STUDIES OF GANODERMA LUCIDUM AND GANODERMA TSUGAE (DELIGNIFICATION, MATING SYSTEMS, ROOT ROT, CULTURAL MORPHOLOGY, TAXONOMY).
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Adaskaveg, James Elliott
STUDIES OF GANODERMA LUCIDUM AND GANODERMA TSUGAE
The University of Arizona PH.D. 1986
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STUDIES OF GANODERMA LUCIDUM AND GANODERMA TSUGAE
by James Elliott Adaskaveg
A Dissertation Submitted to the Faculty of the DEPARTMENT OF PLANT PATHOLOGY In Partial Fullment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA
1 986 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have read James Elliott Adaskaveg the dissertation prepared by Studies of Ganoderma lucidum and Ganoderma tsugae entitled ______~======~=====: ____ ======~==-==~ ______
and recommend that it be accepted as fulfilling the dissertation requirement Doctor of Philosophy for the Degree of ------
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Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation re£:;:P~ Dissertation Director Date STATEMENT BY THE 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 borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement 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 judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. DEDICATION
I would like to dedicate this work in memory of a very special uncle, Michael Venditto, who inspired my agricultural interests and to my mother and father, Fannie and Alex Adaskaveg, whose love was always felt and whose personal sacrifices, constant support, and faith have allowed me to follow my interests and obtain my goals. Although my appreciation was not often spoken it was always heartfelt. Thank you ever so much.
ii i ACKNOWLEDGMENTS
The author wishes to express sincere graditude to the
University of Arizona for providing facilities and financial support for this research. I wish especially to thank: Dr. Robert L. Gilbertson for his supervision and advice during these studies ~ut most of all for his consistent helpfulness, stimulating interest, patience, and great friendship; Dr. Richard B. Hine for his constant support and interest in my behalf and for his kind friendship; and Dr. George B. Cummins for his words of wisdom and sincere friendship. I would also like to thank: Dr. M. Nelson for his support and interest in this research; Dr. M.E.
Stanghell ini, Dr. M.A. McClure, Dr. S.M. Alcorn, and Dr. I. Misaghi for thei r research advice and assistance, support and interest, for thei r critical revie~is of my work, and for their generosity in providing countless resources; Dr. M. Blackwell (Louisiana State University) for her GanodermA collections, literature searches, and unending support and interest; Dr. P.J. Cotty for his technical advice with chemical analyses; Dr. J. Mihail for her critical reviews and advice in statistical analyses; David Bentley for his personal help and support in providing resources and expertise in electron microscopy and photography; Tom Kruk for his critical reviews; Dr. G. Dutt for obtaining grape wood; and Dr. E.A. Mielke for use of field grape plants. Further thanks to Bobbie Malandrone in her memory; Majorie
Braun for all her hel p in the office; Dr. K.H. Yohem for her Ganoderm.ll collections; Plant Pathology Department members for many happy
iv v memories; and for all mentioned their friendship. I would like to also thank family members: Michael and Maryellen for the photographic equipment and all the phone calls that brightened my day; Alex and Debbie for their support and encouragement; Keith W. Leahy, who was more than a great cousin but a sincere and heartful friend whose inpromt collecting of Ganoderma specimens helped me significantly; and Frank and Julia Venditto for their personal sacrifices and inspiration. Most of all I would like to express my greatest thanks to Peg Mauk for all her personal sacrifices, continuing and unending assistance in all aspects of my research, and most sincerely for her steadfast faith and boundless love in standing by me in toubled and triumphant times. TABLE OF CONTENTS Page LIST OF TABLES viii
LIST OF ILLUSTRATIONS • • ix
ABSTRACT xi 1. CULTURAL STUDIES AND GENETICS OF SEXUALITY OF GANODERMA LUClPUM AND ~ TSUGAE IN RELATION TO THE TAXONOMY OF THE ~ LUCIPUM COMPLEX • • • • • • • • • • • • 1
Introduction • • • • • • • • • • • • • • • • • • 1 Materials and Methods •••••••••••••• 5 fruiting Body Collections and Descriptions •••• 5 Macroscopic Cultural and Temperature Studies 5 Microscopic Cultural and Basidiospore Morphology Studies ...... 7 Isolation of Homokaryons, Mating Systems, and Interfertility Studies ••••••••• 8 Results •• • • • • • • • • • • . • • • • . • 9 fruiting Body Descriptions •••••••••••• 9 Macroscopic Cultural and Temperature Studies ••• 10 Microscopic Cultural and Basidiospore Morphology Stud 1es • • • • • • • • • • • • • • • • • • • • • • 14 Mating Systems and Interfertility Studies •• 17 Discussion • • • • • • • • • • • • • • • • • • • • • • • 19 2. IN Y1IBQ DECAY STUDIES Of SELECTIVE DELIGNlfICATION AND SIMULTANEOUS DECAY BY THE WHITE-ROT fUNGI GANOpERMA LUCIDUM AND .G.... TSUGAE • • • • • • • • • • • • • • • 26 Introduction • • • • • • • • • • • • • • • • • 26 Materials and Methods •••••••• 28 fungal Isolates and Wood Samples 28 In ~ Wood Decay Studies ••• 28 Chemical Analyses of Wood Blocks 29 Microscopic Observations ••••• 30 Results ••••••••.••••••• 31 In ~ Wood Decay Studies of Gaooderma luc1dum and ~ tsugae ••••••••••••••• 31 Chemical Studies of Wood Decayed by Gaooderma luc1dllm and ~ tSlJgae • • • • • • • • • • ••• 33 Scanning Electron Microscope Studies ••••• 35 vi vii TABLE OF CONTENTS--Goot1olJed
Page Discuss10n •• ...... 40
3. INFECTION AND COLONIZATION OF YlIlS YINIFERA BY GANOpERMA LUCIPLIM ••••••••••••••••••••• 46
Introduction • • • • • • • • • • • • • • • • 46 Materials and Methods •••••• • • • • • • • •• 47 Grape Cutting and Propagation and Culturing of Gaooderma lucidum collections •••• 47 Greenhouse Studies • • • • • • • •• 48 Field Studies • • • • • • • • • • • • 49 Resu 1ts •• • • • • • • • • • • • • • • • 50 Ident1 f1cat1on of Cultures of a.. lllc1dllm 50 Pathogenicity of a.. lllc1dllm on Grape Plants Grown in the Greenhouse • • • • • • • • • • • 51 Development of a.. lllc1dllm in Field Grape Plants •• 53 Discussion • • • • • • • •• • • • • 58 LITERATURE CITED 61 LIST OF TABLES
Table Page 1. Isolates of Gaooderrna luc1durn and a.. tSlIgae and their sources 6 2. Percent weight loss and percent change of Klason 11gn1n and chlorite holocellulose in control and in ~ de- cayed wood by Gaooderrna luc1durn and a.. tsugae • • •• 32 3. Percent K1ason lignin, chlorite holoce11ulose, and K1ason lignin and holocellu10se ratios in entire samples of controls and wood decayed in ~ by Gaooderrna luc1durn and a.. tsugae •• • • • • • • • • • • • • • • •• 34
viii LIST OF ILLUSTRATIONS
Figure Page
1. L1near growth of Ganoderma lllcidum and G.... tsugae after 14 days at various temperatures and cultural reactions of homokaryons of G.... tsugae in mating system studies 12
2. Growth rates of Ganoderma luc1dum and G.... tsugae iso- lates at temperatures 15-42 C • • • • • • • • • • • • •• 13
3. Growth rates of Ganoderma res1naceum and G.... vales1acum isolates at temperatures 15-42 C • • • • • • • •• 15 4. Microscopic characters of Ganoderma lucidum • • 16 5. Bright-field and scanning electron microscopy of basidio- spores of Ganoderma tSllgae and G... lllc1dum • • • • • • 18 6. Interfertility tests with two compatible mating types re presenting four alleles of each of three Ganoderma lllc1dllm isolates and one G... tsugae isolate • • • • • 20 7. Scanning electron micrographs of white fir and Douglas fir controls and wood decayed by Ganoderma lllc1dum and G..... tsugae . . . • • . . . . . • . . . • ...... 37 8. Scanning electron micrographs of grape and oak controls and wood decayed by Ganoderma luc1dum and G... tSllgae • • • •• 39 9. Scanning electron micrographs of transverse sections of oak wood decayed by Ganoderma luc1dum • • • • • •• 41 10. Two fruiting bodies of Ganoderma lucidllm on a Dog Ridge variety grape plant grown and inoculated in the green- house • • • • • • • • • • • • • • • • • • • •• 52 11. Stages of decay in the development of Ganoderma luc1dllm within Dog Ridge variety grape plants grown and inoc- ulated in the greenhouse • • • • • • • • • • • 54 12. Control plant and plants inoculated with Ganoderma lllc1dllm under field conditions • • • • • • • • 56
ix x LIST OF ILLUSTRATIONS--Coot1nued
Figure Page 13. Transverse section showing natural infection of Gaooderma luc1dum in a field grape plant •••• 57 ABSTRACT
Gaooderma ] IIC i dum and Ji. tsugae are two members of the Ji. lucidum complex that have had a controversial taxonomic relationship. The authenticity of the two widely distributed wood-rotting species was demonstrated by comparative studies. Gaooderma ]ucjduID is restricted to hardwoods. Its "smooth" walled basidiospores were characterized by narrow, numerous inter-wall pillars. Isolates of!h lucjdum produced chlamydospores in culture and had an average growth rate of 7.8 mm/da at their optimum temperature range of 30-34 C. GaoodermA tsugae is restricted to conifers. Its basidiospores were "rough" wal led and had broad inter-wall pillars. Isolates of !h tsugae did not produce chlamydospores in culture and had an average growth rate of 2.1 mm/da at the optimum temperature range of 20-25 C. Mating systems were determined for both species as heterothallic and tetrapolar. Interspecific matings of homokaryons were completely incompatible. Homokaryons of a European Ii.. resjoaceum isolate that fruited in culture were completely interfertile with homokaryons from North American collections of Ii.. lucidwm.
