NewPhytol- (1994), 128, 135-143

Discrimination of some African Armillartaspecies by isozyme electrophoretic analysis

BY A. AGUSTIAN\ C. MOHAMMED^ J. J. GU ILLAUMIN^ANDB. BOTTON^*

^ Laboratoire de Biologie Forestiere associe a VINRA, Groupe de Mycologie et dePhysiologie des Mycorhizes, Umversite de Nancy 7, BP 239, 54506 Vandaeuvre-Les-Nancy Cedex, France^ Oxford Forestry Institute, Department of Plant Sciences, University of Oxford,South Parks Road, Oxford OXl 3RB, U.K.^ Unite de Mycologie, Institut National de la Recherche Agronomique,12, Avenue du Brezet, 63039 Clermont-Ferrand, France

{Received 10 January 1994; accepted 24 May 1994)

SUMMARY

Ten Armillaria isolates, collected from various host plants and widespread geographical origins in tropical Africa,were cultivated on orange fragments in the presence of water and two different culture media in order to optimizeenzyme and mycelial cord production. Seven enzymes involved in the primary metabolism of nitrogen and carbon(glutamate dehydrogenases, aspartate aminotransferase, malate dehydrogenase, glucose 6-phosphate dehydro-genase, 6-phospbogluconatt dehydrogenase and isocitrate dehydrogenase) were extracted from the mycelial cordsand analyzed by polyacrylamide gel electrophoresis. Cluster analy.sis based on calculated similarity i-alues derivedfrom isozyme banding patterns separated the isolates into fi\'e groups. Two isolates considered as belonging to A.metlea ssp. africana (an African species closely related to European A. metlea) were present in a clearly separatedcluster when compared to the other isolate groups. Two Kenyan isolates, belonging to an as yet unnamedbiological species, which w ere characterized by the production of few slow growing mycelial cords, were also foundin a separate cluster with slightly greater similarit\' coefficients to the other isolates. The six other isolates, referredto as isolates of A. heimii (a highly variable species with different sexual systems) fell into three sub^clusters ofvariable homology. The two bomothallic heimii isolates from Tanzania and Malawi, which were \ery closelyrelated, displaying 100 "„ isozyme similarity, exhibited a higher degree of similarit>' with the two other homothalhcheimii isolates from Zimbabwe and Congo, than with the two heterothailic unifactorial heimti isolates fromCameroon and Gabon. The value of isozymes in the classification of African Armtllaria spp. is discussed.

Key words: Armillaria, isozymes, mycelial cords, taxonomy.

Anderson & Korhonen, 1991). Pairing tests, basedINTRODL-CTION ^ ^ sexual behaviour, can establish inter-specific

Certain fungal species of the genus Armillaria (Fr) incompatibility (Korhonen, 1978; Guillaumin et al.,Staude induce root rot disease which can result m 1991) and have been widely exploited to defineextensive economic losses to a diversity of crop biological species or groups m Europe, Australia andplants (Hood, Redfern & Kile, 1991; Kile, North America. Some of these groups corre.spondedMcDonald & Byler, 1991). In Europe, North to known morphologically described species whilstAmerica and Australia, Armillaria spp. are hetero- others represent new biological species (Watling,thallic with a bifactorial mating system (Guillaumin, Kile & Burdsall, 1991). Six species have been

identified in Europe and nine (annulate) NorthAmerican biological species (NABS) are known. All

* To whom correspondence should be addres..ed ^^^ ^^^ ^^^,^ ^^^^^ described as newAbbreviations additional to those listed in the journals " " , i?

'Instructions to Authors'. NBT. Nitro Bluf letrazolium; P5P, species or formally equated to those EuropeanPyridoxal 5-phosphate, PMS, Phenazine methosulphate; PMSF. gpecies with which they are found to be partlyPhenylmethylsulphonylfluoride; PVPP, Polyvinvlpoly- j^j ^ j j ^^^ -^^ f^^^j in Australasia arepyrroJidone; PVP, Polyvinylpyrrolidone. f '^

136 A. Agustian and others

incompatible with European and North Americanspecies.

