BIOLOGY OF SPARASSIS RADICATA (WEIR) IN SOUTHERN ARIZONA
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Authors Martin, Kenneth J., 1942-
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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 74-21,162 MARTIN, Kenneth J, 1942- BIOLOGY OF SPARASSIS RADICATA WEIR IN SOUTHERN ARISM! The University of Arizona, Ph.D., 1974 Agriculture, plant pathology
University Microfilms, A XEROX Company. Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. BIOLOGY OF SBARASSIS RADICATA WEIR IN SOUTHERN ARIZONA
by
Kenneth J Martin
A Dissertation Submitted to the Faculty of the
DEBARTMENT OF PLANT ETHOLOGY
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
19 7 4 THE UNIVERSITY OF ARIZONA.
GRADUATE COLLEGE
I hereby recommend that this dissertation prepared under my direction by Kenneth J Martin entitled BIOLOGY OF SIARASSIS RADICATA WETR IN SOUTHERN
ARIZONA be accepted as fulfilling the dissertation requirement of the degree of Doctor of Philosophy
Dissertation Director Date
After inspection of the final copy of the dissertation, the following members of the Final Examination Committee concur in its approval and recommend its acceptance:-''
fts . Clop*... IX Ma* 1*1*
This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination. STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to bor rowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or re production of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the in terests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: DEDICATION
To Siizanne, Greg, and Kendra
ill ACKNOWLEDGMENTS
I wish to thank Dr. Robert L. Gilbertson, under whose guidance and supervision this work was done, for his patient and expert assis tance and many helpful suggestions. Thanks are also extended to Dr.
George B. Cummins, Dr. Ross M. Allen, Dr. Roger L. Caldwell, Dr. Michael
E. Stanghellini, and Dr. Richard B. Hine for their suggestions and crit ical review of the manuscript, and to J. Rage Lindsey for her assistance and the collections she made. A special thanks to the Department of
Plant Rathology, University of Arizona for providing financial assis tance during this study.
iv TABLE OF CONTENTS
Page
LIST OF ILLUSTRATIONS vii
LIST OF TABLES X
ABSTRACT xi
INTRODUCTION 1
LITERATURE REVIEW 4
MATERIALS AND METHODS 10
Studies on Sparassis radicata 16 Cultural Morphology 16 Oxidase Reactions . 17 Temperature Relations 18 Basidiospore Germination and Jfycelial Development .... 18 Mating System 19 Test for Multiple Alleles for Incompatibility 20 Decay Studies 21 Incidence of ,S. radicata 23 Studies with Cultures Labeled S>. crispa and S. laminosa ... 23 Di-mon Matings 23
RESULTS 25
Morphology of Basidiocarps 25 Cultural Studies of S>. radicata 32 Growth Characters 32 Microscopic Characters 32 Oxidase Reactions 36 Basidiospore Germination and Mycelial Development .... 36 Mating System 40 Asexual Spores 43 Key Pattern 47 Multiple Allele Test for Incompatibility ..... 47 Temperature Relations ..... 50 Decay Studies 50 Decay in Nature 50 Agar-block Decay Studies 55 Incidence of S. radicata in the Santa Catalina Mountains ... 56
v vl
TABLE OF CONTENTS~-Continued
Plage
Studies with Isolates Labeled _S. crlspa and j>. lamlnosa ... 63 Growth Characters 65 Microscopic Characters 65 Di-mon Matlngs 67
DISCUSSION 70
LITERATURE CITED 81 LIST OF ILLUSTRATIONS
Figure Bage
1. Hiotograph of basidiocarp of S. radicata (KJM-274), (X 0.4) "" 26
2. Hiotograph of basidiocarp of _S. radicata (KJM-274) as usually found associated with living trees ...... 26
3. Hiotograph of pseudosclerotium of jS. radicata (KJM-274), (X 0.13) 7 27
4. Camera lucida drawings of subhymenlal and contextual hyphae in basidiocarps of £!. radicata (KJM-468) 29
5. Camera lucida drawings of gloeoplerous hyphae, basidia, and basidiospores in basidiocarps of S. radicata (KJM-468) 30
6. Camera lucida drawings of three types of hyphae in pseudosclerotium of S. radicata (KJM-468) 31
7. Photographs showing growth characters of _S. radicata (RLG-8240) on Nobles agar at 25C in the dark 33
8. Camera lucida drawings of hyphae of the advancing zone and submerged mycelium of J3. radicata (KJM-389) on Nobles agar at 25C in the dark four weeks after inoculation ...... 34
9. Camera lucida drawings of two types of aerial hyphae of £. radicata (KJM-389) on Nobles agar at 25C in the dark six weeks after inoculation 35
10. Hiase contrast photomicrograph of spathulate swollen tips of hyphae of S. radicata (KJM-389) on Nobles agar at 25C in the dark six weeks after inoculation, (X 400) 37
11. Hiotographs showing negative reaction for the production of extracellular oxidase by S. radicata (KJM-274) on gallic and tannic acid media after 10 days at 25C in the dark 38
vii viii
LIST OF ILLUSTRATIONS—Continued
Figure Page
12. Ihotograph showing negative reaction for the production of extracellular oxidase by _S. radicata (KJM-274) with gum guaiac solution 38
13. Camera lucida drawings of germinating basidiospores of JS. radicata (KJM-274) on 3.07. MEA under alternating 12 hr periods of darkness and 85 ft-c illumination .... 39
14. Chart showing results of pairing 23 single basidiospore isolates of Si. radicata (KJM-274) in all combinations ... 41
15. Bipolar pattern of sexuality of j>. radicata (KJM-274) rearranged into mating type groups 42
16. Chart showing results of pairing 20 single basidiospore isolates of £>. radicata (KJM-390) in all combinations ... 44
17. Bipolar pattern of sexuality of j5. radicata (KJM-390) rearranged into mating type groups 45
18. Hiase contrast photomicrograph showing chlamydospores of _S. radicata (KJM-389) produced in Nobles agar at 25C in the dark six weeks after inoculation, (X 400) ... 46
19. Chart showing multiple alleles for incompatibility by pairing 36 homokaryotic isolates representing the two mating types from each of 18 basidiocarps of S. radicata collected in Arizona 48
20. Photograph showing decay caused by S. radicata (JH) in the butt log of a 48" DBH Douglas fir, (X 0.07) .... 53
21. Riotographs showing decay caused by £!. radicata (KJM-389) in the roots of Douglas fir 54
22. Ihotograph showing growth characters on Nobles agar at 25C in the dark of southeastern United States isolates labeled crispa . 64
23. Camera lucida drawings of hyphae of the advancing zone, submerged mycelium, and aerial mycelium of southeastern United States isolates labeled SI. crispa (RLG-3860) . 66 ix
LIST OF ILLUSTRATIONS—Contlnued
Figure Plage
24. Hiotograph showing comparison of growth on Nobles agar at four weeks of £. radicata from Arizona and isolates labeled j>. crlspa from Japan, Germany, and southeastern United States 69 LIST OF TABLES
Table Page
1. Mating type factors found in 18 basidiocarps of S. radicata collected in Arizona ...... 49
2. Growth (mm)* of Sparassis radicata nycelium on 3.0% MIA at various temperatures after 21 days incubation 51
3. Weight loss of Douglas fir and ponderosa pine wood blocks decayed for 24 weeks by Sparassis radicata 57
4. Results of analysis of 50 fallen Douglas fir trees with a brown cubical root and butt rot 58
5. Comparison of cultural characters of jS. radicata and isolates labeled jS. crispa 68
x ABSTRACT
Sparassls radlcata Weir, a basidiomycete currently placed in the Sparassidaceae (Aphyllophorales), was found to be heterothallic with a bipolar type of mating system. Multiple alleles for incompati bility occur at the locus for heterothallism. Tests were negative for the production of extracellular oxidases on gallic or tannic acid media and with gum guaiac solution. The optimum temperature for growth on
3.0% malt extract-agar medium was 23-25C. The thermal death point was approximately 40C. Descriptions of basidiocarps, cultural characters, and the decay are given.
A modified method of Koch's postulates, using agar-block decay tests, proved j>. radicata is capable of causing a brown cubical rot in ponderosa pine (Pinus ponderosa Laws) and Douglas fir (Pseudotsuga men- ziesii (Mirb.) Franco). These tests also showed that both dikaryotic and homokaryotic isolates can decay Douglas fir and ponderosa pine wood.
An incidence study shows that Si. radicata is an important cause of brown cubical root and butt rot of Douglas fir in the Santa Catalina
Mountains of southern Arizona. The failure of 30% of 50 Douglas fir trees was attributed to j3. radicata. 62% to Polyporus schweinitzii Fr., and 8% to both fungus species together. A genetic analysis of mating type factors from 18 basidiocarps gave evidence that j>. radicata moves from diseased to healthy trees through root grafts or root contacts.
xi xii
Cultural studies with Isolates labeled Sparassls crlspa Wulf.
ex Fr. from Europe and Japan and an Isolate labeled Sparassls lamlnosa
Fr. from England show they are extremely similar to S. radicata from
Arizona. Isolates labeled jS. crispa from southeastern United States
differed culturally from the European and Japanese isolates labeled
S. crispa. Apparent dikaryotization of homokaryotic isolates of £5. radicata from Arizona by dikaryotic isolates labeled crispa from
Europe and Japan and jj>. laminosa from England offers additional evidence for the conspecificity of these fungi. xii
Cultural studies with Isolates labeled Sparassls crlspa Wulf. ex Fr. from Europe and Japan and an Isolate labeled Sparassls lamlnosa
Fr. from England show they are extremely similar to S. radlcata from
Arizona. Isolates labeled jS. crlspa from southeastern United States differed culturally from the European and Japanese isolates labeled j>. crlspa. Apparent dikaryotization of homokaryotic isolates of jS. radicata from Arizona by dikaryotic isolates labeled S. crispa from
Europe and Japan and Si. lamlnosa from England offers additional evidence for the conspecificity of these fungi. INTRODUCTION
Sparassis radicata Weir, a basidiomycete currently placed in the family Sparassidaceae (Aphyllophorales), is potentially an impor tant cause of root and butt rot in western conifers. The fungus was described by James R. Weir in 1917, and has been reported in several host-fungus indices as occurring on coniferous trees. The incidence of _S. radicata is not known in any coniferous forest regions, but it may be as significant in causing root and butt rots as Poria weirii
(Murr.) Murr., Fomes annosus (Fr.) Cke., Armillaria mellea Vahl ex Fr.,
Polyporus tomentosus Fr., and Polyporus schweinitzii Fr.
Coniferous forests are the most important source of lumber in
Arizona. Although Arizona is not generally thought of as a forest state, it ranks 13th among the 50 states and 4th among the Rocky Moun tain States in forest area. TWenty-eight percent of the total land area of the state is forest land, occupying 20.6 million acres. Four million acres are classed as commercial. The total sawtimber volume is 27 billion board feet, of which 99% comes from conifer species (Shupe
1965). The annual cut is approximately 300 million board feet. These forests also have a high aesthetic value, especially in recreational areas, and play an important ecological role in watershed protection.
Sparassis radicata occurs on ponderosa pine (Pinus ponderosa
Laws) and Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) in Arizona.
1 2
These are the two major timber producing species in the state (Miller
1964). It has also been collected on white fir (Abies concolor (Gord.
et Glend.) Lindl.) and southwestern white pine (Pinus strobiformis
Engelm.)» both harvested for lumber but on a smaller scale than pon- derosa pine and Douglas fir. All of these tree species are also impor tant for aesthetic value in higher elevation recreational areas in
Arizona.
Root and butt rots of Arizona's coniferous forest species are important because they predispose trees to windthrow and the valuable butt log is destroyed. Large trees weakened with root and butt rots also are potential hazards in recreation areas. Research in hazard evaluation and control has increased greatly with increased use of pub lic recreation areas (Plaine 1971, 1973).
There are no economically feasible ways to control most forest root and butt diseases. More research in the development of practical control measures is needed because of increasing needs for timber and the need to decrease the dangers of falling trees in recreation and summer home areas. Before any such measures can be developed and prac ticed, the biology of the causal organisms and a knowledge of disease cycles must be understood.
Weir (1917) studied a few aspects of the life history of £>. radi- cata. but no other studies have been carried out on the fungus and most of its biology and disease cycle is unknown. My project was initiated to disclose some aspects of the biology of S. radicata and to assess its importance as a cause of decay and windthrow of coniferous trees in southern Arizona.
A review of literature on Sparassis revealed that some European and Japanese specimens and cultures referred to as Sparassis crispa
Wulf. ex Fr. and Sparassis laminosa Fr. are extremely similar to those of j>. radicata from Arizona. These specimens and cultures also differ from those labeled S. crispa from southeastern United States. I felt it was necessary to compare these fungi with S. radicata to determine their taxonomic relationships.
