UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The biology of Flammula alnicola (Fr.) Kummer Denyer, Walter Bruce Glenn 1959

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1959_A1 D3 B4.pdf [ 8.05MB ]
Metadata
JSON: 831-1.0106405.json
JSON-LD: 831-1.0106405-ld.json
RDF/XML (Pretty): 831-1.0106405-rdf.xml
RDF/JSON: 831-1.0106405-rdf.json
Turtle: 831-1.0106405-turtle.txt
N-Triples: 831-1.0106405-rdf-ntriples.txt
Original Record: 831-1.0106405-source.json
Full Text
831-1.0106405-fulltext.txt
Citation
831-1.0106405.ris

Full Text

THE BIOLOGY OF FLAMMULA ALNICOLA (FR.) KUMMER by Walter Bruce Glenn Denver B.Sc, University of Manitoba, 194-3 B.S.F., University of New Brunswick, 194-9 M.A., University of B r i t i s h Columbia, 1951 A thesis submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Department of BIOLOGY and BOTANY We accept this thesis as conforming to the standard required from candidates for the degree of Doctor of Philosophy Members of the Department of Biology and Botany The University of B r i t i s h Columbia April, 1959 i i ABSTRACT Flammula alnicola (Fr.) Kummer, formerly called Flammula conissans Fr. sensu Ricken causes a yellow stringy butt rot of several conifers in Western Canada. It i s usually of infrequent occurrence in conifers, but was found to be the most important butt rot of white spruce in the Smith - Slave Lake area of Alberta. An investigation was conducted to gain information on the biology of the fungus. The decay has a characteristic pattern caused through preference of the fungus for wood rays and early spring wood. A musty odor in decay and cultures i s characteristic. The fungus invades white spruce through pressure wounds caused by root contacts but evidently does not invade recently formed wounds. The average linear rate of decay was one inch per year in two-year-old a r t i f i c i a l infections but probably less in natural infections. The fungus decayed blocks of root and stem wood, and blocks of li g h t and heavy density wood, slowly, but at similar rates. The occurrence of the decay was related to good sites, and non-cal-careous soils with acid f i r s t mineral horizons. The mat of cultures i s fine woolly, white becoming a pale yellow, growth slow. Microscopically cultures are characterized by the presence of clamp connections and a l l o -cysts. Optimum growth on malt agar occurred at 22° C. and at a pH of 4-=4-« Fruiting in culture was induced by aeration with nearly saturated air, a temperature of 5A° F. and illumination of 8 to 30 foot candles. The f r u i t body of F. alnicola i s a yellow mushroom which has a stipe with darker base, adnate lamellae, partial v e i l adhering to. the edge of the pileus, hymenium without pleurocystidia; spores rusty brown, A * 5 - 5 . 5 x 7 - 10 u ovate ellipsoid, apiculate. Spores germinated on culture media only after cold i i i treatment at -7° C. Cold treatment of spores at -18° C. inhibited ger-mination. The fungus shows the tetrapolar type of i n t e r f e r t i l i t y . Parallel experiments with Flammula conissans (Fr.) G i l l e t were con-ducted to establish the two fungi as separate species. F. conissans has similar cultural characters except for a faster rate of growth and the absence of the musty odor characteristic of F. alnicola. Aeration inhibits i n i t i a t i o n of fruiting in F. conissans. Fruit bodies are smaller, the base of the stipe i s concolorous with the cap and pleurocystidia are present on the hymenium. Spores are rusty brown, ^ - i«5 x 5.5 - 8 ji, oval oblong. F. conissans shows the tetrapolar type of f e r t i l i t y . Monosporous mycelia of F. alnicola and F. conissans are inter s t e r i l e . F. alnicola because of i t s slow rate of growth and restriction to heartwood i s relatively unimportant as a cause of wind throw in white spruce. WL\£ PmiiKtrsttg of ^British (Ecrlirmbta Faculty of Graduate Studies 1111 P R O G R A M M E O F T H E FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of WALTER BRUCE GLENN DENYER B.Sc, University of Manitoba B. S. F. , University of New Brunswick M. A., University of British Columbia IN ROOM 33i BIOLOGICAL SCIENCES BUILDING TUESDAY, APRIL 21, 1959 AT 3:00 P. M. C O M M I T T E E I N C H A R G E Dr. W. A. CLEMENS, Chairman T. M. C. TAYLOR R. W. WELLWOOD V. J . KRAJINA C. ROWLES R. BANDONI P. G. HADDOCK J. E . BIER G. W. MARQUIS External Examiner: Dr. MILDRED K. NOBLES Senior Mycologist, Federal Department of Agriculture, Ottawa THE BIOLOGY OF FLAMMULA ALNICOLA (Fr.) KUMMER A B S T R A C T Flammula alnicola (Fr.) Kummer, formerly called Flammula  conissans Fr. sensu Ricken causes a yellow stringy butt rot of several conifers in Western Canada. It is usually of infrequent occurrence in conifers, but was found to be the most important butt rot of white spruce in the Smith-Slave Lake area of Alberta. An investigation was con-ducted to gain information on the biology of the fungus. The decay has a characteristic pattern caused through preference of the fungus for wood rays and early spring wood. A musty odor in decay and cultures is char-acteristic. The fungus invades white spruce through pressure wounds caused by root contacts but evidently does not invade recently-formed wounds. The average linear rate of decay was one inch per year in two-year-old artificial infections but probably less in natural infections. The fungus decayed blocks of root and stem wood, and blocks of light and heavy density wood, slowly, but at similar rates. The occurrence of the decay was related to good sites, and non-calcareous soils with acid first mineral horizons. The mat of cultures is fine woolly, white becom-ing pale yellow, growth slow. Microscopically cultures are character-ized by the presence of clamp connections and allocysts. Optimum growth on malt agar occurred at 22° C. and at a pH of 4. 4. Fruiting in culture was induced by aeration with nearly saturated air, a tempera-ture of 54° F . and illumination of 8 - 30 foot candles. The fruit body of j ; . alnicola is a yellow mushroom which has a stipe with darker base, adnate lamellae, partial veil adhering to the edge of the pileus, hymen-ium without pleurocystidia; spores rusty brown, 4. 5 - 5. 5 x 7 - lOju, ovate ellipsoid, apiculate. Spores germinated on culture media only after cold treatment at - 7 ° C . Cold treatment of spores at - 1 8 ° C . inhibited germination. The fungus shows the tetrapolar type of inter-fertility. Parallel experiments with Flammula conissans (Fr.) Gillet were conducted to establish the two-fungi as separate species. F. coni-ssans has similar cultural characters except for a faster rate of growth and the absence of the'musty odor'characteristic-of F . alnicola. Aera-tion inhibits initiation of fniitmg m£._j^pnissans. Fruit bodies are smaller, the base of the stipe is concolorous with the cap and pleuro-cystidia are present on the humenium. Spores are rusty brown, 4 - 4.5 x 5. 5 - 8, oval, oblong. _F. conissans shows the tetrapolar type of fertility. Monosporous mycelia of F. alnicola and F. conissans are inter sterile. F. alnicola because of its slow rate of growth and restriction to heartwood is relatively unimportant as a cause of windthrow in white spruce. P U B L I C A T I O N S 1952. Denyer, W . B . G . Red stain in Lodgepole pine in Alberta. Canada Dept. Agr. , For. Biol. Div. Bi-Monthly Prog. Rept. 8(2):3. to 1953; Denyer, W . B . G . Cephalosporium canker on Western Hemlock. Can. J . Botany 31:361-366. 1953. Denyer, W . B . G . andRiley, C . G . Decay in white spruce at the Kananaskis Forest Experiment Station. For. Chron. 29:233-247. 1954. Denyer, W . B . G . andRiley, C . G . Decay in white spruce in the Prairie Provinces. Canada Dept. Agr. , For. Bio. Div. , Interim Report. 1958. Denyer, W . B . G . The effect of o-phenylphenol on the growth of some wood-rotting fungi. Canada Dept. Agr. , For. Bio.. Div. Bi-Monthly Prog. Rept. 14(1): 2-3. GRADUATE STUDIES Field of Study: Botany Forest Associations V. J . Krajina Phylogenetics . . V. J . Krajina Mycology F. Dickson Methods in Forest Pathology D. C. Buckland Advanced Forest Pathology D. C. Buckland Principles of Forest Genetics .... A. H. Hutchinson Other Studies: Soil Physics C. A. Rowles Soil Genesis, Morphology and classifications C. A. Rowles Soil Bacteriology D. G. Laird Advanced Wood Technology J. Wilson Statement of rights of publication and loan of thesis entitled, "The biology of Flammula alnicola (Fr.) Kummer". In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my department or, in his absence, by the University Librarian. I t i s understood that any copying or publication of this thesis for financial gain shall not be allowed without my written permission. ¥. B. Go Denyer, Author of the thesis concerned. i v ACKNOWLEDGEMENTS The author wishes to acknowledge support of the investigation as an o f f i c i a l project of the Forest Biology Laboratory, Saskatoon by the senior officers of the Forest Biology Division, Department of Agriculture, Ottawa. The investigation was carried out under the direction of the late Dr. D. C. Buckland who provided technical guidance. Dr. C. G. Riley, 0fficer-5.n-Charge, Forest Biology Laboratory, Saskatoon acted as an advisor throughout the study. Dr. A. H. Hutchinson, former Head, Department of Biology and Botany advised on histological aspects of the study. The author i s indebted to Dr. M. X. Nobles, Senior Mycologist, Botany and Plant Pathology Division, Department of Agriculture, Ottawa for per-mission to include the cultural characters of Flammula alnicola. Dr. J . ¥. Groves, Head, Mycology Unit, Botany and Plant Pathology Division, Ottawa identified the f r u i t body of F. alnicola and provided helpful criticism and advice in the preparation of the technical description of F. alnicola. and F. conissans. Acknowledgement i s also made to the Forestry Branch, Department of Northern Affairs and National Resources, Ottawa for permission to include the site classification of white spruce in the Mixedwood Region of Alberta. V TABLE OF CONTENTS Page I. INTRODUCTION 1 II. THE- DECAY A A. Macroscopic Characters A B. Microscopic Characters . 8 i . Methods 8 i i . Flammula alnicola Decay . . . . 9 i i i . Flammula conissans Decay 1 1 C. Manner of Infection and Extent of Decay in Tdhite Spruce . . . 1 1 D. Laboratory Test of Decay 2 1 E. Inoculation of Living Trees 3 2 F. Occurrence of F. alnicola in Relation to Site Factors . . . . 3 6 III. THE FUNGUS 4 - 3 A. Cultural Characters . . . . . A3 i . Flammula alnicola . . . . . . . . . A3 i i . Flammula conissans 4 6 i i i . Effect of pH on F. alnicola and F. conissans 4 7 iv. Effect of Temperature on F. alnicola in Culture . . . . 4.8 v. Hyphal Fusions • 50 v i . The Growth of F. alnicola in Soil Culture 5 1 B. Fruiting in Nature . 5 3 C. Fruiting in Culture 5 9 i . Flammula alnicola 5 9 i i . Flammula conissans . 6 4 D. Taxonomy and Description 6 6 i . Flammula alnicola . 6 6 i i . Flammula conissans • • 71 E. Spore Germination 7 3 i . Flammula alnicola 7 3 i i . Flammula conissans . . . . . . . . . . 7 5 F. I n t e r f e r t i l i t y Tests 7 5 i . Flammula alnicola . . . . . . . 7 6 i i . Flammula conissans - 7 7 i i i . F. alnicola and F. conissans 7 9 IV. DISCUSSION 80 V. REFERENCES 8 3 vi Page VI. PLATES AMD FIGURES Plate I., Figures 1 - 2 . F. alnicola decay . . . . . 6 Plate II., Figures 3 - 4-. F. alnicola decay 7 Plate III., Figures 5 - 7 . F. alnicola decay . . . . . . . . . 13 Plate IV., Figures 8 - 9 . F. alnicola decay 14-Plate V., Figures 10 - 13. F. alnicola decay . . . . . . . . . . 15 Plate VI., Figure 14.. Source of white spruce wood samples for a decay test of F. alnicola and F. conissans . . . 23 Plate VII., Figures 15 - 17. Cultural characters of F. alnicola and F. conissans 4-5 Plate VIII., Figure 18. Effect of pH on the growth of F. alnicola and F. conissans 49 Plate IX., Figures 19 - 20. Fruiting habit of F. alnicola . . 54-Plate X., Figure 21. Relation of a i r temperature to i n i t i a t i o n of f r u i t i n g of F. alnicola . 56 Plate XI., Figures 22 - 24-. Fruiting habit of F. alnicola . . 58 Plate XII., Figures 25 - 26. F. alnicola fruiting in culture . 6 l Plate XIII., Figures 27 - 30. Fruit bodies and basidiospores of F. alnicola and F. conissans 68 Plate XIV., Figures 31 - 33. I n t e r f e r t i l i t y tests of F. alnicola and F. conissans 78 VII. TABLES Table I. The relation between extent of decay (F. alnicola) and age of wound (infection court) in white spruce. 17 Table II. Percentage loss in conditioned weight of white spruce wood, of heavy and l i g h t density, from stem and root, six months after inoculation with _E. alnicola and _E. conissans 26 v i i Page Table III. The effect of density and type of wood (stem and root) on weight loss of white spruce decayed by F. alnicola and F. conissans; Computation of main effects and interactions 27 Table IV. The effect of density and type of wood (stem and root) on weight loss of white spruce decayed by F. alnicola and F. conissans; Analysis of variance. „ 28 Table V. The effect of density and type of wood (stem and root) on weight loss of white spruce decayed by F. alnicola and F. conissans; Main effects and interactions. . 29 Table VI. The effect of density and type of wood (stem and root) on weight loss of white spruce decayed by F. alnicola and F. conissans; Main effects. . . . . 31 Table VII. The extent of decay in l i v i n g white spruce roots, one and two years after inoculation with F. alnicola and F. conissans. 34 Table VIII. Physiographic site classification of white spruce in the Mixedwood Region of Alberta 38 Table IX. Calculation of Chi square for the relation between site class and occurrence of F. alnicola in white spruce 39 Table X. Calculation of Chi square for the relation between pH of the A£ s o i l horizon and occurrence of F. alnicola in white spruce. 4-0 Table XI. Calculation of Chi square for the relation between liminess of the parent material and occurrence of JL» alnicola in white spruce. 4 1 Table XII. Fruiting of F. alnicola and F. conissans in s o i l wood block cultures as affected by temperature, humidity, and l i g h t . . . . . . . . „ . 