www.fems-microbiology.org

FEMS Microbiology Letters 245 (2005) 221–229

Evaluation of amplified ribosomal DNA restriction analysisas a method for the identification of Botryosphaeria species

Artur Alves a,*, Alan J.L. Phillips b, Isabel Henriques a, Antonio Correia a

a Centro de Biologia Celular, Departamento de Biologia, Universidade de Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugalb Centro de Recursos Microbiologicos, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre,

2829-516 Caparica, Portugal

Received 3 December 2004; received in revised form 15 February 2005; accepted 4 March 2005

First published online 16 March 2005

Edited by B. Paul

Abstract

The polymerase chain reaction was used to amplify a rDNA fragment containing the internal transcribed spacers (ITS1-5.8-

S-ITS2) and the D1/D2 variable domains of the 28S rDNA from 10 species of the genus Botryosphaeria (Fungi, Ascomycota).

Restriction analysis of the amplicons with frequent-cutting endonucleases (amplified ribosomal DNA restriction analysis) allowed

the definition of 12 rDNA haplotypes. Each of the rDNA haplotypes could be unambiguously assigned to a single Botryosphaeria

species, thus allowing clear identification of all the species tested. Intraspecific polymorphism was very low and detected only in

Botryosphaeria parva and Botryosphaeria dothidea. Cluster analysis of banding patterns of the isolates corresponded well with

known species delineations. The method described in this paper provides a simple and rapid procedure for the differentiation

and identification of Botryosphaeria isolates at the species level.

� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Botryosphaeria; rDNA; ITS; D1/D2 domains; Species identification; ARDRA

1. Introduction

Botryosphaeria species (Fungi, Ascomycota) have a

worldwide distribution causing dieback, cankers, shoot

blights, leaf spots, fruit and seed rots and witches�brooms in a wide range of plant hosts of agricultural,

forestry, ecological and economic importance. In addi-

tion, some species are saprophytic while others are endo-

phytic [1–4].

Identification of these fungi at the species level is

complicated by several aspects. Morphological diver-

0378-1097/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2005.03.005

* Corresponding author. Tel.: +351 234370970; fax: +351 234426408.

E-mail address: aalves@bio.ua.pt (A. Alves).

sity among the teleomorphs is often insufficient to al-

low clear species identification. For this reason,

identification of species is based mainly on morpholog-

ical characters of the anamorph, which is the most

common form found in nature [2,5,6]. These charactersinclude size, shape, colour, septation, wall thickness

and texture of the conidia, as well as details of conid-

iogenesis. Cultural aspects, such as colony morphology,

chromogenicity and temperature effects, on mycelial

growth rate have also been used for species recogni-

tion. However, some morphological characters exhibit

extensive plasticity. Thus, size ranges of conidia of dif-

ferent Botryosphaeria species overlap, while age andstate of maturity affect conidial pigmentation and sep-

tation. Taxonomically meaningful morphological char-

acters can also be influenced by the substrate on which

. Published by Elsevier B.V. All rights reserved.

222 A. Alves et al. / FEMS Microbiology Letters 245 (2005) 221–229

the fungus is growing. Moreover, the diversity of hosts

colonized by a single species and the occurrence of dif-

ferent species on the same host precludes the use of

host association as a valid taxonomic character [5,6].

Two closely related species may differ by minor mor-

phological differences making identification a difficulttask for a practitioner without experience of this group

of pathogens. Therefore, there is a need for tools that

can provide accurate and reproducible identifications

of Botryosphaeria species.

Ribosomal DNA (rDNA) sequences are universal

and contain conserved and variable regions allowing

discrimination of fungi at different taxonomic levels

[7]. Non-coding regions, like the internal transcribedspacer (ITS), evolve more rapidly and are consequently

more variable than coding regions. Within coding

rDNA regions some domains like the D1 and D2 of

the 28S rDNA gene are also known to be variable [7].

