Population Structure, and Sporocarp Overwintering
POPULATION STRUCTURE,
SOMATIC INCOMPATIBLïIY,
AND SPOROCARP OVERWINTERING
in
CHONDROSTEREW PURPUREUM
A Graduate Thesis Submitted
in Partial FdWment of the Requirements for the Degree of Masters of Science in Forestry
Faculty of Forestry
Lakehead University
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SCHOOL OF FORESTRY & FOREST ENVIRONMENT LAKEHEAD UNIVERSITY THUNDER BAY, ONTARIO --* 111
ABSTRACT
Patterson, M.L. 2000. Popdation structure, somatic incompatibility, and sporocarp overwintering in Chondrosierecim purpureum. 90 pp. Advisor: Dr. E.C. Setiiff.
Key words: Chondrostereum purpureum, population structure, somatic incompatibility.
An investigation was conducted of the population structure, the somatic incompatibility reaction, and sporocarp overwintering in Chondrostereum purpureum (Pers.:Fr.) Pouzar, a proposed mycoherbicide of undesirable hardwood species, Chondrostereurn purpureum breeding populations of recent cut-over sites in Thunder Bay, Ontario were found to have a high number of individuals possessing different aileles for somatic incompatibility- Up to nine fungal individuds per wood unit (logs, stumps) were found, with the majority of wood uni& baving only one to three individuals. The macroscopic appearance of the somatic incompatibility interaction zone on malt extract agar varied among paired isolates and was occasionally ambiguous. Phenotypic variability of the interaction zone ranged tiom scant mycelia to massai hyphae between the two colonies. Microscopicaiiy the sparse interaction zone had chains of swollen spindle-shaped celis, while the massed interaction zone had distorted hyphae, encoileci hyphae, and hyphal knots. Of overwintered sporocarps coilected in the spring of 1998 and 1999,86% produced viable spores. Basidiospore leve1s in the spring may be greater than once thought, and thus may pose a threat to winter-damaged trees. This shouid be considered in the assessment of the epiderniology of C. purpureurn, and in the assessment of this fungus as a biocontrol agent. iv
CONTENTS
Page
ABSTRACT
TABLES
FIGURES
vii
INTRODUCTION
Research Objectives Chondrostereum purpureum Population Studies of Fungi Population Studies of C. purpureum Microscopic Charactenstics of Somatic Incompatibility Overwintering Capability of C. purpureum Sporocarps
MATERIALS AND METfIODS
Site History Population Study of C. purpureum Microscopic Characteristics of Somatic Incompatibility in C,purpureum Overwintering Capability of C. purpureurn Sporocarps
Population Study of C, purpureum Microscopic Charactenstics of Somatic Incompatibility in C. purpureum Overwintering Capability of C. purpureum Sporocarps
DISCUSSION
Population Study of C. purpureurn Microscopic Characteristics of Somatic hcompatibiiity in C. purpureurn Overwintering Capabiiïty of C. purpureurn Sporocarps
APPENDIX 1 SAMPLE 'INVENTORY FOR CCA, PLOT 59, MBD, AND WBD
APPENDIX II SAMPLE INVENTORY FOR BIRCH PILES
APPEND~Xm PWGS BETWEEN ISOLATES COLLECTED FROM BIRCH LOGS v
TABLES
Table Page
Number of logs in each birch pile, and number and percentage with 39 C. purpureum-
The number of C. puripureum sporocarps collecteci, number of sodc 40 incompatibility groups, A and B face compatibility, and intransitivity of SI reactions for each C. purpuretun infected log in Birch Pile A.
The number of C. purpurem sporocarps coiiected, number of somatic 42 incompatibility groups, A and B face compatibility, and intransitivity of SI reactions for each C,purpureum infected log in Birch Pile B.
The number and percentage of Iogs in Birch Piles A and B having one to nine somatic incornpatibility groups per log.
Sporulation and germination of overwintered sporocarps. 49
Relevant Thunder Bay weather data for winter 1997/98, spring/surnmer/ 50 fa11 1998, and winter 1998199. Figure Page
Map of Thunder Bay indicating the locations of study sites Cascades 25 Conservation Are* Mills Block D, and Williams Block D.
Map of the Cascades Conservation Area study site indicating the 26 locations of the path and penrneter benchrnarks, Plot 59, Birch Pile A and Birch Pile B.
Two-week-old pairings between C. purpureum isolates demonstrating 28 somatic incompatibility and somatic compatibility,
The MBD study site one growing season after poplar and birch harvest 30 at the tirne of the C. purpureurn sporocarp survey.
Birch Pile A and Birch Pile B, from which sporocarps of C. pulpureum 31 were collected one growing season afier the logs were cut.
Pairings between randomly selected C. purpureum isolates collected from 34 the Cascades Conservation Area study site,
Map of Plot 59 with locations of 12 potential wood sites for C. purpureum. 35
Pairing results for C.purpureum isolates collected in Plot 59. 36
Results for pairings between C. purpureum isolates collecteci in Mills 37 Block D.
Pairing results of C. purpureurn isolates fiom WilLiams Block D. 37
Random pairing results of C. purpureum isolates coilected from both birch 38 piles and study site WBD,
Two differing types of C. purpureum interaction zone. 43
Anastomosis between geneticaily dissirnilar hyphae in a non-self pairing. 45
Atypicai spindle-shaped cells with abnormally thickened septa in the 46 interaction zone in a non-self pairing.
The dark hyphal cell at the dysjunction between spindle-shaped ceils 47 and normal cells in the interaction zone of a non-self-pairing.
Base of a suckering balsam poplar stump from which overwintered 48 C. purpureum sporocarps were collected. ACKNO WLEDGEMENTS
Much thanks are due to my supervisor Dr- E-C. SetIiff for his support and encouragement throughout my graduate studies, and to the members of my thesis cornmittee, Dr- P. Tripp, Dr.
W.H. Parker, and Dr. R. Parr for their inspired guidance. 1would Iike to thank Mr. G. Saunders for his generous provision and analysis of the weather data uicluded in this report. 1 am grateful for the laboratory technical help provided by ML L. Sevean and ML S. Eliot, and computer support provided by Dr. U. Runesson, Mi. A. Rudy, Mr. K. Ride, and Mr. P. Leadbitter. 1would iike to thank Mr. V. Laurin, of Hiles-Laurin Contracthg Ltd, and Mr. S. Suke of the Lakehead
Region Conservation Authority for permithg access to the study sites. INTRODUCTION
RESEARCH OBJECTIVES
The main objective ofthis project was to use the somatic incompatibility &on as the criterion in detemuning locai population structure of Chondrostereum purpureum (Pers,:Fr.)
Pouzar [fomierly Steracm purpurmm (Pers.:Fr.) Fr-] (Nakasone 1990) in Thunder Bay, Ontario.
Egger (1992) descrii population strwture as "the patteni of distri'bution of individuals within a species, and their orginkition into groups that share greater overail genetic sunilarity" (p. 193).
Population studies are usefiil in diswvering the means of fhgal dispersal, as the distribution of soddyincompatible individuals in a substrate is a rdleztion of the mode of colonizaîion
(Rayner and Todd 1982a). Worraii (1994) attnbutes the value of understanding fimgai pathogen popuiaîion structures and colonization means to its usefûiness in the establishment of epidemiologicai models ancf disease management strategies. Chondrostereumpurpuratm as a cause of dver leafdisease in £idtrees and with its potential roie in birch deche, is a serious fùngal pest in 6Wt orchards and forests. Because of its role as the cause ofdisease in rnany hardwood species, its eudemic nature in forests, and its proposai use as a mycoherbicide, an understanding of the population structure of CIpurpuracm is important.
Harvested areas provide abundant infection opportunities for this fimgus, with pIenty of dead woody materiai to quickty ampw the population. It may also act as a wound pathogen, causing dieback and decline in the remaining live trees, which are often wounded in the harvesting process. The population structure of C. purpureum includes the incidence of the fiingus and the degree of relatedness within the population. The population structure was obsend at three levels.
At the Grst kvel genetic variability was assessed among isolates found on ùidividual saunps or slash, in order to make detenninations about the prevalence of multiple infections by C-purpureum
within individuai wood units, possibly representing multiple infections in the previously living tree.
The occurrence of multiple infections in live trees may rdt"in a mycotoxin-tolerance threshold
king exceededn (McLauphlin 199 1, p, i 8)- As well, the determination of the population structure
wïthh individual wduni& may shed more Iight on the lifè strategy of C. purpureum. Genetic
vaxiability was determined by pairhg pure culture isolates of C. purpuram, and observing for
reaction lines indicative of somatic incompatibility between the isolates. The second level was a
survey ofinfected wood units and assessnent ofreiatedness between isolates fiom different
individual trees/stumps/slash to determine incidence and genetic variability over a small area (3 14
m2). The incidence and relatedness over the whole clear-cut was the third level. Random painngs
of isoIates collecteci fiom over the study area were used to assess genetic variability.
As the somatic incompaîibility reaction had a variable appearance and was occasionaily
ambiguous, a preliminaxy study of the microscopic appearance of the interaction zone between
paired C-purpureum dzkaryons was undertaken, This included examination of the somatic
incompatibilily interaction zone to detennine if the IIlilfllfestation of somatic incompatibility was
distinguisbable at the microscopic level.
Another objective was to detennine the ability of C,pupreum sporocarps to successfiilly overwinter under naturai conditions. Currently C.purpurmm sporocarps are thought to be of si@cance for only a single season Observations on overwintering success, as detennined by the abiiity to produce viable spores, may contn'bute to our understanding of; and estabikh the possibility of; early spring/smmner hkiion of *ter darnaged trees by basidiospores. The fimgus Chondrostereum purpuram has been found on ail continents except
Antarctica (Charnuris 1988). C. pupreum is the causal agent of silver leaf disease (Brooks and
Moore 1926; Brooks and Storey 1923; Peace 1962) in orchard trees such as apncot, prune, pear, peach, cherry and apple (Aàaskaveg and Ogawa 1990), and causes disease in forest trees such as mountain ash, maple, popiar, wiiiow, buch, hawihome, and shbslike gooseberry, rose, cotoneiister and nanking cheny (Mamyama and Hiratsuka 1985)- The fiingus has been occasionally reported on some conifers (Chamuris 1988; Nakasone 1990). The disease may remain limited to only a few branches, or it may lead to the death of affected branches within a year and tree mortality in as Meas two or three years (Mmyama and Hiratsuka 1985). Sudden silverleafoutbreaks in orchards lead to decreased fruit production (Chaney et al. 1973) and tree mortality causing considerable econornic loss.
The sporocarp of C. purpureurn is characterized by a smoath hymenium, a morphologid characteristic thaï places it in the WlyThelephoraceae (French 1991, p. 170)'. Originally classified as a species of Stereum because of its stemid sporocarp, Reid (197 1) agreed with
Pouzar in the placement ofthis species in the genus Chondrustereum because of its monomitic sporocarp, non-amyloid spores, and vesicIes in the hymenim. The C. putpurmm sporocarp has a beige and tomentose pileus, with a hymdsurface that starts out purpiïsh, but later becornes buff
IIFrench followed Burt's (1920) concept of placing S- purpreum in the artificial fbily
Tbelephoraceae. Later, the ningus was placed in Pil&tYsfàxnily Stereaceae, a family whose delimitations remain uncleaq Charnuris (1988) placed C. purpurmm in the artifïcial fàmily
Corticiaceae sensu lato. to brown (Cartwright and Findlay 1958; Thomas and Podrnore 1953). The sporocarp when
aitacheci to a wood substrate may be nat or bracket shaped (Cartwright and Findlay 1958) and is
snnilar in appearauce to severai Con-olusspp.