The ab 11 ity of Ii.. luc j dum and !h tsugae to decay wood in y;tro was studied using the following woods in agar block decay chambers: grape, oak, mesquite, white fir, and Douglas-fir. Grape wood lost the most weight while mesquite the least. !h ]ucjdum isolates generally caused greater weight loss of all woods than did Ii.. tsugae isolates. Both Gaooderma species caused simultaneous decay in all woods. However, xi xii . chemical analyses of the decayed blocks indicated that selective delignification by both species also occurred in grape and white fir blocks but not in oak or Douglas-fir blocks. Scanning electron microscopy demonstrated selective delignification and simultaneous decay of all woods tested. However, the extent of the delignification varied between the wood species. Isolates of Ganoderma luc1dum infected Dog Ridge variety grape plants, grown in the greenhouse, from below-ground wood block inoculations. The fungus developed in the heartwood and, in later stages, invaded the sapwood. Infected pl ants developed water stress symptoms with leaves wilting, yellowing, and dying. In the wounded plant treatment, one plant died and 3 other plants continued to decline while in the non-wounded plant treatment only one plant developed
symptoms. Reisolations from 24 inoculated plants yielded only ~ lucidum from the five declining plants, demonstrating the pathogenicity of the fungus after 24 mons. Field grape plants inoculated with the fungus developed decay columns as large as 42 cm in 17 mons. Decay was 1 imited to the heartwood and no symptoms were observed on the field plants at the end of the study. CHAPTER 1
CULTURAL MORPHOLOGY AND GENETICS OF SEXUALITY
OF GANOpERMA LUCIDUM AND G. TSUGAE IN RELATION
TO THE TAXONOMY OF THE G. LUCIDUM COMPLEX
Introduction
Ganoderma was establ ishment by Karsten (1881> with G. lucidum
(W. Curt.:Fr.) Karst. as the only species. Karsten mistakenly
attributed the epithet luc1dum to von Leysser, and this error has been
perpetuated in numerous subsequent publications. Patouillard (1889)
listed 48 species worldwide in his treatment of the genus including ~
resinaceum Boud. Boudier and Fischer (1894) and Boudier (1895)
described G. yalesiacum from conifers in Europe. Murrill (1902, 1908) published synopses of species occurring in North America, ommitting G.
lllcidum from his 1908 publication. Murrill described seven new temperate
species including G.. tsugae, G.. sessile, G. zonatum, G.. sulcatum, G.
oregonense, G. sequoj ae, and G. nevadense, a'long with 10 others from
tropical areas. Murrill also accepted G.. cllrtisji polychrom.u.m (Copel.) Murr. as North American species. Murrill considered primary taxonomic characters to be host specificity, geographical distribution, and macromorphology of the fruiting body. The latter included context color, the shape of the margin of the pileus, and whether the fruiting body was stipitate or sessile. Subsequently, Atkinson (1908), Ames (1913), Haddow <1931>, Overholts (1953), Steyaert (1972-80), Bazzalo and Wright (1982) and 1 2 Corner (983) studied selected species of Ganoderm.a.. Each considered some species recognized by Murrill to be valid, whereas others were treated as varieties or synonyms. Haddow 0931> and Steyaert (980), based most of their taxonomy on spore characteristics and morphology of cutis hyphal elements of the fruiting body. Steyaert also emphasized context color. Despite similarities in the characters used, these two workers did not agree on the identity of species present in North America. Overholts (1953) recognized only four North American species in the G... lucidl!m group, placing them in the Friesian genus Polyporus rather than Ganoderma. His taxonomy was based on geographical distribution, host specificity, macroscopic morphology, and spore characteristics. Overholts recognized Polyporus ]ucidus and ~ tsugae as distinct species in North America and considered .G. sessile, G.. po] ychromum, .G. zonatlJrn, and .G. slJlcaturn as synonyms or -varieties of E. luc1dIlS. Other species recognized by Overholts were E. r.lJrtisii and E. oregonense. Karsten attributed the basionym Boletus lucidus, published in 1783, to von Leysser in Flora Halensis. However, William Curtis described and illustrated this taxon as Boletus llJcjdus in 1781- Steyaert <1961> designated plate no. 224 in William Curtis' Flora Londinensis (1781) as the type of the species, in accordance with article 10 of the International Code of Botanical Nomenclature adopted by the 8th International Botanical Congress, Paris, July 1954. An English-Latin description accompanied the drawing and it appeared to Steyaert that Curtis intended publishing a new binomial. Curtis stated 3 clearly in the proto1ogue that a.. lycidus was collected from hazel (presumably Corylys ayellana L.) an elm (!J.l.m.U.s. sp.). However, Steyaert and other authors did not mention the host relationship which is a critical clue to the true identity of Boletys lycidus. Curtis referred to Boletys rugosus Jacq., pub1 ished in 1774, as a possible earl ier name for this taxon, however no host information is included in the description (Jacquin 1774, pl. 169) and therefore the identity of ~ rUQosys is uncertain. Jacquin also described what may be the same fungus as Agaricys pseudoboletys from oak in 1773 (pl. 41), but his description and illustrations are of immature specimens. The identity of 8..... pseudoW:U~.s. is uncertain. Murrill (1902) accepted 8..... pseudoboletus as the correct basionym of the species included here as .Ga. lycidym, but dropped all mention of the former name in North American Flora (1908). Other names prior to 1781 and that may have been based on the fungus included here as .Ga. lycidym include ~ flabelljforme Scopo1i (1772), a.. yariegatus Schaeffer (1774, pl. 263), and a.. obl itjyatys Bull iard (1780, pl. 7). These are of uncerain appl ication due to lack of adequate substrate or morphological data. Corner (1983) summarized the taxonomy of the Ganodermataceae. In his review of Steyaert's classification of Ganoderma, Corner noted gradations of every feature used to describe species. While Corner (1983) accepted Steyaert's basic framework for the subdivisions of Ganoderm.a., he maintained that taxonomy of the species should be simplified to base a new classification on basidiocarp developmental studies in the field. 4 Nobles <1948, 1958) indicated differences in the cultural characteristics of .(h lucidum, .(h tsugae, and .(h oregooeose. Nobles (1965) changed the name of her isolates previously 1 isted as Go.. lucidum to G. sessile, which Steyaert (1972) considered a synonym of Go.. res;oaceum. Stalpers (1978) bel ieved .(h resioaceum to be the correct name for the fungus described as Go.. lucjdum in culture by Davidson et ale (1942) and Nobles (1948). The cultural characteristics of G.... resioaceum given by Bazzalo and Wright (1982) agree with the concept of Nobles (1965) and Stalpers (1978). The description of cultures of G.... lucidllm given by Bazzalo and Wright is very similar to that of G. tsugae as described by Nobles. Further, Stalpers (1978) considered the cultural characteristics of the European G... valesiacum identical to those of .G... tSllgae from North America and listed Go.. tsugae is a synonym of G... yalesjacum. Nobles (1948) suggested interfertility tests be used to determine the identity of species. Homokaryons of Gaooderma species have been difficult to obtain, however, because of the low percentage or total lack of basidiospore germination as indicated in earlier work (Bose 1927; Venkatarayan 1936; Aoshima 1954; Menon 1963; Merrill 1970). Banerjee and Sarkar (1958b) reported a heterothallic, tetrapolar mating system for.G... lucjdllm. However, their descriptions of the spores and basidiocarps of Indian collections (Banerjee and Sarkar 1958a, 1959) differed significantly from those of species in the G... ]ucjdllm complex on hardwoods in North America. Thus, the identity of the taxon in their study is uncertain. The objectives of the study were to determine the mating system of the North America Gaooderma species found on hardwoods and conifersl 5 to investigate interfertility among isolates and between hardwood and conifer isolates, to compare European Gaooderma species with North American species, and to investigate temperature relationships and other cultural characteristics that will provide a more firm basis for clarifying the taxanomy of the North American species of Gaooderma. Materials and Methods Fruiting Body Collections and Descriptions Table 1 lists the isolates used in this research, their sources, and other pertinent collection data. Holotype or isotype specimens of.G.a. resioaceum (Boud.) Pat. (FH), .G.. sessiJe ~'urr., .G.a. oeyadeose Murr., .G.a. seQuoiae Murr., .G.a. oregooeose Murr., .G. sulcatum Murr., and .G.a. zooatum Murr. (NYBG) were examined. The neotype of .G....tsugae ~'urr. (NY), and G... rayeoelii Stey. (K) and ~ ~~ (K) were also examined. Cultures designated .G.a. yalesiacum (from a fruiting body on a conifer) and .G... resioaceum (from a fruiting body on a hardwood), both from Europe (Centraalbureau voor Schimmelcultures, Baarn), were also examined. Macroscopic Cultural and Temperature Studies Cultures were obtained from context tissue of basidiocarps and grown on 2% malt extract agar (MEA) medium at approximately 25 C. Cultural morphology was studied according to Nobles (1948). Tannic and gallic acid agar media and gum guaiac (Davidson et al 1938; Nobles 1958) were used to test for polyphenol oxidases. To study bas1d10carp formation, cultures of RLG 14291 and JEA 6 Table 1. Isolates of GaDcdecma ]ucidum and G.... tsugae and their sources. Species Isolate Host Habit Locality State !hlucldum olEA 245 Quercus Stipltate Madera Canyon AZ hvpoleucoides Santa Rita ~t!. Santa Cruz Coun:, RLG 14291 Quercus Sessile Maderll Canyon AZ hvpoleucoldes Santa Rita Mts. Santa Cruz County JEA 141 ~sp. Sessile Fontainebleau S.P. LA St. Tammany Parish M. Blackwell· JEA 140 Acer saccharum Sessile Cllmp Duessel OM Portage County K. H. Yohem JEA 266 Cercidlum Stlpltate Tucson AZ mlerophyllum Pima County JEA 274 ~negundo Sessile U of RI RI Kingston, RI H. M. Burdsall !h tsugae JEA 207 Ables con color Sessile Bear Wallow AZ Santa Catalina Mts. Pima County JEA 268 ~sp. Sessile Scott River Rd. CA Klamath N.F. Siskiyou County F. Nishida JEA 203 ~sp. Stipitate U of MN Campus MN St. Paul R. A. Blanchette JEA 218 Tsuga canadensis Stipltate Meriden Min. CT Southington HarHord County eNames of c"Uectors other than the authors are also listed here. 7 141 were also grown on silver leaf oak wood blocks approximately 10 x 4 x 4 cm for 6 weeks aseptically in agar-block decay chambers. The colonized wood blocks were then transferred to greenhouse containers and covered with greenhouse soil (1:1:2 peat:soi1:10am Inix) until the blocks were approximately 1 cm beneath the soil surface. Temperature ranges for optimal growth were d?terrr,ined by growing isolates for 2 wk on MEA at eight temperatures ranging from 15 to 42C. The experiment had three replications and was repeated twice. Microscopic Cultural and Basidiospore Morphology Studies Cultural characteristics were determined by bright-field and phase microscopy using phloxine and 2% KOH as mounting media. Microscopic structures from cultures were also examined by scanning electron microscopy (SEM). For this, plugs were cut with a 7 mm cork borer from 2 wk cultures grown on 2% MEA, fixed in 1% glutaraldehyde in a 0.1 M phosphate buffer at pH 7.2 for 6-8 hr, rinsed in 3-10 changes of buffer, postfixed for 2 hr with 2% Os04, and dehydrated in ethanol. The plugs were then critical point dried with carbon dioxide, mounted on SEM stubs, sputter coated to a thickness of 15 nm with an alloy of 60% paladium and 40% gold, and observed with an lSI OS-130 SEM. Basidiospore morphology was examined with bright-field, and