The growing procedure developed by Ronald Ower in 1982 was tried commercially and failed to yield economically practical results. The procedure was cumbersome, requiring sterile conditions and small containers during the early phase, where large masses of sclerotia were produced. And it was unnatural, using sclerotia as a sole source of nutrients for the mycelium. Being abnormal, it was not easily modified or researched.

It also misdirected the science of the subject, as researchers assumed sclerotia was the determining influence over ascocarp formation and the key to solving the mysteries of Morchella.

The patent holders later reported that morels could be produced without sclerotia. Without sclerotia, the theoretical constructions became meaningless.

Nutritional Studies.

A series of nutritional studies based on dry weight measurements were typified by that of Thomas Brock in 1951. He noted that both mycelial growth and sclerotia formation are readily produced on synthetic media.

Pure cultures were started from stipe tissue. A basal medium was used as a derivation of Czapek-Dox medium, consisting of magnesium sulfate, potassium phosphate and ferric sulfate. Carbon was then added, and nitrogen at 250 mg/l.

He first tried measurements of rate of mycelial growth over agar in long tubes, which had been a procedure used in earlier studies. But it did not show as dramatic effects as dry weight with liquid media; so he switched to the later using 125 ml flasks. After six days of growth on liquid media, mycelial mats were removed, dried and weighed.

In comparing carbon sources, he found maximum dry weight from starch, then maltose, d(-)fructose, d(+)turanose, d(+)glucose, sucrose, etc.

With nitrogen sources, the sequence was l-cysteine-HCl, dl-aspartic acid, l(+)asparagine, , urea, etc.

There were numerous limitations to this procedure. Steam sterilization would have resulted in breakdown of many of the media components, as was noted for cysteine-HCl, where H₂S gas evolved. Growth on liquid would have been subject to numerous aberrations including a high density mat that forms between the immersed and aerial phases of growth. Inorganic nitrogen would have caused pH extremes to inhibit growth within a few days, but the author did not say what nitrogen source was used with the carbon.

The primary significance of such studies is that dry weight cannot be interpreted in nutritional or physiological terms. The assumption is that a high dry weight measurement indicates a good nutrient. What is a good nutrient, or what is good growth? Rate of growth was not studied, as only one data point was acquired. Implicitly, the starting point was zero, but neither the starting point nor end point would have been in line with the maximum rate, considering that a 1-2 day lag occurs at the start, and adverse effects usually inhibit growth in less that 6 days.

The dry weight produced in 6 days would result from the ability of the mycelium to overcome obstacles to producing cell mass. The primary factors influencing that result would be differentiation, pH extremes, depletion of nutrients, inhibited oxygen diffusion, dehydration of aerial mycelium and strain variations. Those variables cannot be interpreted with the methodology that was used.

Other studies using a similar methodology show widely varying results, which indicates that the conditions had a greater influence than the nutrients being studied.

The "Barrage" or "Meld."

In attempting to control the growth of Morchella mycelium through nutritional and cultural studies, Hervey, Bistis and Leong (1978) encountered an unusual ridge of growth that forms between mycelial types from different spore strains. They called the line of growth a barrage.

They used single spore isolates, combining two per plate, to test compatibility for possible heterothalism. In all non-self combinations, even with spores from the same ascocarp, a build up of aerial growth developed at the line of contact between the colonies.

The authors attempted to explain the phenomenon in terms of usual patterns of fungal life cycles, stating that it indicates "either (1) the ascus fusion nuclei are heterozygous for certain genetic factors, or (2) the asci of a single ascocarp do not all arise from the same pair of nuclei."

They also noticed patterns of variation in mycelial types which they called downy and granular. They then said, "These data again suggest (but do not prove) that heterokaryosis occurs at some time during the life cycle of M. esculenta."

Volk and Leonard (1989) studied the phenomenon further showing that heterokaryotic mycelium forms in the reaction zone between different types of mycelium. They referred to the reaction zone as a meld rather than a barrage, which emphasizes the new type of mycelium which forms. Drug resistant mutants were used to show that the mycelium in the center of the meld contained genes from both strains.

In comparing several thousand spores strains from the same ascocarp, a small percent (less than 3%) did not form a meld. The mycelia grew together with no visible line between them.

Spores from the same asci were evaluated. A pair referred to as sister spores did not result in a meld, while non-sister spores always did.

The Ower Procedure.

In 1982, Ronald Ower published a brief message stating that he had grown M. esculenta under laboratory conditions. He did not describe the procedure, because he wanted to patent it first.

He claimed to have solved the problem of growing morels by determining the life-cycle. The key factor was later revealed to be the production of sclerotia leading to the formation of ascocarps.

Ower's brief note showed photographs of ascocarp development for M. deliciosa, stating that the species is a synonym for M. esculenta. However, the former tends to be smaller. The size is significant, because other evidence indicates that the procedure will not produce a normal morphology for large morels.

A photograph of laboratory grown morels appeared in Discover magazine in May 1988 (Vogel). The image was deliberately out of focus (referred to as silhouetted), but the morphology was very distinctly abnormal. The caps were large and had a smooth surface unlike the sharply defined ridges that normally occur. Considering that the patent holders were trying to put their best foot forward, they must have been unable to produce a normal morphology.