The objectives of this study were:
1. To make a cultural study of .S. radicata to determine its
cultural morphology, types of extracellular enzyme systems,
temperature relations, and stability of the dikaryon.
2. To determine the sexuality of j>. radicata.
3. To determine the relative decay capabilities of homokaryotic
and dikaryotie mycelium of £>. radicata.
4. To determine if S. radicata parasitizes conifer roots or if
it is a perthophyte.
5. To assess the incidence of .S. radicata on Douglas fir in
the Santa Catalina Mountains.
6. To work out the disease cycle of radicata.
7. To compare basidiocarps and cultural characters of specimens
labeled S. crispa and S. laminosa with those of S. radicata. LITERATURE REVIEW
The genus Sparassis was established by Fries (1821) to include hymenomycetes having a fleshy receptacle with many smooth, flat, dilat ed, anastomosing branches, white spore print, elongated basidia, and an amphigenous hymenium. On the basis of these characters, Fries placed
Sparassis in the family Clavariaceae. After Fries defined the genus, several workers noted an irregularity in the position of the hymenitan and consequently Sparassis has been variously classified.
Quelet (1886) recognized two families based on the position of the hymenial surface. One family, the Clavariaceae, was characterized by a vertical hymenial surface. Sparassis was placed in the second fam ily, the Thelephoraceae, characterized by a horizontal hymenial surface.
Batouillard first (1887) placed Sparassis in the Thelephoraceae but later (1900) in the Clavariaceae. In 1887 Eatouillard did not mention the hymenial development, but in 1900 he described the hymenium of Spa rassis as amphigenous.
Maire (1902) separated Sparassis from the Clavariaceae and made it the type genus of a new family, the Sparassideae. Maire1s classifi cation was used by Lotsy (1907).
Cotton (1911) studied Sparassis. and concluded that the genus was closely allied to Stereum of the Thelephoraceae. He found that the hymenium was on the underside of the exterior free horizontal lobes and
4 5
that it covered both surfaces only in and around the center of the ba-
sidiocarp. He proposed that Sparassis be transferred to the Thelepho-
raceae. Cotton's view was supported by both Lloyd (1913, 1920) and
Buller (1922).
Weir (1917) studied young specimens of jS. crispa and j>. radicata
and found that the hymenium developed more or less on all free surfaces.
He stated that a unilateral hymenium was more pronounced towards the
periphery of the basidiocarp, probably influenced by gravity. Weir
believed that before any transfer of Sparassis from the Clavariaceae
should be accepted, the hymenial development and the permanency of the
unilateral structure needed to be studied.
Kuntze (1891) considered the name Sparassis to be an orthograph ic homonym of Sparaxis Ker-Gawl, a genus of flowering plants in the
Iridaceae. He proposed the generic name Masseeola to replace the name
Sparassis Fr. per Fr.
Donk (1964) placed Sparassis close to the Clavariaceae as a distinct family, the Sparassidaceae Herter. Sparassis is now the only genus in the Sparassidaceae. The following description of the family is from Donk (1964, p. 291).
Fruitbody stalked, branching into a few, or a great mass of, flattened, more or less wavy lobes bearing the hymenium at the physiological underside in horizontally orientated portions (ab- hymenial surface not covered by a crust-like layer) or amphigen- ously in ascending lobes; context tough-fleshy, shrinking and becoming somewhat cartilaginous upon drying, pallid, monomitic; hymenophore smooth; hymenium not becoming layered.
Hyphae thin-walled with a tendency to become firm- to thick-walled; clamps present (scarce to numerous, may occur only at the base of the basidia); also vascular hyphae wnich may end in the hymenium. Basidia club-shaped, chiastic, 2-4 8pored. Spores short-ovoid, ellipsoid, rather small (5-9 ji long), colourless (pale cream or pale ochraceous-cream in a a print; wall smooth, non-amyloid.
Lignicolous (stalk rooting) or humicolous (stalk aris ing from a corticioid patch covering vegetable debris).
There are nine species of Sparassis listed in Saccardo's Sylloge
Fungorum and the Index of Fungi: j3. crispa (Wulf.) Fr., j>. laminosa Fr., j>. spathulata (Schw.) Fr., j>. tremelloides Berk., j>. simplex D. A. Reid,
_S. foliacea St. Amans, _S. herbstii Pk., S. radicata Weir, and JS. kazach- stanica Schwarzman. Sparassis crispa is reported from Europe, Russia,
Japan, and the eastern USA, J3. laminosa from Europe and Russia, £>. foli acea from France, £. simplex from England, and j3. kazachstanica from
Russia. Sparassis spathulata. j>. tremelloides. and S. herbstii are east ern USA species, and j>. radicata is found in western North America.
Several reports indicate that some of these species may be syn onyms. Cotton (1911) states that j3. foliacea is probably synonymous with j3. laminosa and that £5. tremelloides is not a Sparassis. Lloyd
(1913) lists _S. laminosa. SI. spathulata. j>. herbstii. and S>. foliacea as synonyms, and Coker (1921) states that S. spathulata is probably not different from j>. herbstii. Reid (1958) points out that jS. laminosa is possibly only a variety of jS. crispa with a lax growth form. He also points out that the combination of S>. crispa var. laminosa was made by
Quelet in 1886. Further evidence that jj>. laminosa is possibly only a variety of jS. crispa is in a photograph by Buller (1922) of a Sparassis fruiting body showing one side with the characters of j5. crispa and the other side those of S. laminosa. 7
Weir (1917) first described Sparassis radicata. He stated that the chief difference from other species in the genus was, "...in the thinness of its lobes and by its very pronounced perennial sclerotioid rootstalk from which the sporophore develops annually (p. 169)." The type specimen was collected in the Priest River Valley, Idaho. Weir also discovered that the fungus was associated with a decay of roots of conifers in the northwestern United States.
In his investigations on decay, Weir (1917) found yellowish- white mycelial fans under the bark of infected roots. Small rhizomorphs developed from the border of this mycelium after the root had been killed. Hie rhizomorphs penetrated the sapwood and produced a brown or yellowish, laminated, carbonizing rot. The medullary rays were decayed first. Small, narrowly elongated pits, not lined with mycelium, devel oped from shrinkage of the decayed sapwood. Small brown rhizomorphs occasionally developed in the decayed wood either parallel to the tra- cheids or in zigzag lines. The incipient decay was preceded by a red dish discoloration and the boundary of the decayed wood was sometimes marked by a black line. The heartwood was later invaded but was not decayed uniformly. Elongated pits filled with white mycelium formed in different parts of the wood. The pits anastomosed such that long pieces of partially decayed or solid wood could be removed. The decay in the heartwood was brittle but could not be rubbed into a fine powder. The completely decayed root shrank from the bark and the space was filled with a thick mycelial felt. The rootstalk developed from the mycelial felt. Weir concluded that the decay was confined to the roots. The host relationships and geographical distribution of _S. radi- cata reported in the literature are as follows:
BALSAM FIR (Abies balsamea (L.) Mill.)
Locality not given (Seymour 1929)
DOUGLAS FIR (Pseudotsuga menziesii (Mirb.) Franco
Arizona (Gilbertson, Martin, and Lindsey 1974)
British Columbia (Lowe 1969)
Locality not given (Seymour 1929)
Northwest North America (Weir 1917)
Pacific Northwest (Anonymous 1960, Shaw 1973)
ENGELMANN SPRUCE (Picea engelmannii Parry)
Idaho (Anonymous 1960)
Locality not given (Seymour 1929)
Montana (Anonymous 1960)
Northwest North America (Weir 1917)
Oregon (Anonymous 1960)
Pacific Northwest (Shaw 1973)
Washington (Anonymous 1960)
FRASER FIR (Abies fraseri (Pursh) Poir.)
Locality not given (Seymour 1929)
GBAND FIR (Abies grandis (Dougl.) Lind.)
Locality not given (Seymour 1929)
PONDEROSA PINE (Pinus ponderosa Laws)
Arizona (Gilbertson 1974, Gilbertson jet al. 1974) 9
SOUTHWESTERN WHITE PINE (Plnus strobiform!s Engelm.)
Arizona (Gilbertson _et al. 1974)
WESTERN LARCH (Larix occidentalis Nutt.)
Locality not given (Seymour 1929)
Montana (Anonymous 1960)
Northwest North America (Weir 1917)
Oregon (Anonymous 1960)
Pacific Northwest (Shaw 1973)
Washington (Anonymous 1960)
WESTERN WHITE PINE (Pinus monticola D Don)
Locality not given (Seymour 1929)
Montana (Anonymous 1960)
Northwest North America (Weir 1917)
Oregon (Anonymous 1960)
Pacific Northwest (Shaw 1973)
Washington (Anonymous 1960)
WHITE FIR (Abies concolor (Gord. et Glend.) Lindl.)
Arizona (Gilbertson ej: al. 1974)
Locality not given (Seymour 1929)
These records are not all authenticated and include some dubious hosts. Balsam fir and Fraser fir are eastern North America species and no authenticated specimens of jS. radicata have been collected in this area. MATERIALS AND METHODS
Hie Arizona isolates of radicata referred to in the text were from basidiocarps collected primarily during the rainy season in July,
August, and September. Polysporous, single basidiospore, and tissue isolates were obtained from fresh basidiocarps. The basidiocarps were then dried in a forced air dryer and perserved for future reference in the tfycological and Forest Pathology Herbarium at The University of
Arizona. Isolates labeled j3. crispa from Japan, England, and the south eastern United States, an isolate labeled £>. laminosa from England, and some isolates of S. radicata from outside Arizona were obtained from the Forest Products Laboratory, U. S. Forest Service, Madison, Wiscon sin. Isolates labeled S. crispa from Germany were obtained from Dr. R.
Siepmann, Biologische Bendesanstalt, Institut fur Forstpflanzenkrank- heiten, 351 Hann. Miinden, Germany.
The isolates and specimens of JS. radicata are as follows:
JH - Polysporous isolate, basidiocarp on fallen Douglas fir,
Mt. Bigelow, Santa Catalina Mts., Coronado Nat. Forest,
Pima County, Ariz., Coll. Jeff Hubert, Sept. 6, 1971.
RLG-8240 - Polysporous isolate, basidiocarp at base of white
fir, Tub Spring, Chiricahua Mts., Coronado Nat. Forest,
Cochise County, Ariz., Coll. R. L. Gilbertson, Aug. 27,
1968. KJM-274 - Polysporous isolate, basldlocarp at base of living
Douglas fir, Bear Wallow, Santa Catalina Mts., Coll. K. J
Martin, Aug. 11, 1972.
KJM-279 - Polysporous isolate, basldlocarp at base of stump of
ponderosa pine, Mt. Bigelow, Santa Catalina Mts., Coll. K
J. Martin, Aug. 31, 1972.
KJM-389 - Polysporous isolate, basldlocarp at base of living
Douglas fir, Mt. Bigelow, Santa Catalina Mts., Coll. K. J
Martin, Aug. 9, 1973.
KJM-390 - Polysporous isolate, basidiocarp at base of living
Douglas fir, Mt. Bigelow, Santa Catalina Mts., Coll. K. J
Martin, Aug. 9, 1973.
KJM-391 - Polysporous isolate, basidiocarp at base of living
Douglas fir, Mt. Lemmon, Santa Catalina Mts., Coll. K. J.
Martin, Aug. 9, 1973.
KJM-392 - Polysporous isolate, basidiocarp at base of fallen
Douglas fir, Bear Wallow, Santa Catalina Mts., Coll. K. J
. Martin, Aug. 11, 1973.
KJM-450 - Polysporous isolate, basidiocarp at base of living
ponderosa pine, V. T. Lake, Kaibab Plateau, Kaibab Nat.
Forest, Coconino County, Ariz., Coll. K. J. Martin, Aug.
20, 1973.
KJM-461 - Polysporous isolate, basidiocarp at base of living
Douglas fir, Shannon Park, Ptnaleno Mts., Coronado Nat. Forest, Graham County, Ariz., Coll. K. J. Martin, Aug.
31, 1973.
KJM-463 A and B - Folysporous isolates, basidiocarps at base of
living Douglas fir, Treasure Park, Pinaleno Mts., Coll.
K. J. Martin, Aug. 31, 1973.
KJM-467 - Polysporous isolate, basidiocarp growing out of top
of a Douglas fir stump, Hospital Flat, Pinaleno Mts.,
Coll. K. J. Martin, Aug. 31, 1973.
KJM-468 - Polysporous isolate, basidiocarp at base of living
Douglas fir, Moonshine Creek, Pinaleno Mts., Coll. K. J.
Martin, Aug. 31, 1973.
KJM-469 A, B, C, D, E - Polysporous isolates, basidiocarps at
base of living Douglas fir, Moonshine Creek, Pinaleno
Mts., Coll. K. J. Martin, Aug. 31, 1973.
