A Cytological Study of Spore Germination of Volvariella Volvacea*
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Bot. Mag. Tokyo 82: 102-109 (March 25, 1969) A Cytological Study of Spore Germination of Volvariella volvacea* by Shu-ring CHANG* * Received October 9, 1968 Abstract Volvariella volvacea is a tetrasporous fungus of the tropics and subtropics and belongs to the family Amanitaceae of the Basidiomycetes. It has two kinds of spores, one being the sexual basidiospore and the other the asexual chlamydospore. The spores were collected on a permeable cellophane membrane overlying the complete semisolid medium and incubated at 40° for 24-48 hours and at 35° for 24 hours for germination of basidiospores and chlamydospores, respectively. The uninuclear basidiospores and the multinuclear chlamydospores are observed and discussed. Non-septal uninucleate, binucleate, and multinucleate germ tubes and septal multi- nucleate as well as branched germ tubes are also described. Conventional mitotic figures and simple septa as well as suspected dolipore septa are observed in the germ tubes. Special attention was given to the nuclear translocation by migration through the septa. The primary results indicate that this mushroom may be a homothallic fungus. Volvariella volvacea, commonly known as straw mushroom, belongs to the family Amanitaceae of the Basidiomycetes. It is a saprophytic fungus of tropics and sub- tropics, growing on decaying plants and taking up necessary nutrition from them Although the genus V olvariella, in some tropic and subtropic areas, has as much economic importance as Agaricus, the cultivated white mushroom, the Volvariella is not so well studied as the Agaricus. The life history (Sass'', and Kligman2'), mor- phology (Cayley3', and Sarazin4') and cytology (Colson5', Sass6', and Evans7') of the cultivated white mushrooms are fairly well investigated. The life cycle of the straw mushrooms, on the other hand, is incompletely known, and the morphology (Chang8', and Singer9') and cultivation (Singer9', Go10', Hashioka11', and Chang12') of this tropic fungus are only poorly understood. So far as we know there is no information con- cerning the genetics and cytology of this kind of mushroom. Literature on Volvariella is scarce. We have been interested in this fungus for several years in various aspects, and a more intensive study of the cytology and physiology (Chang and Chu13') of spore germination has been carried out in the past year. The cytological study of both basidiospore and chlamydospore germination of the straw mushroom is of considerable interest, since such information may contribute to our further explora- tion of its genetics and breeding. * This work was supported in part by funds provided by the Institute of Science and`. Technology of the Chinese University of Hong Kong. ** Department of Biology , Chung Chi College, The Chinese University of Hong Kong,. Shatin, N. T., Hong Kong. March, 1969 Spore Germination of Volvariella volvacea 103 Materials and Methods The materials used in this investigation were mainly obtained from the cultivated straw bed, as described by Chang12' and modified, grown in laboratory conditions. The space available was relatively small, and for comparison it was found desirable to collect the materials as grown in natural conditions. Because the straw mush- room is a fungus of the tropics and subtropics, it is necessary to have fairly high temperature and relatively high humidity for growth. Straw mushrooms do well during the summer in Hong Kong and can only be grown from May to October. However, in laboratory conditions, we can have the fresh straw mushrooms for the materials of our studies all the year round. (a) Chlamydospore : A block of hyphae bearing chlamydospores from either vegetative cultural mycelium or monosporous progeny was macerated in a blender for two minutes in order to ensure the detachment of spores from the hyphae. The product of maceration was then filtered through glass wool in the funnel. While most of the hyphae were barred by the glass wool, the chlamydospores passed through it easily. The filtrate was a slightly brown spore suspension which was plated onto a cellophane membrane lying flat over the complete semisolid medium (Formula : MgSO4 0.5 g., KH2PO4 0.46 g., K2HPO4 1 g., peptone 2 g., dextrose 20 g., agar 20 g. per liter water). The plates were incubated at 35° for 24 hours for germi- nation. (b) Basidiospore : A normal fruit body at a "mature" stage (Fig. 1), which means that the pileus has completely expanded with brown-coloured lamellae, was hung within a tall, cylindrical glass jar. The spores were collected by placing Petri dish with a permeable cellophane membrane overlying the medium under the fruit body. The time required for spore collection usually took from few seconds to two minutes, depending on the desired spore density and the maturation stage of the fruit body. With this