Plant Pathology (1997) 46, 773784

Geographical cline of DNA variation within the F intersterility

group of Heterobasidion annosum in Italy
N. LA PORTA a b *, P. CAPRETTI a , K. KAMMIOVIRTA b , R. KARJALAINEN b and
K. KORHONEN c
a
Universita` di Firenze, Istituto di Patologia e Zoologia Forestale e Agraria, P. delle Cascine 28, I-50144
Firenze, Italy; bUniversity of Helsinki, Department of Plant Biology, Plant and Forest Pathology Section,
PO Box 28, 00014 Helsinki; and cFinnish Forest Research Institute, Vantaa Research Centre, PO Box18,
01301 Vantaa; Finland
Eighty-six Heterobasidion annosum isolates, mainly belonging to the F intersterility group and obtained from
32 different geographical localities in Italy, were subjected to genetic analysis by the Random Amplified
Polymorphic DNA (RAPD) markers. The similarity between F and S groups was higher than that between F
and P. In UPGMA Cluster Analysis, the F isolates originating from the same locality usually grouped in the
same cluster. The isolates also showed a tendency to group at the level of larger geographical areas. Within
the F group, isolates from the south of the Italian peninsula showed the highest genetic variation and northern
isolates from the Alpine regions showed the lowest. This indicates a gradual cline along the peninsula. The
genetic variability in the Italian F group is discussed in relation to the past and present distribution of the host
species in Italy and Europe.

INTRODUCTION
Heterobasidion annosum is one of the most
important fungal pathogens of coniferous trees in
temperate regions of the world (Stambaugh, 1989).
It is also one of the most important basidiomycetes
causing root rot of forest trees in Italy. Along the
peninsula, this pathogen is distributed from the Alps
in the north to Sicily in the south, although tree
species, climatic conditions, soil types and other
environmental gradients vary widely along the
1500-km length of this country (Moriondo, 1970;
Capretti & Moriondo, 1983; Capretti et al., 1994).
Until the sixties, the damage caused by
H. annosum in Italy was considered to be somewhat
different from that described in central Europe. The
high susceptibility of silver fir to H. annosum was
thought to be related to environmental and climatic
conditions prevailing in Italian territories (Biraghi,
1962; Moriondo, 1970). Later studies, however,
pointed more to the variability of the fungus
(Moriondo et al., 1988), and subsequent mating
* To whom correspondence should be addressed
at Department of Plant Biology, Plant Pathology
Section, PO Box 28, 00014 University of Helsinki,
Finland.
Accepted 15 May 1997.

experiments demonstrated that the fungus infecting

silver fir formed an intersterility group (F group) of
its own (Capretti et al., 1990). Subsequently, F
group isolates were found in other parts of Europe
including France, Switzerland, Austria, Slovenia,
Poland, Hungary, Bulgaria and Greece (Stenlid &
Karlsson, 1994; Capretti et al., 1994; Munda, 1994;
Pagony & Szanto, 1995; Lakomy, 1996; Tsopelas &
Korhonen, 1996; Capretti & La Porta, unpublished
data).
The other two European intersterility groups of
H. annosum, P and S, also occur in Italy. The S
group is closely associated with spruce and has been
found in Italy only in the Alps on the native species,
Norway spruce (Picea abies). The P group can be
found in different parts of the country where it
causes damage mostly to pine species in some
localized areas (Capretti et al., 1994).
The F group is the dominant type of H. annosum
in Italy. Its distribution area covers almost the entire
Italian peninsula and overlaps with the distribution
of the S group in the Alps and the P group in various
parts of the country (Capretti et al., 1994). The F
type is especially common in the Apennines where
the most important host tree is silver fir (Abies
alba); it is found in nearly all silver fir stands older
than 5060 years (Capretti & Moriondo, 1983;
Capretti et al., 1990). However, only in particular