KJM-486 - Tissue isolate, basidiocarp at base of living Douglas
fir, Bear Wallow, Santa Catalina Mts., Coll. K. J. Martin,
Nov. 6, 1973.
JPL-187 - Polysporous isolate, basidiocarp at base of stump of
Douglas fir, Mt. Bigelow, Santa Catalina Mts., Coll. J. P.
Lindsey, Aug. 9, 1973.
JPL-188 - Polysporous isolate, basidiocarp at base of living
Douglas fir, Mt. Bigelow, Santa Catalina Mts., Coll. J. P.
Lindsey, Aug. 9, 1973.
JEL-190 - Polysporous isolate, basidiocarp at base of living
Douglas fir, Mt. Lemmon, Santa Catalina Mts., Coll. J. P.
Lindsey, Aug. 9, 1973. JFL-202 - Polysporous isolate, basidiocarp at base of living
Douglas fir, Mt. Lemmon, Santa Catalina Mts., Coll. J. P.
Lindsey, Aug. 12, 1973.
OKM-4756 - Polysporous isolate, basidiocarp on ground, Priest
Lake area, Kaniksu Nat. Forest, Idaho, Coll. A. H. Smith,
det. and isol. by 0. K. Miller Jr., Sept. 24, 1966.
OKM-4817 - Tissue isolate, basidiocarp on ground, Priest River
Expt. Forest, Kaniksu Nat. Forest, Idaho, Coll. 0. K. Mil
ler Jr., Sept. 29, 1966.
FP133458 x, 2, 4, 5, 6, 9, 10 - Polysporous isolates, basidio
carp at base of Douglas fir stump, Saddleback Mt., Lincoln
County, Oregon, Coll. M. J. Larsen, Nov. 8, 1972.
FP133489 - Polysporous isolate, basidiocarp at base of Douglas
fir stump, Santiam Y, Willamette Nat. Forest, Oregon, Coll.
M. J. Larsen, Nov. 13, 1972.
JLL-9722 - Basidiocarp on pine, Rustler Park, Chiricahua Mts.,
Coll. J. L. Lowe, Sept. 6, 1958.
ABB-1190 - Basidiocarp on Douglas fir, Eldon Mt. Rd., San Fran
cisco Peaks, Coconino Nat. Forest, Coconino County, Ariz.,
Coll. A. B. Budington, Aug. 8, 1969.
RLG-9207 - Basidiocarp at base of living Douglas fir, Bear Wal
low, Santa Catalina Mts., Coll. R. L. Gilbertson, Sept. 16,
1969. KJM-474 - Basidiocarp at base of living southwestern white pine,
Riggs Flat, Pinaleno Mts., Coll. K. J. Martin, Aug. 31,
1973.
RLG-8020 - Basidiocarp on Douglas fir, Fir Camp Ground, Sacra
mento Mts., Lincoln Nat. Forest, Otero County, New Mex.,
Coll. R. L. Gilbertson, Aug. 27, 1968.
6430 - Basidiocarp in duff in Bishop pine (Pinus muricata Don)
woods, Mt. Vision Trail, Inverness, Marin County, Calif.,
Coll. T. T. McCabe, Jan. 27, 1941.
Hie isolates and specimens labeled _S. crispa are as follows:
Splc - Polysporous isolate, received at BFDL from K. Aoshima
from Japan in 1960, no collection data.
JL-181 - Isolated from a basidiocarp on underside of stump of
overturned oak tree, near airport, Beltsville Expt. Forest,
Laurel, Md., Coll. John Lindsay, July 16, 1968.
JL-221 - Polysporous isolate, basidiocarp in duff and oak litter
near dead oak, Cornell Rd., Beltsville Expt. Forest, Laurel
Md.', Coll. John Lindsay, Aug. 8, 1968.
JL-240 - Polysporous isolate, basidiocarp at base of pine stump,
Cornell Rd., Coll. John Lindsay, Aug. 29, 1968.
0KM-3673 - Polysporous isolate, basidiocarp on ground under
Virginia pine (Pinus virginiana Mill.). Beltsville Expt.
Forest, Laurel, Md., Coll. 0. K. Miller Jr., Sept. 30,
1965. 15
OKM-6860 - Bolysporous isolate, basidiocarp under Virginia pine,
ARS Airstrip, Beltsville Expt. Forest, Coll. 0. K. Miller
Jr., Sept. 12, 1968.
RLG-3860 - Polysporous isolate, basidiocarp on pine stump, Bent
Creek Expt. Forest, Asheville, N. C., Coll. R. L. Gilbert-
son, July 31, 1963.
FP-105840 - Basidiocarp tissue isolate, basidiocarp on ground
under pine, Sand Hills State Forest, Patrick, S. C., Coll.
R. P. Broomall, Aug. 13, 1963.
FPRL-312A - Basidiocarp tissue isolate, basidiocarp on Corsican
pine (Pinus nigra calabrica). reed, at Forest Products
Res. Lab., Princes Risborough, Aylesbury, Bucks, England,
1945, reed at BFDL from John Savory, April, 1960.
S265 - Isolated from pine roots, Neidersachsen, Germany, Coll.
R. Siepmann, 1968.
S211 - Isolated from a basidiocarp, Hann. Munden, Germany, Coll.
Zycha, 1952.
Hie following isolate labeled £. laminosa was used:
CBS-Wy-212 - Wilkins isolate, reed, at BFDL from Wyeth Lab. Inc.
in April, 1965, no collection data.
The medium used to maintain cultures and to grow inoculum of the
Sparassis species was 3.0% malt extract-agar (MEA) prepared according to the following formula:
Difco-Bacto Agar 15 g
Difco-Bacto Malt Extract 30 g
Distilled Water 1000 ml 16
Folysporous isolates were made in test tubes on 3.0% MEA slants.
A piece of fresh basidiocarp, 1 cm^, was attached to the inside glass surface directly over the slant, and held in place with a piece of me dium taken from the tip of the slant. The test tube was then positioned so the basidiospores fell onto the slant surface. When a spore print appeared, it was transferred to another MEA slant and incubated at 25C in the dark. Tissue isolates were made from the interior tissue of fresh basidiocarps. Cuts were made to expose this tissue with a sterile scapel. Small pieces were placed on 3.0% MEA slants, and incubated in the dark at 25C. The method for obtaining single basidiospore isolates is described later in the text under the mating system test.
Mycelium from cultures was examined microscopically by mounting small pieces in a drop of 2.0% aqueous potassium hydroxide (KOH) mixed with a drop of 2.0% aqueous phloxine. Free hand sections of basidio carps were examined in KOH-phloxine mixture or in Meltzer's reagent.
Drawings of microscopic characters were made with the use of a camera lucida.
Studies on Sparassis radicata
Cultural Morphology
The method of studying the cultural morphology follows Nobles
(1965). Dikaryotic inoculum of all isolates of S. radicata was trans ferred to the edge of 90 mm glass petri dishes containing 30 ml Nobles agar. Nobles agar consists of 12.5 g Difco-Bacto Malt Extract, 20 g
Difco-Bacto Agar, and 1000 ml distilled water. Three dishes were 17 prepared for each isolate and incubated in the dark at 25C. The morpho logical characters were examined weekly for six weeks. These included the form of both aerial and submerged hyphae of the advancing zone and of the older mat, the rate of growth (expressed as the number of weeks required for the fungus to grow across the agar), the color, topogra phy, and texture of the mat, ability to form fruiting bodies, reverse
(color changes in the agar induced by growth of the fungus), and odor.
Oxidase Reactions
The presence or absence of extracellular polyphenol oxidative enzymes was determined for dikaryotic and homokaryotic isolates on gallic and tannic acid media (Davidson, Campbell, and Blaisdell 1938) and with gum guaiac solution (Nobles 1958). Gallic and tannic acid media are prepared by adding 15 g Difco-Bacto Malt Extract and 20 g
Difco-Bacto Agar to 850 ml distilled water. One hundred fifty ml distilled water is placed in a separate flask. Both are autoclaved for
20 min at 15 lbs psi. While the 150 ml of sterilized water is still hot, 5 g of gallic (or tannic) acid is dissolved in it. This solution is added to the slightly cooled malt extract-agar, thoroughly mixed, and poured directly into sterile petri dishes. Inoculum from each iso late was transferred to the center of 90 mm glass petri dishes contain ing gallic or tannic acid media. After one week at 25C the presence and intensity of a diffusion zone, indicating the formation of extra cellular oxidases, or the absence of a diffusion zone, indicating the lack of extracellular oxidases was recorded. The gum guaiac test was carried out by dropping a 5.0% alcoholic solution of guaiacum (0.5 g 18 gum guaiac in 30 ml 95.0% ethanol (ETOH)) on the mycelial mat on Nobles agar. A blue color indicated the presence of extracellular oxidases
(laccase or tyrosinase). No color change indicated absence of extra cellular oxidases.
Temperature Relations
The optimum temperature for growth and the thermal death point were determined for dikaryotic and homokaryotic isolates of j>. radicata.
Dikaryotic isolates of KJM-274, KJM-279, KJM-389, KJM-391, KJM-450,
KJM-467, and RLG-8240 and homokaryotic isolates representing the two mating types of KJM-274, KJM-389, KJM-391, KJM-450, and KJM-467 were used. Glass 90 mm petri dishes, nearly filled with 3.0% MEA, were inoc ulated in the center and placed in an incubator at 25C in the dark until growth was visible with a 10X hand lens. Replicates were then incubated at 4, 9, 18, 20, 23, 25, 28, 30, 35, 38, 40, 42, and 45C. Growth was measured from the edge of the advancing zone to the edge of the inoculum at the end of 21 days. Measurements represent averages of five repli cations for each isolate. The thermal death point was determined by transferring the isolates showing no growth to fresh 3.0% MEA and re-incubating at 25C for four weeks. Failure to grow indicated that the fungus had died.
Basidiospore Germination and Ifycelial Development
Germination of basidiospores at 25C under 12 hr alternating periods of dark and 85 ft-c illumination was observed during 120 hr after spore prints were made to obtain polysporous isolates. Small 19 pieces of agar with the spore print were taken at 12-24 hr intervals and examined microscopically. Camera lucida drawings were made and pho tomicrographs taken of the germinating basidiospores.
Tests were made to determine if basidiospores would germinate in free water. Sterile distilled water and non-sterile distilled water were used. Loops of sterile or non-sterile distilled water were placed on a spore print resulting in a drop of suspended basidiospores. Loops were taken from the drops of spore suspension and placed in test tubes containing 10 ml sterile or non-sterile distilled water. The basidio spores were examined at 12-24 hr periods for two weeks.
The morphology of mycelium from single basidiospores and from polysporous isolates was examined microscopically to determine the type of septation. The presence of simple septa indicated that each cell was homokaryotic, and the presence of clamp connections that each cell was dikaryotic.
Mating System
Pieces of fresh basidiocarps, 1 cm^, were cut from the lobes and placed on 3.0% MEA slants with the hymenium facing the glass oppo site the slant. Spore prints formed in 6-12 hr. Several loops of ster ile distilled water were placed on a spore print to obtain a drop with suspended spores. A loop of this spore suspension was nonquantitatively diluted in a tube containing 5 ml sterile distilled water. A loop from the dilution tube was put in another tube containing 10 ml of melted
3.0% MEA just above the gelling temperature at 45C. The spores were 20
dispersed in the agar by twirling the tube, and the medium with sus
pended spores was poured into a 90 mm plastic petri dish. The dishes
thus prepared were incubated in the dark at 25C. Mycelial mats from
individual germinating basidiospores were allowed to grow until they were
visible to the naked eye as small white spots. Single spots of myce
lium were transferred to tubes of slanted 3.0% MEA. After 6-7 weeks
at 25C the hyphae were examined to determine if they were homokaryons.
The absence of clamp connections was taken as confirmation of the homo-
karyotic character.
Twenty-three homokaryotic isolates of KJM-274 were retrieved
and paired in all combinations, a total of 253 pairings, in 90 mm
plastic petri dishes nearly filled with 3.07. MEA. The inocula for
each pairing were placed 2-5 mm apart in the center of each dish.
ftfenty homokaryotic isolates of KJM-390 were paired in the same manner.
All matings were incubated in the dark at 25C. After 5-6 weeks, hyphae from the interface of each pairing were examined. Compatible matings showing clamp connections were marked (+) and incompatible matings showing only simple septa were marked (-).
