Alternative reproduction pathway inAspergillus nidulans

Recombinant haploid segregants were recovered in filamentous fungus Aspergillus nidulans (Eidam) G. Winter directly from the heterokaryons instead of diploid segregants (process described earlier as parameiosis). In spite of the reproductive complexity of A. nidulans, parameiosis has only now been observed in this fungus. Since parameiosis was characterized by the occurrence of genetic recombination inside heterokaryotic hyphae, master strains (uvs+) and uvs mutants with high rate of both mitotic exchanges or chromosome nondisjunction were used to form heterokaryons. Two groups of mitotic segregants were recovered directly from heterokaryons--aneuploids and stable haploids. Heterokaryons formed with uvs mutants produced a higher number of parameiotic segregants compared to the heterokaryons formed with uvs+ strains. Segregants were analyzed by nutritional markers, acriflavine resistance and conidial color. Normal meiotic behavior of haploid recombinants was observed.     

Characterization of the Heterokaryotic and Vegetative Diploid Phases of MAGNAPORTHE GRISEA

The heterokaryotic and vegetative diploid phases of Magnaporthe grisea, a fungal pathogen of grasses, have been characterized. Prototrophic heterokaryons form when complementary auxotrophs are paired on minimal medium. Hyphal tip cells and conidia (vegetative spores) taken from these heterokaryons are auxotrophs with phenotypes identical to one or the other of the parents. M. grisea heterokaryons thus resemble those of other fungi that have completely septate hyphae with a single nucleus per cell. Heterokaryons have been utilized for complementation and dominance testing of mutations that affect nutritional characteristics of the fungus. Heterokaryons growing on minimal medium spontaneously give rise to fast-growing sectors that have the genetic properties expected of unstable heterozygous diploids. In fast-growing sectors, most hyphal tip cells are unstable prototrophs. The conidia collected from fast-growing sectors include stable and unstable prototrophs, as well as auxotrophs that exhibit a wide range of phenotypes, including many recombinant classes. Genetic linkage in meiosis has been detected between two auxotrophic mutations that recombine in vegetatively growing unstable diploids. The appearance of recombinants suggests that homologous recombination occurs during vegetative growth of M. grisea. No interstrain barriers to heterokaryosis and diploid formation have been detected. The mating type of the strains that are paired does not influence the formation of heterokaryons or diploids.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

Intraspecific heterokaryon and fruit body formation in Coprinus macrorhizus by protoplast fusion of auxotrophic mutants

Summary Fusion of protoplasts of Coprinus macrorhizus mutants with different amino acid requirements resulted in the production of prototrophic clones at frequencies of 1–4% of the protoplasts surviving the fusion treatment. The frequencies were at least 200 times higher than those of the appearance of revertants. Few prototrophic colonies appeared also when the mutant protoplasts were individually subjected to fusion treatment, or when they were mixedly cultured without fusion treatment. It was thus concluded that intraspecific heterokaryons were formed by protoplast fusion.The auxotrophic mutants did not form fruit bodies when cultured singly or mixedly with each other. In contrast, the heterokaryons produced by protoplast fusion between the mutants of compatible mating types developed into fruit bodies with intermediate morphology of those of the strains from which the mutants were derived. Heterokaryons were also formed by fusion of mutant protoplasts with identical mating genotype, but they failed to form fruit bodies.Abbreviations PEG polyethyleneglycol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid     

Extreme nuclear disproportion and constancy of enzyme activity in a heterokaryon ofNeurospora crassa

Heterokaryons ofNeurospora crassa were generated by transformation of multinucleate conidia of ahistidine-3 auxotroph withhis-3 ⁺ plasmid. In one of the transformants, propagated on a medium with histidine supplementation, a gradual but drastic reduction occurred in the proportion of prototrophic nuclei that contained an ectopically integratedhis-3 ⁺ allele. This response was specific to histidine. The reduction in prototrophic nuclei was confirmed by several criteria: inoculum size test, hyphal tip analysis, genomic Southern analysis, and by visual change in colour of the transformant incorporating genetic colour markers. Construction and analyses of three-component heterokaryons revealed that the change in nuclear ratio resulted from interaction of auxotrophic nucleus with prototrophic nucleus that contained an ectopically integratedhis-3 ⁺ gene, but not with prototrophic nucleus that containedhis-3 ⁺ gene at the normal chromosomal location. The growth rate of heterokaryons and the activity of histidinol dehydrogenase—the protein encoded by thehis-3 ⁺ gene-remained unchanged despite prototrophic nuclei becoming very scarce. The results suggest that not all nuclei in the coenocytic fungal mycelium may be active simultaneously, the rare active nuclei being sufficient to confer the wild-type phenotype.     

