Isozymes of Ganoderma species from Australia

952

Mycol. Res. 104 (8) : 952–961 (August 2000). Printed in the United Kingdom.

Isozymes of Ganoderma species from Australia

B. J. SMITH* and K. SIVASITHAMPARAM Soil Science and Plant Nutrition, Faculty of Agriculture, The University of Western Australia, Nedlands, WA 6907, Australia. E-mail : brendans!pi.csiro.au. Received 26 October 1998 ; accepted 26 November 1999.

Isozymes of five Australian Ganoderma species were studied using cellulose acetate gel electrophoresis (CAGE) and PAGE. Phenetic analysis supported the distinction between the five species, which were separated by unbiased genetic distances of between 0.532 and 3.330. Estimates of heterozygosity and polymorphisms identified considerable genetic variability within populations and species. The putative saprotrophic G. australe, G. incrassatum and G. cupreum had the lowest genetic variability while the pathogen Ganoderma sp. Group 6.3, morphologically similar to G. lucidum, had the highest, followed by G. weberianum. Isolates of G. adspersum, G. applanatum from Europe and G. australe from Australia differed at most loci, thus the commonly accepted synonymy between G. australe and G. adspersum was not supported. Pectic isozymes alone were sufficient to distinguish three laccate (possessing a cutis surface consisting of a palisade of inflated hyphal ends) Australian species, G. weberianum, Ganoderma sp. Group 6.3 and G. cupreum, but they did not distinguish between the non-laccate G. australe and G. incrassatum. The laccate group contains species of greatest economic importance. Australian species were resolved by a single isozyme (glucose-6-phosphate dehydrogenase) using CAGE, demonstrating the potential for this method as a rapid diagnostic tool for identifying species of Ganoderma. This is the first reported population genetics based study of Ganoderma using isozymes.

INTRODUCTION Ganoderma contains species of scientific and commercial importance for their pharmaceutical properties (Jong & Birmingham 1992), as well as for their role as plant pathogens. Despite the importance of the genus world-wide, the morphological taxonomy of Ganoderma is currently in a state of chaos (Ryvarden 1995). Examination of Australian Ganoderma indicated that morphology alone was insufficient to reliably distinguish between all species. A method was also needed to verify the identity of cultures collected without basidiocarps. Isozymes have attracted recent favour in resolving taxonomic conflict in mycology. There are numerous advantages with the use of this technique : genomic differences are detected at the species level, numerous loci are detectable, staining systems are specific and the products are codominant allowing allelic interpretation (Correll 1992). The main drawback of the use of isozymes over DNA based techniques is the need to use a comparatively large amount of fresh material. A second major disadvantage is the number of loci which can reliably be scored is usually limited. There are also

* Current address : CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia.

a large number of parameters such as gel current, ionic strength and pH of running and staining buffers, sample preparation, storage times and activity which can affect the visualisation and resolution of isozymes (Richardson, Baverstock & Adams, 1986). Once experimental protocols are established, however, isozymes are potentially a reliable diagnostic tool, and have been used to routinely identify pathogens (Bonde, Micales & Peterson 1993). With the use of cellulose acetate gel electrophoresis (CAGE) the time needed to conduct electrophoresis can be reduced considerably over that taken with other systems, such as starch or polyacrylamide gel electrophoresis (PAGE) (Correll 1992). The technique has been proven faster than starch gel electrophoresis for identification of Phytophthora infestans isolates (Goodwin, Schneider & Fry 1995) and was considered by Oudemans & Coffey (1991) to have potential for rapid diagnosis of Phytophthora spp. Pectic isozymes using PAGE have been used to distinguish between Sclerotinia spp. (Cruickshank & Wade 1980), anastomosis groupings and species in Rhizoctonia (Sweetingham, Cruickshank & Wong 1986, Burton, Coley-Smith & Wareing 1988, Sweetingham & MacNish 1994), Heterobasidion annosum (Johansson 1988), Armillaria spp. (Mwenje & Ride 1996, 1997), and to distinguish between endophytic fungi (Hutton et al. 1996). Isozymes have also been used in strain and species

