Cultural and other morphological studies of Perenniporia phloiophila and related species
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Cultural and other morphological studies of Perenniporia phloiophila and related species
Flott, James Joseph, M.S.
The University of Arizona, 1990
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106
CULTURAL AND OTHER MORPHOLOGICAL
STUDIES OF PERENNIPORIA PHLOIOPHILA AND RELATED SPECIES
by
James Joseph Flott
A Thesis Submitted to the Faculty of the
PLANT PATHOLOGY DEPARTMENT
in Partial Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
1990 2
STATEMENT BY AUTHOR
This thesis 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 borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction 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 or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
Professor of Plant Pathology 3
DEDICATION
I would like to dedicate this work to my mother and father, Mary Ann and Percy, and to my wife Merri Hartse, whose love, personal sacrifices and constant support allowed me to fulfill my goals. 4
ACKNOWLEDGMENTS
The author wishes to express sincere gratitude to the University of Arizona for providing facilities and financial support for this research. I wish especially to thank: Dr. Robert L Gilbertson for supervision, advice, constant helpfulness and friendship; Dr. Richard B. Hlne for his support, interest and kind words of wisdom; and Dr. Michael Stanghellini for his support and the opportunity to participate in the work of his lab. I would also like to thank Dr. M. Blackweli (Louisiana State
University) for her Perenniporla phloiophila collections and Tom Orum for his personal help and expertise in statistical analysis. Further thanks to the Plant Pathology Department members for many happy memories and for all their cherished friendships. 5
TABLE OF CONTENTS
Page
LIST OF ILLUSTRATIONS 6
LIST OF TABLES 7
ABSTRACT 8
INTRODUCTION 9
MATERIALS AND METHODS 11
Fruiting Body Collections and Identifications 11 Isolation, Cultural Descriptions and Temperature Studies 11 Isolation of Homokaryons and Mating System 13 Vegetative Incompatibility 14 Decay Studies 14
RESULTS 16
Cultural Descriptions 16 Temperature Relationships 22 Mating System 26 Vegetative Incompatibility 33 Decay Studies 33
DISCUSSION 38
REFERENCES CITED 42 6
LIST OF ILLUSTRATIONS
Figure Page
1. Linear growth of Perenniooria ohloloohlla and P. ohiensls after 14 days at various temperatures on 2% malt extract agar 18
2. Growth and reactions of Perennlporla phlolophlla and P. ohiensls on gallic acid medium and tannic acid medium 19
3. Microscopic cultural characteristics of Perenniporia phlolophila MB 2403 20
4. Linear growth of Perenniporia medulla-panis and P. fraxinophila ' after 14 days at various temperatures 21
5. Growth and reactions of Perenniporia medulla-panis and P. fraxinophila on gallic acid medium and tannic acid medium 23
6. Microscopic cultural characteristics of Perenniporia medulla-panis JEA 832 24
7. Growth rates of Perenniporia phlolophila. P. medulla-panis. P. ohiensls and P. fraxinophila at temperatures 10-50°C 25
8. Mating test results of Perenniporia phlolophila MB 2401 28
9. Rearranged results of mating test of Perenniporia phlolophila MB 2401 29
10. Phase contrast photomicrographs of chlamydospores in the interaction zone of a negative mating of single spore isolates of Perenniporia phlolophila MB 2401 ... 30
11. Mating test results of Perenniporia medulla-panis JEA 834 31
12. Rearranged results of mating test of Perenniporia medulla-panis 32
13. Cultural interactions between intraspecific paired dikaryotic isolates of Perenniporia phlolophila and P. medulla-panis 34
. 14. Phase contrast photomicrograph of chlamydopsores in the interaction zone of paired dikaryotic isolates of Perenniporia phlolophila MB 2407 and 2412 35 7
LIST OF TABLES
Table Page
1. Isolates of Perenntoorla species used for this study and their sources ...... 12
2. Summary of macroscopic cultural characteristics of four Perennlporia species in North America 17
3. Percent weight loss In control and In vitro decayed wood and bark by Perennlporia species 36 8
ABSTRACT
Perennlporia phlolophlla (Aphyllophorales: Polyporaceae) colonizes the bark of live oak
(Quercus virainiana Mill.) and is known only in the southeastern United States in this host. Cultural
characteristics and mating systems of P. phlolophlla and P. medulla-oanls. vegetative incompatibility
of P. phlolophlla and temperature relationships and decay capacities of vegetative Isolates of P. phloiophila. P. medulla-panls. P. ohiensls and P. fraxinophila were investigated. Cultural studies indicate macroscopic and microscopic differences between the four species. Antagonistic hyphal interactions developed between different vegetative isolates. Self crosses were compatible.
Optimum temperature ranges and maximum growth temperature differed for all species. Mating test results of both species indicate their heterothallic tetrapolar nature. Woods differed significantly in percent weight loss (PWL) caused by each Perennlporia species. No significant difference occurred between different isolates of the same species tested on the same wood. PWL was greatest on oak wood for all fungal species tested. 9
Introduction
Murrill (1942) described the new genus Perenniporla with P. unita (Pers.) Murr. and P.
niarescens (Bres.) Murr. listed as species. Cooke (1953) and later Donk (1960) recommended that
the first name (P. unita) be considered the type species of the genus. "P. unlta" refers to Polvoorus
unltus Pers., a name now considered to be synonymous with Polvporus medulla-panls Jacq.:Fr..
