Methods, Results and Discussion

The study of differentiation of mushroom mycelium is usually encumbered by a bulky medium in which the mushrooms form. The mycelium of morel mushrooms (Morchella spp.) is readily established in pure culture from ascospores (Hervey, Bistis & Leong, 1978; Gessner, Romano & Schultz, 1987) or stipe tissue (Ower, 1982; Brock, 1985), but ascocarps have only been produced in a soil-like medium (Ower, Mills & Malachowski, 1986). In This study, Morchella mycelium differentiated into an anomaly on agar media providing an unusual opportunity to study differentiation.

The key factor allowing differentiation to occur was suitable nitrogen sources at sufficiently high concentrations. Cultural studies provided information on the physiology of differentiation, and the morphology of the anomaly provided information on the evolution of the genus.

Cultures were started from single, germinating ascospores derived from dried ascocarp tissue of M. esculenta and M. angusticeps, both from Michigan. Tissue was rehydrated in sdH₂O; spores were pressed out with a glass rod; dilutions were made when needed; and a drop of suspension was plated on thin agar without nutrients. Germinating spores were selected in 16-24 h by observing microscopically through the bottom of the plate and marking the location. About 10 mm² of agar was transferred from the marked area.

For subculturing, a basal medium was used. It consisted of the mineral elements of the yeast formula (Wickerham, 1951) without nitrogen at pH 7.0. Organic nutrients were filter sterilized separately and concentrated to prevent heat damage, but dextrose was heat sterilized concentrated in dH₂O. The incubation temperature was 18°C.

Differentiation occurred on agar media containing dextrose 2-4% and casein hydrolysate 0.4-0.8% plus L-arginine HCl 0.4-0.5% or urea 0.2-0.4%. Single nitrogen sources promoting differentiation were L-glutamine 0.8% and L-asparagine 0.8%. The amount of total nitrogen required for differentiation was 1.7 ±0.3 g/L.

The second nitrogen source needed with casein hydrolysate apparently promoted nucleic acid synthesis, and the enhanced metabolism improved the condition of the cells for differentiation. As exceptions, glutamine and asparagine were good starting materials both for amino acids and nucleic acids, as known to be the case with yeasts (Magasanik, 1992).

In agar slants (20x150 mm), a high density mycelium formed at the top after normal growth was completed, which was about 10-15 d. The thin agar below the high density mycelium would normally support little mycelial growth demonstrating that the differentiation was endotrophic. In other words, some of the nutrients were derived from mycelium extending down the slant.

With petri plate cultures and suitable progression of differentiation, ascocarp pigment appeared within the high density mycelium. Morchella esculenta produced gray pigment, but only when a cutout was made in the high density mycelium, as resulted from subculturing (Fig. 1).



The need for a cutout indicated that increased aeration was stimulatory to pigment production or the progression of differentiation. With that mycelial type, an orange pigment sometimes formed below the gray pigment (Fig. 2).

The high density mycelium always formed around the inoculum and extended out from it. It could be distinguished from surrounding mycelium in about 14 d, whereupon a boundary developed around it. The progression is shown with Morchella angusticeps in Figs 3-6.



On day 11, the white disc had a diameter of 82 mm. On day 16, a high density brown disc had a diameter of 65 mm and a boundary around it, while the outer mycelium was thin. The shrinkage of disc diameter, development of a boundary and thinness of extended mycelium are evidence of cell material migrating from the outer mycelium to the high density mycelium in a endotrophic manner. A similar translocation of cell material has been found to occur during sclerotia formation with motion in the direction of greatest nutrient utilization (Amir et al., 1993).

The cell structure of the high density mycelium was filamentous on the upper surface, with cell density increasing toward the agar. On the agar surface below the high density mycelium, a tissue formed having a rubbery, brittle texture similar to that of the ascocarp. The cells extending farthest above the high density mycelium were long and straight with few septa. The septa became more closely spaced at lower depths breaking ultimately into separate polymorphic cells which aggregated into tissue. The agar provided a support base for a layered structure indicating an adaptation to differentiation on a surface. High aeration at the surface along with rich nutrient availability appeared to create the conditions which promoted differentiation.

The onset of differentiation could not be determined. The most objective criterion for differentiation was boundary formation. The differentiated mycelium maintained viability for 4-6 wk from inoculation reforming exotrophic mycelium when subcultured. Mycelium exposed on an agar surface deteriorated due to dehydration and toxic effects requiring frequent reisolation.