B. J. Smith and K. Sivasithamparam identification of Ganoderma (Park et al. 1986, 1994, Shin & Seo 1988, Hseu et al. 1989, Lee & Lee 1991, Miller et al. 1995, Gottlieb, Saidman & Wright 1995, 1998). In this study we use phenetic analysis of isozymes to examine population and species relationships of Ganoderma spp. from Australia. We assessed CAGE as a potential rapid diagnostic tool for identifying Ganoderma spp. in culture. This was compared against pectic isozymes using PAGE which have been used to identify several other genera of fungi. METHODS The Ganoderma cultures used in this study (Table 1) include five Australian species. Representing subgenus Elfvingia were species with non-laccate cutis G. australe and G. incrassatum. Representing subgenus Ganoderma were species with a laccate cutis G. cupreum, G. weberianum and a species referred to as G. sp. Group 6.3, which has not been previously named (Smith

953 & Sivasithamparam 2000), and which is closely related to the Group 6 taxa in Moncalvo et al. (1995). Non-Australian species studied were G. adspersum and G. applanatum. Two systems were used to test for activity in 25 isozymes staining systems (Table 2). Pectin depolymerase and polygalacturonase (pectin methylesterase) enzymes were resolved by horizontal PAGE, while intracellular isozymes were resolved by CAGE. The procedures for preparation of protein extracts, electrophoretic conditions and visualisation of pectin methylesterase and polygalacturonase enzymes have been described previously (Cruickshank & Wade 1980, Cruickshank 1983, Sweetingham & MacNish 1994). Isolates were grown for 8–30 d in bijoux bottles containing 2 ml of a medium consisting of 2n64 g (NH ) SO , 0n34 g K HPO , 14 g MgSO ;7H O, %# % # % % # and citrus pectin 10 g (homogenised in a smaller volume) l−" distilled H O and adjusted to pH 5n5 with NaOH prior to # autoclaving. The optimum time for harvesting was generally

Table 1. Isolates used in this study. Isolates

Locality, host, collector*

Determination

UWA 27, 29, 31, 41–44, DAR 73781 UWA 46, 48, 49, 51, 52, 130 UWA 53, 54, 57, 58, 60 UWA 92 DAR 73782

Grey Block, WA, Eucalyptus diversicolor log, B. Smith

G. australea

1

Grey Block, WA, E. diversicolor log, B. Smith

G. australea

2

Grey Block, WA, Australia, E. diversicolor log, B. Smith Porongurup NP, WA, Dead tree E. diversicolor, B. Smith Leeuwin Naturaliste NP, WA, Dead Banksia seminuda, C. Crane (CC 857.1) Sherbrooke Forest, Vic., collector & host unknown, lodged as G. applanatum John Holmes Jungle Park, NT, Stump rainforest, B. Smith Noosa, Qld, Casuarina sp. lodged as G. lucidum Murwillumbah, NSW, Mangifera sp., lodged as G. lucidum Eungella, Qld, Casuarina sp., lodged as G. lucidum Licuala State Forest Park, Qld, dead wood, lodged as G. chalceum Noosa Heads, Qld, Casuarina sp., T. E. Hunt, lodged as G. chalceum Stony Creek, nr Cairns, Qld, W. Pont F.W.I.T.A., WA, Albizia lebbeck, B. Smith Paddington, Brisbane, Qld, Delonix regia, D. Vanderbyl Lawn Hill NP, Qld, Eucalyptus papuana, C. O’Keefe Mackay, Qld, Cassia sp., L. McVeigh F.W.I.T.A., WA, A. lebbeck, B. Smith Kununurra, WA, Australia, B. Smith Kununurra, WA, Australia, A. lebbeck, B. Smith Tirtaganga, Indonesia, Stump, B. Smith

G. australea G. australea G. australea

3 4 5

G. australea

6

DFP 15749 DAR 73783, UWA 89 DFP 4483 DFP 8405 DFP 8401 QFRI 8678.1 DFP 3896 DFP 4336 UWA 4, 5, 8, 21 QFRI 8147.1 QFRI 8647.1 QFRI 8156 UWA 77, 79, 80, 84 UWA 85 DAR 73779 DAR 73780, UWA 128, 129 IMI 157816 CBS 250.61 CBS 175.30

