A Method to Use Lichen Spores in Quantitative Studies on Germination

Abstract

Lichen spores are discharged onto a parafilm sheet, thoroughly mixed in water, dried, stored, and returned at will into an aqueous suspension to be distributed homogeneously on a culture surface. The randomness of distribution has been verified, allowing the calculation of a confidence interval for the percentage of germinated spores according to the law of binomial distribution. Spores of Xanthoria parietina and Physcia pulverulenta stored on parafilm retain their germinative ability at least one month. The spores of lichen fungi have rarely been used in quantitative experiments on germination because it is difficult to obtain homogeneous samples of such spores. The classical method of collecting them aseptically on an agar nutrient medium has been described by Werner (1927): An agar plate is placed above moistened apothecia a short distance from the epithecium and the spores that are ejected upwards usually adhere to the agar. For monospore cultures, one spore can then be removed with a glass needle and transferred to another agar plate. But for statistical investigations, such as those concerning the average rate of germination, individual transfer of spores is not practical. Germination must be observed directly on the original plate and the drawbacks are the following: a) The spores discharged by one ascus usually remain packed together so that the beginning of germination is often difficult to detect. Later on, especially for spores giving rise to more than one filament, it is even more difficult to see to which spores the hyphae belong. b) Some areas are densely crowded with spores (above the apothecia) while others are nearly devoid of them. Up to now, group effects, commonly described for other kinds of spores, have not been noticed in lichens but they may exist in some cases and it is better to avoid them if possible. c) Spores ejected from different apothecia, and most of all from different thalli (even if these have been collected near to each other), rarely have the same germinative capacity. Therefore, such a distribution of spores is far from random and it becomes necessary to deal with large numbers of spores and to make many replicate experiments to obtain significant results. An improvement of the method is thus required. Scattering spores by gently brushing the surface of the medium with a thin brush should overcome the first difficulties (a and b). However, this procedure is not entirely efficient because it does not suppress the heterogeneity (c) between populations from different culture dishes. The best solution is to mix the spores thoroughly before sowing them and then to scatter them as well as possible. Contrary to other kinds of spores (mosses, ferns), lichen spores are extremely I thank the Institute of Applied Mathematics of Grenoble for providing probability tables of the binomial distribution. I am also grateful to Dr. F. LeBlanc for revising the English text. 2 Laboratoire de Physiologie V~g6tale, Universit6 de Grenoble, Grenoble, France. This content downloaded from 207.46.13.163 on Fri, 08 Jul 2016 05:38:40 UTC All use subject to http://about.jstor.org/terms 1970] KOFLER: LICHEN SPORE GERMINATION 603 hydrophilic. They may adhere to glass surfaces, especially if they have been allowed to dry out. Water itself remains partly on such polar surfaces when drops are rolled across or when it is pipetted. Consequently a smooth hydrophobic surface was looked for, one onto which spores could be ejected and from which these could be easily collected in a suitable liquid. Parafilm, which is commonly used to cap culture vials, was found entirely suitable for this purpose.