These regions are easy to access using universal fungal

primers [8,9] and the polymerase chain reaction (PCR)

and have been used extensively in inter- as well as intra-

specific comparisons of several fungi [7]. Sequence anal-yses of the ITS region alone [2,6,10,11] or in

combination with introns of protein-encoding genes

such as b-tubulin and the translation elongation factor

1-alpha (EF1-a) [12–15] have made a significant contri-

bution to resolving taxonomic problems in the genus

Botryosphaeria. These genomic regions have been used

to study relationships among species and to distinguish

closely related Botryosphaeria spp. [3,11–16].Amplified ribosomal DNA restriction analysis (AR-

DRA) has proved to be a suitable and rapid method

for taxonomic studies of fungi [7]. This method is based

on the PCR amplification of an rDNA fragment,

followed by the digestion of the amplicon with fre-

quent-cutting restriction endonucleases. The resulting

fragments are then analysed by agarose gel electropho-

resis [7,17].The purpose of our work was to develop a rapid pro-

cedure for the identification of Botryosphaeria species

using the ARDRA technique. The method is based on

the PCR amplification of the ITS + D1/D2 variable do-

mains of the 28S rDNA and generation of restriction

fingerprints, and was tested on 35 strains representing

10 species, including the most commonly reported spe-

cies responsible for diseases in plants of economicimportance.

2. Materials and methods

2.1. Fungal strains

The fungal strains used in this study are listed onTable 1. Cultures were maintained on half-strength

Difco potato dextrose agar or oatmeal agar [18].

2.2. DNA extraction

Fungal strains were grown in potato dextrose broth for

5 days at approximately 25 �C. Genomic DNA was ex-

tracted from fresh mycelium following an adaptation of

themethod of Pitcher et al. [19] as published elsewhere [11].

2.3. PCR amplifications

PCR reactions were performed on a Bio Rad iCycler

Thermal Cycler (Hercules, CA, USA). The PCR primers

ITS1(5 0-TCCGTAGGTGAACCTGCGG-3 0) [8] and

NL4 (5 0-GGTCCGTGTTTCAAGACGG-3 0) [9] were

supplied by MWG Biotech AG (Ebersberg, Germany).PCR reactions were carried out with Taq polymerase,

nucleotides and buffers supplied by MBI Fermentas

(Vilnius, Lithuania). The PCR reaction mixtures con-

tained 1 · PCR buffer (PCR buffer without MgCl2:PCR

buffer with (NH4)2SO4; 1:1 v:v), 3 mM MgCl2, 200 lMof each nucleotide, 15 pmol of each primer, 1 U of

Taq polymerase, and 50–100 ng of template DNA. Each

reaction volume was made up to 50 lL with sterilewater. Negative controls with sterile water instead of

the template DNA were used in every PCR reaction.

The amplification conditions were as follows: initial

denaturation of 5 min at 95 �C, followed by 30 cycles

of 30 s at 94 �C, 30 s at 50 �C, and 1 min at 72 �C, anda final extension period of 10 min at 72 �C.

After amplification, 5 lL of each PCR product were

separated by electrophoresis in 1% agarose gels in1 · TAE buffer (40 mM Tris, 40 mM acetate, 2 mM

EDTA, pH 8.0). Gels were stained with ethidium bro-

mide, and visualized on a UV transilluminator to assess

PCR amplification.

2.4. Restriction analysis

The amplicons were digested with one of the follow-ing restriction endonucleases: AluI, AsuI, HaeIII, MboI

(MBI Fermentas, Vilnius, Lithuania); NciI, TaqI (Gibco

BRL, Eggenstein, Germany). The reactions contained

10 lL of the PCR product that was digested with 2 U

of enzyme for 14–16 h following the manufacturer�s rec-ommendations. DNA fragments were electrophoreti-

cally separated in 4% Agarose 3:1 HRBe (Amresco)

gels using 1 · TAE buffer under a constant voltage of80 V for 2.5 h. A molecular weight marker, GeneRulere

100 bp DNA Ladder plus (MBI Fermentas) was run on

both sides of each gel. The procedure (DNA isolation,

PCR amplification and restriction analysis) was per-

formed twice to assess reproducibility of the method.

