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CANADADEPARTMENT OF FORESTRYAND RURAL DEVELOPMENT

Cultural Characteristics ofThree Species of Boletinus

byJohn G. Laut

FORESTRY BRANCH

Reprinted fromCanadian Journal of Botany

Volume 44 (1966)

395

CULTURAL CHARACTERISTICS OF THREE SPECIES OFBOLETINUS'

,JOHN G. LAUTForest Research Laboratory, Department of Forestry of Canada, Winnipeg, Manitoba

Received October 7, 1965

AbstractThree main categories of culture characteristics were examined: gross mor-

phology of the growing colony, microscopic morphology of the mycelium, andchemistry of the mycelium as expressed by color changes induced by specificchemical reagents. The diagnostic characteristics for the three species studiedhave been arranged into a numerical key that is amenable to expansion as datafor more species become available.

IntroductionA number of investigations have been made of the role of ectotrophic

mycorrhizae in the growth and development of forest trees. Many species ofBoletaceae form mycorrhizae with certain tree species; Singer (1962) statedthat all the known species of Boletinus Kalchbr. are mycorrhizal and are ofsome importance in reforestation projects. Three species of Boletinus2 occurin Idaho (Slipp and Snell 1944) and these have been implicated in mycor-rhizal 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. occi-dentalis.

A variety of approaches have been used to identify the fungi involved inmycorrhizae. Observations of carpophore occurrence in association with specifichost trees have formed the basis of many mycorrhizal records. In some casesthese observations have been confirmed by success in synthesizing mycorrhizaeby inoculating aseptically grown seedlings with pure cultures of the suspectedfungi (Trappe 1962). Pantidou (1961a) has pointed out that the results of = :chlaboratory syntheses are "sometimes contradictory and do not appear to beapplicable to forest conditions in many cases." Identification of the fungalcomponent could be most accurately determined by direct isolation fromnaturally occurring mycorrhizae. This procedure has not been adopted in thepast primarily because the mycorrhizal fungi generally do not form carpophoresin culture and thus vegetative mycelium must be used for identification. Thishas presented difficulties because descriptions of mycorrhizal fungi in cultureare rare and workers have lacked a basis for comparisons. Although manyworkers have obtained pure cultures of some bolete species in conjunctionwith studies of mycorrhizae, most of the reports do not include descriptionsof these cultures. Pantidou (1961a, 1961b, 1962, 1964) has studied and de-scribed several species of Boletaceae in pure culture.

'A portion of a thesis presented to the Graduate School, University of Idaho, in partialfulfillment of the requirements for the degree of Master of Science.

2Since the completion of this work Pomerleau and Smith (1962) have transferred B. och-raceoroseus to Fuscoholetinus ochraceoroseus and Smith and Thiers (1964) have transferredB. cavipes to Suillus cavipes and have excluded B. amabilis. The specimens identified here asB. 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 givespecific color reactions with the flesh of carpophores and these have been usedby other taxonomists concerned with boletes (e.g., Dick and Snell 1960;Pomerleau and Smith 1962). These reagents have not been generally used oncultures of Hymenomycetes with the exception of the tests for oxidases incultures of wood-decaying fungi (Lindeberg 1948; Nobles 1958). Pantidou(1961a et seq.) has recorded the reactions of certain chemicals with myceliumin cultures.

The purpose of this study was to examine the characteristics of the Idahospecies of Boletinus in pure culture and to discover which characteristics ofthe mycelium, grown under standardized conditions, might be distinct enoughto permit identification of the fungi, and to arrange these characteristics in asystematic key.

Materials and MethodsThe cultures were obtained originally from fresh carpophores of the three

species of Boletinus collected in Idaho, using techniques similar to thosedescribed by Pantidou (1961a). Voucher specimens for each isolate are filedeither in the University of Idaho Herbarium, Mycological Collections, or inthe Forest Pathology Herbarium, College of Forestry, University of Idaho(see Table I for collection data). Cultures from specimens in the latter werefurnished by Professor F. D. Johnson, who also assisted in identifying someof the specimens used.

The medium used was an adaptation of Modess' (1941) modification ofHagem'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 2 PO 4, 0.5 g MgSO 4 .71-1 20, 0.5 g NH 4C1, 5.0 ml of 0.1 M aqueousCaC1.2H 20, 0.5 ml of 1% aqueous FeCI 3 , 0.5 ml of 1% aqueous MnC12.4H20,

TABLE ICollection data for specimens cultured

SpeciesIsolatenumber

Locationof

collection*

Accessionnumber

of voucherspeciment

Boletinus amabilis 5 Bonner UIFP M7057 Clearwater UIFP M7858 Valley UIFP M7269 Valley UIFP M7316213 Latah ID 780

Boletinus cavipes 14 Clearwater UIFP M7946210 Latah ID 7706211A Latah ID 7826211B Latah ID 781

Boletinus ochraceoroseus 3B Kootenai UIFP M6263C Clearwater UIFP M23716 Benewah UIFP M779623 Valley ID 7836212 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 autoclavedfor 15 minutes at 15 p.s.i. This medium had a pH of 5.0 after autoclaving andcooling. Neither thiamine (100.0 mg/1) nor biotin (5.0 mg/1), when added to themedium, produced any noticeable change in growth of the three species.

