Mycological Society of America

Dinitrogen Fixation Associated with Sporophores of pinicola, , and tinctorium Author(s): M. J. Larsen, M. F. Jurgensen, A. E. Harvey, J. C. Ward Reviewed work(s): Source: Mycologia, Vol. 70, No. 6 (Nov. - Dec., 1978), pp. 1217-1222 Published by: Mycological Society of America Stable URL: http://www.jstor.org/stable/3759320 . Accessed: 31/10/2011 13:17

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http://www.jstor.org DINITROGEN FIXATION ASSOCIATED WITH SPOROPHORES OF FOMITOPSIS PINICOLA, FOMES FOMENTARIUS, AND ECHINODONTIUM TINCTORIUM

M. J. LARSEN U. S. Department of Agriculture, Forest Service, Center for Forest Mycology Research,1 Forest Products Laboratory, Madison, Wisconsin 53705

M. F. JURGENSEN Department of Forestry, Michigan Technological University, Houghton, Michigan 49931

A. E. HARVEY

U. S. Department of Agriculture, Forest Service, Forestry Sciences Laboratory,2 Missoula, Montana 59801

AND

J. C. WARD U. S. Department of Agriculture, Forest Service, Improvements in Drying Technology,1 Forest Products Laboratory, Madison, Wisconsin 53705

SUMMARY

Fixation of atmospheric dinitrogen by bacteria associated with con- textual tissues of sporophoresof Fomitopsis pinicola, Fomes fomentarius, and Echinodontium tinctorium is reported. Since nitrification rates are low, the data indirectly support the view that an autolysis-recycling mechanismof nitrogen from large volumes of woody tissue is the principal means by which wood-destroying fungi obtain nitrogen for sporophore production and sporulation.

In a series of papers on nitrogen requirementsof fungi associated with wood decay summarizedby Cowling (1970), the nitrogen-deficient 1 Maintained in cooperationwith the University of Wisconsin, Madison, Wis- consin. 2 Maintained in cooperation with the University of Montana, Missoula, Mon- tana. 1217 1218 MYCOLOGIA,VOL. 70, 1978 nature of woody materials for fungal growth was emphasized. Merrill and Cowling (1966) and Cowling (1970) indicated that wood-destroying fungi can apparently recycle nitrogen from older fungal tissues and wood (also see Levi et al., 1968) and use it elsewhere. Evidence has been presented (Seidler et al., 1972; Cornaby and Waide, 1973; Sharp and Millbank, 1973; Aho et al., 1974a, b) for the presence or activity of dinitrogen-fixing bacteria associated with decaying wood, indicating that wood-decay fungi may rely, in part, on an extramural source of nitrogen. Herein, we present data that suggest nitrogen requirements of fungal sporophores are apparently met, in small part, by the activity of a nitrogenase function in sporophores of Fomitopsis pinicola (Swartz ex Fr.) Karst., Echinodontium tinctorium (Ell. & Ev.) Ell. & Ev., and Fomes fomentarius (L. ex Fr.) Kickx.

METHODS

Live sporophores of Fomitopsis pinicola, Echinodontium tinctorium, and Fomes fomentarius were obtained from several forest stands on the Coram Experimental Forest in northwest Montana. Each F. pini- cola sporophore was collected from individual dead and fallen boles of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco], of western hem- lock [Tsuga heterophylla (Raf.) Sarg.], and of subalpine fir [Abies lasiocarpa (Hook.) Nutt.]. Fomes fomentarius sporophores were collected from three standing boles of dead paper birch (Betula papyri- fera Marsh.). Three standing live western hemlock trees were selected on the basis of occurrence of E. tinctorium sporophores, which were collected at each of three heights (0-3 m, 6-9 m, and 12-15 m) from each of the three stems. Three conks of each fungal species on each host were sectioned into small pieces (ca. 0.5 cm 3) and placed into 20-ml glass Vacutainer (Becton-Dickinson Div., Becton-Dickinson 3) tubes. Five replicate-tube samples of each conk were prepared. The outer surfaces of the conks were removed to eliminate surface contamination of N2-fixing organisms. Nitrogen-fixation rates in sporophores were estimated using an adaptation of the acetylene-reduction technique described and discussed by Hardy et al. (1968). Within 2 h of sampling, the tubes were evacuated five times with the aid of syringe needles, flushed with argon five times, then filled with a gas mixture of 10% acetylene and 90%