Donk (1967) placed the latter in Perenniporla and Perenniporla medulla-panls (Jacq.:Fr.) Is now
considered the valid name of the type species of the genus.
Today Perenniporla (AphyllophoralesrPolyporaceae) is a large, cosmopolitan genus
occurring on dead and living hardwoods and conifers. Representative species occur throughout
Africa (Ryvarden and Johansen, 1980), Europe (Ryvarden, 1978) and North America (Gilbertson and
Ryvarden, 1987). Some demonstrate host specificity while others occur on a broad range of
substrata. The genus is characterized by generative hyphae with conspicuous clamp connections
in the dikaryotic stage and simple septa in the homokaryotic stage. The basidiospores are ellipsoid,
truncate, usually thick walled and have a variable dextrinoid reaction. Species of Perenniporla cause
white rots, varying from white root and butt rot of heartwood of living hardwoods and conifers, white
trunk rot, decay of bark tissues, to white rot of dead hardwoods and conifers (Gilbertson and
Ryvarden, 1987).
Cultural characteristics of wood-decaying fungi were first studied by Lyman (1907), Long and Harsch (1918), Fritz (1923), and Davidson, Campbell, and Blalsdell (1938). These works provided the basis for cultural studies of wood-decaying Basidiomycetes by other researchers.
Cultural morphology and sexuality were reported for several species of Perenniporla (Campbell,
1938; Nobles 1948,1958,1965; Stalpers 1978), but for other species these characteristics have not been published. Cultural studies provide additional information on the biology of wood decrying
Basidiomycetes that could improve the control of wood destroying fungi and provide Insights into using these fungi for beneficial purposes.
The objectives of this study were to determine the cultural characteristics and mating 10 systems of Perennlporla phloiophlla Gilbertson and M. Blackwell and P. medulla-panis (Jacq.:Fr.)
Donk, to investigate vegetative incompatibility between isolates of £. phloiophlla and to examine temperature relationships and decay capacities of vegetative isolates of P. phloiophlla. P. medulla- panis. P. ohiensis (Berk.) Ryv. and P. fraxinophlla (Pk.) Ryv. in the wood of ash, oak, white fir and the bark of oak. 11
Materials and Methods
Fruiting Body Collections and Identifications
Fruiting bodies of P. fraxlnophlla. P. medulla-panls. P. ohlensls. and P. Dhlolophlla were
obtained from Arizona, California, and Louisiana. Table 1 lists isolates of basldiocarps used in this
research, their sources, hosts, and location of collection for each of the species studied. Isolates
of P. fraxinophila were collected throughout Gardner and Madera Canyons in Arizona from different
trees. P. fraxinophila isolates 7A, 7B, 7C, 7D and P. ohlensls isolates 17890 and 17894 were
obtained from different locations on the same tree. P. phlolophila isolates were obtained from six
different trees at various sites on the campus of Louisiana State University.
Tissue from fresh specimens was prepared for microscopic identification by cutting out small
pieces of basidiocarp and placing them in 2% aqueous phloxine or Melzer's reagent (Ainsworth,
1983). Tissue from dried specimens was mounted either in Melzer's reagent or hydrated with a drop
of 95% ethanol and 3% potassium hydroxide (KOH) then stained with 2% phloxine. Collections by
R. L Gilbertson, James J. Flott, James E. Adaskaveg, Meridith Blackwell and Paul Skelly are designated RLG, JJF, JEA, MB and PS respectively.
Isolation, Cultural Descriptions, and Temperature Studies
Cultures of P. fraxinophila. P. ohlensls and P. medulla-panls were obtained from context tissue of basldiocarps while cultures of P. phloiophila were obtained from context tissue or bark under basldiocarps. Basldiocarps or colonized bark were broken exposing internal tissue, then flamed briefly. Small pieces (5mm X 5mm) of context tissue or colonized bark were cut from exposed inner surfaces with a sterile razorblade, flamed quickly, placed on 2% malt extract agar (MEA) slants and incubated at 25°C. Subcultures of all isolates were maintained at approximately 25*C.
Three isolates of P. phlolophila and three isolates of P. medulla-panls were studied according to
Nobles (1948). Cultural characteristics were examined microscopically by bright field and phase microscopy using 3% KOH, phloxine or Melzer's reagent. Tannic acid and gallic acid agar and gum 12
Table 1. Isolates of Perennlporla species used for this study and their sources
Species Isolate* Host Locality State
P. fraxinoDhila RLG 1-6, 7A, 7B, 7C, 7D Fraxlnus velutina Madera Canyon AZ Santa Rita Mts. Santa Cruz County
JJF 17891, 17892, 17895 Fraxlnus velutina Gardner Canyon AZ 17896, 17898 Santa Rita Mts. Santa Cruz County
P. medulla-Danls JEA832, 834 Prunus SD. Dayton CA J. E. Adaskaveg
PS28B Prunus virens Bear Canyon AZ Santa Catalina Mts. Pima County P. Skelly
P. ohlensls JJF 17890, 17893, 17894 Ouercus Gardner Canyon AZ arizonica Santa Rita Mts. Santa Cruz County
P. DhioioDhila MB 2401, 2402, 2403, Ouercus LSU Campus LA 2405, 2407, 2412 virainiana Baton Rouge Meridith Blackwell
* Isolates of each species listed by authors' or collectors' Initials and number. Collectors other than authors are listed under locality. 13 gualac solution (Davidson et al., 1938; Nobles, 1948) were used to determine the presence or absence of polyphenol oxidase in culture. The plates were inoculated in the center with a 7mm plug of agar from six week old cultures. After one week of growth in the dark at 25*C, the oxidase reactions were rated: negative (-) for no reaction; weak (+) for a reaction light in color and remaining under the mycelial mat or agar plug; moderately strong (++) for a dark reaction extending just beyond the mycelial margin; and strong (+ + +) for a dark reaction extending well beyond the mycelial mat margin.