774

N. La Porta et al.

situations where the inoculum mass is high does the

F group attack other conifer species such as Norway
spruce, sometimes young Douglas fir (Pseudotsuga
menziesii), and more rarely, pine species (Capretti
et al., 1994).
On the basis of climatic indices and geomorphology, the Italian peninsula can be divided into
several phyto-climatic regions, including the Alps
and the northern, central and southern Apennines
(De Philippis, 1937). The effects of different ecological environments, as well as of historical events,
can be observed in the distribution of silver fir.
During the last glacial period silver fir found a
refuge in south Italy. The history of its retreat along
the peninsula and its migration back to the north
after the last glacial period favoured partial
differentiation of this conifer into four ecological
subpopulations: (1) southern Apennines (from
Basento to Aspromonte), (2) central Apennines
(from Lagas Mountains to Basin of Trigno), (3)
northern Apennines (from Abetone to La Verna)
and (4) Alpine (Gellini, 1973). At present, the
highest variation in morphological and physiological traits and the highest proportion of polymorphic
loci of silver fir occurs in the south Italian population and the variation decreases northward (Larsen,
1986a,b; Larsen et al., 1988).
The aim of this work was to investigate the
regional variation of the H. annosum F group in
Italy and to find out whether the variation of the
host tree is reflected in the variation of the
pathogen. For this purpose isolates from different
geographical regions were collected and their
genetic variability was analysed using the
RAPDPCR analysis (Welsh & McClelland,
1990; Williams et al., 1990).
MATERIALS AND METHODS

from heterokaryotic cultures (Korhonen, 1978).

To avoid duplicate sampling of genetically similar
individuals the distance between all collections in
the forest, was at least 50 m, and only one single
basidiospore isolate from a single basidiocarp was
obtained from each sampling point. Homokaryotic
isolates were stored on malt agar slants (1.5% malt
extract) in tubes at 48C prior to the following
experimental work.
The intersterility group of each isolate was
determined with the aid of mating tests using two
homokaryotic testers from each of the groups S, P
and F (Capretti et al., 1990).
DNA extraction
The isolates were cultured for 15 days in Petri
dishes at 218C on Malt agar (1.5%) which had been
overlaid with a sterile cellophane membrane before
inoculation. The mycelium was then scraped off the
cellophane membrane and ground to a fine powder
in liquid nitrogen with a mortar and pestle. The
DNA was purified as described by Lee & Taylor
(1990), except that a further phenolchloroform
extraction step and RNAase treatment were
included (Karjalainen & Kammiovirta, 1994). The
genomic DNA pellet was dissolved in TE buffer
(10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA
concentration was estimated by using ethidium
bromide plates (0.5% agarose, EtBr 2 ng ml1).
Samples of 0.5 mL of the DNA extractions were
applied to draw a visual comparison against l-DNA
intensity standards varying from 10 to 250 ng mL1.
Concentration of DNA extracts ranged between 50
and 250 ng ml1. The samples were diluted in TE
buffer to give a concentration of 10 ng ml1 DNA.
Template DNA and subsequent dilutions were
stored at 208C until used.

Fungal isolates

DNA amplification conditions

The study material was composed of 86 isolates

of H. annosum, collected from 32 different
localities in Italy: 17 isolates from nine localities
in the Alps, 43 isolates from 14 localities in the
northern Apennines and 26 isolates from nine
localities in the southern Apennines (Table 1 and
Fig. 1). The host species for 70 of the isolates was
Abies alba, for nine Picea abies, for three Pinus
nigra and for one each Pinus pinea, Pseudotsuga
menziesii, Cryptomeria japonica and Castanea
sativa.
All isolates were homokaryotic, obtained either
as single basidiospores from fresh basidiocarps or,
in some cases, as homokaryotic conidiospores

Amplification conditions were modified from those

of D. Howland & J. Arnau (University of East
Anglia, 1994, personal communication) and from
Williams et al. (1990) in the following way: 50 mL
reactions were used, including 25 ng of genomic
DNA, 200 mM of each dNTP, 200 nM of primer,
optimized buffer with different concentrations of
MgCl2 to give a final concentration in the PCR
mixture ranging from 1.0 to 5.0 mM, and 1 U
of DynaZyme-polymerase (Finnzymes, Helsinki,
Finland). Two selected primers, which have shown
high polymorphisms and consistent results in
H. annosum (Fabritius & Karjalainen, 1993), were
synthesized by the Biotechnology Institute of the

