Apogamic induction of haploid plasmodia in a myxomycete,Didymium iridis

Interisolate crosses between haploid (mean DNA = 0.32) CR 5-5 (A²) myxamoebae and polyploid (mean DNA = 1.80) CR 2–25 (A⁵) myxamoebae of the myxomycete Didymium iridis result in plasmodia that have the haploid (mean DNA = 0.32) DNA content rather than the predicted polyploid value. F₁ clones possess the mating type allele of the CR 5-5 clone only, and they also have the same mean DNA content as CR 5-5 myxamoebae. Crosses between these F₁ clones and CR 2–25 myxamoebae again resulted in the production of haploid plasmodia. Hence, the polyploid CR 2–25 clone appears to induce the CR 5-5 clone to produce plasmodia without involving itself in nuclear fusion.     

DEVELOPMENT OF APOGAMIC AMOEBAE FROM HETEROTHALLIC LINES OF A MYXOMYCETE,DIDYMIUM IRIDIS

By making appropriate crosses between heterothallic sexual clones of Didymium iridis we can recover apogamic lines in the F₁ generation. In this organism, heterothallic forms typically produce a haploid myxamoebal stage, but recently two diploid myxamoebal clones homozygous for mating types were discovered. When these are crossed, A²A² x A⁵A⁵, tetraploid Plasmodia are produced which later yield diploid F₁ meiospores. Sixty-four percent of the single-spore-derived clones produce both myxamoebae and Plasmodia, while the remainder do not progress past the myxamoebal stage. These results are consistent with the predictions that from tetraploid nuclei, mating types should segregate in the meiospores in a ratio of 1A²A²:4A²A⁵:1A⁵A⁵, and that myxamoebae heterozygous, A²A⁵, for mating type should yield Plasmodia apogamously. As the means for verifying relative ploidy levels of myxamoebae and Plasmodia, nuclear DNA was measured with a scanning microspectrophotometer.     

QUANTITATIVE MICROSPECTROPHOTOMETRY OF NUCLEAR DNA IN SELFING STRAINS OF THE MYXOMYCETE DIDYMIUM IRIDIS

Genetic and cytochemical investigations of the origin, development, nuclear activity, and ploidy level of Plasmodia obtained from selfed clones S-2 and B₁P-33 of the heterothallic myxomycete, Didymium iridis, are presented. To demonstrate that selfing did not result from contamination of the clones, or mutations at the mating-type locus, crosses were made between F₁ clones and clones of known mating types. The data were inconsistent with these two possibilities. DNA was quantified by Feulgen-DNA microspectrophotometry. All cellular phases studied (logarithmic amoebae, swarmers, and encysted amoebae) appear to be haploid, with the nuclear DNA being in the replicated (2C) state. The plasmodia are in all cases diploid; however, the data indicate that the selfed Plasmodia are in an extended G₁ condition. The nuclear DNA content of these is therefore 2C, whereas that of the cross Plasmodium is 4C. Sporangial nuclei exhibit DNA in diploid replicated (4C) category.     

NUCLEAR DNA CONTENT OF MYXAMOEBAE AND PLASMODIA IN SIX NON-HETEROTHALLIC ISOLATES OF A MYXOMYCETE,DIDYMIUM IRIDIS

The nuclear DNA content of six non-heterothallic isolates of the myxomycete Didymium iridis was measured by combining the Feulgen reaction with absorption microspectrophotometry. This allowed us to distinguish between homothallic (sexual) and apogamic (non-sexual) isolates. Four of the isolates studied, Panamanian 4 and 5, California 1, and Missouri 1 are homothallic. Moreover, the average DNA content of the myxamoebal and plasmodial nuclei (0.32 and 0.61 respectively) does not differ significantly from the calculated haploid and diploid values for heterothallic isolates of D. iridis (0.34 and 0.63). Hence, it is concluded that in each of these isolates the myxamoebae are haploid and the plasmodia diploid. In two of the isolates investigated, Georgia 1 and Hawaii 1, the DNA content of the myxamoebal and plasmodial nuclei did not differ significantly. Therefore, in both of these isolates the plasmodia appear to develop apogamically. In addition the mean DNA values recorded for the Ha-1 isolate suggest that it is aneuploid.     