Root rot attributed to Armillaria spp. has beenwidely reported from the wetter regions of Africa,principally in exotic softwood and cash cropplantations (Hood et al., 1991). Experience withtemperate Armillaria suggests that much of thecontradictory information given in the literaturemight be explained by the presence of severaldifferent species. Notwithstanding the size of Africa,the morphology of only two species have beendescribed in any detail: A. mellea (Pegler, 1977) andA. heirnii (Heim, 1963, 1967; Pegler, 1977). Recenttaxonomical studies have shown that A. heimii isindeed the species most frequently found over anextremely widespread area including regions of west.east and central Africa but is highly variable withtwo types of sexual system (homothallism andunifactorial heterothalhsm). A species similar insome respects to temperate A. mellea does existboth in West and East Africa but is still sufficientlydifferent to be considered as a subspecies A. melleassp. africana (Kile et al., 1994). Other more geo-graphically localized biological groups of Armillariahave been differentiated but ha\-e not been assignedto specific species.

Taxonomical studies of African Armillaria haveincluded a wide range of investigative techniquesincluding observations of cultural characteristics(vegetative mat morphology, growth in culture atdifferent temperatures, fruitbodies and monosporeisolates obtained in vitro), pairing tests based on bothsexual and somatic incompatibilit>' and molecularRAPD analyses (Mohammed, Guillaumin &Berthelay, 1989; Mohammed & Guillaumin, 1993;Mohammed, 1994; Mohammed et al., 1994).

Isozyme analysis is also an efficient method fordetermining variation in fungi (Micales, Bonde &Peterson, 1986) and has been widely used in theidentification of species and isolates and populationstructures of a large number of genera includingNeurospora (Spieth, 1975), Heterobasidium (Stenlid,1985; Orotsina et al., 1993), Pleurotus (Magae et al.,1990), Phytophthora (Oudeman & CofTey, 1991),Lentinus (Ohmasa & Furukawa, 1986), Schizo-phyllum (Lily & Charvat, 1987), Tuber (Dupreet al., 1993) and several ecto and endomycorrhizalfungi (Sen & Hepper, 1986; Sen, 1990; Keller,1992). Isozyme patterns has already been used tocharacterize Armillaria species both in Europe(Wahlstrom et al., 1991) and in North America(Morrison et al., 1985; Lin, Dumas & Hubbes,1989). Some Kenyan Armillaria isolates have alsobeen differentiated from both North American andEuropean species, on the basis of morphology,intersterility and their esterase isozyme profiles(Mwangi, Lin & Hubbes, 1989). Most of theseresults are based on isozyme identification on gelsand numerical analvsis. Matrices are constructed

from the absence/presence data and similaritiesbetween isolates are then assessed using unpairedgroup mean average (L^PGMA) cluster analysis.

The aim of the study was to obtain additionalinformation on 10 pre\ious]y characterized isolatesfalling into three groups which could be equatedwith A. mellea, A. heimii and an as yet unnamedbiological species (Mohammed et al., 1994). Inaddition, the suitability of isozyme analysis forroutine analysis of African Armillaria isolates wastested because the presence of homothallic forms ofAfrican Armillaria makes routine identificationbased on pairing tests (as for temperate Armillaria)more difficult due to the absence of the haploid form.

MATERIALS AND METHODS

Source of isolates

The details of geographical origin and host, as well asthe sexual system for the 10 African Armillariaisolates are listed in Table 1. Tw-o isolates (fromdiflerent sides of the African continent) representedA. mellea ssp. africana. The six A. heimii isolatesrepresented geographical and sexual variation in thespecies: Gl (Gabon) and CAl (Cameroon) are WestAfrican, unifactorial and heterothallic, Tl (Tan-zania), M2 (Malawi) and Zl (Zimbabwe) are EastAfrican, homothallic and CI (Congo) is West Africanand homothallic. The remaining two isolates fromthe Highlands of Kenya, KH and K12, wereconsidered as belonging to a biologically distinctgroup of high altitude isolates from this area.Although unnamed, pairing tests have indicated thatthey belong to the Somatic Incompatibility GroupIII, clearly difTerent from A. heimii (group I) and A.mellea (group II) (Mohammed & Guillaumin, 1993).