2.5. Computer-assisted analysis

Agarose gels were scanned on aMolecular Imager FX

(Bio Rad) and recorded as TIFF images. The banding

Table 1

Cultures used in this study

Species (teleomorph) Anamorph Accession no.a Host Locality

Botryosphaeria dothidea Fusicoccum aesculi CMW8000b Prunus sp. Switzerland, Crocifisso

CBS110302 Vitis vinifera Portugal, Montemor-o-Novo

CBS113190 Quercus suber Spain, Sevilla

CBS113191 Quercus ilex Spain, Cordoba

CMW7780 Fraxinus excelsior Switzerland, Molinizza

CBS110300 Populus nigra Portugal, Braga

Botryosphaeria ribis Fusicoccum ribis CMW7772 Ribes sp. USA, New York

CMW7054 Ribes rubrum USA, New York

CMW7773 Ribes sp. USA, New York

Botryosphaeria parva Fusicoccum parvum CMW9081 Populus nigra New Zealand, TePuke

CMW9080 Populus nigra New Zealand, TePuke

CBS110301 Vitis vinifera Portugal, Palmela

Botryosphaeria lutea Fusicoccum luteum CBS110299 Vitis vinifera Portugal, Oeiras

CAP037 Vitis vinifera Portugal, Sintra

Botryosphaeria corticola Diplodia corticola CBS112549 Quercus suber Portugal, Aveiro

CBS112545 Quercus suber Spain, Cadiz

CBS112546 Quercus ilex Spain, Huelva

CBS112552 Quercus suber Portugal, Aveiro

CBS112071 Quercus ilex Spain, Catalonia

Botryosphaeria stevensii Diplodia mutila CBS112553 Vitis vinifera Portugal, Montemor-o-Novo

CBS230.30 Unknown Unknown

Botryosphaeria obtusa Diplodia sp. CBS112555 Vitis vinifera Portugal, Montemor-o-Novo

CAP148 Vitis sp. Italy

Botryosphaeria rhodina Lasiodiplodia theobromae CBS124.13 Unknown Unknown

CBS164.96 Fruit along coral reef coast Papua New Guinea

CAA006 Vitis vinifera USA, California

Botryosphaeria iberica Dothiorella iberica CBS115041 Quercus ilex Spain, Aragon

CBS115035 Quercus ilex Spain, Aragon

CBS115039 Quercus sp. Italy, Lazio

CBS113188 Quercus suber Spain, Sevilla

CBS113189 Quercus ilex Spain, Cordoba

Botryosphaeria sarmentorum Dothiorella sarmentorum IMI63581b Ulmus sp. England, Warwickshire

CBS115038 Malus sp. The Netherlands, Delft

CBS120.41 Pyrus communis Norway

CBS165.33 Prunus armeniaca Unknown

a Acronyms of culture collections: CAA – A. Alves, Universidade de Aveiro, Portugal; CAP – A.J.L. Phillips, Universidade Nova de Lisboa,

Portugal; CBS – Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CMW – M.J. Wingfield, FABI, University of Pretoria, South

Africa; IMI – CABI Bioscience, Egham, UK.b Isolate accession numbers in bold signify cultures ex-type, or from samples that have been linked morphologically to the type material.

A. Alves et al. / FEMS Microbiology Letters 245 (2005) 221–229 223

patterns were analysed with Quantity One (Bio Rad)

software and converted to a two-dimensional binary ma-

trix (1 = presence of a band; 0 = absence of a band). Sim-

ilarity matrices between each pair of combined patterns

were calculated with the Dice coefficient [20], since this

coefficient is the best estimator for calculation of genetic

divergence from DNA fragment patterns [21]. Cluster

analysis of similarity matrices was performed by the un-weighted pair group method using arithmetic averages

(UPGMA). The cophenetic correlation coefficient (r)

was calculated to assess the goodness of the clustering

method. Computer-assisted analysis was performed with

the NTSYSpc2 program for Windows [22].

2.6. DNA sequencing

The D1/D2 regions from representatives of each spe-

cies/haplotype were sequenced with the primers NL1 (5 0-

GCATATCAATAAGCGGAGGA-3 0) and NL4 [9].

Cycle sequencing reactions were as described previously

[11], using the ABI PRISM� BigDyee Terminator Cy-

cle Sequencing Ready Reaction Kit with AmpliTAQDNA Polymerase (PE Applied Biosystems, Foster City,

CA, USA) in a Bio Rad iCycler Thermal Cycler.