When this author later ordered dried morels from the same company, they were a petite variety of a smaller species, M. angusticeps. The caps had normal ridges, but spores did not produce normal mycelium. Sclerotia was heavily encrusted in the mycelium producing a papery surface. Presumably, petite mutants were used, because the small size facilitated a normal appearing surface structure.

Later, a television program on the laboratory production of morels by the same company showed a tray of M. angusticeps. Even though the camera angle was very low showing only the tops, there was a visible ball of growth below each morel. Again, the growers were trying to conceal abnormal morphology.

This evidence indicates that the Ower procedure would not produce normal morphology in most instances. The theoretical reason would be that the mycelium normally spreads over a large amount of space in the soil, and when restricted in a tray, it cannot support complete differentiation with large morels. Producing an abnormal morphology would be impossible with other types of mushrooms, but the morel evolved so recently from an ancestor that lacked macromorphology that it does not have strict control over morphology.

A patent based on the Ower procedure was filed in 1986. The procedure involves producing a large amount of sclerotia under axenic conditions using cooked, sterilized wheat in pint jars. Above the wheat is a medium of soil. The depleted nature of the soil causes a large quantity of sclerotia to form, while the mycelium draws nutrients from the wheat below it. Inoculum is described as ascospores, vegative hyphae or sclerotia. Apparently, the pint jars could not be scaled up, because air must enter around the edge and diffuse into the center.

The sclerotia is removed and placed in nonsterile trays with a medium such as soil and peatmoss. Much water is percolated through. Depletion of exogenous nutrients is emphasized as essential to inducing differentiation. Maintaining complex environmental conditions is critical to preventing abortion of growth.

A later patent (1989) described the use of nutrient rich mycelium grown in liquid media as the spawn in place of sclerotia. In other words, sclerotia is not an essential part of production.

Theorized Life-Cycle.

A cytological study was conducted by Volk and Leonard (1990) and used as a basis for a life-cycle theory. They noted that the ascus mother cell contains a large number of nuclei. A pair of nuclei migrate to the tip of the ascus mother cell and fuse to form a diploid nucleus. Then meiosis occurs followed by four successive mitotic divisions creating eight ascospores per ascus. They reported observing eight nuclei per ascospore, presumably resulting from additional mitosis. (The ascospores contain a large and variable number of nuclei for the presumed purpose of increasing the mass to facilitate dissemination as projectiles.)

As they also reported, hyphae contain porate septa. Typically there are 10-15 nuclei per compartment, but sometimes as many as 50. Anastomosis is highly prevalent. Nuclear pairs are sometimes observed in the undifferentiated hyphae.

The authors theorized a life-cycle where plasmogamy between strain variants creates a heterokaryotic mycelium, which they referred to as secondary mycelium. It then is theorized to form heterokaryotic sclerotia which can either revert back to secondary mycelium or lead to ascocarp formation.

The theory was not tested with the Ower procedure to determine whether monoascosporous mycelium (so-called primary mycelium) would produce ascocarps.

The assumption that heterokaryotic mycelium has a role in the life-cycle is contradicted by much evidence. The unusual mycelium taken from the meld was difficult to grow, which is probably why it was not studied more thoroughly. Forming where two types of mycelium come together, nutrients would be depleted in the area limiting its growth potential. If its purpose were to create genetic interaction, a large amount of growth might not be necessary. But the heterokaryotic mycelium is given the role of leading to ascocarp formation, which would require a large amount of nutrients.

Furthermore, such interactions are not usually known to form a barrage or meld. The unusual growth stems from the cells being walled off after they exchange nuclei. Walling off the cells does not promote their growth but robs them of nutrients. This effect and other evidence indicates that the purpose of walling off the cells is to prevent the exchanged nuclei from diluting necessary variations in each spore strain. The spore strains are adaptations to environmental conditions, as studies by this author indicate.

Even with heterokaryosis, the suggested life-cycle is extremely simple. The sexual stage is limited to karyogamy and meiosis occurring in succession within the developing ascus. No mating types are suggested, and all vegetative growth is haploid. Conidia were added to the life-cycle based upon a misinterpreted study of a leaf mold a century ago. The error has not been studied in recent times, and it is discussed elsewhere.

Multiple Alleles.

The question of genotypes was studied through electrophoresis of isozymes by Gessner, Romano and Schultz (1987). They compared two species: M. esculenta and M. deliciosa. Two or three allotypes were found for several genes, and they varied with spore strains from the same ascocarp. There was more variation between species than within species indicating reproductive isolation between the species.

A large number of additional allotype variations were found within the genomes of Morchella in later studies by Yoon, Gessner and Romano (1990); Royse and May (1990); Jung, Gessner, Kuedell and Romano (1993) and Bunyard, Nicholson and Royse (1994).

The results were interpreted by Gessner, Romano and Schultz to mean that "different parental genomes exist in individual offspring from a single ascocarp." The authors suggest that the differences stem from a Mendelian population which segregates allotypes.