SEM. Spores were observed by both light microscope techniques using Melzer's Reagent or 2% KOH and phloxine. Spore length, width, and index (length/width) (Steyaert 1980) were determined for 25 spores of each of two specimens of both species. For SEM observations, spore prints were obtained from basidiocarps on both hardwood and conifer hosts in the 8 field by taping glass slides to the pore surface for 3 hr. A concentrated suspension of spores from a print was prepared with 0.5 ml sterile distilled water in Pasteur pipette and inserted into a 3 cm strip of 1 cm diam. dialysis tubing. The ends of the tubing were folded and the tubing was placed in a plastic snap cassette. Following fixation, dehydration, and critical point drying of the spores as previously described, the dialysis tubing was opened and the spores were sprinkled onto the tape mounting surface of SEM stubs. Fractured spores were obtained by brushing the spores on the SEM stubs once lightly with paper. The spores were sputter coated and observed under the SEM. Isolation of Homokaryons, Mating System, and Interfertility Studies Basidiospores were obtained from spore prints or extracted from fruiting body tubes. Extractions from tubes were made by aseptically removing several portions of the inner tubes above the pore surface. The section of tubes was then placed in 5 ml of sterile distilled water and shaken on a Vortex Mixer (Scientific Industries) for 30 sec every 5 min for 15 min. The suspension was poured through 3 layers of cheese cloth to remove the tube tissue and spores were then counted using a hemacytometer. Spore concentrations were adjusted to approximately 2000 spores/ml. One hundred ul of the spore suspension were then placed in cooled, liquid 2% MEA and poured into petri dishes. After 1 to 2 wk at 25 C, isolated colonies were transferred to 2% MEA plates. Homokaryons from .G....lucidum (RLG 14291, JEA 141, JEA 245, JEA 140) and .G.... tSLJgae (JEA 207) and .G.... resinaceum (CB 194-76) were obtained by one of the previously described methods. The homokaryons of each 9 isolate were then mated in all possible combinations. At the end of 3-6 wk the mating system was determined for each of the isolates by examining all crosses microscopically for clamp connections and macroscopically for reaction zones (Raper 1966; Esser and Kuenen 1967; and Deacon 1980). The fou r mat i ng types of each of the i sol ates of both spec i es were then crossed with those of the other isolates in all possible combinations. Homokaryons of .G.... luc1dum JEA 140 were also crossed with homokaryons of RLG 14291 and JEA 141. Homokaryons of .G... resinaceum CB- 194-76 were crossed with homokaryons .G... lucidum JEA 141. Reactions were recorded as previously described. Interfertility crosses had three replications and were repeated twice. Results Fruiting Body Descriptions The fruiting bodies of Ganoderma lucidum differed in both color and form. The color of the pileus surface on fruiting bodies RLG 14291, JEA 140, JEA 245, JEA 266, and JEA 274 from hardwoods was· deep red. RLG 14291, JEA 266 and 140 were non-laccate, while JEA 274 and JEA 245 were laccate. The basidiocarp of JEA 140 basidiocarp was alight shade of red and laccate. JEA 245 had a light yellow to white, non-laccate zone present along the margin of the pileus. The context color also varied in the same specimens. The context of the basidiocarps RLG 14291, JEA 140, JEA 141, and JEA 245 was pale buff and slightly darker at the tubes, while the context of JEA 274 and JEA 266 vias dark throughout. The form of these basidiocarps from hardwoods was also variable. As 10 indicated in the material and methods section, some specimens were sessile, while others were stipitate. Further, some of the specimens (JEA 140, JEA 141, JEA 266, and JEA 274) were imbricate, while RLG 14291 and JEA 245 were non-imbricate. The context thickness also varied, ranging from 1-50 mm. Color of pileus surface and context of the fruiting bodies of ~ tsugae from conifers was also variable. The outer margin on specimens JEA 203 and JEA 207 was light yellow to white and non-laccate. Inner zones of the pilei of JEA 218 and JEA 268 were 1 ight red and laccate. All the basidiocarps from conifers were generally deep red and laccate. The context of most conifer fruiting bodies on conifers was pale buff to cream throughout, while that of JEA 203 and JEA 218 was slightly darker at the tubes. Both sessile and stipitate specimens were represented. However, none of the specimens had imbricate pilei. The thickness of the context ranged from 5-30 mm. Macroscopic Cultural and Temperature Studies Macroscopic cultural characteristics and gallic and tannic acid reactions of the dikaryons of Ganoderma lucidum (referred to by Nobles (1965) as 1h. sessile) and G. tsugae were as described by Nobles (1948). The European ~ resinaceum culture was similar to ~ lucidum cultures, while the 1h. yalesiacum culture was similar to 1h. tSllgae cultures. 1h. lucidum and 1h. resinaceum occasionally produced fruiting bodies in culture. In ~~ fruiting has been reported for dikaryons designated 1h. lucidum from India (Banerjee and Sarkar 1956) but we could not determine the identity of the organism from the information presented. 11 Mature ~ luc1dum and ~ res1naceum fruiting bodies in culture appeared as red laccate projections from the white mycelium, and were approximately 5 mm wide and 5 mm high. Pores developed on the upper surface and bas1diospores were produced. G.. tsugae isolates did not fruit in culture. D11,aryotic cultures from sessile fruiting bodies of Ga. lllc1dum (RLG 14291 andJEA 141), when grown on wood blocks in containers of greenhouse soil, produced stipitate fruiting bodies after 6 weeks. Homokaryons of ~ luc1dum and ~ tsugae had cultural characteristics and reactions on gallic and tannic acid similar to those of the dikaryons. However, the mycelial mats never developed yellow or brown pigmented areas like those of dikaryons of both species. Furthe.m0re, the reverse of the homokaryotic cultures were was unstained after several weeks. Homokaryons of G.a. luc1dum, but not those of G.a. tSllgae, occasionally produced aberrant fruiting bodies in culture. These fruiting bodies were coral-like and also produced basidiospores. Temperature studies with ~ luc1dum isolates JEA 140, JEA 141, JEA 141 H7 (Homokaryon No.7), JEA 266, and RLG 14291 and G.a. tsugae isolates JEA 207, JEA 207 H1 (Homokaryon No. 1), JEA 218, JEA 268, and JEA 203 indicated different growth rates and optimal temperature ranges for the two spec i es (F i gs. 1 A,B, and 2). I sol ates of G.a. luc 1dum had an optimal range between 30 and 34 C with growth rates from 7-11 mm/da. Isolates of G.. tSllgae had an optfmal temperature range between 20-25 C, with growth rates from 1-3 mm/da. Temperatu re stud fes of Eu ropean cultures designated G.a. res1naceum (CB 194-76) and G.a. vales1acllm (CB 223-48) indicated that the G.a. res1nacellm isolate had a temperature 12 Fig. 1 A-C. Linear growth of Ganoderm.a. lucjdum and G...._tsugae after 14 days at various temperatures and cultural reactions of homokaryons of~ tsugae in mating system studies. A. Linear growth of~ lucjdum on malt extract agar after 14 da at the following temperatures; (upper row left to right) 15, 20, 25, 27, (bottom row left to right 30, 34, 37, and 42 C. B. Linear growth of~ tsugae on malt extract agar after 14 da at the following temperatures; (upper row, left to right) 15, 20, 25, 27, (bottom row, left to right) 30, 34, 37, and 42 C. C. Mating test reactions of ~ tsugae homokaryons. (Cl ockwise from upper right) Compatible reactions, uncommon A and B factors; barrage reaction, common B factors (uncommon A>; flat barrier reaction, co monA and B factors; and flat reaction, common A factors (uncommon B). '13 " 10 OANODERMA LUCIDUM - OANODBRMA TSUOAB ---- 9 e ~c :E 7 ~ ...x 6 ~ C 0: 5 ...'" o 4 w JEA 201 ~ ~ 0: 3 ---::::a..",_ JEA26BCA ...,_r~ .... :: :: - - ..... - ~ " ....:...... , Hl.~ ~- JEA 201 __ .. __,' 2 _____ .... :::.::. ":E~2O! H1I' ),. '-.. " • -- __ Y - JEA 21B cr -- ":::':::.' . .- - - ...... ~, ,,,,~.... 01~---,r5------,2rO------~25~------3rO----~-.35------4rO~~ TEMPERATURE (·CI Fig. 2. Growth rates of Gaooderm.a. lllc1dum and .G...tslJgae isolates at temperatures 15-42 C; JEA 140 Ohio, JEA 266 Arizona, RLG 14291 Arizona, JEA 141 Louisiana, JEA 141 homokaryon seven, JEA 207 Arizona, JEA 207 homokaryon one, JEA 268 California, JEA 203 Minnesota, and JEA 218 Connecticut. 14 curve similar to the ~ luc1dum isolates in North America. The ~ yales1acum isolate had a temperture curve similar to the ~ tsugae isolates of North America (Fig. 3). Microscopic Cultural and Basidiospore Morphology Studies All !h lucidum and .Ga. resinaceum 1solates produced abundant terminal and intercalary chlamydospores in culture (Figs. 4 A,B). In KOH and phlox1ne chlamydospores were bluish to hyaline, and hyphae were reddish pink. In Melzer's reRgent the chlamydospores had a dextrinoid and were approximately 12-20 x 10-12 urn. SEM studies 1nd1cated that chl amydospores had a smooth or slightl y uneven wall. None of the ~ tSlIgae isolates or the European !h yalesiacum isolates produced ch 1 amydospores in cu 1 tu reo A11 i so 1 ates of .G.a. lllc i dll m, .G... tsugae, .G... resinaceum, and .G.... yalesiacum produced generat1ve and fiber hyphae, as well as staghorn hyphae and cuticular cells. Generative and fiber hyphae were as previously described (Nobles 1948, 1958). Staghorn hyphae were profusely branched clusters of hyphae 0.5 to 1.0 urn in diam., that developed abundantly as aerial mycelium grew (Fig. 4 C). Cuticular cells were thin walled, spherical, 20-25 urn in diam. and ori g i nated from gene rat i ve hyphae. Wall s were smooth with uneven surfaces (Fig. 4 0). Contents of ruptured cuticular cells stained reddish pink in phloxine. In older cultures cuticular cells were empty, hyaline, and compacted. Bas1diospores of .G... luc1dllm and .G... tsugae (Figs. 5 A-F) were brown and ovate, with a truncate to rounded apex, and an eccentric hilar appendix on a rounded base. Spores had a douhle wall with inter- 15 " 10 OI\NODERMI\ RES1NI\CBUM -- OI\HODERMI\ VI\LESIIICUM ---- 9 B Sc :E 7 ~ :l: 6 I- o~ a: 5 Cl ... CI 19~·76 o 4 w ti a: 3 /', / / ", 2 / '-., / ...... / Clm·46 " ,1---~~/~/'----~-----r----~~----~-'--'~~~--~ o 10 15 20 25 3D 35 40 TEMPERATURE (OC) Fig. 3. Growth rates of Gaooderma, resioaceum (CB 194-76) and a.. yalesiacum (CB 224-48) isolates at temperatures 15-42 C. 16 Fig. 4 A-D. Microscopic characters of cultures of Ganoderma lucjdum A. Chlamdyospores of~ lucjdum observed by brightfield microscopy (Bar = 20 urn>. B-D. Scanning electron micrographs; B. Two adjacent chlamydospores of~ lucjdum separated by a clamp connection (Bar = 10 urn). C. Branching network of staghorn hyphae produced in culture by ~ lucjdum (Bar = 2 urn). D. Cuticular cell originating from a clamp connection produced in culture by ~ lucjdum (Bar = 7 urn). 