Several drops of gum gualac solution (GGS) were applied directly onto cultures growing on 2%
MEA. A positive reaction was indicated if the mycelium turned blue within fifteen minutes.
Capitalized color names used In the cultural descriptions are from Ridgway (1912).
Temperature relationships of two dikaryotic isolates of each species (RLG 5 and RLG 6; JEA 832 and PS28B; JJF 17890 and JJF 17894; MB 2402 and MB 2412) were examined. Isolates were incubated for two weeks on 2% MEA at eight temperatures ranging from 10°C to 45*C in intervals of 5°C. Average growth was measured daily in millimeters from the edge of the inoculum to the edge of the advancing zone. Additionally, P. medulla-panis isolates were incubated at 50'C. The experiment had three replications for each isolate and was repeated twice.
Isolation of Homokaryons and Mating System
Baisdiospores of P. phloiophila 2401 were obtained from field-collected basidiocarps. Single spore isolation was obtained by pressing 5mm X 5mm pieces of the basidiocarp on 2% MEA plates, pore surface up, and placing them in a 10°C refrigerator overnight. The plates were inverted the next morning and a spore print was deposited on the lid approximately three days later. The spores were transferred with a loop of sterile water into a sterile water blank and mixed thoroughly. One or two loops of the spore suspension were transferred into a tube of 2% MEA melted in a water bath, cooled to near its gelling point (45°C) and poured into a petri plate. Several loops of spore suspension were also streaked on 2% MEA plates. Approximately two days later white colonies from single spores appeared and were transferred to 2% MEA slants. 14
Thirty single spore isolates were obtained in this manner, allowed to grow for two weeks, and examined microscopically. Isolates with clamp connections were presumed to be dikaryons and not used. Twenty-five homokaryotic isolates lacking clamp connections were then paired in all possible combinations in 60mm x15mm diameter petri plates containing 20ml of 2% MEA. After three weeks, small pieces of agar between inocula were mounted in 2% phloxine and examined microscopically for clamp connections and macroscopically for reaction zones. Positive matlngs
(clamps present) show that no alleles were shared by a particular pairing and negative matings
(clamps absent) indicate that an allele(s) was shared (Raper, 1966; Deacon, 1984). In addition, test blocks of silver leaf Oak (Quercus hypoleucoides A. Camus) in Erlenmeyer flasks were inoculated with actively growing mycelium of isolates MB2401, MB2402, MB2403, MB2405, MB2407, and
MB2412 in attempt to form basidiocarps for single spore isolation.
P. medulla-panls 834 basidiospores were collected from sporulating basidiocarps Induced in culture and field-collected basidiocarps. Homokaryons were isolated by the previously described method.
Vegetative Incompatibility
Dikaryotic isolates of P. phloiophlla were paired on 2% MEA in all possible combinations with three replications of each pairing. Isolates were placed 3cm apart on 25ml of MEA in Petri plates and incubated at 30°C. Self crosses served as controls. Hyphal interactions between opposing cultures were noted after six weeks. P. medulla-panls and P. fraxlnoohila isolates were also treated as above.
Decay Studies
The decay capacity of P. fraxlnoohila. P. medulla-panls. P. ohlensls and E. phloiophlla was evaluated using stock cultures RLG 6, JJF 17896, JEA 832, JEA 834, JJF 17890, JJF 17894, MB
2401 and MB 2407. Test blocks were prepared from silver leaf oak (Quercus hypoleucoides A.
Camus), San Pedro Vista, Santa Catalina Mountains; white fir /Abies concolor (Gord. and Glend.) 15
Until, ex Hlldebr.), Bear Wallow, Santa Catalina Mountains; velvet ash (Fraxlnus velutlna Torr.),
Gardner Canyon, Santa Rita Mountains; and the bark of emory oak (Quercus emorvi Torr.) Old
Prison Camp, Santa Catalina Mountains. The localities listed above are in the Coronado National
Forest, Arizona.