DNA variation of Heterobasidion annosum in Italy

University of Helsinki. The sequences of the
primers and their optimal MgCl2 concentrations
were: 5 0 -CGA TTC GGCG-3 0 (91299) with MgCl2
concentration 3 mM; 5 0 -CGA GGT TCGC-30
(91300) with MgCl2 concentration 2 mM.
Primers and nucleotides were aliquoted before
use to avoid damage caused by repeated freezing
and thawing. Premixtures (without DNA) of each
reaction were made to minimize the risk of
contamination. Samples were overlaid with two
drops of sterile mineral oil to prevent evaporation.
Each amplification reaction was run with a negative
control which contained no template DNA. Amplification was performed in an MJ Research Inc.
Programmable Thermal Controller PTC-100 Model

775

60, programmed for 40 cycles of 1 min denaturation

at 948C, 1 min annealing at 458C and 2 min
extension at 728C. The DNA samples were
repeatedly amplified and after the optimal conditions were established, reproducible results were
obtained.
Amplification products were analysed by
electrophoresis for 5 h at 90 V in 1.4% agarose
gels 20 cm long with 1 TBE (89 mM Tris-borate,
2 mM EDTA) and stained with 200 mg L1 ethidium bromide (Fabritius & Karjalainen, 1993;
Karjalainen & Kammiovirta, 1994). DNAfragments were visualized with a short-wave
ultraviolet transilluminator.
A marker of 7 mL of DNA plasmid Molecular

Fig. 1 Major geographical areas and the collection localities of isolates of Heterobasidion annosum.

N. La Porta et al.

776

Table 1 List of the Italian isolates of Heterobasidion annosum analysed. Locality numbers and geographical
areas are indicated in Fig. 1
Geographical
Localityb
a
area
isolate code
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3

1127
2116
354
4140f
4141f
5119
5123
6142f
6143f
6144f
7146f
842
843
845
8145f
947
948
1088
10130
1196
1211
1212
1213
1330
1331
1332
1315
14122
14132
14151
1595
1641
17131
181
183
185
186
187
188
189
1810
1891
1919
1922
1923
2016
2018
2019
20107
21105
2125
2126
2127

Isolatec
881028.1.2
900712.3.2
881004.2.10
921208.a
921208.b
881004.2.10
880923 5.3
921208.c
921208.d
921208.e
921208.2.1
881121.1.1
881121.1.2
881121.2.1
921208.1.1
881206 1.2
881206.2.2
820528 1.1
89505 1.1
810327.1.1
871024.3
871024.5.1
871024.6.2
871007.2.2
871007.3.1
871007.3.3
881202.1.1
920431.1.1
920830.1.1
920431.1.2
810520.1.1
921206 1.1
920213.1.1
871005.1.1
871005.5.1
871005.7.1
871005.8.1
871005.11.2
871019.1.1
910910.1.8
921205 1.1
910910.1.7
871203 1.4
871203.2.2
871203.3.3
871123 1.1
871123 2.1
871123 3.3
871123 1.3
871215.1.2
871211 1.1
871211.1.3
871211.2.3

Localityd
Bressanone, BZ.
Andalo, TN.
Terlago, TN.
Fiave, TN.
Fiave, TN.
Lavarone, TN.
Lavarone, TN.
Passo Ve`zena, TN.
Passo Ve`zena, TN.
Passo Ve`zena, TN.
Brusson, AO.
Chiusa Pesio, CN.
Chiusa Pesio, CN.
Chiusa Pesio, CN.
Chiusa Pesio ,CN.
M. Gouta, IM.
M. Gouta, IM.
Centocroci, PR
Centocroci, PR.
Pizzorne, LU.
Abetone, PT.
Abetone, PT.
Abetone, PT.
Macchia Antonini PT.
Macchia Antonini PT.
Macchia Antonini PT.
Acquerino, PT.
Romita, PT.
Romita, PT.
Romita, PT.
Pratorsi, PT.
Boccadirio, PO.
Tirrenia, PI.
Vallombrosa, FI.
Vallombrosa, FI.
Vallombrosa, FI.
Vallombrosa, FI.
Vallombros, FI.
Vallombrosa, FI.
Vallombrosa, FI.
Vallombrosa, FI.
Vallombrosa, FI.
P.sso della Futa, FI.
P.sso della Futa, FI.
P.sso della Futa, FI.
Camaldoli, AR.
Camaldoli, AR.
Camaldoli, AR.
Camaldoli, AR.
Varramista, PT.
Varramista, PT.
Varramista, PT.
Varramista, PT.