The Life Cycle of Didymium laxifilum and Physarum album on Oat Agar Culture

The plasmodial slime molds is the largest group in the phylum Amoebozoa. Its life cycle includes the plasmodial trophic stage and the spore‐bearing fruiting bodies. However, only a few species have their complete life cycle known in details so far. This study is the first reporting the morphogenesis of Didymium laxifilum and Physarum album. Spores, from field‐collected sporangia, were incubated into hanging drop cultures for viewing germination and axenic oat agar plates for viewing plasmodial development and sporulation. The spores of D. laxifilum and P. album germinated by method of V‐shape split and minute pore, respectively. The amoeboflagellates, released from spores, were observed in water film. The phaneroplasmodia of two species developed into a number of sporangia by subhypothallic type on oat agar culture. The main interspecific difference of morphogenesis was also discussed.     

STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Field evaluations of systemic fungicides and derivatives of dithiocarbamic acid for the control of clubroot

Strains of Botrytis cinerea and Mucor mucedo germinated and grew over the range 0.25^°C. There were differences in germination rates and growth rates between strains of B. cinerea at any given temperature. Five of the benomyl-resistant strains germinated and grew more slowly than any of the other benomyl-resistant or benomyl-sensitive strains of B. cinerea tested. Strains of Rhizopus stolonifer and R. sexualis germinated and grew between 5 and 25^°C, and although some spores germinated at 2^°C, subsequent growth of the germ tubes and growth from a mycelial inoculum did not occur. Neither species germinated or grew at o^°C. The effect of temperature on mycelial growth in vitro was consistent with the ability of the strains of the four species to infect strawberry fruits.     

RECIPROCAL TRANSFER OF MATERIALS BETWEEN ALGAL CELLS AND MYXOMYCETE PLASMODIA IN INTIMATE ASSOCIATION

Zabka , George G. (U. Iowa, Iowa City), and Waldo R. Lazo . Reciprocal transfer of materials between algal cells and myxomycete plasmodia in intimate association. Amer. Jour. Bot. 49 (2) : 146–148. Illus. 1962.—Pure cultures of Fuligo cinerea, a myxomycete, and Chlorella xanthella, a green alga, were separately permitted to accumulate sodium radiophosphate from an agar medium and come into full association with non-radioactive cultures of the other organism. Manipulation of such mixed cultures indicates that a Plasmodium of Fuligo cinerea and vegetative cells of Chlorella xanthella are both able to absorb radiophosphorus from a nutrient medium and transfer it to each other. It is suggested that these phenomena support the possibility of a symbiotic relationship between the alga and myxomycete in association.     

Multiple Alleles and Other Factors Affecting Plasmodium Formation in the True Slime Mold Physarum polycephalum Schw*

SYNOPSIS. The life cycle of the true slime mold Physarum polycephalum includes 2 vegetative stages: the multinucleate coenocytic plasmodium and the uninucleate amoeba. A clone of amoebae established from a single spore does not normally yield plasmodia. Plasmodia are formed when amoebae from particular clones are mixed; thus plasmodium formation is said to be controlled by a ‘mating-type’ system. Previous work by the author with a sample of P. polycephalum derived from a single source revealed that 2 mating types were present and were determined by a pair of alleles at 1 locus. The present paper reveals the presence of 2 more mating types in a sample of P. polycephalum derived from a different source and provides evidence that these are determined by 2 alleles at the same locus as the other 2. Evidence for the presence of other inherited factors affecting plasmodium formation, the mode of action of these factors and possible explanations for the occurrence of plasmodia in single-spore cultures are also discussed.     