UK, Prunus cerasus, lodged as G. applanatum Austria UK

G. G. G. G. G.

Population

incrassatumb weberianumc weberianumc weberianumc cupreumd

7 8 9 10 11

G. cupreumd G. cupreumd Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp.

12

Group Group Group Group Group Group Group Group

6.3e 6.3e 6.3e 6.3e 6.3e 6.3e 6.3e 6.3e

13 14 15 16 17 18 19 19 20

G. adspersumf G. applanatum G. applanatum

Abbreviations : DAR, Orange Agricultural Institute, Orange, NSW (isolates deposited by BJS) ; DFP, Division of Forest Products, CSIRO, Melbourne, Vic. ; QFRI, Queensland Forest Research Institute, Department of Primary Industries Forestry, Indooroopilly, Qld ; CBS, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands ; IMI, International Mycological Institute, Egham, Surrey, UK ; UWA, author’s (BJS) own collection. * Localities (all Australia unless noted otherwise) : NSW, New South Wales ; Qld, Queensland ; Tas., Tasmania ; Vic., Victoria ; WA, Western Australia ; NP, National Park ; F.W.I.T.A., Frank Wise Institute of Tropical Agriculture ; Pop., population ; aff., affinis ; cpx., complex ; a G. australe cannot be verified as the type is lost. This species may eventually be recognised as G. tornatum ; b Isolate verified against the type specimen ; c Det. by R. L. Steyaert ; d Det. (Steyaert 1967) as G. chalceum with G. cupreum as a synonym. We use G. cupreum, as having nomenclatural priority (Moncalvo & Ryvarden 1997) ; e Previously studied by Hood, Ramsden & Allen (1996), as Ganoderma sp. aff. lucidum. Based on a revision of taxa described from the region, and analysis of rDNA sequence (Smith & Sivasithamparam 2000), this species is apparently yet to be named, and is closely related to the group six taxa in Moncalvo et al. (1995) ; f Held in IMI as G. applanatum, but conspecific with an isolate of G. adspersum, authenticated by V. Demoulin, based on rDNA sequence analysis (Smith & Sivasithamparam 2000) ; g Belongs to the G. resinaceum complex based on rDNA phylogeny studies (Smith & Sivasithamparam 2000).

Isozymes of Australian Ganoderma

954

Table 2. Enzymes examined, pH of staining buffer used and results of initial screening for enzymatic activity. Only isozymes with average or strong staining were subjected to further optimisation and then included in the results.

a ‘ Variable ’

Enzyme

EC number

Stain pH

Result

Glucose-6-isomerase (GPI) Phosphoglucomutase (PGM) Pyruvate kinase (PK) 6-phosphogluconate dehydrogenase (6PGDH) Glucose-6-phosphate dehydrogenase (G6PDH) Isocitrate dehydrogenase (IDH) Hexokinase (HK) Pectin methylesterase (PME) Polygalacturonase (PGN) Carboxylesterase (αEst) Carboxylesterase (βEst) Creatin kinase (CK) Fructose-1,6-diphosphate dehydrogenase (FDPDH) Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) Mannose-6-phosphate isomerase (MPI) Sorbitol dehydrogenase (SDH) Pectin lyase (PL) Alcohol dehydrogenase (ADH) Fumarase (FUM) Lactate dehydrogenase (LDH) Malate dehydrogenase (MDH) Malic enzyme (ME) Menadione reductase (MR) Glycolate oxidase (GOX) Superoxide dismutase (SOD)

EC 5;3;1;9 EC 5;4;2;2 EC 2;7;2;40 EC 1;1;1;44 EC 1;1;1;49 EC 1;1;1;42 EC 2;7;1;1 EC 3;1;1;11 EC 3;2;1;15 EC 3;1;1;1 EC 3;1;1;1 EC 2;7;3;2 EC 3;1;3;11 EC 1;2;1;12 EC 5;3;1;8 EC 1;1;1;14 EC 4;2;2;10 EC 1;1;1;1 EC 4;2;1;2 EC 1;1;1;27 EC 1;1;1;37 EC 1;1;1;40 EC 1;6;9;9 EC 1;1;3;1 EC 1;15;1;1

0n1  Tris, pH 8n5 0n1  Tris, pH 8 0n1  Tris, pH 8 0n1  Tris, pH 8 0n1  Tris, pH 7n5 0n1  Tris, pH 7n5 0n1  Tris, pH 7n5 Variablea variablea 0n1  Na phosphate, pH 7 0n1  Na phosphate, pH 7 0n1  Tris, pH 8 0n1  Tris, pH 8 0n1  Tris, pH 7 0n1  Tris, pH 8 0n1  Tris, pH 8 Variablea 0n1  Tris, pH 8n5 0n1  Tris, pH 7n5 0n1  Tris, pH 7n5 0n1  Tris, pH 8 0n1  Tris, pH 7n5 0n1  Tris, pH 7 0n1  Tris, pH 8 0n1  Tris, pH 8

Strong Strong Strong Average Average Average Average Average Average Weak Weak Weak Weak Weak Weak Weak Weak No activity No activity No activity No activity No activity No activity No activity No activity

staining pH indicates that optimal pH for enzyme staining activity was found by allowing the stain pH to fall slowly over time.

