SUMMARY

Phellinus torulosus (Pers.) Bourd. et Galz. is thecausal agent of white rot that infects especially the rootsand the collar of old trees and shrubs of many species.The random amplified polymorphic DNA (RAPD)technique using polymerase chain reaction (PCR) wasused to explore the genetic variability in 138 P. torulosusisolates from 139 oak woods of Apulia and Basilicata.The use of 16 random primers yielded 150 polymorphicfragments. The dendrograms generated by analysingRAPD data did not show correlations between the clus-ters and other characters such as morphotypes, hostspecies, and geographic origin.

Key words: Phellinus torulosus, white rot, geneticvariability, RAPD-PCR.

INTRODUCTION

Species of Phellinus are lignicolus Hymenomycetesbelonging to the order Hymenochaetales and causewhite rot (Boidin, 1971; Oberwinkler, 1977; Fiassonand Niemelä, 1984). Phellinus torulosus (Pers.) Bourd.et Galz. usually decays heartwood in roots and lowerstems (Gilbertson and Burdsall, 1972; Panconesi et al.,1994). Basidiomes are always found on the lower part ofthe tree trunk, roots and stumps (Bernicchia, 1990). P.torulosus infects a very large range of hosts-more than160 species of plants. In Europe, P. torulosus occursboth as a parasite and saprobe on a wide variety ofbroad leaf trees (Kotlaba, 1975; Isikov and Kuznetsov,1990); its occurrence on conifers is rare. In the UnitedStates, however, P. torulosus seems to be restricted ex-clusively to conifers. In its range of diffusion it is limitedto areas with higher temperatures.

P. torulosus is heterothallic unifactorial with two alle-les at a single mating type, and it is assumed that sexual

Corresponding author: N. LuisiFax: +39.080.5442906E-mail: luisin@agr.uniba.it

reproduction may be important in maintaining and dis-seminating variation within the species (Fischer, 1996).

Two partially intersterile groups of P. torulosus aredocumented by pairing tests and ontogeny of secondarymycelia (Fischer and Bresinsky, 1992). One group con-sists of collections from Europe, whereas the collectionsof the second group were obtained from the Canary Is-lands. No significant differences between the groupswere noted in any cytological and microscopic characterexamined. Two types of mycelia were observed: bleach-type (b-type) and staining type (S-type). The b-type wascharacterised by well-developed aerial hyphae, rapidgrowth (approximately 2-2.5 cm/wk), and weak pig-mentation of the medium. The S-type was characterisedby appressed hyphae with sparse aerial development,slow growth (approximately 0.8-1.1 cm/wk), and deepreddish brown pigmentation of the medium. In a surveyof the genus Phellinus, Larsen and Cobb-Poulle (1990)mentioned 8 “formae” of P. torulosus.

Little is known about the genetic relationshipsamong P. torulosus isolates from different host species,geographic origin, and morphotypes, and it is possiblethat different pathogen genotypes may have evolved toselectively parasitize different hosts. Answering evolu-tionary questions on host-pathogen interactions re-quires studies on the genetics of this pathogen (Mil-groom, 1997; Carter et al., 2000; Harvey et al., 2000).Molecular markers are useful in investigating the genet-ic variation and biology of fungi (Michelmore and Hu-ber, 1987). Restriction fragment length polymorphisms(RFLP) (Appel and Gordon, 1995; Parry et al., 1995;Okoli et al., 1999) of the intergenic spacer (IGS) regionof rDNA and the random amplified polymorphic DNAtechnique (RAPD) were used to examine the existenceof variation within and between fungal species or popu-lations (Welsh and McClelland, 1990; Williams et al.,1990; Smith et al., 1995; Karjalainen, 1996; Koike et al.,1996; Goggioli et al., 1998; Cherrab et al., 2000; Harveyet al., 2000; Santini and Capretti, 2000).

The objective of this study was to establish the de-gree of genetic variability of P. torulosus populations inSouthern Italy through the study of RAPD markers todetermine the polymorphisms associated with all iso-lates studied. Moreover, this study aimed to correlate

Journal of Plant Pathology (2004), 86 (2), 105-115 Edizioni ETS Pisa, 2004 105

GENETIC AND MORPHOLOGIC VARIABILITY OF PHELLINUS TORULOSUS ISOLATESIN SOME OAK WOODS OF SOUTHERN ITALY

G. Campanile, S.L. Giove and N. Luisi

Dipartimento di Biologia e Patologia vegetale, Università degli Studi,Via G. Amendola 165/A, 70126 Bari, Italy

the profiles of genetic variability with the morphologicalcharacteristics of the isolates, host species, and geo-graphic origins.

MATERIALS AND METHODS

Fungal isolates. The isolates were collected in au-tumn of 2000 from 9 oak woods distributed over 4 ar-eas: Northwestern Murge (NWM), Southeastern Murge(SEM), Salento in Apulia, and Monte Vulture in Basili-cata (Fig. 1, Table 1). In each of the woods 2 causal andorthogonal directrices were found, along each of whichabout 150 trees or stumps were analysed. Basidiomeswere sampled at a distance of 50 m.

