1. The frequency of hyphal fusions is strongly affected by various conditions; the main factors have been investigated. a) The frequency of fusions is proportionate to the density of conidia per unit volume of the medium; the higher frequencies were obtained when conidia were less than 10μ apart. b) The concentration and the reaction of the media are important factors; optimal concentrations are between 1/5 and 1/20 of the original concentrations, and optimal reactions are between pH 5.4 and 6.0. c) As concerns the effect of genetic conditions on anastomosis, there was scarcely any difference in the frequency of fusion between the test strain, Asp. oryzae K.B., and different strains of Koji-molds, but there were considerable differences in interspecific and intergeneric crosses. In the Aspergilli, Asp. tamarii, Asp. ochraceus, Asp. wentii, and various black Aspergillus species gave much higher frequencies of anastomosis than other species. In the Penicillia, only the Monoverticillata and some members of the Biverticillata anastomosed with the test strains in comparatively low frequencies. 2. The use of genetic markers (size of conidia, color of conidia, and nutritional requirements) has been examined for their usefulness in the detection of heterocaryons. a) The large size of conidia produced in multinucleate strains such as Koji-molds proved useful in detecting heterocaryons. b) The use of conidial color only as a criterion, without genetic confirmation, does not give reliable results. c) Double nutritional markers give critical evidence for the formation of heterocaryons. 3. Allelism-tests were carried out by using various mimic mutants and the presence of pseudo-allelism was suggested for an arginineless locus. The genetic steps in arginine synthesis were in accordance with those in N. crassa and in P. notatum, but nor in Asp. nidulans; arginine is synthesized from ornithine through citrulline. 4. Heterocaryotic strains are unstable and segregate out into their component strains. The segregation ratios are affected both by genetic back-ground and cultural conditions. 5. The phenotype of heterocaryons between two mutants is generally like that of the wild type, differing from the component strains in both morphology and physiology. a) The increase of conidial size and nuclear number of in heterocaryons is a remarkable characteristic of the Koji-molds. b) Heterocaryons obtained by mixed culture in various combinations showed enzyme activities that were weaker than either parent, intermediate between them, or like one or the other. 6. Heterocaryosis was induced by the UV treatment of homo-type strains in Asp. oryzae aX without hyphal fusion.
1. The artificial production of new stable heterozygous diploid strains was carried out by using mutants of Asp. Sojae with color and nutritional markers. 2. These new strains were obtained through the formation of heterocaryons in the following combinations: y leu+al his, y arg+al his, and y arg+al leu. 3. The frequency of the spontaneous occurrence of diploid strains differs according to the combinations of the component strains (0.2-3.0G per 106 plated conidia). 4. The frequency of the occurrence of the diploids was raised to 2-9 per 102 surviving conidia by UV irradiation-this was an increase of>104 times. 5. The properties of the heterozygous diploids are as follows: a) Conidial heads were uniformly green like the original wild type, while all heterocaryons had, in varying proportions, mixtures of white, yellow, and green conidial chains. b) Growth on M.M. was like that of heterocaryons but unlike either component strain. On C.M., or a synthetic medium supplemented with either or both of the growth factors required by the component strains, they exhibited mostly homogenous green colonies, instead of segregating out into yellow and albino sectors like the heterocaryon. c) They were exceedingly stable and 99.5 per cent of the colonies remained green. After 10-14 days' incubation, they rarely produced spots or sectors of either component color. The majority of the heterocaryons segregated into the two component strains, however, and only about 15 per cent remained heterocaryotic. d) Genetic analysis of the diploids showed the following results. As for the color markers, 0.1-0.5 per cent of yellow or albino segregants occurred. Among these, yellow segregants further gave albinos in low ratio (0.1-0.3 per cent). Concerning the nutritional marker, the diploids were prototrophic and yellow or albino segregants were either prototrophic or required the same substances as the component strains. However, in the combination y arg+al leu, three new segregants have been obtained which differ from the component strains. e) The diploids and their segregants were nearly the same as the original wild type and the component strains in the diameter of their conidia. f) Heterozygous diploids and their segregants had on the average about one half as many nuclei as the original wild type or the component strains. g) The DNA content per conidium was nearly the same as the original wild type and the component strain, but the DNA content per nucleus was about twice as large. h) The new efficient method developed for the production of new strains of asexual fungi promises much in the way of the strain improvement required by industry.