations produced heterokaryons on the basis of nuclear RFLP analyses and/or fruiting trials. The mt genotype of each new intraspecies hybrid was examined by using mt RFLPs as genetic markers. Our results suggest the following. (i) Recombination between the mt genomes was not a common event. (ii) From most individual pairings, all heterokaryons carried the same mt genotype. (iii) Heterokaryons carrying either of the two possible mt genotypes were observed in certain crosses after modification of the pairing procedure. A biparental transmission pattern was demonstrated for some crosses, but there appears to be a preference for one of the mt genotypes to predominate in any specific pairing.     

DNA polymorphisms in commercial and wild strains of the cultivated mushroom,Agaricus bisporus

Summary DNA from the cultivated mushroom, Agaricus bisporus, was cloned into the bacteriophage lambda vector EMBL3 creating a partial genomic library. Ten random clones from the library were used to probe for restriction fragment length polymorphisms (RFLPs). Six of the ten probes detected polymorphisms and were used to demonstrate variation in wild and cultivated strains of the mushroom. These results suggest that RFLPs could form a basis for genetic finger-printing and subsequent strain protection in A. bisporus. In single spore progeny, RFLPs were used to demonstrate normal meiotic segregation and to differentiate between homokaryons and heterokaryons. RFLPs therefore have great potential in the development of the genetics and breeding of this commercially important species.     

Control of fungal development: I. The effects of two regulatory genes on growth in Schizophyllum commune

In Schizophyllum the various events comprising the transition from the homokaryotic to the dikaryotic stage of the life cycle are triggered by two sets of developmental regulatory genes known as the A and B incompatability factors. This paper defines the effects of these genes on growth of surface colonies by establishing the growth curves of dikaryons, comon-A heterokaryons, and strains that morphologically mimic dikaryons or heterokaryons because of constitutive mutations in A and/or B.Like homokaryons, all these developmental stages and genetic mimics have triphasic growth curves with definite exponential phases. Growth curves of dikaryons and common-A heterokaryons are indistinguishable from those of their corresponding genetic mimics. However, the growth rate during exponential phase and the timing of the entire curve are dependent upon developmental type, with the consequence that colonies of different developmental stages harvested either at equal times or at equal weights are not necessarily in the same phase of colonial growth. Data presented allow choice of harvesting times such that colonies of the different developmental types will be within the same growth phase.Biochemical differences between homokaryons and dikaryons must be due to differentiation since the two stages have virtually the same growth rate.     

Homokaryons are more combative than heterokaryons of Hericium coralloides

The homokaryotic stage of the basidiomycete lifecycle is generally considered to be short lived, although there is little experimental evidence relating to their longevity in the field. The vast majority of studies on basidiomycete ecology have used only heterokaryons. The few investigations comparing related homokaryons and heterokaryons have revealed no overall trend in differences of extension rate, wood decay or competitive ability. For a rare species the homokaryotic phase may be of greater importance than in common species as it is likely to last longer. Hericium coralloides, a rare wood decay basidiomycete, was used to investigate differences between homokaryons and heterokaryons in terms of extension rate and combative ability. Fifteen homokaryons from three fruit bodies and five heterokaryons (obtained by fruit body tissue isolation) were compared at 5–35 °C on malt agar for extension rate, and paired against heterokaryons of 13 wood decay species to assess combative ability. Homokaryons were paired to create ten artificial heterokaryons whose extension rate at 10 and 20 °C was compared to parental rates. There were some significant differences in extension rates between homokaryons and natural heterokaryons, between homokaryons and heterokaryons created artificially from homokaryons, and between homokaryons from different fruit bodies, but no consistent trends. Homokaryons proved more combative than heterokaryons, which was assessed quantitatively as well as qualitatively using a scoring system for outcome of each pairing. Results are discussed in relation to previous findings and in an ecological context.     

Heterokaryon formation with homokaryons derived from protoplasts ofRhizoctonia solani anastomosis group eight

Homokaryons were successfully recovered by regenerating protoplasts prepared from vegetative hyphae of field isolates ofRhizoctonia solani anastomosis group (AG) 8, the causal pathogen of bare-patch disease of cereals. A mating type incompatibility system, which is similar to that reported in AG 1 and AG 4, was demonstrated in AG 8. All homokaryons obtained in AG 8 were able to form tufts with their parent isolates and other heterokaryotic field isolates of AG 8 tested. Heterokaryons were readily recovered from tufts of pairing of certain homokaryon combinations. The synthesized heterokaryons formed tufts with both of the contributing homokaryons. The majority of hyphal tip cultures isolated from tufts resembled one of the contributing homokaryons. These nonheterokaryotic hyphae in tufts are attributed to transient heterokaryon effects.     

Biochemical identification of interbreeding between B-type and non B-type strains of the tobacco whiteflyBemisia tabaci

Polyacrylamide gel electrophoresis and a kinetic microplate assay were used to detect heterozygotes resulting from a cross between B-type and non B-type strains of the whiteflyBemisia tabaci. Both strains were homozygous for different esterase and acetylcholinesterase (AChE) enzymes, and heterozygotes were produced in one of two crosses between B-type females (diploid) and non B-type males (haploid). In reciprocal crosses, however, no female offspring were produced, indicating that fertilization had not occurred. Despite the identification of individuals heterozygous for the esterase and AChE markers, there was clearly a significant degree of reproductive incompatibility between the two strains. The biochemical assays provided a vital component of this study and the advantages of their use are discussed.     