Test for Multiple Alleles for Incompatibility
Hie two mating types of isolates KJM-274 and KJM-390 were re
trieved from the mating system test. The two mating types of isolates
KJM-389, KJM-391, KJM-392, KJM-450, KJM-461, KJM-463 A and B, KJM-467,
KJM-468, KJM-469 A, B, C, D, E, JFL-187, and JPL-190 were retrieved as follows: Ten homokaryons were obtained from each isolate using the same method as that for obtaining single basidiospore isolates. One 21 homokaryon was designated as representing one mating type. The other nine were mated with it. The second mating type was selected from a compatible mating of these pairings. A total of 36 homokaryons were obtained from the 18 basidiocarp collections.
To determine if any alleles for incompatibility were shared by the 18 collections, the 36 homokaryons were mated in all conbinations in 60 mm plastic petri dishes nearly filled with 3.0% MEA. A total of
612 pairings were made. After 6-7 weeks, the matings were examined for clamp connections. Positive matings (+) indicated that no alleles were shared and negative matings (-) that an allele was shared.
Decay Studies
The first studies of the decay caused by J3. radicata were of the roots of living trees on which basidiocarps occurred. The roots were exposed, dissected, and the decay examined. Increment borings were taken from the butt to determine the extent of decay above the ground. Dead snags, stumps, and logs of fallen trees with basidiocarps of the fungus were also examined.
Isolations were made from the margin of decay to recover myce lium of jS. radicata. Small pieces of wood taken from the margin were briefly flamed, placed on 3.0% MEA slants, and incubated at 25C in the dark. All isolates obtained from rot were grown in pure culture. The characters of these isolates were then compared with the cultural mor phology previously determined for JS. radicata.
To confirm field observations on the type of rot caused by S. radicata. a series of agar-block decay tests were made. These tests also determined the relative decay capability of homokaryotic and di-
karyotic mycelium.
Test blocks measuring 5 x 2 x 2 cm were cut from sapwood slabs
of ponderosa pine and Douglas fir. The blocks were numbered with a
soft pencil, dried at 105C for 24 hr, and placed in a dessicator for
24 hr to equilibrate their weight. Each block was then weighed to the
nearest hundredth of a gram. After soaking in water for 24 hr, the
blocks were placed on paper toweling for six hr to drain off excess
water, and autoclaved for 30 min at 15 psi. The blocks were then
placed, with the transverse cut surface up, on V-shaped glass rods on
slanted 3.0% MIA in 8 oz French square bottles.
Four dikaryotic isolates (RLG-8240, KJM-274, KJM-279, and JH) and homokaryotic single basidiospore isolates representing the two mating types of KJM-274 were used in the decay test. Inoculum from
each isolate was placed on the medium near the wood block and the cham
bers incubated for 24 weeks at 25C in the dark. Five chambers were
prepared for each isolate for both ponderosa pine and Douglas fir blocks, and five noninoculated control chambers were prepared for each of the two types of wood. After 24 weeks the blocks were removed from the chambers and the surface mycelium was carefully removed with a soft nylon brush. The blocks were oven dried at 105C for 24 hr and placed in a dessicator for 24 hr. The blocks were then weighed, and the per cent weight loss, based on original oven dry weight, was used as the criterion for the decay capability of each isolate. After the percent weight loss was determined, the blocks were broken open, and the decay compared with the type of decay found in nature. Incidence of £>. radicata
After the decay caused by jS. radicata could be recognized, the incidence of the fungus on Douglas fir was studied. These studies were made in Bear Wallow, Mt. Bigelow, Mt. Lemmon, Summerhaven, and
Marshall Gulch of the Santa Catalina Mountains. Fifty fallen Douglas fir trees that had a brown rot of roots and butts were examined. Only trees were counted that had fallen so recently that secondary organisms had not become established to a great extent. Hie diameter breast high
(DBH) of each tree, the presence or absence of basidiocarps, the way in which the trees had broken, the decay and mycelial characteristics, and the aspect of the trees were noted. Isolations were attempted from all trees suspected of being decayed by j3. radicata.
Studies with Cultures Labeled J3. crispa and S. laminosa
The method of studying the cultural morphology, and the type of extracellular oxidative enzymes was the same as that described for
_S. radicata. The method of studying the temperature relations was also the same except measurements were taken at one week for the southeastern
United States isolates labeled j3. crispa. All of the previously listed isolates labeled S. crispa and laminosa were used.
Di-mon Matings
A series of dikaryotic x homokaryotic (Di-mon) matings were made with homokaryotic isolates of j>. radicata and dikaryotic isolates labeled S. crispa and £. laminosa. Inoculum of each homokaryotic iso late of S. radicata representing the two mating types of KJM-274, 24
KJM-390, KJM-468, and KJM-450 were individually placed at the agar mar
gin in three 90 mm glass petri dishes containing 3.0% MEA. These cul
tures grew at 25C in the dark until the mycelium had covered half of
the medium surface. Inocula from dikaryotic isolates labeled S. crispa
(S265, S211, Splc, FPRL-312A, and RLG-3860), and S. lamlnosa (CBS-Wy-212)
were transferred individually to the medium 5 ran from the advancing
edge of each homokaryon of S. radicata. The matings were incubated at
25C in the dark. After the isolates met, hyphae in the advancing zone
of the original homokaryotic mats were examined periodically for clamp
connections. The presence of clamp connections on original homokaryotic mycelium indicated that dikaryotization of the homokaryons possibly oc curred. RESULTS
The basidiocarps of j3. radicata (Figure 1) were usually found
on the ground within a six foot radius from the base of living trees.
Figure 2 shows a basidiocarp of the fungus as it is commonly found asso
ciated with a living tree. All basidiocarps under living trees were
connected to the roots by an elongated, underground pseudosclerotium
(Figure 3). Of four basidiocarps not associated with living trees,
two were found at the base of stumps, one on the top of a stump, and
one on the top side of a log. Those growing directly out of wood were
not associated with a pseudosclerotium.
Morphology of Basidiocarps
Based on the specimens previously listed, the following descrip
tion of S. radicata is given:
Basidiocarps annual, arising from a perennial, elongated, under ground pseudosclerotium as a rounded mass of many anastomosing and sub divided horizontal and vertical petaloid branches with thin, wavy margins; sometimes growing directly out of wood and lacking a pseudo sclerotium; becoming quite large, mostly 10-30 cm in diam, 10-20 cm high; creamish-yellow, fleshy-tough when fresh, drying cartilaginous, creamish-yellow to yellowish-brown; odor strong, becoming -slightly disagreeable; pseudosclerotium a mass of soil and humus particles held together by mycelium, up to 70 cm long and 10 cm in diam, attached to roots of living or dead conifers, odor somewhat like smoke-cured bacon.
25 Figure 1. Riotograph of basldiocarp of j3. radlcata (KJM-274), (X 0.4).
Figure 2. Hiotograph of basldiocarp of _S. radlcata (KJM-274) as usually found associated with living trees. Figure 3. Hiotograph of pseudosclerotlum of _S. radlcata (KJM-274), (X 0.13). 28
Hyphal system monomltic; subhymenial hyphae (Figure 4a) com
pactly arranged and interwoven, thin-walled, 2-8 p in diam with irreg
ularly inflated portions up to 16 fi in diam, not clearly differentiated from contextual hyphae, mostly with clamp connections and with some simple septa; contextual hyphae (Figure 4b) similar, slightly larger,
4-8 jj in diam, irregularly swollen and inflated with swollen portions
up to 20 (i in diam, mostly with clamp connections and with some simple septa, clamp connections sometimes contorted and difficult to discern; aseptate, thin-walled gloeoplerous hyphae (Figure 5a) scattered through out, 6-12 fi in diam, most abundant towards base of basidiocarp; hyphae of the pseudosclerotium compact, of three types; some (Figure 6a) 2-4 ji in diam, thin-walled, contorted, with clamp connections; some (Figure 6b)
6-12 p in diam, thin-walled, with clamp connections, sometimes appear ing aseptate; some (Figure 6c) 12-16 p in diam, thick-walled, the walls highly refractive and the contents staining deeply in KOH-phloxine, with clamp connections and simple septa.
Hymenial layer distinct, mostly amphigenous, sometimes uni lateral depending on position of the branch and age of the basidiocarp; cystidia or other sterile hymenial elements none; basidia (Figure 5b) closely packed, clavate, 4-sterigmate, 40-60 X 4-8 p; basidiospores
(Figure 5c) ellipsoid, hyaline, smooth, negative in Melzer's reagent,
1-guttulate, 5-7 X 3-5 p; spore print white. Figure A. Camera lucida drawings of subhymenial and contextual hyphae in basidiocarps of _S. radicata (KJM-468).— (a) subhymenial hyphae; (b) contextual hyphae. 30
<§> 0 c
10 20
Figure 5. Camera lucida drawings of gloeoplerous hyphae, basidia, and basidiospores in basidiocarps of S. radicata (KJM-468). -- (a) gloeoplerous hyphae; (b) basidia; (c) basidiospores. Figure 6. Camera lucida drawings of three types of hyphae in pseudosclerotium of j>. radicata (KJM-468).— (a) thin- walled, contorted hyphae with clamp connections; (b) thin-walled hyphae, some aseptate, some with clamp connections; (c) thick-walled hyphae with clamp con nections and simple septa. 32
Cultural Studies of jS. radicata
Growth Characters
Growth very slow, radius from edge of inoculum to edge of ad vancing zone 6-12 mm at 14 days, 20-35 mm at 30 days, not covering sur face of agar in 90 mm petri dish at six weeks; margin of advancing zone even, appressed, thin and translucent; aerial mycelium developing sparsely 1-2 mm behind advancing edge, white, thin-floccose; mat devel oping first around inoculum, becoming woolly, thicker portions some times developing in distinct radiating zones, later mounding up in piles around area of inoculum, becoming creamish, convoluted, tough and difficult to separate; small, scattered white mounds sometimes developing between inoculum and advancing zone; mat growing on the wall of the petri dish white, woolly at first, becoming creamish, thick, rubbery, and convoluted; reverse unchanged; odor peppery-cinnamon; no fruiting observed in inverted dishes at the end of six weeks.
Figures 7a, 7b, and 7c show characteristic growth on Nobles agar at
25C in the dark at two weeks, four weeks, and six weeks, respectively.
Microscopic Characters
Hyphae of advancing zone (Figure 8a) 2-4 p in diam, hyaline, thin-walled, with clamp connections, occasionally branched, branches often arising from a clamp, rounded to slightly tapered at tips; aerial mycelium hyaline, of two types; smaller hyphae (Figure 9a) 2-4 p in diam, with clamp connections, thin-walled, frequently branched; larger hyphae (Figure 9b) hyaline, thin- to moderately thick-walled, irregularly Figure 7. Hiotographs showing growth characters of _S. radicata (RLG-8240) on Nobles agar at 25C in the dark.—(a) two weeks; (b) four weeks; (c) six weeks. 20
Figure 8. Camera lucida drawings of hyphae of the advancing zone and submerged mycelium of radicata (KJM-389) on Nobles agar at 25C in the dark four weeks after inocu lation.—(a) hyphae of advancing zone; (b) submerged mycelium. 35
20
Figure 9. Camera lucida drawings of two types of aerial hyphae of j3. radicata (KJM-389) on Nobles agar at 25C in the dark six weeks after inoculation.—(a) smaller, thin- walled, frequently branched hyphae with clamp connec tions; (b) larger, thin- to moderately thick-walled, irregularly swollen hyphae with clamp connections and simple septa. 36 inflated and contorted, mostly 6-10 ji in diam with swellings up to
16 p in diam, mostly with clamp connections and occasional simple septa, frequently ampulate at septa; hyphae of submerged mycelium
(Figure 8b) hyaline, mostly with clamp connections, with occasional simple septa, frequently ampullate at septa, thin- to slightly thick- walled, with occasional to frequent branching, branches frequently arising from a clamp, with spathulate tips (Figure 10) abundant in older areas, with scattered chlamydospores, associated with numerous crystals.
Oxidase Reactions
A diffusion zone was absent on both gallic and tannic acid media for all dikaryotic and homokaryotic isolates of radicata (Figure 11).
The gum guaiac test was negative for all isolates tested (Figure 12).
These tests indicate that j>. radicata does not produce extracellular polyphenol oxidative enzymes. Growth on gallic acid was 3-14 mm from the edge of the inoculum to the edge of the advancing zone at 14 days, and 18-25 mm at 30 days. No isolate grew on tannic acid medium.
Basidiospore Germination and Mycelial Development
Germination of basidiospores of j3. radicata on 3.0% MEA slants at 25C under 12 hr alternating periods of dark and 85 ft-c illumination began between 36 and 48 hr. At 36 hr germinating basidiospores were slightly swollen and at 48 hr (Figure 13a) they had swollen and elon gated to about twice the length and diameter of non-germinating ones.