In live trees hiting occurs at the base of affected branches or just above the root coiiar
(Maruyama and Hiratsuka 1985). Fniitùig may be extensive over any exposed surface of dead woody material (WaU 1997)-
New basidiocarps of C. purpuracm are produced in spring and autumn when it is cool and damp (Wall 1991). Wail(1997) found th& the production of sporocarps mdd be expeded to last as Meas two years following infection by C. purpuram, when they are then displaced by other cornmon qrophytic hyrnenomycdes (Fritz 1954; Rayner 1978; Rayner 1979; Rayner and Todd -. 1979; True and MacDonaId 1973). Conversely Ebtkka (1993) found that new C. purpurmm basidioçarps were hdon stumps five to men years 014 which may have occurred due to resistance such that the tree's moxtaiity was exîended and sporocarp production continued.
- * C-puTpureum is tetrapolar and heterothallic Wbak 1942), wiîh cuiturai characteristics as describeci by Stalpers (1978). Waii et al, (1996) found dopatric homokaryon isohfkom different regions of Canada were matiog compatible, thus C purpurem readily outcrosses and there is Meimpediment to gene flow among Canadian popiiiations.
C-purpureum is a primary saprophyte that produces a white rot (Adaskaveg and Ogawa
1990) of recently wouuded and freslily Ialled hardwood material (Fritz 1954; H.1993;
Rayner and Wdy 1986). Although it pwsrapidly, C. ppreum has a slow rate of decay
(Rayner and Boddy 1988 p, 269; Rishbeth 1976), which has been suggested to be the result of its ab* to use oniy nutrient reserves of live parenchyma ce& (Guinier 1933, cited in Cartwright and
Findlay 195 8). Luttreli (1974) has characterîzed C. purpuram as having hemibiotrophic behaviow, where tissue dies folIowing infection, thereby conkrring to the fiingus the advantage of prïmary occupation of the food resoure at the time of de&. In this way C. purpuracm may pre-
empt other saprophytes in the cornpetition for recently4ead wood substrates (Fritz 1954; Luttreli
1974)- Spiers and Hopcroft (1988a) postuiated that in a similar manner C-pu'pureum might lie as
a latent infection that becornes active when host susceptiibility increase.3 or cornparimen~onis
overcome, Wail(l99 1) found that C. pupreum could survive for at least six years in
succes~ycomparîmenîaiïzed discoIored wood, and in this way Iiving trees may act as the
reservoîr for the bgus, and thereby provide the inocuium for Merinfection (Spiers and
Hopcrofi 19%8a)-
McLaughiin's (1 99 1) observation of the re-emergence of a canker fâr rernoved fiom the
point of original infection and cankering, demonstrated thai C. purpreum could be an aggressive
paîhogen on birch seedlings. This indication that the fiingus could overcome the strongest ofthe barrier wds, the extenial waU surrounding the decay (Shigo 1984, cited in Mchphlin 199 l), is supported by Wall's (1986) conclusions that C-purpuracm may invade iive cambial tissue and associated sapwood.
C-prpureum may spread by root grafting (Maniyarna and Hiratsuka 1985) although wounds infected via aihome basidiospores are usually the route ofnew infections (Brooks and
Moore1926; Brooks and Storey 1923; Grosclaude et al- 1973; Maruyama and Hiratsuka 1985).
Gadgil and Bawden (198 1) found C-purpureum in 12,4% of experimental pnrning wounds, making it the most fhquently isoM of decay.causing fungi in a New Zealand orchard.
There have been several seemingly contradictory saidies on seasonai susceptibility to C. purpureurn, with différent mearchers finditg winter (Stanïsktwek et al, 1987), late winter/early spnuig (Brooks and Moore 1926), sprUig/earIy sumrner (Beever 1970), mid-summer (Spiers et al.
1998; Waü 199 1), and Iate swnmer @umas et al- 1997) to be the time of increased tree susceptibility. There appears to be a general wllse~lsusthat suscepbiility peaks at some time in spring or summer. Wd(199 1) describeci purpureum as fiuituig at times when tree suscepti%ility is low, noting that with thiç tendency, and the ab* of healthy trees to comparûnentalize infection, this firngus, under normal conditions, may be a threat only to trees compmmised by stress. Supporting this, de Jong et al. (1996) found C. puqmreum basidiocarps on live trees, were associated only with injuries-
Silverïng ofthe Ieaves ofaffiected trees is the optical result produced when a polygalacturonase toxin produced by purpuratm (Miyairi et al. 1977) causes the palkade mesophyU ceik to separate fiom the epidexmïs and each other (Peace 1962)- Silverïng may not occur in all species affected by this fiingus (Peace 1962; Spiers and Hopcroft 1987), or even m some epidemic siaiatiom where Swift mortality may preclude the advent of the silvexing symptom
(Setliffand Wade 1973). In birch seedlings McLaughh (1991) found symptoms of C. purpureum inf'ection included srnail, unevedy pigmented, and lackluster leaves, and silverkg scarcely noteworthy in large trees.
Darkening and discoloration of affiwood caused by fùngal production of laccase
(Miyairi et al. 1982, cited in Charnuris 1988) and black zone lines may be observed in trees infected with C-purpumm (Setliff and Wade 1973). Plugging of the vascular system by wood gums (WCiIliams and Cameron 1956, cited in Chamwis 1988) may be evidence of invasion of the xylem tissue by the fiuigus (h4cLaugih 199 1). When a rapid decay course occurs, a chacteristic reaction zone may nd be present (Pearce et al, 1994), which may be due to the ab* ofC. purpumm to "either invade hctional xylem rapidly, or propagate xyIem dysfùnction ahead of the infection fkont, whiist avoidùig, deto- or suppressing host defensive responses"
(Pearce L996, p. 227). Thomas and Podmore (1953) characterïzed early decay by C. purpumm in black cottonwood as a pale-brown stain, where wood strength appears &ected- Advanchg decay resuits in the fading of the stain as the wood becornes bleacbed, britùe and light weight
(Thomas and Podmore 1953; Spiers and Hopcrofi 1988a).
A massive birch dieback phenomenon occurd hm1937 and 1949 in southeastem
Canada and northeastern United States (Braaîhe 1995). Birch dieback characteristics desmi by
Pomerleau (1953a) included ccdiscoiorationand wilting ofthe Ieaves, smaiier and thinner foliage, dyïng of twigs and branches, which progressively extends fiom the top to the base of the crown, and Myto the death ofthe entire treei7(p, 147). Although recent work by Braathe (1995) indicated thai sprhg thaw and subsequent frost at a critical point in bud burst may have led to this phenomenon, symptoms of birch dieback suggest a systemic injury (Balch 1953), not iinlike what is observed in C. purpurmm infêcted birch. The large scale dieback of birch has long been posaiiated to be a redt of changes in tree water economy due to disruption in normal water conduction (Greenridge 1953); and vascular dysfiuiction is a leadhg cause of the sympt0m.s seen in trees infécted with C.pupureurn (h4cL.a~~199 1; Spiers and Hopcrofi 1987). In C. pupreurn infected trees, the translocation of mycotoxins produce symptorns fiu fiom the point of active infection (Cartwright and Fiudiay 1958). McLaughh (199 1) suggested this as a reason for the fàilute of the fiingus to be isolated in early studies of birch dieback (Hansbrough 1953; Horner
1953; Pomerleau 1953b; Redmond 1953a; 19536; Stillweil 1955).
More recently some studies have found an association between C. purpreurn infection and birch with deche symptoms or discoiored wood (Allen 1996; McLaughlin 1991; McLaughlin and Sem1990; Setliffand McLaughh 1991; Morawski et al. 1958, cited in Allen 1996).
Because wood discoloration due to damage or disease is most rapid in birch wrnpared to other hardwoods (Shigo 1965, cited in Allen 1996), wddecay and discoloration in birch is a serious problem that bas lead to its decreased value as timber. Because white birch is a cornmon ornamentai tree in mmy Canadian cities, the importance ofdieback and decay in birch is not limited to its decre;tsing commercial value for the forestry ind-.
McLaughh (199 1) postulated that C-purpurmm may act on infected trees in one of two ways. It may be a primas. pathogen wiîh the capability of kiilïng parts or aii of a tree quickly, or it may act as a predisposing ktor, with decline symptoms a dtofmycotoxins and secondary organkms, Factors that inûuence tree hedth may be Iargely based on the success of cornpartmentakationofinfection within the tree, as in&echial cornpartmentalidon may allow rnycotoxin seepage into the sap flow or idkdon to overcome the compmentalization walls and to spread (h4cLauphlin 199 1). Vascular disruption causai by the mycelium plugging the xylexn
(Spiers and Hopcroft l988a) and compartmentalization of ~afef+xnductingtissue (McLauphlui
1991) are beiieved to dis- vascuiar translocation leading to wilting and other deche symptoms.
Scheepens and Hqerbnigge (1 988, cited in de Jong et al- 1990) found tbat C. purpureum had a two-year mortaiity efficacy rate of 6 1%, aptiy demonstratulg the potenna1 of C. purpurem as a mycoherbicide of brd-teaved weeds, Winder and Shamoun (199 1)note a paradie shift within forestry with the goai of using biocontrol to decrease cornpetition fiom weeds rather than to reduce their numbers. Application of C. purpureum to stumps has been demonstrated to reduce sprouting or sprout viability in Acer rubrurn L. (Wall 1990), Alrms simata
(Reg-)Rydb. (Comeau and Harper 1996), Alnus rubra Bong. (Prasad L996), Populza tremuloides
Michx., P. grandidentuta Michx. @umas et ai. 1997), Betulapapynj5era Marsh., and Pmm pensylvanica LX (Jobidon 1998; Waii 1990)- Wall (1994) found that coupiing Mihg treatment ofu~wantedAlmcs rubra with application of C. purpuracm cultures increased tree rnortalay, and
Prasad (1996) found that this increased foliage mortality. At this time certain C. purpuram isolates are king tesied for their environmentai impact, so that eventually a debiologicai control prduct can be used for conifer release treatments and for clearing utility rights-of-ways (lobidon
1998).
POPULATION STUDIES OF FUNGI
There are a variety of methods that may fK used in the study of fimgai populations and their structure. These techniques involve directly or indirectiy assessing genetic variability within the intraspecsc gemme- Isoenzyme, protein, and vinilence studies may detennine biochemkai pathway cornmonalities within a population, and DNA analyses enable observation of the variabdity at specific loci and regions of the gamme. The number and distribution ofmating-type factors and somatic incompabbility groups may be us4to examine genetic variability a loci governing these behaviours.
Sen (1990) states that isoenzymes may be supenor to the somatic incornpati'bility technique, because the degree of genetic relatedness among closely reiaîeû individuais might be assessed- Although Burdon (1993) has suggested that analysîs with isoenzymes may not sufiicidy reflect variability wittÜn lower population levels for many species. As isoenzyme markers are limited to the coding regions of DNA (Egger 1992; McDermott and McDonald 1993), this reasoning may extend to any meamrable cbrack&ic such as vinilence CLeung et ai. 1993;
McDermott and McDonald 1993). As weli, because they may be subject to environmental, and thus evolufionary influences, characteristics such as vinilence and isoenzyrnes may repriesent convergent evolutionary pathways rather than actual population structure (Lemg et al- 1993;
McDddand McDermott 1993)- Hence analytical techniques that reflect variation in universai or regionai spedïc characteristics may not be ideai for determinaaon of population structure at regid or local levels. Restriction DNA analyses "provides for large numbers of markers £rom coding and non- coding regions of the DNA" (Egger 1992, p. 193, possbly circumventîng detection of variation found only in regions of genic (coding) DNA which is more highly conserved, thus less variable
(Egger 1992; McDonald and McDermoû 1993)- Greater levels of variation may be found within intervening DNA (transcribed DNA excluded following RNA procresshg), and intergenic DNA
(sequemes &ch are non-transcribed) (Egger 1992)- Thus, general or specific DNA markers appropriate for exposing genetic variation at the desired popuiaîion hierarchicai level are possible
(Egger 1992; Tan et al. 1994).
