Carbon Nutrition of some Mycorrhizal Boletus Species

Notes and Brief Articles

795

REFERENCE

R. T. A. & WHEELER, B. E.]. (1967)' Overwintering of cleistocarps, and infection by ascospores, of Erysiphe cichoracearum on Arctium lappa. Trans. Br. mycol. Soc. 50, 625-63°.

COOK,

E. C. CUTrER AND B. E. J. WHEELER, Imperial College Field Station, Silwood Park, Sunninghill, Berks.

CARBON NUTRITION OF SOME MYCORRHIZAL BOLETUS SPECIES

Except for certain Tricholoma species (Norkrans, 1950) the carbon nutrition and relevant metabolic pathways of mycorrhizal basidiomycetes, have not been studied. Some non-mycorrhizal basidiomycetes have been investigated more thoroughly, notably Marasmius (Lindeberg, 1944) and Coprinus (Fries, 1955). The metabolic pathways involved in carbon nutrition have been examined in Polyporus brumalis (Casselton, 1966). Work done so far with Boletus (Melin, 1925; How, 1940; Ferry, 1967) has concerned those species which form mycorrhizas with gymnosperms. In every case glucose proved to be the best carbon source, although mannose was frequently almost as good. Fructose was invariably poor, as was galactose and the only pentose sugar tried, xylose. Growth with disaccharides was fair, maltose usually being superior to sucrose. Growth was very poor with acetate and fair with malate. Attempts were made to measure growth with various polysaccharides including cellulose, starch, pectic substances and lignin. Slight responses to pectic compounds and to starch were observed for some species. This pattern ofcarbon nutrition of mycorrhizal Boletus species conforms with results for other groups, notably mycorrhizal Tricholoma species (Norkrans, 1950), and differs from that of non-mycorrhizal basidiomycetes especially in utilization of polysaccharides. The present investigation constitutes a more thorough survey of soluble carbon compounds as carbon sources for mycorrhizal Boletus species, prior to investigation of the metabolic pathways associated with carbon nutrition in these fungi. It is considered that carbon nutrition and metabolism in these fungi may be in some way related to host ranges. Four Boletus species were used, namely B. elegans Schum. ex Fr., B. bovinus L. ex Fr., B. luteus L. ex Fr. and B. variegatus Sow. ex Fr. The first two fungi have been reported to form mycorrhizas with species of Larix and Pinus respectively, and the last two fungi with both Larix and Pinus. Pure cultures of the fungi were obtained by transplanting pieces of sporophore tissue on to agar slopes of a medium containing, per litre of distilled water: glucose, 20 g; NH4CI, 0'5 g; MgS04.7HzO, 0'5 g; KH zP04, 0'5 g; Oxoid malt extracty g g, and Oxoid ionagar no. 3, 15 g. The sporophores were collected from different localities in south-east England. Experiments were conducted in a medium containing, per litre of distilled water: carbon source, 20 g; ammonium tartrate, 0'5 g; Trans. Br. mycol. Soc. 5I (5), (1968). Printed in Great Britain

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Isolate 102 109 128 110 III 121 123 13° 116 122 lSI 100 112 113 127

None 2'O±O'1 1'2±O'1 I'S±o'6 1'7±o'4 1'3±o'l O,6±O'4 1"4±O'2 2'1±o'4 I'O±O'I 1'1 ±o'3 o'g±o'l o'7±o'3 1'2±O'S I'I±O'S 1'3±o'l

Glucose 28'7± 1"0 31'S± 1'2 2S'S± 1'1 II'S±O'2 IS'7±2'7 19'4±o'3 12'3± 1'2 15'3±o'9 21'g±2'3 IS,6±o'7 30'g±2,8 21'1±1"0 23'S±2'2 17'1±o'g 16'2±o'g

7'3±o'9 6'S±o'7 1I,6±o'l S'2± 1"6 3'9±o'l 3'3±O,6 2'4±o'3 2'2±O'3 12'2+0'1 S,6±O'2 13,6±o,6 2'g±O'2 4,6±o'2 2'9±o'4 3'7±o'4

26'3± 1'3 33'6± 1'7 21'S± 1'2 1I,6± 1,6 g'l±o'g lI'g±o'S g,6±o'3 1O'4±o'9 12'3± 1'2 16'S±o'7 1O,8±o'4 8'o±o'7 14,8±I'3 14,6±o'2 13'2± 1'1

Galactose 6,8+ 1'1 13'O±o'8 6'g±o'3 2'S±o'3 4'1 ±o'3 2,8±o'4 3'1 ±o'S S'I±O'S 3'1± I'g S'2±o'6 3'S±O'2 2'9±o'4 2'2±O'S 1"7±o,6 2'S±o,6