The cultures in petri dishes were incubated at 20 °C in the dark and weresubcultured periodically on fresh medium. After various incubation periodscultures were examined for characteristics of possible taxonomic significanceincluding: mat characters (growth rate, color, and type of colony); hyphalcharacters, both general and special; and chemical characteristics as shownby color changes of the mycelium induced by specific reagents. The first twogroups of characteristics have been outlined and discussed by Pantidou (1961a).

The chemical reagents used in this study were selected from those listed bySinger (1962) and were made up in the recommended concentrations (pp.82-94). Small sections (approximately 0.5 cm 2) of unpigmented mycelial matof a 4-week-old culture were removed and placed on a white porcelain spot-plate. A drop of reagent was placed on a section and, since some of the reac-tions 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 culturewas mounted on standard microscope slides in either Amman solution(Johansen 1940), Amman solution plus 0.1% cotton blue, or stained with0.1% aqueous cresyl blue and mounted in glycerine.

The color terms, in quotations, and the code numbers, in parentheses, arebased on Maerz and Paul (1950). The special descriptive terms for both grosscharacteristics and microscopic features, unless otherwise noted, are used asdefined by Nobles (1948).

ResultsThe three species of Boletinus grew fairly well on the medium used in this

study. There were noticeable differences between the species, especially inregard 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 alsovaried in their response to subculturing to maintain viability. Growth rates,measured as diameter of colony, varied considerably between species. Colonypigmentations were of some value in recognizing species.

Microscopic examination of the mycelium showed that all three weregenerally similar in their morphology. There was very little differentiationof the hyphae, and no special structures such as chlamydospores, rhizomorphs,or cystidia (allocysts of Singer 1962, p. 16) were observed. The branchinghabit of the hyphae proved to be of some specificity. Branches were, as out-lined by Pantidou (1961a), of two types, simple or whorled. The term simplewas used if only one branch arose from a particular point on a hypha. Theterm "paarige", although originally applied to a pair of branches formed inthe same position on a hypha (Pantidou 1961a), has been adopted here tomean a whorl of two or more branches (Fig. 1). Clamp connections werepresent in all three species of Boletinus studied, but there were measurabledifferences between the species in the abundance and position of the clamps.Septa were present in all hyphae examined.

!irK

M N 0


A

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 mycel-ium and many gave the same results on all three species. Those reagents thatgave 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 areaction or merely staining of the mycelium); sulfoformol, light yellow;formalin, no reaction; Melzer's iodine, yellow; alpha naphthol, pink; KOHand NH 4OH, pink; chloro-vanillin, orange; phenol, dark pink. Guaiac, ferric

TABLE IIComparison of chemically induced color reactions on mycelium of three species

of Boletinus

Reagent B. amabilis B. cavipes B. ochraceoroseus

Gum guaiac Orange Light blue PinkFe2(SO4)3 Brown None Light blue-greenAniline oil Red Red NonePhenol aniline Dark red Dark red None

LAUT: BOLETINUS 399

sulfate, aniline oil, and phenol aniline gave reactions (Table II) which weresufficiently different among the three species to be of diagnostic value.

Cultural CharactersBoletinus amabilis (Peck) Snell apud Slipp and Snell 1944 (30 cultures ex-

amined)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 to15A6) in 2-4 weeks except for the advancing zone, which remains white.Aerial mycelium produced close to margin, cottony to woolly, often cominginto contact with the cover of petri dish, exuding drops of clear brown liquid.Reverse light yellow under the margin of growth, becoming dark brown underthe central portion of the mat. Some cultures have a yellow pigment diffusedthrough the medium. No distinctive odor produced.

Hyphal characters.—Hyphae septate, averaging 3.0 ,u in diameter, contentsgranular, occasionally hyphae larger (4.5-6 ,u) with homogeneous contentsappearing fluid-filled. Simple clamps extremely rare, in young cultures only,often formed opposite a branch. Clamps with branches emerging from thetop of clamp somewhat more frequent. Paarige very common in all cultureswith various configurations, branching habit often obscuring the presence ofclamp connections (Fig. 1 F, H, I, L, M, P). Some hyphae, or cells withinhyphae, when stained in cresyl blue, stain light pink, while most are stainedlight 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 fewhyphae only. These were seen only in mycelium taken from the margin of thecolony and were not present in other isolates of B. amabilis.

