L t4 7 CANADA DEPARTMENT OF FORESTRY AND RURAL DEVELOPMENT Cultural Characteristics of Three Species of Boletinus by John G. Laut FORESTRY BRANCH Reprinted from Canadian Journal of Botany Volume 44 (1966)
395 CULTURAL CHARACTERISTICS OF THREE SPECIES OF BOLETINUS',JOHN G. LAUT Forest Research Laboratory, Department of Forestry of Canada, Winnipeg, Manitoba Received October 7, 1965 Abstract Three main categories of culture characteristics were examined: gross morphology of the growing colony, microscopic morphology of the mycelium, and chemistry of the mycelium as expressed by color changes induced by specific chemical reagents. The diagnostic characteristics for the three species studied have been arranged into a numerical key that is amenable to expansion as data for more species become available. Introduction A number of investigations have been made of the role of ectotrophic mycorrhizae in the growth and development of forest trees. Many species of Boletaceae form mycorrhizae with certain tree species; Singer (1962) stated that all the known species of Boletinus Kalchbr. are mycorrhizal and are of some importance in reforestation projects. Three species of Boletinus 2 occur in Idaho (Slipp and Snell 1944) and these have been implicated in mycorrhizal associations with some important host species in the region : B. amabilis (Peck) Snell on Pseudotsuga menziesii (Mirb.) Franco; B. cavipes (Opat.) Kalchbr. on Larix occidentalis Nutt.; and B. ochraceoroseus Snell on L. occidentalis. A variety of approaches have been used to identify the fungi involved in mycorrhizae. Observations of carpophore occurrence in association with specific host trees have formed the basis of many mycorrhizal records. In some cases these observations have been confirmed by success in synthesizing mycorrhizae by inoculating aseptically grown seedlings with pure cultures of the suspected fungi (Trappe 1962). Pantidou (1961a) has pointed out that the results of = :ch laboratory syntheses are "sometimes contradictory and do not appear to be applicable to forest conditions in many cases." Identification of the fungal component could be most accurately determined by direct isolation from naturally occurring mycorrhizae. This procedure has not been adopted in the past primarily because the mycorrhizal fungi generally do not form carpophores in culture and thus vegetative mycelium must be used for identification. This has presented difficulties because descriptions of mycorrhizal fungi in culture are rare and workers have lacked a basis for comparisons. Although many workers have obtained pure cultures of some bolete species in conjunction with studies of mycorrhizae, most of the reports do not include descriptions of these cultures. Pantidou (1961a, 1961b, 1962, 1964) has studied and described several species of Boletaceae in pure culture. 'A portion of a thesis presented to the Graduate School, University of Idaho, in partial fulfillment of the requirements for the degree of Master of Science. 2Since the completion of this work Pomerleau and Smith (1962) have transferred B. ochraceoroseus to Fuscoholetinus ochraceoroseus and Smith and Thiers (1964) have transferred B. cavipes to Suillus cavipes and have excluded B. amabilis. The specimens identified here as B. amabilis are probably Suillus lakei (Murrill) Smith and Thiers. (cf. Singer 1965). Canadian Journal of Botany. Volume 44 (1966)
396 CANADIAN JOURNAL OF BOTANY. VOL. 44, 1966 Certain chemical properties of carpophores have recently received attention. Singer (1949) advocates and summarizes the use of certain chemicals that give specific color reactions with the flesh of carpophores and these have been used by other taxonomists concerned with boletes (e.g., Dick and Snell 1960; Pomerleau and Smith 1962). These reagents have not been generally used on cultures of Hymenomycetes with the exception of the tests for oxidases in cultures of wood-decaying fungi (Lindeberg 1948; Nobles 1958). Pantidou (1961a et seq.) has recorded the reactions of certain chemicals with mycelium in cultures. The purpose of this study was to examine the characteristics of the Idaho species of Boletinus in pure culture and to discover which characteristics of the mycelium, grown under standardized conditions, might be distinct enough to permit identification of the fungi, and to arrange these characteristics in a systematic key. Materials and Methods The cultures were obtained originally from fresh carpophores of the three species of Boletinus collected in Idaho, using techniques similar to those described by Pantidou (1961a). Voucher specimens for each isolate are filed either in the University of Idaho Herbarium, Mycological Collections, or