Correlation between DNA and SO-c incompatibility (SI) analyses of f'ungal genetic vanation vary £iom poor (Jacobsen et al. 1993; Vdgaiys and Godez 1990), to good (Kohli et al-
1992; Mar@ et al- 1998; Rizu, et al. 1995a; Roy et al- 1997), and to perfect (Charnuris and
Falk 1987; Descenu, and kmk@on 1994; Garbelotto et al. 1997; Hoher et al. 1994; Kohn et al- 1991; Smith et al- 1994; Vasiliauskas and Stenlid 1W8a; 1998b)- Roy et al. (1997) observed that although their DNA profiles for Phlebiopsis Wntea(Fr.) Jül. isolates corresponded largely with their SI rd&,the two techniques are based upon "helatedgenetic cnteriay7(p. 2 10 1). It seems llkely that unless the restriction sites, or regions of sequenced DNA are within loci coding for the SI reactiog the wrrelation between DNA anaiyses and SI within a population may be coincidental.
Worrall(1997) listed tbree incoqatiiility systems in the basidiomycetous £Ûn.gï- The first system, intersteriliry, is between 'biological species"; thus hyphai fùsion or mating may not nodyOCCUC~ The sexuai Ïncompatibility system is based on genetic siroilarity where wmmon mating type aiieles will prevent mating e.g. AIB & or AIB - in cases where the mating type alleles are not in cornmon., mating may OCC~~between two hornokqons to produce a heteroiciuyon (Worraii 1997); aLso called a dikaryon in the basidiomycetes (Tocid and Rayner 1980)- WoW (1997) explains that the heterokaryon forms the seco on^ rnyceiium~'which wntains geneticaily dzfferent paired nuclei in each ceil, wÏth one fiom each homdraryoa The third system, somatic incompatibiliîy, aisa caüed vegetative or heterokaryon incompatiiility (Carlile
1987) occurs with genetic dissirnilarity (Worraii 1997). When heterokaryons of différent genotypes meet, recognition of non-sel£ occurs, and hyphal &sion does not take ph. If on the other hand the hyphae recognize each other as "cself',anastornosis (hyphal fision and exchange of cytoplasmic and nuclear contents) occurs and the two individuais wili grow together fonning one thallus of intermingling hyphae (Worrall 1997)-
The "'unit myceliwn" concept vodd and Rayner 1978; 1980) ailowed intraspecific hyphal fùsion between multiple genetically dissimilar dikaryons enabling iarge fnùting structures otherwise thought impossible (Builer 193 1, p. 155- 169). Because evidence for this phenornenon in nature is lacking, it has been replaced by the cCindîMdualisticm~iïum" concept, where somatic incompatibility serves to delimit fiingal individu& within an interbreeding popuiaîion (Rayner
1991a; 1991 b; Rayner and Boddy 1988, p. 205; Rayner and Todd 1979; Rayner et al. 1984; Todd and Rayner 1978; 1980). SI aiso acts to secure food resources for the individuai (Rayner and
Todd 1982b), thus this mechanism is a fonn of territonality (Rayner 1991b; Rayner ef al, 1984).
The most signifiant evolutionary benefit of SI is that it prevents negation of sexualiy denved genetic variation within populations, which wouid happen if somatic compati'bïiity were the nile
(Lane 198 1; Todd and Rayner 1978; 1980; Worrail 1997).
Fusion, or amsîumnsis, of somatïcally compatiale fùngi has its advanîages as it produces a chirnera, which may be more fit than its individual components due to "agreater store of genetic variability with which to tespond to . , . environmentai change" (Buss 1982, p. 5339).
Anasîomosis also benefits bath individuals as survivorship may be higher among larger individuals, and may allow the reproductive stage to be reached fiaster (Buss 1982; Worrali 1997). Buss (1982) has suggested that in the determination of somatic compatibility, Ian selection may
result, as in rnany species the SI loci are Me-to fertility loci, thus reducing the costs of somatic
firsion,
,bastomosis cornes with some practid disadvantages, incluning somatic cell parasitism,
where one genome benefits to the detriment of the derin the somatic association (Buss 1982)-
Even more serious is the phenornenon of genomic replacement, m which one dikaryon set
completely replaces the other after anastomosis (Ainsworth et al- 1990; Carlile 1987; Rayner
199la; Rayner et al- 1984). As well, transmission of mycoviruses and deragents of disease may
be facilitated by hyphal fitsion (Anagnostakk 1992; Caten 1972; Carme 1987; Rayner and Todd
1979; Wod1997). In Canadian isolates of C-pupreum there have been studies on an association between virus-like particles of double stranded RNA and hypoMrulence (Shamoun and
Valverde 1994; Shamoun et al. 1996).
Worrall(1997) defines somatic hcompatibility as the fidure to pproduw a stable heterokaryon by anastomosis of two non-self individuals, when contact between self hyphae usually produces a stable heterokaryon. Somatic incompanbility x~y~ccur rarely in fiingi as monincomptibiliîy, where fiision fails to take place3or more commonly as pst-+ion incomparibility, where hyphal f'usion and cytopIasmic mixing occurs followed by the characteristic macroscopic SI reaction (Carlife 1987). Heterokaryon self-incompatibility, where sbtürrs Lack the ability to fonn a stable heterokaryon (Correll et al. 1989; Hyakumachi and Ui 1987; kiie 1993) is distinguished fiom somatic incompatibility by dtingeom pre-hion events, which prevent anastornosis fiom occurring. Correil et al. (1989) posailated that seIf-incompatibility might occur due to a mutation a-a single gene that wntrols the anactnmosis fiinction.
The somatic inc~rnpatïilityreadon on artifid media where two colonies interface has been described as a '%ne of demarcation" (Schmitz 1925), "he of aversion" ('ounce 1929, cited in Adams and Roth 1967), ccbarrage~~(Vandenciries and Brodie 1933, cited in Adams and Roth
1967), and '%ne of antagonism" (Wonall 1997). As the actuai mechanism of SI is poorly
understood, Barrett and Uscuplic (197 1) described the SI reaction on qpr as the "intefaction
zone", &ch is the tenninology used in this report. This reaction has been correlated in some
species to "hnelines" in infécted wcxxj, whîch rnay serve to delimit the individual and its substrate
territory (Rayner 199la; Rayner and Wdy 1988, p, 430; Todd and Rayner 1978; 1980; Williams
et al. 198 1); altbough zone lines were not observable in wiiiow infected wÏth multiple C. purpreurn basidiospores (Spiers et al. 2000). The appearance of the SI irrteraction unie rnay vary
among hgal species and even among isolates of species (Marçais et al. 2000; Rayner and Boddy
1988 p- 205; Worrall 1997). Appearances range £iom thickened walls of aenal mycelia on one or
both sides, sometimes accompanied by pigmentation, to a zone of scant mycelia (Worraü 1997).
The pigments are thought to be melanin or meb-like oxidation produe due to the presence of
phenoloxidases and peroxidases produceci by one or bdh isolates in the interaction zone (Li 198 1).
These pigments rnay provide hyphal cek with physicd resistance to lysis by the other isolate (Li
198 1). The vanation in intensity ofnegatîve somatic reactions may be an additive result of many
dBèrent genetic charaders (Marçak et al. 2000; Worrali 1997).
The chromosomal sites determining SI are termed vegetative compatibility, v-c loci, or
heterokaryon compatibility, h-c loci (Rayner and Boddy 1988 p. 209), het loci (Jacobsen et al,
1998), or Mc loci (Leslie 1993). Somatic incompatibility groups (SIGs,also dedvegetatively compatîble groups VCGs) contain members who are somaîidy compatiMe because they have
identicai aileles at loci that govern this behaviour (Leslie 1993). Sornatically compatible isolates
are not genetically homogeneous as they may exhiïit differing maîïng aileles (Mdett and Harrison
1988), cultural characteristics (Adams and Roth 1967; Rayner and Turton 1982), isoenzyme
patterns (Rodrigues et al. 1995), and protein profiles (Lewis and Hansen 1991 ), and so they might merely have cornon aiieles at the SI loci. Therefore, it shouid be emphasized that somatically co~~lpatibleisolates represent manbers of sodcincompatibility groups rather than genetidy unique individuals.
For many researchers the inference that somaîic incompatibilay may be used to differentiate genotypes is cornmon practice (Leslie 1993; Shaw and Roth 1976; Todd and Rayner
1980), and has been used as the sole criterion in many studies of fùngal populations (Anagnostakis
1992; Barrett and Uscuplic 1971; Dahlberg and Stenlid 1990; Fries 1987; Holmer and Stenlid
1991; Kile 1986; Thompson and Rayner 1982; Williams et al. 1981). The usefülness of sornafic incompatibility in population studies is that "such identifications cmbe important in detemùning the number ofgenddy distinct individuals withùi a population" (Leslie 1993, p. 1411, &ou& it is "net us& in determining the degree of relatedness ifthe two isolates are not identical" (Leslie
1993, p. 136). Rayner and Todd (1 979) concur that anîagonism occurs on the basis of fimgai genetic clifference without regard for degree of relaîedness, yet there is considerable evidence that the SI reaction decreases in intensity with increased relatedness (Adams and Roth 1967; Adams et al. 198 1; Anagnostakis 1984; Childs 1937, cited in Rayner and Todd 1979; Coates et al. 198 1;
Hansen et al- 1993a; Kay and Viigalys 1992; Kiie 1983; Leslie 1993; May 198 8; Rayner and
Tcuid 1979; 1982a; Rayner and Turton 1982; Rayner et al. 1984; Stenlid 1985; Todd and Rayner
1978; 1980; Wod1997), and mating allele shihity(Adams and Roth 1967; Wilson 1991).
In hgalpopulation saidies, isolates are transitive when th& compatibility is in concordance e-g. isolate A is compatible with isolate B, B is compatible with isolate C, and A and
C are companble (Murphy and Miller 1993). A concem in using SI as a determinant of genetic identity appears when a la& of transitiveness is mcouutered (lacobson et al. 1993), where a deikîte pattern of compatibility or incornpatibibty cannot be established. Marçais et al. (2000) postulated tbat SI discrepancies rnight be the result of ~~in distinguishing weak incompatibility from cornpaîibility. It has been suggested tbat Merences at only a fav SI loci can
lead to these ambiguous somatic incompatiiïlky reactions which may confounà interpretatioa of SI
resuhs (Anagnostakis 1984; Malik 1996, cÏted in Wod1997).
Mallett and Hamson (1 988) with Marasmius oreades (BoltrFr,) Fr. and Stealid (1985) with Heterobasidion annomm Fr,) Bref found all painngs between sibfings were sornafically incompatible. Kay and Vilgaiys (1992) found tbt 90% of PZeurotu.~ostreatus (Jacq,:Fr,)
Kummer pairings betwm £bilsib heterokaryous and their parent were soniancdy incompatible.
In similar experirnents by Murphy and Miller (1993) 98% of Collybia submrda (Ellisgk) Gilliam and 15% ofMarasmielhispraeacutus (EIlis) Halling sib heterokaryon and parent pairings were incompatible- Thus it seems that the ability to distinguish among reiated individuals using the somatic incornpatiiility reaction varies fkom one fiingal species to another. Hence when SI is the sole criterion in population studies, genotypic densities are likely to be underestimated (Adams and
Roth 1969; Murphy and Miller 1993).
Leung et al- (1993) stated tbat the somatic incompatibility ctiaracter is not dciently polymorphic to be used as a tool in investigating genetic variability, yet for many hgal species SI is thought to be a poiygenic and muitiaiielic characteristic (Anagnostalas 1987; Rayner 19Q la;
Rayner and Boddy 1988, p. 209; Rayner et al, 1984; Smith et al. 1994; Worrail 1997).
Neurospora crassa Shear & B.0-Dodge wiîh at least ten known genes for somatic incornpatibiiify
(Mylak 1976) has been caiculated to have 1024 possible SI genotypes (CarMe 1987), and
Podospora anserina (Rabenh.) Niessl calcuiated to have 7680 (Anagnostakis 1987) making the SI characteristic for these species exîremely polporphic. Unlike these and other ascomycete species,
Marçais et al. (2000) has indicated that somaîic incompatibiiity in basidiomyçetes is not weU- understood. in the basidiomycetes SI is believed to be under the control of fewer (one to four genes), hence the difErentiation among individuals based on somatic incornpatibiiity is expected to
be lower (Marçais et al. 2000).