Agar-block decay chambers were prepared by a modified method as described by Gilbertson and Canfield (1972). All wood and bark blocks were cut 2 x 2 x 4cm with a large transverse face, oven dried at 110*C for 24 hours, placed in a desiccator for 24 hours and weighed. Blocks were rehydrated in a water bath for 2 hours and then sterilized at 120°C, 16psi for one hour in an autoclave. The blocks were then placed on V-shaped glass rods on 25ml of sterilized 2% MEA in eight ounce French square decay chambers. Decay chambers were inoculated with a 7mm plug of actively growing mycelium for each fungal isolate-wood combination. Five replications were prepared for each Isolate-wood combination. One chamber of each wood type was not inoculated as a control. The chambers containing wood blocks were incubated in the dark at 25*C for twenty weeks. Chambers containing bark blocks were incubated for sixteen weeks. Additionally, wood decay chambers containing isolates MB 2401 and MB 2407 on velvet ash and MB 2401 on silverteaf oak were also incubated at 30°C to determine temperature effect on wood decay capacity.
Following the incubation period, the decayed wood and bark blocks were carefully brushed clean of mycelium, oven dried and weighed as previously described. Percent weight loss was determined as a fraction of the oven dry weight lost over the original oven dry wood or bark block weight and multiplied by one hundred. Statistical analysis of wood decay results were performed using the
SPSS/PC statistics program. 16
Results
Cultural Descriptions
As no cultural descriptions of Perennlporia phlolophlla have appeared in the literature, one Is
given here. Additionally, a description of Perennlporia medulla-panls Is provided. Comparative data
of macroscopic cultural characteristics of species used in this study are given in Table 2.
Perennlporia phloiophila
Macroscopic. Growth rate moderately rapid, 2% MEA plates covered in three weeks at 25*C
(Fig. 1); margin of advancing zone even and appressed, aerial, white mycelium extending to the
edge of the advancing zone; mat white and remaining so for six weeks, tomentose to chamois-like,
becoming slightly yellowish around the inoculum after four weeks, then Chartreuse Yellow; mycelium
homogeneous over plate; tough, peeling readily from agar surface, reverse unchanged; no fruiting
before the end of six weeks; reaction on gallic acid agar medium strong, growth 0-5mm In four
weeks; reaction on tannic acid agar medium strong, growth 35-45mm in four weeks (Fig. 2); positive
reaction in gum guaiac solution after fifteen minutes; optimum temperature range of 30 to 35*C on
2% MEA, growth rate 5 to 6mm per day; some growth at 40-C, no growth at 45'C.
Microscopic. Hyphae of advancing zone 1-3/»m in diameter, thin walled, nodose-septate, dextrinoid, branched frequently, forming a fine network ending in thin whip-like tips; hyphae of aerial mycelium as in advancing zone; submerged hyphae as in advancing zone but coiled occasionally; numerous, rhomboidal randomly occurring crystals present; chlamydospores not present (Fig. 3).
Cultures examined: MB 2401, 2403 and 2407. Species code according to Nobles' (1965) system: 2, 3, 8, 32, 34, 37, 38, 43, 53, 54, 60.
Perennlporia medulla-panis
Macroscopic. Growth rate rapid, 2% MEA plates covered In two weeks at 25-C (Fig. 4); margin of advancing zone even, appressed or submerged; aerial mycelium extending to within 4mm of edge, white; portions of mat turning a light yellow at four weeks, then Martius Yellow, downy; 17
Table 2. Summary of macroscopic cultural characteristics of four Perennlporla species In North America
Optimum Growthf Maximum Growth/reactionsS Growth temperature rate growth Chlamydo- Species Color* habitt range (*C) (mm/day) temperature (°C) spores Gallic Tannic
P. fraxinoDhlla White Even, 25-30 4-5 35 + 0/+ + 20-35/+ + felty
P. medulla- White to Even, 30-35 6-6 45 + 5-20/+ + + 35-45/ + + + panis pale yellow downy raised
P. ohlensis White Even, 20-25 1-3 30 - 0/++ 0-1/+ + + felty
P. DhloloDhlla White to Even, 30-35 5-6 40 - 0-5/+ + + 35-45/+ + + pale yellow tomentose
* Macroscopic growth characteristics after 4 weeks. Color and growth habit as described by Nobles (1948). 4 Growth habit refers to margin and texture of the cultural mat. 4- Growth rate at optimum temperature. 5 Radial growth in millimeters after 4 weeks. Oxidase reaction recorded as: negative(-); weak, reaction light in color, and remaining under mycelial mat or agar plug (+); moderately strong, reaction dark and extending just beyond mycelial margin (+ +); and strong, reaction dark and extending well beyond mycelial margin (+ + +). 18
Figure 1 A-B. Linear growth of Perenniporia phloiophila and P. ohiensls after 14 days on 2% malt extract agar at following temperatures: (upper row, left to right) 10*. 15*, 20*, 25*C, (bottom row, left to right) 30*, 35*, 40*. 45*C. A. Linear growth of P. phloiophila. B. Linear growth of E. ohiensls. ~ HIS
m
Figure 2 A-B. Growth and reactions of (A) Perenniporia phlolophila MB 2412; and (B) P. ohlensis JJF 17890 on gallic acid medium (left) and tannic acid medium (right) in 90mm diameter Petri 20
Figure 3 A-C. Microscopic cultural characteristics of Perenniporla phloiophlia MB 2403. A. Generative hyphae. B. Slender branching hyphae with whip-like tips. C. Rhomboidal crystals. 21
Figure 4 A-B. Linear growth of Perenniporia medulla-panls and P. fraxinophlla after 14 days at various temperatures. A. Linear growth of P. medulla-panls on 2% malt extract agar (MEA) at following temperatures: (upper row, left to right) 10\ 15\ 20«C, (middle row, left to right) 25', 30*, 35"C, (bottom row, left to right) 40*, 45*, 50*C. B. Linear growth of P. fraxinophlla on 2% MEA at following temperatures: (upper row, left to right) 15\ 20*, 25-C, (bottom row, left to right) 30*, 35% 40*C. 22
mycelium continuous over plate, forming randomly raised (2-4mm), irregular shaped, dense tufts at
four to six weeks, reverse bleached; no fruiting before the end of six weeks; reaction on gallic acid
agar medium strong, growth 5-20mm in four weeks; reaction on tannic acid agar medium strong,
growth 35-45mm in four weeks (Fig. 5); positive reaction in gum guaiac solution after fifteen minutes;
optimum temperature range of 30 to 35° C on 2% MEA plates, growth rate 6-8mm per day, some
growth at 10-C and 45-C, no growth at 50*C.