67 I. INTRODUCTION Flarnmula alnicola (Fr.) Kummer was isolated in Canada f i r s t from decay in Tsuga heterophylla (Raf.) Sarg. in the Queen Charlotte Islands of B r i t i s h Columbia, in 194-6. Its identity at that time was unknown, but because f r u i t body primordia, suggesting an agaric, developed in culture the fungus was designated "Hemlock agaric" by Dr. M. K. Nobles"*-. The fungus was isolated from decay in western hemlock and white spruce between 194-6 and 1950 but i t s identity remained unknown. In 1950, an agaric, associated with a yellow stringy decay in Picea glauca (Moench) Voss var. albertiana (S. Brown) Sarg., was collected by Dr. C. G. Riley and myself at Seebe, Alberta. This collection was identified, tentatively, as Flammula  conissans Fr. sensu Ricken by Dr. J. ¥. Groves . Cultures from the context of the mushrooms were identical with "Hemlock agaric" cultures isolated from decay. In the course of this study, f r u i t bodies produced in a cul-ture of Flammula conissans (Fr.) G i l l e t , from Europe, were compared with those of the "Hemlock agaric". They were found to be separate species and the identity of the "Hemlock agaric" was established as Flammula a l n i - cola (Fr.) Kummer. F. alnicola had been isolated from decay in several coniferous species in B r i t i s h Columbia (17); Tsuga heterophylla in the Upper Columbia, Kitimat, and the San Juan regions; Picea glauca (Moench) Voss in the Prince George region; Abies amabilis (Dougl.) Forb. in the Kitimat region; Abies l a s i o - carpa (Hook.) Nutt. in the Prince George region; and Picea Engelmanni Senior Mycologist, and ^ Head, Mycology Unit, Botany and Plant Pathology Division, Department of Agriculture, Ottawa. - 2 -Parry in the Kamloops region. Although the fungus i s widely distributed in B r i t i s h Columbia i t i s quite rare on a l l hosts. Out of 1307 isolations from decay in western hemlock in the Columbia region only seven were F. alnicola, and in amabilis f i r in the Kitimat region two out of 367 i s o l a -tions yielded the fungus. Fo alnicola was isolated more frequently from decay in white spruce in the Prairie Provinces. At Seebe, Alberta, ( l l ) F. alnicola was obtained in 13 out of 67 isolations from decay in Picea glauca var. albertiana. and in two out of 21 isolations from decay in Pinus contorta Dougl. var. l a t i f o l i a Engelm. In white spruce i t was the second fungus in frequency of occurrence, and accounted for 21% of the total decay volume. Only Fomes pini (Thore) Lloyd and Polyporus tomentosus Fr. were responsible for greater losses in volume. In another decay study of white spruce (Picea  glauca) in Alberta, Saskatchewan, and Manitoba (12), F. alnicola was the fourth most frequently isolated fungus from butt rots. I t was obtained in 32 out of 513 isolations from butt and trunk rots. Twenty-four out of 207 isolations from spruce on medium sites were F. alnicola. but on poor sites only four out of 296 isolations were J_. alnicola. In the white spruce sampled in Alberta F. alnicola was the most frequent cause of butt rot (22 out of 109 isolations) and accounted for 17% of the total decay volume. I t was less common in Saskatchewan (8 out of 364. isolations) and Manitoba (two out of 40 isolations). F. alnicola occurred in a l l three l o c a l i t i e s sampled i n Alberta (Fawcett Lake, Lesser Slave Lake, and Calling Lake), in two of the four sampled in Saskatchewan (Beaupre Lake and Dore Lake), and in one of the two l o c a l i t i e s sampled in Manitoba (Duck Mountain). - 3 -F. alnicola has never been found in the Candle Lake area of Saskatchewan although this area has been rather intensively sampled. The fungus also occurred in Picea mariana (Mill.) B.S.P. in Saskatchewan. In general the spruce in the sample in Alberta occurred on better sites than in the other two provinces, which may, in part, account for the more frequent occurrence of the decay in Alberta. Butt rot caused by Fj. alnicola has a wide dis-tribution but usually occurs infrequently on conifers in Western Canada. In certain regions, notably the Smith-Slave Lake area.of Alberta i t was the most important butt rot. Wood i s not uniformly decayed by F. alnicola. only some of the wood tissues are affected (Fig. 1 ) . This suggested that the rate of decay might be rapid and the fungus, therefore, an important cause of wind throw. The present study was undertaken to gain knowledge of the decay process and rate of decay, the relation of site conditions to occurrence, the cultural characters, fruiting habits, and taxonomy of the fungus. The f i e l d work of the investigation was carried out in the Smith-Slave Lake area of Alberta. - u-II. THE DECAY A. Macroscopic Characters Butt rot caused by Flammula alnicola in l i v i n g white spruce i s easily distinguished from other kinds of decay by i t s typical pattern and odor. In the cross section of a log or the surface of a stump, i t exhibits a pattern of narrow radial and concentric yellow lines or streaks (Fig. l ) . The radial pattern i s caused by decay originating in the wood rays and the concentric pattern by decay following the inner spring wood of annual rings. The pattern i s usually centred at the pith, but occasionally i t may be eccentric. In radial section, decay has a smooth mottled pattern of v e r t i -cal stripes (decayed spring wood) connected by horizontal bars (decayed rays) (Fig. 2). In tangential section decay occurs as stringy or striate sheets which tend to separate the annual rings. Typically decay i s yellow, varying from a rather vivid yellow to yellow brown (Munsel color equivalents (27) of dried samples 2.5Y 8 / 6 to 10.0YR 6 / 6 ) ; the color i s brighter in fresh material. ¥ood decayed by this fungus has a penetrating musty odor which serves as an excellent diagnostic character. Samples of decay that had been dried and kept for eight years s t i l l emitted the characteristic odor on wetting. Very rarely the odor may be absent from freshly exposed decay. Unlike most kinds of decay, there i s no incipient stage in the sense of an area of firm discolored wood surrounding, or in advance of breakdown of wood structure. Wood between the streaks of decay i s firm and not - 5 -discolored. At the tip of the decay column the wood may show a faint dis-coloration, more evident on wetting, in the same pattern as later stages. Wetting also brings out discoloration of a narrow zone surrounding the decayed streaks, which in this case indicates the presence of the fungus sli g h t l y beyond the visible l i m i t of decay. As decay progresses, wood between the radial streaks becomes decayed and the streaks coalesce. Similarly the concentric streaks expand, and eventually occupy a l l of the spring wood of annual rings. Summer wood i s broken down more slowly, and especially in compression wood i t may appear unaffected when spring wood on either side i s completely decayed. The pattern of decay in cross section may vary considerably (Fig. 3) f for example: I t may be composed almost entirely of either radial streaks or concentric lines of decay, or these may occur separately in different parts of the same decay area. The predominantly radial pattern occurs more frequently in wood with wide rings and the concentric one in wood with narrow rings. In decayed wood with narrow rings the radial streaks are confined within the annual rings. The summer wood in this case appears to be a barrier against the radial advance of the fungus. In narrow-ringed wood, with a concentric decay pattern, decay i s usually most advanced in the inner part of the spring wood. Occasionally, however, i t i s most advanced in the central region, and rarely, in the outer spring wood. Checks and shakes in the heartwood afford avenues of extension for the fungus well in advance of the main decay column. In cross section these breaks are surrounded by a narrow zone of decay. - 6 -PLATE I, Figures 1 - 2 PLATE I. F. alnicola Decay Figure 1. Typical decay pattern in white spruce. Figure 2. Radial longitudinal section of decay in white spruce. - 7 -PLATE II, Figures 3 - 4 PLATE II, F. alnicola Decay Figure 3. Variation of decay pattern in white spruce. Concentric pattern in narrow-ringed wood, l e f t ; typical pattern, centre; radial pattern in wide-ringed wood, right. Figure J+. Hyphae of F. alnicola in black spruce; A, network of hyphae in a wood ray; B, network of hyphae in first-formed spring wood showing clamp connections, and bore holes in the tangential walls of tracheids. - 8 -The decay pattern in black spruce and lodgepole pine i s similar to that in white spruce; other species from which the fungus has been reported have not been examined in this investigation. B. Microscopic Characters i . Methods ¥ood decayed by Flammula alnicola was sectioned on both a rotary and a sliding microtome. Serial sections were more useful for tracing the hyphae in wood 'tissues. In one embedding schedule^, material was fixed in propionic acid and propyl alcohol, softened i n a propyl alcohol solution of urea, and embedded in paraffin by the usual method. Carbowax embedding (26) provided a more rapid method of softening and embedding woody material, and was- also employed. Staining hyphae in wood presented d i f f i c u l t i e s in that dyes which stain the hyphae also stain partly broken-down l i g n i f i e d tissues and many of the hyphae, even in recently invaded wood, are without contents and w i l l not stain. The following stains were used: Cartwright's (6) picro aniline blue and variations of i t , methyl green, Pianese I l l b , Waterman's (34) revised Pianese I l l b , acid fuchsin, Haidenhain's haema-toxylin, rose bengal, thionin, and azure B. The best results were obtained with picro aniline blue and methyl green in combination, and azure B alone. Azure B was an interesting stain in that i t stained tracheids green, and wood rays bordered pits and occasional hyphae blue. Hyphae of F. alnicola were examined microscopically in sections of naturally infected white and black spruce. Sections from a r t i f i c i a l l y 'A schedule devised for embedding hard resinous material by Dr. A. H. Hutchinson, University of B r i t i s h Columbia. - 9 -infected white spruce were examined to study the very early stages of decay. Wood a r t i f i c i a l l y infected with F. conissans was also sectioned to compare the microscopic characters of the two fungi. i i . Flammula alnicola Decay Hyphae of F. alnicola are common in wood showing the earliest signs of decay and are abundant in wood showing the typical decay pattern. They are 1 to 3 M in diameter, and clamp connections are regularly present. Hyphae occur mostly in the spring wood and rays, and much less commonly in summer wood; they pass from c e l l to c e l l through pits or penetrate the walls. Bore holes in tangential walls of tracheids are narrower than broad hyphae, which are constricted at the point of passing through the walls. Hyphae are often swollen on either side of the bore holes. Fine hyphae penetrate the walls without constriction. Presumably, fine hyphae enlarge to produce broad hyphae with constrictions. Bore holes in radial walls, however, are usually larger than the diameter of the hyphae and somewhat irregular in outline. Allocysts (pear-shaped, thin-walled, chlamydospore-like c e l l s ) , characteristic of the fungus in culture, do not occur on hyphae in decayed wood in nature. The earliest stage of decay, only evident on wetting the wood, i s characterized microscopically by the presence of single strands of hyphae in the lumen of the first-formed spring wood tracheids and ray ce l l s . At this stage the hyphae penetrate the end walls of ray ce l l s to pass from c e l l to c e l l , but penetrations of tracheid walls were observed only rarely. Later when the wood shows the typical pattern of decay, a branching network - 1 0 -of hyphae occurs in the lumen of the largest spring tracheids and in the ray cells (Fig. U)• This network of hyphae i s commonly continuous through several annual rings, connecting the hyphae in the spring wood of succes-sive rings. Up to this point the summer wood, especially the late summer wood, may be devoid of hyphae. When a dense network of hyphae i s present in the spring wood, hyphae pass from i t 'into the summer wood through bore holes in the tangential walls. Hyphae often traverse the small summer wood tracheids without branching. At this stage rays and spring wood are partly broken down, but the summer wood i s s t i l l firm, resulting in the charac-t e r i s t i c macroscopic pattern of decay. The concentration of hyphae in the spring wood and rays appears to act as a base for the invasion and destruction of the summer wood in the f i n a l stage of decay. Lumen diameter of small summer wood tracheids was only half that of the largest spring wood tracheids where hyphae occurred abundantly. Air and moisture proportions may be different in summer and spring wood, but size of lumen alone does not account for the usual absence of hyphae from summer wood, since ray cells have lumens of similar diameter. Among ray cells , broad hyphae occur more frequently in larger cells or cells with contents, while narrow hyphae usually occur in smaller ray c e l l s . Characters of value in the identification of F. alnicola in wood are, concentration of hyphae in the spring wood and rays, clamp connections regularly present', and constriction of broad hyphae at the point of passing through tangential walls of tracheids. - 11 -i i i . Flammula conissans Decay Hyphae of F. conissans in white spruce are similar to those of F. alnicola in size and appearance, in the occurrence of clamp connections and constriction of broad hyphae where tracheid walls are penetrated. They are dissimilar in that allocysts occur infrequently but f a i r l y con-stantly on the hyphae of F. conissans. Hyphae appear to colonize the rays f i r s t , but have no special pre-ference for the largest spring tracheids. As a result the spring and summer wood of annual rings i s decayed more uniformly than F. alnicola. G. Manner of Infection and Extent of Decay in White Spruce Butts and root systems of trees infected by F. alnicola. in the Smith-Slave Lake area of Alberta, were dissected to explore the extent of decay and to determine the means of entry of the fungus. For this purpose the stumps were sawn into a series of vertical sections, and a l l decay was traced out in the roots. Typical conditions were as described below for tree nos. 1 and 2. Tree No. 1 The tree was a dominant white spruce, 135 years old, 92.3 f t . in height, and 19.1 ins. D.B.H. The stump and main l a t e r a l roots, the extent of decay in the roots, the points of infection, and the position of two of the vertical cuts are shown diagramatically i n Fig. 5« Another system of smaller l a t e r a l roots occurred lower; these were a l l sound and are not - 12 -shown in the diagram. Fig. 6 (arrow on Cut 4- in Fig. 5) shows the point of entry of infection A, which was a pressure wound between root E and the base of the stump. Fig. 7 i s a closeup of the infection court. The dead bark of the pressure wound (right side of root E) was in contact with the s o i l . Decay was most advanced at this point and as this was the only place where decay extended to the bark i t was clearly the point of infec-tion. Throughout the rest of the infection decay was confined to the heartwood. A count of the rings in root E showed that the cambium had been k i l l e d 80 years ago (tree age 55 years). Charred spruce roots, decayed with a yellow stringy rot were common under the stump, one of these was close to but not in contact with the point of infection. Decay was too far advanced in these charred roots "to isolate the causal fungus, but this could have been the source from which root E became infected. From the point of infection decay extended five feet upward into the trunk, but only 1.5 feet outward into root E. The stump contained a second discrete infection. Entry of the fungus was through the dead bark of a pressure wound in root J (Rot B, Fig. 8 and arrow on Cut 5 in Fig.' 5) in a similar manner to infection A. Age of this pressure wound was 95 years (tree age 4-0 years). Although this wound was 10 years older than that associated with infection A, decajr extended only 2.5 feet up into the butt and one foot into root J from the point of entry. - 13 -PLATE III, Figures 5 PLATE III. F. alnicola Decay Figure 5 . Diagram of stump and main roots of tree no. 1 (white spruce). The hatched areas show the extent of decay in the roots, and the counter-hatched areas within the stump show the area of decay on the stump surface. The broken lines show the position of vertical cuts, and the arrows indicate the loca-tion of pressure wounds (infection courts). Figure 6 . Vertical section of tree no. 1 (Cut 4- in Fig. 5 ) showing the point of entry of the fungus at a pressure wound between root E and the stump. Figure 7 . Enlarged view of Fig. 6 . - I N -FLATE IV, Figures 8 - 9 PLATE IV. F. alnicola Decay Figure 8. Vertical section of tree no. 1 (Cut 5 in Fig. 5), showing the-point-of-entry of the fungus (arrow) at a pressure wound. The two infections (Rot A and Rot B) were discrete. Figure 9. Diagram of stump and main roots of tree no. 2 (white spruce). The hatched area shows the extent of decay in the roots, end the counter-hatched area within the stump shows the area of decay on the stump surface. The broken lines indicate the position of the vertical cuts and the arrows i n d i -cate the location of pressure wounds (infection courts). - 15 -PLATE V, Figures 10 PLATE V. F. alnicola Decay Figure 10. Decay on the stump surface of tree no. 2. Figure 11. Stump of tree no. 2, with s o i l removed from the main roots. A pressure wound in the crotch of the two large roots in the l e f t foreground formed the infection court for the fungus. Figure 12. Vertical section of tree no. 2 (Cut 3 in Fig. 9) showing the point of entry of the fungus (arrow) at a pressure wound in the crotch of roots F and G. Figure 13. Vertical section of tree no. 2 (Cut 7 in Fig. 9) showing a possible second point of entry of the fungus (arrow) at a pressure wound. - 1 6 -Tree No. 2 A dominant white spruce, 110 years old, 91.0 f t . in height, 1 4 - . 