In some the apiculus had disappeared and vestiges of a germ tube could be detected. At 72 hr (Figure 13b) the germ tubes were 2-4 ji in diam 37
r
A • ^ / * / •'* ;\ r,0 V I
• t i' f v' r •' V <'
, /
Figure 10. Hiase contrast photomicrograph of spathulate swollen tips of hyphae of _S. radicata (KJM-389) on Nobles agar at 25C in the dark six weeks after inoculation, (X 400). 38
Figure 11. Hiotographs showing negative reaction for the production of extracellular oxidase by £>. radicata (KJM-274) on gal lic and tannic acid media after 10 days at 25C in the dark.- - (a) gallic acid medium; (b) tannic acid medium.
r
I
Figure 12. Hiotograph showing negative reaction for the production of extracellular oxidase by radicata (KJM-274) with gum guaiac solution. r i
^ 3
^ Q <3? 0 /P ® (fp
o>
20
Figure 13. Camera lucida drawings of germinating basidiospores of S. radicata (KJM-274) on 3.0% MEA under alternat ing 12 hr periods of darkness and 85 ft-c illumina tion.—(a) 48 hr; (b) 72 hr; (c) 96 hr. 40 and up to 40 jli long. Septa formed in many, and some branching began.
The droplet had moved down the germ tube in most. By 96 hr (Figure 13c) the droplet had disappeared and distinct branching was visible. Ihe hyphae were 2-4 ja in diam with swellings up to 10 p. At 120 hr hyphae could no longer be traced to original basidiospores.
Throughout the 120 hr nongerminating basidiospores and basidio spores in various stages of germination could be seen. Stages of ger mination became less distinct and nongerminating basidiospores became more difficult to find after 96 hr.
Basidiospores did not germinate in either sterile or non-sterile distilled water within two weeks. This suggests that an exogenous nutrient source may be required to stimulate germination of the basidio spores.
Single basidiospore isolates required 10-12 days for growth to become visible as small white spots. The hyphae from single basidio spores showed only simple septa. Clamp connections formed on hyphae in polysporous isolates in 4-9 weeks. Clamp connections on hyphae from polysporous isolates and in basidiocarps, and only simple septa in single basidiospore isolates indicate that jS. radicata is hetero- thallic.
Mating System
The results of pairing 23 randomly numbered single basidiospore isolates of KJM-274 in all possible combinations are shown in Figure 14.
The pattern rearranged into mating type groups is shown in Figure 15. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1 \— — — 4 4- 4 — 4- 4- 4- — 4- 4- + 4- — 4- 4 4- 4- — — 2 — — — 4 + 4 — + 4- 4- — 4- 4- 4- + — 4- 4- 4- 4- — — 3 — —\ 4 4- + — 4- 4- 4 — 4 4- + 4- — 4 4- 4- 4- — — 4 — — —\ 4- 4- 4- — 4- 4- 4- — 4- 4 4- 4- — + 4- 4- 4- — — 5 + 4- + +\ — — + — — — 4- — — — — + — — — — 4 4- 6 -h 4- 4- 4" —\ — + — — — + — — — — 4- — — — — 4- 4- 7 + 4- + 4- — — \4- — — — 4- — — — — 4- — — — — 4- 4* — 8 — — 4- 4- 4-\+ 4- 4 — 4- + 4- 4- — 4- 4- 4- 4- — — 9 + 4- + 4- — — — + \ — — 4 — — — — 4- — — — — 4- 4- 10 -f + + 4- — — — 4- — \— + — — — — 4" — — — — 4- 4- 11 4- 4- 4- 4- — — — 4- — — \,4- — — — — 4- — — — — 4- 4- 12 — — — — 4- 4- 4- — 4- 4- + \4" 4- 4- + — 4- 4- 4- 4- — — 13 4- 4- 4- + — — — 4- — — — 4- \ — — — 4- — — — — 4 4- 14 + 4- 4- 4- — — — 4- — — — 4- — \— — 4- — — — — 4- 4- 15 + 4- 4- 4- — — — + — — — 4- — — \— + — — — — 4- 4- 16 + 4- 4- 4- — — — + — — — 4- — — — \ 4- — — — — 4- 4- 17 — — — 4- + 4 — -h 4- + — 4" 4- 4- 4* 4- 4- 4- + — — 18 +I+ + 4- — — — 4- — — — 4- — — — — 4- — — — 4- 4- — 19 44+ + 4- — — — 4- — — 4- — — — — 4- — \— — 4 + 20 + 4- + + 4- 4- 4- \ 4" 4- 21 4- 4- + 4- 4- + 4- \ 4- + — 22 — — — 4 4- + — •j- 4- 4~ — 4- + 4- 4- — 4 -l- 4- +\ 23 — — — — + 4 — 4- 4- 4 — 4 4 4* 4 — 4- 4- + 4" — \
Figure 14. Chart showing results of pairing 23 single basidiospore isolates of S. radicata (KJM-274) in all combinations. 42
K A' y < A2 n 1 2 3 4 8 12 17 22 23 5 6 7 9 10 11 13 14 15 16 18 19 20 21 1 \ + 4 4 4 4 + 4 4" 4- 4 4 + 4 + 2 \ + 4 4 4 4 + 4 4 4 + + 4* + + 3 — —\ — — — — — — 4" 4 4 4 4- -4 4 4 + 4 4- 4* 4 + 4 — — —\ — — — — — + 4 4 + + 4 4 + + + -4 + + + 8 — — — —\ — — — — 4* 4 + 4 + 4 4 + 4- + 4" 4 + + 12 — — — — — — — — 4 4 4 4 + + 4 + + 4 4- + 4- + 17 — — — — — \ — — + 4 + + 4- 4 4 4 + 4 + + -F 4 22 \— + 4 4 4 4 + 4 4 4 4 -F 4 4 + 23 \+ 4 4 4 4 4 4 4- -F 4 + 4- 4- 4- 5 + + + + + + 4- -F 4 \ 6 + 4" 4- 4" 4 + + + 4 — \ 7 + 4- 4 + 4 + + + + — — \— — — — — — — — — — — 9 + + 4- + + 4-I4 4 4 — — — \ — — — — — — — — — — 10 + + 4 + 4 + + + 4 — — — — \ — — — — — — — — — 11 + 4- 4- + 4 -H -F 4- 4 — — — — — \— — — — — — — — — 13 + + 4- + 4 + 4 + 4 — — — — — — \ — — — — — 14 + + 4- + 4 4- + + \ 15 + 4- + 4" 4 + 4 4- + — — — — — — — —\ — — — — — 16 4- 4- 4- + 4 4- + + 4- — — — — — — — — — \ — — — 18 4 4- + + 4 -H 4 4 4 — — — — — — — — — —\ — — — 19 + 4- 4- + 4 4- 4 + -H — — — — — — — — — —\ — bo + 4- + + 4- + + + + \ 21 4" 4- + + 4 4- 4 + 4 \
Figure 15. Bipolar pattern of sexuality of jJ. radicata (KJM-274) rearranged into mating type groups. 43
The two figures show that isolates 1, 2, 3, 4, 8, 12, 17, 22, and 23 represent one mating type and were compatible with isolates 5, 6, 7,
9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, and 21 which represent a second mating type. The presence of two mating types shows that jS. radicata has a bipolar type of mating system.
Crosses of 20 single basidiospore isolates of KJM-390 in all combinations also gave a typical bipolar pattern of sexuality. Figure
16 shows the result of this cross with the rearranged results shown in Figure 17. Isolates 1, 2, 3, 5, 7, 10, 11, 12, 13, 15, 16, 17, and
18 represent one mating type, and isolates 4, 6, 8, 9, 14, 19, and 20 represent the second mating type.
Asexual Spores
Both homokaryotic and dikaryotic isolates of j3. radicata produced intercalary and terminal asexual chlamydospores in culture (Figure 18).
The chlamydospores were ellipsoid to subglobose and 8-12 X 6-10 ji. In tercalary chlamydospores were cut off squarely at both ends. Those produced terminally were cut off squarely at one end. All of the septa were simple. No clamps were seen at the septa delimiting chlamydospores produced on dikaryotic mycelium. The spathulate swellings found on the ends of many hyphae were probably developing chlamydospores. Several had septa, but did not have the thick walls of mature chlamydospores.
The thick wall apparently develops after the septa form. Chlamydo spores developed on older mycelium. They were often abundant around the area of inoculum, less numerous towards the advancing zone, and absent in the advancing zone. 44
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 — + — + 4" + — — — — + — — — — 4" 4- 2 — — + — + + + — — — — + — — — — •4 4- 3 — + — + + + — — — — + — — — — 4 4- 4 + + + + — + — — 4- 4 + 4 — + 4 4" 4- — — 5 — — +\ + "b + — — — — 4- — — — — + 4- 6 4- + + — + + — — + 4 4- + — 4- 4 4- 4" — — 7 — — + — + 4- + — — — — 4* — — — — 4- 8 + + + — + — + — 4- 4 4- + — 4- + 4- 4- — — 9 + + + — + — + — 4~ 4 + 4- — 4- + 4- 4- — — 10 — — + — + + +\ — — — — — — 4- 4- 11 — — + — + + + \ — — — — — — 4" 4- 12 — — 4- — + + 4- — — \ -f — — — — 4- 4- 13 — — + — + + — — \-f — — — — 4- 4- 14 4- + + — 4- — + — — + 4 •4 4* + + + — — 15 — — 4- — 4" -h + — — — — "h — — — 4- 4- 16 — — + — + + + — — — — •4 — — — 4- 4- 17 — — + — + + + — — — — -h — — — 4- 4- 18 — — — — — — — — — — + + + + 4* l+ 4- 19 4- 4- + — 4- — + — — 4- 4 4* 4 — 4* 4- 4- — 20l— — — + — + + + — — — — + — — — — +\
Figure 16. Chart showing results.of pairing 20 single basidiospore isolates of S. radicata (KJM-390) in all combinations. 11 A3 * A4 1 2 3 5 7 10 11 12 13 15 16 17 18 4 6 8 9 14 19 20 1 4" 4- + + + 4" + 2 — 4* 4- 4- 4~ + + + 3 4- 4" 4- 4- 4- + 4- 5 4- 4" 4- 4- 4- 4- 4- 7 — — — —\ — — — — — — — — + 4- 4- 4- 4- 4- 4~ 10 4" 4" 4- 4- 4- 4- 4- 11 4- 4- 4- 4- 4- 4- 4- 12 4- 4- 4- 4~ 4- 4- 4* 13 — — — — — —\ — — — — 4- + 4~ 4~ 4- 4- 4- 15 — — — — — — — — — — — — 4- 4- 4- 4- 4- -f- 4- 16 — — — — — — — — — \ — — + 4- 4- 4- 4" 4- 4- 17 — — — — — — — \— + 4" 4- 4- 4* 4- 4- 18 \+ 4- 4" 4- 4- 4- 4- 4 + 4- 4- + 4- 4- + 4- + + 4- 4" 4-\ 6 -i- 4- 4- + 4- 4- 4* 4- 4- 4- + 4- + — \ 8 4- 4- + + 4 4- 4~ + 4- + 4- + 4- — \— — — — 9 4- 4- + 4- 4- 4- 4- 4- + 4- + 4- + — — — — — 14 4- 4- 4- 4- 4- 4- + 4- 4- 4- + -f 4- — — — — — — 19 4- 4- 4- 4- 4- 4" 4- 4- + 4- 4 H- 4- — bo +i+ 4- + 4" 4" +i4- 4- 4- + + + —
Figure 17. Bipolar pattern of sexuality of JS. radicata (KJM-390) rearranged into mating type groups. 46
>
s
' -)
•
Figure 18. Hiase contrast photomicrograph showing chlamydospores of .S. radicata (KJM-389) produced in Nobles agar at 25C in the dark six weeks after inoculation, (X 400). Key Battern
Based on the results of the cultural studies, the key pattern for j>. radicata is 1. 3. 26. 34. 36. 38. 47. 53. 55. 59. These code symbols (Nobles 1965) are: (1) results negative in tests for extra cellular oxidase, (3) thin-walled hyphae consistently nodose-septate,
(26) noteworthy swellings on hyphae, (34) chlamydospores present, (36) hyphae hyaline and mats white or pale in color, (38) reverse unchanged in color, (47) plates not covered in six weeks, (53) odor noteworthy,
(55) associated with decay in coniferous trees, and (59) heterothallic, with a bipolar type of mating system.
Multiple Allele Test for Incompatibility
Hie results of testing for multiple alleles for incompatibility at the locus for heterothallism in radicata are shown in Figure 19.