3 Use of trade or proprietary names is for informational purposes only and does not imply any endorsement by the Forest Service of the U. S. Department of Agriculture. LARSEN ET AL.: DINITROGEN FIXATION 1219 argon. Final oxygen concentrations in the tubes approximated 0.05 %. Acetylene was omitted from one tubed sample of a sporophore of each species for monitoring endogenous ethylene production; these values were subtracted from those obtained from samples incubated with acetylene. The sample tubes containing sporophore tissues were incubated in the dark for 24 h at ambient field temperature (12-15 C). Gas sub- samples were removed by 2-ml Vacutainer tubes and analyzed for acetylene and ethylene levels using a Varian Series 2800 gas chromato- graph fitted with an 80-100 mesh Poropak R column. The column was run at 50 C using nitrogen as the carrier gas. After gas sampling, the sporophore samples were dried at 105 C to determine dry weight and moisture contents. Tissue isolates were made from all sporophores before the was cut into pieces for the acetylene-reduction test. Pieces of context tissue were placed on malt agar (2% w/v) and incubated at 18 C. Fun- gal growth typical of F. pinicola, F. fomentarius, and E. tinctorium indi- cated the presence of viable and active hyphae within the sporophores tested.

RESULTS AND DISCUSSION

Our data (TABLESI and II) demonstrate a nitrogenase function in fungal sporophores by the acetylene-reduction technique. No sig- nificant differences were detected between Echinodontium tinctorium sporophores (TABLEII) horizontally or vertically. Differences (P = 0.001) were significant between sporophores of F. pinicola on hemlock and all other fungus-substrate associations. However, these differences were not reflected by moisture contents of the various species because F. fomentarius and E. tinctorium contained significantly less moisture (P = 0.001 and P = 0.01, respectively) than F. pinicola on Douglas-fir, subalpine fir, and hemlock. Moisture contents of sporophores were not highly correlated with increased dinitrogen fixation. The fixation rates reported here are similar to those reported for "soil" from Georgia by Cornaby and Waide (1973) as 1.18 x 10-9 mol/ g/24 h and by Larsen et al. (1978) as 7 X 10-9 mol/g/24 h from a variety of woody substrates in various stages of decay in western Montana. Whether or not the amounts of dinitrogen fixed actively contribute to the nitrogen regimes of actively sporulating sporophores of F. pini- 1220 MYCOLOGIA, VOL. 70, 1978

TABLE I

MEAN NUMBER MOLES (X10-9) PER G OVENDRY WEIGHT OF FUNGAL TISSUE OF NET ETHYLENE PRODUCEDIN 24 H IN SPOROPHORESOF Fomitopsis pinicola ON DOUGLAS-FIR, WESTERN HEMLOCK, AND SUBALPINE FIR, AND Fomes fomentarius ON PAPERBIRCH

Percent moisture Moles produced Fungus and associated content in 24 h tree species 2 i SD ±- SD

Fomitopsis pinicola Douglas-fir 166.6 + 25.4 0.98 i- 0.40 - Subalpine fir 162.15 i 12.13 0.75 0.20 Western hemlock 176.3 - 19.9 2.20 i- 0.30 Fomes fomentarius White birch 111.4 ± 95.8 0.98 ± 0.12 cola, F. fomentarius, and E. tinctorium is a question that awaits resolu- tion. If we assume from our work an average rate of net ethylene produc- tion for sporophores as 1.12 X 10-9 mol/g odw (ovendry weight) fungal tissue/24 h, then for 200 da at that level of fixation in a 300-g sporophore (assumed average weight) 0.6 x 10-3 g of N would be fixed annually. Upon reviewing the figures of Merrill and Cowling (1966) on the re- gimes and the amounts of nitrogen they reported for fungi, the amount of nitrogen fixed in our experiments appears to represent only a small fraction of the nitrogen requirements for sporophore formation and sporulation. Merrill and Cowling (1966) reported that 15.6 g of N would be required for 100 da of sporulation by Ganoderma applanatum (Pers. ex Wallr.) Pat., and if sporulation occurred for as long as White (1920) observed (6 mo), 28 g of N would be required annually. The data of Meyer (1936), as interpreted by Merrill and Cowling (1966), are even more impressive because she reported the astounding figure of more than