Host
Picea abies
Abies alba
Abies alba
Picea abies
Picea abies
Abies alba
Picea abies
Picea abies
Picea abies
Picea abies
Picea abies
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Pinus nigra
Abies alba
Abies alba
Picea abies
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Pinus nigra
Abies alba
Abies alba
Abies alba
Pinus pinea
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Castanea sativa
Abies alba
Abies alba
Cryptomeria japonica
Abies alba
Abies alba
Pseudotsuga menziesii
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba

ISGe
S
F
F
S
S
F
S
S
S
S
S
F
F
F
F
F
F
F
P
F
F
F
F
F
F
F
F
F
P
F
F
F
P
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F

DNA variation of Heterobasidion annosum in Italy

777

Table 1 continued
Geographical
Localityb
areaa
isolate code
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5

2234
2336
2337
2338
2339
2340
23134
2486
2486a
2487
2577
2579
2580
2581
2599
25102
25115
2684
2686d
2782
2783
2874
2875
2876
2970
2971
3059
3061
3063
30133
3172
3265
3289

Isolatec
890501.1.1
921204.2.1
921204.3.1
921204.6.1
921204.6.2
920601.5.1
921204.4.1
920701.3.3
920701.3.1
920701 3.4
921218 3.1
921218.4.2
921218.4.3
921218.4.4
921218.3.4
921218.3.6
921218.3.5
920701.2.4
920701.2.5
920701.1.2
920701.1.4
921217 1.5
921217.1.10
921217.2.1
881011.13.2
881011.14.3
881011.2.1
881011.5.1
881011.9.1
921006 3.4
890628 2.1
890626.2.1
890626.2.1

Localityd
Greve in Chianti, FI.
M. Amiata, GR.
M. Amiata, GR.
M. Amiata, GR.
M. Amiata, GR.
M. Amiata, GR.
M. Amiata, GR.
M. Taburno, BN.
M. Taburno, BN.
M. Taburno, BN.
Gargano, FG.
Gargano, FG.
Gargano, FG.
Gargano, FG.
Gargano, FG.
Gargano, FG.
Gargano, FG.
M. Vulture, PZ.
M. Vulture, PZ.
Monticchio, PZ.
Monticchio, PZ
Marsico Nuovo, PZ.
Marsico Nuovo, PZ.
Marsico Nuovo, PZ.
Fagnana, CS.
Fagnana, CS.
M. Cocuzzo, CS.
M. Cocuzzo, CS.
M. Cocuzzo, CS.
M. Cocuzzo. CS
Serre Calabre, CS.
Serra S. Bruno, RC.
Serra S. Bruno, RC.

Host

ISGe

Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Abies alba
Pinus nigra
Abies alba
Abies alba
Abies alba

F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
P
F
F
F

1, eastern Alps; 2 , western Alps; 3, northern Apennines; 4, central Apennines; 5, southern Apennines.
Locality numbers correspond to numbers on Fig. 1. c Isolate numbers are the stock numbers in the collection of
the Finnish Forest Research Institute (METLA) of Vaanta, Finland. d The two letters after the locality names
represent the Italian district codes. e ISG, intersterile group. f Isolates were collected by G. Nicolotti.

Marker VI (Boehringer Mannheim, 1062-590) at

a concentration of 40 ng ml1 was also included on
each gel.
Data analysis
Readings were made from photographic prints of
the gels. Molecular weight markers were run in
duplicate on each gel and against these, presence or
absence of amplified fragments were scored. Each
amplification was performed at least twice and only
the reliable, consistent and reproducible amplified
fragments were scored for analysis.

A binomial data matrix was constructed on the

basis of presence (1) or absence (0) of RAPD
fragments for each isolate. The data for both primers
were combined. The similarity coefficient between
pairs of isolates (SCxy) was then calculated from the
formula of Nei & Li (1979): Scxy 2Nxy/Nx Ny;
where Nxy is the number of common fragments
between the two isolates, and Nx and Ny are the
numbers of the fragments in isolates X and Y,
respectively. To estimate genetic differentiation in
the Italian F group, SC was calculated between
sample populations originating from the five geographical ranges shown in Fig. 1. Isolates from the