These data show that the fungus is multi-allelic for incompatibility and that a duplication of mating type factors occurred in several iso lates. Seventeen alleles for incompatibility were found in the 18 basidiocarps (Table 1). The allele designated as A^g occurred six times,
A^y five times, A4 four times, A3 four times, Ag three times, A7 two times, A2 two times, and the remaining 10 occurred one time each. The allele designated as A2 was shared by KJM-274 and KJM-391; A3 was shared by KJM-391, JPL-187, KJM-389, and KJM-390; A4 was shared by KJM-392,
JFL-187, KJM-389, and KJM-390; A6 was shared by KJM-463A, KJM-463B, and
KJM-461; A7 was shared by KJM-463A and KJM-463B; A^g was shared by KJM-
469 A, B, C, D, and E, and KJM-450; A^y was shared by KJM-469 A, B, C,
D, and E. 1 2 3 4 5 6 7 8 9 1C 11 12 13 <4 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 1 f 4-f 4 f 4 4 4 4 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4- 2 1-4 -1- 4 f 4 4 4 4 4 4 4 4 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 ... 4-[-4-f 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ... 4-f 4-h 4 4 4 4 4 4 4 4- 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 4 5 + • 1- 4-1-4 4 4 4 4 4* 4 4 4 4 4 4 4 4 — — 4 4 4- 4 4 4 4 4 6 4- b 4-1-4 4 4 4 4 + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -- 7 4 L4-b 4 4 4 4 4 4 — 4 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 4 4 -- e 4- - + --4 -r 4 4 4 4 4 4 4 4 4 -4 4 4 — 4 4 4 4 4 4 4 4 4 4 4 4 -- 9 4-b 4 -1-4- -f- 4 - 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -- 10 4-I—I—I -4-1-4- 4 4 4 + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 11 t"-4- -4--4--4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4" 12 4H-4-4 -4- -4--4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 - 4 4* -- 13 + H -4--44-4 --4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4- 4 4 4 4 4- 14 4" -4-b4--4--4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -- 15 + -h 4-H*44 -4 4 4 4 4 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 4 4 4+ 16 4"L 4-4- 4 --4-b 4 4 4 4 4 4 4 *4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -4 17 + -—h --4-b44b 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 4 4 + 18 + -—h -"4"b4- -4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 — — 4 4 4 — 4 19 f —I— -44-4-4 -4 4 4 4 4 4 4 4 4 4 4 4 — 4 4 — 4 4i4 4 4-4 4 4 4 + 20 + • -4-4 -4--4 4 4 4 4 4 4 4 4 4 4 4 4 — — 4 4 4 4 4 4 4 4 4 4 4- 21 t" -4-4 -4--4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + 22 4" -4-4-4-4 — 4-4 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4-+ 23 •f H-4-- •4-H -44-4 4 4 4 4 4 4 4 4 4 — 4 4 4 4 — 4 4 4 4 4* 4 4 4 4 + 1 | | 24 i _ J_FT _L -J r ^ t I 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4- J A | I | | | i | | 2C Ii ii 1 I T" I I I i 4 4 4 4 4 4 4 4 4 4 4 + 26 •-H-4- •44-44 • 4- 4 4 4 4 4 4 4 4 4 — 4 4 4 — 4 4 4 4 4 4 + 4+ 4 + 27 --4-4-4 •4-4 ---4 -4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 — 4 4 4 26 - 4 -4 4 4•44 -4 4 4 4 4 4" 4 4 4 — 4 4 4 4 4 4 4 4 — 4 4 4 — + — 29 -4 -4•44 -4 "4 4 4 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 — 4 4 — 4* —i 30 -4 --4-44 -4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 r1 i -L 31 -4•44 -44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 — 4 4 — 4 __ 4i 32 -4 --4-44 4 4 4 4 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 — — 4 4 — + — 33 4 -4 -4•44 -4 4 4 4 *4 4 4 4 4 4 4 4 4 4 4 4 4 4 — 4 4 — — 4 _ + 34 44-4-4 •4 4 4 4 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 — — 4 4 -4 35- --4-44 -4 4 4 4 + 4 4 4 4 4 4 4 4 4 4 4 4 4 — 4 4 — — 4 4 36 - — — f —h•44 4 4 4 4 4 4 4 4 +1 4 4 4 4 4 4 4 4 4 4 4 4 -
19. Chart showing multiple alleles for incompatibility by pairing 36 homokaryotic isolates representing the two mating types from each of 18 basidiocarps of _S. radicata collected in Arizona. 49
Table 1. Mating type factors found in 18 basidiocarps of j>. radicata collected in Arizona.
Basidiocarp Isolate Allele Location Mountain Range
KJM-274 1 A1 Bear Wallow Santa Catalina 2 A2 KJM-391 3 A2 Mt. Lemmon Santa Catalina 4 a3 KJM-392 5 a4 Bear Wallow Santa Catalina 6 a5 KJM-463B 7 a6 Treasure Park Pinaleno 8 a7 JPL-190 9 a8 Mt. Lemmon Santa Catalina 10 a9 KJM-468 11 A10 Moonshine Cr. Pinaleno 12 A11 KJM-467 13 A12 Hospital Flat Pinaleno 14 a13 KJM-461 15 a6 Shannon Bark Pinaleno 16 a14 KJM-450 17 a15 V. T. Lake Kaibab Plateau 18 a16 JEL-187 19 a3 Mt. Bigelow Santa Catalina 20 a4 KJM-463A 21 a7 Treasure Bark Pinaleno 22 a6 KJM-390 23 a3 Mt. Bigelow Santa Catalina 24 a4 KJM-389 25 a4 Mt. Bigelow Santa Catalina 26 a3 KJM-469B 27 a17 Moonshine Cr. Pinaleno 28 a16 KJM-469D 29 a16 Moonshine Cr. Pinaleno 30 a17 KJM-469C 31 a17 Moonshine Cr. Pinaleno 32 a16 KJM-469A 33 a17 Moonshine Cr. Pinaleno 34 a16 KJM-469E 35 a17 Moonshine Cr. Pinaleno 36 a16 50
Temperature Relations
Average growth of JS. radicata from the edge of inoculum to the edge of the advancing zone at 13 constant temperatures at the end of
21 days is shown in Table 2. The optimum was 23-25C for all isolates tested except one. Isolate KJM-279 had an optimum growth temperature of 28C. All isolates showed appreciable growth from 18-28C. At 30C there was a sharp drop in growth rate and only a trace at 35C. None of the isolates grew above 35C. All showed a trace of growth at 4C and 9C. There was no apparent difference in growth rate between homo- karyotic and dikaryotic mycelia of individual isolates at any tempera ture.
The thermal death point was approximately 40C. Isolates not growing after three weeks at 38, 40, 42, and 45C were transferred to
25C and checked for growth after four more weeks. All isolates taken from 38C, except one dish of KJM-274 and one dish of KJM-391 A2, grew within the four weeks at 25C. One dish of KJM-279 was the only isolate taken from 40C that was growing after the four weeks. None grew taken from temperature settings above 40C.
Decay Studies
Decay in Nature
Sparassis radicata was found to produce a yellowish-brown to brown carbonizing rot in the roots and butts of Douglas fir in Arizona.
The decay of the butt (Figure 20) was confined to the heartwood and was found extending up to 12 feet above the ground level. Decay in the roots
(Figure 21) occurred in both heartwood and sapwood. The incipent decay Growth (mm)* of Sparassis radicata mycelium on 3.0% MEA at various temperatures after 21 days incubation.
Temperature
4C 9C 18C 20C 23C 25C 28C 30C 35C 38C 40C 42( 45C
T 15.3 15.7 18.7 18.7 16.3 2.0 T 0 0 0 0
T T 14.7 15.5 17.4 18.0 16.7 2.4 T 0 0 0 0
T T 15.6 15.9 18.1 18.4 17.1 3.1 T 0 0 0 0
T T 21.3 22.1 27.0 27.9 21.7 3.1 T 0 0 0 0
T T 20.7 22.9 27.4 27.6 22.1 2.9 T 0 0 0 0
T T 20.9 22.6 26.8 27.1 20.2 5.7 T 0 0 0 0
T T 20.0 24.1 26.8 26.9 20.2 3.4 T 0 0 0 0
T T 19.4 24.2 27.1 26.5 20.9 5.1 T 0 0 0 0
T T 18.6 23.6 26.3 26.8 21.3 4.4 T 0 0 0 0
T T 12.1 14.6 20.2 19.8 17.5 2.7 T 0 0 0 0
T T 11.4 14.2 19.5 20.3 17.1 4.3 T 0 0 0 0
T T 12.0 15.1 19.8 20.7 17.0 3.2 T 0 0 0 0 Table 2, Continued
Temperature
Isolate 4C 9C 18C 20C 23C 25C 28C 30C 35C 38C 40C 42C 45C
KJM-467 T T 17.8 17.6 24.4 24.1 20.8 6.6 T 0 0 0 0
A12 T T 15.4 16.3 25.3 26.1 21.2 4.1 T 0 0 0 0
A13 T T 17.1 17.1 22.7 23.9 19.7 2.9 T 0 0 0 0
KJM-279 0 T 13.1 14.6 15.2 16.8 20.2 9.1 T 0 0 0 0
RLG-8240 T T 16.9 21.7 25.3 25.1 18.4 4.2 T 0 0 0 0
* Hie average of five replications of each isolate measured from the edge of the inoculum to the edge of the advancing zone.
** T • Trace (less than 2 mm growth).
*** The series of MA" isolates represent the two mating types of each dikaryotic isolate. Figure 20. Riotograph showing decay caused by S. radicata (JH) in the butt log of a 48" DBH Douglas fir, (X 0.07). Figure 21. Photographs showing decay caused by _S. radlcata (KJM-389) in the roots of Douglas fir.—(a) decay confined primar ily to the heartwood (X 0.18); (b) decay in both heart- wood and sapwood (X 0.6). 55 was a dull reddish-brown. Advanced decay was yellowish-brown to dull brown. Freshly exposed decay was soft and "cheesy", becoming brittle and broken up into cubes only in very advanced stages. A dark purple to black pitchy zone sometimes occurred between the heartwood and sap- wood in the roots. This dark pitchy zone was not found in the decay of the butt. Yellowish to light brown, almost transparent, resinous crusts often occurred in shrinkage cracks of very advanced decay of the butt.
Elongated pits formed in the decay of the heartwood in roots became filled with white mycelium. Mycelium in the butt was in thin scattered patches in shrinkage cracks. The mycelium had clamp connec tions and was often incrusted with crystals. The spathulate swellings on the tips of many hyphae, as previously described in the cultural morphology, were common. Only the very advanced decay could be rubbed into a fine powder. Decay in the roots had a strong turpentine odor.
Agar-block Decay Studies
Both dikaryotic and homokaryotic isolates of jS. radicata pro duced a brown rot in blocks of ponderosa pine and Douglas fir. The decay was the same for both types of mycelium and was similar to that associated with basidiocarps. The wood in early stages of decay showed a slight reddish-brown discoloration. Advanced decay was yellowish- brown. After the blocks dried, shrinkage cracks developed in advanced decay such that the wood sometimes broke up into cubes. Fresh decay could not be rubbed into a fine powder. The advanced decay could be rubbed into a powder after the blocks were dried. Microscopic examina tion revealed hyphae running longitudinally along tracheid walls and 56 horizontally in the parenchyma of the rays. Hyphae entered adjacent tracheids through bore holes and pits. No constrictions of the hyphae in bore holes were seen. Clamps were not observed on homokaryotic hyphae but were present on dikaryotic hyphae.
The results of the relative decay capabilities of homokaryotic and dikaryotic mycelium in agar-block decay tests are presented in
Table 3. The data show that both types of mycelium are capable of causing decay in Douglas fir and ponderosa pine. There were no appar ent differences in the decay capabilities of the two types of mycelium in either type of wood.
Incidence of S. radicata in i ttti — the Santa Catalina Mountains
The study of the incidence of £. radicata in the Santa Catalina
Mountains indicates that the fungus is an important cause of root and butt rot of Douglas fir in this area. The results are summarized in
Table 4. Sparassis radicata and Polyporus schweinitzii were the only species that caused a brown root and butt rot in the 50 trees examined.
One tree also had areas of a white pocket rot caused by Polyporus tomen- tosus.
Of the 50 trees, the failure of 15 (30%) was attributed to £. radicata. 31 (62%) to _P. schweinitzii. and 4 (8%) to S. radicata and
P. schweinitzii together. The highest incidence of S. radicata was in
Marshall Gulch and Bear Wallow, followed by Mt. Bigelow, Mt. Lemmon, and Summerhaven, respectively. The species responsible for the failure of some of the trees could not be determined with certainty. In these trees basidiocarps were absent or most of the decay was in the advanced Table 3. Weight loss of Douglas fir and ponderosa pine wood blocks decayed for 24 weeks by Sparassls radicata.