17 wall pillars separating the two walls. The apex contained a germ canal from which germ tubes were observed to emerge. Most spores contained a large vacuole and all were negative in Melzer's reagent. Basid1ospores of a... luc1dum were 10.6-<11.5)-11.8 x 6.8-<7.4)-7.8 um (mean in parentheses), while a... tsugae basidiospores were 10.9-<11.2)-12.5 x 6.6-(7.8)-8.5 um. The widths, but not the 1 engths, of the basidiospores were significantly different (P=O.05). The mean of the spore index (length/width ratio) of .G.a. luc1dum was 1.50, significantly different from that of a... tsugae, which was 1.57 (P=O.Ol>. a... t'sLlgae isolates had inter-wall pillars that were distinct partitions between the inner and outer walls, while the inter-wall pillars of .G.a. lucidum were not as obvious (Figs. 5 A,B). Vacuoles were more conspicuous in spores of a...lucfdum than in spores of a...tsllgae. Basidiospores of a... lucidum had a surface with shallow depressions, as seen with the SEM (Figs. 5 C-F). Numerous small inter-wall pillars were observed in fractured spores. Basidiospores of.G.a. tsugae had a rougher surface on the outer-wall with pronounced depressions. The inter-wall pillars of fractured spores were broader and less numerous than those of a... lucidum. Basid1ospores from .in illm fruiting bodies of .G.a. resfnaceum were similar to bas1diospores in field collection of .G.a. luc1dum. Mating Systems and Interfertility Studies Four macroscop1c mating reactions were observed (Fig. 1 C). These included: compatible, heterozygous A and B factors; flat barrier (incompatible), common A and B factors; flat (incompatible), common A factors; and barrage (incompatible), common B factors. Each pairing 18 Fig. 5 A-F. Bright-field and scanning electron microscopy of basidiospores of Ganoderma tsugae and~ lucjdum. A-B. Brightfield microscopy (Bar= 5 um). A. Basidiospores of~ tsugae. Inter-wall pillars (arrow) are well defined; B. Basidiospores of~ lucjdum Inter-wall pillars (arrow> are not as apparent as in spores of~ ~u~a~. C-F. Scanning electron micrographs (Bar = 2 urn); c. Basidiospore of .G._ tsugae. Outer wall surface with prominent depressions; D. Basidiospore of~ lucjdum. Outer wall surface with shallow depressions; E. Fractured basidiospore of ih_tsugae exposing large, inter-wall pillars (arrow}; F. Fractured basidiospore of~ lucjdum exposing small, numerous inter-wall pillars (arrows). 19 was examined microscopically for clamp connections. Compatible reactions produced abundant clamp connections on generative hyphae 3-6 wk after pairing. Incompatible matings produced no clamps. G.... lucjdum, G.... resjnaceum, and G....tsugae proved to be heterothallic and tetrapolar. All four mating types were retrieved from G.... lucidum specimens RLG 14291, JEA 141, JEA 245, and Ga. tsugae specimen JEA 207. Interfertility studies were conducted with mating types of each of the above isolates. In Fig. 6, the two compatible mating types from each dikaryotic isolate carry the two alleles of each factor present in that dikaryon. Self crosses were incompatible. Homokaryons of all three Ganoderma lucidum isolates were completely interfertile. indicating that the species is multiallelic and 4 dikaryons shared no alleles. Clamp connections and macroscopically visible compatible reactions were readily observed. Undesignated mating type homokaryons of JEA 140 were also completely interfertile with the other G....lucjdum homokaryons. None of the Ga. tsugae homokaryons were interfert 11 e with the Ga. 1 ucidum homokaryons. Homokaryons of the European .G&. resjnaceLlm isolate CB 194- 76 were completely interfertile with homokaryons of .G&. lucjdu[o JEA 141. Discllssion Basing species of Ganoderm.a. within the G.... lucidum complex on macroscopic basidiocarp characters has resulted in the description of many superfluous species. Examinations of types and neotypes and of recentl y coll ected spec i mens of Ganoderma in North America revealed gradations of these characters, including presence or absence of a stipe, the color of the basidiocarp cuticle, its degree of varnish, 20 GL.14291 GL 141 GL 245 GT 20':7 'I 2 1 2 1 2 1 2' - - - GL 14291 + + + + + 2 + - + + + + -- - - - GLI41 + + + + + 2 + + + - + + - - - - - GL245 + + + + + 2 + + + + + ------GT207 + 2 ------+ - Fig. 6. Interfertllity tests with two compatible mating types representing four alleles of each of three GanQderma luc1dum (GL) isolates and one G.. tsugae (GT> isolate. Plus equals interfertlle crosses. GL 14291 RLG Arizona: 1 = A B ; 2 = A B ; GL 141 JEA Louisiana: 1 = A B ;2 = A B ; GL 245 JEA Arizona: 1 = A B ; 2 = A B ; GT 207 JEA Arizona: 1 = A B ; 2 = A B • 21 context color, and context and tube thickness. Microscopically, basidiospores of the two species differed in outer wall surface roughness and inter-wall pillar dimensions and number. However, there were degrees of variation between basidiospores among specimens from a population that were apparent when comparisons were made of a number of collections. A similar problem arose when basidiospore biometerics were considered. Although the spore width and spore index of one specimen of each species was shown in this study to be significantly different, the range of these measurements had considerable overlap. Distinguishing species by these statistics is not a practical technique due to the natural variation within the population. Steyaert (1975) also recognized the pleomorphic nature of species of Ganoderm~ in his analyses of spore sizes of ~ tornatum. Steyaert found that with increasing latitude or elevation spore size incr·eased; he further noted that context color became darker with southern latitudes and lower altitudes. These observations suggested the i nfl uence of te"mperatu re on morphol og ical characters. The possiblity exists that other characteristics, such as color and form of the pileus, are influenced by environmental conditions present during basidiocarp development. Also, collections made during early stages of development may not represent the characteristics of a mature specimen. The collections we studied can be divided into two groups on the basis of cultural morphology, basidiospore morphology, interfertility studies, and temperature relationships. Isolates designated ~ luc1dum and ~ resinaceum produced chlamydospores in culture, had an optimum 22 temperature range of 30-34 C, were capable of growing above 34 C within 2 wk, had "smooth" basidiospores with thin inter-wall pillars, and were interfertile regardless of geographical distribution of macroscopic morphological differences. Thus, the information obtained in our study indicates that {h lucidum and {h resinacellm are conspecific. Ganoderma tSllgae and .Ga. valesi~m isolates did not produce chlamydospores in cultures, had an optimum temperature range of 20-25 C, and were not capable of growing above 34 C within 2 wk. Ganoderm.a. tsugae specimens had "rough" basidiospores with broad inter-wall pillars. Ganoderm.a. lllcidum and .G..... tsugae had a heterothallic tetrapolar mating system similar to those previously described for other Ganoderm.a. species (Aoshima 1953; Banerjee and Sarkar 1958b). Basidiospores of only one specimen of .Ga. tsugae were germinated but these homokaryons were not interfertile with the homokaryon of .G..... lucidum. All "smooth" spored specimens (.G.....l!lcidum) were from hardwoods, while all "rough" spored specimens ({h tsugae) were from conifers. Since biological species or non-interfertile sub-populations have been reported for Armillaria mellea (Anderson and Ullrich 1979; and EomUopsis pin1cola (Mounce 1929), the criterion of non-interfertll ity may not be an absolute method to distinguish otherwise morphologically similar organisms. However, based on the erwise morphologically similar organisms. However, based on the other evidence of basdiospore morphology and cultural characteristics (morphology and temperature studies) we considered {h l!lcidum and {h tsugae separate species. A review of the literature on the names of the species of Ganoderm.a. indicated that G... lucidllm and G... resinacellm were possible 23 names for specimens from hardwoods, having smooth basidiospores, and with chlamydospores in culture. Ganoderma vales1ru:JJlIl and .Ga. tsugae were possible names for specimens obtained from conifers, having rough basidiospores, and lacking chlamydospores in culture. Steyaert <1961> designated the 1781 Curtis plate 224 as the type of .Ga. luc1dum. A Latin English description accompanying Curtis' .plate states that his original specimen of Boletus ~~ was collected from hazel and that the species also occurs on elm, both hardwoods. This would suggest that our specimens collected from hardwoods from several locations in North America and Europe which were completely interfertile, would be correctly referred to as G.... luc1dum with G.... res1naceum as a synonym. However, no microscopic information about spore characters is given in Curtis' description. Karsten (1889) reported G... lllc1dllm to occur on both hardwoods and conifers (oak, alder, and spruce). Since he was unaware that· there were 2 species involved, one on hardwoods and one on conifers, he would have considered specimens of either to be G... luc1dllm because they are macroscopically very similar. Karsten's Fungi Fenniae Exsiccati (1865) contains a specimen (No. 239, annotated from oak) under the name fa. lllc1dlls with rough basid10spores. Consequently, subsequent workers including Haddow (1931) and Steyaert (1972) have mistakenly developed the concept of G.... luc1dum with rough basidiospores and occurring on both conifers and hardwoods. This concept has led Haddow <1931> and Steyaert (1977) to consider G.... tSlIgae to be a synonym of G.... lllc1dum, although late,', Steyaert (1980) considered them to be separate species with G... luc1dum not occurring in North America. Overholts (1953) 24 had described G... lucidum with "smooth" walled basidiospores and occurring on hardwoods and conifers. Jahn ,et al. (980) believed the taxon described by Overholts (1953) as E..... lllc1dus was G... resinacellm since the type of ~ resinaceum has smooth basidiospores. None of these workers have placed emphasis on host information or cultural characteristics. Based on cultural information, Nobles (1965) had corrected the name ~ lllcidum to ~ sessl1e for cultures that produced chlamydospores. Stalpers (1978) and Bazzalo and ~/right (1982) agreed with this description under the name ~ resinacellm. Stalpers (1978) suggested the species designated as ~ lucidus by Davidson et ale (1942) was probably ~ res1oacellm. The isolates Davidson et ale had studied had an optimum temperture of 30 C and had chlamydospores. This agrees with our concept of ~ luc1durn. Haddow <1931>, Overholts (1953), and Bazzalo and Wright (1982) considered specimens with rough basidiospores to occur on both hardwoods and conifers. Bazzalo and Wright described ~ lllcidllrn without chlamydospores in culture, hoeever no host data is given for the specific cultures observed. Apparently, they were describing what we have referred to as G... tSllQae or some other Gaooderrna species. Stal pers (1978) described ~ yalesiaclJrn in culture without chlamydospores, and as previously mentioned, he considered ~ tsu~~ a synonym of ~ yales1acllm. In our studies, all of the hardwood isolates of Gaooderm.a produced chlamydospores in culture and were interfertile regardless of macroscopic morphology. The conifer isolates of Gaooderm.a did not produce chlamydospores in culture. Interfertility tests should be conducted between the European and North American isolates from conifers 25 when homokaryons are obtained. If they are interfertile ~ yalesiacum would be the valid name with .G.a. tSlIgae a synonym. Overholts (1953) considered ~ curtsii and ~ lucidlls (.G.a. lllc1dllm) separate species based on pileus color and stipitate habit of ~ curt1s1i. Davidson et al. (1942) found no significant cultural differences between the isolates designated as'~ lucidlls and ~ cllrt1s11. Our studies indicated that homokaryotic isolates from specimens differing in these morphological characters (JEA 245 stipitate x RLG 14291 sessile), were interfertile. Further, dikaryotic cultures from sessile fruiting bodies (RLG 14291 and JEA 141) produced stipitate basidiocarps on subterranean wood blocks in the greenhouse studies. Stud i es of type spec i mens of .G.a. cu rt 15 11 and .G... rayeneJl11 p rov i ded no basts for distinguishing these species from .G... lllcidum. Therefore, based on this information, the .G...curt1sii description coincides with the ~ lucidum concept. We suggest that future species designations in the .G.a.luc1dum complex should be considered val id only after cultural and interfertility studies are conducted. CHAPTER 2 l.N