in the Forest Pathology Herbarium, College of Forestry, University of Idaho (see Table I for collection data). Cultures from specimens in the latter were furnished by Professor F. D. Johnson, who also assisted in identifying some of the specimens used. The medium used was an adaptation of Modess' (1941) modification of Hagem's agar. The formulation was 1 liter distilled water, 12.0 g Difco agar, 20.0 g dextrose, 5.0 g Difco malt extract, 5.0 g casein hydrolysate (enzymatic), 1.0 g KH 2PO 4, 0.5 g MgSO 4.71-1 20, 0.5 g NH 4C1, 5.0 ml of 0.1 M aqueous CaC1.2H 20, 0.5 ml of 1% aqueous FeCI 3, 0.5 ml of 1% aqueous MnC12.4H20, Species TABLE I Collection data for specimens cultured Isolate number Location of collection* Accession number of voucher speciment Boletinus amabilis 5 Bonner UIFP M705 7 Clearwater UIFP M785 8 Valley UIFP M726 9 Valley UIFP M731 6213 Latah ID 780 Boletinus cavipes 14 Clearwater UIFP M794 6210 Latah ID 770 6211A Latah ID 782 6211B Latah ID 781 Boletinus ochraceoroseus 3B Kootenai UIFP M626 3C Clearwater UIFP M237 16 Benewah UIFP M779 623 Valley ID 783 6212 I,atah ID 784 *County in Idaho where collection was made. I'DIFP indicates University of Idaho Forest Pathology Herbarium (College of Forestry), furnished by Prof. F. D. Johnson. ID indicates University of Idaho Herbarium, Mycological Collection (Department of Biological Sciences).
LAUT: BOLETINUS 397 0.5 ml of 1% aqueous ZnSO 4. The mixture of all ingredients was autoclaved for 15 minutes at 15 p.s.i. This medium had a ph of 5.0 after autoclaving and cooling. Neither thiamine (100.0 mg/1) nor biotin (5.0 mg/1), when added to the medium, produced any noticeable change in growth of the three species. The cultures in petri dishes were incubated at 20 C in the dark and were subcultured periodically on fresh medium. After various incubation periods cultures were examined for characteristics of possible taxonomic significance including: mat characters (growth rate, color, and type of colony); hyphal characters, both general and special; and chemical characteristics as shown by color changes of the mycelium induced by specific reagents. The first two groups of characteristics have been outlined and discussed by Pantidou (1961a). The chemical reagents used in this study were selected from those listed by Singer (1962) and were made up in the recommended concentrations (pp. 82-94). Small sections (approximately 0.5 cm 2) of unpigmented mycelial mat of a 4-week-old culture were removed and placed on a white porcelain spotplate. A drop of reagent was placed on a section and, since some of the reactions were slow to appear, the section was kept under observation for 2 hours. Color changes during this period were recorded. For microscopic examination mycelium from different areas of a culture was mounted on standard microscope slides in either Amman solution (Johansen 1940), Amman solution plus 0.1% cotton blue, or stained with 0.1% aqueous cresyl blue and mounted in glycerine. The color terms, in quotations, and the code numbers, in parentheses, are based on Maerz and Paul (1950). The special descriptive terms for both gross characteristics and microscopic features, unless otherwise noted, are used as defined by Nobles (1948). Results The three species of Boletinus grew fairly well on the medium used in this study. There were noticeable differences between the species, especially in regard to the time lag between inoculation and the first visible signs of growth, both at the time of original isolation and after subculturing. The species also varied in their response to subculturing to maintain viability. Growth rates, measured as diameter of colony, varied considerably between species. Colony pigmentations were of some value in recognizing species. Microscopic examination of the mycelium showed that all three were generally similar in their morphology. There was very little differentiation of the hyphae, and no special structures such as chlamydospores, rhizomorphs, or cystidia (allocysts of Singer 1962, p. 16) were observed. The branching habit of the hyphae proved to be of some specificity. Branches were, as outlined by Pantidou (1961a), of two types, simple or whorled. The term simple was used if only one branch arose from a particular point on a hypha. The term "paarige", although originally applied to a pair of branches formed in the same position on a hypha (Pantidou 1961a), has been adopted here to mean a whorl of two or more branches (Fig. 1). Clamp connections were present in all three species of Boletinus studied, but there were measurable differences between the species in the abundance and position of the clamps. Septa were present in all hyphae examined.
