STIMULATION OF ASEXUAL REPRODUCTION OF PILOBOLUS BY MUCOR PLUMBEUS

PAGE, ROBERT M. (Stanford U., Calif.) Stimulation of asexual reproduction of Pilobolus by Mucor plumbeus. Amer. Jour. Bot. 46(8): 579-585. Illus. 1959.-The fact that a culture of Pilobolus kleinii which was contaminated with Mucor plumbeus produced far more sporangia than had ever been observed before on a synthetic medium suggested that M. plumbeus produced a substance which stimulated asexual reproduction in Pilobolus. When grown on a medium containing asparagine, sodium acetate, hemin, thiamine, and salts, P. kleinii consistently produced more trophocysts and sporangia when grown with M. plumbeus than when grown alone. Sterilized filtrate of culture medium in which M. plumbeus had grown also stimulated sporangium formation by P. kleinei. By the use of 2 types of 2-membered culture in which there was no liquid connection between the 2 fungi, it was shown that the stimulatory substance was volatile. In addition to being volatile at room temperature, the material was soluble in acid but not in alkali, and it gave a positive reaction with Nessler's reagent. Since (1) the material had some of the properties of ammonia, (2) the intensity of asexual reproduction in 2-membered cultures with absorbents was correlated with the distribution of ammonia, (3) M. plumbeus produced ammonia when grown on asparagine, aspartic acid, or NaNO3, and (4) the effect of the substance could be imitated either with gaseous ammonia or with ammonium salts at appropriate concentrations, it seems highly probable that the substance produced by M. plumbeus which stimulates asexual reproduction of P. kleinii and some other species of Pilobolus is ammonia. PILOBOLUs, a fungus which belongs to the phycomycetous order Mucorales and whose natural habitat is the dung of herbivorous animals, is characterized by its black, heavily cutinzed sporangia which are shot away from the sporangiophore at maturity. The genus is also distinctive in that each sporangiophore arises from a hyhal swelling known as a trophocyst. Pilobolus has been cultivated successfully on a synthetic medium which contains asparagine, sodium acetate, and hemin as its principal ingredients (Page, 1952). All of the species of Pilobolus which have been tested have grown on this medium, and stock cultures of P. kleinii van Tiegh., P. sphaerosporus Palla, P. crystallinus Fries, and P. umbonatus Buller, as well as several undescribed or unidentified strains have been maintained on it for as long as 3 yr. by weekly transfer of hyphal tips. In addition to vigorous mycelial growth, all strains have produced sporangia on the synthetic medium, and several strains have formed zygospores. That this synthetic medium was not the optimal one for Pilobolus was shown by a laboratory accident. In the spring of 1957, a fungus appeared as a chance contaminant in a culture of Pilobolus kleinii which was growing on the synthetic medium. In this contaminated culture, P. kleinii produced far more sporangia than had ever been observed on an agar medium. It seemed possible that the contaminant produced some substance which 'Received for publication March 7, 1959. The author wishes to express his gratitude to Dr. Samson R. Gross for many helpful suggestions received during the early part of this investigation and to Prof. G. M. Smith for reading the manuscript. greatly stimulated sporangium production by Pilobolus. The contaminant was isolated and sent to Dr. C. W. Hesseltine who very kindly identified it as Mucor plumbeus Bonorden. It is the purpose of this paper to report the results of experiments designed to elucidate the mechanism of this stimulation of asexual reproduction of Pilobolus by Mucor plumbeus. MATERIALS AND METHODS.-The strain of Pilobolus used for most of these studies was isolated from cow dung collected in 1956 in Marin County, California. This strain, which had both asexual and sexual characteristics typical of Pilobolus kleinii van Tiegh., was grown on synthetic medium (Page, 1952). This medium had the following composition: L-asparagine, 7.55 g.; anhydrous sodium acetate, 6.03 g.; hemin, 10 mg. (dissolved in 37.5 ml. 0.1 N NaOH); thiamine HCl, 10 mg.; KH2PO4, 0.5 g.; K,HPO4, 0.6 g.; MgSO4 7H.0, 0.5 g.; NaCl, 0.1 g.; CaCl2, 0.1 g.; stock solution of micronutrients (Ryan et al., 1943), 1.0 ml.; agar, 20 g., and distilled water, 962 ml. Stock cultures of Mucor plumbeus were maintained on potato-dextrose agar. The number of trophocysts or sporangia formed by Pilobolus was used as a measure of the intensity of asexual reproduction. In the usual procedure, cultures were inoculated with hyphal tips contained in agar blocks cut from the periphery of an actively growing mycelium. Cultures were incubated in the dark at 25?C. for 3 days and then were exposed briefly to light (150 ft.-c. for 100 sec.) from fluorescent tubes to induce the formation of trophocysts (Page, 1956). Following exposure, the cultures