Y..lIB.Q. DECAY STUDIES OF SELECTIVE DELIGNIFICATION AND SIMULTANEOUS DECAY BY THE WHITE-ROT FUNGI GANODERMA LUCIDUM AND ~ TSUGAE Introductjon Ganoderma lucjdum (Curt. ex. Fr.) Karst. Pat. and Ganoderm.a tsugae Mur~ are wood decaying fungi that cause white rots, removing lignin as well as cellulose and related polysaccharides from wood of many species in North America and Europe (Blanchette 1984b). Ganoderma lucjdum decays hardwoods, and common hosts include oak, maple, sycamore, and ash (Hepting 1971). Other hosts in the southwestern United States and Northern ~1exico include mesquite, 01 ive, and grape (Gilbertson et al. 1979). Ganoderma tsugae decays stumps or dead standing and fallen conifers (Hepting 1971; Gilbertson et al. 1979). In the north-central and northeastern United States this fungus is commonly found on eastern hemlock (Hept ing 1971). In the southwestern Un fted States it is commonly found of white fir (8bie..s concolor (Gord. and Glend.) Lindl. ex Hildebr.) and rarely on Douglas-fir (Pseudotsuga menzjesji (~1irb.) Franco) (Gil bertson et al. 1979). White rot fungi degrade the wood components lignin, cellulose, and hemicellulose. Many white rot fungi have been reported to be either simultaneous or selective in decaying the various components (Kirk and Moore 1972; Kirk and Highley 1973; Ander and Eriksson 1977; Blanchette 1984b). Simultaneous decay is characterized by removal of all the wood components at a uniform rate. In selective decay 1ign1n and 26 27 hemicellulose are removed preferentially. Further, some white-rot fungi are capable of both types of decay in the same wood or in different wood species (Meier 1955; Blanchette 1984a,b). Ander and Eriksson (1977) reported higher delignification of pine wood blocks (pinus silyestrjs l.) or Kraft 1 ignin by pycnoporlls c1nnabar1nus (J acq.:Fr.) Karst. in the presence of malt extract. Kirk and Moore (1972) found that the rates of lignin removal varied in relation to the removal of carbohydrates depending on the wood used as a sUbstrate. These results suggest that selective or simultaneous decay of wood component may be influenced by the nutritional requirements of the fungus, of the structural arrangement and the chemical composition of the wood. Ganoderma lucidum (Curt.:Fr.) Karst. and ~ curticjj (Berk.) Murr. have been reported to cause simultaneous decay of naturally decayed red and black oaks (Quercus rub ra l. and Ua. .n..i.Q..eJ:. lam.) (Blanchette 1984b). The taxon referred to as ~ luciduID in the paper has been considered to include G... curtisii as the species occurring on hardwoods in North America (see chapter 1). ~ tsugae has been reported to selectively delignify field decayed eastern hemlock (Isuga canadensis (l.) Carr.) by removing middle lamellae and defibrating cells (Blanchette 1984a). The purpose of this study was to determine the decay capabilities of the two Ganoderma species in YitLQ on several wood types and to determine the presence of simultaneous decay or selective de11gnif1cation of each wood type studied. 28 Mate r 1a J s .and. .M.e.:t.ho.ds. Fungal Isolates and Wood Samples Basidiocarps C?f,(h Jycidum and ,(htsllgae were collected from several locations in North America. Isolates were obtained from context tissue of specimens in the following collections: ,(h lycidum (GL) RLG 14291, Madera Canyon, Santa Rita Mountains, Coronado National Forest, Arizona, from silver leaf oak ( (~ sp.) collected by M. Blackwell; ,(h tsygae (GT) JEA 201, Upper bear Wallow, Santa Catalina Mountains, Coronado National Forest, Arizona, from white fir; GT JEA 203, University of Minnesota Campus, St. Paul, Minnesota on spruce (picea sp.) collected by R.A. Blanchette; GT JEA 207, Upper Bear Wallow, Santa Catalina Mountains, Coronado National Forest, Arizona, on white fir. Two homokaryons obtained by germinating basipiospores of GL 141 and GT 207 in malt extract agar were also used in this study. The homokaryons were designated GL 141 H7 and GT 207 H1. Sound wood for test blocks was obtained from silver leaf oak, Bear Canyon, Santa Catal ina Mountains, Coronado National Forest, Arizona; mesquite (prosopis veJut1na Woot.), Saguaro National Monument Wast, Tucson, Arizona; and grape (Y.1.:t1s. yinifera L.), Willcox, Arizona. Sound wood of white fir and Douglas-fir was obtained from Upper bear Wallow, Santa Catalina Mountains, Coronado National Forest, Arizona. In ~ Wood Decay Studies Agar-block decay chambers were prepared by a modified method as described by Gilbertson and Canfield (1972). All wood blocks were cut 29 2x2x4 crn with the large face transverse, oven dried at 110 C for 16 hours in a recirculating oven, and weighed. Blocl Chemical Analyses of Wood Blocks Control and decayed wood blocks were each ground to pass through a 40 mesh screen (Cowling 1960, 1961). The ground wood samples were oven dried for 16 hr as previously described and extracted first by ethanol-benzene and then by 95% ethanol (Tappi 1975). Chemical analysis of acid insoluble lignin (Klason lignin) was performed using samples from the extracted wood as described by Effland (1977). Apparent acid soluble lignin determinations were made from the Klason filtrate by UV spectrophotometry. Maximum absorbance occurred at approximately 280 nm for all samples. Filtrates from control and decayed wood were equally diluted with distilled water and measured at peak absorbance with a UV 30 spectrophotometer (Cowling 1960; Pearle and Busche 1960). Chlorite holocellulose was also d~termined as described by Seifert (1983). Extracted wood samples of approximately 400 mg each were placed in SO ml Erlenmyer flasks. Seven milliters of a buffer solution consisting of 60 ml glacial acetic acid, 1.3 g sodium hydroxide, per 1000 ml distilled water was added to each flask. Three milliliters of a 20% (w/w) aqueous solution of sodium chlorite was immediately added and the flasks were sealed with parafilm and alunlinum foil. The flasks were placed in an orbital shaker at 110 rpm at 4S C for 36 to 40 hr. After the incubation period, the flasks were placed in an ice bath to stop the reaction. The contents were then transferred to preweighed 60 ml coarse por~sity scintered glass crucibles using 100 ml of 1% acetic acid. The holocellulose was washed with S ml of acetone three times and oven dried at lOS C for 4 to 6 hr before weighing. Klason lignin and chlorite holocellulose analyses were replicated three times for each treatment. Data were subjected to analysis of variance and mean separation was done by Duncan's multiple range test (P=O.OS). The micro-Kappa number was determined to estimate the residual lignin for the holocellulose from each original hardwood and conifer wood for 12 random samp 1es (Berz i ns 1966). Microscopic Observations Light microscope and scanning electon microscope (SEM) studies were conducted with controls (sound wood) and each wood type decayed in ill.I:.c. by G... lucidum and G... tSlJgae. For SEM studies, wood was fixed in a mixture of S% formaldehyde and 10% gulteraldehyde in a 7% phosphate 31 buffer (pH=7.2). Solutions were changed once daily for 3 days or until clear. Samples were dehydrated in a 10-100% ethanol series and then subjected to critical point drying with carbon dioxide. Sections were cut with a razor blade and mounted on SEM stubs. The samples were then sputter coated to a thickness of 15 nm with an alloy of 60% paladium and 40% gold, and observed using an lSI DS-130 SEM. Results In ~ Wood Decay Studies of G... Jllcjdllm and G... tsugae The woods varied in percent weight loss caused by each Ganoderm~ species (Table 2). The controls of each wood type lost a small percentage (0-4%) of the original weight during the preparation and sterilization procedures. The precent weight loss (PWL) of the wood shown for each isolate was not corrected for the control weight loss. The woods arranged from the highest to lowest PWL were grape, oak, white fir, Douglas-fir, and mesquite. Isolates of the two Ganoderma species varied in their abll ity to decay each wood studied with no consistent differences observed between homokaryons and dikaryons. Percent weight loss ranged from 73.18 (Grape GL 147 H7) to 1.69 (Mesquite GL H7). The G... lucjdum isolates consistently caused greater weight loss in the grape and oak than did the G.,..tsugae isolates, except for GL 141 H7, which was not significantly different from the .G.... tSllgae isolates in decaying the silver leaf oak. GL 141 caused greater weight loss in the non-host coniferous wood than did any other isolate. The other G.... lucidllm dikaryon, GL 14291, caused weight loss of conifer wood 32 Table 2. Percent weight loss and percent change of K1ason lignin and chlorite ho1oce11u1ose in control and in YitrQ decayed wood by Ganoderma 1uc1dum and a.. tsugae Percent P'!!reent Wood Type Isolate Weight LoS5t1 Cha~;~~~~Lb Change In cne Grape Control 4.02 !. 0.18 • D. lucldum 141 81.9B ! S.11 .,t - 12.50 . 10.59 O. lueldum 14291 53.84 ! 2.6S c,d - 5.47 5.63 G. lu.ldum 14IH7 13.18 ! 3.19 r - 1.95 3.85 O. tsugae 201 21.38 !. 6.55 b - 5.40 8.22 O. IsIIg •• 203 ·60.06 !. 5.28 d •• - 2.36 5.25 G. IslIgo.201111 41.66 !. 12.32 • - 8.22 8.34 Sliver leaf oak Control 0.92 ! 0.50 • O. lueldum 141 45.88 ! 3.62 • - 4.40 6.H O. lueldum 14291 47.84 !. 2.14 0 - 8.65 1.67 O. lu.ldum I41H7 19.38 ! 8.61 b . 0.01 1.20 Q. tsugae 20t U.48 !. 4.00 b - 1.61 3.78 O. IsIIg.' 203 n.n !. 4.73 b - 1.85 2.77 Q. bUilO 201Hl 15.30 ! 2.70 b - 2.09 0.42 __ c Mnqulte Control 1.41 ! 0.23 • G. lueldum 141 2.24 ! 0.46 a,b O. lueldum 14291 2.65 ! 0.99 a,b O. lu.ldum I4IH7 1.69 ! 0.44 0 O. tsuge.e '2:0t 4.62 ! 3.'" b O. tsU(/ •• 203 2.35 ! 0.31 I.b O. tsugee 207Hl 3.08 ! 2.53 .,b White Plr Control 0.48 ! 0.10 0 O.lueldum 141 15.18 ! 1.81 d - 3.10 9.49 O. lu.ldum 14291 10.53 !. 1.19 0 - 4.44 11.41 O. tueldurn 141H7 8.07 ! 0.99 b - 4.43 . 12.19 O. bugae 201 4.09 ! 3.20 b - 1.53 0.68 0. IsU(/ae 203 8.15 ! 0.69 c - 2.62 0.91 O. Isug .. 207111 4.39 ! 0.75 b - 7.93 . 11.89 Douglu Plr Control 0.31 ! 0.19 I O. lueldum 141 9.98 ! 4.90 c - 2.93 3.47 O.lueldum 1429\ 8.12 ! 3.87 b,ll 0.01 1.03 Q. lucldum 141117 $,66 ! 3.'66 b · 0.63 0.30 O. Isug •• 201 · 1.37 O.H 3.61 !. 2.18 •• b · O. Isug •• 203 5.26 ! 1.47 b - 0.61 0.12 G. tsurae 'lOTHl 3.31 ! 0.91 alb - 0.85 6.t6 • Anrage percent welsht Ion determined (rom :5 replications .fter 20 wk. weight losses followed hy the same letter are not slgnlCaelintly different at P1lI1.0S (O\,lnCM AMly,I,). b Average percent change in Klason lianin and chlorile holoeellulose of the wood decayed by el\ch Isol"le was determined Irom the Ivtrage of each component from controls of sound wood aher '20 wk In deeay chambers. a Klason litoln and chlorite holoe-allulose wu not rtelermlnecS tor the mesquite ",000 block,. 33 comparable to that caused by GL 141. However, because ~f the variation within the treatment, differences were not significant. Al'l isolates caused the least amount of weight loss in mesquite. Chemical Studies of Wood Decayed by .G... luc1dum and .G... tsugae The acid insoluble lignin (Klason lignin) and chlorite holocellulose values presented are the averages for tohe 'Isolates of each species (Table 3). The Klason lignin and the chlorit~ ho16cellulose were not corrected for the percent total extracts s1nc~ the correction did not change the relative values of each componen~. M~cro-Kappa number determinations indicated an average of 0.963 ± 0.308% residual lignin present in the holocellulose. The percent changel of Klason lignin and chlorite holocellulose for each wood decayed by each isolate when compared to the control is presented in Table 2. Portions of both the Klason lignin and chlori~e holocellulose wood fractiQns w.re removed by the fungi based on total amounts in controls an~j dec'ayed wood. However, the percentage of Klason lignin generally deer/eased while the holocellulose incr-eased based on the oven dried weight of the decayed wood sample in relation to the percentage of each comp~nent determined for the control blocks. The percent change of each wood fraction varied between each isolate depending on the wood being decayed. No isolate consistently caused a greater change in either the Klas~n lignin or the holocellulose among all the woods studied. The percent Klason lignin and chlorite holocell~losel levels for the grape and white fir decayed by .G.a.. luc1dum wer~ sig,nHicantly different from those of the controls (Table 3). Th~ le~el of each 34 Table 3. Percent Klason lignin, chlorite holocellulose, and Klason lignin and holocellulose ratios in entire samples of controls and wood decayed in Yit.I:.a. by G.arloderma 1ucidum and G.... tsuQae Wood Type Treatment 96 KLa 96 CHC KL/CHC Grape Control 32.72 :!: 0.54 a 69.59 :!: 2.28 a 0.47 :!: 0.02 a G.lucidum 26.08 :!: 5.37 a,b 76.28 .:!:. 