6 ins. D.B.H. that was located 25 feet from tree no. 1. The stump and main roots, the extent of decay, the position of two vertical cuts, and points of entry of the fungus (arrows) are shown diagramatically in Fig. 9. The decay on the stump surface i s shown in Fig. 10. Infection occurred through a pressure wound in the crotch of roots F and G (Fig. 11, the two roots in the l e f t foreground, and arrow on Cut 3, Fig. 9)• The age of the pressure wound (Fig. 12) was 75 years (tree age 35). From the point of infection decay extended 3.5 feet up into the butt and 1.5 feet out into root G. Fig. 13 (arrow on Cut 7, Fig. 9) shows a possible second point of entry at a 93-year-old pressure wound. Decay under this wound was con-tinuous with the main column of decay, but did not extend outward into root J beyond the wound. I t i s doubtful, therefore, that the fungus entered here. I t i s more l i k e l y that the fungus advanced outward to the bark at this point through the dead wood under the wound. Busgen and Munch (5) point out that, "An alteration of the wood exactly similar to this normal heartwood formation also occurs as a pathological phenomenon in the v i c i n i t y of wounds". Tree Nos. 3 to 13 Thirteen trees in a l l were examined, photographed, and described in a similar manner. The data are shown in Table I. Trees U to 8 were located in the sub-luvial valley of a creek that drained into Lesser Slave Lake from the south, and trees 9 to 13 were higher on the side of the same valley. TABLE I. THE RELATION BETWEEN EXTENT OF DECAY (F. ALNICOLA) AND AGE OF WOUND (INFECTION COURT) IN WHITE SPRUCE Tree number and l o c a l i t y D.B.H. Age of tree Age of Wound Tree when wounded Diameter of decay at stump height (ins.) Extent of decay from point of infection (ft.) In stump In roots Average rate of growth per year (ins.) Fawcett Lake 1 19 .1 135 80 5 5 3 5 . 0 1 . 5 . 7 5 9 5 4 0 2 2 . 5 1 . 0 no 2 U . o 1 1 0 7 5 3 5 5 3 . 5 1 . 5 . 5 6 )tter Creek 3 12 .7 1 2 4 8 5 - 9 0 3 5 - 4 0 trace 1 . 5 1 . 0 . 2 1 Slave Lake Valley bottom 4 2 3 . 3 178 92? 8 6 ? 6 . 5 1 0 . 0 2 . 8 1 . 3 0 5 * 178 9 5 8 3 6 ? ( 2 + ) 1 . 5 ? 6 * 1 6 5 117 4 8 1 1 ? ( 3 + ) 2 . 5 7 * 190 1 0 6 8 4 9 ? ( 2 + ) 10 .0 1 .13 8 * 157 1 0 4 . 5 3 5 ? ( 2 + ) < 1 Valley side 9 1 2 . 9 1 2 4 8 6 3 8 trace 1 . 5 0 . 8 .21 10 15.1 1 2 4 8 5 3 9 2 2 . 0 1 . 8 .28 82 4 2 - 0 . 9 1 . 0 .13 1 2 * 1 2 3 71 5 2 trace 2 . 0 2 . 2 . 3 4 1 3 * 1 2 5 8 6 3 9 5 2 . 0 1 . 0 .28 6 7 5 8 < 1 < 1 *Tree had been logged, stump only examined. - 18 -In tree nos. 3, 5, 6, 7, 8, 10, 12, and 13 the fungus entered through clearly defined pressure wounds between lateral roots and the stump or crotch wounds in la t e r a l roots. Tree no. 7 was of interest because decay extended outward in one root for 10 feet from the point of infection. At the extremity of decay the root was only three quarters of an inch in diameter. Decay extended outwards only a short distance in other roots of the same tree. In tree no. 9 the entrance of the fungus was traced to a completely healed wound on a root that had been one inch in diameter when the wound occurred. Cause of this wound could not be ascertained though i t could have been pressure. Entry of the fungus in tree no. 4- appeared to be through several small root stubs. Cause or time of death of these small roots could not be discovered. Decay was associated with a 92-year-old pressure wound in this tree, but this appeared to be either a secondary infection or another instance where decay had extended outward to the wound. The entry point of the fungus could not be established in tree no. 11. Four infections of Armillaria mellea Vahl ex Fries and one infection of Coniophora puteana (Schum. ex Fries) Karst. which were found in these trees, had also entered through pressure wounds. In one and two-year-old a r t i f i c i a l infections of F. alnicola, the average linear rate of decay was one inch per year (see section Inocula-tion of Living Trees). In natural infections (Table I) the average annual rate of decay was assumed to be, the distance between point of entry of the fungus and extremity of the decay column, divided by the age of the wound. This apparent rate varied greatly, and was much slower than i n - 19 -a r t i f i c i a l infections except in tree nos. U and 7 . This calculation serves to i l l u s t r a t e that rate of decay in nature cannot be determined in a simple manner. Fire-Killed Trees Twelve white spruce trees ( 7 to 1 6 ins. B.B.H.) k i l l e d by f i r e two years previously were examined to learn i f F. alnicola could survive a severe forest f i r e . Trees in the burn were severely charred, lateral roots had been burned off, and vegetation and duff consumed, leaving bare mineral s o i l . Typical decay was found in only one tree ( 1 3 ins. D.B.H.), and F. alnicola was isolated from this. The decay was confined to the heartwood, and extended above stump height and into several lat e r a l roots, conclusive evidence that the fungus had entered the tree long before the f i r e . The stump of this tree was charred and one l a t e r a l root had been burned off. JL* alnicola i s able, evidently, to survive a hot f i r e . Folyporus tomen- tosus also was isolated from decay in one of these f i r e - k i l l e d trees. Young Trees Up to this time the disease had been studied only in advanced stages in old trees. Table I shows that most of the associated pressure wounds had occurred when the trees were 35 to 58 years old. Young trees within this range of ages, therefore, were examined with the intent of studying the disease in i t s early stages. For this purpose two young stands were selected adjacent to old stands where the disease was known to occur. - 2 0 -In the f i r s t stand, six trees 35 years old were dissected. Pressure wounds and especially crotch wounds 2 to 5 years old were common on the roots. A l l had a yellow stain, suggesting incipient decay, in the wood immediately under the dead bark. Charred stumps and logs containing brown cubical and yellow stringy decays were found under the trees examined. In the second stand, 18 trees, 30 to 4-0 years old, were dissected. The previous generation had been a mixed stand of spruce and poplar, of which charred rotting trunks and stumps remained. Many trees were growing in rotting wood. Crotch wounds were particularly abundant in trees that originated as seedlings on rotting logs. Roots of these trees tended to be oriented along the log, nearly parall e l . This obviously had promoted the development of root pressure wounds which were from 2 to 5 years old (one was 1 4 years old). As in the other stand, yellow stain was present in wood beneath the wounds. Stained wood under the wounds was cultured. Of 70 platings from 2 4 wounds, 4 1 remained sterile, 2 2 were purple agar fungus (Coryne sarcoides (Jacq.) Tul.), and seven were miscellaneous molds. The yellow stained wood under some wounds was completely s t e r i l e . It seems probable that the yellow stain was not due to fungus activity of any kind, and i t almost certainly was not the incipient stage of any wood-rotting fungus. Coryne  sarcoides has often been isolated, along with the causal fungus, from a variety of decays and even from "sound" wood. I t was surprising to find that root wounds below ground, and in contact with rotting wood, were not invaded by wood-rotting fungi for up to 1 4 years. - 21 -F. alnicola was not isolated from any of these trees. I f i t i s a general rule that pressure wounds on roots remain sterile or are colonized only by fungi not antagonistic to F. alnicola. then wounds could remain a potential infection court for many years. Indeed, i t may be that F. alnicola i s incapable of infecting newly formed pressure wounds. F. alnicola i s a heartwood inhabiting fungus which gains entrance to below ground parts of the tree through pressure wounds or otherwise k i l l e d bark. There was no evidence that the fungus can invade l i v i n g bark or sapwood. The fungus decays the roots and butts of l i v i n g trees and i t s apparent restriction to the butt may be due only to the fact that i t enters through roots and i t s rate of advance i s slow. D. Laboratory Test of Decay Dissection of the stumps of white spruce trees infected with F. a l n i - cola had shown that from the point of infection, decay consistently extended further up into the base of the tree than outward into the roots. An experiment was designed to test whether stem wood was decayed more rapidly than root wood, that i s , i f this phenomenon in nature i s due to differences in the wood i t s e l f . The experiment was also designed to test whether density in terms of rings per inch affected the rate of decay. Two fungi, F. alnicola and F. conissans which at the time of the experiment were thought to be Canadian and European isolates of the same species, were tested to compare their a b i l i t y to decay spruce wood. - 2 2 -A sound white spruce windfall near Slave Lake, Alberta, was chosen as the source of test wood. Stem wood was taken from the tree at a distance of seven feet from the root collar and root wood was taken from a main lateral root close to the butt. Strips of wood 1.5 by 3.5 cm. in end section were cut from the heartwood of the sections of root and stem and planed to exact size. As far as possible replicate samples were cut from the same group of annual rings. Fig. 1 4 shows the position of the samples cut from stem and root. The average number of rings per sample were: Stem wood l i g h t density (SL) 13, root wood l i g h t density (RL) 1 0 , stem wood heavy density (SH) 2 1 , and root wood heavy density (RH) 2 4.5. Root samples of l i g h t and heavy density were taken from similar groups of annual rings, but this was not possible on the stem section., Stem samples of heavy density were taken from an area of compression wood, and from an older group of annual rings than those of l i g h t density. The strips of wood were cut into blocks measuring 1.5 x 3.5 x 5 cm. One block was used to determine the moisture content after conditioning and the other three were used in the test. ¥ood blocks were conditioned to a moisture content of 13.5% of oven dry weight (except root l i g h t density which came to an equilibrium of 13.0% under the same conditions) in a modification of an apparatus suggested by Mr. J. ¥. Roff (32) as follows: The wood blocks were placed in chambers through which humid air was circulated. The a i r was f i r s t bubbled through a saturated solution of sodium chloride to bring i t to the desired rela-tive humidity. Spare blocks were removed from time to time and tested for moisture content, until the blocks had reached an equilibrium moisture - 23 -PLATE VI, Figure 14. PLATE VI. Source of White Spruce Wood Samples for a Decay Test of F. alnicola and F. conissans Figure I 4 . Cross section of stem (left) and root (right) showing the position, in relation to the annual rings, of samples used in a decay test. - 24- -content. The culture medium consisted of 1 6 oz. jars, with vented caps ( 1 4 . ) , half f i l l e d with moist garden s o i l . A thin feeder strip of spruce sapwood was placed on top of the s o i l in each jar. The jars of s o i l were ster i l i z e d in an autoclave and inoculated with F. alnicola and F. conissans. The s o i l was inoculated with F. alnicola (isolate DA0M25874-) four months before addition of test wood blocks, but with F. conissans (isolate DA0M22888) only three months prior because of the more rapid rate of growth of F. conissans. The purpose of this was to insure that s o i l cultures of both fungi were in similar states of development at the start of the decay test. The conditioned wood blocks were weighed, then held in a jet of steam and replaced in the chamber. The blocks were surface s t e r i l i z e d with propylene oxide vapor for 24- hours. Filtered a i r was then passed through the chamber to remove the vapor. One wood block was placed on the feeder strip of each s o i l jar. Twenty ml. of sterile water were added to each jar. The experiment was set up on a 3 x 2 x 2 fac t o r i a l design with four replicates (35). The jars were incubated at room temperature for six months. Surface mycelium was removed from the blocks, which were then oven dried and re-weighed. The oven dry weight was converted to conditioned weight, i.e., O.D. wt. x 1.135 = Conditioned wt. A conversion factor of 1.130 was used for li g h t density root wood blocks because they had reached an equilibrium at 13.0$ moisture content. - 25 -Results Table II shows the percentage loss in conditioned weight of the wood blocks. Losses in weight due to the action of the fungi were small, and generally not more than the lower one-third of the blocks was decayed. Larger losses would have occurred with thinner blocks or a longer period of incubation. Decay in the blocks did not exhibit the pattern found in nature where early spring wood and wood rays are decayed f i r s t . The decay appeared as a uniform yellow stringy decay. Decay caused by the two fungi was similar. Controls which theoretically should have shown no loss in weight varied from -1.7 to 1.8%. Some of this variation was caused through contamination of some control blocks with mold fungi. Otherwise, the variation in the controls i s indicative of the accuracy of moisture con-tent determination by the method used. Table III shows the computation of the main effects and interactions from which the analysis of variance, Table IV, for the experiment was calculated. There was no significant difference between replicates but a highly significant difference between treatments. Among treatments the effect of part of the tree, i.e., stem, or root (R) and density (H) were significant at the 5% l e v e l . The effect of fungi was highly significant and one interaction between fungi and density (FH) was also significant. Table V shows the main effects and interactions between part of the tree and fungi, and density and fungi. The effect of the fungi on root as opposed to stem wood was significant, i.e., 4.62 - 3.22 = 1.40* (necessary difference 1.32), i.e., stem wood was decayed more rapidly than root wood but only by F. conissans, i.e., 9.10 - 5.45 = 3«65** (3.08). The difference * Significant at the 5% l e v e l . Significant at the 1% l e v e l . - 2 6 -TABLE II. PERCENTAGE LOSS IN CONDITIONED WEIGHT OF WHITE SPRUCE WOOD, OF HEAVY AND LIGHT DENSITY, FROM STEM AND ROOT, SIX MONTHS AFTER INOCULATION WITH F. ALNICOLA AND F. CONISSANS Replicate Totals I II III IV Stem light density Control -0.1 -0.1 -1.7 1.1 -0.8 F.a. 0.8 6.3 3.5 5.1 15.7 F.c. 8.8 7.0 11.5 1 6.8 44.1 59.0 Root light density Control 0.7 1.3 0.9 -0.2 2.7 F.a. 3.4 3.9 1.8 6.6 15.7 F.c. 11.4 10.1 10.7 4.2 36.4 54-8 Stem heavy density Control 1.8 1.2 0.4 0.1 3.5 F.a. 5.1 4.6 3.3 6.7 19.7 F.c. 4.6 7.4 6.8 10.0 28.8 52.0 Root heavy density Control 0.0 -0.6 0.2 -0.9 -1.3 F.a. 2.3 5.3 4.3 2.0 13.9 F.c. 1.4 1.0 5.2 -0.4 7.2 1 9.8 Totals 4 0.2 47.4 46.9 51.1 185.6 TABLE i n . THE EFFECT OF DENSITY AND TYPE OF WOOD (STEM AND ROOT) ON WEIGHT LOSS OF WHITE SPRUCE DECAYED BY F. ALNICOLA. AND F. CONISSANS: COMPUTATION OF MAIN EFFECTS AND INTERACTIONS Decrease in conditioned weight (per cent) Effects Control F.a. F.c. Total Control F.a. F.c. Total , Control F.a. F.c. Total (1) -0.8 1 5 . 