The sequences were obtained with the ABI PRISM�

310 Genetic Analyzer (PE Applied Biosystems, Foster

City, CA, USA) and were read and edited with

224 A. Alves et al. / FEMS Microbiology Letters 245 (2005) 221–229

Chromas 1.45 (http://www.technelysium.com.au/chro-

mas.html). All sequences were checked manually and

nucleotide arrangements at ambiguous positions were

clarified using both primer direction sequences. Se-

quences were deposited in the GenBank public data-

base with the following Accession Nos: AY928043 toAY928054.

2.7. Restriction map analysis

Sequence data of the ITS + D1/D2 regions of selected

isolates representing each species or haplotype were ana-

lysed with the software Webcutter 2.0 available online

(http://rna.lundberg.gu.se/cutter2/) to identify polymor-

Fig. 1. ARDRA patterns of the PCR-amplified rDNA fragment obtained by

separated in 4% Agarose 3:1 HRBe gels. Each letter corresponds to a differ

phisms of restriction sites. Restriction maps (Fig. 3)

were constructed for the six enzymes tested.

3. Results

3.1. Restriction analysis of PCR amplicons

PCR reactions using primers ITS1 and NL4 yielded a

single amplicon of about 1200 bp for each of the 35

strains tested. All amplicons were digested with each

of the six restriction enzymes used, resulting in two to

six fragments, depending on the enzymes and strains.

In some cases, no restriction fragments were produced

digestion with the enzymes AluI, AsuI, HaeIII, MboI, NciI, and TaqI

ent restriction pattern. M-GeneRulere 100 bp DNA Ladder plus.

Table 2

rDNA haplotypes generated by restriction analysis of the PCR-amplified rDNA among Botryosphaeria isolates

Species rDNA haplotype ARDRA patterns

AluI AsuI HaeIII MboI NciI TaqI

B. lutea 1 A A A A A A

B. ribis 2 A B A A A B

B. parva 3 A C B A A B

B. parva 4 A C B A A C

B. dothidea 5 B D C B B D

B. dothidea 6 B D C B F D

B. stevensii 7 A E D B C E

B. obtusa 8 A F E B D F

B. corticola 9 C F E C C B

B. sarmentorum 10 D G F D D G

B. iberica 11 D H F D D G

B. rhodina 12 E F G E E H

A. Alves et al. / FEMS Microbiology Letters 245 (2005) 221–229 225

with the enzyme NciI. The sum of the restriction frag-

ments sometimes gave a size smaller than that of theundigested PCR products. This is because restriction

fragments smaller than 50 bp were not taken into con-

sideration since they were not clearly resolved by elec-

trophoresis in 4% agarose gels. Also, some bands may

represent two restriction fragments of the same size

(doublets).

Previous tests revealed that restriction analysis of

purified and unpurifed PCR products yielded the sameresult and that the components of the PCR reaction

did not affect the digestion reaction (data not shown).

Eliminating the PCR product purification step renders

Fig. 2. Dice/UPGMA cluster analysis of combined ARDRA fingerprints.

indicated to the right of the dendrogram. The numbers in parentheses indica

the method faster and cheaper and so all subsequent

enzymatic digestions were performed with unpurifiedPCR products. Enzymatic digestion of amplicons ob-

tained from independent analyses always yielded the

same ARDRA patterns, thus showing the reproducibil-

ity of the procedure.

The enzymes MboI and AluI generated five distinct

restriction patterns, NciI generated six, HaeIII gener-

ated seven, TaqI and AsuI generated eight restriction

patterns (Fig. 1). Each unique fingerprint produced byan endonuclease was designated by a letter. The AR-

DRA patterns obtained with the six restriction enzymes

were combined into 12 rDNA haplotypes (Table 2),

Cophenetic correlation coefficient r = 0.94. Anamorphic genera are

te the haplotype.

