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.     

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.     

Why does the bioluminescent fungus <Emphasis Type="Italic">Armillaria mellea</Emphasis> have luminous mycelium but nonluminous fruiting body?

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin—the key component of fungal bioluminescent system—was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.     

Heterokaryosis inFusarium moniliforme

Ultraviolet light readily induced both auxotrophic and morphological mutants in isolates ofF. moniliforme from corn and sorghum. Sexual analysis of these mutants yielded a genetic map with several instances of linkage. Forced heterokaryons formed readily between auxotrophs of the same strain but not between auxotrophs of different strains. At least four nuclear gene loci, but not the mating-type locus, controlled heterokaryon formation in this fungus. In the auxotroph pairings, heterokaryosis was confined to the anastomosed cells; there was no evidence for nuclear migration through established hyphae. Genetic complementation occurred in anastomosed cells, and the products of this complementation were then translocated to the homokaryotic cells of the colony. No heterozygous diploids were recovered from the heterokaryons, although there was evidence for rare somatic gene recombination. Heterokaryosis in isolates ofF. moniliforme in nature is probably rare.     

Improvement in citric acid production by haploidization of Aspergillus niger diploid strains

Segregation of diploid strains by a haploidizing agent was used to improve citric acid producing strains of Aspergillus niger. Stable diploid strains were obtained via protoplast fusion between two citric acid-producing strains from different genealogies, one for shaking culture and the other for solid culture. Diploid strains were treated by benomyl as a haploidizing agent, and many segregants were obtained. Prototrophic segregants were selected and their haploidy was confirmed by their conidial size and DNA contents. The prototrophic segregants were very variable in their citric acid productivities, some of them better either in shaking culture or in solid culture than both the parental strains. The presence of methanol stimulated citric acid production by the parental and the diploid strains. However, all prototrophic segregants derived from one diploid strain had higher productivities in solid cultures without methanol than in those with methanol.     

Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii.     

Heterozygous diploids ofPenicillium Chrysogenum and their segregation patterns

Several heterozygous diploids were made between genetically labelled derivatives of two strains ofPenicillium chrysogenum which produced relatively large amounts of penicillin and were of divergent lineage. The derivatives were labelled with spore colour and nutritional mutations. The diploids, although uniform in having wild type spore colour and being prototrophic, ranged from types having penicillin yields close to that of the original parents to types having less than a quarter of this titre level. Intermediate types had titre levels of about half to threequarters that of the high yielding diploids. Segregants were selected which had arisen naturally and also after nitrogen mustard treatment; most had the spore colour and auxotrophic phenotype of one or other immediate parent. From diploids of low and intermediate titre only haploid segregants with the genetical markers of one parent could be recovered with intact penicillin yield; haploids with the genetic markers of the other showed a marked reduction in yield. However, from diploids of high yield, both parental types could be recovered showing no loss of their original penicillin yield. The bearing of these results is discussed on the suggestion that different degrees of homozygosity between diploids may account for the titre variation observed. An alternative suggestion that mutations suppressive to penicillin titre might cause such variation is also considered.     

Reproductive biology of the fernPhegopteris decursive-pinnata

It has been revealed that gametophytes of diploid plants ofPhegopteris decursive-pinnata have a low capability for intragametophytic selfing (Masuyama, 1979). In the present study, intergametophytic mating tests were conducted for the self-sterile gametophytes of four diploids to demonstrate the genetic factors responsible for such a low capability for selfing. The results of the tests indicated that the gametophytes carried two or more kinds of recessive embryonic lethal factors which were non-allelic with each other and that the occurrence frequency of the gametophytes with an identical recessive lethal factor was 13% to 27% in the gametophyte families of these four diploids The karyological study of a diploid sporophyte suggested not the tetraploid but the diploid constitution of somatic chromosomes. Based on these data, the diploid inheritance of two or three special deleterious genes with a synergistic interaction responsible for the embryonic lethality was hypothesized to elucidate the self-sterility in the diploids of this species.     