3.49 b 0.34 :!: 0.08 b G. tsugae 27.39 :!: 2.93 b 76.86 :!: 1.75 b 0.31 :!: 0.05 b Oak Control 21.01 :!: 0.99 a 76.49 :!: 2.37 a 0.27 :!: 0.02 a G.lucidllm 16.67 :!: 4.33 Il 81.94 :!: 3.84 a 0.21 :!: 0.06 a G. tsugae 19.17 .:!:. 0.24 a 78.57 :!: 2.12 a 0.24 .:!:. 0.01 a White Fir Control 32.10 .:!:. 0.85 a 64.25 .:!:. 2.12 a 0.50 .:!:. 0.02 a G.lucidum 27.91 :!: 0.42 b 74.61 :!: 1.58 b 0.37 .:!:. 0.01 b G. tsugae 28.07 .:!:. 3.42 a 68.76 .:!:. 6.39 a,b 0.40 .:!:. 0.07 a;b Douglas Fir Control 32.84 .:!:. 1.16 a 64.64 .:!:. 0.63 a 0.51 .:!:. 0.02 a G.lucidum 32.14 .:!:. 1.98 a 65.55 .:!:. 2.31 a 0.49 .:!:. 0.05 a G. tsugae 32.81 .:!:. 1.22 a 66.61 .:!:. 3.01 a 0.49 .:!:. 0.03 a a Percent Klason lignin, chlorite holocellul~lse, and the ratio of the percent of each component are the average of 3 replications. b Values followed by the same letter are not significantly different at P=0.05 (Duncan Analysis). 35 component changed significantly (P=O.OS) in grape decayed by ~ tsugae compared to those of the controls. Although the oak wood decayed by each species had lower Klason lignin and higher holocellu10se levels than the control, there were no significant differences due to the variation in the analyses of the entire wood blocks. Levels of the two wood fractions in the Douglas-fir blocks decayed by each Ganoderm~ species were relatively equivalent to those of the control blocks. The ratio of the percent Klason lignin (%KL) and the percent chlorite ho10ce11u10se (%CHC) is presented in Table 3. Decreases in the %KL/%CHC ratio indicate selective de1ignificat1on. The statistical analysis indicated that the grape and white fir wood decayed by ~ lyc1dum and ~ tSIJgae were significantly different from the controls in the ratio of the two wood components. The change in the decayed oak blocks appeared to be similar to that in the decayed grape and white fir blocks, however, there were no sign 1ficant d Hferences. The decayed Douglas-fir wood was virtually unchanged in the ratio of these components compared to the controls. The apparent acid soluble lignin for each treatment of each wood showed no significant differences between the controls and decayed grape, oak, and Douglas-fir blocks. However. the absorbance of the K1ason lignin filtrates from the decayed white fir wood from both Ganoderma spec1es were reduced by 25% ± 5% from the control (P=O.05). Scanning Electron Microscope Studies SEM studies were conducted of the in ~ controls and decayed wood to determine if the chemical changes observed in the analyses of 36 the entire wood blocks were accompanied by apparent morphological changes. Macroscopically the white fir and Douglas-fir wood decayed by each species appeared more bleached 1n color than the control, with some areas appear1ng undecayed. The radial section of undecayed wood of white fir (Fig. 7A) shows the closely packed tracheids and the thickened walls of the border pits. Ray parenchyma can be seen running perpendicular to the tracheids at the top of the micrograph. The white fir wood decayed by each Gaooderma species had evidence of simultaneous decay typical of white rots (Figs. 7B and 7C). Microscopically this decay was apparent with tracheid walls eroded, giving a 1aminated or str1ngy appearance to the once cellular t1ssue. The un1serate rays were disrupted. Small areas within the decayed wood varied from apparently undecayed to having the middle lamella removed between the tracheids (Fig. 7B). The trache1ds appeared to be separated from each other when compared to the control. The control of Douglas-fir wood is shown in a tangential section in Fig. 70. Macroscopically the Douglas fir wood is shown in a tangential section in Fig. 70. Macroscopically the Douglas-fir wood decayed white fir wood. However, microscopically a general breakdown of the wood cells was observed (Fig. 7F) with intermittent localized areas that appeared less altered (Fig. 7E). The tracheids of the less altered areas had enlarged pits with the middle lamella and possibly the primary wall removed, exposing the spirally oriented macrof1brils of the secondary walls. The grape wood decayed by either Gaooderma species, unlike the decayed conifer woods, macroscopically appeared uniformly bleached and Figs. 7 A-F. Scanning electron micrographs of white fir and Douglas-fir controls and wood decayed by Ganoderm.a luc1dllm and !i..tsugae. F'jg. A, radial face of undecayed white fir wood with tracheids ttl, ray cells (r), and bordered pits with thickened wall s (arrow) (Bar = 100 um). Fig. B, tangential face of decayed white fir wood (GT 203) showing delign1fied zones (d) within simultaneously decayed areas (s). Fiber tracheids within delignified areas were loosened from each other by the removal of middle lamella (arrow) (Bar = 100 um). Fig. C, tangential face of decayed white fir wood (GT 207 Hl) with a uniserate ray (arrow) severel y decayed. Pl ate-like remnants of fiber tracheid wall s with outer layers (double arrow) of the secondary walls being removed (Bar = 50 um). Fig. D, tangential face of undecayed Douglas-fir wood with uniserate rays (r), tracheids (t), and bordered pits (arrow) (Bar = 100 um). Fig. E, delignified tracheids in decayed Douglas-fir wood (GL 141) with bordered pits enlarged (arrow) and the middle lamella removed. Primary wall s (double arrow) were partially removed exposing spi rally ariented macrofibrlls of the secondary wall (Bar = 50 um). Fig. F, simultaneous decay in Douglas-fir wood with tracheid pits coalasced (arrows) and walls degraded (Bar = 100 um). 37 Figs. 7 A-F. Scanning electron micrograyhs of white fir and Douglas fir controls and wood decayed by Ganoderma ucjdum and ~ tsugae. 38 decayed throughout. No zones or variation in color were evident in the decayed wood blocks. The ray parenchyma and the fiber tracheids of the controls are uniformly cemented together (Fig. 8A). In large areas of the wood decayed by either Goa. 1 yc1dum or .G... :tsugae. the ray parenchyma appeared s1multaneously decayed (Fig. 89). In these areas the fiber tracheids were selectively delignif1ed with the middle lamella and the primary wall removed leaving the skelatal secondary wall undecayed. Other areas thought to represent further stages of decay had the ray parenchyma almost entirely replaced with mycelium. Scalar1form pits of the fiber tracheid secondary walls were enlarged and segments of the wall were removed (Fig. 8C>. Macroscopically the oak wood decayed by either species appeared more uniformly discolored and decayed than either of the conifer ,,·oods. However, unlike the grape wood, there were apparently undecayed zones present in the blocks. Further, within the decayed zones, there were localized 1 rilm bleached areas. Some oak blocks also had black streaks or flecks within the deca~ed wood which were not observed in any of the other wood types. Part of a multiserate ray and uniserate rays in between fiber tracheids were observed in the tangential section of the oak wood control (F ig. 80). Locali zed bleached areas withi n decayed wood appeared as loosely packed delignified tracheids without middle lamella (Fig. 8E). Other areas appeared simultaneously decayed (Fig. 2F), with trache1 d walls fragmented. Un 1serate ray parenchyma p its were enlarged and coalesced, with portions of the secondary wall decayed. Figs. 8 A-F. Scanning electron micrographs of grape and oak controls and wood decayed by Ganoderma lllc1dum and G... tSlJgae. Fig. A, tangential face of a portion of undecayed grape wood with fiber tracheids (t) and a partially exposed vessel element (v) between two multiser1ate rays (r). Fig. B, tangential face of grape wood with simultaneous decay of multiser1ate rays (s) (GL 14291) and del ignif1ed adjacent fiber tracheids (d). Fig. C, tangential face of decayed grape wood (GT 207 HI) showing advanced simultaneous decay with mycelium and completely disrupted mul t 1seri ate rays (r) eventuall y observed as voi ds (v). Scalariform pits of the fiber tracheids coalesced (arrow) as the secondary walls were decayed. Fig. 0, tangential face of undecayed. oak wood through a portion of a mult1seriate ray (r) and fiber trache1ds (t) with dispersed un1serate rays (arrow). Fig. E, face of decayed oak wood (GL 14291) showing del1gnif1cat1on resulting in separating fiber tracheids (arrow) without middle lamellae. Fig. F, tangential face of simultaneously decayed oak weod (GL 14291) with portions of fiber trache1d walls removed, uniserate ray pits enlarged and coalescing (arrow), and secondary wall s decayed (double arrow) (Bar = 100 urn). 39 Figs. 8 A-F. Scanning electron micrographs of grape and oak controls and wood decayed by Ganoderma lucidum and ~ tsugae. 40 Most of the rays were disrupted or severely decayed. Vessels usually became fill ed w1th mycel ium <>ther areas appeared undecayed or delignified. In cross section, gaps between fiber tracheids where middle lamellae had been removed were observed in delignified areas (Fig. 9B). Primary walls and secondary walls were partially decayed in some cells, indication simultaneous decay. Discussion The Ganoderm..a lucidumand {h tsugae isolates decayed all the wood species tested. Although G.... luc1durn is not found on conifers or G.... tsugae on hardwoods in nature, isolates of both species caused substantial percent weight losses in all the woods except mesquite. The mesquite wood had the highest density of any of the woods studied and neither Ganoderrna species colonized the mesquite wood blocks to the extent of the other woods. The G.... Jucidurn isolates generally caused a greater percent weight loss in the hardwood and the conifer wood blocks than the G,,- tSllgae isolates. The rapid growth rate of {h Jucidurn in relation to G... tsugae (2.0 to 3.9 times) as reported in chapter 1 may account for the variation observed. The percent weight loss caused by G... Juc1durn or G...tsugae in anyone wood was nearly proportional to its growth rate. Further, both Ganoderrna species decayed the conifer blocks less than the hardwoods (except for mesquite). The range of the PWL varied with the wood being decayed. As previously noted (Kirk and Moore 41 Figs. 9 A-B. Scanning electron micrographs of transverse sections of oak wood decayed by Ganoderma lucjdum (GL 141). Fig. A, vessel elements with portions of the secondary wall removed (arrow) and filled with mycelium and chlamydospores; voids (v) where fiber tracheids were simultaneously decayed and completely degraded (Bar = 100 urn). Fig. B, spaces (arrow) between fiber tracheids where del ignification resulted i n rem o v a 1 of m i d d 1 e 1 am e 1 1 a , a n d d ec aye d sec on d a r y w a 1 1 s res u 1 t i n g from simultaneous decay (double arrow> (Bar = 10 urn). 