These isolates, conserved as mycehal stockcultures, were cultivated in the dark on 2'^\, maltagar medium at 24 °C.

Alycelial cord production and enzyme extraction

A number of the isolates studied produced in Petridishes only a limited amount of \ egetative myceliumwhich was rapidly melanized and transformed into abrown crustose mat where most of the enzymeacti\ities were not detectable. Preliminary resultshave shown that enzyme activities were more easilydetected in mycelial cords developed in liquidculture (Mohammed et al., 1989). The materialgrown has been designated in this paper as 'mycelialcords' instead of the more traditional 'subcorticalisrhizomorphs' to highlight the fact that the organs inquestion are poorly differentiated in contrast withthe true cylindrical and melanized rhizomorphsinitiated by Armillaria species in the soil. Mycelialcords were grown from an inoculated segment oforange supported above a column of water or culturemedia, in test tubes, as already described by

Isozymes in African Armillaria 137

Table 1. Isolate designation, species, geographic origin and sexual system of the studied African Armillariaisolates

Isolates

STIK6GlCAlTlM2ZlClKl lK12

origin

Sao ThomeKenyaGabonCameroonTanzaniaMalawiZinnbabvveCongoKenyaKenya

Host plant

Theohroma cacaoTea sinensisManihot sp.unknozcnPinus elliottiiPinus elliottiiBrachystegia utilisManihot sp.Vitex keniensisPinus patula

Type

me I leamelleaheimiiheimiiheimiiheimiiheimiiheimiiUnnamedUnnamed

Longitude-latitude

0M9'N6°43'E0°22'S35M9-E6M5'N lOMl'E6° OO'X 9° 2O'E6° 19'N 10°20'W

15°22'S35°22'EJ8°44'S32M9'E4°30'S i r 3O'EO=23'S37° 12'E0^24'N 35°27'E

Altitude(m)

1000-1500> 2000

0-100< 100

1550-20001000-1500tOOO-1500

0-200> 2000> 2000

Pattern ofsexuality

HomothallicHomothallicHeterothallicHeterothallicHomothallicHomothatlicHomothaJIJcHomothallicUnknownUnknown

Mohammed et al. (1989). Culture media werePachlewski's medium or Bertrand's medium. Com-position of the Pachlewski's medium per litre was asfollows: maltose: 5 g; glucose: 20 g; ammoniumtartrate: 2-3 g; KH^PO,: lOg ; MgSO,.7H,O:0-5 g; thiamine: 10//g; microelements, includingZnSO,,.7H,>O: 0-63 mg; MnSO^.H-.O: l-54mg;CuSOj.5H,0: 0 25 mg; MoXXNH,.4H,0:0-05 mg, Fe, EDTA(III): 344 mg. The pH wasadjusted to 5'5 with NaOH giving a pH of .S-3 afterautoclaving. Composition of the Bertrand's mediumper litre was as follows: sucrose: 45 g; tartaric acid:2-6 g; NH.NO, : 3g; (NH^l.SO,: 0 2 g; KH.TO^:0-6 g; K,,CO,: 04 g; MgSOj.7H,O: 0-8 g;FeSO,.7H",O: 86 mg; ZnSO4.7H,O: 41 mg;MnSO, .H,O: 8-5 mg; CaCl2.2H,O: 14-7 mg. ThepH was adjusted to 54 with NaOH giving a finalpH of 5'6 after sterilization.

Mycelial cords were harvested 3 wk after in-oculation of the orange fragment and washed withdistilled water. They were ground in the presence ofacid washed sand in a pre-chilled mortar with cold50 mM K-phosphate buffer (pH 7-0) containingseveral protectants: lOmM Na glutamate, lOmMMgSOj, 1 mM EDTA. 1 mM PMSF, 14 mM f^-mercaptoethanol, 10 mM dithiothreitol. lO",, gly-cerol, 20% insoluble PVPP and 3 °'o soluble PVP.The ratio of buffer to cord material was 4 or 5 m! g"'fresh w-eight. Samples were centrifuged at 40000 £for 20 min and the resulting supernatant constitutedthe crude extract which was used for enzyme assaysand gel electrophoresis.