P. torulosus was isolated from basidiomes by placing

small pieces on a selective medium for Basidiomycetes(Kuhlman and Hendrix, 1962). After 6 days of incuba-tion in diffuse light at 22±3°C, hyphal tips were trans-ferred on 2% malt extract agar (MEA). This procedurewas repeated as often as necessary to obtain pure iso-lates. Isolates were identified as P. torulosus in accor-dance with key of Stalpers (1978). For morphologicalstudies Petri dishes containing MEA were inoculatedwith a piece of mycelium. Plates were incubated in dif-fuse light for 6 days at 22±3°C and examined at 5-daysintervals. The isolates were scored for type and develop-ment of aerial mycelium, diameter growth, and colourof colony.

DNA isolation. Fungal colonies were grown on cel-lophane sheets placed on MEA for 4 days in diffuselight at 22±3°C. Mycelium was collected, transferred to

Eppendorf tubes, and ground under liquid nitrogen.DNA extraction and purification was carried out ac-cording to the method of Murray and Thompson(1980), as modified by Rogers et al. (1989) and Kim etal. (1990). Briefly, mycelium (200-300 mg fresh weight)was suspended in 600 µl of cold CTAB buffer (100 mMTris-HCl, pH 8.0; 1.4 M NaCl; 20 mM EDTA, pH 8.0;2% cetyldimethylethylammonium bromide; 0.2% b-mercaptoethanol). The mixture was transferred three-fold to liquid nitrogen, then to warm water (75°C) andafterwards maintained at 75°C for 1 hour. After extrac-tion with 600 µl chloroform, nucleic acids were precipi-tated with isopropanol (at –20°C for 2 h) and recoveredby centrifugation. The pellet, washed with 70% ethanol,was dissolved in 200 µl TE (10 mM Tris-HCl, 1 mMEDTA, pH 8.0). The solution was amended with 0.1 mgml–1 DNAase-free pancreatic RNAase and kept for 2 hat 37°C. DNA was precipitated with 2 vol. absoluteethanol in the presence of 0.6 vol. 5 M ammonium ac-etate, recovered by centrifugation, and dissolved in wa-ter at a final concentration of 50 ng µl-1. The concentra-tion and purity of the extracted DNA were determinedby spectrophotometric measurement.

RAPD analysis. To assess genotypic diversity be-tween isolates of P. torulosus, RAPD profiles were pro-duced for all isolates. Arbitrary random amplification ofDNA sequences was performed with a set of 20 primersobtained from Operon Technologies (Alameda, CA,USA) (Table 2). RAPD-PCR reactions were performedin a total volume of 25 µl containing 10 mM Tris-HCl,pH 9.0; 50 mM KCl; 0.1% Triton X-100; 2 mM MgCl2;75 µM each of dATP, dGTP, dCTP, dTTP (Promega,Madison, WI, USA); 0.5 µM primer (10-mer, OperonTechnologies, Alameda, CA, USA); 50 ng of genomicDNA and 1.5 units of Taq-polymerase (Promega, Madi-son, WI, USA). Reactions were carried out in a thermalcycler (Gene Amp PCR System 9700; Perkin–Elmer,Norwalk, USA) programmed as follows: 5 minutes at94°C; 40 cycles of 30 sec at 94°C, 30 sec at 35°C, 30 secat 72°C; a conclusive extension phase of 7 minutes at72°C. DNA amplification products were separated on1.5% agarose gel (Bio-Rad Laboratories, Hercules, CA,USA). The molecular size of the amplification productswas estimated using the 100 bp DNA Ladder (NewEngland Bio-Labs, Beverly, USA). Fragments were visu-alized under 300 nm of UV light using the Gel Doc2000 system (Bio-Rad Laboratories, Hercules, CA,USA) after staining with ethidium bromide, pho-tographed, and scored for the presence or absence ofbands. The most intense bands were scored with the as-sumption of positional homology. Only fragments thatwere reproducible in at least 2 replicate PCR reactionsand reproducible using different DNA extracts fromthe same isolates were included for further analysis.

106 Variation in Phellinus torulosus Journal of Plant Pathology (2004), 86 (2), 105-115

Fig. 1. Map of Apulia and Basilicata regions showing the ar-eas (NWM, SEM, Salento and Monte Vulture) where basi-diomes of Phellinus torulosus were sampled.

1. Atella2. Andria3. Corato4. Ruvo di Puglia5. Mottola6. Martina Franca7. Tuturano8. Maglie9. Scorrano

Journal of Plant Pathology (2004), 86 (2), 105-115 Campanile et al. 107

Table 1. Isolates of Phellinus torulosus (Pt) and Fomitiporia punctata (FP) used in this study.