GENETICAL AND CYTOLOGICAL EVIDENCE FOR CHROMOSOMAL ELIMINATION IN A TRUE SLIME MOLD,DIDYMIUM IRIDIS

In crosses involving a polyploid myxamoebal clone. CR 2-25*, F₁ plasmodia and myxamoebae display a variety of unexpected ploidy levels as indicated by nuclear DNA measurements. Genetic analyses of the F₁ generations reveal either complete elimination of certain genetic markers or greatly skewed segregation ratios. On the basis of these two kinds of evidence, it is assumed that chromosome elimination occurs at some stage (or stages) following karyogamy between parental nuclei. The possible significance of polyploidy in relation to myxomycete speciation and evolution is discussed.     

Genetic Analysis of Resistance to Benzimidazoles in Physarum: Differential Expression of β-Tubulin Genes

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a β-tubulin with noval electrophoretic mobility, in addition to a β-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for β-tubulin, and that at least two β-tubulin genes are expressed in myxamoebae. Comparisons of the β-tubulins of wildtype and benD210 strains by gel electrophoresis revealed that, of the three (or more) β-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia.     

CYTOPHOTOMETRIC MEASUREMENT OF NUCLEAR DNA IN SEVEN HETEROTHALLIC ISOLATES OF DIDYMIUM IRIDIS,A MYXOMYCETE

Absorption cytophotometry was used to measure nuclear Feulgen-DNA content of myxamoebae and Plasmodia in seven heterothallic isolates of Didymium iridis. Measurements of myxamoebal nuclei from clones of four isolates (Hon 1, Pan 1, Pan 2, and CR 5) gave a mean DNA value of 0.34, whereas the nuclei of Plasmodia which develop from each of the four intraisolate crosses had a mean value of 0.63. These values correspond to the 2C haploid level in myxamoebae and the 4C diploid level in Plasmodia. DNA values in two additional isolates (Pan 3 and CR 2) are much higher than the mean for the other five. Accordingly, it is proposed that these may be polyploid. The question of polyploidy in D. iridis and in other myxomycetes is evaluated. The seventh isolate, Ky 1, is taxonomically very close to D. nigripes and was not included in calculations of mean values for D. iridis.     

Agarose Medium for Germination of Oospores of Phytophthora cactorum

When oospores of Phytophthora caetorum from 30-day-old culture were treated with 0.25% KMnO₄ for 20 min and incubated at 24°C under light for 10 days, 65–75% germinated on water agar and water agarose but only 1–21% germinated on V-8 agar and S+L agar. Water agarose was selected because germinated oospores formed restrieted colonies on this medium that could be isolated easily. KMnO₄ treatment killed sporangia, chlamydospores and mycelial fragments present in oospore suspensions. Under the above conditions, approximately 44% of oospores from 10-day-old culture germinated and the optimum germination rate of about 75% was obtained when oospores reached about 20 days old.     

A DEVELOPMENTAL STUDY OF GUTTULINOPSIS VULGARIS (ACRASIALES)

The cellular slime mold, Guttulinopsis vulgaris E. W. Olive, is a common and widely distributed species. Its myxamoebae have lobose pseudopodia and aggregate, not by streaming, but by movement singly or in small groups into the aggregation center. The resulting shield-shaped pseudoplasmodium gives rise to one (or possibly more on occasion) whitish, simple or compound sorocarp. Sorocarps are 95–465 μ in height, typically with relatively short, stout stalks and with mucilaginous spore heads that measure 75–350 μ broad. The spores are mostly irregular, with flattened or concave sides, and are surrounded by a thin spore wall. They measure 3.8–9.2 μ diam., or 2.6–7.8 × 3.9–9.2 μ. During sorocarp development the myxamoebae become grouped within membranous compartments both in the sorocarp and pseudoplasmodium. In the upper part of the sorocarp they develop almost entirely into spores, while in the lower stalk region of the sorocarp and in the pseudoplasmodium, some myxamoebae develop into spores while others degenerate. The phylogenetic position of Guttulinopsis is unclear, but the genus is probably not closely related to the Dictyosteliaceae. This investigation has been supported by National Science Foundation Grants G–44263 and GB-501. The writer is grateful to Miss Carmen Stoianovitch for her assistance.     