Morphological and cytological aspects of oidium formation in a basidiomycete,Pholiota nameko

Pholiota nameko produced abundant oidia on aerial hyphae from monokaryotic and dikaryotic test stocks, but oidia were rare on submerged hyphae. The oidia from the former stocks had a layer of hydrophobic protein between the cell wall and the inner cell membrane which was absent in the oidia from the latter. The only remarkable differences in the morphological features of the oidia from monokaryotic and dikaryotic mycelia was the slightly larger size of the latter. Observation of various test stocks on slide cultures revealed that about 80% of oidia were produced from the secondary branched hypha, and about 20% from the terminal hyphal, cell of the main hypha. In the former, the secondary hyphae were segmented to form several oidium cells; in the latter, a single or several oidia were formed at the terminal end of the main hypha. Most oidia from monokaryons and dikaryons had only one haploid nucleus, while the remainders were multinucleate. Among the stocks tested, most oidia had a DNA content with a haploid amount at the G1 phase of the cell cycle, but a few contained twice that amount corresponding to the G2 phase     

Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (= A. bisporus)

A new technique for the production of hybrid strains of the cultivated mushroom Agaricus brunnescens is described. Homokaryons were recovered from regenerated protoplasts obtained from several heterokaryotic strains. A total of 16 novel hybrids were produced in 63 attempted crosses between paired homokaryons. Recovery of both homokaryons and hybrids was verified by analysis of restriction fragment length polymorphisms. Three of four hybrids fruited in small-scale tests, further confirming that the isolates were true hybrids. Colony morphology alone was found to be a poor indicator of hybrid status. In two instances, three homokaryons crossed successfully in all combinations, suggesting that there are at least three alleles at the putative mating-type locus. Crosses between homokaryons from commercial and wild-collected isolates indicated that these strains belong to the same biological species.     

Interspecific competitive ability of homokaryotic and heterokaryotic wood decay basidiomycetes

The role of the homokaryotic life stage in the dynamics of fungal communities is relatively unknown. However, homokaryons are thought to be only a temporary stage and are therefore not generally used in ecological experiments with fungi. In this study, the relative competitive ability and growth rates of homokaryons and heterokaryons of wood decay fungi were tested to assess the potential role of homokaryons in community dynamics. A homokaryon and a heterokaryon of each of four species (Aleurodiscus lividocoeruleus, Peniophora sp. 1, Peniophora sp. 2 and Pereniporia medulla‐panis) were assessed for their competitive abilities on an agar medium. The relationship between nuclear status and competitive ability varied between species. The homokaryon of Peniophora sp. 2 was competitively superior to its heterokaryon, whereas the homokaryon of Peniophora sp. 1 was inferior to its heterokaryon. A hierarchy of competitive abilities of each isolate revealed that Pereniporia medulla‐panis homokaryon = P. medulla‐panis heterokaryon > Peniophora sp. 1 heterokaryon > Peniophora sp. 2 homokaryon > Peniophora sp. 2 heterokaryon > A. lividocoeruleus heterokaryon = A. lividocoeruleus homokaryon. This experiment indicates that homokaryons as well as heterokaryons have the potential to influence community structure through competitive effects.     

The location and analysis of two heterokaryon incompatibility (het) loci in strains of Aspergillus nidulans

The heterokaryon incompatibility system in Aspergillus nidulans has been investigated by parasexual methods. The use of complementary auxotrophs with a repeated serial transfer method or with a protoplast fusion technique has enabled heterokaryons and diploid strains to be recovered from heterokaryon incompatible combinations of strains. The effects of allelic interaction at heterokaryon incompatibility (het) loci on the morphologies of the heterokaryon and diploid colonies isolated are described. Parasexual analyses conducted among strains belonging to the heterokaryon compatibility groups, h-cGl and h-cB, and the two recombinant compatibility classes, have located the hetA and hetB genes to linkage groups V and VI respectively.     

Research on basal stem rot (BSR) of ornamental palms caused by basidiospores from <Emphasis Type="Italic">Ganoderma boninense</Emphasis>

Basidiospores were isolated from the fruiting bodies of Ganoderma infecting oil palms from an estate in Johor and from ornamental palms (including oil palms) from Singapore. The spores were then germinated to obtain homokaryotic mycelia. Based on clamp connection formation in paired hyphal fusions, tester strains were identified from the homokaryons isolated. Compatibility tests were then carried out using these testers to determine the relatedness of the homokaryotic Ganoderma isolates, both from Johor and from Singapore. Results from the compatibility tests showed that Ganoderma from both locations belong to the same species, while the Ganoderma isolates from Singapore share some common alleles. The pathogenicity tests carried out on Chrysalidocarpus lutescens seedlings using inoculum growing on rubber wood blocks showed that dikaryotic mycelia can cause basal stem rot infection.     

Genetic variability of the wild incompatibility alleles of the tetrapolar basidiomycete Agrocybe aegerita

Summary The variability of the sexual incompatibility genes of Agrocybe aegerita was investigated in the homokaryotic progeny of 13 wild dikaryotic strains from five distinct European geographic origins. Results of mating tests allowed identification of 18 A alleles and 16 B alleles out of a possible 26 different alleles for each in the sample. The determination and the comparison by a contingency ² test of the frequencies of allele replications between intra- and interregional matings showed no departure from a random distribution of incompatibility alleles. The allelic series estimated for the incompatibility genes of the entire population of A. aegerita, 30 A and 25 B aleles, are significantly less extensive than those already hypothesized for other tetrapolar hymenomycetes. However, the low variability of incompatibility genes has little effect on the outbreeding efficiency (92.6%) of this mushroom. The low variability of the incompatibility alleles and the apparent absence of intrafactorial recombination could relate to a single-locus structure of the incompatibility genes in A. aegerita.