Accordiog to Anagnostakk (1992) the wide use of somatic incompatibm in estimating
fimgai population diversity is because like intergenic DNA, SI "is alieged to be under no seldon
pressure" (p, 183). Although the mechanism by which SI genes &kt the SI reaction is largely
unknown, the genes are likely to be under some kind of selecticm pressure. Fincham et al- (1979,
p. 195) postulated tbat a density dependent mechanism controls the population of somatic
compati'bïiity deles, promotïng a polymorphic poptdaîion of aiieles, as the maimemuce of sodc
incompatibility prevents situations where one nuclear genotype exploits another (Hart1 et al. 1975).
Because the nirturirl population structure of many fiingal species is niiiintained by somatic
incornpatibility (Rayner and Todd 1979), somatically inwrnpatible colonies "behave as individuals
and thus may represent the primary units of selectionm(Kay and Vdgaiys 1992, p.178). Heace the natural fiinciion of SI aiiows it to rdect variability within populations to a degree that depends upon the species. Although SI is poorly understood, and its &il@ to distinguish between related isolates for many species is poor or unlaiown, the somatic incompatiiility reaction is generally accepted as a means of detecting genetic variation within füngai populations.
POPULATION STUDIES OF C. PmUREW
Biochemid testing and DNA analyses have beeo used to develop our CUIT& understanding of the population sûucture of Chondrostereurn purpureum. The global C. purpureum popdaîion is discoatinuous in some biochernical and DNA patterns as a result of geographic sepamiion. Shamoun et al. (1995) detemiined Little variation in sensitivity to cycloheximide among isolates fiom Canada, Europe and New Zealand, aithough Caoadian isolates dïffêred in their reaction to L-Dopa, Shamoun et al- (1995) interpreted these findings as low hmspedic varïability in the biochemical pathways of C. purpureum. indirirtinp that these pathways may be homogenous over the global or continental scde. Ramsfield et al. (1998) found isolates fiam British Columbia, Switzerland and Finland exhibited uniformity in their restriction fiagrnent length polymorphism (RFLP) patterns suggesting that vdonin C. purpuram mitochondnal DNA is low, Shamoun et al, (199 la) fodlittle variation among restriction pa#erns of the intemai tramcribed spacer region of the ribosomal DNA repeat, among isolates fiom Canada and Europe; however the New Zealand isok showed a uniqye pattern,
Research by Ramsfield et al. (1996a; 1996b; 1997) of the RFLPs of the highly conserveci large non-transcnbed spacer in ribosod DNA indicated that there were three nuclear types in C- purpureum. Type I is found in al1 regions studied (New Zealand, Europe and North America), while type ïI was found ody in North Amerîca, and type Ill found only in Europe and New
Zealand. They betieve that geographic isolation has allowed the fomiation of distinct nuclear types. From their study it was conciuded that gene flow across North America occurs, as there was an almost equal distribution oftype 1and type II in the central portion of the continent, while type 1predominiitpil in the easkm part of the continent and Spe II in the western part (Ramsfield et al. 1996a; 1996b; 1997).
Initial isoenzyme analyses of C. pu'pureum isolates indicaîed that biochemical vanation might be sufficient to charllctenze isolates (Shamoun et al. 199 1b). Yet in Merstudies it was found that the chamdmhiics protein CO- enzymatic activity, isoenzyme patterns (Shamoun and Wail 1996), vinilence, and growth temperature ~oddoullahet al. 1993) were sMar among C. purpureum isoiates fiom New Brunswick Vancouver Island, and south-eastern British
Columbia, Based on these specific markers, there appears to be lMe distinctness among Canadian populations of C. purpureum. Sïrdarly Gosselin et al. found, with the random amplSed polymorphic DNA (RA1PD) technique, that there was little significant genetic differentiation
between ecoregions of Quebec (1995) and ecozones of Canada (1999)- Yet a high degree of genetic variation at the local level has been found at the sub-population level within Canada
(Gosselin et al. 1995; 1999) and New Zealand (Spiers et al, 2000), as the use of RAPD markers enabled differentiation eeenisolates, Biochemical (Shamoun and Wail 1996) and RAPD anaiyses (Gosselin et al- 1995; 1999; Spiers 2000) have indicated that variation among isolates of
C-pupreum does not correlate with gwgmphic origin or demonstrate host speciakation. In
Canada Gosselin et al. (1999) concluded ttiat the local vanation within geographlc subpopulations of C. purpuratm occurred on a fine scale, with this variability evenly distributed across the wuntsr-
As C. pupreum spore dissembaîion is usiially local beause the spores are not likely resilienî enough for dispersai over long distances (Grosclaude 1969, cited in WaU 1997),
Ekramoddoullah et al, (1993) hypothesized that transport of forest products and nursery stock have contributed to the homogenization ofvariability among Canadian subpopuiations of C- purpureum. Because of the genetidiy homogeneous nature of the Canadian population, and the outcrossing ability putthg Merestriction on gene flow within the national population (Wail et al.
1996), CO- thus fàr is that the use of C. pu'pureum as a mycoherbicide is not a threat to genetïc varialion within local C. purpureurn populations.
Pupuialion saidies that detemine the spatiai distribution of fùngai isohmay provide evidmce of life strategy and mode of colonization of the fimgal species. Wind disseminated spores ohresuft in a mosaic of individuaïs occupying the same wood substrate (Rayner and Boddy
1986; Rayner et al. 1984; Williams et ai. 1981); for example Coates and Rayner (1985) found an average of 7.1 somatidy incompaîibie C. purpureurn isolates in 0.28 cm3of wood substrate.
Alternately, sole occupation of the wdresource may indicate an asexual means of colonization (Anderson et al- 1979; Carruthers and Rayner 1979; Rayner and Todd 1982a), or a latent infection
previoudy compartmentalized and oniy corne to the fore as a dtof lowered tree defbes
(Rayner and Boddy 1986; Rayner et al, 1984). Rayner and Boddy (1 986) characterize deral life
ssategies with fleebng occupation ofthe resource where rapid assimiiiition of nutrien& allows a
hasty reproductive stage to be reachd. A form of the stress-tolerant Life strategy is persistence of
the fûngus in heaithy sapwood untii conditions becorne fàvourable for latent iofection, Costes
(1984, cited in Rayner and Boddy 1986) found that C. purpureum grew swiftly on cut beech, with
isolates fiom the same log king mmatidy compati-ble thus appearing to be the resuit of Iatent
infection (Rayner and Boddy 1986). Thus it seems that C. purpuracm may cornbine ruderal and
stress tderant life strategies, as Ïî is capable of causing niderai and Iatent infections, both . . culminabng in early reproduction and replacement by other species.
Rayner and Todd (1982b) found with autoradiographic studies involving rubidium-86, that
active translocation of cytopl-c contents occurs following hyphal hion betwee~lcompatible
isolates of Coriolus versicolor @,.:Fr.) Quél,, and that this did not happen between incompati-ble
isolates. Rizm et al. (1995b) observed in Phellinus giZ~(Schw.) Pat, that seK-paired hyphae
intermingled and hlyanastomosed, while in non-selfpairings, hyphae demonstrated initia1 anastomosis and çubsequent hyphal lysis, which produd a sparse zone observable at both the micro and macroscopic level. Following anastomosis ccincompatiblecombinations initiate a sequence of events that dtimately ends in de& of the hion ce1.i" (Jacobson et al. 1998, p. 45).
These "senescencepathways7' (Rayner and Todd 1979) of programmai cell death may be similar to apoptosis found in animai and plant ceiis (Jacobson et aL 1W8), with sysûzms involving proteases and phenoloxidase initiaihg hyphal ceil de& (Rayner and Boddy 1988, p. 209; Rayner
and Coates 1987; Rayner et al. 1984). Rayner (199 1a) noted that interhyphal interactions
occurred on a spectnun with "responses fkom nonantact, to contact interference, to encoiling and
petration giving physiologicai access, to true fiisim giving physiological and genetic access
which can be restricted by rejection and septai maintenance or blockage" (p. 55). AIthough the
range of reactions observed might be a fùnction ofthe polygenic and mdtiallelic ch.of
somatic incornpatibïkty, Jacobsen et a' (1998) suggested ce11 deaîh was the result regardes of the
pathway initiated by individual somatic inwmpathilÏty genes,
A "'nuclear replacement reaction" has been observed in hyphal fùsion of C. versicolor,
where a dikaryon set of the donor hyphe replaces by conjugak division, the disintegrated nuclear
sdof the recipient hyphae (Aylmore and Todd 1984; Todd and Aylmore 1985)- In non-self
fhsions this phenornenon was seen for up to four days doutany change; thus they speculated a
delayd SI reaction might have occurred subsequent to their observations. Nuclear replacement
niay not be involved in SI at ail, as the phenornenon was observed equaliy in self and non-self
frisions. The nuclear repIacement reaction has also been obsewed in C.purpurmm (Ainsworth
and Rayner 1989).
Spindle-shaped ce& have been observed in the interaction zones of non-seifpairings of C. versicolor, Bjerkandera adusta (Fr,) Kar. (Rayer and Tdd 1982b), and Phlebia radiata Fr.
(Boddy and Rayner 1983), while adjacent hyphal cells may becorne vacuolated (Aylmore and Todd
1984) and die leaving hyphal ccgh0sts77@dd and Rayner 1980). Tdd and Rayner (1980) postulated that spindle celis formed because uncontrolled lysis within the cells softened the ceIl w&, and turgor pressure caused the cells to swell and distort the softening ce11 walls. Although in later studies Aylmore and Todd (1984) speculated that spindle ce& arose fiom hyphal fùsion between somaticaily inmmpatible hyphae but had no associabon with fytic advity. Ayhore and Todd (1984) suspected that the septa between spide cells were plugged, and in this way they hctioned to separate the hioncek fkom the rest of the rnycelium (Tdd and Rayner 1978)-
Rayner and Todd (1979) postuiated that spindle celis ""aithoughsparse, neverthetess efficiently dose off the immediate area fiom the rest of the mycelial system and act effectively as a trap for hyphal tips" (p- 374).
Vacuolation of non-seif fusion cek has been reported in H. annosum (Hansa et al-
1993b) and Phanerochaete velutina @C.Tets .) Parmasto (Aylmore and Todd 1986). Adams et al, (198 1) observed cek that became granuIar "'and often dk3ppear les- hyphal 'ghosts' " (p.
5 11), while merback they observed hyphae ""sweiiinto vesicles, fonnuig chah of interwmected chlamydospore-like ceils which evenaiauy proliferate to form the pseudosclerotial antagonistic zone" (p. 5 11). Barrett and Uscuplic (1971) also observed abnormally short hyphaI ce&, chlamydospore formation, and darkened ceiis with celiuiar disruption in the interaction zone of Polyporus schweinitzii Fr. Aylmore and Todd (1986) concluded that the SI reacbon is, at least initially, an orderly prc3cess of autophagie vacuolation,
Knots of hyphae were observed in the SI interaction zone ofM. oreades (Mallett and
Harrison 1988) and P. schweinitzii (Barrett and Uscuplic 1971). Coiling reactions were seen occasionally between self and non-self hyphae of Stereum hirsuhtm (W-ïlld.:Fr.) S .F.G- (Ainsworth and Rayner 1989) and H- annosum (J3imm et al, 1993b), and m di-mon paùings of
Schizophyilum commune Fr. (Nguyen and Niederpruem 1984); hence this reaction may not necessarily be a somaîic inwmpatibility reaction. Hyphal coiling bas most fiequentiy been observed as an interspecific reaction (Boddy and Rayner 1983; lkediugwu and Webster 1970;
Macre 1967; Rayner and Webber 1984). Macre (1967) observed the winduig of hyphae around opposing hyphae forming hyphal bots and cellular detenoration at the contact point betweeu species of Hirschiponrs. Hyphal coiling may be a fonn of mycoparasitism (Rayer and Boddy
1988, p. 2 19) or part of a replacement reaction (Rayner and Webber 1984).