Microscopic. Hyphae of advancing zone 1-2.5^m in diameter, thin-walled, nodose-septate,
dextrinoid, branching frequently, forming a fine network ending in thin whip-like tips; hyphae of aerial
mycelium as in advancing zone, submerged hyphae as above but 1-3.5/im in diameter and coiled
occasionally; chlamydospores few to numerous, terminal and intercalary, thick walled, ovoid to
ellipsoid, 4.0-5.0/im X 6.0-12.0/im; crystals lacking (Fig. 6).
Cultures examined: JEA 832, 834, and PS 28B. Species code according to Nobles' (1965)
system: 2, 3, 8, 34, 37, 40, 42, 53, 54, 60.
Temperature Relationships
Temperature studies with Perenniporia fraxinophlla isolates RLG 5 and 7, Perennlporla medulla-
panls isolates JEA 832, 834 and PS28B, Perenniporia ohiensis isolates JJF 17890 and 17894 and
Perenniporia phloiophila isolates MB 2402 and 2412 indicated different growth rates and optimal
temperature ranges for the four species (Fig. 7). Isolates of P. fraxinophlla have an optimal
temperature range between 25 and 30*C, with average growth rates from 2-6mm per day. Campbell
(1938) reported optimum growth at the same temperature range. Some growth of P. fraxinophlla isolates also occurred at temperatures of 15,20 and 35*C while no growth occurred at 10, 40 and
45-C. Isolates incubated at 10*C were transferred to 30*C after fourteen days and showed significant growth the following day. Isolates Incubated at 40 and 45*C were also transferred to
30*C after fourteen days but did not show growth after seven days. The thermal death point for all
E. fraxinophlla isolates tested was 40-C. 23
Figure 5 A-B. Growth and reactions of (A) Perenniporia medulla-panls JEA 834 and (B) P. fraxinophila JJF 17895 on gallic acid medium Oeft) and tannic acid medium (right) in 90mm diameter Petri dishes. 24
Figure 6 A-C. Microscopic cultural characteristics of Perennlporia medulla-panls JEA 834. A. Generative hyphae. B. Slender branching hyphae with whip-like tips. C. Chlamydospores. 25
A P. phtoiophiia P. medufla-pania
M 90 M H c P. ohiensis P. fraxinophila
M 44 to
Figure 7 A-D. Growth rates of Perennlporia isolates at temperatures 10-50*C. A. P. phiolophila MB 2402 and 2412. B. P. medulla-panis JEA 832.834 and PS 28B. C. P. ohiensis JJF 17890 and 17894. D. £. fraxinophila RLG 5 and 6. 26
Isolates of P. medulla-panls had an optimum temperature range and the fastest growth rate
between 30 and 35*C. At optimum temperature, growth rates ranged from 6-8mm per day and all
isolates covered the petri plates within ten days. No significant difference in growth rate was
observed at temperatures of 30 or 35°C. P. medulla-panls Isolates grew at the widest temperature
range of all species tested. Growth occurred from 10 to 45°C. No growth was observed at 50*C and isolates did not survive exposure to 50*C.
The temperature optimum of Perenniporia ohlensls Isolates observed in this study ranged from
20 to 25°C. This agrees with the earlier report by Campbell (1938). Average daily growth at optimum temperature ranged from 1 to 3.5mm per day. P. ohlensls isolates demonstrated the narrowest growth range of all species tested. Growth occurred at 15, 20, 25 and 30*C. However growth was very slow at 30°C after fourteen days. No growth was observed at 10°C and above temperatures of 35°C. Isolates incubated at 10°C were transferred to 25*C after twelve days and showed an average growth of 2mm at the end of two days. Isolates transferred from 35°C and
40°C to 25°C incubators after eight days demonstrated no growth following seven days.
The temperature optimum of Perenniporia phlolophlla Isolates observed in this study ranged from
30 to 35'C. Average daily growth at optimum temperature ranged from 5 to 8mm per day. P. phlolophlla isolates also showed growth at 15,20,25 and 40*C while no growth occurred at 10 and
45"C. Isolates transferred from 10 to 30'C following twelve days showed an average growth of
2mm at the end of twenty-four hours. P. phlolophlla isolates incubated at 45-C for seven days did not grow when transferred to 30° C.