Isolate Geographic Origin Location Host Morphotype Isolating Year

Pt1 SEM Martina Franca Crataegus monogyna Jacq. G September 2000

Pt2 SEM Martina Franca Quercus coccifera L. C September 2000

Pt3 SEM Martina Franca Crataegus monogyna Jacq. F September 2000

Pt4 SEM Martina Franca Crataegus monogyna Jacq. F September 2000

Pt5 SEM Martina Franca Crataegus monogyna Jacq. F September 2000

Pt6 SEM Martina Franca Quercus trojana L. G September 2000

Pt7 SEM Martina Franca Crataegus monogyna Jacq. B September 2000

Pt8 SEM Martina Franca Quercus trojana L. B September 2000

Pt9 SEM Martina Franca Crataegus monogyna Jacq. B September 2000

Pt10 SEM Mottola Quercus coccifera L. B September 2000

Pt11 SEM Mottola Crataegus monogyna Jacq. D September 2000

Pt12 SEM Mottola Quercus trojana L. I October 2000

Pt13 SEM Mottola Quercus cocciferaL. F October 2000

Pt14 SEM Mottola Quercus coccifera L. H October 2000

Pt15 SEM Mottola Crataegus monogyna Jacq. B October 2000

Pt16 SEM Mottola Quercus trojana L. B October 2000

Pt17 SEM Mottola Quercus coccifera L. B October 2000

Pt18 SEM Mottola Quercus coccifera L. F October 2000

Pt19 SEM Martina Franca Quercus trojana L. H October 2000

Pt20 SEM Martina Franca Quercus coccifera L. H October 2000

Pt21 SEM Martina Franca Quercus coccifera L. H October 2000

Pt22 SEM Martina Franca Quercus trojana L. B October 2000

Pt23 M. Vulture Atella Pistacia terebintus L. B October 2000

Pt24 SEM Martina Franca Quercus trojana L. B November 2000

Pt25 SEM Martina Franca Quercus trojana L. I November 2000

Pt26 SEM Mottola Crataegus monogyna Jacq. G November 2000

Pt27 SEM Martina Franca Quercus trojana L. B November 2000

Pt28 SEM Martina Franca Quercus trojana L. B November 2000

Pt29 NWM Corato Crataegus monogyna Jacq. B November 2000

Pt30 NWM Corato Crataegus monogyna Jacq. B November 2000

Pt31 NWM Corato stump of Quercus sp. D November 2000

Pt32 SEM Mottola Quercus trojana L. C November 2000

Pt33 M. Vulture Atella Pistacia terebintus L. D November 2000

Pt34 NWM Andria Crataegus monogyna Jacq. G November 2000

Pt35 NWM Andria Pinus halepensis L. E November 2000

Pt36 NWM Andria Pinus halepensis L. E Novembre 2000

Pt37 NWM Andria Crataegus monogyna Jacq. I Novembre 2000

Pt38 SEM Mottola Quercus trojana L. G November 2000

Pt39 SEM Mottola Quercus trojana L. G November 2000

Pt40 SEM Mottola Quercus trojana L. C November 2000

Pt41 M. Vulture Atella Crataegus monogyna Jacq. I November 2000

Pt42 SEM Martina Franca Quercus trojana L. C November 2000

Pt43 SEM Mottola Quercus trojana L. C November 2000

Pt44 M. Vulture Atella Cornus mas L. C November 2000

Pt46 M. Vulture Atella Crataegus monogyna Jacq. F November 2000

Pt47 M. Vulture Atella Cornus mas L. D November 2000

Pt48 M. Vulture Atella Crataegus monogyna Jacq. I November 2000

108 Variation in Phellinus torulosus Journal of Plant Pathology (2004), 86 (2), 105-115

Isolate Geographic Origin Location Host Morphotype Isolating Year

Pt1 SEM Martina Franca Crataegus monogyna Jacq. G September 2000

Pt2 SEM Martina Franca Quercus coccifera L C September 2000

Pt49 SEM Martina Franca Quercus trojana L. I September 2000

Pt50 NWM Ruvo di Puglia stump of Quercus sp. B September 2000

Pt51 NWM Ruvo di Puglia stump of Quercus sp. B September 2000

Pt52 NWM Ruvo di Puglia stump of Quercus sp. E September 2000

Pt53 NWM Ruvo di Puglia stump of Quercus sp. A September 2000

Pt54 NWM Ruvo di Puglia Quercus pubescens Willd. A September 2000

Pt55 NWM Corato stump of Quercus sp. F September 2000

Pt56 NWM Corato Pistacia lentiscus L. I September 2000

Pt57 Salento Tuturano Quercus ilex L. B September 2000

Pt58 NWM Andria Cupressus sempervirens L. E September 2000

Pt59 NWM Andria Crataegus monogyna Jacq. C September 2000

Pt60 NWM Ruvo di Puglia Cupressus sempervirens L. A September 2000

Pt61 NWM Ruvo di Puglia Crataegus monogyna Jacq. H October 2000

Pt62 NWM Ruvo di Puglia Cupressus sempervirens L. H October 2000

Pt63 NWM Ruvo di Puglia Cupressus sempervirens L. D October 2000

Pt64 NWM Ruvo di Puglia Cupressus sempervirens L. D October 2000

Pt65 NWM Corato Crataegus monogyna Jacq. D October 2000

Pt66 NWM Corato Crataegus monogyna Jacq. D October 2000

Pt67 NWM Corato Cupressus sempervirens L. I October 2000