Hyphai swehgs as weil as excessive branchiog have been observed in the interaction zone between incompaîiile isolates of Ganoderma spp. (Adaskaveg and Gilbertson 1987) and
Peniophora rufa (Fr.:Fr.) Boidon (Charnuris and Falk 1987). Maliett (1989) found thai somatidy incompatihie isolates of Armillaria spp, surrounded themselves with a pseudosclerotiai plate (PSP) cornposed of 'bladderlike ceils", Although the PSPs serve to prevent contact between the isolates, they observed sparse hypbae with bulbous sweiiïngs, within the inter-PSP region.
Little hypld interaction observed between non-self hyphae of fihinodontiurn tirzctoriurn
Ellis & Everh, (W-in 1991), and unnumm (Hansen et al. 1993b), indiriitinp that diffiisibie substances might also mediate the somatic incompatibility response.
The dousmorphologies of interaction mnes observed in Mirent species might indiCate that mechanisms for the SI phenornenon are not universai, with the somatic incompatibility reaction dlfferhg among hgal species.
OVERWINTEFUNG CAPABILITY OF C. PURPUREW SPOROCARPS
The sporocarp of C,pttpreum has a wide range of morphologies tbat are dependent upon humidity and other environmental factors. With high relative hurnidity and moisture content the basidiocarps are flexible and leatheq, and under desiccatirtg conditions they are rigid and brittie. Spiers and Hopcroft (1988b) found that C. pupreum sporocarps could survive to water contents as low as 22% with the cytophbecoming wndensed and vacuotated. The thin walls of the basidia allow rapid rehydration such that spore production and release follow several hours iaîer (Spiers and Hopcroft 198 8b)- C. purpureum sporocarps are long thought to be annuai structures, viable for only one
growing season and thereafter displaced by other decay (Wall 1997)- Wall (1991) observed
that C. purpuratm sporocarps were sloughed off durhg the dry or winter period, wbiie de Jong
(1988, cited in de Jong et al, 1990) found that they die foilowing moderate fiost, These -dies
indicate that overwintered sporocarps are not sources of basidiospores in the sprhg.
Mazur (1960) descxibed how fk&g damage to ceils mïght occur in one oftwo ways,
Slow cooling leadîng to dehydration and extracelidar i6reezing may cause an increased solute
concentrafion, precipitation of solutes, ceii shnnkage?and plasmolysis, which may result in
irreversible protein denahration. Rapid cooling may lead to intraceliular ice forming in the
qtoplasm, which invariably leads to damage ofthe ceiiuiar membrane systems, Hace damage to the ce4 due to dehydraîïon and formation of intracellular ice, may occur on a continuum depending upon "cooling velocity and permeability of the cell to water'' (Maau 1960, p, 444). Deveraii
(1965) suggests that the repeating cycle of kzing and thawing of fiingal cells is detrimental to their survival in the wild, However Mazur (1968) ascri'bes the monsuxvivai of fbqi in situ to the "rarity of intraceliular fieezing" (p- 383), due to the low cooling velocities found in nature. 24
MATERIALS AND METHODS
SITE HISTORY
The main site used in this study is adjacent to the north side of the Cascades CollseNatim
Area (CCA), located no* of the city of Thunder Bay, Ontano (Figures 1 and 2)- This approximately forty acres of land, is owned prïvately by Vic La- Sr-,and was barvested in the winter of 1995/96 by Hiles-Laurin Contracting Limited. Prim to harvesting, the tree composition was roughiy 90% poplar and 10% buch with a damount ofrnisceIlane0us coniférs, Followiug harvesting ofmost of the poplar on site in the winter of 1995/96, sorne of the residual birch was harvested or cut down and l& in the winter of 1997/98. The method ofharvesting was feiier- buncher and skidder.
Study areas Mills Block D (MBD)and WiBlock D (WBD) are both 5 ha. cut blocks located on land overseen by the Lakehead Region Conservation Authonty (Figure 1)- They had a similar species composition as the CC& and were harvested of th& poplar and birch in the winîer of 1997/98 by cut and skid method. These settings, following partial cuüing ofmixed wood stands, with residual live hardwd and abundant slash and stumps, ideally supports the dual yet integrated ecological roles of C. purpureum. Woody debrk and stumps provide ample fimd resources on which the fungus can act saprophytically, and remaining live trees, which are ob wounded in the harvesting process, may becorne bfécted- CCA
Finure I. Uap of Thunder Bay (Guide f rinting and Publishing 1993) indicating the locations of study sites Cascades Conservation Area (CCA),Mills Block D (MBD),and WilLiams Block D (WBD). Fimre 2. Map of the Cascades Conservation Area (CCA) study site indicating the locations of the path and perimeter benchmarks, Plot 59, Birch Pile A (BPA) and Birch Pile B (BPB). POPULATION STUDY OF C. PURPUREW
In October and November 1997, basidiocarps of C. purpureurn were randomiy coilected
flom around the primeter and central path in a survey of the whole CCA study ara- Sporocarps
were aven an identiSling number (Appendÿr 1) based on the order in which they were found de
in the vicinity of the nearest benchmark on the rnap of the CCA study area (Figure 2). A whole or
piece of the sporocarp was coilected fiom each distinct grouping ofsporocarps on slash and
stumps. In the labotatory the sporocarps were rehydrated for 4-24 hours in a humidor of moist
paper towel in a glass petn dish. They were then stuck to the underside of pWcpetri dish iîds
with petro1eu.m jeliy, fiom which they cast spores onto 1% dtextract agar (MEA) overnight.
For each sample an approramately 5x5 mm area of agq with spores fiom the edge of the spore
priut, was transferred to another MEA ph. In a da& 24°C incubator the spores were allowed to
grow into a multi-spore heterokaryotic culture, These individuai cultures were used to conduct
compaîibility tests been29 (20% of 144) randomly chosen isolates paired in aU combinations
(406 pairings). To pair all ofthe 144 CCA isolates in aU combinations would have requkd
10,296 pairings. Round mycetiai plugs (5 mm diameter) of multi-spore cultures were placed 1-3
cm apart and observeci over one to two months for somatic companiilÏty. Where the paired
isolates grew into a single colony, the isolates were record4 as somaticaily compatible (+). Where this did not occur, but an interaction zone demarcating the pwthofthe two isolates appeared, the isolates were detexmineci to be somatically incompatible (-) (Figure 3)- Random ~e~pairingswere used as positive controls. There was repetition of pauiags when resuhs were inconsistent or ambiguous.
In October 1997, Plot 59 was istensively harvested of any C. purpureum sporocarps, with each coilection given an iden- number (AppenVr I) based on the order in which the Fime 3. Two-week-ld pairings between C. purpurarm isolates demonstrating somatic incompati'bility O&) and somatic compatibility (nght). wood substraîe was observed (Figure 7 in the Resuits section), Laboratory treatment was the same as for the CCA sporocarps except that compatibility testing was umducted amcmg ail 20 isolates in di possible combinations (190 pairings). in the fidl of 1998, sporocarps were çoUeçted fkom a tmnsect traversing each study area MBD and WBD (Figure 4), and given numbers accord.@ to the order in which they were found (Appendix 1). The suMBD and eight WDisolates were paired among themselves in aii combinations (15 and 28 pairings respeCtive1y)-
In the fàii of 1998 sporocarps were intensively surveyed and collected fkom two piles of birch logs, found in the CCA study area that were cut in &ter 1997/98 (Figure 5). Logs in Birch
Pile A (BPA) and Birch Pile B @PB) were gïven either BPA or BPB designaîions, and then numbered acwrding to the order in which they were observeci- The south king end ofthe log was desi@ A, and the north fàce B, and the side of the log was given the designation S. Sporocarp isolates were given an identifjing number with the birch pile, Iog number, A, B, or S indichg exposure, and a number indicating the order in which it was found clockwise fbm the top of the
Fice; or if on the side fiom north to south (Appendix II). Laboratory mentwas as above, except isolates collecteci firom the same log were paired among themselves in ail wmbinations (40 1 pairings). Of the 19 1 isolates collected fkom the buch piles, MBD, and WBD, 36 (19% of 19 1) randomly chosen isolates were paired in all combinaiions (630 pairings). The exclusion of MBD isolates was random.
Dïkaryon mdti-spore stock cultures fiom each finubng body have been saved in 1% MEA slant tubes, and are stored at 4OC in the Lakehead UniversÏty Forest Pathology Laboratory. As well, the sporocarps themselves are stored in paper bags at room temperature in the same locationOfl
Some ofthe fhîtkg bodies wiiected in the fidl of 1997 were hot air dried while the rest were dned at momtemperature. Finure 4- The MBD study site one pwing season after popkir and birch harvest at the time of the C. purpureum sporocarp survey. Figure 5- Birch Pile A (BPA), in the fareground, and Bkch PiIe B @PB), fiom which sporocarps of C-purpureurn were coliected one growing season after the logs were cut, MICROSCOPIC CHARACTERISTICS OF SOMATIC INCOMPATJBILITY IN
c*PURPUREW
Seif and non-seif pairings were conducteci on coverslips dipped in water agar and placed on 1% MEA plates. Non-self pairing isolates BPA-34-A3/BPA-34-A4 and BPB-7-Al/BPB-7-
B lwere observed in five piairings each, while BPA-344.3and BPB-7-AI were obsefved in five self-pairings each, Five mm round plugs of myceliurn were used in all pairings, except in one set where 3 mm round plugs were used, Sets ofpairuigs always used the same group of isolates, although coverslïp width and length were varied. The isolate plugs to be paired were pldon opposing &es ofthe agar water coverslip, with part of the plug overtiaaging the MEA plate to provide the nutrient for growth, Followiug 3-6 days of incubation in the dark at 24OC, the coversiips and hyphae growing on than were cut fiom the surrounding growth and media. The coverslips were placed upsidedown on glas slides, in a drop of 2% glutaraldehyde used as a cytoplasmîc fixative. The interaction zones for the pairings were observed using brightfield microscopy at 400x and lOOk(oïl immersion), and black and white photos were taken to document the results.
Overwintered C. pupreum sporocarps were coilected in the spring of 1998 and 1999 f?om the CCA study area. Sampiing ofsporocarps was not random, as it was biased towards overwïntered sporocarps that had a healthy appearance. They were moisteneci and held over 1%
MEA to coUect any spores, as desmi eariier. The spores were observed rnicroscopically (400x) for genniriation foliowing ovemight incubation at 24OC. RESULTS
POPULATION STUDY OF C. PUIWUREW
Random pairings of isolates coiiected fiom the CCA study site in the fàil of 1997 were ail
somatically incornpatibie, evm among those collected hmthe saxe wood unît (Figure 6).
However, one painog Weenisolates found not fhr fkom each other had an ambiguous SI reaction
thaî could not be detennined even after several repetitions. This pairing was said to have
indetenninate wmpatibiiity.
Plot 59 isolates collected in the fidl of 1997 were collected fiom 9 of 12 wood units
(stumps or Iogs) (Figure 7). No compati'ble isolates were found, even among isolates collectai
fiom the same wood unit (Figure 8). There were 1-8 sporocarps collecteci on the wdunits in
Plot 59, wiiere each sporocarp represented a distinct somatic incompaiï'bility group (SIG).
Isolates collected fiom MBD and WBD in the fhii of 1998, paired against ali isolaies
within their block were found to be incompatiile except for one pairing between WBD isolates
witb indeterminate wmpatibility (Figures 9 and IO). Random pairing of isolates collected fiom
Wl3D (by chance MBD was excluded) and tbe birch piles fomd compatible isolates in only three
pairings where they were ail fiom the same log in BPA (Figure 11).