7 4-4.1 59.0 1 . 9 3 1.4 80.5 1 1 3 . 8 4.1 65.0 1 1 6.5 185.6 Sum r 2 . 7 15 .7 3 6.4 54.8 2.2 33 .6 3 6.0 7 1.8 -1.3 -5.8 - 2 9 . 3 - 3 6 ; 4 R h 3.5 19 .7 28.8 52.0 3.5 0 - 7 . 7 -4.2 0.3 2.2 -44.5 - 4 2 . 0 H rh -1.3 13 .9 7.2 19.8 - 4.8 -5.8 -21 .6 -32.2 -8.3 -5.8 -13 .9 -28.0 RH R = Root or stem wood, H = Heavy or light density, F = Fungi - 28 -TABLE IV. THE EFFECT OF DENSITY AND TYPE OF WOOD (STEM AND ROOT) ON WEIGHT LOSS OF WHITE SPRUCE DECAYED BY F. ALNICOLA AND F. CONISSANS: ANALYSIS OF VARIANCE D.F. Sum of squares Mean scjuare F F . 0 5 . 0 1 Correction for mean 7 1 7 . 6 Replicates 3 5 . 2 1 . 7 0 . 3 2 . 9 0 4 . 4 6 Treatment s 1 1 5 9 4 . 2 5 4 . 0 1 0 . 8 * * 2 . 1 0 2 . 8 6 R 1 2 7 . 6 2 7 . 6 5 . 5 * 4 . 1 5 7 . 5 0 H 1 3 6 . 8 3 6 . 8 7 . 4 * EH 1 1 6 . 3 1 6 . 3 3 . 3 F 2 3 9 5 . 7 1 9 7 . 9 3 9 . 6 * * 3 . 3 0 5 . 3 4 FR 2 28 . 3 1 4 . 2 2 . 8 FH 2 8 7 . 3 4 3 . 7 8 . 7 * * FRH 2 2 . 2 1 . 1 0 . 5 Error 3 3 1 6 4 . 6 5 . 0 Total 4 7 7 6 3 . 9 * Significant at the 5 $ level R = Root or stem wood ** Significant at the 1% level H = Heavy or l i g h t density F = Fungi - 29 -TABLE V. THE EFFECT OF DENSITY AND TYPE OF WOOD (STEM AND SOOT) ON WEIGHT LOSS OF WHITE SPRUCE DECAYED BY F. ALNICOLA AND F. CONISSANS; MAIN EFFECTS AND INTERACTIONS MEANS Control F.a. F.c. Light Heavy Stem 0 . 3 4 4 . 4 3 9 . 1 0 4 . 6 2 4 . 9 2 4 . 3 3 Soot 0.18 3 . 7 0 5 . 4 5 3 . 2 2 4 . 5 7 1 . 6 5 0 . 2 6 4 . 0 7 7.28 3 . 9 2 4 . 7 5 2 . 9 9 Light 0 . 2 4 3 . 9 2 1 0 . 0 6 4 . 7 4 Heavy 0 . 2 8 4 . 2 0 4 . 5 0 2 . 9 9 Nec. d i f f . between 2 4 plot means; 1 . 3 2 (5$ level) 1 . 7 9 {ifo l e v e l ) . 1 6 1 . 6 1 2 . 1 8 1 2 1 . 8 6 2 . 5 0 8 2 . 2 9 3.08 - 30 -in the action of F. alnicola on stem and root wood was not significant, i.e., 4-43 - 3.70 = 0.73 (2.29). The action of the two fungi on wood was significantly different, i.e., F.c. - F.a. = 7.28 - 4-.07 = 3.21** (2.18), but a significant difference occurred only in their action on stem wood, i.e., 9.10 - 4.4-3 = 3.67** (3.08). The effect of density was significant, i.e., 4.74- - 2.99 = 1.75* (1.32), and lighter wood was decayed more rapidly, again, however, the difference was only significant with F. conissans (10.06 - 4«50 = 5.56** (3.08)). F. conissans decayed lighter wood more rapidly than F. alnicola (10.06 - 3-92 = 6.14** (3.08)) but there was no significant difference in their a b i l i t y to decay heavier wood (4.50 - 4.20 = 0.30 (2.29)). The interaction between fungi and density (FH Table IV) has been shown to be significant. This was caused by the greater loss in weight of light wood decayed by J_. conissans (Table V) and the much smaller loss in heavy root wood (Table V i ) . Table VI shows that there i s no significant difference in the a b i l i t y of F. alnicola to decay l i g h t and heavy stem wood or l i g h t and heavy root wood. In contrast there are marked differences in the a b i l i t y of F. conis- sans to decay wood of different densities or root and stem wood. In addi-tion although J_. conissans decayed l i g h t and dense stem wood and l i g h t root wood more rapidly than F. alnicola. this rate was reversed with heavy root wood. - 31 -TABLE ¥.1. THE EFFECT OF DENSITY AND TYPE OF WOOD (STEM AND ROOT) ON WEIGHT LOSS OF WHITE SPRUCE DECAYED BY F. ALINCOLA AND F. CONISSANS: MAIN EFFECTS ~ MEAHS Control F.a. F.a. Stem Light -0.20 3.93 11.03 Heavy 0.87 4.93 7.20 Root Light 0.68 3.93 9.10 Heavy -0.33 3.68 1.80 Nec. d i f f . between 4 plot means: 3.23 (5$ level), 4.35 (1% level) - 32 -Conclusions The two fungi, which at the time of the experiment were thought to be strains of the same species, d i f f e r significantly in their rates of decay. The observation which prompted the experiment, that F. alnicola grew more rapidly i n the stem than in the roots (as evidenced by linear extent of decay) was not borne out by the experiment. This difference in the extent of decay in root and stem i s not due to inherent differences in the wood i t s e l f . No conclusions may be drawn from difference in decay rate of F. conissans on heavy root wood as opposed to l i g h t root wood and stem wood. This fungus has not been isolated from decay in Canada, so that nothing i s known about the decay in nature. E. Inoculation of Living Trees The roots of white spruce, (6 to 12 inches D.B.H.) were inoculated with Flammula alnicola and F. conissans at Slave Lake, Alberta. Spruce dowels (five-eighths by two inches) decayed by the two fungi were placed in holes d r i l l e d into the heartwood of roots, and the holes f i l l e d with melted grafting wax. Sound spruce dowels were used as controls. Two lateral roots of each tree were inoculated with the same fungus and a third treated as a control. Four trees were inoculated with F_. alnicola and four with F. conissans on August 27, 1955. One year later on September 22, 1956, half of the inoculations, one from each tree was taken up. The rest and the controls were l e f t for a second year and harvested on September 18, 1957. A section of the root containing the - 33 -inoculum was removed, s p l i t open for examination, and cultured where there was visible evidence of decay. F. alnicola was recovered in culture from the inoculum plug and adjacent wood of a l l four inoculations harvested after one year. Generally the yellow decay extended from 1 to 3 mm. into the wood surrounding the inoculum, but in two roots decay had extended for 5 cm. in the spring wood of several rings. F. conissans did not establish i t s e l f as well. In two roots the fungus caused no visible decay, in a third the l i n i n g of the d r i l l e d hole only was decayed, and in a fourth the fungus was recovered from the wood 2 mm. from the inoculum. After two years F. alnicola decay extended proximally and d i s t a l l y for 10 cm. from the inoculum in one root. The maximum cross sectional area of this column was 4 by 10 cm. Decay columns in the other three roots extended for 7, 5 and 1 cm. respectively from the inoculum. Pure cultures o r" £• alnicola were recovered from a l l four roots. F. conissans caused nearly as much decay, the largest decay column was also 10 cm. in length and had a maximum cross sectional area of 3 x 5 cm. Decay columns in other roots extended for 3.5 and 0.5 cm. Pure cultures of F. conissans were recovered from the successfully inoculated roots. The inoculum plug of one root, in which there was no take, yielded a mixed culture of F. conissans and bacteria. The average linear rate of decay of a l l successful inoculations of F. alnicola was 2.7 cm. per year (Table VII) while that of F. conissans was only 1..4 cm. per year. The maximum extent of decay in individual inoculations, however, was similar. In contrast F. conissans decays TABLE VII. THE EXTENT OF DECAY IN LIVING MITE SPRUCE SOOTS, ONE AND WO YEARS AFTER I^OOTJIATION WITH F. ALNICOLA AND F. CONISSANS F. alnicola F. conissans Topographic Inoc. Extent of decay (cm.) Inoc. Extent of decay (cm.) position number 1 year 2 years Average per year number 1 year 2 years Average per year Level 2 + 1 . 0 . 2 5 1 + 0 . 5 . 1 3 Slope A + 5 . 0 1 . 2 5 5 + - + Slope 6 5 . 0 7 . 0 4 . 2 5 7 3 . 5 1 . 7 5 Slope 9 5 . 0 1 0 . 0 5 . 0 8 1 0 . 0 2 . 5 - 35 -sterilized wood blocks more rapidly and grows faster on culture media. The decay pattern of the most successful inoculation (no. 9 Table VII) of Flammula alnicola was typical. In this inoculation the fungus spread along rings 8 to 34- years old. The youngest rings may, therefore, have been sapwood. Decay had the characteristic musty odor of natural infections of the fungus. In another inoculation the fungus was recovered from wood in which the only evidence of decay was a slight color differen-tiation of the wood rays when the wood was moistened. Decay produced by inoculation of F_. conissans was similar in color and in stringy nature to F. alnicola. but did not have the musty odor, nor the pattern of F. alnicola decay. Within annual rings decay was more uniform throughout the spring and summer wood. Trees were inoculated in the same creek valley where the natural infections of F. alnicola were dissected. Trees 1 and 2 (Table VII) were on a terrace on the side of the valley while the remaining inoculated trees were more or less in a li n e up the slope immediately above the terrace. The rate of decay was least on the terrace for both species (inoculations 1 and 2) and the rate was increasingly greater up the valley side. This trend was more evident in inoculations l e f t for two years. This suggested a possible inverse relation between moisture conditions of the site (within rather narrow limits) and rate of growth. The terrace represented a moister site than the slope above i t and this was shown by the ground vegetation. Although the slope was drier than the terrace i t represented a moist s i t e . The successful inoculation of the roots of l i v i n g white spruce with pure cultures of F. alnicola, resulting in the characteristic type of - 3 6 -decay and the subsequent reisolation of the fungus In pure culture from the decay i s proof, according to Koch's postulates, that the fungus causes the decay with which i t is associated in nature. Inoculation of trees with F. alnicola and F. conissans has shown that though they both may cause yellow stringy decays there are differences in the pattern of the decay. F. Occurrence of F. alnicola in Relation to Site Factors Data on the relation of F. alnicola decay to a number of site factors were obtained incidentally to a study on site classification of white spruce in the Mixedwood Region of Alberta conducted by the Calgary office of the Forestry Branch, Department of Northern Affairs and National Resources. The Saskatoon Laboratory of the Forest Biology Division, Department of Agriculture participated in the study to investigate decay of white spruce in relation to site. The occurrence of decay was based on the examination of four dominant white spruce per fifth-acre plot.. The small number of trees examined per plot imposed severe restrictions on interpretation of the data. Thus i t could not be stated that the decay did not occur on plots where i t was not found. Plots were divided into those with F. alnicola and those without. The two groups were then classified according to a site factor, for example, pH of the A2 horizon, and the Chi square test (10) applied to see i f there was a significant difference between the two groups. If the difference was significant then the occurrence of the fungus in relation to the site factor could be considered, with some - 37 -assurance that the relation was not due merely to chance. F. alnicola was present on 25 out of 251 plots established on a wide variety of sites. From the above mentioned study, Quaite (31) drew up a provisional site classification for white spruce in the Mixedwood Region of Alberta based on physiographic features. This classification i s shown in Table VIII. The calculation of the Chi square test for plots with F. alnicola and without, classified according to site, i s shown in Table IX. The two groups differed significantly (at the 1% l e v e l ) . Therefore, i t i s concluded that F. alnicola occurred more frequently on the better sites (especially the best sites) and this occurrence i s not due to chance. Denyer and Riley (12) found a similar relation, based on site index, in the same region. Individual site factors were then tested for significance in relation to the occurrence of the fungus. The Chi square test for the occurrence of the fungus in relation to pH of the Ag or f i r s t mineral s o i l horizon i s shown in Table X. The pH of the A£ horizon differed at the five per cent level between plots with and without F. .alnicola. Jj\ alnicola occurred more frequently on plots with an acid A^ horizon. Similarly liminess of the parent material (Table XI) was found to be significant. Liminess was determined by the reaction of the parent material to hydrochloric acid. The fungus occurred more frequently on non-calcareous s o i l s . This result i s in agreement with that for pH of the A2 horizon since the amount of lime in the parent material would have an influence on the other s o i l horizons derived from i t . Aspect (significant at the eight per cent level) and moisture regime (significant at the 12 per cent level) of - 3 8 -TABLE PHYSIOGRAPHIC SITE CLASSIFICATION OF WHITE SPRUCE IN THE MIXEDWOOD REGION OF ALBERTA Av. dominant height at Site Moisture 60 years class S o i l origin Material regime (feet) A Lowland alluvium Subluvial Sands, s i l t s 3 , U, 5 7 7 B T i l l Clay-loam, clay* u, 5 7 2 C T i l l slope Clay-loam, clay 2 , 3 7 0 D T i l l Lacustrine f l a t Ponded Clay-loam, clay* 6 7 0 E Highland alluvium Sands, s i l t s 1 , 2 6 1 F T i l l ridges Stoney, clay-loam 1 — G Grassy swamp Clay 7 6 5 H Sand dunes Fine to coarse sand 0 , 1 , 2 5 5 Telluric water may raise site quality. - 39 -TABLE IX. CALCULATION OF CHI SQUARE FOR THE RELATION BETWEEN SITE CLASS AND OCCURRENCE OF F. ALNICOLA IN WHITE SPRUCE Site class A B C D E F G H Total Plots with F. alnicola 6 6 10 2 1 0 0 0 25 Plots without F. alnicola 9 59 6 l 26 30 8 11 17 221 Total 15 65 71 28 31 8 11 17 246 X 2 = 23.31 D.F. = 16 - 9 = 7 X 2 > 0 1 - 18.48 X2. 