Pt68 NWM Andria Cupressus sempervirens L. I October 2000

Pt69 NWM Andria Quercus pubescens Willd. B October 2000

Pt70 NWM Corato Cupressus sempervirens L. F October 2000

Pt71 NWM Ruvo di Puglia Crataegus monogyna Jacq. C October 2000

Pt72 NWM Corato Quercus pubescens Willd. E December 2000

Pt73 NWM Corato Cupressus sempervirens L. C December 2000

Pt74 NWM Corato Quercus pubescens Willd. G December 2000

Pt75 NWM Corato Quercus pubescens Willd. B December 2000

Pt76 NWM Corato Quercus pubescens Willd. B December 2000

Pt77 NWM Corato Quercus pubescens Willd. I December 2000

Pt78 NWM Corato Quercus pubescens Willd. I December 2000

Pt80 NWM Andria Crataegus monogyna Jacq. D December 2000

Pt81 NWM Andria Quercus pubescens Willd. D December 2000

Pt82 NWM Andria Quercus pubescens Willd. H December 2000

Pt83 NWM Andria Quercus pubescens Willd. C December 2000

Pt84 NWM Andria Quercus pubescens Willd. C December 2000

FP85 NWM Andria Vitis vinifera L. - December 2000

Pt86 NWM Ruvo di Puglia Arbutus unedo L. B December 2000

Pt87 NWM Ruvo di Puglia Quercus pubescens Willd. B October 2000

Pt88 NWM Ruvo di Puglia Quercus pubescens Willd. E October 2000

Pt89 NWM Ruvo di Puglia Pistacia lentiscus L. D October 2000

Pt90 NWM Ruvo di Puglia Quercus pubescens Willd. H October 2000

Pt91 NWM Ruvo di Puglia Arbutus unedo L. A October 2000

Pt92 Salento Maglie Quercus ilex L. B October 2000

Pt93 NWM Ruvo di Puglia Crataegus monogyna Jacq. D October 2000

Pt94 Salento Maglie Quercus ilex L. D October 2000

Pt95 Salento Maglie Quercus ilex .L. D October 2000

Pt96 NWM Ruvo di Puglia Arbutus unedo L. B October 2000

Pt97 NWM Corato Arbutus unedo L. B October 2000

Journal of Plant Pathology (2004), 86 (2), 105-115 Campanile et al. 109

Isolate Geographic Origin Location Host Morphotype Isolating Year

Pt1 SEM Martina Franca Crataegus monogyna Jacq. G September 2000

Pt2 SEM Martina Franca Quercus coccifera L C September 2000

Pt98 Salento Maglie Quercus ilex L. A October 2000

Pt99 Salento Maglie Quercus ilex L. B December 2000

Pt100 Salento Tuturano Quercus ilex L. B December 2000

Pt101 Salento Tuturano Quercus ilex L. E December 2000

Pt102 Salento Tuturano Quercus ilex L. A December 2000

Pt103 Salento Scorrano Arbutus unedo L. A December 2000

Pt104 NWM Ruvo di Puglia stump of Quercus sp. B December 2000

FP105 NWM Ruvo di Puglia Vitis vinifera L. - December 2000

FP106 NWM Corato Vitis vinifera L. - December 2000

FP107 NWM Andria Vitis vinifera L. - December 2000

Pt108 Salento Scorrano Quercus ilex L. E December 2000

Pt109 NWM Andria stump of Quercus sp. A December 2000

Pt110 NWM Corato stump of Quercus sp. A October 2000

Pt111 NWM Andria Arbutus unedo L. A October 2000

Pt112 NWM Corato Pinus halepensis L. G October 2000

Pt113 NWM Andria Arbutus unedo L. B October 2000

Pt114 NWM Andria Arbutus unedo L. C October 2000

Pt115 Salento Tuturano Arbutus unedo L. H October 2000

Pt116 NWM Corato Pinus halepensis L. G December 2000

Pt117 Salento Maglie Viburnum tinus L. A December 2000

Pt118 Salento Maglie Arbutus unedo L. D December 2000

Pt119 Salento Maglie Viburnum tinus L. B December 2000

Pt120 Salento Scorrano Viburnum tinus L. E December 2000

Pt121 Salento Scorrano Viburnum tinus L. F December 2000

Pt122 NWM Andria stump of Quercus sp. F November 2000

Pt123 Salento Tuturano Viburnum tinus L. F November 2000

Pt124 NWM Ruvo di Puglia stump of Quercus sp. D November 2000

Pt125 NWM Ruvo di Puglia stump of Quercus sp. E November 2000

Pt126 NWM Ruvo di Puglia stump of Quercus sp. F December 2000

Pt127 NWM Ruvo di Puglia Pinus halepensis L. F December 2000

Pt128 NWM Corato Pinus halepensis L. B December 2000

Pt129 Salento Tuturano Viburnum tinus L. B December 2000

Pt130 NWM Andria Pinus halepensis L. F December 2000

Pt131 M. Vulture Atella stump of Quercus sp. F December 2000

Pt132 NWM Corato Pinus halepensis L. B December 2000

Pt133 NWM Corato Pinus halepensis L. F December 2000

Pt134 M. Vulture Atella stump of Quercus sp. D November 2000

Pt135 Salento Tuturano Arbutus unedo L. I November 2000

Pt136 NWM Ruvo di Puglia Pinus lentiscus L. C November 2000

Pt137 NWM Ruvo di Puglia stump of Quercus sp. F November 2000

Pt138 NWM Ruvo di Puglia Pinus lentiscus L. E November 2000

Pt139 NWM Ruvo di Puglia Pinus lentiscus L. F November 2000