778

N. La Porta et al.

same geographical area were considered to belong to

the same population.
The relationship between populations can be
presented more clearly by using similarity matrices.
The average of the similarity coefficients (ASC)
among each geographical area within the F group
was calculated as the arithmetic mean of SC for all
isolate comparisons between any two populations
(Gilbert et al., 1990). In the construction of the
matrix it was assumed that corresponding bands of
the same size were homologous, i.e. they had arisen
by amplification of the same genomic locus
(Cognato et al., 1995).
The similarity coefficients
were transformed
into angular values (sin 1 %) and processed by
ANOVA; to compare the statistical significance
between ASCs, Duncans multiple range test
(P < 0.05) was used. The standard deviation was
used to quantify the error of the estimate of mean
value.
Data analyses were carried out using the
numerical taxonomy package NTSYS-pc version
1.70 (Rohlf, 1988). The program SIMQUAL was
used to produce a similarity matrix via the DICEs
similarity coefficient equivalent to the band
sharing coefficient. The phenogram was produced
with the program TREE. Cluster analysis was

performed on a similarity matrix by Sequential

Agglomerative Hierarchical Nested (SAHN) clustering (Sneath & Sokal, 1973) using the
Unweighted Pair-Group Method with arithmetic
Averaging (UPGMA).
RESULTS
Mating test
Mating tests with the homokaryon testers from S, P
and F groups indicated that out of a total of 86
isolates, four isolates were P group, eight were S,
with the remaining 74 confirmed as F intersterility
group.
Representatives of the different intersterility
groups of H. annosum were found to be strongly
associated with their specific hosts (Table 1). All
the eight S isolates came from Norway spruce and
four P isolates from Pinus species. Seventy of the F
isolates were from 70 silver fir and, in four cases,
the F group was found on hosts other than fir,
including one Norway spruce. These exceptions
were found only in localities in the northern
Apennines where atypical hosts had been planted
after silver fir had been harvested or were growing
in mixed stands with silver fir.

Fig. 2 RAPD profile of genomic DNA obtained with the primer 91299 (CGA TTC GGCG) from P, S and F groups of
H. annosum from Italy. Numbers on lanes 213 correspond to the isolate codes shown in Table 1. Lanes marked M show
the molecular weight marker with fragment sizes in base pairs on the left side.

DNA variation of Heterobasidion annosum in Italy

779

Fig. 3 UPGMA cluster phenogram of the genetic relationships among 86 Italian isolates of H. annosum. Based on DICEs
similarity coefficients, the Sequential Agglomerative Hierarchical Nested (SAHN) clustering was performed using
NTSYS-pc program (Rohlf, 1988). Underlined isolates belong to P group, those italicized to the S group, and all the others
to the group F. The three parts of each isolate number represent in order: a unique isolate code, the locality number and the
geographical area (see Table 1 and Fig. 1).

780

N. La Porta et al.

Fig. 4 UPGMA cluster phenogram of the relationship between 74 Italian F group isolates of H. annosum grouped in five
geographical areas.

Relationship among F and other intersterility

groups
Out of the 86 isolates, a total of 47 unique DNA
fragments were scored following PCR amplification with two primers, ranging from 0.4 to 2.3 kb in
size. Only one band was shared among all 86
isolates. Another two bands were common
between all the F and S isolates. An example of
a RAPD profile after amplification by primer
91299 with examples of all three intersterility
groups is shown in Fig. 2.
UPGMA clustering analysis (Fig. 3) indicated
that the P group is genetically more distant from the
F group than the S group (similarity coefficients
about 25% and 57%, respectively). The separation
achieved between the groups F and S was not
absolute; two S isolates, 140-4 and 142-6 from
Trentino, grouped with other isolates from the
southern Apennines (Fig. 3).

Variation within F-group

Seventy-four F isolates collected from different
geographical regions of Italy showed a large
variation in RAPDPCR banding profiles (Fig. 3).
A total of 38 different bands were produced with the
two primers from these isolates, and only three
bands were common to all isolates. Genetic
variability among the F isolates, as revealed by
similarity values based on the two primers, ranged
from 63% to 100% (Fig. 3).
The phenogram based on UPGMA clustering
revealed that most isolates yielded a unique pattern.
However, 16 exceptions were observed where two
isolates gave rise to the same amplification
products. They formed eight groups with two
isolates in each: group 1, 10-18.3 and 91-18.3;
group 2, 1-18.3 and 41-16.3; group 3, 11-10.3 and
34-22.3; group 4, 32-12.3 and 95-15.3; group 5, 1820.3 and 39-23.3; group 6, 40-23.3 and 47-9.2;
group 7, 134-23.3 and 48-9.2; group 8, 96-11.3 and
74-28.5 (Fig. 3). The isolates of group 1 came from
the same locality (no. 18, Vallombrosa). For groups
25 the isolates came from different localities but