Protein contents of the extracts w ere determmedby the method of Bradford (1976), usmg BovineSerum Albumin as a standard.

Electrophoresis

Fnzymes were separated by electrophoresis in ver-tical 6"o polyacrylamide slab gels (7x10 cm) ac-cording to Laemmh (1970) with an electrode buffer(pH 8'3) containing 79 niM Tris and 27 mM glycine.Electrophoresis was performed at a constant voltage

of 200 V and runs were terminated when thebromophenol blue band reached the bottom edge ofthe gels. About 40/Ag of proteins (crude extract)were added to each well of the gels.

Enzyme assays

The seven enzymes, listed with their abbreviationsin Table 2, were analyzed from extracts of 21-d-oldmycelial cords. The dehydrogenase acti\'ities weremeasured spectrometrically by recording, at 340 nmand 30'^C, the disappearance of NADPH (NADP-GDH) or NADH (XAD-GDH, MDH) as well asthe appearance of NADPH (IDH and G6PDH).The assay mixture included the respective cofactorsat a final concentration of 0'2 mM.

Activities of NAD and NADP-GDH were assayedas described by Dell et al. (1989) w ith saturatingconcentrations of ammonium sulphate (128 mM) anda-ketoglutarate (10 mM) in O'l M phosphate buffer,pH 7 0. Activity of MDH was estimated according tothe method of Vidal et al. (1977). The assay mixturecontained lOmM oxaloacetate and 5 mM EDTA in50 mM Tris-HCl buffer, pH 7'6. Activity of IDHwas assayed according to the procedure of Ni,Robertson & Ree\ es (1987) in the presence of 2'5 mMisocitrate in 50 mM Tris-HCl buffer, pH 8-3, con-taining 1 mM MgCl.^. Activity of G6PDH was as-sayed according to Muto & Uritani (1970). Thereaction mixture contained 50 mM glucose 6-phos-phate and 50 mM MgCl., in 50 mM Tris-HCl buffer,pH 7-6. Activity of AAT was estimated according toDell el al. (1989) by using a coupled assay reactioncatalysed by NAD-MDH.

Staining of enzyme activities on gels

On completion of electrophoresis, gels were im-mediately stained in enzyme-specific reactionmixtures containing dyes and catalysts as listed inTable 2. Gels were incubated in the dark at roomtemperature until visualization of the enzyme band-ing patterns. All the enzyme-staining solutionswere composed of 10 ml 0-1 M Tris-HCl buffer.

138 A. Agustian and others

Table 2. Enzyme characteristics and staining methodology

EC.No

Oxidoreductases1.4.1.4

1.4.1.2

1.1.1.371.1.1.44

1 .1.1.421.1.1.49

Transferase2.6.1.1

Name

Glutamate dehvdrogenase(NADP)

Glutamate dehydrogenase(NAD)