AluIB. lutea / ribis / parva 1172-1174 bp

B. dothidea 1177 bp

B. corticola 1184 bp

B. sarmentorum / iberica 1156-1157 bp

B. rhodina 1136 bp

45 331/332 252/253 14 166 30 137 80 117

378 255 14 166 30 217 117

50 333 257 14 166 30 334

45 316/317 251 14 166 30 217 117

43 292 257 14 166 30 334

AsuIB. lutea 1173 bp

B. ribis 1174 bp

B. parva 1172-1173 bp

B. dothidea 1177 bp

B. stevensii 1172 bp

96 7/27/22 11 495

50

48 63 35 499 49511 17/4

17/4494

96 7/49 11 495 17/4495

96 7/48 11 495240 17/4253/254

7/14/37 1002 17/446

B. obtusa 1174 bp112 4999/27 49511 17/4

B. corticola 1184 bp115 49911/32 49511 17/4

B. rhodina 1136 bp110 499 49511 17/4

B. sarmentorum 1156 bp96 533 49511 17/4

B. iberica 1157 bp96 493 49511 17/441

HaeIIIB. lutea / ribis 1173-1174 bp

B. parva 1173 bp

B. dothidea 1177 bp

B. obtusa 1174 bp

B. stevensii 1172 bp

98 34/4 511/512 203 157

98 37 258 253/254 203 157 5

51 47/16/4/20 716 157

113 10 525 203 157 5

49 63 534 83203 157

5 83 58/20

83 58/20

5 83 58/20

83 58/20

5 58/20B. corticola 1184 bp

116 12 530 83203 157 5 58/20B. rhodina 1136 bp

101 509 203 157 5/30/53 58/20B. sarmentorum / iberica 1156-1157 bp

98 11/12 521 203 157 58/205 83

B. stevensii 1172 bp371 257 14 166 30 217 117

B. obtusa 1174 bp373 257 14 166 30 217 117

Fig. 3. Restriction fragment length polymorphism maps of the PCR amplicons of Botryosphaeria spp. digested with the enzymes AluI, AsuI, HaeIII,

MboI, NciI, and TaqI. The total length of each fragment (in base pairs) is given in brackets and fragment lengths are given below each line.

226 A. Alves et al. / FEMS Microbiology Letters 245 (2005) 221–229

which are defined by the combination of the patterns ob-

tained with the restriction enzymes. However, three en-

zymes (AsuI, TaqI and NciI) were sufficient to resolve

the same 12 rDNA haplotypes. Moreover, any of the

following two enzyme combinations: AsuI/AluI, AsuI/MboI, AsuI/NciI and AsuI/TaqI allowed for Botryosp-

haeria species differentiation. Intraspecific polymor-

phism was detected only in Botryosphaeria parva and

Botryosphaeria dothidea. The three B. parva strains stud-

ied produced two different TaqI restriction patterns

while B. dothidea strain CBS110300 produced an NciI

restriction pattern distinct from all other B. dothidea

strains.

3.2. Cluster analysis

A cluster analysis was performed on the combination

of ARDRA fingerprints. Using the combined data ob-

tained with the six enzymes, a total of 56 restriction frag-

ments was analysed. The dendrogram generated with

the UPGMA method is shown in Fig. 2. A cophenetic

correlation coefficient of 0.94 was found between the

cophenetic value matrix deduced from the dendrogram

and the similarity matrix. An r value greater than 0.9

can be interpreted as a very good fit of the cluster anal-ysis to the data [22].

The clustering was consistent with the division of the

isolates into 12 different rDNA haplotypes and the cor-

responding 10 species tested. Species with Fusicoccum

anamorphs were divided into two groups, namely, a

cluster of B. dothidea isolates well separated from a clus-

ter containing Botryosphaeria lutea, Botryosphaeria ribis

and B. parva (approx. 90% similarity). Within this lastgroup B. lutea and B. ribis formed a sub-cluster at

approx. 94% similarity. The species with Diplodia ana-

morphs formed a cluster containing the Botryosphaeria

stevensii and Botryosphaeria obtusa isolates (approx.