Ofthe 102 buch Iogs in BPA and BPB censused in the of 1998,50 logs, or 49% had
C. purpurmm sporocarps. The larger pile, BPA, with 86 logs, had 43% logs bearing the
sporocarps. Conversdy, of the smaU pile with 16 logs, 81% of the logs in BPB bore C. purpureum sporocarps (Table 1).
Of the 125 sporocarp isolates collected over the 37 purpureum infècted logs in BPA, 93
diiErent SIGs were observed (Table 2)- And in BPB 43 SIG were observed among 52 Fiwre 6. Paurings between randomiy selected C. purpurmm isolates coiiecîed fkom the Cascades Conservation Area study site. AU paUings were incompatible, except for the indetenninate reaction between P-E-9and P-F-4.
Fiwe 8. Pairing dtsfor C, purpuracm isolates coliected in Plot 59. They were all somaticaily incompatible. Fi-mre 9. Resuits for pairings between C-purpurmm isolaîes coilected in Mills Block D. Ali of the six isolates were somaîicaliy incompatible.
Finure IO. Païring results of C. purpureum isolates fiom Williams Block D. AU but one pairing was somatically incompatible; WBD-5-3 and WBD-6 had indetexminate wmpaîibility. Fieure 11. Randorn pairhg results of C. purpureum isolaies coilected fiom both birch piles in and study site WBD (MBDwas randomly exctuded). The three compatible isolates onginated fiom the side of the same log in BPA Table 1- Number of logs in each birch pile, and number and percentage with C. purpureum.
# of Logs % of Logs # of Logs with C, purpureum with C, purpureum Birch Pile A 86 37 43 Bimh Pile B 16 13 81 102 50 49 Table 2. The number of C. purpureurn sporocarps ooileded, number of somatic incompatibility groups (SIGs), A and B face compatibility, and intransitivity of SI reactiom for each C. purpumm infectexi log in Birch Pile A. (Y = yes).
LW # iso lates # of SlGs compatibility Intransitive BPA-9 5 5 isolaîes in just 13 C. purpureum infected logs gable 3). Compatibility arnong isolates fiom both
A and B fàces of logs appears to be less conmon than for isolates fiom A and B to be
incompatible, as over both piles, and 50 infected log, ody nine, or 18% had AB compatibdity
(Tables 2 and 3).
Most of the pairings were easy to score after one month of incubation because of the intensity of the reaction, akhough several were re4uated after Merincubation, and ifd ambiguous the painng was repeated up to two more times. intransitive pairings were &O repeated twice, Wtiile most painngs between isolates collected fkom the same log were found to be fiilly transitive, three logs in BPA and one in BPB, had isolates that did not meet this criterion (Tables 2 and 3)- Inhransitive isohîes were detenajILed to belong to separate SIGs, Of 1640 unique pairings there were ody 8 ((1%) inintransitive or indeterminate pairings overail, The number of SIGs per log ranged fiom 1-9, with 68% ofthe C, purpureum infected logs in both birch piles contaïxüng 1-3
SIGs (Table 4). Painng charts for isolates coliected from the birch piles can be found m Appendïx m.
MICROSCOPIC CZEARACTERISTICS OF SOMATIC INCOMPATIBILITY IN C.
Pmmw
The macroscopic somatic incumpatibility reaction varied in its appearance. It appeared as a zone of scat mycelia separating the two colonies, such as between isolates BPA-344.3 and
BPA-34-A4, and m=uiifèsted as a zone of built up hyphae with acc~mpanyingpigmentation, as is seen behveen BPB-7-ALand BPB-7-B 1 (Figure 12)- There were interaction zones observed that had macroscopic appearances intennediate to these manifestations of SI, Hyphal characteristics Tabie 3. The number of C-purpuracm sporocarps milected, number of somatic incornpatibility groups (SIG), A and B face compatibility, and intransitivity of SI donsfor each C. purpurmm infécted log in Birch Pile B, (Y = yes).
LW # isolates Groups Compatibility Intransitive BPB-1 11 9 Y
Table 4- The number and percentage of logs in Birch Piles A and B having one to nine sornatic incompatiidity groups (SI&) per log.
# of SIGMog # of logs Oh of logs Fimre 12, Two diBering types of C. purpureum intexaction zone. Isolates BPA-3443 and BPA-34A4 interacted to fonn a sparse zone of scanty myceiia (arrowhead), and BPB-7-At and BPB-7-B 1 developed masseci hyphe with accompanying pigmentation (arrow) at the interaction zone. obsewed microswpidy of Chondrostereum puTpureum in dnUe hcluded vesicles, inreguiady spaced clamp connections, and hyphal whoris with and *out clamp connections.
Microscopically, initial anastomosis appeared similar in self and non-seif fiisions (Figure
13). Subsequent microscopie reactions withui the interaction zone seem to dBër among pairings of isolates of C. purpureum. In the interaction zone of pairings of BPA-3443 and BPA-34A4 were found inflated ceüs that wuld be descnï as "spindle" shaped (Figure 141, arising fiom a darkened celi (Figure 15), and occasional degradeci hyphal celis. The interaction zone of BPB-7-
Al x BPB-7-B1 exhiited distorted and deforrned hyphae and hyphal coiling fiequently fonning hyphal hots. Hypbal wiling with a random and disorganized appearance may occur as an artifâct of slide preparation,
Of the sporocarps coilected in Aprii of 1998, 10 of 11 produced spores tbat genninated on
1% In Apnl of 1999, 12 of 15 sporocarps prduced viable spores- F- Second and Third
Collection sporocarps fiom Apd 1999, Collections A and B fiom May 1999, and Collections Az and & fiom June 1999 were ali collectai fiom the same group of sporocarps at the base of a balsam poplar stump and were found to produce viable spores (Figure 16). In total 32 of 37, or
86% of the ovefwintered sporocarps collected in the spring of 1998 and 1999 produced spores that genninated (Table 5). The average temperature for thïs period was higher than normal. RaidU was lower than normal hmApril to September 1998, especially Apd, and higher than normal in
October 1998 uable 6). Fimue 13. Anastomosis (arrow) between genetically dissimilar hyphae in a non-self pairing between isolates BPA-3443 and BPA-34-A4. (1 000~)- Fb- 14. Atypical spindle-shaped cek with abnonmally thickened septa in the interaction zone of a non-self pairing of isoiates BPA-3443 and BPA-34-A4- (1000x)- Figure 15. The dark hyphal ceii at the disjuncbon between spincile-shaped cek and normal cells in the interaction zone of a non-selfpairing of BPA-34-A3 and BPA-34-A4 (1000~). Figure 16, Base of a suckerhg balsam poplar saimp, pictured in May 1999, fiom which overwintered C. purpureum sporocarps were collectai, Viable spores were produced fiom sporocarps colieded in April, May and June 1999. Table 5. Sporulation and germination of overwintered sporocarps, (Y = yes)-
Collection Date lsolate Sporulation Germination - April30 1998 SPI Y Y SP~ SP3 Y Y sp4 Y Y sp6 Y Y Sp7 Y Y SP~ Y Y Sp9 Y Y Spl O Y Y Spl 1 Y Y SPI2 Y Y 1st coll, Y Y 2nd coll. Y Y 3rd COIL Y Y w-1 Y Y w-2 Y Y WP-3 Y Y WP4 Y Y w-5 Y Y WP-6 Y Y w-7 Y Y WP-8 WP-1 O WP-11 Y Y WP2-SI Y Y WP2-N-3 May 23 1999 Coll- A Y Y Coll. B Y Y WP-12 Y Y w4 Y Y wp-52 Y Y WP-62 Y Y w-82 WP-20 Y Y TA Y Y June 141999 Coll, A2 Y Y COIL B2 Y Y 37 32 32 Table 6. Relevant Thunder Bay weather data* for winter 1997198, spring/surmner/M 1998, and winter 1998199.
Temperature (OC) Average snow Rainfall (mm) Relative humid@ (%) Average onn na? Low on qround (cm) Total ~ormafMm-mum Minimum
Nov, '97 Dec. '97 Jan. '98 Feb, '98 Mar. '98
Apr. '98 May '98 June '98 July '98
Aug ,'98 Sept. '98 Oct. '98
Nov, '98 Dec, '98 Jan- '99 Feb. '99 Mar. '99 Apr. '99 4.4 2-7 -6.7 10.6
*fiorn Tranqdo Ridge C~1ogica.iStatio~ courtesy of Graham Saunders. # Environment Canada 30 Year Nonnals. DISCUSSION
POPULATION STUDY OF C. PURPURECM
The genetic variabilïty at the somatic incoropaîibiiity loci appears to be high over both the
CCA study area, including Plot 59, and the birch piles- Within the limited samples available at the
MBD and WBD sites, there also seems to be high SI variability. The numkr of individual SI genotypes detected approached the number of isoIates, representing the number of discrete fnuting clusters representative of the study areas. Where isolates were found to be compati'ble they origktd fiom the same wood unit and ahhough may only represent individuais of the same SIG, they most likely were isohrepresentinp the same individual. Sïrdar to the studies by Gosselin et al- (1995; 1999) the large nuruber of SIG5 detected in tbis study indicates that local populations of Chondrostereum purpuram are composed of many individual genotypes, which rnay refiect a high genetic divers@ witbin the species, This is supportai by the literaaire, which indicaies that within populations seeded by wind-blown basidiospores there is high genetic diversity, with fiequent genets occupying dareas (Chamuris and Falk 1987; Kay and Vdgalys 1992; Marçais et al. 1998; Murphy and Miller 1993; Rayner and Todd 1982a; Wiet al. 198 1).
As indeteminate or intransitive reactions occurred in less tham 1% of the painngs, the SI miction in C. purpureum seems fàir1y reiïable. The intransitive pairïqp between some isolates may be attributable to close relaîedness arnong the intxansitive set of isolates. Sïmilarly, the indeteminate pairings may be due to only small differences at the SI loci (Malik 1996, CMin
Worrall1997) denisolates difFkhg at only a single SI gene (Anagnostakis 1984) or a few SI genes (Kile 1983) may have a reaction almost indistinguishable fkom a sodcally compatible doa. Because there is a higher likelihwd of neighbouring fimgal individuals to be closely reIated (Lane 198 1; Wolfénbarger 1959, cite-in Kay and Vilgaiys 1WZ), it may be expected that
intransitive and indetenninate reactions would occur more fiequently beîween isolates found close
to each other, as was the case here.
Like genetic markers that are not variable enough, markers that show too much differentiation may not be usefiil in reveahg relationships (Leung et al, 1993)- When the SI character shows hi@ variabfity, as seen here Mth C-purpureum, the genetic relationship among the isolaies is unclear (Leung et al. 1993)- The polygenic and muitiallelic character of somatic incompatibility may lead to situations where inevitable rejection between isolates is the nom
(Rayner and Boddy 1986).
Although 144 samples were collectecl over the whole CCA study area, 20 in Plot 59,6 fiom MBD, 8 fiom WBD,no more than 29,20,6, and 8, respectively, were paired against one another. Of the 191 pooled isolates fiom BPq BPB, MBD and WBD,36 samples were paired.
The birch pile isolates were also paüed in all cornbidons with &ers colle& fiom the same wood unit with no pairing set exceeding 13 sarnples. AU of these pairhg experiment sample sizes feu weU below the sample size limit of 60 for detection of a genotype thaî has a firequency of 10 % with 95% confidence (Leung et al. 1993). This may explain why so fav somatidy compatible isolates were detected, A larger study cddresolve this dilemma, Conversely, the large number of
SIGs detected may be representative of a wealth of possible SI genotypes within the C. purpuram population.
Although all isolates used in this stuciy were subcultured up to two times, changes in SI reactivity in previous shidies were usually noted after severai muitiple su- (Hawker 1950), hence it is iinlikely that the high number of SIGs observeci in this study was the result of degradation of the somatic response. Some studies indicate somatic incompatiiility remains stable in vitro (Leslie 1993; Murphy and Miller 1993) and in vivo (Barretî and Uscuplic 197 l), yet progressive changes in characteristics, such as virulenceyfecundity, growth rate7 and fermentaiion or metaboiic activity, have been documented following successive subcutturing (HA* 1950).