Douglas fir ponderosa pine
Range of % * % weight ** Range of % % weight Isolate weight loss loss weight loss loss
RLG-8240 10.5 - 19.5 15.8 9.7 - 14.9 12.6
KJM-274 13.7 - 18.9 17.2 17.1 - 18.3 17.4
Ai *** 14.6 - 21.2 19.8 13.5 - 15.5 14.5
A2 15.8 - 17.8 17.1 17.4 - 22.8 19.4 KJM-279 8.8 - 24.9 16.9 10.3 - 19.6 15.2
JH 11.9 - 15.6 13.5 13.5 - 18.8 15.1
Control 0.5 - 1.0 0.6 0.4 - 1.1 0.7
* Lowest and highest % weight loss determined for five test blocks exposed to each isolate, based on initial oven dry weight.
** "A weight loss determined for five test blocks exposed to each isolate, based on total initial oven dry weight and total weight loss.
*** and A2 represent the two mating types of KJM-274. Table 4. Results of analysis of 50 fallen Douglas fir trees with a brown cubical root and butt rot.
Decay and Failure Tree Basidiocarps* Breakage** mycelial Jfycelium attrib number Location DBH present type characteristics isolated uted to
1. Mt. Bigelow 48" Ps A Ps none Ps
2. II 48" Sr B Sr Sr Sr
3. II 30" A Ps none Ps !t1 4. II 60" Pi A Ps none Ps
5. II 48" none A Ps none Ps
6. II 36" Ps A Ps none Ps
7. II 24" none B Sr none Sr
8. II 60" none A Ps none Ps
9. Bear Wallow 50" Ps B Ps none Ps Sr Sr
10. it 30" Ps A Ps none Ps
11. it 28" none B Sr none Sr M H < ti 34" Ps A Ps none Ps• Table 4. Results of analysis of 50 fallen Douglas fir trees with a brown cubical root and butt rot, Continued.
Decay and Failure Tree Basidiocarps* Breakage** mycelial fycelium attrib- number Location DBH present type characteristics isolated uted to
13. Bear Wallow 36" Ps A Ps none Ps
14. 36" none B Sr Sr Sr
15. 38" none B Sr none Sr
16. 40" Sr B Sr none Sr Ps Ps Ps Pt Pt
17. 30" none B Sr Sr Sr
18. Marshall Gulch 40" Ps A Ps none Ps
19. 32" none A Ps none Ps
20. 24" none B Sr Sr Sr
21. 36" none B Sr none Sr
22. 38" Ps A Ps none Ps
23. 36" Ps A Ps none Ps Table 4. Results of analysis of 50 fallen Douglas fir trees with a brown cubical root and butt rot, Continued.
Decay and Failure Tree Basidiocarps* Breakage** mycelial Ifycelium attrib- number Location DBH Present type characteristics isolated uted to
24. Marshall Gulch 30" none B Sr none Sr
25. 30" none B Sr Sr Sr
26. 40" none B Sr none Sr
27. Summerhaven 40" none B Sr none Sr
28. " 30" none A Ps none Ps
29. " 32" Ps A Ps none Ps
30. " 30" Ps A Ps none Ps
31. " 60" none A Ps none Ps
32. " 36" none A Ps none Ps
33. " 42" Ps A Ps none Ps
34. Mt. Lemmon 40" Ps B Ps none Ps Sr Sr
35. 38" Ps Ps none Ps Table 4. Results of analysis of 50 fallen Douglas fir trees with a brown cubical root and butt rot, Continued.
Decay and Failure Tree Basidiocarps* Breakage** mycelial tfycelium attrib- number Location DBH present type characteristics isolated uted to
36. Mt. Lemmon 36" none B Sr Sr Sr
37. 38" none B Sr none Sr
38. 40" Ps A Ps none Ps
39. 40" Ps A Ps none Ps
40. 30" none A Ps none Ps
41. 40" Ps A Ps none Ps
42. 36" Ps A Ps none Ps
43. 40" Sr B Sr Sr Sr
44. 48" Ps A Ps none Ps
45. 36" Ps B Ps none Ps Sr Sr
46. 38" Ps A Ps none Ps
47. 40" Ps A Ps none Ps Table 4. Results of analysis of 50 fallen Douglas fir trees with a brown cubical root and butt rot, Continued.
Decay and Failure Tree Basidiocarps* Breakage** mycelial Mycelium attrib number Location DBH present type characteristics isolated uted to
48. Mt. Lemmon 38" Ps A Ps none Ps
49. M 36" none B Sr Sr Sr
50. ii 36" none A Ps none Ps
* Sr • Sparassis radicata. Ps • Polyporus schweinitzil. Pt • Polyporus tomentosus
** A « Trees broken 1-12 feet above ground line with no roots exposed. B • Trees broken at or below ground line with parts of the root system exposed. stage and isolation attempts failed. In such cases, the most probable causal agent was listed, based on decay and mycelial characteristics and breakage type. The type of mycelium present in the decay and the breakage type were two characters found to be different in trees decayed by jS. radicata and those decayed by P. schweinitzii. The trees with decay attributed to JS. radicata were always broken at or below the ground line with part of the root system exposed. Those attributed to
P. schweinitzii were always broken from 1-12 feet above the ground line.
The mycelium of JS. radicata was white and clamp connections were present.
Clamp connections were absent on the hyphae of P. schweinitzii and the mycelium was yellowish-white.
All of the trees, except three, had a DBH over 30 inches. Two of the trees less than 30 inches DBH had rots attributed to j3. radicata and one to P. schweinitzii. All trees were on north facing slopes.
Studies with Isolates Labeled S. crispa and laroinosa
The isolates labeled crispa from Germany (S265 and S211),
England (FFRL-312A), and Japan (Splc), and the isolate labeled 55. lami- nosa from England (CBS-Wy-212) did not differ in their cultural morphol ogy, temperature relations, and oxidase reactions from those described for S. radicata. The cultures labeled J3. crispa from the southeastern
United States were easily distinguished macroscopically and microscopi cally from S[. radicata and from the European and Japanese isolates la beled S. crispa. The growth (Figure 22) and microscopic characters on
Nobles agar of the southeastern United States isolates labeled j3. crispa Figure 22. Riotograph showing growth characters on Nobles agar at 25C in the dark of southeastern United States isolates labeled S. crispa.- - Left: RLG-3860; right: 0KM-6860. 65
(JL-181, JL-221, JL-240, OKM-3673, OKM-686O, RLG-3860, FP-105840, and
FP-105855) is as follows:
Growth Characters
Growth moderately rapid, radius from edge of inoculum to edge of advancing zone 33-57 mm at two weeks, completely covering surface of agar in 90 mm petri dish at 3-4 weeks; margin of advancing zone slightly bayed, white, raised aerial mycelium extending to limit of growth; aerial mycelium white, at first woolly-floccose, with tufts merging to form large continuous patches, sometimes in concentric zones, often becoming appressed or slightly raised around area of inoculum; reverse unchanged; odor faintly musty; diffusion zones absent on gallic and tannic acid medium; no growth on tannic acid medium; growth on gallic acid medium
7-16 mm from edge of inoculum to edge of advancing zone at two weeks; reaction negative with gum guaiac solution; fruiting in inverted dishes in 4-6 weeks.
Microscopic Characters
Hyphae of the advancing zone (Figure 23a) 2-8 p in diam, hyaline, thin-walled, simple-septate, occasionally to frequently branched, rounded to blunt at the tips; aerial mycelium (Figure 23c) hyaline, 2-8 p in diam, thin- to thick-walled, mostly simple-septate with rare to occa sional clamp connections, some large, 12-16 p in diam, occasionally branched, simple-septate, hyphae scattered throughout; submerged mycelium
(Figure 23b) hyaline, 2-8 p in diam, thin- to thick-walled, simple- septate, often with irregular swellings, smaller hyphae often coming off larger hyphae, with larger, 12-16 fi in diam, simple-septate, 66
i
i
20
Figure 23. Camera lucida drawings of hyphae of the advancing zone, submerged mycelium, and aerial mycelium of southeastern United States isolates labeled S. crispa (RLG-3860).— (a) hyphae of advancing zone; (b) submerged mycelium; (c) aerial mycelium. thick-walled, hyphae scattered throughout, associated with numerous
crystals.
A comparison of the cultural characters of _S. radicata and the
isolates labeled crispa is given in Table 5 and shown in Figure 24.
Based on cultural characters, the isolates of Sparassis used in this
study can be placed into two groups. One group includes Arizona iso
lates of S. radicata. European and Japanese isolates labeled j>. crispa. and isolates labeled £>. laminosa from England. The second group in cludes the isolates from the southeastern United States labeled crispa.
Di-mon Matings
Matings made with dikaryotic isolates labeled crispa from
Europe and Japan and homokaryotic isolates of j5. radicata indicate that
the homokaryotic mycelium was apparently dikaryotized by the dikaryotic mycelium. Clamp connections were found on hyphae of all the original homokaryotic mats 4-6 weeks after inoculation with dikaryotic isolates labeled £5. crispa from Germany (S265 and S211), England (FPRL-312A), and Japan (Splc). The clamps were found in the advancing zone up to
15 mm from the area of contact with the dikaryotic mycelium of isolates labeled J3. crispa. No clamps were found in the homokaryotic mats of any matings made with homokaryons of S>. radicata and isolates labeled
S. crispa from the southeastern United States. Table 5. Comparison of cultural characters of S. radicata and isolates labeled S. crispa.
Growth (2 weeks) Septation Basidiospores Chlamydo- Isolate at 25C of hyphae produced spores
S. radicata 6-12 mm mostly nodose- not produced ellipsoid septate with at end of six to subglo some simple weeks bose, 8-12 septa X 6-10 u j£. crispa (Japan) 8-10 mm mostly nodose- not produced ellipsoid (splc) septate with at end of six to subglo some simple weeks bose, 8-14 septa X 8-10 u
S. crispa (Germany) 7-11 mm mostly nodose- not produced ellipsoid (S265, S211) septate with at end of six to subglo some simple weeks bose, 8-12 septa X 8-10 u
S. crispa (England) 6-9 TTTTTI mostly nodose- not produced ellipsoid (FPRL-312A) septate with at end of six to subglo some simple weeks bose, 8-12 septa X 8-10 u
_S. crispa (south 35-58 mm mostly simple produced in not pro eastern U. S.) septate with 4-6 weeks duced (RLG-3860, OKM-3673, rare clamp OKM-686O, JL-181, connections JL-221, JL-240, FP- 105840, FP-105855) Figure 24. Hiotograph showing comparison of growth on Nobles agar at four weeks of jS. radicata from Arizona and isolates labeled j>. crispa from Japan, Germany, and southeastern United States.--Top row, left to right: S. radlcata (JPL-202), j>. crispa from Japan (Splc), crispa from Germany (S265); bottom row, left to right: crispa from southeastern United States, (OKM-3673), (RLG-3860), (OKM-686O). DISCUSSION
Host index records indicate that S,. radicata is associated only with members of the Pinaceae and that Douglas fir is the main host, tfy study supports these data. The basidiocarps collected in Arizona and those I examined from other regions were primarily associated with
Douglas fir. Of the 28 Arizona basidiocarps of _S. radicata. 23 were associated with Douglas fir, two with ponderosa pine, and one each with southwestern white pine, white fir, and an unidentified pine. The col lection data on the basidiocarps and isolates of j3. radicata from out side the state show that three were on Douglas fir, one was associated with Bishop pine, and for two the substratum was not given. The fungus was not found in southern Arizona on hardwoods or on the lower elevation members of the Pinaceae such as Apache pine (Pinus latifolia Sarg.),
Chihuahua pine (Pinus leiophylla var. chihuahuana (Engelm.) Shaw), and pinyon pine (Pinus edulis Englm.). It apparently does not occur much below 8000 feet elevation in southern Arizona.
Basidiocarps of j>. radicata have been associated with decay in living conifers, but there are no reported attempts to prove that the fungus is the actual cause of the decay. It is difficult to use living trees in nature to carry out Koch's postulates to establish such proof with absolute certainty that other wood-rotting fungi are not present.
I used agar-block decay tests to provide a modified method of Koch's postulates in proving £>. radicata is capable of causing a brown cubical
70 71 rot in Douglas fir and ponderosa pine wood. This proof is summarized
below:
1. Basidiocarps of -S. radicata were always found associated
with a brown cubical root and butt rot.
2. Polysporous isolates and tissue isolates from basidiocarps
of j>. radicata were grown on artificial medium and the
characters described.
3. Ifycelium was isolated from the decay associated with ba
sidiocarps of £>. radicata. grown in pure culture on arti
ficial medium, and its characters described.
4. Ifycelium growing in pure culture was inoculated on wood
blocks of the same tree species in which the decay occurred
and the same type of decay was produced in the blocks.
5. Jfycelium was isolated in pure culture from the decayed
blocks and its characters were the same as those isolated
from decayed trees, from polysporous isolates, and tissue
isolates.