Inhibition of Aflatoxin B1 Production by Aspergillus parasiticus Using Nonaflatoxigenic Strains: Role of Vegetative Compatibility

The effect of vegetative compatibility on the inhibition of aflatoxin B₁ production by Aspergillus parasiticus was examined using nonaflatoxigenic strains. Nonaflatoxigenic white-conidial mutants were paired in different proportions on an agar medium with aflatoxigenic yellow-conidial mutants belonging to the same isolate, to the same vegetative compatibility group but with the original wild types differing in phenotype, and to different vegetative compatibility groups. Heterokaryosis as a result of hyphal anastomosis was detected by the presence of conidiogenous structures with a mixture of green and parental (white and/or yellow) chains of conidia. Sclerotium production (number and dry weight) was significantly greater in pairings of compatible strains that formed heterokaryons than in pairings of strains from different vegetative compatibility groups. In contrast, there were no consistent differences in aflatoxin B₁ inhibition by nonaflatoxigenic strains in pairings from the same vegetative compatibility group and pairings from different groups. Therefore, the composition of vegetative compatibility groups within a population may be of minor importance in predicting the efficacy of a particular nonaflatoxigenic strain for the biological control of aflatoxin contamination of crops.     

Genetic Recombination in Colletotrichum sublineolum

Genetic recombination without typical parasexuality (parameiosis) was shown in the fungus Colletotrichum sublineolum, causal agent of anthracnose on sorghum. The cross among auxotrophic mutants and mutants resistant to benomyl and cycloheximide confirmed the occurrence of heterokaryosis in this species. Aneuploid and haploid recombinants were obtained from heterokaryons. Some segregants released sectors continually after several replication cycles, and were considered aneuploids in the process of haploidization. Heterokaryons derived from crosses among mutants that presented low pathogenicity produced more virulent recombinants. Parasexuality with parameiosis may represent a natural mechanism for genetic variability amplification explaining the rapid appearance of new C. sublineolum physiological races.     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

Parasexuality in Race 65 Colletotrichum lindemuthianum Isolates

ABSTRACT. Heterokaryosis is the initial step of the parasexual cycle, a process that provides genetic variability in filamentous fungi through the production of heterozygous diploid nuclei. To characterize the parasexual cycle in Colletotrichum lindemuthianum, we evaluated the presence of heterokaryosis, vegetative compatibility reactions, and diploid formation among isolates of Race 65 collected from different Brazilian states. Vegetative compatibility groups were identified among the isolates according to their ability to form heterokaryons. Two heterozygous diploids were selected from compatible heterokaryons, which were characterized by the segregation of the parental auxotrophic markers and by RAPD profiles.     

Recombination after protoplast fusion in the yeast Candida tropicalis

Candida tropicalis protoplasts obtained by snail enzyme treatment were induced to fuse by the use of polyethylene-glycol. Heterokaryons formed by two auxotrophic strains were selected by complementation on minimal medium. These heterokaryons were unstable and readily dissociated into their nuclear components. Under appropriate conditions, the parental nuclei of an heterokaryon fused. The homokaryon so obtained was unstable and segregated into various types of auxotrophic and prototrophic recombinants.List of Abbreviations Used MM minimal medium - YEA yeast extract agar (complete medium) - YPGT yeast-peptone-glucosethiol (medium for protoplast preparation) - PTP medium for cell pretreatment (used before the action of snail enzyme) - PEG polyethylene glycol - p-FPA para-fluorophenylalanine - 5-FC 5-fluorocytosine     

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.     

Interspecific hybridization between Penicillium chrysogenum and Penicillium cyaneo-fulvum following protoplast fusion

Summary Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.     

High-frequency heterokaryon formation by Mucor racemosus.

Heterokaryons from Mucor racemosus were produced from two auxotrophic strains of the fungus. Germlings were converted to spheroplasts by using commercial chitinase and purified Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed and fused in solutions of polyethylene glycol and CaCl2. Under optimal conditions, prototrophic heterokaryons were formed at a frequency of about 5%.     

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate

A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.     