Although serial trader of developing colonies is cornmon practice @Say and Vilgdys 1992), it is possible that multiple subculauing events couid riiminish the ability to forrn a stable anastomosis product (Barrett and Uscupiic 197 1; Hyakumachi and Ui 1987), or decreaçe the intensïty of the SI reaction (May 1988) thus produchg the erroneous appearance ofa high or low number of SI&-
Any mitochondrial effion the SI & motbe accomted for, as pfecautions against mitocbondnal incompatibïiÏty were not taken in this study. The mitochondrim is suspected to play a role in ceil de& and thus be involved in the mechaaism of somatic uicompatibihty (Rayner
199 la; 199 1b). Mitochondria do not coaiigrate with exchanged nuclei in homokaryon pairings
(Casselton and Economou 1985; Hintz et al. 1988; May and Taylor 1988; Specht et al. 1992) and in &mon pairings (May and Taylor 1988); therefore it seems unlilcely Ifiat they CO-migratein somatic reactions between heterokaryons. May (1988) found that 2 of 8 pairings between heterokaryons with monnuciei but difkiog mitochondrial poipuiaîions were intoleraut of dis difEerence and were somatically incompatible. Rdet al- (1995b) produced sib-reW heterokaryons by pairing single spore cuhres against a standard single spore cuIture7 and producing stock cultures only from the side ofthe standard culture to nep.te any possible mitochondnal inauence on subsequent dccompaihbility tests. Although this seems prudent, it was not done here, and may account for the high number of soddyincompatible individuals observed. This phenornenon needs to be researched in C- purpureum, as it may be a serious confounding fàctor in this population study,
Multiple pairing is c(311lrmoniypracticed in somatic incompati'biLÏty studies, and has thus fir been found to have no effion SI rdts(Charnuris and Faik 1987)- in this study with Chondrostereumpurpurmm an interaction efféct resultïng fiom multiple pairings on single plates
seems imlikely as many pairings were repeated in diffèrent arrangements wïth the same outcorne.
There appears to be no correlation betwee~lthe number of individuals on a wood unit and
A and B face compatibility. Thus AB compati'bility does not indic& îbat one individuai
dominated the resource unit deterring infection by other individuals, The number of isolates per
log within the birch pila was as high as 9 isolates per log. Yet over 68% ofthe logs bearuig C. purpureurn sporocarps were lunited to 1-3 SIGs per log- That C. purpumm gains its primary
occupation adyanfage by arising fiom previously cornp- ~omhas been suggested
(Coates 1984, cited in Rayner and Boddy 1986; Fritz 1954). Thai îhere was a low number of
SLGs in most of the infeded logs, and ihat within the fjrst growing season following resource availability C. pu'pureurn became active, suggests that it was pre-established in the living tree
(EMdy and Rayner 1982; Rayner L979b). Akhough these resutts do not conclude tbat infëdon of the birch Iogs was latent, they do not precfude this.
Over both birch piles 49% of the logs had C. purpureum sporocarps, Yet, ifbroken down into the two piles, BPA had a sporocarp incidence of 43%, while BPB had an incidence of 8 1% ahost twice that of BPA. The différence may be that BPA had more logs thus the pile consisteci of many logs witb less avaiIable surface area exposed and available as Monsites for airborne spores, pointing towards spores as the most SiguifiCant source ofinféction- Study of the bitch piles would have yielded more idormation ifthe C. purpureurn population could have been observed over seved years, as Fritz (1954) fbmd C. prpzcreum Eniiting on pop& logs in sturage reached aimost 100% withia the £kttwo seasons foUowing cu#ing.
Bdh birch piles and study sites MBD and WBD were in their fi& season following harvest, yet they had differïng population sizes of C. purpirreum, with MBD and WBD having low numbers of the fÙngus. The hger population on the birch piles may be the dtof the increased levels of inocuIum due to prevîous harvesting on the CCA site providing much infécted wdy
debris as a spore source,
From this study, one can conclude that Idpopulations of Chondrostereum purpureum
are composed of high numbers ofindividuals with difFîerent aiieles for soIIlilfic incolllpatibdity-
Population studies of tbis small size are sigdicant as "in sexual organisrns tbe unit of greatest
importance is the breeding population &ch can be defined as that gmpof individuais wbich,
because of their proximity in space and time, are potentially capable of interbreeding" (Rayner and
Todd 1979, p- 346). It is important to remthat popiilat;on shidies based on the SI cnterion are
inherdy underestimates of genotypic fkquency.
MICROSCOPIC CHARACTERISTICS OF SOMATIC INCOMPATIBILITY IN C.
PURPUREW
Verticillaie or ccwhorIed"clamp co~ectionswere observed as weil as hyphal whorls with hyphe arising from them, Ciamp wmections occurring m whorls have been exîemïvely reporteci in Stereurn spp. (Ainsworth 1987; Boidon 197 1 ;Boddy and Rayner 1982; Rayner and Turton
1982), and little reported in Chondrostereum purpurmm (Chamuis 1 988;Nakasone 1990;
Stalpers 1978), except by Cartwright and Findiay (1958). This phenornenon is not associated with the somatic inwmpatiiility reaction as it was observed in both unpaired and paired cultures of the fiingus and is mentioned here only because it seems under-reported. Boddy and Rayner (1982) have posailated that whoried clamp connections may be "'an adaptation to rapid radial spfeadn @,
345), which would be f3îing in such a fâst-growing species as C. purpureum.
The BPA-3443 and BPA-34-A4 mteraction zone macroscopically appeared as a sparse zone between the two isotates. in the interaction zone, sparse populations of spindle-shaped hyphal tek, such as mas described for C. versicolor and B. adusta by Rayner and Todd (L982b), were observed in chains, Degraded hyphae were also observed and were most likely the remnants of
hyphae in later stages of the incompatiily reaction, The degradation ofthe hyphal ce& and
subsequent cessation of growth into the interaction area may lead to the sparse interaction zone
between these two isolates. h each pairing, hyphae were observed at diifërent stages of the somatic incompatibility reaction, and because the hyphae were fïxed pnor to microscopie observation the sequence of SI events was inferred-
The macroscopic appearance of the interaction zone between isolates BPB-7-A1and BPB-
7-BI was a barrier composeci of hyphae at the interface- Where there is dense mycehat the interaction zone, this may indicate a mutual deadlock in which the hyphal interaction involves ccgrcssmyceiïal contactn (Rayner and Webber 1984, p. 402). Microscopie fwfound in the somatic interaction zone were vesicles and distorted hyphae that may be undergoing an initial somatic reaction. These hyphae may follow an antagonistic SI paîhway leading to hyphai coïling where the hyphae becorne entangied in a relatively ordered manner. Hyphal coiling may lead to hyphal knotting and may be anaIogous to the hyphal bots in the SI intefaction zone ofP. schweinitzii (Barrett and Uscupiic 197 1) and M oreudes (Mallett and Harrison 1988). It seems likely that interacting hyphae die ensurhg mycelial discontinuity between somaticaüy incompati'ble isolates,
It must k acknowledged that the hyphal coihg phenornenon could result fiom twisting of the hyphae during slide preparation; thus disorderiy wrapping may be an while dehi coiling may be the result of a somatic reaction, Cooke and Rayner (1984, cited in Rayner and
Webber 1984) suggest that combat hyphaI ktemdïons, lïke hyphal coiling, are used in defénse of territory, or for secondary resource capture or replacement. However Rayner and Boddy (1988, p.
249) suggest it may occur as self or non-self parasitism when there is a low nutrient supply.
Although low nutrients are an unlikely factor here because the buik of the mycelium was growing on MEA, the mycelium in the interaction zone was growing dong water agar to facilitate
observation of the reaction. Further studies using low nutrient agar need to be done-
The somatic inwrnpatibiiity reactiuz m C- prpureum occurred with variaîïon that ranged
betwee~lthe two extremes examined here, yet in ail pairings the &on occurred equally on both
sides, giwig the appearance of the interaction zone maniCiestation being dependent upon the two
isolirtes initiating equal responses. The initial confionthg self and non-self hyphae of C.
putpureum isolates anastomose in the same way, probably to aUow somatic compatibility to be
assessed. Subsequent somatic reactiions dïffknd among somatically incompatiMe painngs,
possibly depending upon which SI loci are dissimilar. Anagnostakis (1987) postdates that each SI
gene may regdate a diffèrent mechanism by *ch SI is efkted, hence the macroscopic
appearance of the interaction zone may correspond with Me~gmodes of asserting somatic
incompatriiiity.
Barretî and Uscuphc (1971) suggest two independent rnechanisms for somatic
incompatiiility, with one early mechaniSm "'restrictingclose association of opposing hyphae", and
the other following intermingling "resultbg in the death of certain hyphey'(p, 596). The existence
of such systems may explain the rarity of obsedolls of non-selfhyphal interactions in C. purpureum, and other species that may pracbce non-self hyphal avoidaace over hyphai interaction
(Hamen et al. 1993b; WiIson 199 1). However, the iow nurnbers of interactions seen in tbis study
rnight bave been a fiinction of the srnail nurnber of non-self pairings (10) observed microscopically.
The conclusions reached in this prebmïnaxy study are speculative, thus Merstudies are needed to detexmine if différent mechanhm of somatic incumpatibiLity are the cause of the Merences
seen at the C.purpureum SI interaction zone- Chomï"ostereumpurpuracm sparocarps can succesdidly overwinter as 86% of the sampled sporocarps produced viable spores. Sampling was not random, as sporocarps thai were exîremely weathed in appearance or were parasitized by derfungi were not cdected. As sporocarps with this type ofappearance seemed in the minority, it may be that many that did not survive the winter were ccsloughedoff7 as descrïbed by Wall (1991)- Spore production in C. purpureum has been reporteci to last 12 (Spiers 1985) to 16 rnonths (Dye 1974, cited in Spiers
1985), with the number of spores pduced decreasing wÏth sporocarp age (Spiers 1985)- The spore germination rate approached 100% in those sporocarps that did produce spores; hence when the iifé of the sporocarp is intemipted by winter weather and it Sunives, viabie spore production is likely.
Spore production is stllnuhted following rabMi, and continues as long as the temperature is between 0°C and 2S°C, and the relative humidity does not fkll beiow 90% according to de Jong et al- (1990), or 75% according to Spiers (1985). These weaîher cooditions were found to be nd mcoxnmon in spring in the Thunder Bay area,
The fïnding that C. purpurmm overwintered sporocarps ca.successfùily produce viable spores, indicates their potential for causing sp~ginfection of Winter-wounded trees. This may be signiflcant iftree susceptibility is high in the spring due to hïgh xylem nutrient Ievells, as has been postulated by Beever (1970). This also increases the amount of inoculum thought to be produced by C. pupreum sporocarps in the fbrest environment, occumiq at a time when winter damage is fi-esbiy exposed. These £âctors should be considered when assessing the mycoherbicidal uses of tllîs fiingus. Adams, DH. and L-F. Roth, 1967, Demarcation lines in paired cultures of Fomes mianderi as a basis for detechg genetidy distinct mycelia. Can f . Bot, 45: 1583- 1588,
Adams, DH. and L.F. Roth, 1969. Intraspecific cornpetition among genotypes of Fomes cajandeeri decaying young-growth Douglas fir. For, Sci. 15:327-33 1.
Adams, TJH, N-K, Todd, and AD.M, Rayner. 198 1. AntagoniSm between dïkary011~of Pipfoprus bef~Iinus.Tram, Br. Mycol. Soc, 76:5 10-5 13.
Adaskaveg, J.E- and RL,Gilbertson, 1987. Vegetaîive incompatibiiity between intraspeciific dikaryotic pairings of Ganodenna Zucidum and G tsugae. Mycologia 79:603-6 13.
Adaskaveg, J.E_and I-M, Ogawa 1990. Wood decay pathobgy of- and nut trees in California Piant Dis, 74:341-352.
