Physiology of mycelial growth of the mycorrhizal mushroom Cantharellus cibarius Fr.

In this thesis in vitro growth of mycelium of Cantharellus cibarius Fr. is described as the first step in the domestication of this edible mycorrhizal mushroom. A collection of pure cultures was obtained and the requirements for vegetative growth were determined.IsolationA culture collection of 33 strains was obtained by isolation from spores or by cultivation from fruit body tissue. Isolation was technically not difficult. However, many attempts were required because only very few spores germinated and only few tissue explants remained free from contaminants. In addition the incubation time required for successful isolation is long, viz. three months. DNA/DNA hybridization experiments proved the identity of the cultures. The biological variation in the strain collection was Illustrated by differences in growth parameters such as relative growth rate.Growth requirementsThe objects of the experiments were the development of an optimal nutrient solution for mycelial growth and the elucidation of root effects on growth. In these studies two kinds of inoculum were used: mycelial tufts and hyphal fragments. These inocula showed different requirements. Mycelial tufts grew strongly in Fries medium supplemented with micro- minerals. Hyphal fragments showed more extensive requirements; their growth was limited to the development of small and thin mycelia. The modified Fries medium and incubation at 20° appeared almost optimal for growth of mycelial tufts. In sterilization of the nutrient solution, filtration is of major importance. Emersed growth was found to be better than standard, submersed incubation. Fungal growth reacts strongly to various additions to the medium. The specific growth rate was low. It could not beIncreased by the use of other media or by addition of possible growth factors to modified Fries medium. The specific growth rates of the tested strains increased in the course of the studies independently of nutrition or incubation conditions.Development of hyphal fragments into strongly growing mycelium at first required the presence of a living root in the nutrient solution. Three observations indicated that the factor(s) involved could be produced by the fungus itself:- the specific growth rates of mycelial tufts in the absence of roots and of hyphal fragments in the presence of roots were identical;- kinetics of growth of hyphal fragments showed that a root effect was limited to the duration of the lag phase;- hyphae brought into intimate mutual contact grew as strongly as a mycelial tuft.The unknown growth factor(s) was volatile in nature. It could be replaced by carbon dioxide. A 0.5% concentration of CO 2 in the incubation atmosphere is sufficient for strong growth of hyphal fragments.CO 2 could be replaced by a mixture of malic acid, thymine, and Tween 80. This suggests that CO 2 fixation reactions involved in the replenishment of Krebs cycle intermediates, in the synthesis of pyrimidines, and in the synthesis of fatty acids are growth limiting for hyphal fragments.On the one hand, the homogeneous growth of hyphal fragments, described in this thesis, allows the second step in the domestication of this mushroom, viz. the study of fruit body formation. On the other hand, the analysis of the growth requirements of these fragments further elucidates the ecological nature of the mycorrhizal relationship between C. cibarius and plant roots. The view that the fungus is supplemented with specific growth factor(s) is not supported. The production of carbon dioxide by roots enables the establishment of the fungus in the rhizosphere.