Pt140 Salento Tuturano Viburnum tinus L. E September 2000

Pt141 NWM Andria Pinus halepensis L. F September 2000

Pt142 M. Vulture Atella stump of Quercus sp. G September 2000

Pt143 NWM Corato Pinus halepensis L. I September 2000

Pt144 SEM Martina Franca Crataegus monogyna Jacq. I September 2000

Analysis of genotypic diversity. Manipulation of gelimages and elaboration of data were carried out usingthe software package Diversity DatabaseTM (Ver. 2.1 forWindows; Bio-Rad Laboratories, Hercules, CA, USA).The banding pattern of each isolate was scored for thepresence or absence of each marker. To provide a quan-titative measure of relatedness among isolates, geneticsimilarity between all pairs of isolates was estimated ac-cording to the following formula: 2bij/(bij+bi+bj), wherebij is the number of amplicons shared by 2 isolates i andj, and bi and bj are the numbers of unshared bands(Dice, 1945; Nei and Li, 1979). Similarity matrices werethen used to construct a phylogenetic tree according tothe Unweighted Pair Group Method using ArithmeticAverages (UPGMA).

RESULTS

The diploid colonies of P. torulosus showed pheno-typic variability in diameter growth, colour, type of aeri-al mycelium (fluffy, cottony or powdery) and its devel-opment (moderate, abundant or very abundant), whichallow the identification of 9 morphotypes designated asfollows: A, B, C, D, E, F, G, H and I (Fig. 2; Tables 1and 3).

Of the 20 primers screened, 16 produced polymor-phic profiles useful for classification of the fungal iso-lates and were selected for further analysis (Table 2).Each of the selected primers resulted in different RAPDpatterns; representative banding patterns from 3 differ-ent primers (OPA-03, OPA-13, OPA-18) are illustrated

110 Variation in Phellinus torulosus Journal of Plant Pathology (2004), 86 (2), 105-115

Fig. 2. Morphotypes identified among colonies of Phellinus torulosus (Pt).

A B C

D E F

G H I

Pt 7 Pt 59 Pt 90

Pt 125 Pt 137 Pt 142

Pt 134 Pt 115 Pt 77

in Fig. 3. The number of polymorphic fragments pro-duced from amplification of each of the genomic DNAranged from 5 to 12, with molecular weights rangingfrom approximately 200 to 1,400 base pairs (Table 3).In total, 150 polymorphic fragments were obtained andused to determine genetic relationships among P. torulo-

sus isolates. Similarity coefficients (mean 60.8%) werecalculated from the RAPD banding pattern and used toconstruct 2 distinct dendrograms: I and II, according tothe UPGMA method.

Dendrogram I (Fig. 4) includes isolates from treespecies. The isolates were grouped into 2 main clusters:A and B. Cluster A includes 4 isolates of Fomitiporiapunctata (Fr.) Murrill (outgroup), whereas cluster B con-tains P. torulosus isolates subdivided into 5 subclusters:B1, B2, B3, B4 and B5. Subcluster B1 includes isolatesfound on Pinus halepensis and Quercus spp. from NWMand M. Vulture, respectively. Subcluster B2 includes iso-

Journal of Plant Pathology (2004), 86 (2), 105-115 Campanile et al. 111

Table 2. Sequence of 20 primers and number of polymorphicfragments obtained with each primer in random amplifiedpolymorphic DNA analysis.

Primer Sequence (5' to 3') N. polymorphicfragments

OPA01 CAGGCCCTTC 10

OPA02 TGCCGAGCTG 7OPA03 AGTCAGCCAC 11OPA04 AATCGGGCTG 10OPA05 AGGGGTCTTG 6OPA06 GGTCCCTGAC 6OPA07 GAAACGGGTG 10

OPA08 GTGACGTAGG 12OPA09 GGGTAACGCC 12OPA10 GTGATCGCAG 10OPA11 CAATCGCCGT 5OPA12 TCGGCGATAG 8OPA13 CAGCACCCAC 10

OPA14 TCTGTGCTGG 0OPA15 TTCCGAACCC 4OPA16 AGCCAGCGAA 15OPA17 GACCGCTTGT 0OPA18 AGGTGACCGT 14OPA19 CAAACGTCGG 0

OPA20 GTTGCGATCC 0

Totale 150

Table 3. Characteristics of morphotypes identified within Phellinus torulosus populations in Southern Italy.

Arial Mycelium Morphotype

Type Development Colour of Colony

A Fluffy Moderate Dark brown

B Fluffy Abundant Creamy

C Fluffy Very Abundant Dark brown

D Cottony Moderate Dark brown

E Cottony Very Abundant Creamy

F Cottony Abundant Creamy

G Cottony Abundant Dark brown

H Cottony and Fluffy Abundant Creamy

I Powdery Abundant Dark brown

Fig. 3. Amplification products generated from Phellinus toru-losus isolates with primers OPA-03, OPA-13 and OPA-18.The first lane on the left and the last lane on the right containa DNA marker (100 bp DNA Ladder, New England Bio-Labs, Beverly, USA).