42 1972) the rate of decay of conifer wood by white rot fungi is generally slow. Therfore, under the conditions of our study the fungi appeared to be reacting only to the spec1fic wood in anyone treatment. The host specificity of each species observed in nature may depend on the mode of infect'lon, host resistance, or other ecological requi rements. The gross chemical analyses of the decayed grape and white fir wood blocks by both Ganoderma species indicated significantly reduced Klason lignin percentages and accompanying elevated ho10cellulose percentages when compared to the levels of each corllponent in control wood blocks (Table 3). The apparent acid soluble lignin (AASL) fraction was reduced for the decayed ~hite fir blocks but unchanged for the other decayed woods in relation to the control levels. This may indicate reductions in the methoxyl content of the decayed lignin (Musha and Goring 1974) and suggests different methoxy1ated components of lignin may be removed in a possible step-wise breakdown process. The reduction in %KL/%CHC indicated that selective delignif1cation had taken place in the grape and white fir wood blocks. However, the over-all analyses did not indicate significant lignin reduction within the decayed oak blocks indicated they were delignified. However, due to the high variation, no statistically significant difference was demonstrated. Blanchette <1884a,b) and Blanchette et ale (1985) indicated that gross analyses of decayed wood may mask small delignif1ed areas within large areas of undecayed or simultaneous decayed wood. SEM studies demonstrated that delignified wood could be identified morphologically (Eriksson et ale 1980a; Blanchette 1984a,b; Blanchette et ale 1985). Our SEM studies indicated that the oak and Douglas-fir blocks had delignified areas 43 adjacent to simultaneously decayed wood as in the grape and white fir blocks. However, the extent of del1gnified ~reas varied greatly between the woods. Therefore, the analyses of the entire wood blocks did not indicate de1ignification in all the woods studied and the method depended on large amounts of de11gn1f1ed wood being present within the blocks sampled. No relation was found between growth rates and selective de1ignification. Setliff and Eudy (1980) also found no relation between de1ignification and growth rate with the fungi they studied. SEM studies of all decayed woods indicated the general occurrence of simultaneous decay and selective del1gn1f1cat1on. Selective del1gnification was indicated by removal of middle lamellae and primary walls. High levels of lignin have meen shown to be present in the middle 1ama11a, while the cellulose concentration increases in the secondary walls (51 and S2) (Pansh1n 1970). In all the woods both the K1ason 11gn1n and ho10cellulose we-re decayed. However, SEM studies indicated that rates of removal of each component a.ppeared to vary with K1ason lignin degradation generally occurring before the ho10cellulose breakdown. Since ho10ce11u10se consists of both the cellulose and hemicellulose fractions, a selective removal of either component may have occu rred. 81 anchette (l984a) noted that .G... tSlIgae removed hemice11u10ses before the cellulose in naturally decayed hemlock. Structural models of the arrangements of cell wall components have been proposed (Keegstra et ale 1973; Kerr and Goring 1975). The de1ign1ficat10n process observed in our studies may possibly depend on 44 the spatial arrangement and accessibility of lignin in relation to the cellulose macrofibrlls and other cell wall components in each of the woods studied. As mentioned above, the SEM studies indicated that all of the wood blocks were de11gnif1ed to various extents. Grape wood was delignified the most, while the Douglas-fir was primarily simultaneously decayed with small localized areas of de1ignified wood. Ander and Eriksson (1977) indicated that fJlcnoporus c1nnaba'r1Dus caused greater de1ign1fication of pine wood in the presence of malt extract. In our studies, the malt extract may have enabled the fungi to cause in ~ de1ign1f1cat10n of wood. However, the de1ign1fying process caused by both Ganoderma species appeared to be influenced by the wood type as was the percent weight loss. The influence of wood type on selective de1ignifications was observed in studies with other fungi (Meier 1955; Kirk and Moore 1972; Kirk and Highley 1973; Blanchette 1984b). Kirk and Moore (1972) found that lignin was removed only slightly faster than the carbohydrate in Douglas-fir wood decayed by Fornes ulmarius (Sow. ex. Fr.) Gill. Our results were similar and indicate the possible structural or nutritional influence of wood on the processes of decay. In anyone wood various stages of decay appeared. SEM stUdies indicated that rays were generally simultaneously decayed while tracheids were de1ignified. In the grape, oak, white fir, and Douglas fir wood types, de1ignified areas appeared to be completely degraded in later stages of decay (Figs. 8A, 8B, and 8e). The rate of the two processes appeared to vary between the wood types and the wood tissue. For example, the grape wood had its large mu1tiserate rays sim1utaneous1y decayed While adjacent fiber tracheids were de1ignified. 45 The Douglas-fir had localized areas of tracheids delignified while most of the wood appeared simlutaneously decayed. As mentioned above, the structural arrangement of the wood components may account for the decay differences observed between tissue. Otjen and Blanchette (1984) have indicated simultaneous decay of rays of oak by the white pocket-rot fungus Iooootus dryophllus (Berk.) Murr. They have suggested from other studies (Merrill and Cowling 1966; Kirk et al. 1976) that the elevated nitrogen content of ray parenchyma may promote simultaneous decay in these areas. This may be another possibe explanation of the decay differences observed within different tissues and different wood types. Blanchette (1984b) considered GAn~m~ ~~um (Pers.:Wallr.) Pat. capable of both simultaneous decay and selective delignification depending on the wood species being decayed. Although he considered Ga. lllcidurn (Curt.:Fr.) Karst. and .Ga. cllrtis1i (Berk.) Murr. as simultaneous decay fungi, he thought that these fungi had the potential of selective delign1fication. Our studies indicate that Ga. luc1dum (Ga. curt1si1) and Ga. tsugae are capable of in Yitro selective delignification of wood. The potential inportance of selective delignification of wood for industria1 applications has been reviewed (Kirk and Moore 1972; Ander and Eriksson 1977; Kirk et al. 1980; Blanchette 1984a,b) of white rot fungi have been shown to have the ability to selectively delignify wood. Our studies indicate that the in vitro use of Ga.lucidum or G... ~~~ may clarify on the enzymatic mechanisms of selective delignificatfon. CHAPTER 3 INFECTION AND COLONIZATION OF YlIl£ YINIEERA BY GANODERMA LUCIPUM Introduction Ganoderma lucidllm (Curt.: Fr.) Karst. is a wood decaying basidiomycete that occurs on many species of hardwoods in North America (Hepting, 1971). There are numerous reports on the association of ~ lucidum with dying or declining trees including oak, sycamore, honeylocust, ash, sassafras, and maple (Nickell, 1952; Edgerton, 1954; Pirone, 1957; Peace, 1962; Parris, 1966; Toole, 1966; Schmidt and Seymour, 1972; and McCracken, 1977). It is also reported from tropical areas where hosts include 011, areca, and coconut palms and hardwood species such as white mulberry, Dalbergia sp., Acacia sp., and Casuarina sp. (Venkatarayan, 1936; Turner, 1965; Mahmood, 1971; and Bakshi et al., 1968, 1976). In general, these reports consider ~ lucfdym to be a wound parasite. Only a few studies have been made on the pathogenicity of ~ lucidum using Koch's postulates (Pirone, 1957; Navaratnam and Leong, 1965; and Toole, 1966). In these latter studies ~ lucidum was shown to be weakly pathogeniC with slow development once established within the host. Infected plants seemed water-stressed and usually died after a year or more. In 1979, a report from northern Mexico was made on the association of fruiting bodies of a Ganoderma species with a disease of grape (V1tlls yfn1~era L.) called Colapso (Teliz, 1979). The disease symptoms included severely stressed plants with leaves that wilted, 46 47 turned brown, and died within 3 days. Fruiting bodies of the fungus were found on adjacent support posts and occasionally on the trunks of the grape vines. The fungus was thought to be brought in on infected posts. However, Ga.. lucidym 1s indigenous to the Sonoran Desert reg~on, occurring on native hosts such as mesquite and palo verde (Gilbertson et a1., 1979) and is presumab1y the causal organism of Co1apso. Presently, northern Mexico has approximately 40,000 ha of commercial grape vineyards in production, mainly in the states of Sonora, Durango, Coahuila, and Baja California. Common grape varieties grown are Thompson Seedless and Perlette (Te1iz, 1979). Arizona also has a developing grape industry with 2,000 ha. Since Ga..lucidum occurs naturally on native hosts in northern Mexico and Arizona and may be a pathogen of grape, there appears to be a potential for economic loss. The objectives of this r~searth were to determine the pathogenicity of .G... luc1dum on grape based on Koch's postulates, to determine the rate of development of the disease, and to elucidate on the development of the fungus from infection to establishment and colonization within the host plant. Materials And Methods Grape Cutting Propagation and Culturing of .G... lucidum Collections Vitys vinifera softwood cuttings of the variety Robin and rootstock cuttings of the variety Dog Ridge were made following Winkler et a1. (1974). Seventy cuttings were directly planted with no root hormone treatment into sand with bottom-heating of 23-26 C. Cuttings were automatically misted every 90 sec on a 12 hr day/night schedule. 48 After two wk, cuttings were transplanted into eight-liter containers using a peat-sand-loam (1:1:2 ratio) pasteurized mix and watered 2-4 times/wk throughout the two yr study. Temperatures ranged in the summer from 32-35 C during the day and 24-27 C during the night, while in the winter temperatures were 24-27 C during the day and 21-24 C during the night. Grape pl ants were pruned and fertil hed \II ith Osmocote 19-6-12 (Sierra Chemical Co., Milpitas, CA ) 4 times/yr. Greenhouse fumigations with insecticides were conducted periodically; however no fungicides were used. Basidiocarps of G....luc1dum were collected from Arizona, Ohio, and Louisiana. Isolates were obtained from context tissue and grown on 2% malt extract agar (MEA) from specimens in the following collections: G.... luc1dum RLG 14291, Madera Canyon, Santa Rita Mountains, Coronado National Forest, Arizona, from silver leaf oak (Quercus hypolellco1des A. Camus); JEA 140, Camp Duessel, Portage County, Ohio, from sugar mapl e (~ saccharum Marsh.), collected by K.H. Yohem; and JEA 141, Fontainebleau State Park, St. Tammany Parish, Louisiana, from maple (~ sp.) collected by M. Blackwell. Greenhouse Studies Inoculum was prepared for above-ground inoculations of ~ lllc1dllm on grape cuttings grown in the greenhouse. For this, wood chips (ca. 0.2 X 0.5 X 1.5 cm) were cut from 3-yr old, woody grape canes and placed on cultures of isolates RLG 14291 and JEA 141 growing on MEA at 25 C. After 2 wk the wood chip inocula were completely colonized. Inoculations were made by placing inoculum into a 2-cm, xylem-exposing 49 graft-cut, made approximately 13 cm above soil level. The outer bark was folded over the inoculum and wrapped with Paraf11m-M (American Can Co., Greenwich, cn. Six plants of each variety were inoculated with each fungus isolate while six uninoculated plants and six plants inoculated with uninfested grape chips were maintained as controls. For root inoculations, silver leaf oak wood blocks (ca. 6 x 6 x 10 cm) were cut with the large face tangential and placed on 3 mm 'V' shaped glass rods on 100 m1 of MEA in O.S L glass chambers. Media were then inoculated with either the RLG 14291 or the JEA 141 isolates of ~ lyc1dum. After incubating for 10 wk the completely colonized wood blocks