Mating System
Attempts to induce basidiocarp formation of P. phlolophlla isolates in culture or on oak wood test blocks were not successful. However, single basidiospores collected successfully from the field basidiocarps of isolate MB 2401 were used to determine the type of mating system present In £. phlolophlla. The bipolar or tetrapolar nature of the species is indicated by the pattern of compatibility or incompatibility shown by the mating of single spore isolates. Results of pairing 27
twenty-five single spore homokaryotic Isolates of MB 2401 are shown in Figure 8. The matlngs fall
into a typical tetrapolar pattern, which when rearranged in Figure 9, tell into four distinct groups as
follows: A1B1-2, 3, 6, 7, 8 19, 20; A2B2-1, 5, 9, 11,14, 18, 21, 25; A1B2-10, 13, 23; and A2B1-4, 12,
15, 16, 17, 22, 24. These data confirm the heterothallic, tetrapolar nature of the species. Four
macroscopic reactions in the mated homokaryons were observed. These included: compatible,
heterozygous A and B factors; flat, (incompatible) common A factors; flat, (incompatible) common
A and B factors; and barrage, (incompatible) common B factors. Compatible reactions produced
abundant clamp connections on generative hyphae after two to three weeks. Incompatible matings
produced no clamp connections, but abundant chlamydospores previously not reported for P.
phloiophila were detected. The chlamydospores were not observed in six week old dikaryotic
cultures or in the compatible matings of single spore isolates. Chlamydospores were concentrated
in the region of the interaction zone. Chlamydospores were smooth, thick-walled, mostly intercalary,
ovoid to ellipsoid, 2-4 X 4-12/im and dextrinoid in Melzer's reagent (Fig. 10).
Attempts to induce basidiocarp formation of P. medulla-panis in culture were successful.
Basidiospores were collected from sporulating basldiocarps in culture and from field collected
basidiocarps of isolate JEA 834. Results of pairing twenty-five single basidiospore isolates of JEA
834 in all combinations are shown in Figure 11. When rearranged in Figure 12, the homokaryons
fell into four mating types as follows: A1B1-1, 8, 10, 11, 18, 26, 27; A2B2-5, 6, 7, 15, 17, 23, 24;
A1B2-2, 9, 19, 22; and A2B1-4, 12, 13, 14, 16, 20, 25. The matings and macroscopic reactions
confirm the heterothallic, tetrapolar nature of the species. Macroscopic reactions of homokaryotic
matings of isolate JEA 834 were as described for P. phloiophila above. Chlamydospores, present
only in the interaction zones of P. phloiophila. occur throughout the homokaryotic isolates of P.
medulla-panis. They are smooth, thick-walled, mostly intercalary, ovoid to ellipsoid, 2-4/im X 5-
8Mm and dextrinoid in Melzer's reagent. 28
II 12 (3
Figure 8. Results of mating test with 25 single spore isolates of Perennloorla ohlolophila MB 2401. 29
2 3 fcl7lfl
Figure 9. Rearranged results of Perenniporla phlolophlla MB 2401 mating test. . Indicates barrage effect. 30
Figure 10. Phase contrast photomicrographs of chlamydospores in the Interaction zone of a negative mating of homokaryotic isolates 5 and 11 of Perennlporia phlolophlia MB 2401. Bar = 20M- 31
19 zcJHEJlW IS\ZL 27
Figure 11. Results of mating test with 25 single spore isolates of Perennlporia medulla-panls JEA
i 32
A.B< AaBi AcBz v AaBi //|/8lz^l27t5l 1 \1s\n\12\vi\2. 1 11 22 n\l3 li 20 +- + 4- -H-H4-I-H — +-I+-I4-I 10 II 14-14- n H-N* +4+J+-I-H 21, I+I+- 27 + + +*1+1
IS •H+-1 +~ /7 23 4- 14-|4-
4*14-| 4- 4- 4-|4-|4- & 4-4-4- 22 4- 4-14- 4-
|4-4-
Flgure 12. Rearranged results of Perennlooria medulla-panls JEA 834 mating test. ^ Indicates barrage effect 33
Vegetative Incompatibility
All pairings between different dikaryotic Isolates of P. phlolophlla resulted in similar antagonistic interaction zones, indicating they are different genotypes. The results are shown in Figure 13. P. phlolophila isolates characteristically develop tough, thick mycelia mats on 2% MEA. When paired, opposing cultures grew within one to five millimeters of one another. Hyphae were submerged and abundant in the dark pigmented zone between each isolate. The interface margins of each opposing mat were characterized by a buildup of mycelium, often brown to dark brown in color.
All self paired isolates resulted in complete intermingling of isolates with no interaction zone.
Compatible reaction zones between self paired isolates were characterized by uniform mat color and the absences of dark interaction zones and chlamydospores.
Hyphal interaction between opposing isolates resulted in an abundant production of chlamydospores in the interaction zone (Fig. 14). Chlamydospores were not observed in either isolate of opposing isolates or in self-paired compatible isolates. The chlamydospores were smooth with thin or thick walls, dextrinoid, ovoid to ellipsoid and ranged in size from 6-10/zm X 6-18/im.
