The morphology and cultural characters of Pisolithus tinctorius (Gasteromycetes) in South Africa

Sporocarp morphology as well as surface ornamentation of the basidiospores of South African collections of Pisolithus tinctorius agree in respect of those of specimens from Australia. Cultural characters and micromorphology of isolates made from certain of these collections correspond with those of P. tinctorius cultures obtained from sporocarps of strains forming mycorrhizal associations with pines in France and the U.S.A. It is concluded that only strains of the fungus which are unable to form mycorrhizas with pines occur in South Africa.


Introduction
The Gasteromycete, Pisolithus tinctorius (Mich.: Pers.) Coker & Couch (1928) was first reported in South Africa by van der Bijl (1918). He noted that fruit-bodies were common in Eucalyptus plantations near Pretoria and, on the basis of careful microscopic observations, concluded that this fungus lived symbiotically on the roots of Eucalyptus trees. Subsequently Ashton (1976), Marx & Bryan (1970) and Gibson (1969) among others, reported experimental confirmation of this association. In a survey of the host range and world distribution of P. tinctorius, Marx (1977) reported its occurrence in 33 countries of the world . Its ability to form ectomycorrhizas on the roots of 11 species of Eucalyptus , 30 of Pinus, two of Quercus and a number of other coniferous and broad-leaved tree species has been demonstrated experimentally (Marx 1977). It has been reported to form mycorrhizas under natural conditions with nine additional species of Pinus , two of Eucalyptus and eight of Quercus as well as with a number of other tree species (Marx 1977). P. tinctorius has been shown to have great potential use in afforestation projects because of its wide host range, adaptability to different sites, and ability to improve tree growth in the nursery and survival in the field. Practical techniques for its artificial introduction into nursery soils are also available (Marx 1976;). However, Marx (1981) later reported significant variation in the ability of P. tinctorius isolates from oak or pine hosts south of the Tropic of Cancer , to form mycorrhizas with Pinus taeda. Some isolates failed to form mycorrhizas under experimental conditions . South African P. tinctorius is common and widely distributed tn association with Eucaluptus spp.
BO TANY -a (Bottomley 1948;Doidge 1950;van der Westhuizen & Eicker 1987). It has also been found in association with wattle, Acacia mearnsii De Wild. , by the present authors. Its fruit-bodies commonly appear under Eucalyptus spp. planted as firebelts around pine plantations but rarely occur under the pines. It is known to occur in association with pines on a site in the vicinity of George, Cape Province, that had previously been planted with Eucalyptus sp . (van der Westhuizen & Eicker 1987). It has also been seen in an open stand of Pinus radiata in Tokai State Forest (Prof. D.T. Mitchell, University College Dublin , pers. comm.). However, fungi isolated from ectomycorrhizas on roots of Pinus spp. seedlings grown in a potting mixture containing basidiospores from fruit-bodies of P. tinctorius collected from the pine stand near George , did not include isolates of P. tinctorius (Viljoen 1987). Mixing basidiospores of P. tinctorius into soil of seedling beds is an accepted practice for promoting mycorrhizal formation by this fungus with pines . These observations together with the general absence of P. tinctorius fruitbodies from pine stands in South Africa , strongly indicate an absence of mycorrhizal relations with pines . These observations raise doubts about the identity of the fungus known asP. tinctorius in South Africa . However, they also tend to support a suggestion by Dring (1973) that both saprophytic and mycorhizal varieties of P. tinctorius may exist.
The sporocarp morphology of P. tinctorius has been described from South Africa by van der Bijl (1918) and Bottomley (1948) , from Australia by Cunningham (1944), and from North America by Coker & Couch (1928) , Grand (1976) and Mims (1980). The two latter authors also described the surface ornamentation and ultrastructure respectively, of the basidiospores from collections in North America. Bronchart et al. (1975) and Hansen (1986) described and figured basidiospores of Pisolithus arhizus and P. tinctorius respectively from Europe .
The cultural characters and micromorphology in pure culture have been studied by Hile & Hennen (1969) and Miller et al. (1983).
It was decided to investigate the identity of South African specimens designated as P. tinctorius in an effort to find a possible reason for the absence of its association with pines in this country . This paper reports on the results of the investigation .

Materials and Methods
Conventional mycological methods were used to study sporocarps of 22 collections of P. tinctorius, including one from New South Wales , Australia, obtained from the herbarium of the National Collection of Fungi (PREM), Pretoria . Eleven more were collected by the authors from different localities in South Africa during the summer of 1986/87. Fruit-bodies from these collections were deposited in the Fungus Collections (PRUM) of the H.C.W.J. Schweickerdt Herbarium , University of Pretoria. In order to obtain cultures, fresh sporocarps were cut open by means of a sterilized scalpel. From the freshly exposed surfaces , tissue from the lower parts of the gleba was removed by means of sterilized forceps and transfered to plates of Melin-Norkrans agar (MMN) as modified by Marx (1969) .
Cultures of eight isolates of P. tinctorius as well as basidiospores from the basidiocarps from which six of these cultures had been made, were received from the Department of Forest Science, University of Stellenbosch and were included in the present study. Two of these cultures had been isolated from sporocarps of strains forming mycorrhizas with pines in France and in Georgia, U ,S.A .
All cultures were incubated in the dark at 25°C. For comparative studies, five cultures of each isolate were examined after 6 weeks incubation according to the methods described by Miller et al. (1983) and van der Westhuizen (1971) .
Basidiospores mounted in lactophenol were examined and photographed by light microscopy . Spore size was determined by measuring 25 spores from each collection. Spore ornamentation was studied by means of scanning electron microscopy (SEM) after mounting spores on adhesive-coated bosses and coating with gold in a sputter coater to about 120 A. Colour names given in quotation marks are in accordance with the notation by Rayner (1970) .