The study on the incidence of ,S. radicata indicates that the fungus is one of the important causes of a brown root and butt rot in
Douglas fir in the Santa Catalina Mountains. Polyporus schweinitzii and _S. radicata are the only two fungi known to be associated with a
brown cubical root and butt rot in living Douglas fir in southern
Arizona. Polyporus balsameus Fk. is known to cause a brown root and
butt rot, and basidiocarps of the fungus have been found on Douglas fir in southern Arizona. However, it has not been found on living trees and appears to occur only on stumps and slash. 72
It is possible that radicata is more important in other
Douglas fir regions of western North America than has been reported, but due to the rapid deterioration of the basidiocarps and the similar ity of the advanced decay in the butt to that caused by J?, schweinitzii. it may have been overlooked. Bawsey (1971) found this to be the case with S. crispa and J?, schweinitzii in conifer plantations in Great
Britain. He reports that a portion of the decay previously attributed entirely to P. schweinitzii was actually caused by j>. crispa. He also states that in Sitka spruce (Picea sitchensis (Bong.) Carr.) plantations, the incidence of butt rot caused by S3, crispa is less than that caused by _P. schweinitzii and that both fungi may occur in the same tree. The incidence of root and butt rot caused by J>. radicata in Douglas fir in my study is similar to that described in P&wsey's report. Polyporus schweinitzii and _S. radicata were found together in four of the 50 trees examined. The incidence of P. schweinitzii was greater than j3. radicata
(62% by P. schweinitzii compared to 30% by S. radicata). Schonar (1970) reports the incidence of J>. crispa was greater than P. schweinitzii in
Scots pine (Pinus sylvestris L.) in Baden-Wurttemberg, Germany. Spa- rassis crispa was isolated from 27.2% of the decayed trees compared to
16.2% for P. schweinitzii.
The decay characteristics of j3. radicata in the trees I exam ined do not differ much from those described by Weir (1917). Weir states that the fungus is parasitic on the roots of conifers and in vades the heartwood after the living tissues of the root have been killed. He also reports he did not find any evidence of a butt rot caused by j3. radicata. Butt rot was common in the Douglas fir trees I examined. Ity evidence also indicates that the heartwood is often de cayed while the sapwood is still sound. This is especially true in upper portions of the roots near the butt of the tree. Decay in the butt appears to be confined to the heartwood. The fungus probably exists mainly as a perthophyte in the heartwood of the butt and is capable of being parasitic on the living tissues of the deeper roots.
The breakage type of trees decayed by j3. radicata offers addi tional evidence that much of the root system is completely decayed by the fungus. The windthrown trees attributed to j>. radicata were al ways broken at or below the ground level with parts of the root system exposed. This breakage type was not found in windthrown trees attrib uted to _P. schweinitzii. These were broken off 1-12 feet above the ground with no roots exposed. The action of P. schweinitzii is con fined mainly to the heartwood of the roots and butt (Weir 1917, Boyce
1961). Roots with only the heartwood decayed would still have a great deal of strength. Roots would be considerably weaker if both the heartwood and sapwood were decayed. This might explain the differences in breakage type between j3. radicata and P. schweinitzii.
The ability to kill living tissues of the roots indicates that ii* radicata could kill trees. I did not find any trees that definitely had been killed by the fungus. Weir (1917) found only four trees, two
Douglas fir, one western white pine, and one Engelmann spruce, that he thought had been killed by £. radicata. His conclusion that the trees were killed by the fungus was "...because of the absence of any other fungus or factor which has heretofore been accredited as causing the 74
death of the trees (p. 176)." More work is needed to prove the ability
of j>. radicata to kill trees.
Some evidence on portions of the disease cycle of _S. radicata has been accumulated but many aspects are still unexplained. It is not known how the fungus initially gets into living trees. Weir (1917, p. 177)
states, "mycelium attacks the bast of the roots and later the wood pro ducing a yellow or brown carbonizing rot." This indicates that direct
penetration of the roots by the fungus may be possible but proof is lacking.
The role of basidiospores in infection is not known. evi dence indicates that fire scars play little role as infection courts.
Douglas fir, which appears to be the main host of _S. radicata. has a thick layer of bark and is highly resistant to fire damage. No visible fire scars or any evidence of callosed over scars were present on the living Douglas fir on which basidiocarps of J3. radicata were found.
Southwestern white pine has thin bark and is extremely susceptible to fire damage. One basidiocarp of S. radicata was found on southwestern white pine but no fire scars were evident.
The multi-allele test for incompatibility gives evidence that
JS. radicata moves from diseased trees to healthy trees through root grafts or root contacts. These results showed that the basidiocarps of JPL-187, KJM-389, and KJM-390 came from dikaryotic mycelium carry ing the same incompatibility factors. KJM-389 and KJM-390 were col lected on living Douglas fir trees that were three feet apart. JPL-187 was collected on the roots of a Douglas fir stump that was 30 feet from the two living trees. Because the three isolates carried the same in compatibility factors, they were probably from the same dikaryotic mycelium. The proximity of the trees was such that root grafts could easily have occurred. The probability is high that all three isolates came from the same dikaryotic mycelium that grew into the roots of the trees from a single infection.
Spread of root-rotting fungi through root grafts or root con tacts has been shown in several of the major root-rotting fungi. Wallis and Reynolds (1965) showed that Poria weirii moves through root contacts of Douglas fir. Whitney (1962) found that Polyporus tomentosus entered healthy roots in contact with infected roots of white spruce. It is established that Fomes annosus moves from tree to tree through root contacts (Boyce 1961, Hepting and Fowler 1962). None of the studies proving that the fungi move through root grafts or root contacts was done by genetic analysis of the mycelium. It should be pointed out that the genetic analysis method I used can be carried out only on species that are heterothallic and that form clamp connections. Host of the major root-rotting fungi (Fomes annosus, Poria weirii, Armil- laria mellea. Polyporus tomentosus, and Polyporus schweinitzii) do not have clamp connections and different methods would have to be used to identify dikaryons and to prove they move through root grafts or root contacts by a genetic analysis of mating type factors.
Three of the basidiocarps of j>. radicata were collected on stumps. Two (KJM-467 and JPL-187) were on Douglas fir and one (KJM-279) was on ponderosa pine. This indicates that the fungus can remain vi able in decayed stumps. The evidence showing that S. radicata probably 76 moves through root grafts or root contacts indicates that vegetative mycelium surviving in roots on stumps and dead trees could provide an important source of inoculum for surrounding healthy trees as well as for the next rotation of trees.
The agar-block decay tests show that in 24 weeks there is no apparent difference in the decay capability of homokaryotic and dikary- otic mycelium of j>. radicata. It is possible that homokaryotic mycelium from a single basidiospore could infect and cause decay in living trees.
However, I did not isolate any homokaryotic mycelium from trees with decay attributed to j>. radicata. More work needs to be carried out to determine how significant homokaryotic mycelium of S. radicata is in causing decay in nature.
I did not find evidence that different dikaryotic mycelium with different mating type factors occur in the same tree. Two trees were found with more than one basidiocarp of S. radicata. Isolates KJM-463
A and B were collected on the same root of a living Douglas fir. These shared the same mating factors, indicating that both basidiocarps orig inated from the same dikaryotic mycelium. Basidiocarps of KJM-469 A, B,
C, D, and E were collected around one living Douglas fir. All shared the same mating factors indicating that the basidiocarps originated from the same dikaryotic mycelium. It was not determined whether the five basidiocarps were all on roots of the same tree. Their proximity to the tree suggests that they probably were.
I did not observe any crown symptoms that could be attributed directly to j>. radicata. Most of the living Douglas fir on which the fungus was found were also highly infected with Douglas fir mistletoe 77
(Arceuthoblum douglasli Engelm.) making any crown symptoms which might have been caused by SI. radicata difficult to detect. The most obvious symptom is windthrown trees containing a brown cubical root and butt rot with parts of the root system exposed.
The only sign of the fungus on living trees is basidiocarps connected to the roots by an elongated pseudosclerotium. Basidiocarps in southern Arizona are usually found only during the rainy season in summer months. The earliest date fresh basidiocarps were collected in southern Arizona was August 9th and the latest was September 16th. The basidiocarps deteriorate rapidly and also are eaten by animals. White mycelium occurring in elongated pits in roots and in thin scattered
patches in shrinkage cracks in the butt are signs usually found in windthrown trees. The hyphae have clamp connections, and the swollen spathulate tips are fairly characteristic of the fungus. Basidiocarps are sometimes found growing directly out of the wood on windthrown trees.
The literature indicates that, with few exceptions, wood-rotting basidiomycetes with a bipolar type of mating system are associated with negative polyphenol oxidase reactions and a brown rot. These combina tions in _S. radicata are therefore not unusual. The presence of mul tiple alleles for incompatibility at the locus for heterothallism is also common in wood-rotting basidiomycetes. I assumed that the alleles for incompatibility found in j>. radicata were randomly distributed.
Random distribution is demonstrated by the number of common mating factors found in geographically separated isolates. This phenomonem has been shown in a number of basidiomycetes (Raper, Krongelb, and Baxter 1958, Neuhauser and Gllbertson 1971, Whitehouse 1949). I did not prove random distribution statistically because of the small number of isolates used.
The allele designated as A16 was shared by isolates KJM-469 A,
B, C, D, and E and KJM-450. These were the farthest geographically separated isolates that shared a common mating factor. KJM-450 was collected at V. T. Lake on the Kaibab Plateau and KJM-469 A, B, C, D, and E were collected at Moonshine Creek in the Pinaleno Mountains, areas separated by about 285 miles. Several alleles for incompatibility were shared by isolates collected in different locations in the same mountain range. In the Santa Catalina Mountains, the allele designated as A£ was shared by KJM-274 (Bear Wallow) and KJM-391 (Mt. Lemmon) separated by approximately four miles. A3 was shared by KJM-391 (Mt. Lemmon) and
KJM-389, KJM-390, and JFL-187 (Mt. Bigelow). These locations are approx imately five miles apart. KJM-389, KJM-390, and JPL-187 were discussed previously as probably orignating from the same dikaryotic mycelium.
These three isolates also shared an allele for incompatibility (A4) with KJM-392. The latter isolate was collected in Bear Wallow, about one mile from KJM-389, KJM-390, and JPL-187. The other isolates that shared alleles for incompatibility were collected in the Pinaleno
Mountains. Isolates KJM-469 A, B, C, D, and E from Moonshine Creek were previously discussed. The allele designated as Ag was shared by
KJM-461 (Shannon Park) and KJM-463 A and B (Treasure Park). These isolates were collected about one mile apart.
The similarities in the cultural characters of j>. radicata from
Arizona and isolates labeled j>. crispa from Europe and Japan and j3. laminosa from England indicate that a single species is probably repre sented. The key pattern for the cultural characters of Arizona isolates of _S. radicata was slightly different from that reported by Siepmann
(1970) for isolates labeled j3. crispa from Germany. His key was 1. 3.
10. 34. 36. 38. 47. (48). 55. 59. The key I obtained for jS. radicata was 1. 3. 26. 34. 36. 38. 47. 55. 59. Hie key for Arizona isolates of
S. radicata did not include code symbol 10 (hyphae differentated to form cuticular cells, closely packed together to form a pseudoparenchyma) or code symbol (48) (fruiting bodies produced in some isolates and not in others before the end of six weeks) that Siepmann obtained for iso lates labeled S. crispa. Siepmann did not include code symbol 26 (note worthy swellings on hyphae) for isolates labeled j>. crispa that I in cluded in the key for S, radicata. I received two of the isolates labeled J3. crispa (S265 and S211) that Siepmann used in his study. I examined their cultural characters and would include code symbol 26 in their key pattern, but exclude code symbols 10 and (48). The latter two symbols are associated with production of fruiting areas which I did not observe in the isolates labeled S. crispa from Europe and Japan,
laminosa from England, or j>. radicata.
The cultural characters of isolates of jS. radicata from Arizona are similar to those described for _S. crispa isolated from Larix kaem- feri Sarg. by Kamei and Igarashi (1959) in Japan. They do not give a key pattern, but the description given for the fungus on malt agar is the same as that observed for j>. radicata.
Hie apparent dikaryotization of homokaryons of radicata from
Arizona by dikaryons of isolates labeled j>. crispa and S>. laminosa 80 provides additional evidence for the conspecificity. The Di-mon mating test does not offer rigorous proof that the homokaryons were dikaryo- tized because the induced dikaryon was not analyzed for new combinations of mating type factors. Failure to produce fruiting in culture prevented this analysis.
The Sparassis from the southeastern United States has been generally referred to as £>. crispa but comparative studies show it to be specifically distinct from European specimens under that name. At least two distinct species of Sparassis are present in North America.
The nomenclatural problem concerning these have not been 3olved. It is likely that j3. radicata will become a synonym of an older name from the
European literature. Another name will probably be found to be valid for the southeastern United States Sparassis. LITERATURE CITED
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