all within the same geographical area (the northern

Apennines). In groups 6 and 7, the isolates came
from different but adjacent geographical areas (the
western Alps and the northern Apennines). However,
in the last group, there was no geographical
relationship between the two isolates.
The UPGMA cluster phenogram generated by
the NTSYS-PC program indicated that the F
isolates did not form distinct groups according to
their collection locality. Isolates from a single
locality (e.g. Vallombrosa, no. 18) were spread over
several clusters of the dendrogram (Fig. 3).
However, there was a slight tendency for isolates
to group according to the major geographical areas.
After empirical testing of the grouping, UPGMA
was performed from similarity averages within and
between the five populations of H. annosum isolates
collected from the same major geographical areas
where silver fir is naturally present (Fig. 1). The
result of this clustering analysis is shown in Fig. 4.
The five populations were separated at various
levels between 75% and 85%. However, there was
variation in the number of isolates in different areas
which may cause bias in distance estimations and
derived subgroupings. For example, the sample
population from the eastern Alps consisted of only
three F isolates and that of the western Alps only
seven. Therefore, at this stage these geographical
subgroupings should be considered tentative. The
variation within the five geographical areas, as
shown through the ASC, indicated that the southern
areas have the highest amount of genetic variation
and that variation appears to decrease from south to
north.
ANOVA revealed that the geographical area factor
was highly significant ( P < 0.001). Isolates from the
central and southern Apennines showed the highest
degree of variability in the number of polymorphic
bands, 0.777 and 0.760, respectively. In contrast,
the lowest degree of variation (0.841) was found in
isolates from the eastern Alps. Duncans multiple
range test revealed significant differences between
ASCs for different areas (Table 2). The ASCs
among the isolates from the northern Apennines
and western Alps were intermediate between the

DNA variation of Heterobasidion annosum in Italy

781

Table 2 Means and standard errors (in parentheses) of similarity coefficients calculated for 74
F-type isolates of H. annosum, grouped according to five geographical areas of Italy
Geographical areaa
Geographical
area
E Alps

E Alps

W Alps

N Apen.

C Apen.

S Apen.

0.841c
(0.0218)

0.848c
(0.0157)
0.813bc
(0.0245)

0.847c
(0.0030)
0.734ab
(0.0033)
0.811bc
(0.0032)

0.764b
(0.0073)
0.707a
(0.0079)
0.765b
(0.0023)
0.777b
(0.0087)

0.799b
(0.0104)
0.754b
(0.0092)
0.779b
(0.0027)
0.766b
(0.0057)
0.760b
(0.0139)

W Alps
N Apen.
C Apen.
S Apen.

The five geographical areas are shown in Fig. 1. Means followed by a different letter differ
significantly using Duncans Multiple Range test (P < 0.05).

ASCs of the other geographical regions and not

significantly different (Table 2). In order to
explore further the relationship between geographical separation of the five populations and
the degree of genetic similarity between them, a
regression analysis was carried out on the basis of
the linear distances between the populations. This
indicated that coefficients of similarity were
inversely related to geographical distance, with a
highly significant ( P < 0.001) regression coefficient
of 0.34 (Fig. 5).
DISCUSSION

Similarity index (%)

The genetic distances between the F, S and P

90
85

B B

y = 81 318 - 0.00572 x
r 2 = 0.34***

80

B
B

75

B
B

B
B

70
65

200

400 600 800 1000 1200

Distance (km)

Fig. 5 Relationship between similarity index and geographical distance between (V) and within (W) populations. The similarity index for a population pair was
calculated as the mean of all interpopulation comparisons
of individual isolates within the population pair.