Malate dehydrogenase6-phosphog] uconate

dehydrogenaseIsocitrate dehydrogenaseGlucose 6-phosphatedehydrogenase

Aspartate aminotransferase

Symbol

NADP-GDH

NAD-GDH

MDH6PGDH

IDHG6PDH

AAT

Coenzyme

NADP

NAD

NADNADP

NADPNADP

P5P

Substrate

L-glutamic acid

L-glutamic acid

Malic acid6-PhosphogIuconicacid

Isocitric acidGlucose-6-phosphate

L-Aspartic acida-ketoglutaric acid

Dye andcatalyst

NBT, PMS

NBT, PMS

NBT, PMSNBT, PMS

NBT, PMSNBT, PMS

Fast Blue BBsalt or FastViolet Blue

pH 8 (except for IDH, which was at pH 8 8)containing 3 mg NAD or NADP, 8 mg NBT and1-4 mg PMS. For each of the enzymes, the substratesincluded in the staining solutions were as follows;GDH: 56 mg glutamate; MDH: 167 mg malic acid,lOmg MgC'L; 6PGDH: 12 mg 6-phosphogluconicacid, lOmg MgCl^; IDH: 103 mg isocitric acid,lOmgMgCU; G6PDH: 14 mg glucose 6-phosphate,10 mg MgCI>,; AAT: 13 mg aspartate, 7 mg a-ketoglutarate, 4 /d P5P. After incubation of the latterenzyme at room temperature for 30 min, bandingpatterns were revealed under the same conditionswith Fast Blue BB salt or Fast Violet Blue (30 mg10 ml"' distilled water). The relative electrophoreticmobilities (R^) were calculated between the bromo-phenol blue dye front and the visualized enzymeactivity bands. Isozymes, expressed as individualand reproducibly detected bands in the gels, werenumbered sequentially from the most anodal to themost cathodal. Isozyme patterns for each enzymewere divided into band sets which were tentativelyregarded as being determined by a single geneticlocus.

Calculation of similarities

To compare the isolates in term of all seven enzymes,an estimate of similarities was calculated using aDice-type coefficient defined as: S("o) =[2A\B/(iV^-FN,,)]xlOO, where S("o): similarityvalue for each enzyme, A' ^ : number of bands withsame 7?,, A' and N^: number of bands of isolates Aand B, respectively (Sneath & Sokal, 1973). Themean value of the seven S values was presented asthe similarity index (Sm) between isolates A and B.A dendrogram based on calculated similarity valueswas constructed using the unweighted pair groupmean average (UPGMA) cluster analysis (Sneath &Sokal, 1973) to provide further insights into therelationships between isolates.

RESULTSMycelial cord production

Mycelia] cord production of the different Armillariaisolates grown using the 'orange' method wasestimated on a fresh weight basis after 21 d ofincubation. Culture media dependent growth of themycelial cords was clearly apparent (Fig. 1). Ingeneral, mycehal cord production was highest onPachlewski's medium, w'hilst growth was consider-ably reduced in the presence of water and Bertrand'smedium, particularly in the case of isolates CI andZl and STl , T l , K l l and K12, respectively. Thetwo Kenyan isolates, K l l and K12, produced veryfew, fairly slow growing, mycelial cords on allculture media tested, but among the other isolates, itwas not possible to establish any grouping based ontheir mycelial cord production.

Enzyme activities

Enzyme activities of the 21 d-old mycelial cordswere both isolate and culture media dependent(Table 3). Eor six isolates, specific activity of

STl K6 Gl CAl Tl M2 ZlIsolates

CI K l l K12

Figure 1. Rhizomorph production in 3 wk-old Armillariaisolates cultivated on water (D). Bertrand's medium ( • )and Pachlewski's medium ( • ) .

Isozymes in African Armilkria 139

Table 3. Aspartate aminotransfcrase and NAD~malate dehydrogenasespecific activities (nkat mg"' protein) of the Armillaria isolates cultix^ated inthree different culture media*

Enzyme

Aspartateaminf)transf erase

NAD-malatedehydrogenase

Isolates

STlK6GlCAlTIM2ZlClK l lK12

S T lK6G lCAlT l\ I2ZlClKl lKt2

Culture media

Water

23-1 ±4-524'3 + 6-212-4 + 4-117-8 + 3-220-7 + 3-816-4+ 2-421'3 + 4-l18'5±3-537-1+6-837-5 + 8-3

239-2 + 21'6278-b±28-3l^s-^ i iM14'7±3-2

174-6 ±19-6196-1 + 12-795-3 + 174

117'6±2l-5299-0±25-7278'0±31'2

Bertrand

17-9 + 3-222-2 + 2-720-6 ±3'58-8 + 1-6

17-4 + 2-3!8'2±4-l7'2±|-59'3 + 2'64-5+M

15-7 + 2-4tid

ndndndndndndndndnd

Pachlewski

24-9 ±3-937-6 ±4-118-9±2-627±0'4

15'2±3-625-7 ±4-84-7 ±0-9

ll-4±2-37'6±i-4

18'0±3-739!-8±21'5464-5 ± 34-3540-3 ±40-9206'0±1843I9-2±21-4536-7 ±25-2213-9±17-6398-2±21-8610-8±28-9.S91'2±35-7

* Enzynne activities were determined on 21-d-old mycelial cords, nd: notdetermined. N'alues are means of three measurements±st.