398 CANADIAN JOURNAL OF BOTANY. VOL. 44, 1966 A!ir K M N 0 FIG. 1. Simple branches and clamp connections (A J) and paarige configurations (K P) in cultured mycelium of three species of Boletinus (diagrammatic). Most of the chemical reagents used gave some color reactions on the mycelium and many gave the same results on all three species. Those reagents that gave similar results on all the cultures were sulfovanillin, dark brown to black (this reagent is very dark and it was difficult to ascertain whether there was a reaction or merely staining of the mycelium); sulfoformol, light yellow; formalin, no reaction; Melzer's iodine, yellow; alpha naphthol, pink; KOH and NH 4OH, pink; chloro-vanillin, orange; phenol, dark pink. Guaiac, ferric TABLE II Comparison of chemically induced color reactions on mycelium of three species of Boletinus Reagent B. amabilis B. cavipes B. ochraceoroseus Gum guaiac Orange Light blue Pink Fe2(SO4)3 Brown None Light blue-green Aniline oil Red Red None Phenol aniline Dark red Dark red None
LAUT: BOLETINUS 399 sulfate, aniline oil, and phenol aniline gave reactions (Table II) which were sufficiently different among the three species to be of diagnostic value. Cultural Characters Boletinus amabilis (Peck) Snell apud Slipp and Snell 1944 (30 cultures examined) Mat characters. Average diameter of mat 4.0 cm in 4 weeks. Advancing margin even or slightly bayed, mat white at first, becoming brown (14E7 to 15A6) in 2-4 weeks except for the advancing zone, which remains white. Aerial mycelium produced close to margin, cottony to woolly, often coming into contact with the cover of petri dish, exuding drops of clear brown liquid. Reverse light yellow under the margin of growth, becoming dark brown under the central portion of the mat. Some cultures have a yellow pigment diffused through the medium. No distinctive odor produced. Hyphal characters. Hyphae septate, averaging 3.0,u in diameter, contents granular, occasionally hyphae larger (4.5-6,u) with homogeneous contents appearing fluid-filled. Simple clamps extremely rare, in young cultures only, often formed opposite a branch. Clamps with branches emerging from the top of clamp somewhat more frequent. Paarige very common in all cultures with various configurations, branching habit often obscuring the presence of clamp connections (Fig. 1 F, H, I, L, M, P). Some hyphae, or cells within hyphae, when stained in cresyl blue, stain light pink, while most are stained light blue. Chemical reactions. See Table II. This species appears to be very stable under culture conditions. All of the isolates studied showed only slight variations in the characteristics mentioned. One culture of isolate No. 8 had vesicle-like, intercalary swellings on a few hyphae only. These were seen only in mycelium taken from the margin of the colony and were not present in other isolates of B. amabilis. Boletinus cavipes (Opat.) Kalchbr. Bot. Zeitschr. 25:182 (12 cultures examined) Mat characters. Maximum diameter approaching 4.0 cm in 4 weeks. Margins even. Mat white at first, becoming light brown in older, central portion. Mycelium cottony throughout the mat. Reverse dark brown; agar becoming brown before growth from the inoculum is visible. Distinct pungent odor produced. Hyphal characters.--hyphae septate, averaging 3.0 (1.5-5.0) in diameter, contents mostly granular but occasional hyphae with homogeneous contents. Strands of hyphae common in older parts of the mat. Simple clamps were seen in one of the original isolates, but were extremely rare, and were never seen in subsequent transfers or in cultures from other specimens. Simple branches rare to common (Fig. 1, A-D); paarige not present. In cresyl blue some hyphae stained pink; some stained light blue-green. Chemical reactions. See Table II. B. cavipes was the most difficult of the species to maintain in culture and required transfer more frequently than once a month. Cultures, although attaining approximately the same diameter as the other species after a month,
400 CANADIAN JOURNAL OF BOTANY. VOL. 44, 1966 were extremely slow in beginning growth. Most transfers took more than a week to show any signs of growth and many of them failed to grow at all. The mycelium, as indicated by the description above, shows much less differentiation into special structures than does that of the other two species. There are notable discrepancies between the description of B. cavipes offered here and that of Pantidou (1961a). Her cultures exhibited frequent paarige and clamps; hyphae commonly with homogeneous contents and rare strands. She also reported a red color appearing when the mat was touched with a needle. None of my cultures exhibited this phenomenon. This suggests that the B. cavipes found in Ontario may be a different variety, or strain, from that found in Idaho. Boletinus ochraceoroseus Snell. Mycologia, 33:35 (Fuscoboletinus ochraceoroseus Pomerleau and Smith, 1962) (15 