The formation of a tissue, as found on agar, would not be advantageous during normal growth, which demonstrates that the differentiated mycelium was an anomaly. Considering the structured nature of the growth, including zoned ascocarp pigment and a tissue, the high density mycelium appeared to be a rudimentary formation of a mushroom as it existed earlier in evolution. The primitive morphology including a lack of strict controls indicates evolution from a yeast. The ability to revert to a primitive form indicates that the evolution was very recent. Such a formation could have been found at the base of trees during the cold conditions of an ice age (Erickson, 1990), which would reduce dehydration. The base of trees could provide soluble nutrients as exudate and an interface between above and below ground growth for the transitional state of evolution.

Two likely genera from which Morchella evolved are Nadsonia and Schizosaccharomyces--the first based on physiology, and the second based on morphology. Nadsonia includes species found in tree exudate (Miller & Phaff, 1984). Those species have adapted to survival on tree bark through endotrophic sporulation (Novak, 1981), which allows survival after being washed over the surface by rain.

Nadsonia has life cycle characteristics similar to those of Morchella, both being haploid (Gessner et al., 1987; Yoon, Gessner & Romano, 1990; Miller & Phaff, 1984) with karyogamy and meiosis occurring in succession during ascospore formation (Volk & Leonard, 1990, Miller & Phaff, 1984; Phaff, Miller & Mrak, 1966).

If, however, morphology is weighed more heavily than physiology, the ancestor may be in the genus Schizosaccharomyces, which contains filamentous yeasts and sometimes shows 8 spores per ascus (Yarrow, 1984), as does Morchella (Volk & Leonard, 1990). The life-cycle characteristics of Schizosaccharomyces are similar to those described above, except that somatic conjugation of vegetative cells usually precedes ascus formation (Yarrow, 1984; Phaff et al., 1966).

Nitrogen depletion appeared to have a major effect upon differentiation and was therefore studied in some detail. A stimulatory effect by nitrogen depletion was tested in agar slants. The high density mass formed more readily at the top of slants, where agar was thin and nutrients depleted, than lower down. This effect was studied with M. angusticeps using dextrose at 3% and urea 0.2% with casein hydrolysate 0.5%. The high dextrose concentration was stimulatory to differentiation on thin agar.

In this test, the boundary, being an objective criterion for differentiation, formed only where the agar depth was moderately thin, which meant around the inoculum when placed on the upper half of the slant but not when placed lower down. The agar depth determined the amount of nitrogen available and its rate of depletion indicating that depletion of nitrogen was stimulatory to differentiation. A similar effect was observed with slanted petri plates. The boundary was sharp where agar was thin, but it became invisible where agar was thick.

Nitrogen depletion was not however totally necessary. Differentiation including pigment production sometimes occurred with thick agar (The plate in Fig. 1 had an agar depth of 15 mm.), where growth was not proportionately heavier, and nitrogen was apparently not totally depleted. Another point of evidence was in the need for nitrogen availability to be extended for some time to produce black pigment, which was formed late in the progression of differentiation. Dextrose had to be used at the moderate level of 2%, not 3%, to produce black pigment effectively. The lower dextrose level reduced the rate of differentiation which would delay the depletion of nitrogen. Stated differently, a high nitrogen to carbon ratio promoted black pigment production; but nitrogen was near the maximum level tolerated, and only a reduction of carbon would change the ratio suitably. Similarly, arginine promoted black pigment formation, and it is known to not be metabolized rapidly (Magasanik, 1992). The need for extended nitrogen availability for black pigment production indicates that nitrogen was not depleted at least during the earlier part of differentiation.

The partial influence of nitrogen depletion indicates that the direct inducer of differentiation was an energy peak which was promoted by inhibited synthesis due to nitrogen deficiency. This mechanism was found to control the sporulation of yeasts, both exotrophic (Croes, 1967) and endotrophic (Novak, 1981), where the physiology has been studied in some detail. Evidence indicates, as described below, that endotrophic differentiation controlled by the energy level is the method by which soil mushrooms in general form.

With yeasts, the test of endotrophism is sporulation in distilled water (Novak, 1981). In this study, the test was differentiation on thin agar, where nutrients were depleted. The general mechanism of endotrophism for mushroom formation would be the accumulation of cell mass within the mycelium before it is transferred to the sporocarp as nutrients, preformed molecules and cell components.

Evidence for endotrophism with soil mushrooms in general is in the need for a build up of mycelial mass before mushrooms form (Stamets & Chilton, 1983). Evidence for a link to the energy level is in the need for a casing layer above the mycelium (Stamets & Chilton, 1983). The casing layer would restrict oxygen availability thereby limiting vegetative growth while causing reduced energy to accumulate as NADH. As the mycelium approaches the surface of the casing, an increase in oxygen availability would produce a sharp rise in the level of ATP inducing mushrooms to form.

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