aspartate amino transferase was highest when theywere cultivated in water. For three isolates,Pachlewski's medium led to a better activity of theenzyme, whilst in many cases, Bertrand's mediumgave intermediate \-alues. Similar results were toundwith NAD and NADP-GDH, G6PDH and IDH(not shown). Specific activity of MDH in all the 10isolates was high when they were cultivated onPachlewski's medium and low when incubated inwater. Enzyme activities did not allow any cleargrouping of the isolates. As Pachlew-ski's mediuminduced a better grow th of the mycelial cords and ledto a higher activity of certain enzymes, the extractsfrom mycelial cords grown on this medium wereused for gel isozyme analysis.

Isozyme banding patterns

Presentation. A diagrammatic representation of theisozyme banding patterns and their respective i?,values are shown in Figure 2 and the aspect of theAAT isozymes on gel is given in Figure 3.

Glutamate dehydrogenases. NADP-GDH isozymeactivity was expressed as a single band (Rf 0-43) in al!the isolates mvestigated. NAD-GDH was alsoexpressed as a single band in alt isolates with Rfvalues of 0-46, 0-52 and 0-57. The former band w-ascommon to TI and M2 and the latter to STl and K6{R, 0-57), whilst the intermediate band was shared bythe other six isolates.

Malate dehydrogenase. Isolates T l and M2displayed a single common band witb a R, value of0-83, whilst an additional band at R, 0-52 and anotherat i?, 0-69 were respectively found in the groups Zl,Cl and K l l , K12. The isolates Gl and CAl werealso closely related, displaying a set of three commonbands. STl and K6 were slightly different as theband at R^ 0-83 was not found in the latter isolate.

6-Phosphogluconate dehydrogenase. This enzyme wasexpressed as a single band, at a R, value of 0'54 withthe isolates K6 and CAl, and at a i?, value of 0-50with all the other isolates,

Isocitric dehydrogenase. IDH isozyme activity wasexpressed as three common closely migrating bandsin SIX isolates, whilst a smgle band was found in theisolates CAJ and CJ (R^ OS), as well as in the isolatesKl l and K12 {R, 0-45).

Glucose 6-phosphate dehydrogenase. Tbe isolates Gl ,T l and M2, as well as Cl, and K12 exhibited asingle common band {R^ 0-47) and an additionalband (R^ 0-51) was detected in STl , K6, CAl andZl. The isolates Kl l exhibited a single band at R,0-49. With the exception of groups STl , K6 and T l ,M2, this enzyme seems to be expressed as variablebands.

Aspartate aminotransferase. The 10 isolates allshowed a single banding pattern for AAT which

140 A. Agustian and others

STl K6 Gl CAT Tl IVI2 Zl CI KIT K12 STl K6 Gl CAl Tl M2 Zl Cl KIT K12

Figure 2. Diagrammatic representation of isozyme banding patterns of (a) NADP-GDH, (b) NAD-tiDH, (c)MDH, (d) 6PGDH, (c) IDH, (/) G6PDH and (g) AAT m rhizomorph extracts of the Armitlaria isolates.

CAl Kl l K12 ZlI I I I

Cl

Figure 3. Uniform (6 '\,) polyacrylamide gelelectropboresis of AAT extracted from the Armillariaisolates. Each well was loaded with about 40/^g of proteins(crude extract).

separated the isolates into four fairly well definedgroups (Figs 2, 3). The A. mellea isolates (STl , K6)with a Rj value of 0-42, and the Kenyan isolates(Kl l , K12) with a R, value of 0-37, were clearlydistinguishable from the others. The groupcomposed of Gl and CAl {R^ 0-48) was very closelyrelated to the four other isolates, these latterexhibiting similar AAT banding pattern at a R^ valueof 0-47.