Isolate 102 109 128 110 III 121 123 13° 116 122 131 100 112 113 127

Sucrose

Maltose IS'2± 2'4 S'9±o'7 7'o±o'S 3'1 ± 0'2 6'4±o,8 S'2±O'S S'J±O'2 7'2±O'g J 1'8± 0'1 lO'g± 1'5 14'2±I'1 4'1±o'4 S'2±O'3 4'4±O'4 IS'3± 1'2

Cellobiose IS'8±o'g g'g±o'S 13'3± 1'5 4'S±O'S 6'7±O'S 8'4± 1'0 6'4± 1"2 IO'2±O'g 9'3± 1'5 14'4±2'3 14'9±o'g 1O,6±o'8 9'7±o'9 S,6±o'l 7'7±O'1

Lactose 8'1 ±o'g 3'3±o'S 6'3±O'3 2'4±O'4 4'2±I'1 3'9±O'3 2,6±o'2 2'9±o'3 3'o±o'l 8,8±o'g 4'3±o'3 S'3±o'4 2'g±O'2 3'8±o'S 3'1±O'1

Mannitol 13'8±I'8 12,8± 1'0 g'9±o'4 2'7±O'S S'S±O'J S'4±O'4 3'J±O'S S'6± 1"0 13,6±o'2 14'7±o'9 18'1 ± o'g 4'o±o'S 2'7±O'3 2'9±o'3 6'7±O'4

Fructose

Mannose

Xylose 4'O±O'4 7'2±O'S 12'3±o'2 S'4±O'2 3'4±o'3 3'3±o'S 3'1±o'3 4,6±o'l 2'4±o'4 3'4±o'2 2'9±o'3 3'1±o'3 2'g±O'2 2'g±o,6 1,8±o'2

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1I'4± 1'4 1O,8±o'S JO'O± 1"0 6'1±O'2 7'9±o'7 13'3±o'9 6'2±O,8 4'3±O,6 7'9±o'8 IO'4±o,6 IS'2±O'S S'O±O'2 7'4±O,8 7'7±o'3 7'4±I'1

Glycerol 2'S±O'S 3'J ±O'J 4'o±o'g l'g±O'2 3'S±O'1 3'2±O'S 2'9±o'3 4'3±o,6 2'g±o,6 7'S±o'S 2,8±o'3 2'S±o'3 1,6±o'2 1"9±o'4 I'S±O'I

t-Arabinose

Ribose

17'7±o'4 8'7±o'S 3'4±o'S 2,6±o'2 3'1 ±O'I 3'3±o'3 2,6±o'2 2'1 ±o'4 2'1 ±O'I S,6±o'l 2'7±O'S S,6±o'S 2'2±O'1 2'8±o'3 2,6±o'2

7'1 ±o'7 7'1 ±o'3 I'S±O'2 2'o±o'4 3'9±o'3 3'3±o'2 2'o±o'4 2'O±O'2 S'3±o'4 6'3± 1'1 S'o± 1'3 2,8±o'S 2'S±O'S 2'O±O'2 2'4±o'2

Sod, acetate 3'2±O'8 2'3 ± 0'4 1,8±o'l

Sod, malate 3,6±o'8 J'g±o'l II'S± 1'0 S'g±o'l 4'8±o'S 3'9±o'l 2,8±o'7 4'9±o'g 3'1±o'6 3,8±o'2 2,8±o'S 3,8±o'2 4,6±o'l 3'O±O'2 2'S±O'1

1'9±o'3 S'2±O'1 S,8±O'2 J'7±O'J 4,8±o'3 2'4±o'3 2'9±o'4 S'o±o'3 2'7±o'3 I"g±O'2 1'7±o'4 2'2±o'g