intersterility groups of H. annosum in Italy,

revealed from DNA analysis, were generally in
agreement with those found in earlier studies in
Europe (Fabritius & Karjalainen, 1993; Kasuga
et al., 1993; Stenlid et al., 1994; Karlsson, 1994;
Vasara & Karjalainen, 1994). Within the F group,
the genetic similarity between populations from
different geographical areas showed a slight inverse
relationship with increasing distance between them.
This observation is in accordance with findings for
the European S group populations by Stenlid (1994)
and Stenlid et al. (1994).
Our data revealed the highest degree of DNA
polymorphism in F isolates originating from central
and southern regions of the Italian peninsula, with
the lowest degree of variability in isolates from the
Alpine region (Table 2). This association between
geographical location and the amount of variability
may be related to genetic differences found in the
populations of silver fir.
It is generally agreed that the amount of genetic
variation in the silver fir populations in Europe
changes along a northsouth continuum (Larsen,
1989; Bergmann et al., 1990). Furthermore, within
the Italian peninsula, different indigenous populations of silver fir show a cline of significant genetic
differences (Ciampi & Di Tomaso, 1973; Kramer,
1984) and they can be grouped, following ecological
criteria, into four subpopulations (Gellini, 1973).
The differentiation in silver fir according to
geographical location may to be related to historical
events. During the last glacial period, silver fir

782

N. La Porta et al.

almost totally disappeared from Europe, surviving

only in certain small enclaves in southern Europe.
In response to early postglacial climate warming
silver fir moved northward to recolonize central
Europe. According to Kral (1982) and Kramer
(1984) the re-immigration from the southern Italy
refuge was the main mechanism in this process.
Konnert & Bergmann (1995) suggest that some
migration also took place from refuges in central or
eastern France and in the southern Balkan Peninsula. The consecutive postglacial cooling and
warming periods during the Holocene were
reflected in stops and expansion phases of silver
fir along the migration route (Huntley & Birks,
1983).
Because of the longer migration distance and the
competition by beech (Fagus sylvatica) in the
course of the colonization process, the northern
populations were genetically more deprived and
thus have lost some genetic variation compared
with the source population (Bernetti, 1995). The
subsequent fragmentation of range caused a
decrease in the gene flow and amplification of the
divergence among the developing fir populations
(Gellini, 1973). In addition, at the beginning of the
historical period, silver fir was present only in small
spots along the Italian peninsula, in mixed stands
with beech (Chiarugi, 1938). These small original
populations might have contributed to the population differentiation (founder effect) in silver fir.
However, since Medieval times this tree has been
planted along the peninsula, mainly on former
agricultural lands (Bernetti, 1995), and the amount
of the genetic variability may thus have been
partially reduced in the present fir populations.
The observation that F-isolate grouping based on
RAPDPCR analysis fits quite well with the
divisions based on both host tree and climatic
regions is interesting. It seems possible that the
separation of the silver fir range into small
fragments, well apart from each other, may also
have induced a certain differentiation in the
pathogen population. Our data, although preliminary, suggest that parallel loss of variability has
taken place in both host and pathogen during their
co-evolution. Climatic and geographical barriers,
due to the presence of the Apennine mountains, have
apparently been important evolutionary forces by
restricting gene flow and thereby promoting population differentiation of both the host tree and
associated pathogen. This seems to be particularly
evident for the isolates collected along the peninsula.
Some of these genetic differences may simply reflect
the history of postglacial colonization and migration
patterns. Others, however, may be of adaptive

relevance and thus have competitive importance for

H. annosum in different environments.
The available data for the distribution of the F
type in Europe indicates that this fungus is also
present in central Europe, but less frequent and less
aggressive towards silver fir than in the Italian
peninsula (Capretti et al., 1990; Munda, 1994;
Lakomy, 1996). This is somewhat surprising,
because declining silver fir populations in central
Europe might be especially susceptible to a root
pathogen like H. annosum. The less aggressive
nature of the F group in central Europe may be due
to a number of factors, but it may also be related to
reduced genetic diversity of H. annosum in this
area.
ACKNOWLEDGEMENTS
This research was funded by a EU grant (contract
no. ERBCHBICT 930668) awarded to Dr Nicola La
Porta. The authors are grateful to Dr Giovanni
Nicolotti, University of Turin, for providing some
cultures, to Professor Francesco Moriondo and
Professor Giovanni Bernetti of the University of
Florence and to Professor Sandro Pignatti of the
University La Sapienza of Rome for their helpful
suggestions. We wish to thank Mr Donald Smart
and Dr Daniel Austin for the revision of the English
language.
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