Cluster analysis

Cluster analysis, based on similarity indices of theseven isozyme banding data, was used to summarizethe variations between the isolates and is presented

Isozymes in African Armillaria 141

40Similarity index (%)

58.3

60

6S

81.0

73-9

9

64-9

—r80

RO 1

- • S T I

• K6

D Gl

D CAT

D Tl

a 21

00. i

• Cl

85.7

0 K12

1too

A. mellea sspafricana

A. heimiiheterothallic

A. heimiihomothallic

Unnamedspecies

Figure 4. Dendrogram derived from a matrix of isozyme similarity values following UPGMA cluster analysisol the 10 African Armillaria isolates. ( • , A. mcllea; Q, A. lieimii\ [r]], unnamed species). Isolate designationsare those given in Table 1.

in the form of a dendrogram (Fig. 4). The 10 isolateswere c\ear\y separated into fi-ve groups, each con-taining two isolates, with the Armillaria mellea ssp.africana group (STI, K6) showing the mostpronounced dissimiJarity with the otber isolategroupings. The isolates belonging to the SomaticIncompatibility Group 111 (Kl 1 and K12) also forma distinct cluster which is only loosely related to theother isolates, and w-ithin the A. heimii isolates agradual decrease in homology is apparent. Thehomothallic isolate M2 showed lOO",, similaritywith another homothallic isolate, T l . Thehomoiogies between the remaining isolates weresmaJler, with similarities of 73-9",, between the twoheterothallic west African isolates, Gl and CAl, and88-1 ",) between two homothallic isolates fromdifferent sides of the continent, Cl (Congo) and Zl(Zimbabwe). It is to be noted that the twohomothallic isolates from Tanzania and Malawi, hada greater similarity (77-4"„) with the two otherhomothallic isolates from the Congo and Zimbabwe,than with the two heterothallic isolates Gl and CAl(69-9' 'o similarity). The seven enzymes resolved inthe present study may be interpreted as being codedby at least seven putative loci for all the isolates witha total of 17 alleles. The total number of alleles in A.mellea ssp. africana, A. heimii, and the group K l land Kl2 are 12, 15 and 9 respectively. Consequently,the a\'erage number of alleles per locus for eachspecies is 1-71 ± 0 76, 214±0-69and 1 28 + 0-49 i n ^ .mellea ssp. africana, A. heimii, and the two isolates

from the Highlands of Kenya, respectively. Thelargest variability is found in the heimii species,although a larger number of isolates should beanalyzed in each group before any definite conclusioncan be drawn.

DISCUSSION

In the Armillaria isolates tested, production ofmycelial cords, displaying the highest enzyme ac-tivities, was most pronounced on Pachlewski'smedium. It is likely that glucose supplied in thismedium was the most suitable carbohydrate forpromoting rhizomorph growth in Armillaria. Pres-ence of thiamine in the Pachlewski's medium couldalso be responsible for greater mycelial cord de-\elopment. In contrast with the other isolates,de\*elopment of mycelial cords at 24 °C was limitedin the Kenyan group composed of K l l and K12,which appeared, thus clearly distinguishable fromthe other isolates. Tbis is in agreement with thepre\'ailing climatic conditions, as the Armillariagroup of the Highlands of Kenya, including Kl 1 andK12, is adapted to fairly low temperatures, withoptimal growth in vitro at 20 °C (Mohammed et al.,1994).

The enzyme activity levels did not seem to reflectthe genetic variability of the isolates. However,isozyme banding patterns are relati\'ely more in-dependent of any variations in cultural conditionsand thus provide more reliable taxonomic infor-

142 A. Agustian and others

mation for grouping of the fungal isolates. Indeedresults with this method separated the ten isolatesinto three major clusters specific for A. mellea,heimii, and the group including Kl l and K12.