112 Variation in Phellinus torulosus Journal of Plant Pathology (2004), 86 (2), 105-115

Fig. 5. Dendrogram II obtained using the UPGMA method containing Phellinus torulosus isolates from shrub species. The iso-lates were grouped into 2 main clusters: C and D, subdivided into 5 subclusters: C1, C2 and D1, D2, D3 respectively.

Fig. 4. Dendrogram I obtained using the UPGMA method. The isolates were grouped into 2 main clusters: A and B; the first con-taining 4 isolates of Fomitiporia punctata (Outgroup) and the second containing Phellinus torulosus isolates from tree species.Cluster B was subdivided into 5 subclusters: B1, B2, B3, B4 and B5.

lates from SEM and NWM and was found on Q. trojanaand Quercus spp., respectively. Subcluster B3 containsisolates found on Q. coccifera and Q. trojana from theSEM area. The isolates found on Q. ilex from the Salen-to area were included in cluster B4. Samples found on Q.pubescens, Quercus spp., and Cupressus sempervirensoriginating from NWM were grouped in cluster B5.

Dendrogram II (Fig. 5) includes isolates from shrubspecies. The isolates were classified into two main clus-ters: C and D. Clusters C and D were subdivided into 5subclusters: C1, C2 and D1, D2, D3, respectively. Isolatesfound on Cornus mas and Pistacia terebintus from M.Vulture were included in subcluster C2. Samples foundon Crataegus monogyna from SEM, NWM, and M. Vul-ture were grouped into different subclusters, C1, C2,and D1, whereas isolates found on Pistacia lentiscusfrom the NWM area were included in subcluster D2.Subcluster D3 includes isolates from NWM and Salentothat were found on Arbutus unedo and Viburnus tinus,respectively.

DISCUSSION

The results of the analysis of the P. torulosus popula-tion indicated a high level of genetic and morphologicvariability. Based on their morphological characteristics,the isolates were classified into 9 morphotypes not cor-related with RAPD-PCR banding patterns. Morpholog-ic variability among the P. torulosus isolates was previ-ously reported (Fischer and Bresinsky, 1992; Luisi et al.,1998). Fischer and Bresinsky (1982) distinguished twotypes of mycelia, the bleaching type (b-type) and stain-ing type (S-type), in P. torulosus isolates from Europeand the Canary Islands. In a study on biological andepidemic aspects of P. torulosus in Southern Italy, Luisiet al. (1998) observed remarkable variability in colonymorphology, colour, and development of aerial myceli-um. Correlations were also not reported between clus-ters in the dendrograms and morphological characteris-tics for other fungi, such as: Verticillium dahlie (Cherrabet al., 2000) and Cryphonectria parasitica (Wronski et al.,1997) (fungi not ecologically similar to P. torulosus). Thegenetic differentiation found using laboratory methodswas not necessarily correlated with differences in phe-notype (Wronski et al., 1997).

Comparisons of RAPD profiles and host speciesshowed no evident correlations. To date, there are nostudies on the variation in P. torulosus isolates associatedwith their host species. It is hypothetically possible thatP. torulosus isolates from multiple host species are morevariable than those isolated from a single host, implyingthat absence of intense host-mediated selection pressuremight also maintain genetic variation within popula-tions. Exposure of pathogen populations to alternativehosts (which lack resistance) can cause relaxation or

shifts in selection pressure, resulting in greater geneticdiversity (Burdon, 1993; Burdon and Silk, 1997).

P. torulosus may have co-evolved with its host speciesgiven its ability to grow as a saprobe or a parasite (Fis-cher and Bresinsky, 1992). The co-evolution hypothesissuggests a new genetic base, with diversity arising dueto the age of the pathosystem and diversity of the host(Bentely et al., 1995). A more complete understandingof the evolution of P. torulosus could be obtained bystudying isolates collected world-wide (Bentely et al.,1995; Fischer, 1996; Johannesson and Stenlid, 2003).

Among the collected isolates of P. torulosus, therewere no correlations between RAPD markers and geo-graphic origin. For example, isolates within subclustersB1 and C2 produced similar RAPD-PCR banding pat-terns irrespective of geographic origin, with the excep-tion of the isolates within subclusters B4 and C1. Thelack of a relationship between genetic identities and geo-graphic origin implies that there exists a considerableflow of genes among population of P. torulosus in con-trast to the observation made by Fischer and Bresinsky(1992). The authors identified two intersterility (IS)groups of P. torulosus. IS groups represent an example ofspeciation in fungi in which distinct morphological andanatomical differentiation is preceded by partial geneticisolation. In their work gene flow between the two ISgroups was low; thus, partial reproductive isolationwould seem to protect gene pools adapted to differentconditions.

The high level of genetic variability observed in thisstudy suggests that local propagation was probablycaused by basidiospores, whereas vegetative growththrough host root systems was low (Kile, 1983; Pollastroet al., 2000). The sampling methodology supported thishypothesis; in fact, the basidiomes were obtained fromtrees and shrubs at 50-m distances.