Fig. 1. CAGE gels showing banding patterns resulting from quaternary protein structure. From left to right ; Gel 1, stained for 6PGDH (single locus, monomeric protein), lanes 1–3 are, UWA 29, DAR 73781, UWA 21, (all homozygous alleles) ; Gel 2, stained for PGM (single locus, monomeric protein), lanes 1–3 are, UWA 130 (homozygous), QFRI 8647.1 (heterozygous), UWA 53 (homozygous) ; Gel 3, stained for PGI (single locus, dimeric protein), lanes 1–3 are, UWA 84 (homozygous), UWA 80 (heterozygous), UWA 79 (heterozygous). The middle band present in dimeric heterozygous loci (Gel 3, lanes 2, 3) is a heterodimer, the functional product of the combination of the two different alleles, the homodimeric phenotypes of which are the slower and faster bands. The cathodal loading zones, not shown, are above the bands.

when the mycelium had filled the available volume. Cultures were stored at k80 mC prior to electrophoresis. The cast gel dimensions were 70i150i3 mm with 18 loading slots (25 µl) along the length of the gel and 35 mm from one edge, and such that the loading slots did not fully penetrate the thickness of the gel. The gel was cast between a lower plate which supported the gel and an upper plate which formed the wells. The formulation of the gel was citrus

pectin 0n08 g, and N,N methylene-bis-acrylamide 0n2 g " dissolved in 80 ml of 38 m Tris-citrate buffer, pH 8n7. This was followed by 8 g acrylamide, 80 µl N,N,N ,N tetramethyl" " ethylenediamine and then 0n08 g ammonium persulphate added and mixed immediately prior to casting. The upper plate was removed approx. 15 min after pouring. Small aliquots of the pectin broth cultures were mixed 4 : 1 (v\v) with Sephadex G-150 before transferring 20 µl of the solution into the gel loading slots. Electrophoresis was carried out for 100 min at 20 mA using a 0n158  borate tank buffer (pH 8n7) with the loading slots at the cathodal end. After electrophoresis the gels were rinsed with tap water, soaked in 0n1  D\L-malic acid for 60 min, then 0n03 % ruthenium red at 4 m for 12 h, rinsed in cold water and oxidised in cold 0n1 % ammonium persulphate for 20 min to improve contrast. Pectin lyase was not assessed as activity was inconsistent and not well resolved. Gels were scored prior to and after contact photography as the pectin lyase isozymes often overlapped the activity of polygalacturonase from which it is indistinguishable by black and white photography. Intracellular enzymes for CAGE were extracted from mycelia grown in 10 ml of 5 % malt extract broth in McCartney bottles. Mycelia were spooled into 2 ml microcentrifuge tubes, centrifuged and the excess liquid removed with a pipette. The samples were then suspended in 40 µl of extraction buffer and frozen at k80 m prior to grinding. The extraction buffer consisted of 0n4 g polyvinylpyrrolidone (PVP), 1 g sucrose, 17 mg EDTA, 2 mg ascorbic acid, 10 mg bovine serum, 5 mg NAD, 2n5 mg NADP and 7 mg dithiothreitol. After grinding, the samples were centrifuged a final time leaving a supernatant containing the crude protein

B. J. Smith and K. Sivasithamparam

955

0

20

10

Latitude (°S)

7 13 11

14, 18, 19

20

16

10 17 8 15

30

12

9

1, 2, 3 5

40

4

G. australe G. incrassatum G. weberianum G. cupreum G. sp. Group 6.3

1000 km

110

120

6

130

140

150

Longitude (°E)

Fig. 2. Distribution of Australasian isolates and species used in this study. The numbers given for each site refer to populations in Table 1, which also contains information on individual isolates.

Table 3. Voltage, run time and results of electrophoresis for the seven enzymes systems chosen for allozyme analysis. Good or poor resolution (R or r) and separation (S and s) are presented. Where not indicated Ed or Sm indicates electrodecanting or smearing of bands respectively. Dash indicates not attempted. The assessment of separation and resolution are for comparison between the three buffers for each enzyme system, and not for comparison between the different systems. For key to abbreviations of enzymes see Table 2. The composition of the TEC and TGC running buffers is given in the methods.