Ainsworth, A.M. 1987. Occurrence and interactions of outcrossing and non-outcrossing popuiations in Srereum, Phanemchaete, and Coniophora. Pp, 285-299 in Rayner AD-M-, CM,Brasier, and D. Moore (eds,) Evolutionslry biology of the fùngi, Cambridge University Press, Cambridge, UK 465 pp.
Ainsworth, A.M. and A-D-M, Rayner, 1989. Hyphal and mycelial responses associated with genetic exchange within and between species of the basidiomycete genus 2ereurn. J. Gen. Microbiol, 135:1643-1659-
Ainsworth, A.M., GD.M- Rayer, SJ- Broxholme, and J.R Beeching- 1990- Occurrence of uniiilted genetic transfïer and genomic replacement between strains of Stereum hirsuîurn fkom non-outcrossing and outcrossing populations New Phytol. 115: 119-128.
Auen, E- 1996, Decay and wood utiIization problerns in red alder and paper bisch, Pp. 139-145 in Cameau, P.G., G.I. Harper, ME, Blache, J-O. Boateng and K.D. Thomas (eds,) Ecology and Management of B.C. Hardwoods Workshop proceedings, December 1 and 2, 1993, Richmond, BC. FRDA Report No. 255.
Anagnostakis, S .L. 1984. The mycelial biology of Endothia parasitica IL Vegetative incompat%iiity. Pp- 499-507 in Jennjngs, DHand AD,M- Rayner (eds.) The ecology and physiology of the fimgai myceiium, Cambridge University Press. Cambridge, UK. 564 PP-
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CASCADES CONSERVATION AREA (CCA) lsolate Date Collected Source October 5 1997 Poplar s :ump -just above root collar II Poplar s ;ump -just above root wllar II Poplar s :ump -side -superior to 1BI u Poplar s ump -side -superior to 1B2 II Poplar s ump -cut surface II Poplar s :ump -cut surface n Poplar s ump -cut surface n Poplar s :ump -cut surface II Poplar s :ump -cut surface II Poplar s :ump -cut surface II Poplar s :ump -cut surface I Poplar s :ump -side -inferior to 1C2 11 Poplar s :ump -side 4nferior to 1Cl II Poplar s :ump - just above root wllar -inferior to 1Dl n Poplar s :ump -just above root collar n Poplar s ump -just above mot collar -superior to 1El Il Poplar s :ump -cut surface II Poplar s :ump -cut surface n Poplar s ump -cut surface n Poplar s ump -cut surface #I Poplar s :ump -just above root collar -inferior to 4A2 II Poplar s :ump -side 4nferior to 4A1 October 7 1997 Poplar s ump -cut surface n Poplar s ump -tut surface II Poplar s :ump -cut surface II Poplar s :ump -side n Poplar s ump -just above mot collar n Poplar s :ump -cut surface Il Poplar s :ump -cut surface u Poplar s :ump -cut surface n Poplar s ump -mot mllar n Poplar s ump -cut surface Il Poplar s :ump -side November 5 1997 Poplar s :ump -cut surface II Poplar s ump -cut surface n Poplar s ump -tut surface II Poplar s :ump -cut surface II Poplar s :ump -cut surface n Poplar s :ump -cut surface n Po~lars :umD -cut surface lsolate Date Collected Source 12-A-1 Novernber 5 1997 Poplar stump -side 12-El n Poplar stump -cut surface 12-B-2 n Poplar sturnp -side 12-B-3 II Poplar stump -side 12-C-1 n Poplar sturnp -side 12-C-2 n Poplar sturnp -side 17-A-1 November 3 1997 Poplar sturnp -side 17-A-2 II Poplar stump -side 18-A-1 Il Poplar stump -side 18-A-2 II Poplar sturnp -side 19-A-? II Poptar stump -side 19-A-2 11 Poplar sturnp -side 19-A-3 Il Poptar sturnp -side 20-A-1 n Poplar stump -cut surface 20-A-2 n Poplar sturnp -side 20-A-5 11 Poplar stump -side 20-A-6 Il Poplar stump -side 21-A-1 n Popfar stump -side 21-8-1 n Poplar stump -cut surface 22-A-'! 11 Poplar stump -east side 22-A-2 II Poplar stump -west side 23-A-1 n Poplar stump -side 23-B-1 A Poplar stump -side 24-A- 1 Il Poplar sturnp -wt surface 24-A-2 It Poplar stump -side 24-51 n Poplar stump -side A-A-1 Novernber 2 1997 Poplar stump -side B-A-1 11 Poplar stump -cut surface B-A-2 II Poplar stump -side B-A-3 Il Poplar stump -side BA-4 n Poplar stump -side 6-RI II Poplar sturnp -cut surface GA-1 II Poplar stump -cut surface GA-2 n Poplar sturnp -cut surface GA-3 II Poplar stump -cut surface GA-4 tl Poplar stump -ut surface GA-5 II Poplar sturnp -cut surface GA-6 II Poplar sturnp -side GA-7 n Poplar stump -side GA-8 al Poplar stump -side GA-9 II Poplar stump -side GA-1O u Poplar stump -side GA-1 1 w Poplar stump -side C-A- 12 II Poplar stump -side C-A-73 Il Poplar stump -side lsolate Date Collected Source November 3 A 997 Poplar stump -root collar Il Poplar stump -side 11 Poplar stump -just above mot collar O Poplar stump -cut surface II Poplar stump -cut surface II Poplar stump -cut surface n Poplar stump -side Il Popiar stump -cut surface October 13 1997 Poplar stump -cut surface n Poplar stump -cut surface n Poplar stump -side II Poplar stump -side Il Poplar stump -just above mot collar n Poplar stump -tut surface l* Poplar stump -cut surface II Poplar stump -cut surface Il Poplar stump -cut surface n Poplar stump -cut surface n Poplar stump -cut surface $1 Poplar stump -wt surface 11 Poplar stump -cut surface II Poplar stump -cut surface lm Poplar stump -side (I Poplar stump -side Il Poplar stump -side n Poplar stump -cut surface n Poplar stump -tut surface Il Poplar stump -cut surface Il Poplar stump -wt surface n Poplar stump -cut surface 11 Poplar stump -cut surface II Poplar stump -side II Poplar stump -side n Poplar stump -side 11 Poplar stump -side II Poplar stump -side u Poplar stump -just above mot collar n Poplar stump -cut surface October 17 1997 Poplar stump -side II Poplar stump -side II Poplar stump -side n Poplar stump -side n Popiar stump -side II Poplar stump -side Il Popfar stump -side lsolate Date Collected Source P-1-1 October 17 1997 lar stum s -cut surface P-1-2 I1 lar stum 3 -cut surface P- 1-3 I1 lar stum 3 -side P-14 II iar stum 3 -side -infen'or to Pl3 P- 1-5 IV lar stum 3 -side -inferior to Pl4 P-1-6 n ar stum 3 -side -infen'or to Pl5 P-1-7 n lar stum s -side -inferior to Pl6 P- 1-8 II ar stum 3 -side P-1-1 O II ar stum 3 -side P-1-12 II ar stum 3 -mot collar P-3-1 n ar stum s -tut surface X-A- 1 November 2 1997 ar stum 2 -cut surface x-C-1 n ar stum 2 -cut surface X-G2 11 Poplar stump -wt surface
PLOT 59 lsolate Date Collected Source B-1 October 3 1997 Poplar stump -root collar C-1 11 Poplar stump -just above root collar D-1 la Poplar stump -cut surface E-1 a$ Poplar slash E-2 n Poplar slash E-3 $1 Poplar slash E-4 11 Poplar slash E-5 u Poplar slash E-6 II Poplar slash E-7 IV Poplar slash E-8 11 Poplar slash F-1 11 birch log G-1 1I birch stump -cut surface H-2 n birch stump -root collar 1-1 n birch slash 1-2 II birch slash 1-3 II birch slash 14 VI birch slash K-1 II birch slash K-2 n birch slash MlLLS BLOCK D (MBD) lsolate Date Collected Source MBD-1 October 2 1998 Poplar stump MBû-2 81 Poplar sturnp MBD-3 11 Poplar stump MBD-4 *1 Poplar sturnp MBD-5-1 l* Poplar slash MBM n Poplar stump
WILLIAMS BLOCK D WBD) lsolate Date Collected Source WBD-1 October 3 1998 Poplar sturnp 91 Poplar stump 11 Poplar stump 1* Poplar stump n Poplar slash ** Poplar slash n Poplar slash VI Poplar slash SAMPLE INVENTORY FOR BIRCH PILES
Log # lsolate # Date collected SourceJComrnents - October 17 1998 no C-purpumum no C-purpumum no C. purpueum no C-purpumum no CIpurpumum no C. purpumum no C. purpumum no C-purpumum sapwood sapwood sapwood heartwood sapwood no CI purputeum sapwood sapwood sapwood/heartwood sapwood/heartwood no C. purpumum no Ccpurpumum no C-purpurirum sapwood heartwood no C- purpumum no C. purpureum no C. purpunwm no C. purpureum no C. purpurirum sapwood sapwood sapwood heartwood sapwood no C. purpurirum no C-purputtmm no C- purpureum sapwood/heartwood sapwood/heartwood no C- purpumum no C. purpumum sapwood Log # fsoiate # Date collected Source/Cornments BPA-30 A-2x1 October 17 1998 heartwood heartwood sapwood sapwood no C. purpureum no C. purpureum no C. purpumum sapwood sapwood sapwood sapwood side side side no comment no CIpurpureum no CIpurpureum heartwood sapwood sapwood no CI purpureum sapwood sapwood sapwood sapwood sapwood sapwood sapwood sapwood no C. purputeum no C. purpumum no C. purpureum sapwood sapwood sapwood sapwood sapwood sapwood sapwaad sapwood heartwaod no C-purpureum sapwood sapwood sapwood Log # lsolate # Date collected Source/Comments October 17 1998 sapwood II sapwood n sapwood Il sapwood II heartwood n sapwood October 23 1998 sapwood II sapwood U sapwood II heartwood n heartwood n sapwood/bark Il sapwoodlbark 11 wound in side 1r sapwoodfbark Il sapwood n heartwood II side II side Il side n side Il side - Il side II side II side IL side II sapwood n no C. purpumum n sapwood/bark Il sapwood II sapwood II sapwood II side n side Il sapwood II sapwoodibark U sapwood n hearhrvaod II sapwood II sapwood Pl sapwood 11 heartwood a* heartwood II sapwood n no C. purpu~?um Log # lsolate # Date collected Source/Comments - October 23 1998 no C. purpufeum no C. purpureum heartwood heartwood no C- purpureum no C- purpureum heartwood sapwood heartwood sa pwoodfheartwood no C. pupuEum sapwood heartwood no C- purputeum sapwood sapwood sapwood sapwood sapwood sapwood sapwood no C. purpumum heartwood sapwood sapwood heartwood heartwood sapwood sapwood sapwood no C. purpu~urn no C. purpumum no C-pupumum sapwood/heartwood no C. pupurirum no C. pupumum sapwood heartwood no C. purpureum heartwood heartwood no C. pupureum no C-pupumum sapwood sapwood Log # lsolate # Date collected Source/Comments BPB-1 6-1 October 23 1998 sapwood sapwood sapwood side side side side side side sapwood sapwood heartwood sapwood heartwood side side sapwood sapwood sapwood side side sapwood sapwood sapwood sapwood no C-purpumum no C-purpumurn sapwood sapwood sapwood no C. purpumurn sapwood sapwood heartwood sapwood sapwood sapwood heartwaod sapwood/heartwood heartwood side side sapwood sapwood heartwaod Log # lsolate # Date collected Source/Comments BPB-13 October 23 1998 sapwood II II sapwood heartwood sapwood sapwood heartwood heartwood heartwood PAIRINGS BETWEEN ISOLATES COLLECTED FROM BIRCH LOGS
BPA-34 A-1 A-2 A-3 A-4 A-I - - - A-2 - -
A-I 1 A-2 / A-3 1 51 /