Similar observations of abundant chlamydospore production were also seen in the interaction zones of negative matings of homokaryotic isolates of P. phlolophlla.
Decay Studies
The results of wood decay studies are presented in Table 3. Average percent weight loss (PWL) for the five blocks of each wood-isolate combination was determined. The woods differed significantly (P=0.05) in PWL caused by each Perennlporia species. The controls of each wood type lost a small percentage (0.0-3.2%) of their original weight during the preparation and sterilization procedures. The PWL of the wood shown for each isolate was not corrected for the control weight loss. There was no significant difference between different Isolates of the same species tested on the same wood species at the same temperature. PWL was greatest on oak wood for all species tested. P. ohiensis isolates caused consistently less weight loss in all woods tested. P. medulla-panis isolates caused the greatest weight loss on white fir while P. fraxinoohlla 34
Figure 13 A-B. Cultural interactions between intraspecific paired dikaryotic isolates of Perennlporia phloiophlla and P. medulla-oanis on 2% malt extract agar. A. Three isolates of E. phlolophlla (MB 2402, 2403 and 2405) paired in all combinations. B. Three isolates of P. medulla-panls (JEA 832, 834 and PS 28B) paired in all combinations. Self-pairings in A and B are the first plates of each row. Figure 14. Phase contrast photomicrographs of chlamydospores in the interaction zone of paired dikaryotic Isolates of Perennlporia Dhlolophlla MB 2407 and 2412. Bar = 20M. 36
Table 3. Percent weight loss in control and in vitro decayed wood and bark by Perennlporia species.*
Wood type Isolate Percent Weight Loss*
White fir Control 0.97:t 0.06a P. fraxinophila 6 12.31 i : 1.34d P. fraxinophila 17896 16.46 i: 0.98c P. medulla-panis 832 32.45 it 1.74e P. medulla-panis 834 29.73 i s 1.72e P. ohiensis 17894 11.58 i: 0.66b P. ohiensis 17890 13.18 i t 0.73b
Velvet Ash Control 1.13 :t 0.10a P. fraxinophila 6 19.65 j; 1.75d P. fraxinophila 17896 15.87 i: 1.35C P. medulla-panis 832 19.50 i: 1.82d P. medulla-panis 834 10.29 i: 1.64d P. ohiensis 17894 6.60:t 0.63b P. ohiensis 17890 6.88:t 0.45b P. phloiophila 2401 7.63:t 0.66b P. phloiophila 2401* 14.43 i: 1.42C P. phloiophila 2407 9.20:t 0.80b P. phloiophila 2407# 15.74 i: 1.33c
Silverleaf Oak Control 1.48:t 0.15a P. fraxinophila 6 46.84 i: 4.77f P. fraxinophila 17896 41.32 i: 6.40f P. medulla-panis 832 41.20 i: 4.03e P. medulla-panis 834 30.88 i: 4.07e P. ohiensis 17894 8.45:1 0.85b P. phloiophila 2401 21.63 i: 2.10C P. phloiophila 2401* 35.88i: 3.59d
Emory Oak bark Control 2.02 :t 0.21a P. phloiophila 2403 6.74:t 0.34b P. phloiophila 2405 4.26:t 0.56a
* Average percent weight loss determined from replications after 20 weeks at 25'C. • Weight losses for the same wood type followed by the same letter are not significantly different at P=0.05 (Duncan Analysis). 4 Incubated at 30*C. 37 caused the greatest PWL on oak and ash wood types. Significant differences occurred in PWL when P. phlolophila isolates 2401 and 2407 were tested at different temperatures on oak and ash wood. Increasing the temperature from 25*C to 30*C caused a significant increase in PWL
Mycelium of P. phlolophlla isolates colonized the emory bark blocks heavily, but only isolate MB
2403 caused a significant PWL 38
Discussion
A cultural description for P. phlolophila has not been previously reported in the literature.
Distinctive macroscopic cultural characteristics of the four species studied include mat color and mat texture. Two species, P. phlolophila and P. medulla-panls can be differentiated from the other two species based on these characteristics. £. phlolophila has a tomentose mat texture and mostly white to pale yellow mat color while P. medulla-panls has a raised downy mat texture and a white to pale yellow mat color. Mat color and texture were similar In the other two species. Although some macroscopic characters of these fungi can be distinguished, similarities in morphology limit their sole use in the identification of species.
Distinctive microscopic features of P. phlolophila include the presence of rhomboidal crystals and the apparent lack of chlamydospores. Distinctive microscopic characteristics of P. medulla- panls include the presence of chlamydospores and the lack of crystals. Generative and coiled and slender hyphae forming a network ending in whip-like tips were present In both P. phlolophila and