The reproductive process of P. torulosus is both sexu-al and asexual. Sexual reproduction and migration ofgenotype by basidiospores are thought to be importantin maintaining the high level of genetic variability ob-served (Hsiang and Mahuku, 1999). Thus, sexual re-combination and/or reassortment of genetically differ-ent nuclei could contribute to the observed DNA poly-morphism (Milgroom, 1996). The other conceivablemeans by which new genotypes might arise is by accu-mulation over time of mutations within the vegetativemycelia (nucleotide changes, deletions and insertions)(Kile, 1990).

The results of the present study show that RAPDanalysis is suitable for detecting and measuring geneticrelatedness and variation within populations of P. toru-losus and for increasing the understanding of the ecolo-gy and biology of this fungus. A high level of geneticvariability of the fungus may be associated with differ-ent characteristics, such as its pathogenic ability(Dobinson et al., 1996), its adaptation to new hosts

Journal of Plant Pathology (2004), 86 (2), 105-115 Campanile et al. 113

(Okoli et al., 1994) or degree of resistance toward cer-tain hosts (Strausbaugh, 1993). Moreover, asexual and,in particular, sexual reproduction could play a role inthe population structure of P. torulosus. Consequently,sexual recombination between genotypes may be im-portant in maintaining and disseminating variationwithin the species and might have implications for pre-dicting the durability of disease control measures.

ACKNOWLEDGEMENTS

The authors are grateful to F. Faretra and S. Pollastrofrom Dipartimento di Protezione delle Piante e Micro-biologia Applicata of the University of Bari for their col-laboration in the development of molecular techniques.

REFERENCES

Appel D.J., Gordon T.R., 1995. Intraspecific variation withinpopulations of Fusarium oxysporum based on RFLP analy-sis of the intergenic spacer region (IGS) of the rDNA. Ex-perimental Mycology 19: 120-128.

Bentley S., Pegg K.G., Dale J.L., 1995. Genetic variationamong a worldwide collection of isolates of Fusarium oxy-sporum f.sp. cubense analysed by RAPD-PCR fingerprint-ing. Mycological Research 99: 1378-1384.

Bernicchia A., 1990. Polyporaceae s.l. in Italia. Istituto di Pa-tologia Vegetale, Università degli Studi, Bologna, Italy.

Boidin J., 1971. Nuclear behavior in the mycelium and theevolution of the Basidiomycetes. In: R.H. Peterson (ed.).Evolution in the higher Basidiomycetes, pp. 129-148. Uni-versity of Tennessee Press, Knoxville, Tennessee, USA.

Burdon J.J., 1993. The structure of pathogen populations innatural plant communities. Annual Review of Phytopathol-ogy 31: 305-328.

Burdon J.J., Silk J., 1997. Sources and patterns of diversity inplant-pathogenic fungi. Phytopathology 87: 664-673.

Carter J.P., Rezanoor H.N., Desjardins A.E., Nicholson P.,2000. Variation in Fusarium graminearum isolates fromNepal associated with their host of origin. Plant Pathology49: 452-460.

Cherrab M., Serrhini M.N., Charest P.M., 2000. Characteriza-tion of Moroccan isolates of Verticillium dahliae Kleb usingRAPD markers. Journal of Phytopathology 148: 243-249.

Dice L.R., 1945. Measures of the amount of ecological associ-ation between species. Ecology 26: 297-302.

Dobinson K.F., Tenuta G.K., Lasarovits G., 1996. Occurenceof race 2 of Verticillium dahliae in processing tomato fieldsin south-western Ontario. Canadian Journal of PlantPathology 18: 55-58.

Fiasson J.L., Niemelä T., 1984. The Hymenochaetales: a revi-sion of the European poroid taxa. Karstenia 24:14-28.

Fischer M., Bresinsky A., 1992. Phellinus torulosus: sexualityand evidence of intersterility groups. Mycologia 84: 823-833.

Fischer M., 1996. On the species complexes within Phellinus:Fomitiporia revisited. Mycological Research 100: 1459-1467.

Gilbertson R.L., Burdsall H.H., 1972. Phellinus torulosus inNorth America. Mycologia 64: 1258-1269.

Goggioli V., Capretti P., Hamelin R.C., Vendramin G.C.,1998. Isozyme and RAPD polymorphisms in Heterobasi-dion annosum in Italy. European Journal of Forest Pathology28: 63-74.

Harvey P.R., Butterworth P.J., Hawke B.G., Pankhurst C.E.,2000. Genetic variation among populations of Pythium ir-regulare in southern Australia. Plant Pathology 49: 619-627.

Hsiang T., Mahuku G.S., 1999. Genetic variation within andbetween southern Ontario populations of Sclerotinia ho-moeocarpa. Plant Pathology 48: 83-94.

Isikov V.P., Kuznetsov V.N., 1990. Biological characteristics ofPhellinus torulosus (Pers.) Bourd. et Galz. and Ganodermaapplanatum (Pers. et Wall.) Pat. in the Crimea. Mycologyand Phytopathology 24: 513-519.

Johannesson H., Stenlid J., 2003. Molecular markers revealgenetic isolation and phylogeography of the S and F inter-sterility groups of the wood-decay fungus Heterobasidionannosum. Molecular Phylogenetics and Evolution 29: 94-101.

Karjalainen R., 1996. Genetic relatedness among strains ofHeterobasidion annosum as detected by random amplifiedpolymorphic DNA markers. Journal of Phytopathology144: 399-404.