0 5 = H . 0 7 Significant at the Vfa level,, TABLE X. CALCULATION OF CHI SQUARE FOR THE RELATION BETWEEN pH OF THE A2 SOIL HORIZON AND OCCURRENCE OF F. ALNICOLA IN WHITE SPRUCE pH of A 2 horizon 4.0 - 4.9 5.0 - 5.9 6.0 - 6.9 7.0 - Total Plots with F. alnicola Plots without F. alnicola 13 8 43 4 14 0 17 19 87 Total 20 5 1 18 17 106 X 2 - 8.09 D.F.= 8 - 5 = 3 '.Oi " 1 1 - 3 4 ^ . 0 5 = 7 ' 8 1 Significant at the 5$ level, TABLE XI. CALCULATION OF CHI SQUARE FOR THE RELATION BETWEEN LIMINESS OF THE PARENT MATERIAL AND OCCURRENCE OF F. ALNICOLA IN WHITE SPRUCE Very Liminess of Non slightly Slightly Mod. Strongly Total parent material calc. calc. calc. calc. calc. Plots with F. alnicola 2 0 8 0 * 0 1 2 4 Plots without F. alnicola 1 2 5 1 1 2 3 3 6 2 3 2 1 8 Total 1 4 5 1 4 2 3 3 6 2 4 2 4 2 X 2 = 1 1 . 8 9 D.F. - 1 0 - 6 = 4 X 2 = 1 3 . 2 8 . 0 1 X 2 . 0 5 = 9 . 4 9 Significant at the 5 $ level. - 42 -plots with F. alnicola did not d i f f e r significantly from the rest of the plot samples. Flammula alnicola occurred on soils derived from a f a i r l y vide range of geologic materials including: lowland alluvium and sub-luvial deposits, lacustrine material, glacial t i l l and highland alluvium. I t occurred on moisture regimes of 2 to 6, that i s , generally under moist to wet condi-tions and generally on non-calcareous soils with an acid kg horizon. - A3 -III. THE FUNGUS The fungus, isolated from decay in spruce and other conifers in Western Canada, had been identified as Flammula conissans Fr. sensu Ricken, but the identification was not positive. A culture of Flammula  conissans (Fr.) G i l l e t was obtained from the Centraalbureau voor Schimmel-cultures, Baarn, Holland (deposited there by Prof. R. Kuhner), for compari-son with Canadian isolates. Microscopic characters of the two fungi were similar, but the European one lacked a musty odor and had a faster growth rate. I t was thought, then, that they might be European and American strains of the same species. Most of the cultural studies with the Canadian iso-lates, therefore, were carried out in parallel with F. conissans from Europe. A comparison of f r u i t bodies produced in culture, and pairings of monosporous mycelia proved them to be separate species. A. Cultural Characters The cultures have been described in accordance with Nobles' (28) technique. Dr. M. K. Nobles supplied the description of F. alnicola. which has been checked and modified sli g h t l y by the author. i . Flammula alnicola (Fr.) Kummer Key Pattern; (1,2) 111 (7,9) 2224-22 CULTURES EXAMINED: CANADA.- Brit i s h Columbia: Queen Charlotte Islands, on Tsuga hetero- phylla. DA0M17168; Alberta: Seebe, on Picea glauca var. albertiana. DA0M25874- (K329), and other isolates from P. glauca. - AA -Cultural Characters Growth Characters.- Growth very slow, radius up to 6.5 cm. in six weeks (Fig. 15). Advancing zone even, slightly raised, aerial mycelium to l i m i t of growth. Mat white at f i r s t and remaining so or becoming pale yellow (Munsell (27) color equivalent: 2.5Y 8/6) after two to three weeks, slightly raised, fine woolly, aggregated in small tufts so arranged as to give a zonate or radiate appearance in some cultures, occasionally with compact yellow balls (abortive f r u i t body primordia) usually appressed to the wall of the Petri dish. Reverse unchanged or pale yellow. Odor pene-trating, musty. On ga l l i c and tannic agars, diffusion zone very weak to moderately strong, no growth on either medium. Gum guaiac test (30) positive. L Hyphal Characters.- Advancing zone: hyphae hyaline, nodose-septate, branched at and between septa, 1.5 - 3.1 V- diameter. ! Aerial mycelium: (a) hyphae as in the advancing zone, occasionally with golden yellow contents; (b) allocysts^- (Fig. 17) very numerous, even 2-3 mm. from l i m i t of growth, usually terminal (formed singly or in a chain of two or three), occasionally intercalary, thin walled, at f i r s t hyaline with granular or oi l y contents deeply staining in phloxine, later rarely yellow, more frequently empty, subglobose to ovoid, or pyriform, 7.5 - 15.6 x 7.5 -23.5 u. Submerged mycelium: (a) hyphae and (b) allocysts as in aerial mycelium. Allocysts numerous in monosporous cultures. 'See Snell, ¥. H. and Dick, E. A. A glossary of mycology. Harvard University Press, Cambridge, Massachusetts. 1957. p. 5. PLATE VII, Figures 15 PLATE VII. Cultural Characters of F. alnicola and F. conissans Figure 15. Culture of F. alnicola on malt agar, six weeks old. Figure 1 6 . Culture of F. conissans on malt agar, three weeks old. Figure 17. Hyphae from advancing zone, and allocysts of F. alnicola ( l e f t ) , and F. conissans (right). - Ab -Type of Rot: yellow stringy root and butt rot with laminate and radiate character. Isolated and identified from decay in: Tsuga heterophylla,  Abies balsamea (L.) M i l l . , A. amabilis. A. lasiocarpa. Picea glauca. P. glauca var. albertiana. P. mariana. P. Engelmanni, Pinus Banksiana Lamb., P_. contorta var. l a t i f o l i a . Betula lutea Michx. f. and Populus sp. from Br i t i s h Columbia, Alberta, Saskatchewan, Manitoba, Ontario, Quebec, New Brunswick and Nova Scotia. i i . Flammula conissans (Fr.) G i l l e t Key Pattern: 1111 (7,9) 222222 CULTURES EXAMINED: HOLLAND: Locality and host not known, DA0M22888. Cultural Characters Growth Characters.- Growth moderately rapid, plates covered in three to four weeks (Fig. 16). Advancing zone white, even, raised, 0.5 cm. in extent with aerial mycelium to l i m i t of growth. Mat white at f i r s t and remaining so, or with patches of pale yellow (2.5Y 8/4, 2«5Y 8/6), f e l t y . Reverse unchanged. No odor. Reaction with gum guaiac positive. Hyphal Characters.- Advancing zone: hyphae hyaline, nodose septate, branched at and between septa, 1.5 - 5.0 u diameter. Aerial mycelium: (a) hyphae as in advancing zone; (b) allocysts (Fig. 17) very numerous even 2 - 3 mm. from l i m i t of growth, terminal, occasionally intercalary, mostly solitary, occasionally in chains of two or three> at f i r s t hyaline with granular or o i l y contents deeply staining in phloxine, later rarely yellow, more frequently empty, subglobose to ovoid or pyriform, 6.3 - 8.8 x 11.3 -20o0 u. Submerged mycelium: (a) hyphae and (b) allocysts as in aerial mycelium. Allocysts usually numerous in monosporous cultures. - 47 -Type of Rot: The fungus has not been isolated from decay in Canada. A yellow stringy decay was produced in roots of Picea glauca inoculated with the fungus. Lange (25), and Kuhner and Romagnesi (24) reported the f r u i t bodies on Salix cinerea L. The descriptions show that the microscopic characters of the two fungi are similar. Cultures, however, may be distinguished readily by (l) the strong musty odor of F. alnicola. lacking in F. conissans. (2) the faster rate of growth in F. conissans, and (3) the difference in the appearance of the mat. i i i . Effect of pH on F. alnicola and F. conissans Flammula alnicola (isolate DA0M25874) and Z« conissans (isolate DA0M22888) were grown on media buffered to different pH values to deter-mine the optimum pH for each, and to compare their behavior. Double strength Difco malt agar was sterilized, cooled and mixed with an equal volume of Mcllvaine's standard buffer solutions (7) to give theoretical pH values of: 2.4, 3.0, 3-4> 4«0, 4«4> 5.0, 5.4> 6.0, 6.4, and 7.0. The pH of the buffered agar was measured with a Beckman pH meter, which indicated actual values for the series of: 2.8, 3«3, 3.8, 4.4> 4»9, 5.4» 5.8, 6.1, 6.3, and 6.8. Inocula cut from the advancing zone of actively growing cultures with a number two cork borer were trans-ferred to the centre of Petri dishes containing the buffered agar. Five replicates of the two fungi at each pH value were incubated at 25° C. The area of the mat in square centimetres was recorded at the end of two, four, and six weeks. This was done by tracing the outline of the culture - 48 -on tracing paper held against the reverse of the Petri dish. The area of the mat was determined with a planimeter. Net growth was determined by subtracting the area of the inoculum. The pH of the agar at the periphery of the mat and under the mat was measured at the end of the test to deter-mine changes in pH caused by the fungi. The growth of the cultures at the end of four weeks i s shown in Fig. 18. The arrows indicate, quantitatively, the change in pH during the period of growth. Optimum growth of F. alnicola and F. conissans on buffered agar occurred at pH 4«4» Growth of F. alnicola was greatest at 4»A through-out the four week period. In F. conissans. at the end of two weeks, growth was greatest at pH 4*9, but thereafter at 4.A* The growth of F_. alnicola on Difco malt agar buffered to 5.4 was much slower (0.2 sq. 'cm.) than on unbuffered (pH 5.5) Difco malt agar (6.9 sq. cm.). This was probably due to the effect of the buffering solutions apart from pH. F. conissans grows much more rapidly than F. alnicola on normal culture media. This was the case also on buffered agar at pH 4*4 and above, but not for lower pH values. At the end of two and three weeks F. alnicola grew faster at pH 4«4 and below than F.. conissans. but above pH 4»4 F. conissans grew faster. F. alnicola appears to be more tolerant of pH values below 4»4 and F. conissans more tolerant of values above 4.4. The maximum (final) pH tolerated by F. alnicola was 6.0, and for F. conissans i t was 6.55. iv. Effect of Temperature on F. alnicola in Culture. Inocula cut with a number two cork borer from the advancing zone of a culture (DA0M25874) on malt agar incubated at 22° C. were transferred to - 4.9 -PLATE VIII, Figure 18 • ( PLATE VIII. Effect of pH on the Growth of F. alnicola  and F. conissans ... Figure 18. The growth of cultures of F. alnicola (broken line) and F. conissans (solid line) in four weeks, on malt agar buffered with Mcllvaine's standard buffer solutions. The arrows indicate, quantita-tively, the change in pH of the medium during the test. 2 3 4 5 'pH (BUFFERED MALT AGAR) - 50 -the centre of Petri dishes containing Difco malt agar. Five cultures were incubated at each of the following temperatures: 7°, 12°, 17° and 22° C. Other temperatures were not available at the time of this test. The area of the mat was recorded in square centimetres (the same method as in the preceding section) at weekly intervals for four weeks. Average growth (sq. cm.) at the end of four weeks was: 7° - 0.25, 12° - 1.34, 17° - 4.63, and 22° - 6.96. Subsequently Etheridge (15) tested the effect of temperature on the growth of Flammula conissans (F. alnicola) in culture. Cultures incubated at 4°, 10°, 20°, 25°, 30°, and 35° C. showed a temperature optimum at 20° C j a slower rate of growth at 25° C , and no growth at 30° C. The optimum temperature for growth in culture i s evidently close to 22° C. v. Hyphal Fusions The formation of hyphal fusions in pairings of dikaryotic mycelia has been used to demonstrate that fungi from different geographical locations belong to the same species. Buller (4) paired mycelia of a luminous form of Pan us stypticus Fr. from North America with a non-luminous form from England. He considered hyphal fusions of the two mycelia to be strong evidence that they belonged to the same species. Flammula alnicola (DA0M25874) (at this time tentatively identified as F. conissans) and J_. conissans (DA0M22888) from Europe were tested in a similar manner. Employing the techniques described by Bourchier (3) and J^rgensen (21) mycelia of the same isolate f i r s t , were paired to determine i f fusions occurred between the hyphae. Hyphal fusions occurred readily between - 51 -hyphae of the same isolate but no fusions occurred between mycelia of F. alnicola and F. conissans. which supports their identification as separate species. v i . The Growth of F. alnicola in Soil Culture. Dissection had revealed that F. alnicola entered spruce roots through wounds below ground. As infection did not appear to take place through direct contact of an infected root with a healthy one, laboratory experi-ments were designed to assess the a b i l i t y of the fungus to grow in s o i l . The occurrence of the fungus in white spruce appeared to be related to s o i l pH and this also was tested. A modification of the technique described by Chinn (8) was used to study the behavior of the fungus in s o i l . Mycelium of F. alnicola (DA0M25874.) with a l i t t l e water, was ground in a Waring blendor and mixed with cooled (but not solidified) one per cent water agar. Sterile micro-scope slides were dipped in the suspension, and after the agar had s o l i -dified, placed upright in 1 6 0 2 . jars of moist s o i l so that the slides were half buried. Four slides were placed i n each jar. Three soils (from the f i r s t mineral horizon) from Slave Lake, Alberta, which had been air dried for five months, with pH values of 4-.6, 5.3, and 7.1 were used as the growth media. Three jars containing s o i l of each type were ste r i l i z e d before addition of the slides and served as controls. Soil jars were incubated at 7°, 12°, and 17° C. Control jars were incubated at 12° C. Slides were removed at intervals over a period of a month, stained in carbol rose bengal and examined microscopically. Slides from the three - 52 -sterilized soils a l l showed that an extensive mycelium had developed from the hyphal fragments. Clamp connections and allocysts were common on the hyphae, permitting identification of the fungus as F. alnicola. The original hyphal fragments could be distinguished usually from the new growth by an abrupt change in the diameter of the hyphae. The new hyphae were narrower. Slides from the three types of s o i l (non-sterile) incubated at the three temperatures failed to reveal renewed growth of F. alnicola except that one slide from the pH 4.6 s o i l incubated at 7° C. showed evidently renewed growth of a single hypha with clamp connections. Soil fungi, bacteria and actinomycetes had grown on these slides. Soil fungi developed most profusely in the s o i l of pH 5.3 at a l l three temperatures. In most slides the original inocula had disappeared. It i s concluded that F_. alnicola w i l l not grow in natural soils in the range of pH 4°6 to 7.1, and that this i s due to the effect of micro-organisms comprising the normal s o i l flora rather than lack of nutrients or unfavorable pH, Another experiment was designed to test whether hyphae of F_. alnicola would grow out from a food base through non-sterile s o i l . For inoculum, spruce dowels (5/8 i n . dia. and one i n . long) well rotted by F. alnicola were s p l i t lengthwise aseptically, and one half placed with the f l a t side against a slide coated with sterile one per cent water agar. A piece of sterilized spruce root of similar size and shape was also placed on the slide about three quarters of an inch from the dowel. The slide, with adhering wood blocks, was inserted into a jar of moist s o i l (the short - 53 -axis of the slide was vertic a l ) , and s o i l placed around the slide so that i t was nearly buried,, The same soils were used as in the previous experi-ment and s o i l jars were incubated at 