P. medulla-panls. All hyphal types mentioned above have ciamp connections.
Humphrey and Siggers (1933) emphasized the importance of a knowledge of the growth rate of wood destroying fungi at'different temperatures and the possible manipulation of temperatures as a control measure for decay. Temperature relationships of the species studied correlated with geographical distribution of these fungi (Gilbertson and Ryvarden, 1987). P. phlolophila has a high optimum temperature range between 30 and 35*C, grew at 40-C and is consistently associated with live oak (Quercus virqiniana Mill.) in the southeastern United States (Gilbertson and M. Blackwell,
1984). P. medulla-panls isolates demonstrated the widest growth range (10-45'C) of the four species and a high optimum temperature range of 30 to 35cC. This may account for its' wide geographical distribution throughout temperate and tropical climates (Ryvarden and Johansen, 1972;
Gilbertson and Ryvarden, 1987). P. fraxlnophlla is found throughout North America (Gilbertson and
Ryvarden, 1987) and has an optimum high temperature range between 25 and 30"C. and grew well at 35-C. P. ohiensis has the lowest optimum temperature range (20-25'C) and the narrowest 39
growth range (15-30*C) of the four species in this study. However, it occurs throughout North
America (Gilbertson and Ryvarden, 1987) and collections for this study are from low elevation forests
in Arizona.
Perennlporia phlolophila isolates failed to form basidiocarps in culture or on silverleaf oak wood
blocks. However, basidiospores collected from the field basidiocarps were used to determine the
mating system of the species. Mating test results confirm the heterothallic tetrapolar nature of £.
phlolophila. This agrees with reports of sexuality (Montgomery, 1936; Nobles, 1957) for other
species of the genus. Typical macroscopic reactions in the homokaryotic matings were observed.
Microscopic observation of the reaction zones of negative matings of homokaryons revealed an abundance of chlamydospores. Although germination was not tested, chlamydospores observed microscopically contain cytoplasm and appear viable. Chlamydospores were not observed in homokaryotic and dikaryotic cultures of P. phlolophila nor in the reaction zone of positive homokaryotic matings. The presence of chlamydospores in the reaction zone between negative homokaryotic matings of P. phlolophila could aid cultural identification studies.
Vegetatively incompatible dikaryotic isolates of P. phlolophila. P. medulla-panls and P. fraxinophlla resulted in antagonistic interaction zones. All non-self pairings between dikaryotic isolates of the same Perennlporia species obtained from different trees of natural populations resulted in antagonistic reactions. The formation of interaction zones between heterokaryons has been reported and Interpreted by other researchers (Adams and Roth, 1967; Caten, 1972; Rayner and Todd, 1977; Hansen, 1979; Goldstein and Gilbertson, 1981; and Adaskaveg and Gilbertson,
1987,1989) to indicate the presence of different genotypes within a population of wood decay fungi.
Compatible reactions occurred in the self-pairings of isolates of the same species and between four isolates of P. fraxlnophila collected from the same tree. Barrett and Uscuplic (1971) reported that individual trees of Sitka spruce populations are usually colonized by one strain of Phaeolus schweinitzil (Fr.) Pat. not normally found in any other tree. Adaskaveg and Gilbertson (1987) made similar observations for Ganoderma lucidum (W.Curt.:Fr.) Karst. on silverleaf oak. This suggests 40 one dikaryon of P. fraxinophlla colonized the tree and produced multiple fruit bodies as no genotype was found to be present in different trees of host populations studied. From this occurrence of different strains, even in adjacent trees, it can be suggested that basidiospores are important in the infection process and these fungi exist as populations of different genotypes. As reported earlier
In this study, P. phlolophila does not produce chlamydospores in culture. However, chlamydospores were produced only In the interaction zone of Intraspeciflc dikaryotlc incompatible pairings of P. phlolophila. Chlamydospores observed microscopically contain cytoplasm and appear viable.
All Perennlporla isolates decayed all wood and bark species tested. With the exception of P. medulla-panis. percent weight loss (PWL) was generally lower on velvet ash than sllverleaf oak or white fir. The P. medulla-panls isolates generally caused a greater PWL than the other species tested while P. ohiensls Isolates generally caused the least PWL The rapid growth rate of P. medulla-panis and the slow growth rate of P. ohiensls in relation to the other species at the temperature selected in this study may account for this observation. Merrill (1970) reported the optimum temperature for decay may be the same or a few degrees lower than the optimum temperature for enzyme activity and mycelial growth. This was demonstrated in this study with P. phlolophila isolates tested on sllverleaf oak and velvet ash wood at different temperatures. As mentioned earlier in this report, optimum growth range for P. phlolophila is 30' to 35*C. A significant (P=0.05) increase in PWL occurred for all wood-isolate combinations of P. phlolophila tested when the temperature was increased from 25* to 30*C.
The bark of live oak is the natural nutrient source of P. phlolophila. P. phlolophila isolates caused substantial PWL on all wood types tested. All emory oak bark blocks were heavily colonized by the mycelium of all P. phlolophila isolates tested. However, there was no significant difference between weight loss in bark decayed by P. phlolophila isolate MB 2403 and the control.
Additionally, in vitro decay tests of P. phlolophila isolates resulted in significantly less PWL on bark than on all woods tested. Chang (1955) determined significant differences between the chemical 41 composition of wood and bark. Martin (1969) expressed doubt about whether the cellulose .of bark is the same as that of wood and that lignin in bark may vary in chemical structure from that found in wood. This may account for the difference in PWL of P. phlolophila isolates on bark and wood.
Greater knowledge of the degradation of wood and bark should provide information needed not only to improve the control of wood decay fungi but also to provide insights into using these fungi for beneficial purposes and new biotechnological processes. 42
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