In this study, the mellea group is represented bytwo isolates from Sao Thomtf and Kenya.Mohammed & Guillaumin (1989) have found that indiploid/dipioid confrontations, the isolates STl andK6 are intercompatible but incompatible with iso-lates of the other groups. Additional data based onhost specialization, partial compatibility with Euro-pean and American A. mellea haploid testers,similar cultural characteristics, protein and esteraseanalyses, suggest that STl and K6 are very similar toEuropean A. mellea (Mohammed & GuiUaumin,1993). Howe\'er, RAPD analysis carried out byMohammed (1994) has indicated that this group issomewhat different from the temperate speciesA. mellea. Moreover, this group of African isolates ishomothallic whereas the temperate A. mellea isheterothallic. It has been proposed that the tem-perate and African taxa should be reconsidered asthe respective subspecies; A. mellea ssp. mellea andA. mellea ssp. africana (Kile et al., 1994).

l^he isolates belonging to the Somatic Incom-patibility Group III, represented by K l l and K12,both coming from high altitude locations, also appearas very different from the other isolates. In somaticpairings, the isolates of this group show incom-patibility reactions (w-ith black lines) with the otherisolates and may represent a group sufficientlydifferent to be considered as a new species. Aspreviously mentioned, in comparison with isolatesfrom the other groups, they are more sensitive, invitro, to elevated temperatures (24 °C and above).They also differ from both A. mellea ssp. africanaand A. heimii in a number of morphologicalcharacteristics (Mohammed et aL. 1994). In thepresent study, the isolates of this group differed fromthe others both in the development of cords at 24 °Cand in their isozyme patterns.

All the six other isolates had been identified asbelonging to the large species A. heimii on the basisof morphological characteristics, vegetative com-patibility and especially from the laboratorysynthesized fruit bodies which display a typical A.heimii morphology. Analysis of the resulting singlespore derived haploid cultures have shown that Gland CAl are heterothallic with a unifactorial matingsystem, while T l , M2, Zl and Cl are homothaliic.Slight differences have also been observed betweenthe homothallic and heterothallic isolates of . heimii(culture mat morphology, growth at differenttemperatures). The present study separates theisolates of A. heimii in accordance with their sexualsystem; indeed, the dendrogram of this heimii groupis divided into three subclusters, each of two isolates,with two homothallic isolates (Tl and M2) moreclosely related to the homothallic isolates (Cl and

Zl) than to the other heterothallic isolates (Gl andCAl). The isolates T l and M2 originating fromfairly high altitudes in Tanzania and Malawi wereboth isolated from Pinus elliottii and in this studyalso show identical isozyme phenotypes. Tbe twounifactorial isolates CAl and GI from neighbouringgeographical locations and at elevations below100 m, are also logically regrouped. Tbe similaritybetween Zl and Cl, isolates which originate fromwidely separated geographical areas (Zimbabwe andthe Congo) was less expected. However, these twoisolates both grow at high temperatures above 25 °C(Mohammed et aL, 1994).

This study demonstrates that isozyme analysis is auseful supplement to other methods for phylogeniccharacterizations of isolates. The polymorphismamong enzyme loci is somehow related to metabolicfunction. As has been established from the numerousstudies of plant isoz\'mes, the constitutivelyexpressed isozymes (those that are invoked in theprimary metabolism and consequently vitalprocesses such as glycolysis, pentose phosphatepathway, Krebs cycle and amino acid production)are preferentially used as gene markers (Triest,1992). Indeed, contrary to enzymes of broadspecificity which utilize substrates originating fromthe external environment (phosphatases,peroxidases) and are coded by a large number ofgenes, the enzymes investigated here utilize specificmetabolically produced substrates and are generallyconsidered as not modified by environmentalvariations. However, evaluation of the phylogeneticrelationships betw^een the three groups constitutedby A. mellea, A. heimii -and the isolates Kl 1 and K12,will probably require additional methods based onvariation in the genomic DNA, such as restrictionfragment patterns and direct sequencing of highlypolymorphic regions of DNA, as already carried outwith North American species of Armillaria(Anderson, Bailey & Pukkila, 1989; Anderson &Stasovski, 1992). Nevertheless, isozyme analysis isstill very useful m clarifymg taxonomic problems atthe interspecific or intraspecific levels and fordeveloping a routine identification test for AfricanArmillaria spp.

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