cultures examined) Mat characters. Average diameter of mat 6.0 cm in 4 weeks. Advancing margin even, mat white at first becoming very light pink (3B1) after a month. Central zone slowly turning darker pink and then light brown; all colors becoming darker when exposed to light. Mycelium cotton y throughout but somewhat woolly in older portions of the cultures. Reverse yellow (11G2) beneath the margin shading into dark "cocoa" brown (7E12) in center; diffusing pigment turning agar yellow. No distinctive odor. Ilyphal characters. Hyphae septate, averaging 4.0 p. (3.0-6.0) in diameter, contents granular. Strands of hyphae rare. Simple clamp connections common to abundant. Simple and dichotomous branching common; paarige rare (Fig. 1 B, C, E, J, N, 0). Chemical reactions. See Table 1I. This species was the most vigorous in culture. Subcultures grew more rapidly than did the original isolates; aerial mycelium frequently reached the cover of the dish. It was also much faster to respond to fresh medium after subculturing and growth from inoculum was often clearly visible 48 hours after transfer. Discussion As indicated by the results of this study, and others previously cited, the morphological characteristics of the mycelium of many species of Boletaceae differ mainly in degree rather than in type. For example, all three species studied here produced clamp connections in culture. Clamps on Boletinus cavipes hyphae were rare and then only of the simple type. They were common to abundant in the other species and were usually modified to some degree. These modifications often obscured the clamps to the point where they were difficult to recognize in older cultures. The chemical reactions of the cultured mycelium have been more or less ignored as a taxonomic tool in the past. Reagents that combine with part of the chemical system of the cells to give visible reactions (color) have been shown here to be of value in distinguishing between certain species of one genus. It may well be that certain of the reagents used will produce different reactions when tested on species of different genera and thus be of value at higher taxonomic levels.
LAUT: BOLETINCS 401 TABLE III Numerical key to cultures of six Boletinus species Species 1 2 3 4 5 6 7 8 9 10 11 B. cavipes 0(1) 1 1 2 1 0 3(0) 2 0 1 1 B. amabilis 1 1(2) 3(2) 0 2(3) 1 1 2 3 1 1 B. ochraceoroseus 2(1) 3 1(2) 1 0 2 2 0(1) 0 1 1 B. grisellus* 1(2) 2 1 0 1 0 0 0 0 1 B. pictus* 1 2 2 2(3) 1 2 1 1 1(0) 1 B. spectabilis* 1 2(1) 3 3 0 0 0 2(1) 2 2 *Data from Pantidou (1961). These observations of culture characters of three species of Boletinus could be incorporated into a dichotomous key for identification purposes. However, to facilitate the addition of more species of mycorrhizal fungi as the data become available, the following numerical key is suggested (Table III). It is similar to, and functions in the same manner as, those developed by Nobles (1948) and others. The three species studied here are listed first; these are followed by three species studied by Pantidou whose data have been interpreted to fit the proposed code and included here for comparison. The meaning of the numerical values is as follows. Column 1, mat color: 0 white, 1 brown, 2 pink. Column 2, clamp connections: 0 not seen, 1 rare, 2 common, 3 abundant. Column 3, paarige: same as column 2. Column 4, strands: same as column 2. Column 5, "fluid" hyphae: same as column 2. Column 6, reaction with Fes(SO 4) 3 : 0 no reaction, 1 brown, 2 blue-green, no data available. Column 7, reaction with gum guaiac: 0 no reaction, 1 orange, 2 pink, 3 blue. Column 8, reaction with phenol aniline: 0 no reaction, 1 pink, 2 dark red. Column 9, reaction with formalin: 0 no reaction, 1 pink, 2 red, 3 yellow. Column 10, reaction with KOH: 0 no reaction, 1 pink, 2 purple. Column 11, reaction with NII 4OH: 0 no reaction, 1 pink, 2 purple. No attempt has been made here to determine the substances within the mycelium which reacted with the reagents. Lack of this information at the present time should not be of particular concern to those who wish to use such reactions as taxonomic tools. As Singer (1949) points out, it matters only that a color appears specifically under defined conditions for that reaction to be useful. Characteristics of the mycelium of some species of Boletaceae grown in pure culture under standardized conditions can be used to identify the fungi. The procedures presented here should be of use to those who desire to identify the fungi involved in mycorrhizae of forest trees by direct isolation from the organ. They can also be of value in resolving the taxonomy of the boletes and, by extension, of other taxonomically difficult groups of Agaricales. References D ICK, E. A. and SNELL, W. H. 1960. Notes on boletes. XIII. Mycologia, 52, 444-454. J OHANSEN, D. A. 1940. Plant microtechnique. McGraw-Hill, New York.
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