Enzyme

No. loci

Type

TGC 8.1

GPI PGM PK 6PGDH G6PDH IDH HK

1 1 1 1 1 1 1

Monomer Dimer Monomer Monomer Monomer Monomer Monomer

135 V, 135 V, 135 V, 140 V, — 140 V, 135 V,

30 min, 20 min, 30 min, 20 min,

TEC 8.1 R, S r, S R, S Ed, Sm

20 min, Sm 15 min, r, s

extract. The extracts were blotted onto the cellulose acetate gels using the Super Z-12 applicator system (Helena Laboratories, Texas, USA). Where subsequent staining activity was low double or triple applications were used. Six continuous buffer systems were tested. These were a 0n248  Tris-glycine buffer at pH 8n5 (TG 8n5), and the same buffer adjusted to pH 8n1 (TGC 8n1) and 7n5 (TGC 7n5) with citric acid. And buffers consisting of 80 m Tris, 1 m EDTA, 13n3 m glycine, 6n4 m histidine, 3 m glutamic acid, 3n8 m aspartic acid and 0n5 m MgCl . This was then buffered to pH 8n1 # (TEC 8n1) or 7n5 (TEC 7n5) with citric acid, or 8n2 (TEC 8n2) with NaOH after first bringing the pH to 7n6 with maleic acid. Staining protocols followed have been described previously by Richardson et al. (1986) and Herbert & Beaton (1993). Putative loci and quaternary structure were inferred from previously published studies, or characteristic banding patterns

— 200 V, 200 V, 200 V, 200 V, — 200 V,

30 min, 15 min, 15 min, 15 min,

TEC 7.5

R, S R, s r, S R, S

15 min, r, s

200 V, 200 V, 200 V, 200 V, 200 V, 200 V, 200 V,

30 min, 30 min, 30 min, 15 min, 15 min, 15 min, 10 min,

R, R, R R, R, R, R,

s S S S S S

produced by heterozygous loci (Fig. 1). Following the recommendations of Richardson et al. (1986), alleles were differentiated when they were separated by half a bandwidth. For each locus alleles were scored alphabetically starting with the allele with the greatest electrophoretic mobility. Analysis of population allelic frequencies of Australian isolates were made using Biosys-1 (Swofford & Selander, 1981). Each population (Fig. 2) consisted of isolates obtained from basidiocarps at a single site ( 0n25 ha). Heterozygosity, mean polymorphic loci and two distance measures, the unbiased genetic distance (D) of Nei (1978) and the modified Rogers distance (DT) of Wright (1978), were calculated. Formulae described below are discussed in Swofford & Selander (1981), Nei (1978) and Wright (1978). DT l [(1\L)ΣLD#]"/#,

(1)

Isozymes of Australian Ganoderma

956

Table 4. Isozyme alleles of Ganoderma spp. For key to abbreviations of enzymes see Table 2. Isolates allocated population (Pop.) numbers were subjected to phenetic analysis (Fig. 3). Isolate

Species

Pop.

PGN

PME

G6PDH PGM

6PGDH PGI

PK

IDH

HK

UWA 27 UWA 29 UWA 31 UWA 37 DAR 73781 UWA 41 UWA 42 UWA 43 UWA 44 UWA 46 UWA 48 UWA 49 UWA 51 UWA 52 UWA 130 UWA 53 UWA 54 UWA 57 UWA 58 UWA 60 UWA 92 DAR 73782 DFP 15749 DAR 73783 UWA 89 DFP 4483 DFP 8405 DFP 8401 QFRI 8678.1 DFP 3896 DFP 4336 UWA 4 UWA 5 UWA 8 UWA 21 QFRI 8147.1 QFRI 8647.1 QFRI 8156 UWA 77 UWA 79 UWA 80 UWA 84 UWA 85 DAR 73779 DAR 73780 UWA 128 UWA 129 IMI 157816 CBS 250.61 CBS 175.30

G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. australe G. incrassatum G. incrassatum G. weberianum G. weberianum G. weberianum G. cupreum G. cupreum G. cupreum Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. G. adspersum G. applanatum G. applanatum

1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 4 5 6 7 7 8 9 10 11 12 13 14 14 14 14 15 16 17 18 18 18 18 19 19 20 20 20 — — —

BC BC — — — — — — BB — BC — BC — DD — BB BB BC BC — BC — — — BD BD BB AA AA AC BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB DD BC BC

AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA BB BB BB AA AA AA DE DE DE DD EE EE DE DE DE DE DD DD DE DD DD DD BB AA AA

BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB AA — BB BB BB DD DD DD CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC AA DD CC

AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA — AA AA AA AA AA BB BB CC BB BB BB CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC AA BB BB

BB BB BB BB BB BB BB BB BB BB BB BB BB BB CC BB BB BB BB BB CC BB AA AA AA AA BB BB AA AA AA CC CC CC CC CC CC CC BB BB BB BB CC CC CC CC CC AA AA AA

CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC — CC CC CC AA AA CC CC CC BB BB BB CC CC CC CC CC CC CC CC CC CC CC CC — CC CC CC AA DD BB

BB — BB BB — — BB BB BB BB BB BB BB — BB BB BB — BB BB BB BB BB BC BC CC BB CC CC CC CC BG BG BG — BG BG BG — BG BG BG BG BG DG DG DG BD — EE

Group Group Group Group Group Group Group Group Group Group Group Group Group Group Group Group

6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3

CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC AA AA AA DD DD DD BD BD BD DD CD CD BB DD DD DD BD BD BD BD BD BD DD DD DD

FF FF FF FF FF FF FF FF FF FF FF DF FF FF FF FF FF FF FF CF FF FF FF EE EE AD AA DD AD AE AA EE EE EE EE DD EE AD — AD AD EE EE EE EE EE AD AA CC CC

where L l loci, (for a given locus) Dl [0n5Σk(qX(i)kqY(i))#]"/#, where q l allelic frequency, i l the ith allele at a locus, X and Y are two populations (Wright 1978).

Cluster analysis was performed on these distances (DT and D) using UPGMA. Species were identified using the genotypic cluster definition (Mallet 1999).

unbiased D lkln[GXY\NGXGY],

RESULTS

where GX l (2nX JXk1)\(2nXk1), GY l (2nY JYk1)\ (2nYk1), GXY l JXY, JX l Σxi#, JY l Σy#i , JXY l Σxi yi, and where xi and yi are sample frequencies of the ith allele in populations X and Y respectively (Nei 1978).

Enzymes which gave weak or no staining activity under the conditions tested are shown in Table 2. Nine loci, based on eight enzyme staining protocols, were sufficiently active to be reliably scored (Table 2). The responses of the seven loci

B. J. Smith and K. Sivasithamparam

957 1 G. australe 2 G. australe 3 G. australe 5 G. australe 4 G. australe 6 G. australe 7 G. incrassatum 8 G. weberianum 9 G. weberianum 10 G. weberianum 14 G. sp. group 6.3 19 G. sp. group 6.3 20 G. sp. group 6.3 16 G. sp. group 6.3 17 G. sp. group 6.3 15 G. sp. group 6.3 18 G. sp. group 6.3 11 G. cupreum 13 G. cupreum 12 G. cupreum

2.0

1.6

1.2 0.8 0.4 Nei’s (1978) unbiased genetic distance (D)

0.0

Figure 3. UPGMA cluster analysis of populations of Australian Ganoderma based on eight putative loci using Nei’s (1978) unbiased genetic distance. Numbers correspond to populations identified in Table 4.

stained following CAGE, to running buffer and voltage conditions, are summarised in Table 3. PAGE of pectic isozymes polygalacturonase and pectin methylesterase alone were sufficient to distinguish three laccate Australian species, G. weberianum, Ganoderma sp. Group 6.3 and G. cupreum. The Australian non-laccate G. australe and G. incrassatum, and isolates of G. applanatum from Europe were not distinguished at these loci. Both G. incrassatum isolates failed to produce activity in polygalacturonase, as did many isolates of G. australe (Table 4). Isolates from outside Australia were all characterised by unique isozyme patterns (Table 4). For CAGE no single buffer\voltage combination gave consistent staining activity and resolution for all enzymes tested. The high ionic strength and acidic TEC 7n5 buffer resulted in the best staining and resolution of isozymes, but was not suitable for visualising many enzyme systems as there was greatly reduced separation of isozymes. This is a characteristic of higher ionic strength buffers. Better separation was found in the TEC 8n1 buffer, but at the expense of resolution especially for PGD and HK. The TGC 8n1 buffer gave good separation and resolution of isozymes for the systems PGI and PK. All other enzymes systems suffered from electrodecanting, smearing, poor resolution and poor separation with this buffer. Of the other buffer systems TG 8n5 and TGC 7n5 gave the poorest results and the TEC 8n2 buffer was similar but not as good as the TEC 8n1. The results of differing buffer systems are summarised in Table 4. As some isolates of G. australe and all of G. incrassatum