82% similarity) that were well separated from another

cluster containing only Botryosphaeria corticola. The

two species with Dothiorella anamorphs (Botryosphaeria

MboIB. lutea / ribis / parva 1172-1174 bp

B. dothidea 1177 bp

B. stevensii / B. obtusa 1172-1174 bp

B. corticola 1184 bp

22 200/201 307/308 347/348 232

22/32 170 310 348

22 195/197 312

22 207 312 348

232 63

348 232

232B. rhodina 1136 bp

22 159 312 348B. sarmentorum / iberica 1156-1157 bp

22 185/186 306 348 232

63

63

63

295

63

NciIB. lutea / ribis / parva 1172-1174 bp

B. dothidea 1177 bp

B. dothidea 1177 bp

B. obtusa 1174 bp

B. stevensii 1172 bp

1172/1173/1174

128 956

128 853

334 747

1079 93

103

93

B. corticola 1184 bp1091 93

B. rhodina 1136 bp296 747 93

B. sarmentorum / iberica 1156-1157 bp322/323 741 93

93

93

B. lutea 1173 bp

B. ribis / parva 1173-1174 bp

B. parva 1172 bp

B. dothidea 1177 bp

B. stevensii 1172 bp

41/12 59 63

243

173 63 5359 92432 292

364189

41/12 188/189 53 428 859

41/12/71 53 427117 9259

450 92

B. obtusa 1174 bp51 63124 5359 432

B. corticola 1184 bp

B. rhodina 1136 bp39/12 149 5359 432

B. sarmentorum / iberica 1156-1157 bp41/12 173/174 5359 426

TaqI

53 8 92 292

92 291

8 292

59 53 8 292

8

8 92 292

63185 53 432 8 92 29259

8 92 292

8 92 292

Fig. 3 (continued)

A. Alves et al. / FEMS Microbiology Letters 245 (2005) 221–229 227

sarmentorum and Botryosphaeria iberica) grouped to-

gether at 96% similarity level. Botryosphaeria rhodina

(anamorph Lasiodiplodia theobromae) formed a cluster

separated from all the other species.

3.3. DNA sequence analysis

The D1/D2 region of the 28S rDNA of Botryosphae-ria spp. as determined by DNA sequencing with primers

NL1 and NL4 consisted of 614 bp. No size variation of

the D1/D2 region was detected between or within spe-

cies. The nucleotide sequences obtained were combined

with the nucleotide sequences of the ITS regions re-

trieved from GenBank database allowing the accurate

determination of the size of PCR amplicons: B. lutea

CBS 110299 (1173 bp), B. ribis CMW7772 (1174 bp),B. parva CMW9081 and CBS110301 (1172–1173 bp),

B. dothidea CMW8000 and CBS110300 (1177 bp), B.

stevensii CBS112553 (1172 bp), B. obtusa CBS112555

(1174 bp), B. corticola CBS112549 (1184 bp), B. sarmen-

torum IMI63581b (1156 bp), B. iberica CBS115041

(1157 bp) and B. rhodina CBS124.13 (1136 bp).

3.4. Restriction map analysis

Using the DNA sequence information from com-

bined ITS + D1/D2 regions of representatives from each

species/haplotype restriction maps for the six enzymes

tested were constructed to determine precisely the

restriction sites and the size of the fragments generated

by each enzyme. This way it was possible to identifybands corresponding to doublets in AsuI restriction pat-

terns (Fig. 1), and several small fragments undetected by

agarose gel electrophoresis were also identified.

4. Discussion

The amplified rDNA fragment (approx. 1200 bp) in-cluded the ITS1-5.8S rDNA-ITS2 regions and the first

614 bp of the ribosomal large subunit gene (28S rDNA).

The ITS region has proven useful in the phylogenetic

analysis of Botryosphaeria species [2,6,10] and other fun-

gal taxa, e.g. [23,24]. The D1/D2 domains of the 28S

rDNA, although never studied in Botryosphaeria spe-

228 A. Alves et al. / FEMS Microbiology Letters 245 (2005) 221–229

cies, are frequently used for phylogenetic analysis of

yeasts [29] and less frequently for filamentous fungi

[30]. The ARDRA procedure has been widely used for

the identification of fungal phytopathogens, e.g.

[25,26,31] and clinical fungi, e.g. [27,28]. This method

was recently applied to Botryosphaeria species [33].However, in that report it consisted of restriction analy-

sis of the ITS region alone and was tested on a small set

of species with Fusicoccum anamorphs.

The ARDRA procedure presented here allowed a

clear differentiation of the isolates at the species level.

Twelve rDNA haplotypes revealed by the restriction

analysis could be clearly assigned to each of the 10 Bot-

ryosphaeria species. Cluster analysis is consistent withthe division of the isolates into 12 haplotypes and 10

species which are readily identified in the dendrogram.