12° C. Slides were removed, stained with carbol rose bengal and the area between inoculum and root section examined under the microscope for evi-dence of hyphae growing out from the inoculum. The slide from st e r i l i z e d s o i l of pH 4-oD only, showed that hyphae had grown from the inoculum through s o i l to the sterilized root. No hyphae of F. alnicola were found on slides from non-sterile s o i l . It was concluded that F. alnicola cannot grow through natural soils of pH 4-«6, 5.3, and 7.1 from a small food base. • Inoculum potential as defined by Garrett (18) was not investigated. B. Fruiting in Nature The mushrooms of F. alnicola usually develop on dead wood, and rarely on l i v i n g trees. They occur most commonly on infected wood that has been torn apart or exposed, such as the surface of stumps or butts and roots that have been broken or exposed by windfall and road building machinery. Occasionally they are found in the duff with their bases appressed to the bark of stumps (Fig. 19). From 1951 to 1956 the f r u i t body has been collected from August 2 to September 22, mostly from the middle to the end of August. Late fruiting, or a second crop of mushrooms has been observed in protected locations, e.g., mushrooms were found on roots in the p i t of a p a r t i a l l y excavated stump (Fig. 20) on August 2, 1956 and a second crop was produced by September 8 of the same year. - 5A-PLATE IX, Figures PLATE IX. Fruiting Habit of F. alnicola Figure 19. F. alnicola fruiting in the duff at the base of a white spruce stump. The duff covering the bases of the mushrooms has been removed. Figure 20. F. alnicola fruiting on the roots of a white spruce stump partially excavated the pre-ceding year. - 55 -Hygrothermograph records were obtained for the summer months of 1954- and 1955, and thermograph records for 1956 to see i f fruiting in nature could be related to temperature and relative humidity. The hygro-thermograph was established in a standard shelter six inches above ground beside the stump of a logged white spruce containing an infection of F. alnicola. No mature f r u i t bodies were produced, but in August 1955, mushroom primordia of F. alnicola developed on the stump surface. No correlation between relative humidity and fruiting was discernable. Humidity was con-sistently high at night.. The site was low lying and moist (moisture regime 7), and water was present on the ground for most of the summer. Mean daily temperature, calculated by averaging the temperature at 2, 5, 8, 11, 14., 17, 20 and 23 hrs. was used as the criterion to relate tempera-ture to fr u i t i n g . The curves (Fig. 21, solid line) were smoothed (five-point moving average) to obtain a better i l l u s t r a t i o n of trend, since a plot of mean daily temperature exhibited considerable fluctuation from day to day. The temperature curve for 1955 shows that after a peak in mid-July the temperature remained below 60° F. for a three week period followed by development of primordia of the fungus. No such period occurred in 1956 and no primordia developed. The tree was logged in 1954-* less than a month before temperature observations were started. Although temperature for the recorded period of this year was also below 60° F. no primordia were formed. Sporophores have been found on the stump surface of trees logged the previous winter, but not on trees logged in the current summer, - 56 -PLATE X, Figure PLATE X. Relation of Air Temperattire to Initiation of  Fruiting of F. alnicola Figure 21. A five point moving average of mean daily-temperature (six inches above ground) in rela-tion to i n i t i a t i o n of fruiting in F. alnicola (solid line) and Polystictus circinatus (broken l i n e ) . The graph for P_. circinatus i s not for 1955. - 57 -suggesting a period of time i s necessary for the fungus to prepare for fruiting. I t i s concluded that a period, when mean daily temperature remains below 60° F., i s necessary f o r the i n i t i a t i o n of f r u i t i n g . Gosselin (20) recorded maximum and minimum temperature in connection with the fr u i t i n g of Polystictus circinatus (Polyporus tomentosus) in nature. P. circinatus also causes a root rot in spruce. Mean daily temperature calculated from Gosselin's data by averaging the maximum and minimum temperature, and smoothed by the five-point moving average method is shown in Fig. 21 (broken line) for comparison with the 1955 temperature curve in connection with fr u i t i n g of F. alnicola. The two sets of data bear striking similarities: temperature peaks followed by periods of nearly three weeks with temperature below 60° F. after which fruiting occurred. Gosselin suggested that a temperature of 60° F. was c r i t i c a l for fruiting. .This tends; to support the findings for F. alnicola. Where f u l l y developed f r u i t bodies of F. alnicola have been found on the surface of stumps, the surface has been rough as a result of breakage, or i t has been partly covered with l i t t e r (Fig. 22), that i s , on surfaces that would retain moisture better than smooth ones. The stump kept under observation had a surface which was smooth and presumably became too dry to permit further development of the primordia. These results in them-selves are not conclusive but they were corroborated subsequently by experi-ments in culture which were prompted by them. Temperature i s one factor that appears to cause variation in the time of fruiting from year to year, and even the absence of fruiting in certain years. - 58 -PLATE XI, Figures 22 PLATE XI. Fruiting Habit of F. alnicola Figure 22. F. alnicola fruiting on the litter-covered surface of a white spruce stump. Figure 23. F. alnicola fruiting in a Badcock f r u c t i f i -cation flask. Humidified a i r was introduced into the flask via the rubber tube. Figure 24.. Detail of the f r u i t body in Fig. 23. - 59 -C. Fruiting in Culture i . Flammula alnicola Attempts to induce fruiting in culture were made originally to obtain additional basidiospores for germination studies. As fruiting occurred in nature only for a short time, which varied from year to year, in compara-tively inaccessible locations (the nearest known infection of F. alnicola was 500 miles from the laboratory), a method was sought to provide a supply of fresh spores throughout the year. Later attempts were made to verify the observations on fru i t i n g with respect to temperature. Badcock fru c t i f i c a t i o n flasks (2) containing either white spruce or Scots pine sawdust and various amounts of "accelerator" (l) were inoculated with the fungus (DA0M25874-) and incubated at room temperature. Repeated t r i a l s from 1952 to 1954- were unsuccessful. Occasionally, primordia formed at the mouth of the test tube or within the medium, but did not develop further. A mushroom (Figs. 23 and 24.) developed in one fruc t i f i c a t i o n flask of a series at the University of Br i t i s h Columbia in January, 1955. These flasks had been inoculated a year previously, and incubated a/t room temperature; but a month and a half prior to fruiting, they were placed on a window ledge and aerated. This experiment was repeated but no other mushrooms were produced. Later, at Saskatoon, jars containing s o i l and feeder strip of spruce wood, inoculated with F. alnicola (DAOM25874-) a year previously, had been used in a decay test. After the test was completed, the jars were retained because in a number, mushroom primordia of the fungus had formed in the s o i l - 60 -at the edge of the feeder strip. No further development of the primordia took place at room temperature. To test the observation that a period of low temperature i s necessary for fruiting in nature, the jars were incubated at 54-° F. in a B.O.D. incubator. Continuous circulation of fresh humid a i r was provided by a Phywe diaphragm pump, which f i r s t pumped a i r through a gas dispersion tube in a flask of water (Fig. 25). Jars that were not aerated served as controls. The ligh t in the incubator was l e f t on, exposing the jars to a l i g h t intensity of approximately eight foot candles (measured with a Norwood Director) for eight hours per day i n i t i a l l y , but continuously later on. This method was highly successful and the f i r s t mushroom was produced in three weeks. Subsequently, mushrooms were produced in shorter periods under continuous illumination (Fig. 26).' No mushrooms, developed in the controls. . . Another experiment was designed to test the necessity of incubation at a higher temperature preceding incubation at 54° F. . Thermograph records for 1955 had shown a temperature peak in mid July before the period when temperature remained below 60° F. in connection with the i n i t i a t i o n of fruiting in nature. Two jars containing year-old s o i l wood block cultures of F. alnicola. were given different treatments. One jar was incubated at 4-5° F. and the other at 63° F. for two months. Then both were incubated at 54° F., with continuous aeration as described above and continuous illumination of eight foot candles. A mushroom developed in the jar pre-treated at 63° F. in six weeks, but eleven weeks were required for development of a mushroom - 61 -PLATE XII, Figures PLATE XII. F. alnicola Fruiting in Culture Figure 25. Apparatus used to induce fruiting of F. alnicola. In operation, the culture jar was incubated at 54-° F. in a cabinet. Figure 26. F. alnicola fruiting on a s o i l wood block medium. - 62 -i n the culture pre-treated at 45° F. The la t t e r mushroom was half the size of the former. Other s o i l wood block cultures of F. alnicola (DA0M25874-) were incubated under similar conditions of temperature and aeration, but the source of illumination (an incandescent 25 watt bulb) was level with the base of the s o i l jars. No mushrooms developed in these cultures in several weeks. In the belief that lig h t intensity was too high, the l i g h t was replaced with a 15 watt bulb, and when this f a i l e d after several weeks to induce fruiting, a 10 watt bulb was used which produced an intensity of 10 foot candles at the surface of the jar. No mushrooms developed. Another s o i l wood block culture of the fungus was set up in the original B.O.D. incubator in which the light source was above the jars and lig h t f e l l on them at an angle. Illumination at the surface of the jars was 50 foot candles (measured with a Weston Illumination Meter, Model 756). Mushrooms developed in one month in the shade of the jar cap only ( i l l u -mination 30 foot candles). Mushrooms also developed in the former incu-bator when the l i g h t source was raised so that l i g h t f e l l on the surface of the cultures from above. Conclusions. Nutritional requirements for fruiting were not.investigated. The media used, sawdust plus "accelerator", and wood plus s o i l , were intended only to provide a suitable medium for fruiting. Initiation of fru i t i n g (the development of primordia) does not occur in actively growing cultures at room temperature, but primordia develop when the medium appears to be - 63 -nearly exhausted. The stimulus here i s probably the near exhaustion of the medium. Under environmental conditions suitable for fr u i t i n g , p r i -mordia develop in actively growing cultures. The stimulus in this case i s not exhaustion of the medium for more than one crop of mushrooms may be grown in the same culture. A similar condition exists in nature where successive crops of mushrooms have developed on the same stump for a number of years. In culture, although f r u i t body primordia may develop at room temperature, further development i s dependent on, among other factors, temperature below 60° F. The observation that f r u i t i n g occurs in nature only at temperatures below 60° F. has been verified by cultural experiments described above. Even under favorable conditions of humidity, l i g h t and aeration, fruiting did not occur at room temperature. In the experiments at the University of Br i t i s h Columbia, fruiting occurred only after the fruc t i f i c a t i o n flasks were placed on a window ledge in winter where the temperature was lower. When the experiment was repeated later in the year, no fruiting occurred, presumably because the suitable low tempera-ture no longer existed. Prior incubation at a temperature above 60° F. followed by lower temperature i s not essential for fruiting, but appears to favor development since a larger f r u i t body developed in a shorter time as compared with prior incubation at a lower temperature. Optimum tempera-ture for fruiting in culture appears to be 50 - 55° F. Aeration i s necessary for f r u i t body development, but whether this i s related to an adequate supply of oxygen, removal of carbon dioxide, or both was not investigated. Fruiting did not occur in capped jar cultures under conditions of favorable li g h t , temperature, and humidity, but did in aerated cultures beside them. Humidity of the a i r stream was recorded at 100$ with an electric hygrometer. While this instrument was not completely dependable for readings of 100$, condensed vapor on the inside of aerated jars for a long period indicated that humidity within the jars was close to saturation. The lower l i m i t of humidity permitting f r u i t body forma-tion was not investigated. The need for maintaining moisture within the medium in addition to a near saturated atmosphere was demonstrated in a culture in which a mushroom had developed to a stage where cap expansion had begun. Further development ceased unt i l water was added to the medium. Then development was completed rapidly. Fruit bodies were produced in l i g h t intensities of 8 to 30 foot candles where li g h t f e l l on the surface of the cultures from above. No fruiting occurred when the ligh t source (at similar intensities) was at the same level as the cultures. Whether angle of incidence of the l i g h t i s c r i t i c a l , in addition to intensity, was not studied. Any one of the environmental factors, temperature, moisture, aeration or l i g h t may act as a limiting factor preventing f r u i t body development when deficient. i i . Flammula conissans In conjunction with experiments to induce fr u i t i n g in cultures of F. alnicola. parallel t r i a l s were run with cultures of F. conissans. P r i -mordia did not develop in cultures at room temperature or under the condi-tions in which mushrooms of F. alnicola developed in s o i l wood block cultures, that i s , a temperature of 54-° F., circulation of nearly saturated air through the jar, and illumination of eight foot candles. After one of - 6 5 -these t r i a l s a s o i l wood culture was l e f t in the incubator but not aerated. Several months later a small mushroom had developed. An experiment was set up to reproduce the fortuitous circumstances that led to fru i t i n g of F. conissans. Two well developed s o i l wood block cultures (DA0M22888) were incubated at 5 4 ° F. The source of illumination (an incandescent bulb) was level with the base of the jars. Light inten-s i t y was 10 foot candles at the surface of the jars, but less in the i n -terior because mycelium had grown up the jar sides. One culture was aerated with near