Kile G.A., 1983. Identification of genotypes and the clonaldevelopment of Armillaria luteobubalina Watling-Kile ineucalypt forests. Australian Journal of Botany 31: 657-671.

Kim W.K., Mauthe W., Hausner G., Klassen G.R., 1990. Iso-lation of high molecular weight DNA and double-strandedRNAs from fungi. Canadian Journal of Botany 68: 1898-1902.

Koike M., Fujita M., Nagao H., Ohshima S., 1996. Randomamplified polymorphic DNA analysis of Japanese isolatesof Verticillium dahliae and V. albo-atrum. Plant Disease 80:1224-1227.

Kotlaba F., 1975. Geographical distribution and ecology ofthe polypore Phellinus torulosus (Pers.) Bourd. et Galz.with special regard to Czechoslovakia. Ĉeska Mykological29: 5-24.

Kuhlman E.G., Hendrix F.F., 1962. A selective medium forthe isolation of Fomes annosus. Phytopathology 52: 1310-1312.

Larsen M.J., Cobb-Poulle L.A., 1990. Phellinus (Hi-menochaetaceae). A survey of the world taxa. Fungiflora,Oslo, Norway.

Luisi N., Perlini C., Lerario P., 1998. Aspetti biologici ed epi-demiologici di Phellinus torulosus in Italia meridionale. Mi-cologia Italiana 2: 19-25.

Michelmore R.W., Hulbert S.H., 1987. Molecular markers forgenetic analysis of phytopathogenic fungi. Annual Reviewof Phytopathology 25: 383-404.

114 Variation in Phellinus torulosus Journal of Plant Pathology (2004), 86 (2), 105-115

Milgroom M.G., 1996. Recombination and the multilocusstructure of fungal populations. Annual Review of Phy-topathology 34: 457-477.

Milgroom M.G., 1997. Genetic variation and the applicationof genetic markers for studying plant pathogen popula-tions. Journal of Plant Pathology 78: 1-13.

Murray H.G., Thompson W.F., 1980. Rapid isolation of highmolecular weight DNA. Nucleic Acids Research 8: 4321-4325.

Nei M., Li W.H., 1979. Mathematical model for studying ge-netic variation in terms of restriction endonucleases. Pro-ceedings of the National Academy of Sciences USA 76: 5269-5273.

Oberwinkler F., 1977. Das neue System der Basidiomyceten.In: Frey W., Hurka H., Oberwinkler F. (eds.). Beitrag zurBiologie der niederen Pflanzen, pp. 59-105. G. Fischer,Stuttgart, Germany.

Okoli C.A.N., Carder H.J., Barbara D.J., 1994. Restrictionfragment length polymorphism (RFLPs) and the relation-ships of some host-adapted isolates of Verticillium dahliae.Plant Pathology 43: 33-40.

Panconesi A., Santini A., Casini N., 1994. Phellinus torulosuson Cupressus sempervirens in Italy. European Journal ofForest Pathology 24: 238-240.

Parry D.W., Rezanoor H.N., Pettitt T.R., Hare M.C., Nichol-son P., 1995. Analysis of Microdochium nivale isolates fromwheat in the UK during 1993. Annals of Applied Biology126: 449-454.

Pollastro S., Abbatecola A., Dongiovanni C., Faretra F., 2000.Usage of molecular markers (PCR-RAPD) for studying ge-

netic variability in Phellinus (Fomitiporia) sp.. Phyto-pathologia Mediterranea 39: 107-111.

Rogers S.O., Rehner S., Bledsoe C., Muller G.J., AmmiratiJ.F., 1989. Extraction of DNA from Basidiomycetes for ri-bosomal DNA hybridization. Canadian Journal of Botany67: 1235-1243.

Santini A., Capretti P., 2000. Analysis of the Italian popula-tion of Ceratocystis fimbriata f.sp. platani using RAPD andminisatellite markers. Plant Pathology 49: 461-467.

Smith D.R., Stanosz G.R., 1995. Confirmation of two distinctpopulations of Sphaeropsis sapinea in the north centralUnited States using RAPDs. Phytopathology 85: 699-704.

Stalpers J.A., 1978. Identification of wood-inhabiting fungi inpure culture. Studies in Mycology 16: 1-248.

Strausbaugh C.A., 1993. Assessment of vegetative compatibil-ity and virulence of Verticillium dahliae isolates from Idahopotatoes and tester strains. Phytopathology 83: 1253-1258.

Welsh J., McClelland M., 1990. Fingerprinting genomes usingPCR with arbitrary primers. Nucleic Acids Research 18:7213-7218.

Williams J.G.K., Kubelik A.R., Livak K.J., Rafalski J.A.,Tingey S.V., 1990. DNA polymorphisms amplified by arbi-trary primers are useful as genetic markers. Nucleic AcidsResearch 18: 6531-6535.

Wronski R., Kudera U., Wilhelm E., 1997. Characterization ofCryphonectria parasitica strains by random amplified poly-morphic DNA (RAPD) technique and conventional meth-ods. European Journal of Forest Pathology 27: 95-103.

Journal of Plant Pathology (2004), 86 (2), 105-115 Campanile et al. 115

Received 29 August 2003Accepted 6 April 2004