failed to produce activity for polygalacturonase, this locus was excluded from the population phenetic analysis. Most other loci consistently stained for activity. Only the Australian isolates were included in the analysis as there were too few isolates of the non-Australian material to constitute a representative sample. The UPGMA cluster analysis based on genetic distance (Nei 1978) of the Australian isolates (Fig. 3) identified each of the five species included in the study (cophenetic correlation l 0n842). Cluster analysis based on the modified Rogers distance of Wright (1978) and Nei’s (1978) genetic distance gave a tree structures differing only in branch lengths. Nei’s unbiased genetic distance (Table 5) varied between 0n000–0n135 for G. australe, 0n342–0n470 for G. weberianum, 0n032–0n034 for G. cupreum and 0n000–0n345 for Ganoderma sp. Group 6.3. Nei’s unbiased genetic distances between species were least between isolates of G. australe and G. incrassatum (0n532–0n783) and greatest between G. cupreum and Ganoderma sp. Group 6.3 (1n698–3n330). Heterozygosity and polymorphisms (Table 6) were much higher in Ganoderma sp. Group 6.3 than the other species. Populations of G. cupreum, G. weberianum and G. incrassatum were represented by single individuals skewing estimates. The mean percentage of polymorphic loci for populations was 6n3 % for G. australe, 12n5 % for G. incrassatum, 4n2 % for G. weberianum, 8n3 % for G. cupreum and 35n7 % for Ganoderma sp. Group 6.3. Percentage of polymorphic loci for each species was 25 % for G. australe, 12n5 % for G. incrassatum and G.

Isozymes of Australian Ganoderma

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B. J. Smith and K. Sivasithamparam

959

Table 6. Genetic variability in Australian Ganoderma populations. Measures are unbiased mean heterozygosity per locus (.. in parenthesis), percentage of polymorphic loci for each population, mean of population polymorphisms and mean percentage of polymorphic loci for each species.

Population

Species

Mean heterozygosity (unbiased)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

G. australe G. australe G. australe G. australe G. australe G. australe G. incrassatum G. weberianum G. weberianum G. weberianum G. cupreum G. cupreum G. cupreum Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp. Ganoderma sp.

0n0 0n059 (0n41) 0n25 (0n25) 0n0 0n0 0n0 0n083 (0n83) 0n125 (0n125) 0n0 0n0 0n125 (0n125) 0n125 (0n125) 0n0 0n209 (0n102) 0n25 (0n164) 0n25 (0n164) 0n375 (0n183) 0n273 (0n116) 0n229 (0n133) 0n225 (0n11)

Group Group Group Group Group Group Group

6.3 6.3 6.3 6.3 6.3 6.3 6.3

cupreum, 50 % for G. weberianum and 62n5 % for Ganoderma sp. Group 6.3. DISCUSSION Genetic analysis of population structure of the five Australian species supported the taxonomic distinction between the isolates studied. The results were consistent for population analysis using both unbiased genetic distance (Nei 1978) and the modified Rogers distance of Wright (1978). Following the genotypic cluster species definition (Mallet 1995), five distinct species could be identified, typified by a lack of intermediates (Fig. 3). The results of the isozyme analysis confirm taxonomic conclusions obtained previously by rDNA sequence analysis (Smith & Sivasithamparam 2000). Isolates previously determined as G. applanatum and G. lucidum were found to belong to G. australe and Ganoderma sp. Group 6.3 and G. webarianum. Previous studies of Ganoderma have often failed to distinguish species. Miller et al. (1995) found a large number of Ganoderma isolations from the host Elaeis guineensis, with a wide geographical distribution, fell within a single cluster based on pectin isozymes. Unfortunately they did not attempt to assign names to isolates, to delineate taxa on the basis of the cluster analysis, or identify isolates used in the study in the cluster analysis. Although Miller et al. (1995) concluded that existing species definitions were of little value in interpreting disease processes, they did not determine the species relationships of any of the isolates they examined. In our study, G. australe and G. incrassatum could not be distinguished on the basis of pectic isozymes alone, highlighting the limitations of using limited numbers of enzymes systems to distinguish taxa. Gottlieb et al. (1998) used eight enzyme systems codified