TABLE II

MEAN NUMBER MOLES (X10-9) PER G OVENDRY WEIGHT OF FUNGAL TISSUE OF NET ETHYLENE PRODUCED IN 24 H IN SPOROPHORES OF Echinodontium tinctorium ON WESTERNHEMLOCK

Percent moisture Bole position of sporophore content Moles produced (m above ground level) E ± SD 2 +- SD

0-3 97.9 ± 34.4 1.07 ± 0.14 6-9 131.6 ± 20.7 1.0 i 0.09 12-15 132.7 i 35.9 0.98 ± 0.13 Total, all sporophores 120.75 ± 34.33 1.01 i 0.12 LARSEN ET AL.: DINITROGEN FIXATION 1221

1 kg of spores produced in a 20-da period by F. fomentarius, requiring 35 g of N (assuming N content of spores to be 3%). We may conclude that the autolysis-recycling mechanism(s) of nitrogen from large volumes of woody materials proposed by Merrill and Cowling (1966) is the primary means by which wood-decay fungi are provided with the necessary amounts of nitrogen for sporophore formation and subsequent sporulation. Although we have not demonstrated the physical presence of N2- fixing bacteria in sporophore tissue, we may reasonably assume the fixation phenomenon is not caused by the fungus (Millbank, 1968), but instead by an intimately associated bacterial population. Pre- liminary results from isolations of sporophores and examination by scanning-electron microscopy of contextual tissues indicate that a bac- terial population does exist. Our inability to date to cultivate the orga- nism(s) indicates that it is extremely fastidious in its requirements for axenic culture.

ACKNOWLEDGMENT

The capable and responsible technical assistance of Ms. K. R. Slattery is cited.

LITERATURE CITED

Aho, P. E., R. J. Seidler, H. J. Evans, and P. N. Raju. 1974a. Distribution, enumeration, and identification of nitrogen-fixing bacteria associated with decay in living white fir trees. Phytopathology 64: 1413-1420. - , --, - and A. D. Nelson. 1974b. Association of nitrogen-fixing bac- teria with decay in white fir. Proc. First Internat. Symp. Nitrogen Fixationl V. 2: 629-640. Cornaby, B. W., and J. B. Waide. 1973. Nitrogen fixation in decaying chest- nut logs. PI. & Soil 39: 445-448. Cowling, E. B. 1970. Nitrogen in forest trees and its role in wood deterioration. Acta Univ. Upsal. (Abstr. Uppsala Diss. in Science. 164) : 1-19. Hardy, R. W. F., R. D. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethyleneassay for N2 fixation: laboratory and field evaluation. PI. Physiol. (Lancaster) 43: 1185-1207. Larsen, M. J., M. F. Jurgensen, and A. E. Harvey. 1978. N2 fixation asso- ciated with wood decayed by some common fungi in western Montana. Canad. J. For. Res. (in press). Levi, M. P., W. Merrill, and E. B. Cowling. 1968. Role of nitrogen in wood deterioration VI. Mycelial fractions and model nitrogen compounds as substrates for growth of versicolor and other wood-destroying and wood-inhabiting fungi. Phytopathology 58: 626-634. Merrill, W., and E. B. Cowling. 1966. Role of nitrogen in wood deterioration: Amount and distributionof nitrogen in fungi. Phytopathology 56: 1083-1090. 1222 MYCOLOGIA,VOL. 70, 1978

Meyer, H. 1936. Spore formation and discharge in Fomes fomentarius. Phyto- pathology 26: 1155-1156. Millbank, J. W. 1968. Nitrogen fixation in moulds and yeasts-a reappraisal. Arch. Mikrobiol. 68: 32-39. Seidler, R. J., P. E. Aho, P. N. Raju, and H. J. Evans. 1972. Nitrogen fixa- tion by bacterial isolates from decay in living white fir trees [Abies concolor (Gord. & Glend.) Lindl]. J. Gen. Microbiol. 73: 413-416. Sharp, R. F., and J. W. Millbank. 1973. Nitrogen fixation in deteriorating wood. Experientia 29: 895-896. White, J. H. 1920. On the biology of Fomes applantaus. Trans. Roy. Canad. Inst. 12: 133-174. Accepted for publication June 26, 1978