were removed and placed approximately 1 cm below the soil surface within 1-2 cm of the plant stem. Each plant was inoculated with one wood block. Cuttings of Dog Ridge cultivars (cv.) but not Robin cv. were divided into wounded and non-wounded treatments. Plants for the wounded treatment were injured with a grafting knife by exposing 2 cm of xylem tissue in a tangential cut of the stem 4 cm below the soil surface on the side facing the inoculum block. Both wounded and non wounded treatments also had minor injuries to the secondary roots when the inoculum was bur1ed. Controls were plants with non-inoculated wood blocks and plants without wood blocks. All treatments were replicated six times in the 2-yr experiment. At the end of 24 months, the plants were harvested, cut in half longitudinally, and reiso1at1ons were conducted. Field Studies Fifteen 17-yr old Robin variety grape plants maintained in 50 production since 1965 at the University of Arizona Campbell Ave. Field Station, Tucson were inoculated with either of the Gao luc1dum isolates. The vines were approximately 15 cm in base diam. by 125 cm in height and were grown on a 'T' trellis system. Viticultural practices included spring applications of 900 gm of ammonium sulfate/plant/yr, fall pruning, and furrow irrigations once/2 wk in summer and once/4 wk in winter. Insecticides were occasionally used, however fungicides were not used during this 2-yr study. Inoculum was prepared (as described for above-ground inoculations) using isolates JEA 140, JEA 141, and RLG 14291. Inoculation were accomplished using sterile techniques which included flame sterilization of the drill bit and surface sterilization of the trunk area to be drilled with 95% ethanol. A l-cm diam. hole was d r111 ed to the center of the heartwood at the base of each trunk. The wood chip inoculum was inserted into each cavity, which was then plugged with a cork. Five inoculations were done in January and another five again in March when the plants were dormant. Five actively rowing plants were inoculated in June. Controls were uninoculated plants and plants inoculated with non-infested wood chips. Two yr after the first inoculations, plants were removed from the field and cut into 10-14 cm long transverse sections. The sections were then cut in half 10gitud1nally. Decay columns were measured and reisolations were conducted. Results Identification of cultures of Ga. lucidum Isolates of Gao luc1duoo had cultural characteristics as described 51 by Nobles (1948). Chlamydospores, cuticular cells, stag horn hyphae, and clamp connections were readily identifiable in culture when observed by brightfield microscopy. Pathogenicity of .G,.. lllcidllm on Grape Plants Grown in the Greenhouse Fruiting bodies of .G,..lllc1dum started to developed on the soil surface from below-ground wood block inoculum within 3 mo after inoculation; most fruiting occurred within 16 mo. A total of 31 fruiting bodies developed (one/inoculum block) from the 36 infested blocks placed below ground. Fruiting bodies typically were stipltate (3- 6 cm) with an eccentr1c pileus (2-4 cm). Occasionally aberrant fruiting bodies developed with tubes on the upper surface. All inoculated wood blocks were in advanced stages of decay at the end of the 2 yr study while those in the checks were undecayed. During the first year of the experiment plants generally appeared healthy, with rapid new shoot development after each pruning. However, about 12-18 mo after inoculation three plants inoculated below ground developed water stress symptoms and had slower shoot development after pruning. After 19 months one of these plants failed to produce new growth after pruning. Two fruiting bodies developed on this plant within 1 mo after death (Fig. 10). After 24 mo, the number of plan::s infected was determined. Four of the 12 inoculated cv. Dog Ridge plants in the wounded plant treatment became infected. One of these plants had died and developed fruiting bodies. None of the controls were infected and the plants with uninoculated wood blocks showed no symptoms of decline. Only one plant was infected in the treatment with 52 Figure 10. Two fruiting bodies of Ganoderm~ Jucjdum on a Dog Ridge variety grape plant grown and inoculated in the greenhouse (arrows). 53 non-wounded plants. The five infected plants were sectioned and three stages development were observed and compared to a healthy control plant (Fig. 11): 1) the fungus entered through the wound or callus tissue on the below-ground portion of the stem; 2) the decay column developed vertically through the heartwood of the stem; and 3) the decay columne developed laterally into the sapwood in later stages (Fig. 11 8-0). Decay was identified by bleached, spongy, wood typical of white-rots. Ganoderma luc1dum was reisolated from these areas. The control plants had a darkened heartwood with a uniform, lighter colored sapwood (Fig. 11 A). Primary and secondary roots were not infected by the fungus. Non-wounded Robin cultivars were not infected in the below ground inocul ations. All above-ground i nocul at ions on both cul t ivars also were unsuccessful. The fungus apparently died and was compartmentalized by the host since the fungus was not reisolated from these areas and the wood was darkened around the inoculation site. Development of ~ luc1dum in Field Grape Plants Three plants were inoculated for each of the three G....luc1dum isolates. The remaining six were controls. Ganoderma lllc1dum was reisolated from the three of the nine plants inoculated at the end of the study. Two of these three plants developed decay columns 25 and 42 cm in height from the initial point of inoculation within 17 months. The fungus grew mostly upward in the heartwood with some growth (5-10 cm) in the downward direction (Fig. 12). The fungus was not reisolated from the sapwood. The third inoculated plant from which G.... luc1dum was 54 Figure 11 A-D. Stages of decay in the development of Ganoderma lucjdum within Dog Ridge variety grape plants grown and inoculated in the greenhouse. Fig A. Control plant with dark heartwood (h) and 1 ighter colored sapwood (s). Fig. B. Early stage of decay limited to the heartwood (arrow) and entering at wound-callus area (double arrow). Fig. C. Heartwood (h) completely decayed, macroscopically detectable white-rot. Fig. D. Sapwood (s) and heartwood (h) decayed in an advanced stage (Mag.=1.2X) 55 re1solated also had another wood decaying basidiomycete, pbel11nus texanus (Murr.) A. Ames, fruiting on an upper branch. Zone 1 ines separating two decay columns were evident in longitudinal sections. pbe1l1nus texanus was isolated from the fruiting body and the decay column that originated from the top of the plant, while.G.. luc1duoo was reisolated from the lower decay column (Fig. 12). Susceptibilities could not be related either to time of inoculation or to the isolate of .G.....luc1dum. Ganoderma luc1dllm was not reisolated from the other six inoculated plants. Two of these remained healthy, the other four had established advanced decay columns of either E.a.. texanus, pbel11nus bad1Us (Berk.) G.H. Cunn., or pen1opbora albobad1a (Schw.: Fr.) Soidin. Fruiting bodies of the three species were found on the upper branches of the grape plants. Cultures from decay columns were identified by comparison with cultures from basidiocarps. Six controls. three uninoculated and three inoculated with an uninfested wood chip, were used in the field study. No fungi were recovered from one plant from each set of controls. Control plants normally had dark heartwood and lighter colored sapwood (Fig. 12). Three other control s had decay from either E.a.. texanus or E.a.. bad 1 us in the upper trunk, while the middle to lower trunk remained undecayed. Control plants that were inoculated with a healthy wood chip were not adversely affected by the treatment. One remaining control plant, that was not inoculated, had a small decay column (approximately 8 cm) that originated from a soil-line shoot injury. Reisolations from the decayed area indicated a natural infection of .G..... luc1dum (Fig. 13). 56 Figure 12 A-C. Control plant and plants inoculated with Ganoderma lucjdum under field conditions. Fig. A. Control plant with no decay. Fig. B. Field grape plant inoculated with isolate JEA 141. Decay column limited to the heartwood (arrows indicate upper and lower limits of decay; i =inoculation point). Fig. C. Field grape plant with mixed infection of~ lucjdum and Phellinus texanus. Zone lines separate the two decay columns (arrows). 57 Figure 13. Transverse section showing natural infection of Ganoderma lucjdum in a field grape plant. Decay originates from a soil-line shoot injury and continues through the pith of the stem into the heartwood of the trunk (arrows). 58 The decay column developed from the wound and advanced from the branch pith to the trunk heartwood. Discussion Ganoderma lucidum is an indigenous wood-decaying fungus on hardwood species in North America (Adaskaveg and Gilbertson, unpublished). Under optimal temperature conditions of 30-34 C, the fungus can rapidly colonize and decay suitable wood substrates in ~ (Adaskaveg and Gilbertson, unpublished). The environmental conditions of the treatments in the greenhouse-grown grape plants were conducive for both pl ant and fungus growth. Control pl ants had well developed root systems and healthy top growth with no symptom of nutrit ional deficiencies while Ganoderma lucidum grew as evidenced by fruiting body development from the buried, infested block and by the continued decay of these inocula. Although the incidence of infection was low, we have established by Koch's postulates that ~ lucidum can infect grape plants grown in the greenhouse. Infection occurred in the callus tissue of the grape stem, primary roots or secondary roots (mostly less than 5 mm in diam.) were not infected. Although 8akshi et al. (1975) considered it necessary for G... lllc1dllm to be in direct contact with a healthy root for infection to occur, our study indicated that ~ lucfdum can grou short distances (1-2 cm) through the sol1 from an inoculum source to cause infection of the grape plant. Many reports suggest that G.a. lucidum either is a wound pathogen or an organism infecting weakened plants (Vankartarayan, 1936; Bagchee and Bakshi, 1950; Bakshi, 1951; Pirone, 1957; Peace, 1962; 59 McCracken, 1977). However, other investigators have reported it to be pathogenic on unwounded plants (Navaratnam and Leong, 1965 and Toole, 1966). Ganoderma luc1dum was found to be pathogenic regardless of the treatment. In our studies, the wounding treatment favored infection. suggesting the possibility of transmission of the fungus from a below ground infested wood source (such as a support post or another infected plant) to a healthy plant through wounds created by management practices. Basidiospores may also be a source of inoculum since one of our field plants was naturally infected through a soil-line shoot injury. Although Bakshi et a1. (1976) concluded that basidiospores were not involved in infection of stumps of khair, Turner (1965) and Adaskaveg and Gilbertson (unpublished) have found different genotypes present in a population of 1i.. lucidum from closely distributed hosts in a specific area. This would indicate the involvement of basidiospores in the distribution of the fungus. GanQderma lucidum develops within the heartwood and sapwood of hardwood host plants (Bagchee and Baksh1, 1950 and Pirone, 1957). Although the development of the fungus within our greenhouse grown grapes occurred vertically in the heartwood, with lateral development into the sapwood in later stages, our field inoculated grapes had decay columns limited to the heartwood. This suggests that the sapwood is decayed by the fungus only in the later stages of the disease. 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