The effect of volatile and gaseous metabolites of swelling seeds on germination of fungal spores

Effects of volatile and gaseous metabolites of swelling seeds of pea, bean, wheat, corn, cucumber, tomato, lentil, carrot, red pepper and lettuce on germination of spores of five genera of fungi were found to depend rather on the fungal than on the plant genus. Germination of spores ofBotrytis cinerea, Mucor racemosus andTrichoderma viride was most severely inhibited. Spores ofVerticillium dahliae were less sensitive and germination of spores ofFusarium oxysporum was inhibited only in two cases. On the other hand, exudates of pea and bean stimulated germination of spores ofFusarium oxysporum. Also spores ofTrichoderma viride germinated better in an atmosphere enriched with exuded metabolites of swelling lettuce seeds. When carbon dioxide produced by the swelling seeds was absorbed in potassium hydroxide, spores ofTrichoderma viride andVerticillium dahliae did not germinate at all, the inhibitory effects of volatile and gaseous exudates on germination of spores ofMucor racemosus were accentuated, and also the percentage of germinated spores ofFusarium oxysporum decreased. Germination of spores ofBotrytis cinerea was not influenced. Absorption of volatile and gaseous metabolites in a solution of potassium permanganate decreased in most cases their inhibitory effects, particularly inBotrytis cinerea.     

Genetics of somatic fusion in a myxomycete: F2 studies

Summary Extensive genetic studies have been undertaken on the control of asexual cell (plasmodial) fusion inDidymium iridis. At least 6 loci control asexual cell fusion and mutual exchange of protoplasm in the Pan. 1 strain. In this study it was determined that labor-saving use of backcrosses provides reliable genetic data which is in accord with more extensive F₂ studies. Additional fusion data on 45 Pan. 1 clones are reported and possible biochemical control mechanisms of the fusion loci are discussed.     

Apparent bisexual behavior of yeast strains obtained from hybridization of industrial yeasts of the genusSaccharomyces with auxotrophic diploids

During a genetic study of some hybrids of brewer's and distiller's yeast strains with impaired sporulation characteristics and genetically marked auxotrophic aa and alpha alpha diploids, strains which showed positive mating reactions with both a and alpha haploid tester strains were observed. These strains proved to be homothallic and sporulated freely. The original hybrids, which appeared to be tetraploid, usually yielded sporulating single-spore clones on dissection of asci formed from them, with few or no mating strains among them. Dissection of asci from these clones yielded some single-spore clones which showed mating reactions with one or the other or both haploid tester strains, and further selection produced strains which on sporulation and dissection yielded single-spore clones which were apparently bisexual and sporulated freely. These strains proved to be homothallic, yielding single-spore clones which were all of the a mating type, and in which the mating reaction and, possibly, the action of the genes for homothallism were impaired, so that sporulating, non-mating diploids and haploids of both mating types were present in cultures originally obtained as single-spore clones.     

Life Cycles of Myxogastria Stemonitopsis typhina and Stemonitis fusca on Agar Culture

Myxogastria is a group of protozoa characterized by cellular uninucleate amoeboflagellates (myxamoebae and flagellated swarm cell), acellular multinucleate plasmodia, and stationary spore‐bearing sporocarps. The Stemonitales is a large order in the Myxogastria and contains approximately 230 species, but only 13 species have their completed life cycles observed so far. Here, we described the life cycles of two species in Stemonitales, Stemonitopsis typhina and Stemonitis fusca by culturing in water agar medium and observing the morphogenesis of their spore germination, plasmodium, and sporocarp development. The spore‐to‐spore life cycles of Ste. typhina and S. fusca were completed in approximately 67 and 12 d, respectively. Both species possessed an aphanoplasmodium. However, the spores of Ste. typhina and S. fusca germinated by the V‐shape split and pore methods, respectively. Unlike S. fusca with an evanescent peridium, Ste. typhina produced a shiny persistent peridium which was continuous with the membrane surrounding its stalk. The information will contribute to a better understanding of their taxonomy and phylogeny.