Heterokaryosis and the role of cytoplasmic inheritance in dark resting structure formation in Verticillium spp

Summary Auxotrophic and morphological mutants of Verticillium albo-atrum (producing darkly pigmented resting mycelium) and V. dahliae (forming dark microsclerotia) were isolated after treatment of conidia (haploid and uninucleate) with ultraviolet light. Hyphal tip and conidial analysis revealed that complementation between pairs of auxotrophs on minimal medium was due to a mosaic of homokaryotic and heterokaryotic regions with some hyphal tips growing syntrophically. A degree of incompatibility was observed in a few intraspecific, but in most of the interspecific, heterokaryon tests. Heterozygous diploid conidia (6–11 in length compared with 3–6 for haploids) were recovered at a frequency of 1 in 8x10⁶ by plating spores at high density on MM. Young diploid colonies segregated to give haploid and diploid sectors, some of which were recombinant types (parasexual cycle). Heterokaryons between complementary auxotrophs which were wild-type for dark pigmentation (hyl⁺) resembled wild-type and only darkly pigmented colonies were recovered by conidial analysis. Heterokaryons between hyl⁺ and hyaline (hyl) auxotrophs again resembled hyl⁺ morphology and usually only hyl⁺ colonies of both auxotrophic genotypes were recovered. Conidia from heterokaryons formed by stable hyl auxotrophs produced only hyl colonies of both auxotrophic genotypes. The important role played by cytoplasmic factors in the inheritance of darkly-pigmented resting structures in Verticillium was strongly confirmed by the present work.     

Recombinogenic and mutagenic effects of the antitumor antibiotic bleomycin in Aspergillus nidulans

Bleomycin, an antibiotic and antineoplastic drug that inhibits DNA synthesis and causes several types of chromosomal aberration, was found to increase mitotic recombination in Aspergillus nidulans. Heterozygous prototrophic diploid strains grown on media containing bleomycin produced significant increases of yellow and white sectors compared with controls. Further, the increased colour segregants were due to mitotic crossing-over, whereas the non-dis junctional segregants remained at the control level. Bleomycin also induced point mutations in the methionine-suppressor system of the methGl biAl strain of Aspergillus nidulans. Conidia treated in suspension with various concentrations of bleomycin increased the methionine-independent mutants 30-fold and more.     

Genetic analysis of polyauxotrophy and early mitotic progeny of Saccharomyces cerevisiae zygotes. II. Spore-forming segregants in the mitotic and meiotic progeny of polyauxotrophic clones

This article continues the investigation of polyauxotrophic (PA) clones formed in early mitotic progeny of zygotes. Cloning and segregation analysis of PA progeny suggest an unusual state of diploid genome in these strains, which is expressed as elimination of the dominance effect of the wild allele and as suppression or conversion of either of two loci of mating type. In PA progeny, except for recombinant haploids, sporulating diploids and unstable clones were detected. The tetrad analysis of the diploids points to homozygotization for individual markers. Over-replication of diploid set of chromosomes, prior to meiosis, and replacement of the haploid nucleus (the product of meiosis) for the diploid nucleus may explain the appearance of sporulating segregants in the diploid meiotic progeny. Unstable segregants may be considered as heterokaryons with complex interaction of nuclei.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

Mating-Type Differentiation by Transposition of Controlling Elements in SACCHAROMYCES CEREVISIAE

The nonfunctional mutation of the homothallic gene HML alpha, designated hml alpha, produced two mutant alleles, hml alpha-1 and hml alpha-2. Both mutant clones were mixed cultures consisting of a mating-type cells and nonmating haploid cells. The frequencies of the two cell types were different, and a few diploid cells able to sporulate were found in the hml alpha-2 mutant. Conversions of an a mating-type cell to nonmater, and vice versa, were observed in both mutants. The conversion of an a mating phenotype to nonmating is postulated to occur by alteration of the a mating type to the sterile mating-type allele in the hml alpha-1 mutant. In tetrad dissection of prototrophic diploids that were obtained by rare mating of hml alpha-1 mutants with a heterothallic strain having the MATa ho HMRa HMLa genotype, many mating-deficient haploid segregants were found, while alpha mating-type segregants were observed in a similar diploid using an hml alpha-2 mutant. The mating-type-deficient haploid segregants were supposed to have the sterile alpha mating-type allele because the nonmating genetic trait always segregated with the mating-type locus. Sporogenous diploid cells obtained in the hml alpha-2 mutant clone had the MATa/MAT alpha HO/HO HMRa/HMRa hml alpha-2/hml alpha-2 genotype. These observations suggested that the hml alpha-1 allele produces a transposable element that gives rise to the sterile alpha mating type by transposition into the mating-type locus, and that the hml alpha-2 allele produces an element that provides alpha mating-type information, but is defective in the structure for transposition.     

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate

K undu , P.N. & D as , A. 1985. A note on crossing experiments with Aspergillus niger for the production of calcium gluconate. Journal of Applied Bacteriology 59 , 1–5.
A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N-nitro-N-nitrosoguanidne (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.