Heterogenomic recombinants from compatible nocardiae

Recombinants obtained from matings of Nocardia erythropolis x N. canicruria were tested for their genetic stability by comparing phenotypes from direct selection with the same population after unselected growth. Contraselective loci were employed in various combinations in order that all of the mapped characters might be subjected to unselected analysis. Some recombinant class types appeared as stable haploids, whereas others behaved as heterozygous diploids, segregating out new phenotypes. All regions of the parental genomes were found to be involved in segregation, implying that the entire mapped region can become merozygotic under standard mating conditions. On the basis of segregating phenotypes, the genetic potentials of these compatible nocardiae were ascertained as follows: the formation of a diploid with subsequent segregation of parental or haploid recombinant genomes or both; persistence of the diploid through many generations; continuing reassortment of genetic information by multiple matings between parental or recombinant organisms; and, very probably, second-round recombinations within the diploid. A considerable difference in the nuclear division time between the parental organisms was postulated to have significant effects on the nature of the unselected segregants.     

The Genetic System Controlling Homothallism in Saccharomyces Yeasts

There are four types of life cycles in Saccharomyces cerevisiae and its related species. A perfect homothallic life cycle (the Ho type) is observed in the classic D strain. Two other types show semi-homothallism; one of them shows a 2-homothallic diploid:2alpha heterothallic haploid segregation (the Hp type) and another, a 2-homothallic:2a segregation (the Hq type). In the segregants from these Ho, Hp, and Hq diploids, each homothallic segregant shows the same segregation pattern as its parental diploid. The fourth type has a heterothallic life cycle showing a 2a:2alpha segregation and the diploids are produced by the fusion of two haploid cells of opposite mating types. The diploids prepared by the crosses of alpha Hp (an alpha haploid segregant from the Hp diploid) to a Hq (an a haploid from the Hq diploid) segregated two types (Type I and II) of the Ho type homothallic clone among their meiotic segregants. Genetic analyses were performed to investigate this phenomenon and the genotypes of the Ho type homothallic clones of Type I and Type II. Results of these genetic analyses have been most adequately explained by postulating three kinds of homothallic genes, each consisting of a single pair of alleles, HO/ho, HMalpha/hmalpha, and HMa/hma, respectively. One of them, the HMalpha locus, was proved to be loosely linked (64 stranes) to the mating-type locus. A spore having the HO hmalpha hma genotype gives rise to an Ho type homothallic diploid (Type I), the same as in the case of the D strain which has the HO HMalpha HMa genotype (Type II). A spore having the a HO hmalpha HMa or alpha HO HMalpha hma genotype will produce an Hp or Hq type homothallic diploid culture, respectively. The other genotypes, a HO HMalpha hma, alpha HO hmalpha HMa, and the genotypes combined with the ho allele give a heterothallic character to the spore culture. A possible molecular hypothesis for the mating-type differentiation with the controlling elements produced by the HMalpha and HMa genes is proposed.     

Aspects of penicillin production in Aspergillus hybrids

Hybrids have been obtained by protoplast fusion of penicillin-producing strains of Aspergillus nidulans with strains of A. rugulosus which do not produce the antibiotic at detectable levels. Induced haploidization of the hybrids allowed the recovery of stable segregants, which were screened for penicillin production. Approximately half of the segregants from each cross produced penicillin, indicating segregation of one or several linked genes which are essential for penicillin biosynthesis. These essential genes were found to be located on linkage-group VI of A. nidulans by observing segregation in multi-marked strains. The penicillin-producing segregants showed a wide range of titres. Some segregants, which had recombinant phenotypes, produced significantly more penicillin than the A. nidulans parent.     

Vegetative Incompatibility and the Mating-Type Locus in the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM

The genetic basis of vegetative incompatibility in the cellular slime mold, Dictyostelium discoideum, is elucidated. Vegetatively compatible haploid strains from parasexual diploids at a frequency of between 10^-6 and 10^-5, whereas "escaped" diploids are formed between vegetatively incompatible strains at a frequency of ~10^-8. There is probably only a single vegetative incompatibility site, which appears to be located at, or closely linked to, the mating-type locus. The nature of the vegetative incompatibility is deduced from parasexual diploid formation between wild isolates and tester strains of each mating type, examination of the frequency of formation of "escaped" diploids formed between vegetatively incompatible strains, and examination of the mating type and vegetative incompatibility of haploid segregants obtained from "escaped" diploids.     

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.     

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.     