The ARDRA procedure tested appears to be an excel-

lent tool for the differentiation of even cryptic species

such as B. ribis/B. parva and B. sarmentorum/B. iberica.

For example,Botryosphaeria ribis and B. parva have been

the subject of much discussion [6,12]. These two species

are difficult to separate on morphology and reliable iden-tifications should be based on molecular methods. Se-

quence data from the ITS region alone cannot clearly

discriminate between these species and it is necessary to

combine these data with partial sequence of the EF1-agene [12]. Differentiation of these two species was impos-

sible by ARDRA analysis of the ITS region alone [33].

Interestingly, these two species are quite easily separated

using the ARDRA procedure described here. Both spe-cies have distinctive HaeIII and AsuI patterns. Our ap-

proach differs from the one previously reported [33] in

using a larger portion of the ribosomal cluster. Digestion

of this PCR amplicon produced restriction patterns with

larger and more numerous fragments, making the pat-

terns more discriminative and easier to score.

Botryosphaeria sarmentorum and B. iberica, two re-

cently described Botryosphaeria species with Dothiorella

anamorphs [15] can be readily distinguished based on tel-

eomorph features. However, this form is extremely rare,

and the anamorphic forms (Dothiorella sarmentorum and

Dothiorella iberica) are the ones most frequently encoun-

tered and can be separated only by minor differences in

the size and shape of the conidia [15]. In the same way

as B. ribis and B. parva these two species can be differen-

tiated by sequence analysis of the ITS and EF1-a regions[15]. As shown in the present work, these two species can

be distinguished easily by the ARDRA patterns gener-

ated with the enzyme AsuI.

Intraspecific variability was detected only in B. parva

and B. dothidea with the enzymes TaqI and NciI, respec-

tively. For all the other species no polymorphisms were

detected amongst the isolates tested. The strain

CBS110300 was described previously as Botryosphaeriapopuli [32]. However, phylogenetic analysis placed this

isolate within the B. dothidea clade [14,15] and thus B.

populi is considered a synonym of B. dothidea. The same

can be seen on the dendrogram where isolate

CBS110300 clusters together with all the B. dothidea iso-

lates. Although this isolate differs from the other

B. dothidea isolates in the NciI restriction patterns, this

merely represents intraspecific variation as seen withthe B. parva isolates.

The results of the present study demonstrate that

ARDRA is a useful tool for the identification of the ma-

jor species in the genus Botryosphaeria. Several new spe-

cies have been described recently in the genus [13,14,33],

these as well as other species already described [3,16]

would need to be studied to confirm that the ARDRA

procedure is a valid method for species identificationin Botryosphaeria. Although the technique distinguishes

even some cryptic species it is possible that it does not

differentiate between others that have not been tested.

In this case, the procedure can be further improved by

testing different restriction enzymes.

Sequencing of ITS as well as other genes (e.g., EF1-a,b-tubulin) has proven useful for the discrimination of

Botryosphaeria species. ARDRA fingerprinting is aninexpensive and simple alternative to sequencing, and

is especially appropriate when dealing with large sets

of isolates [7,17]. In this respect, it is noteworthy that

it was possible to discriminate between the 10 Botryo-

spheria spp. by using combinations of only two restric-

tion enzymes. This makes the identification procedure

faster and easier, and greatly reduces the cost that would

otherwise be close to those of DNA sequencing. The ma-jor advantages of the method are its simplicity, the uni-

versal availability of PCR primers, its reproducibility,

and amenability to computer database analysis. It is

possible to build a database of ARDRA patterns that

can be used for routine identification of isolates by sim-

ple comparison of fingerprints.

Acknowledgements

This work was financed by Fundacao para a Ciencia

e a Tecnologia (FCT) under project POCTI/AGR/

44348/2002 (Portugal). A. Alves and I. Henriques were

supported by Grant numbers SFRH/BD/10389/2002

and SFRH/BD/5275/2001 from FCT. M.E. Sanchez

and A. Trapero (University of Cordoba, Spain), J. Lu-que (IRTA, Spain), M.J. Wingfield (FABI, University

of Pretoria, South Africa) and T.J. Michailides (Univer-

sity of California, Davis, USA) donated some of the

strains included in this work.

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