saturated a i r . The cap of the other jar was vented ( 1 4 ) . Excess moisture was maintained on the fungus mat of the l a t t e r to provide an atmosphere of high relative humidity. Primordia developed on the wood blocks in the vented jar within a month and a half. Numerous small mushrooms (largest cap diameter 7 mm.) formed within two months. No primordia or mushrooms developed in the aerated jar. Aeration of this culture was continued to see i f f r u i t i n g would occur later. A frozen compressed air line, however, halted aeration of the culture for two weeks. In this period primordia developed. Aera-tion was then resumed and mushrooms developed. The largest mushroom (cap diameter 2 cm.) developed in this culture (Fig. 28). Fruiting had not occurred during several months of aeration, but when aeration was stopped conditions became favorable for primordia development. Further develop-ment of f r u i t bodies was favored by aeration. In another test, a s o i l wood block culture (DA0M22888), in a vented jar, was incubated at 5 4 ° E« in a B.O.D. incubator. Cultures were i l l u -minated from above and li g h t intensity at the outside of the jar was 50 - 66 -foot candles, but less in the interior because of mycelium growing on the jar sides. Mushrooms were produced in one month from mycelium which had grown from a wood block onto the wall of the jar. Larger mushrooms were produced than in the f i r s t t r i a l , but they were not as large as those produced in the culture aerated after primordia development. Conclusions The conditions for fruiting of F. conissans in well established s o i l wood block cultures ares Incubation at 50 to 55° F., l i g h t intensity of 8 to 30 foot candles, an atmosphere of high humidity and l i t t l e to no aeration u n t i l primordia develop. Aeration favors further development. Thus the environmental conditions for fruiting of F. conissans are distinct from those for F_. alnicola. and neither fungus w i l l f r u i t under conditions favorable to the other. Mushrooms of F. alnicola invariabljr developed on the s o i l of the culture, and F. conissans on wood or from mycelium growing from the wood blocks of the culture. Conditions for fr u i t i n g are compared in Table XII. Fruit bodies of the two fungi produced in culture are shown in Figs. 27 and 28. D. Taxonomy and Description^ i . Flammula alnicola (Fr.) Kummer, Der Fuhrer in die Pilzkunde p. 82. 1871. Agaricus alnicola Fr.. Syst. Myc. Is250. 1821. Dryophila alnicola Quel.. Enchir. Fung. p. 71. 1886. Flammula conissans sensu Ricken, Die Blatterpilze p. 205. 1915. ^The fungi were identified by Dr. J. ¥. Groves, Head, Mycology Unit. Botany and Plant Pathology Division, Department of Agriculture, Ottawa, who also furnished the references. - 6 7 -TABLE XII. FRUITING- OF F. ALNICOLA AND F. CONNISSANS IN SOIL ?/OOD BLOCK CULTURES AS AFFECTED BY TFJ1PEPATURE, RUICEDITY, AND LIGHT Environment F. alnicola F. conissans Temperature (a) 5 0 - 5 5°F. + + (b) over 6 0°F. 100% humidity: (a) aerated + (b) not aerated - + Light ( 8 - 3 0 f.c.) (a) from above + + (b) horizontal - + - 68 -PLATE XIII, Figures 27 PLATE XIII. Fruit Bodies and Basidiospores of F. alnicola  and F. conissans Figure 27. F. alnicola fruiting on a s o i l wood block medium. Figure 28. F. conissans. fruiting on a s o i l wood block medium. Figure 29. Basidiospores of F. alnicola, X 720. Figure 30. Basidiospores of F. conissans, X 720. - 69 -Pileus 1 . 5 - 7 (11) cm. rounded or conical at f i r s t , becoming subumbonate, convex or plano-convex, sometimes with reflexed margin. Clear brown (Munsell color 10YR 5/6) in the button stage with paler margin (2.5Y 8/4), becoming yellow (2.5Y 8/4 at the centre to 2.5Y 8/3 at the margin); darker (brown) with age. Viscid in the button stage, becoming dry, glabrous. Traces of a partial v e i l adhering to the margin for some time. Context yellowish with a watery green line in the flesh just above the lamellae in fresh specimens. Lamellae adnate to subdecurrent, pale yellow becoming brown due to deposits of spores. Stipe 3 - 1 4 cm. long, 5 - 7 (20) mm. in diameter, terete, striate, pale yellow (2.5Y 8/3), darker at the base (10YR 5/6). Annulus inconspicuous, superior, f i b r i l l o s e , sometimes absent. Spores rusty brown, smooth, 4»5 - 5.5 (6) x 7 - 10 u, ovate ellipsoid, flattened on one side, apiculate (Fig. 2 9 ) . Basidia hyaline, four-spored, 6 - 7.5 x 25 - 30 u. Pleurocystidia absent. Cheilocystidia clavate, obclavate 5 - 8 u broad. G i l l trama of broad parallel hyaline hyphae 7 - 12 u broad; vesicular cells 12 - 20 u in diameter; and occasional narrow hyphae 5 - 10 u broad with yellow contents. Pileus trama composed of a thin p e l l i c l e , 60 - 125 ]i in thickness of prostrate interwoven or parallel hyphae 3 - 6 u broad; flesh proper of short, broad, interwoven hyphae 10 - 12 u broad, without clamp connections. Stipe without caulocystidia. Habitats Usually caespitose on stumps, roots of windfalls or in the duff at the base of stumps of white spruce, rarely on l i v i n g trees. Other hosts are l i s t e d under cultural characters. - 70 -SPECIMENS EXAMINED: CANADA: Alberta: Seebe, DAOM 40076 , 25874 on Picea glauca var. albertiana. Slave Lalce, DASFB 13857, 1386, 1387, 1388, 1389 on P. glauca. Fruit bodies developed in culture from isolate DAOM 25874: DASFB 1390, 1391, DAOM 60219. Donk (13) pointed out that Kummer had raised Flammula to the rank of genus in 1871, antedating Qu'elet who had raised i t to a genus in 1872. Kummer i s , therefore, the authority for the genus name. The above description agrees closely with that of Runner and Romagnesi (24) for Dryophila alnicola. They note "Odeur speciale (un peu de bonbons anglais, H. R. pas d'Hypholoma)". I t i s not clear what this odor i s , but cultures of F. alnicola have a penetrating musty odor, which is also evident in decayed wood with which f r u i t bodies are associated, but not in the f r u i t bodies themselves. They also note that pleurocystidia ("cyst, faciales") are absent and that "ne pas prendre pour des chrysocys-tides les vieux elements hymeniens collapse's, qui sont * jauhatres, mais ne fixent pas le bleu lactique". It i s noteworthy that these authors state that the forms on hardwoods and softwoods are similar for, the above description of the f r u i t body i s based on collections from softwoods, while the descriptions of European authors were based on collections from hardwoods. They consider F. conissans sensu Ricken to be the same as Dryophila alnicola. Herbarium numbers, Mycology Unit, Department of Agriculture, Ottawa. Herbarium numbers, Forest Biology Laboratory, Saskatoon. - 71 -The description of Flammula alnicola (Fr.) Kummer by Konrad and Maublanc (23), as well as the figure for the mushroom, matches the above description, except that a greenish tinge to the pileus has been noted, only rarely. Lange (25) also notes a greenish color at the edge of the pileus. His reference to cystidiat "Cystidia hair-shaped, clavate, apex about 6 - 7 u", presumably refers to cheilocystidia. Apart from this his description i s in agreement. The i l l u s t r a t i o n of Agaric us (Flammula)  alnicola Fr. by Cooke (9) matches the f r u i t bodies collected by the author. i i . Flammula conissans (Fr.) G i l l . , Champ. Fr. p. 525. 1876. Agaricus conissans Fr.. Epicr. Myc. p. 187. I838. Dryophila conissans Quel.. Enchir. Fung. p. 71. 1886. Pileus 0.5 - 2 cm., rounded becoming convex, furfuraceous then glabrous, yellow (2.5Y 8/6), paler at the margin (2.5Y 8/4.). Lamellae subdecurrent, paler than the cap. Veil fugaceous. Stipe 2.5-6 cm. long, 2 - 3 ram. in diameter, terete, furfuraceous at f i r s t , becoming finely striate, paler than the cap (2.5Y 8/3). Spores rusty brown, smooth, 4 - 4.5 x 5.5 - 8 n, oval oblong, sli g h t l y flattened on one side (Fig, 30). Basidia hyaline four-spored, 4 - 5 x 13 - 20 u. Pleurocystidia (chrysocystidia^) abundant, clavate to ventricose with obtuse apex (some with papillate apex), 8 - 14 x 24 - 34 u with golden yellow contents, deeply staining in lactophenol-cotton blue, embedded in the hymenium. Cheilocystidia f i l i f o r m , filiform-clavate, or wavy f i l i f o r m 3 - 5 u broad. G i l l trama of broad parallel hyaline hyphae 5 - 12 u broad. See Snell, ¥. H. and Dick, E. A. 1957. A- glossary of mycology. Harvard University Press, Cambridge, Massachusetts, p. 29. - 72 -Pileus trama composed of a thin p e l l i c l e (not viscid) 75 - 200 u in thick-ness of prostrate interwoven hyphae, 3 - 6 u broad; flesh proper of short, broad interwoven hyphae 5 - 20 u broad without clamp connections. Stipe without caulocystidia. Habitat: Kuhner and Romagnesi (24) reported i t on the stumps of willows, especially Salix cinerea and Lange (25) reported i t as occurring exclusively on S. cinerea. SPECIMENS EXAMINED: Fruit bodies developed in culture from isolate DA0M22888 obtained from Centraalbureau voor Schimmelcultures, Baarn, Holland and deposited there by Kuhner: DASFB 1392, DAOM 60217, 6024-7. Fruit bodies developed in culture are generally smaller than those developed in nature, consequently the upper l i m i t of pileus diameter in this description i s less than that in published descriptions. The above, description agrees quite well with that of Kuhner and Romagnesi (24) except,for spore shape which they state i s "subcylindracees a. reniformes". Spores with a reniform shape were not found. Lange's (25) description i s in accord except that his measurements of cystidia (cheilocystidia) are larger. Lange does not refer to chrysocystidia which are a striking feature of the hymenium. The f r u i t bodies of the two species of Flammula di f f e r in that F. alnicola i s larger, darker colored, the base of the stipe i s darker than the upper part, and chrysocystidia are absent from the hymenium. - 73 -E. Spore Germination i . Flammula alnicola From 1951 on, numerous attempts were ma.de to induce germination of basidiospores discharged from fresh f r u i t bodies of Flammula alnicola. Until a satisfactory method of producing f r u i t bodies in culture was dis-covered, this was dependent on finding f r u i t bodies in nature. The routine method consisted of affixing pieces of the hymenium to the l i d of a Petri dish and allowing spores to be discharged onto the agar below. Single spores were isolated and transferred to slants of malt agar. The plates and slants were kept at room temperature or in a refrigerator (approxi-mately 0° C.). Plates were kept unt i l they became contaminated. No isolated spores germinated, but two polysporous transfers produced cultures after several months of refrigeration. In other (unsuccessful) methods, spores were plated on: hot and cold s o i l extract agar, hot and cold water extracts of wood plus agar, sterile cellophane over alkaline and acid humus, 2.5$ phosphoric acid, and malt broth in which ctiltures of F. alnicola had been growing. Spores in the above t r i a l s were kept at 3° and 7° C. Basidiospores from a f r u i t body collected in September, 1957, were suspended in d i s t i l l e d water and kept at 3° C. At weekly intervals a drop or two of spore suspension was transferred to plates of malt agar which were then kept at 7° C. The plates were examined for spore germination at weekly intervals and kept u n t i l they became contaminated. A few spores germinated after five months treatment at 3° C. Germination was roughly - 74 -0.2%. Single germinating spores were isolated and transferred to slants of malt agar, but none continued to grow. Ho spores germinated in subse-quent platings. A spore print obtained from a f r u i t body produced in culture (DAOM25874-) in September, 1958, was cold-treated at -7° C. as recommended by Kneebone (22). After five weeks, spores were scraped from the print, suspended in water and plated on malt agar. The plates were incubated at room tempera-ture in a saturated atmosphere. Within two weeks a number of spores had germinated (about a dozen per heavily seeded plate). Spores were plated, similarly, after 10 and 12 weeks of cold treatment at -7° C. Germination was 0.4% in both cases. A spore print, cold-treated at -7° C. for 12 weeks, was then cold-treated at -18° C. Spores were plated after one and a half weeks at -18° C , and examined a week later. Germination was very low compared with that of spores cold-treated at -7° C. for 13 weeks. Spores plated after six and a half weeks at -18° C. failed to germinate completely, but spores cold-treated for 18 1/2 weeks at -7° C. germinated as well as those cold-treated at -7° C. for 10, and 12 weeks. Basidiospores, cold-treated at -7° C. for 11 weeks, were suspended in cooled one per cent water agar. Slides were dipped in the suspension and then placed upright in jars of moist s o i l (pH 4*6). Four slides were placed in each of four jars, two of which had been st e r i l i z e d . The jars of s t e r i l e and non-sterile s o i l were incubated at 7° and 17° C. Slides from the jars were examined for spore germination at one, three, four and five weeks. Ho spores germinated. - 7 5 -i i . Flammula conissans Basidiospores of F. conissans were obtained from f r u i t bodies developed in culture (DA0M22888). Spores, plated from a spore print kept at - 7 ° C. for three weeks, germinated in good numbers. Spores, suspended in two per cent malt broth, were cold-treated at - 7 ° C. for 11 weeks and then incubated at 7 ° C Platings showed germination of 4 1 % « Spores in malt broth kept at room temperature did not germinate. Twenty single spores, isolated from a fresh spore print and transferred to slants of malt agar, were kept at - 7 ° C. for six weeks. The slants were then incubated at room tempera-ture but no spores germinated. Under similar conditions polyspore transfers produced cultures. The above tests indicate that spores of F. alnicola and F. conissans require cold treatment for germination, but spores of F. alnicola require longer cold treatment and lower temperatures than F. conissans for germina-tion. The very low numbers of spores of F. alnicola that germinated at - 7 ° C , and the inhibition of germination at -18° C. suggest that factors other than cold treatment are involved in spore germination in this species. F. I n t e r f e r t i l i t y Tests The standard method of determining i n t e r f e r t i l i t y type, in which a l l possible combinations of a series of monosporous cultures are paired, has been followed. The presence of clamp connections after fusion of two monosporous mycelia denotes compatibility and their absence denotes - 76 -incompatibility. Rare clamp connections in resulting mycelia are consi-dered to be illegitimate pairings. Following Nobles et a l (29), "The conventional symbols for the alleles governing i n t e r f e r t i l i t y , A^A^B^B^ in tetrapolar species, have been used and assigned in an arbitrary manner". i„ Flammula alnicola Basidiospores from a f r u i t body developed in culture (isolate DA0M25874-) on September 16, 1958, were kept at -7° C. for seven weeks and then plated on malt agar. Isolated colonies had developed on the heavily seeded plates in two weeks. Transfers of the colonies were made to slants of malt agar. As soon as cultures were