Boletinus cavipes (Opat.) Kalchbr. Bot. Zeitschr. 25:182 (12 cultures ex-amined)

Mat characters.—Maximum diameter approaching 4.0 cm in 4 weeks.Margins even. Mat white at first, becoming light brown in older, centralportion. Mycelium cottony throughout the mat. Reverse dark brown; agarbecoming brown before growth from the inoculum is visible. Distinct pungentodor 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 wereseen in one of the original isolates, but were extremely rare, and were neverseen in subsequent transfers or in cultures from other specimens. Simplebranches rare to common (Fig. 1, A-D); paarige not present. In cresyl bluesome 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, althoughattaining approximately the same diameter as the other species after a month,


were extremely slow in beginning growth. Most transfers took more than aweek to show any signs of growth and many of them failed to grow at all. Themycelium, as indicated by the description above, shows much less differentia-tion into special structures than does that of the other two species.

There are notable discrepancies between the description of B. cavipesoffered here and that of Pantidou (1961a). Her cultures exhibited frequentpaarige and clamps; hyphae commonly with homogeneous contents and rarestrands. She also reported a red color appearing when the mat was touchedwith a needle. None of my cultures exhibited this phenomenon. This suggeststhat the B. cavipes found in Ontario may be a different variety, or strain, fromthat found in Idaho.

Boletinus ochraceoroseus Snell. Mycologia, 33:35 (Fuscoboletinus ochraceoroseusPomerleau and Smith, 1962) (15 cultures examined)

Mat characters.—Average diameter of mat 6.0 cm in 4 weeks. Advancingmargin even, mat white at first becoming very light pink (3B1) after a month.Central zone slowly turning darker pink and then light brown; all colorsbecoming darker when exposed to light. Mycelium cotton y throughout butsomewhat woolly in older portions of the cultures. Reverse yellow (11G2)beneath the margin shading into dark "cocoa" brown (7E12) in center; dif-fusing 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 commonto 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 thecover of the dish. It was also much faster to respond to fresh medium aftersubculturing and growth from inoculum was often clearly visible 48 hoursafter transfer.

DiscussionAs indicated by the results of this study, and others previously cited, the

morphological characteristics of the mycelium of many species of Boletaceaediffer mainly in degree rather than in type. For example, all three speciesstudied here produced clamp connections in culture. Clamps on Boletinuscavipes hyphae were rare and then only of the simple type. They were commonto abundant in the other species and were usually modified to some degree.These modifications often obscured the clamps to the point where they weredifficult to recognize in older cultures.

The chemical reactions of the cultured mycelium have been more or lessignored as a taxonomic tool in the past. Reagents that combine with part ofthe chemical system of the cells to give visible reactions (color) have beenshown here to be of value in distinguishing between certain species of onegenus. It may well be that certain of the reagents used will produce differentreactions when tested on species of different genera and thus be of value athigher taxonomic levels.

LAUT: BOLETINCS 401

TABLE IIINumerical 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 1B. amabilis 1 1(2) 3(2) 0 2(3) 1 1 2 3 1 1B. ochraceoroseus 2(1) 3 1(2) 1 0 2 2 0(1) 0 1 1B. grisellus* 1(2) 2 1 0 1 — 0 0 0 0 1B. pictus* 1 2 2 2(3) 1 — 2 1 1 1(0) 1B. 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 couldbe incorporated into a dichotomous key for identification purposes. However,to facilitate the addition of more species of mycorrhizal fungi as the databecome available, the following numerical key is suggested (Table III). It issimilar to, and functions in the same manner as, those developed by Nobles(1948) and others. The three species studied here are listed first; these arefollowed by three species studied by Pantidou whose data have been inter-preted to fit the proposed code and included here for comparison. The meaningof 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 thepresent time should not be of particular concern to those who wish to usesuch reactions as taxonomic tools. As Singer (1949) points out, it mattersonly that a color appears specifically under defined conditions for that reactionto be useful. Characteristics of the mycelium of some species of Boletaceaegrown in pure culture under standardized conditions can be used to identifythe fungi. The procedures presented here should be of use to those who desireto identify the fungi involved in mycorrhizae of forest trees by direct isolationfrom the organ. They can also be of value in resolving the taxonomy of theboletes and, by extension, of other taxonomically difficult groups of Agaricales.

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LINDEBERG, G. 1948. On the occurrence of polyphenol oxidases in soil inhabiting Basidio-mycetes. Physiol. Plantarum, 1, 196-205.

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tania, 14, 156-172.SINGER, R. 1949. The "Agaricales" (mushrooms) in modern taxonomy. Lilloa, 22, 1-832.

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Suillus. Mycologia, 57, 669-674.SLIP?, A. W. and SNELL, W. H. 1944. Taxonomic-ecologic studies of the Boletaceae in northern

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