There is an unwarranted assumption in that conclusion. It is that differences in allozyme types represent differences in genotypes. Macrogenetics indicates that gene exchange homogenizes the gene pool of local populations. The mechanism involves a loss of half of all genes during recombination. The lost genes reduce variations within an interbreeding group. External sources of variation are required to increase diversity of genomes. These mechanisms eliminate heterokaryosis as a source of diverse genes circulating through the mycelium.

Therefore, such a large number of allotypes cannot represent differences in genotypes. The correct interpretation of the results, particularly when combined with a large amount of additional evidence, is that the differences are phenotypic but not genotypic. This result is similar to embryonic development, where cells which are genotypically identical differentiate into phenotypically different cell types. They do that by turning genes on or off in a manner which is stable through mitosis.

In all of these studies, the authors tried to shoe Morchella into a pattern similar to the fungi which they were familiar with. The pattern did not fit, and the authors often noted the unusual nature of their results. Yet they doggedly persisted in constructing illogical explanations which avoided any indication of Morchella evolving recently from a yeast.

The Future.

Work by this author indicates that controlled growth of morel mushrooms will require liquid nutrients, which of course must be sterilized. Sterilization should be advantageous when mechanized, because it allows greater control and speed of production and eliminates problems with pests. The same technology would have great advantages with Agaricus, because compost could be replaced with liquid nutrients including corn syrup. Such results are readily produced under laboratory conditions.

But unlike Agaricus, Morchella will not grow adequately in batch culture. The primary reason is because Morchella is adapted for spreading, while Agaricus is adapted for clumping. The use of extracellular enzymes to break down solid substrates requires a clumping mycelium, because the enzymes must be concentrated, and the nutrients are concentrated. But Morchella feeds on bacteria which are spread widely through the soil. And Morchella is accustomed to finding a continuous supply of such nutrients.

Therefore, liquid nutrients need to be sprayed over Morchella mycelium on a periodic basis. Between spraying, oxygen must be available, which requires good drainage, because liquids seal out oxygen.

The medium would have to be much more open than usual, because the spreading mycelium of Morchella seals the medium rapidly when using liquid nutrients. The medium might be shredded hardwood or a fibrous material like jute or hemp.

There is an ideal inoculum for the purpose. Morchella forms arthrospores when rich liquid nutrients are combined with high aeration. The arthrospores result from mycelium breaking into small blocks. The mechanism appears to create a method of water-borne dissemination, when mycelium grows to the surface of the soil during heavy rains. A critical characteristic of arthrospores is that they could all be of the same spore strain, which would prevent competition between different spore strains.

The breakdown of mycelium into arthrospores would probably not be a problem when spraying on liquid nutrients. If it is, temporary reduction in aeration might be required.

Induction may require covering the mycelium with a medium. However, a continuum of growth and direct control over differentiation would be preferable. Theory indicates that the mycelium would naturally differentiate when the mass got large and oxygen became restricted below the surface. The patent holders indicated that a depletion of nutrients was required for morel formation, but studies of yeast physiology indicate that only nitrogen needs to restricted to promote differentiation.

References.

Brock, T. D. (1951). Studies on the nutrition of Morchella esculenta Fries. Mycologia 43, 402-422.

Bunyard, B. A., Nicholson, M. S. & Royse, D. J. (1994). A systematic assessment of Morchella using RFLP analysis of the 28S ribosomal RNA gene. Mycologia 86, 762-772.

Gessner, R. V., Romano, M. A. & Schultz, W. R. (1987). Allelic variation and segregation in Morchella deliciosa and M. esculenta. Mycologia 79, 683-687.

Hervey, A., Bistis, G. & Leong, I. (1978). Cultural studies of single ascospore isolates of Morchella esculenta. Mycologia 70, 1269-1274.

Jung, S. W., Gessner, R. V., Kuedell, K. C. & Romano, M. A. (1993). Systematics of Morchella esculenta complex using enzyme-linked immunosorbent assay. Mycologia 85, 677-684.

Ower, R. (1982). Notes on the development of the morel ascocarp: Morchella esculenta. Mycologia 74, 142-144.

Ower, R. D., Millls, G. L. & Malachowski, J. A. (1986). Cultivation of Morchella. U.S. Patent No. 4,594,809.

Ower, R. D., Millls, G. L. & Malachowski, J. A. (1989). Cultivation of Morchella. U.S. Patent No. 4,866,878.

Royse, D. J. & May, B. (1990). Interspecific allozyme variation among Morchella spp. and its inference for systematics within the genus. Biochemical Systematics and Ecology 18, 475-479.

Vogel, S. (1988). Taming the Wild Morel. Discover, May, 1988, 9, 58-60.

Volk, T. J. & Leonard, T. J. (1989). Experimental studies on the morel, I. Heterokaryon formation between monoascosporous strains of Morchella. Mycologia 81, 523-531.

Volk, T. J. & Leonard, T. J. (1990). Cytology of the life-cycle of Morchella. Mycological Research 94, 399-406.

Yoon, C., Gessner, R. V. & Romano, M. A. (1990). Population genetics and systematics of the Morchella esculenta complex. Mycologia 82, 227-235.

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