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Notes and Brief Articles

797

MgSOt.7H20, O·S g; KH 2PO t, I g; FeSOt.7H20, S mg; ZnSOt.7H20, S mg, and thiamine HCI, 0'05 mg. The control flasks contained no carbon source. Tartrate is not utilized by any of these fungi. All carbon sources were sterilized separately from the remainder of the medium. Fructose and the pentose sugars were sterilized by millipore filtration, the remaining carbon sources were steam-sterilized. The pH was adjusted before sterilization, to give a final value of S'o in the medium. Each treatment in an experiment was represented by four replicates and experiments were carried out at 2S °C in the dark, growth being permitted for 8 days (IS days for B. elegans). Flasks, containing 20 ml of medium, were inoculated with mycelium on agar disks cut from colonies growing on the experimental medium plus Oxoid ionagar no. 2, IS gil. The disks were leached by floating on sterile distilled water for 24 h prior to use. Mycelia were harvested and dried to constant weight at 70°. The dry-weight values obtained for the control flasks represent approximately the weight of the inoculum. The results are summarized in Table I and show that glucose is invariably the best carbon source, although mannose is, for some isolates, equally good. Fructose is generally poor except for isolates of B. luteus. Galactose is a poor carbon source for all isolates. Growth with pentose sugars is poor, isolates of B. elegans responding significantly better than isolates of B. variegatus. Isolates of B. luteus grow well with n-ribose which gives the poorest growth with most other isolates. Of the disaccharides tested lactose is invariably the poorest carbon source. Cellobiose, sucrose and maltose range from good to very poor, cellobiose being preferred by most isolates. Response to mannitol ranges from poor to very poor except for isolates of B. luteus which grow well with this sugar alcohol. Glycerol is invariably poor. Of the organic acids malate is generally preferable to acetate but both are poor carbon sources. These results agree in general with those obtained by other workers for mycorrhizal Boletus species and for other mycorrhizal basidiomycetes. The use of several isolates to represent each species examined is clearly important, if meaningful conclusions are to be drawn from such experiments. Probably isolates should be obtained from more widely separated geographical areas than those in the present investigations, in order to obtain a representative sample of a species. Nevertheless, the considerable variability in the present isolates emphasizes the need for more than a single isolate to represent a species (see Table I, growth of B. elegans with t-arabinose). This is perhaps more important in basidiomycetes than other fungi, because of the dikaryotic state and high frequency of hyphal anastomoses. Dry-weight values represent growth attained in 8 (or IS) days and correspond to points on the rapid growth phase of the growth curve for all isolates with the readily utilizable carbon sources. For some of the poor carbon sources it is possible that synthesis of inducible enzymes is going Trans. Br. mycol. Soc. 5I (5), (1968). Printed in Great Britain

798

Transactions British Mycological Society

on, and that the dry-weight values therefore correspond to points on the lag or acceleration phases of the growth curve. Nevertheless, it is considered that dry-weight values obtained by this method are as meaningful as maximal dry-weight values attained in a definite amount of medium. Clearly some quite significant differences in the utilization of various carbon sources exist in the four Boletus species examined. Of particular interest is the relatively good growth of B. luteus isolates with D-fructose, mannitol and n-ribose when compared with isolates of the other species. REFERENCES CASSELTON, P.]. (1966). Enzymes of the Embden-Meyerhoff and pentose phosphate pathways in Polyporus brumalis extracts. ]. expo Bot. '17, 579-589. FERRY, B. W. (1967). Ph.D. Thesis, London. FRIES, L. (1955). Studies in the physiology of Coprinus. I. Svensk bot. Tidskr·49, 475-535. How,]. E. (1940). The mycorrhizal relations oflarch. I. Ann. Bot. 4, 135-150. LINDEBERG, G. (1944). Uber die Physiologie ligninabbauender Boden-Hymenomyceten. Symb. bot. upsal, 8, 1-183. . MELIN, E. (1925). Untersuchungen iiber die Bedeutung der Baummykorrhiza. Eine 6kologische physiologische Studie.] ena: G. Fisher. NORKRANs, B. (1950), Studies in growth and cellulolytic enzymes of Tricholoma. Symb. bot. upsaliens. n, 1-126. B. W. FERRY and NEELA DAS, Department of Botany, Bedford College, London

Two NEW SPECIES OF WARDOMYCES During extensive investigations into the mycoflora of wheat field soils at Kitzeburg and Kiel, several dark aleuriosporic hyphomycetes were isolated. In three of them a second hyaline, annellophoric spore-form was observed. Amongst more than 23000 isolates the following species were identified: Mammaria echinobotryoides Ces. (20 isolates), Wardomyces columbinus (Demel.) Henneb. (5 isolates), and two new species to be described in this paper (34 and 3 isolates respectively). Hennebert (1968) has, by the inclusion of Wardomyces columbinus (Demel.) Henneb. comb.nov. expanded this genus, so that it comprises species with usually simple conidiophores and also with a second, annellophoric, state. The two following species with both spore forms are closely related to W. columbinus. They could not be identified with any known species of the dark aleuriosporic genera. Wardomyces ovalis sp.nov, (Fig. I) Mycelium in agaro maltoso tarde crescit, albidum, grisescens, parce floccosum; sporulatio duobus modis. Aleuriosporae primum globosae, demum ovales vel ellipticae, brunnescentes, pariete crasso, plerumque pallido sulco praeditae, 8-11 X 4-5 p, solitariae vel gregariae, sessiles vel hyphis aleuriophoris plerumque simplicibus, interdum apice inflatis portatae. Conidia Scopulariopsis modo ovoidea, quasi hyalina, levia, 5'5-8 ( - 10) x 3'5-5( - 6) p, e cellulis annellophoris brevibus oriunda, basi 6-10 x 2-3 Trans. Br, mycol. Soc. 51 (5), (1968). Printed in Great Britain