Aspects of genetic interaction in hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion

Hybrids were produced by protoplast fusion between strains of Aspergillus rugulosus and mitotic master strains of Aspergillus nidulans with a genetic marker on each linkage group. Analysis of segregants induced by growth on benomyl revealed recombination between every pair of unlinked markers. Parental combinations of markers were often recovered at significantly higher frequencies than expected. This aberrant segregation was not correlated with any particular pair of linkage-groups and was attributed to inter-species incompatibility. The segregation of genetic markers of A. rugulosus from the hybrids suggested that A. nidulans and A. rugulosus may differ in haploid chromosome number and chromosome size. In sexual crosses between A. nidulans and strains containing chromosomes of mixed parental origin recombinants were recovered. The results support the classification of A. nidulans and A. rugulosus as separate species.     

Genetics of CHLAMYDOMONAS REINHARDTII Diploids I. Isolation and Characterization and Meiotic Segregation Pattern of a Homozygous Diploid

A strain of Chlamydomonas reinhardtii has been investigated which, when mated with known wild-types, produces very few viable germination products and transmits its Mendelian markers to more than half of those products. Cytogenetic observations, fluorometric measurements of DNA and genetic data all suggest that the strain, d mt^-ery-M3a sr-u-1 is a stable homozygous diploid. This strain has twice as many nuclear chromatin bodies at metaphase and twice as much DNA as its haploid progenitor, and the phenotypes of its meiotic progeny are consistent with predictions based on triploid meiosis. Data from crosses involving d mt^-ery-M3a sr-u-1 and from crosses involving hybrid diploids indicate that the frequency of second division segregation increases in triploid zygotes and that mitotic segregation following triploid meiosis is a frequent event which may more often result from mitotic recombination than from chromosome loss.     

Systems and results of tests for chemical induction of mitotic malsegregation and aneuploidy in Aspergillus nidulans

In Aspergillus several types of test systems have been developed for detection of chemicals which induce aneuploidy and/or malsegregation of chromosomes. Results from 23 papers were reviewed in which numerical data for 42 chemicals had been reported. The test systems fall into two groups. One group includes all purely genetic tests that detect euploid mitotic segregants from heterozygous diploids and identify these either as products of malsegregation of chromosomes or as products of crossing-over (13 papers, several reviewed in detail previously; K?fer et al. (1982) and Scott et al. (1982)). The other group includes tests that treat haploid or diploid strains and detect aneuploids as unstable abnormally growing segregants which can be identified as specific disomics or trisomics by their characteristic phenotypes. In addition, such tests characterize abnormal segregants from heterozygous diploids by correlating phenotypes with patterns of genetic segregation in spontaneous euploid sectors. This analysis makes it possible to distinguish between induced primary aneuploidy of whole chromosomes and partial tri- or monosomy resulting from chromosome breakage and secondary spontaneous malsegregation (10 papers). Based on results of both types of tests, it is postulated that chemicals which cause increases of euploid malsegregants, but not of crossovers, normally induce aneuploids as primary products (as shown for 7 of the 14 cases). These include compounds which damage spindles or membranes (especially the well-known haploidizing agents) and generally are effective only when growing cells are exposed. (8 chemicals that may belong in this category could not be classified for certain, because information was insufficient.) On the other hand, chemicals which cause increases of all types of euploid segregants (11 cases), mostly induce drastic mutations and aberrations as primary effects and cause spontaneous malsegregation or crossing-over only as secondary events (as demonstrated for radiation-induced abnormals). In addition, a few chemicals were negative, because they increased only crossing-over or showed no increased segregation at all at concentrations which reduced survival or growth rate (9 cases). Recommendations are made for standardization of methods and protocols. New tester strains and specific procedures are outlined which should be useful for conclusive tests of chemicals that may induce aneuploidy.     

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.     

Asexual recombination in a uvsH mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.     

Alternation of nuclear phase in the filamentous basidiomycete,Helicobasidium mompa

Variation in the number of nuclei and cellular ploidy were observed in eight strains ofHelicobasidium mompa. The basidiospores, single-spore isolates and field-isolated strains were all dikaryons. The cellular ploidy, which was assessed by analyzing the fluorescence emitted by DAPI-stained nuclei, was unstable: monokaryotic strains derived from the original dikaryotic strains by successive subcultures were mainly tetraploid, although the original dikaryon was in most cases diploid. On the other hand, a dikaryotic strain derived by treatment with benomyl was haploid. These results suggest that diploid dikaryon is a normal nuclear phase ofH. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.