established on the slants they were propagated to other slants. The cultures were examined for the presence of clamp connections, and, i f none were present, they were presumed to be mono-sporous cultures. Of twelve cultures, two had clamp connections and a third was a contaminant. Transfers of developing colonies contained many ungerminated spores, but because of the very low percentage of germination retransferring resulted in monosporous cultures. This was verified by the pairing behavior of the cultures. Since monosporous cultures were obtained by this method the isolation of single germinating spores was unnecessary. The nine monosporous cultures were paired in a l l possible combinations and were examined for the presence of clamp connections after three weeks. The results are shown in Fig. 31. Flammula alnicola shows the tetrapolar type of i n t e r f e r t i l i t y . - 77 -i i . Flammula conissans Basidiospores from a f r u i t body produced in culture (isolate DA0M22888 from Europe) on September 29, 1958, were kept at -7° C. for five weeks and then plated on malt agar. Two days later single germinating spores were isolated under the microscope and transferred to slants of malt agar. Out of 4-0 isolations only 12 developed into cultures. The 12 monosporous cultures were paired in a l l possible combinations and examined for the presence of clamp connections. Results are shown in Fig. 32. Although the monosporous cultures f e l l into four groups, and F. conissans is. therefore tetrapolar, mating occurred between supposedly incompatible.groups.' > Pairings of a l l combinations of A-jB-^  and A 2B 2 > r e r e f e r t i l e and Aj_B]_ would not mate with any other group. .. Pairings o f - a l l .possible combinations of AjB^ and A^B^ were f e r t i l e and'A^B^ would not mate with any other group. Pairings of a l l possible' combinations of A2B2 and A j j ^ were also f e r t i l e . Pairings between the latter two groups were not illegitimate pairings, since clamp connections in the fused mycelia were just as common as in legitimate pairings, although they took longer to develop. A probable explanation of the anomalous behavior of A2B2 cultures i s that they were a mixture of A2B2 and AgB^. This mixture would not be f e r t i l e in i t s e l f but would be f e r t i l e in combination with both A-jB-]_ and A^B^. In spite of the care exercised to isolate single germinating spores two of the isolations resulted in cultures that were mixtures of identical composition. An additional 4-0 single germinating spores were isolated, but only four continued to grow when transferred to slants of malt agar. The four - 78 -PLATE XIV, Figures 31 - 33 PLATE XIV. I n t e r f e r t i l i t y Tests of F. alnicola and F. conissans Figure 31. Pairing of nine monosporous mycelia of F. alnicola in a l l possible combinations. Figure 32„ Pairing of 12 monosporous mycelia of F. conissans in a l l possible combinations. Figure 33. Pairing of four monosporous mycelia of F. alnicola and F. conissans in a l l possible combinations. I N T E R F E R T I L I T Y T A B L E F l o m m u l a o l n i c o l o ( Fr . ) K u m m e r S ing le S p o r e C u l t u r e s A , B | A 2 B 2 A , B 2 A 2 B | A , B , < A 2 B 2 . A , B 2 < A 2 B , ^ 3 |7 8 1 |l0|l2|5 6 9 ^3 — 4 4 - -f + + i 4 4 4-10 4 4 4 _ • \z J  - — + 4 5 4 4 6 4 4 — 9 - - 4- 4- — 31 I N T E R F E R T I L I T Y T A B L E F l a m mulo c o n l s s o n s (Fr.) G i l l e t S ing le S p o r e C u l t u r e s A j B j A , B , A , B , A , B , *l°2 A 2 B 2 A , B 2 A 2 B | 1 2 4 7 9 10 I I 3 5 6 8 12 Z_ 4_ J_ _9 i d 4 4 4 -i-h + + H  + + - - 4 - 4 -4-4-4-4-4- - 4 4 4 -4 -4 -4 - - 4 -4 -4 - -h+ h 4- + - - 4 h 4 4 4 - 4 4 4 4 + -I N T E R F E R T I L I T Y T A B L E F lammula olnicolo (Fr.) K u m m e r and F l o m m u l a c o n i s s a n s (Fr.) Gillet S i n g l e Spore C u l t u r e s F, o ln ico lo F, con i ssans 3 1101 5 | e 1 1101 3 | 8 3 10 _5_ 6 4 4 4 V _ l_ _I0 _3_ 8 4 4 4 -- 4 4 r - f - T rue Clomps - J - F a l s e C l a m p s monosporous cultures vrere each paired with cultures, 2(AjB2), 10(A2B2 + A 2 B 1 3 ^ A l B 2 ^ * 8 1 1 ( 1 8 ( A 2 B l ) * ^ r e s v l t s indicated two of the addi-tional monosporous cultures belonged to the A^ Bg group, and the other two belonged to the A2B-^ group. Pure monosporous cultures of the A2B2 group were not obtained. In any event Flammula conissans shows the tetrapolar rather than the bipolar type of i n t e r f e r t i l i t y . The tetrapolar condition has been reported for this species by Yen (36). i i i . F_. alnicola and F. conissans Monosporous mycelia of the four mating types of F. alnicola and F. conissans were paired in a l l possible combinations. Fertile combinations, i.e., mycelia with clamp connections would indicate that only one species was involved. The results of the pairings are shown in Fig. 33. No f e r t i l e com-bination resulted from any pairing between F. alnicola and F. conissans, and i t is concluded, therefore, that they are distinct species. Within • species clamp connections resulted from pairings of compatible monosporous mycelia. The anomaly in pairings of F. conissans was the same as that noted i n section i i . - 80 -IV. DISCUSSION Theoretically, Flammula alnicola may be spread from tree to tree by the dissemination of basidiospores or by vegetative infection of neigh-boring trees. Basidiospores are released in large numbers from sporophores. To cause infection directly, basidiospores would have to be washed into the s o i l and deposited on the dead bark of root pressure wounds which pro-vide infection courts. Germination of spores i s favored by low temperature and high humidity. Cold-treated basidiospores w i l l germinate on culture media, but not on st e r i l e or non-sterile s o i l ; nor w i l l hyphae grow on a medium of non-sterile s o i l . I f spores germinated in the surface layers of s o i l they could not be washed down to infection courts. Soil appears to have an inhibitory effect on germination; this may act as a mechanism to prevent germination in locations unfavorable for further growth. Good and Spanis (19) have shown that spores of Fomes igniarius var. populinus (Neuman) Campbell germinate i n higher percentages on aged wound extracts of poplar wood than on fresh wood extracts. The bark of aged root pressure wounds in spruce may induce spores of F. alnicola to germinate. Twenty-four pressure wounds, 2 to 14 years old (mostly 2 to 5 years) on the roots of spruce growing on rotting stumps and logs were not invaded by heart rot fungi, suggesting that recently formed wounds do not foster infection. Vegetative infection of trees from f i r e - k i l l e d or dead members of the preceding generation evidently occurs also. Trees, established as seedlings, on rotting stumps or logs are potential hosts for F. alnicola, which i s capable of surviving in trees k i l l e d by f i r e . The fungus, perhaps, may be - 8 1 -able to persist in dead trees for 30 to 50 years or more without exhausting i t s food supply, or being overrun and k i l l e d by competing fungi. Infection of the trees could then take place through root wounds in contact with or close to decay. Fruit bodies have been found that could have been pro-duced only by hyphae that had grown for one to two feet through s o i l from a large food base of an infected tree. Nearby trees may be infected in such extension of hyphae through the s o i l . F. alnicola w i l l not invade sapwood presumably because i t i s too wet. Similarly, wood under recently formed wounds in roots may be too wet for invasion. ¥hile wood under wounds on aerial parts of trees dry out quickly, the wood under root wounds would dry out much more slowly. The time required for the wood to reach a mois-ture content at which i t could be invaded would be influenced by the wetness of the site. Vegetative infection from generation to succeeding generation of the host would result in very limited spread of the fungus, and i t i s doubtful i f a l l infections would survive. Basidiospores appear to be the only means by which F. alnicola can be dispersed. The isolated occurrence of some decay infections in stands may be the result of chance deposition of basidiospores in places where they can cause infection. Determination of the time of entry of the fungus i s d i f f i c u l t . Infection through wounds in the roots provided a means of estimating the age of infections. Assuming the age of infection to be the same as the age of the wound permitting entry of the fungus, the rate of decay was very slow. The rate varied considerably even between different infections in the same tree. The actual rate was probably somewhat faster because of an apparent lapse of several years, which also may vary, between wound - 82 -formation and infection. Wagener and Davidson ( 3 3 ) , in discussing the progress of decay in individual trees, point out that the i n i t i a l rate of extension of a heart rot fungus may be much more rapid than that pre-vailing later. The maximum rate of extension of two-year-old a r t i f i c i a l infections of F. alnicola was nearly twice the maximum calculated rate in natural infections. Etheridge (15 and 16) found that the average moisture optimum for decay was higher in butt-rotting fungi (including F. alnicola) than trunk-rotting fungi; and that moisture content of the heartwood at 1.5 feet was higher than at 20 feet or above. Decreasing moisture content of heartwood with height may result i n a decreasing rate of extension upward into the trunk. Differences in the moisture content among trees may account for different over-all rates of decay. F. alnicola decay occurred more frequently on acid soils and on better sites; conditions which are probably related to infection. The s o i l microflora of acid soils may be less antagonistic to the germination of basidiospores on root wounds. Good sites were moist, a condition that appeared to favor infection. Dryer or wetter s o i l conditions resulted in lower site quality. Flammula alnicola, Polyporus tomentosus, and Coniophora puteana are the most frequent cause of butt-rot in boreal white spruce. P_. tomentosus and C_. puteana are common causes of wind throw; but F. alnicola because of i t s slow rate of growth, particularly in roots, and restriction of the decay to heartwood i s less important as a cause of wind throw. V. REFERENCES 1. Badcock, E. C. 1941. New.methods for the cultivation of wood-rotting fungi. Trans. B r i t . Mycol. Soc. 2£:200-205. 2. Badcock, E. C. 1943. Methods for obtaining fructifications of wood-rotting fungi in culture. Trans. B r i t . Mycol. Soc. 26:127-132. 3. Bourchier, R. J. 1957. Variations in cultural conditions and i t s effect on hyphal fusions in Corticium vellereum. Mycologia 49:20-28. 4. Buller, A. R. H. 1924. Researches on fungi. Vol. III. Longmans, Green & Co., Ltd., London, p. 413. 5. Busgen, M. and Munch, E. 1929. The structure and l i f e of forest trees. Trans, by T. Thomson. Chapman and Hall, Ltd., London. 6. Cartwright, K. St. G. and Findlay, ¥. P. K. 1946. Decay of timber and i t s prevention. His Majesty's Stationery Office, London. 7. Handbook of chemistry and physics. 1956. 38th ed. Chemical Rubber Publishing Co., Cleveland, Ohio. p. 1615. 8. Chinn, S. H. F. 1953. A slide technique for the study of fungi and Actinomycetes in s o i l with special reference to Helminthosporium sativum. Can. J. Botany 31:718-724. 9. Cooke, M. C. 1885. Illustrations of Br i t i s h Fungi (Hymenomycetes). Vol. TV. Williams and Norgate, London, p i . 443. 10. Cox, C. E. 1954. Handbook on s t a t i s t i c a l methods. Processed Pub. No. 3> Canada Dept. Agr. 11. Denyer, W. B. G. and Riley, C. G. 1953. Decay in white spruce at the Kananaskis Forest Experiment Station. For. Chron. 29:233-247. 12. Denyer, W. B. G. and Riley, C. G. 1954- Decay of white spruce in the Prairie Provinces. Canada Dept. Agr., For. B i o l . Div. Interim Rept. 13« Donk, M. A. 1949. New and revised nomina generica conservanda proposed for Basidiomycetes (Fungi). B u l l . Bot. Gard. Buitenz., Ser. I l l 18:83-168. 14. Eades, H. W. and Roff, J. W. 1953. The regulation of aeration in wood s o i l contact culture technique. J. For. Prod. Res. Soc. _3_:68-71, 94-95. 15. Etheridge, D. E. 1957. Moisture and temperature relations of heartwood fungi i n subalpine spruce. Can. J. Botany 35:935-944. - 84 -1 6 . Etheridge, D. E. 1958, The effect on variations in decay of moisture content and rate of growth in subalpine spruce. Can. J . Botany J6:187-206. 17. Foster, R. E. 1959. Forest Biology Laboratory, Victoria. Personal correspondence. February 24.. 18. Garrett, S. D. 1956. Biology of"root-infecting fungi. Cambridge University Press, London, p. 79. 19. Good, H. M. and Spanis, ¥. 1958. Some factors affecting the germina-tion of spores of Fomes igniarius var. •po-pulinus (Neuman) Campbell, and the significance of these factors in infection. Can. J. Botany 36 : 4 2 1 - 4 3 7 . 20. Gosselin, R. 1944-. Studies on Polystictus circinatus and i t s relation to butt-rot of spruce. Farlowia 1_: 528-568. 21. J0rgensen, E. 1955. A method for the study of mycelial anastomoses. Friesia 75-79. 22. Kneebone, L. R. 1951. An investigation of basidiospore germination in the Hymenomycetes, especially in the Agaricaceae. University Micro-films, Ann Arbor, Michigan. 23. Konrad, P. and Maublanc, A. 19.30. Icones selectae fungorum. Vol. I. Fasc. VI. Edited by Paul Lechevalier, Paris, p i . 64. 24-. Kuhner, R. and Romagnesi, H. 1953. Flore analytique des champignons superieurs (Agarics, Bolets, Chanterelles). Masson et Cie, Paris, p. 330. 25. Lange, J. E. 1939. Flora Agaricina Danica. Vol. IV. Recato, Copenhagen, p. 10. 2.6. Miles, A. E. ¥. and Linder, J. E. 1952. Polyethylene glycols as histo-logical embedding media. J. Roy. Micro. Soc. 72:199-213. 27. Munsell Book of Color. 1929-194-2. Munsell Color Co., Inc., Baltimore, Maryland. 28. Nobles, M. K. 1948. Studies in forest pathology. VI. Identification of cultures of wood-rotting fungi. Can. J. Research C 26:281-431. 29. Nobles, M. K., Macrae, R. and Tomlin, B. P. 1957. Results of inter-f e r t i l i t y tests on some species of Hymenomycetes. Can. J . Botany i i : 377-387. 30. Nobles, M. K. 1958. A rapid test for extracellular oxidase in cultures of wood-inhabiting Hymenomycetes. Can. J. Botany 3,6;91-99. - 85 -31. Quaite, J. 1955. The evaluation of site and growth of white spruce in the Mixedwood Section of Alberta. Project A:29. Canada Dept. of Northern Affairs and National Resources, For. Br. Interim Rept. 32. Roff, J. ¥. 1955. For. Prod. Lab., Vancouver. Personal correspon-dence. October 18. 33. Wagener, ¥. ¥. and Davidson, R. ¥. 1954. Heart rots i n l i v i n g trees. Bot. Rev. 20:61-134-. 34-« ¥aterman, A. M. 1955. A stain technique for diagnosing b l i s t e r rust in cankers on white pine. For. Sci. 1:219-221. 35. Yates, F. 1937. The design and analysis of factorial experiments. Imp. Bureau Soil Sci. Tech. Comm. No. 35. Example 9d. 36. Yen, H. C. 1947. Note prlliminaire sur l a polarite sexuelle et sur les caracteres du mycelium haploide de plusieurs Homobasidiomycetes. Compt. Rend. 224.: 1239-124-0. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0106405/manifest

Comment

Related Items