method, spores could be scattered uniformly on the membrane. A temperature of 20° or above is necessary for the discharge of spores and the fruit body continued to discharge spores for about 18 hours after it was placed in the jar at 25°. The plates were then incubated at 40° for 24-48 hours for germination. The cellophane membranes with germinated spores as well as ungermi- nated ones were cut into small pieces about 5x 10 mm and fixed and stained by slight modification of the method described by Raper14'. These cellophane pieces were dropped in 1% (w/v )HgC12 in absolute methanol chilled to -70° in dry ice and left in the fixing solu- tion maintained at this temperature for three to four days. Then they were brought to room temperature and washed for three hours in three changes Fig. 1. Completely expanded basidiocarps of the straw mushroom with the remainder of absolute methanol, passed through of the volva around the base of stalk. Viewed 1% (w/v) collodion dissolved in a 1:1 both from above and below. x about 1/4.5. mixture of absolute ethanol and ethyl 104 CHANG, S. T. Vol. 82 ether and carried through a graded ethanol series to water, stained for 4 minutes in fresh solution of Mayer's haematum which contains hematoxylin 0.1 g., sodium iodate 0.02 g., and potassium aluminum sulfate 5 g in 100 ml water. The stained materials were mounted in 50% glycerine in water and kept at room temperature for two to four days. Then the microscopical studies were made. Results Volvariella volvacea has two kinds of spores. One of them is the sexual basi- diospore and the other is the asexual chlamydospore. The chlamydospores are brown- coloured and thick-walled. They are not formed by the direct transformation of certain cells of a hypha, but are borne on the specialized spore-bearing branches which usually consist of several swollen cells (Fig. 2). The spores are detached from the bearer at maturity. The most common shape of chlamydospore is a spheri- cal one with an average diameter of about 58.8,1 (Fig. 3a). One germ tube (Fig. 3b and c) or several ones (Fig. 3d) can emerge out of the spore at any point of the wall. Fig. 2. Aerial hyphae bearing swollen cells The chlamydospores are in multi- and the chlamydospores which are separaed from nucleate condition (Fig. 3a). Since the specialized bearer at maturity. Fig. 3. Chlamydospores and germination of the spores. a) Spherical chlamydospore in multinucleate condition. b) Papilla of chlamydospore. c) Chlamydospore germinating in one germ point, d) Chiamydospore germinating in several germ points and with multinucleate germ tubes. March, 1969 Spore Germination of Volvariella volvacea 105 the wall of this kind spore is of uniform thickness and the rupturing of the papillae can randomly occur on the surface. The germ tubes are also in multinucleate condi- tion (Fig. 3d). The spawn developed from monochlamydosporous propeny produces normal fruit bodies on the regular straw beds. The basidiospores of this fungus per basidium are four in number as reported by Chang15' and are asymmetrical in shape. They tend to be egg-shaped, but spherical or ellipsoidal ones are not uncommon. The outline of the spore depends on the direc- tion of observation. The average length of egg-shaped spores is 7-9~c ; the widest part is 5-6,u and narrowest part 3-4i across. One nucleus in each basidiospore was observed (Fig. 4a). Occasionally, more than one nuclei were found in the spore. The wall is relatively thick and brown in colour when spores shed. Germination of spores is not observed at room temperature (22-24'). Protrusion of the germ tube is always at the hilum, which is a slightly protruded part with thinner wall, and also believed to be the point of attachment of the spore to sterigma (Fig. 4b). Germination of the basidiospore commences with the appearance of a small clear rounded vesicle which emerges from a minute pore wall (Fig. 4c). At this stage of germination the nucleus still appears in the spore. The germ tubes, after protrusion Fig. 4. Basidiospores and their germination, a) Mature thick-walled spores containing one nucleus. b) Mature spores indicating the hilum from which the vesicle emerges out. c) The vesicle emerging through the germ pore. d) Non-septate, uninucleate germ tube. Note the absence of the nucleus in the spore. e) Non-septate germ tube with two nuclei. f) Non- septate multinucleate germ tube. g) Septal germ tube, with several nuclei. Note one nucleus still in the spore. h) Non-septate germ tube with nuclear division. i) Branched germ tube with conventional mitotic divisions in the arms (see text). 106 CHANG, S. T. Vol. 82 Fig. 5. Monosporous mycelium is shown forming two suspected dolipore septa (arrows). from the spores, usually extend to a certain length before branching, or occasionally they may branch at the germination point in two directions (Fig. 4g). In some cases, germ tubes may remain unbranched even at a length of 835p.