Results
Sporocarps of 33 collections were examined . These varied in size, shape and colouring of the exoperidium but were very similar in anatomical characters (Figure 1).
Basidiospores from 39 sporocarps were studied . They varied somewhat in size , but otherwise appeared very similar under the light microscope (Figures 2 & 3).
Cultures obtained from seven of the fruit-bodies collected by the authors , as well as eight isolates received from the University of Stellenbosch, grew at different rates and showed slight variation in colour of the mat and colour intensity on the reverse of the plates . However, they were very similar in general appearance , colour , texture of the colonies, and in micromorphology (Figure 7).

Cultural characters
Colonies on MMN slow growing reaching a radius of 20-35 mm after 6 weeks ( Figure 7) . Margin even, thin , mycelium raised to limit of growth, sparse , forming numerous thin strands, 'pale luteous' ; aerial mycelium thin over marginal zone, becoming progressively more dense to woolly and raised over the older parts , finally soft felty over the oldest parts and inoculum plug, darkening to 'honey' . The mat remains soft and easily torn. The reverse is unchanged at first but darkening under the inoculum to 'isabelline' then 'greying sepia' to finally 'fuscous black' , the darkened area gradually increasing radially .

Microscopic characters
Advancing zone: hyphae hyaline , unbranched at first , later branching, thin-walled , septate , with simple clamps at the septa, often branching behind a septum with the septum and clamp close to the main hypha, or branching from a clamp with a septum and clamp formed close to the origin, 2,0-3 ,5 J-lm in diameter (Figure 8) .
Aerial mycelium : hyphae as in the advancing zone, occasionally showing paarige branching ( Figure 9) or with multiple branches and clamps at some septa on older hyphae 2,0--5,6 J.Lm diam. (Figure 10) and small granules on some of the older hyphae ( Figure 11). Strands of 3-5 hyphae were present in older parts of the mat. Submerged mycelium: hyphae as in the advancing zone but more tortuous and more frequently branched with short, tortuous branches , 2 ,0--3,0 J.Lm (Figure 12).

Discussion
The sporocarps of collections examined during the present investigation varied widely in size , shape and general appearance but corresponded with regard to anatomical characters. All correspond with descriptions of sporocarp morphology of Pisolithus tinctorius by Bottomley (1948), Cunningam (1944, Grand (1976) and van der Bijl (1918). Basidiospores from all collections from which they were available , were very similar in morphology when viewed under the light microscope although they were mostly smaller than the sizes given by Grand (1976). Basidiospores from South African sporocarps are larger and more coarsely echinulate than those of the related species Pisolithus microcarpus (Cunningham 1944). Spore size of the South African collections are within the ranges given by Bottomley (1948), Cunningham (1944), Mims (1980) and van der Bijl (1918). Under the light microscope, basidiospores of the Australian specimen, PREM 46185, agree in size and morphology with those of the South African collections. Their surface ornamentation also corresponds with that of the South African collections when examined at 5 000 x magnification by SEM. Characters of these spores in turn correspond with the descriptions and electronmicrographs of spores from North American collections (Grand 1976;Mims 1980). They differ however, in surface ornamentation from spores of a collection of P. tinctorius reported from Denmark by Hansen (1986) which has sharp-pointed spines . Bronchart et al. (1975) also figured spores with sharp-pointed spines from sporocarps of the European species, Pisolithus arhizus, and suggested that this character may make it possible to separate European Pisolithus from a similar species frequently occuring in Africa. However Grand (1976) figured scanning electron micrographs of basidiospores of Pisolithus which show both sharp-pointed and blunt spines from various localities in the U.S.A . He further reported no differences in spore size and ornamentation related to location, habitat or habit of 167 sporocarps of P. tinctorius.
Cultural characters and micromorphology of isolates obtained from South African sporocarps are closely similar to those of isolates from Pinus pinaster from France and Pinus taeda from Georgia , U .S.A., respectively. All the isolates agree in respect of micromorphological and cultural characters with the descriptions by Hile & Hennen (1969) and Miller et al. (1983). However, inflated hyphae as described and figured by the latter authors were not observed in our cultures although this may be due to the use of different media.
The South African collections thus agree in morphology and cultural characters with specimens and cultures that had been referred to P. tinctorius from localities outside of this country. Furthermore the isolates from Eucalyptus were indistinguishable from those obtained from Pinus spp. These observations together with descriptions by Grand (1976) , Hile & Hennen (1969) and Mims (1980) of P. tinctorius associated with Pinus spp. , reveal no morphological differences between sporocarps of P. tinctorius associated with pines and those associated with eucalypts. The absence of P. tinctorius sporocarps from pine stands in South Africa can therefore not be ascribed to the occurrence of a different Pisolithus species in this country. Grand (1976) and Marx (1977) reported sporocarps of P. tinctorius as being widely distributed in the U .S.A. , occurring mostly on poor, acid, sandy or clayey soils and usually in associatiOn with various tree species. Occasionally sites without trees such as gardens , lawns, pastures and sand dunes were cited as localities . This led Grand (1977) to support the view put forward by Dring (1973) that mycorrhizal as well as saprophytic forms (varieties) of P. tinctorius may exist. Marx (1981) later reported experimental results supporting this view. On the basis of these reports together with observations described in this paper it appears that strains of P. tinctorius not generally capable of forming mycorrhizas with pines , generally occur in South Africa.