Polymorphisms (%) 0n0 25n0 12n5 0n0 0n0 0n0 12n5 12n5 0n0 0n0 12n5 12n5 0n0 37n5 25 25 37n5 50n0 37n5 37n5

for presence or absence of bands to examine the relationship of laccate and non-laccate species of Ganoderma from South America. While the results were inconclusive in resolving species boundaries due to the inconsistency of the taxonomic determination of isolates, the retention of the G. applanatum and G. lucidum complexes were supported by cluster analysis. In the G. applanatum complex four of several closely related clusters contained interfertile isolates. They concluded that the binomials applied to isolates were of very closely related species or were in need of revision. A feature of several isozyme studies of Ganoderma has been the absence of allelic interpretation (Shin & Seo 1988, Hseu et al. 1989, Park et al. 1986, 1994, Lee & Lee 1991, Gottlieb et al. 1995, 1998, Miller et al. 1995). Esterases, examined in all but the study of Hseu et al. (1989), may have multiple loci and possess quaternary structure which give rise to complex banding patterns (Herbert & Beaton 1993). In these cases progeny studies are necessary in order to identify loci and alleles. Additionally with the exception of Gottlieb et al. (1995, 1998), these studies were restricted to three or less enzymes systems. In our study we have focused on isozymes which are amenable to allelic determination. This combined with a greater number of loci studied in comparison with other studies resulted in clear delineation of the species in the analysis. The use of esterases and other enzymes with banding patterns resulting from quaternary enzyme structure and multiple loci are probably more suited to diagnostics of well characterised species, rather than solving taxonomic dilemmas in poorly known species. Nevertheless, the application of a few enzymes systems as diagnostic markers in Ganoderma has been successfully demonstrated by Hseu et al. (1989) using laccase and Park et al. (1994) using a combination of esterase, malate dehydrogenase and phosphoglucomutase. The clusters represented by the isolates of G. australe, G.

Isozymes of Australian Ganoderma cupreum, Ganoderma sp. Group 6.3 and G. weberianum were probably closely representative in terms of their geographic occurrence. G. incrassatum was only represented by a single Australian collection and further collections are needed. The heterozygosity observed in the Australian Ganoderma was particularly high in Ganoderma sp. Group 6.3 and G. weberianum, which are more commonly reported as pathogenic. In contrast the wood rotting G. australe showed lower variability across populations separated by as much as approx. 4000 km. Genetic variability in Ganoderma may be influenced by factors such as spatial distribution, fructification frequency and the numbers of alleles involved in the heterothalic mating system restricting inbreeding. The Ganoderma sp. Group 6.3 isolates collected for this study from Kununurra and nearby tree plantations (in arid-tropical north Western Australia) certainly satisfy the model of an organism with a frequent reproductive cycle with outbreeding potential. In contrast G. australe from temperate south west forests fruit rarely and populations are sparsely distributed. G. applanatum and G. adspersum have a distribution which includes Europe, where the type specimens of these species were collected. The G. adspersum isolate (IMI 157816, lodged as G. applanatum) used in this study was authenticated against an isolate originally determined by V. Demoulin (pers. comm.) using rDNA sequence (Smith & Sivasithamparam 2000). G. adspersum was considered a synonym of G. australe by Ryvarden & Gilbertson (1993), but this was not supported by our data which shows dissimilarity between the isozymes of these two species. Although we have used the name G. australe the status of this species is uncertain, as the type (originally collected from the Pacific region) is apparently lost (Moncalvo & Ryvarden 1997). Furthermore, the only authentic material identified to date is of European origin (Moncalvo & Ryvarden 1997) and outside the probable distribution of G. australe. There are also problems with the current concept of G. australe since Yeh & Chen (1990) demonstrated that Taiwan specimens fitting the modern concept of G. australe were comprised of two biological species, based on intersterility and temperature-growth rate studies. Given the broadly accepted synonymy between G. australe and G. tornatum (Ryvarden & Johansen 1980, Gilbertson & Ryvarden 1986, Ryvarden & Gilbertson 1993, Steyaert 1967, 1972, 1975a, b), G. tornatum could replace G. australe should the typification problem remain unresolved. Considering the findings of Yeh & Chen (1990), however, we consider it to be premature to apply this name without taxonomic or molecular studies of relevant material from the Australasian region. The species identities of Australian Ganoderma isolates were resolved by a single isozyme glucose-6-phosphate dehydrogenase, demonstrating the potential for CAGE, using a small number of isozyme loci, as a simple diagnostic tool. Pectic enzymes using PAGE showed promise in identifying laccate species, which contain species of economic importance. Isozymes are especially suited for making inferences about population genetics. This study has identified a number of isozymes which would be suitable for measuring genetic diversity, founder effects, and the mode of infection by populations of Ganoderma species such as those pathogenic to oil palm.

960 A C K N O W L E D G E M E N TS We gratefully acknowledge Dr J. M. Moncalvo (Duke University) ; Dr I. A. Hood & Mr M. Ramsden (Department of Primary Industry, Queensland) ; Dr R. N. G. Miller (International Mycological Institute, Egham, Surrey) and Fungi Perfecti (Olympia, USA) for allowing us to use their cultures.

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