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709

An initial assessment of genetic relationships

among populations of Fusarium circinatum in
different parts of the world
Karen Wikler and Thomas R. Gordon

Abstract: Fusarium circinatum Nirenberg & O’Donnell, the fungus responsible for pitch canker disease, is a destruc-
tive pathogen of Pinus spp. Pitch canker was first described in 1946 in the southeastern United States, and since 1987
has been reported in numerous other locations including California, Mexico, Japan, and South Africa. To make a pre-
liminary assessment of relationships between populations of F. circinatum in these different locations, we compared al-
lele and genotype frequencies based on eight polymorphic regions of DNA from 76 isolates of the fungus. Patterns of
relatedness indicate that the California and Japanese populations of the fungus share lineages with the southeastern
U.S.A. population. Genetic diversity is highest in Mexico, implicating it as the center of origin for the fungus. The as-
sociation of multiple vegetative compatibility groups with a common multilocus genotype suggests that vegetative com-
patible group diversity may be generated by mutation, rather than through recombination resulting from sexual
reproduction.
Key words: genomic subtraction, tree disease, genetic distance.
Résumé : Le Fusarium circinatum Nirenberg & O’Donnell, champignon responsable du chancre poisseux, cause une
maladie grave chez les Pinus spp. Le chancre poisseux a été décrit pour la première fois en 1946 dans le sud-est des
États-Unis, et a été rapporté dans de nombreuses autres localités depuis 1987, incluant la Californie, le Mexique, le Ja-
pon et l’Afrique du Sud. Afin d’effectuer une évaluation préliminaire des relations qui existent entre les populations du
F. circinatum, dans ces différentes localités, les auteurs ont comparé les fréquences alléliques et phénotypiques basées
sur des régions polymorphes de l’ADN provenant de 76 isolats du champignon. Les patrons de rapprochement indi-
quent que les populations de la Californie et du Japon, de ce champignon, ont des lignées en commun avec celles du
sud-est des États-Unis. La diversité génétique est plus élevée au Mexique ce qui en fait le foyer d’origine du champi-
gnon. L’association de nombreux groupes de compatibilité végétative possédant un génotype multiloculaire commun
suggère que la diversité des groupes de compatibilité végétative puisse être générée par mutation, plutôt que par recom-
binaison d’origine sexuelle.
Mots clés : soustraction génomique, maladie des arbres, distance génétique.
[Traduit par la Rédaction] Wikler and Gordon 717

Introduction by pitch canker in coastal pine forests of California is remi-

Pitch canker was first described as a pine disease in the
southeastern United States (SE U.S.A.) in 1946 (Hepting
and Roth 1946), but it was not reported to be highly damag-
ing until severe epidemics occurred in slash pine plantations
in Florida in the mid 1970’s (Schmidt 1978; Dwinell et al.
1985). More recently, the pathogen, Fusarium circinatum
Nirenberg & O’Donnell (= F. subglutinans (Wollenw. &
Reinking) Nelson et al. f.sp. pini (Correll et al.)), has been
identified in California (CA) (McCain et al. 1987), Japan
(Kobayashi and Muramoto 1989), South Africa (Viljoen et
al. 1994), and Mexico (Guerra-Santos 1999).
In California, the pitch canker fungus is now well estab-
lished, colonizing and killing pines in urban and native for-
ests in 18 coastal counties. The extensive mortality caused
Received October 20, 1999.
K. Wikler and T. Gordon.1 Department of Plant Pathology,
University of California, Davis, Davis, CA 95616, U.S.A.
1
Author to whom all correspondence should be addressed
(e-mail: trgordon@ucdavis.edu).
Can. J. Bot. 78: 709–717 (2000)
niscent of the damage caused by chestnut blight in the hard-
wood forests of the eastern United States (Anagnostakis
1987), only the host range of pitch canker is much wider.
In South Africa, pitch canker has been confined to nurser-
ies, but even so, the scale of the epidemic has been large,
affecting over six million seedlings in a single infestation
(M.J. Wingfield, personal communication). Pines are not na-
tive to South Africa, so the disease there is likely a recent in-
troduction. In Japan, pitch canker is affecting native Ryukyu
pine (Pinus luchuensis) on six of the southern, subtropi-
cal islands, and some consider the disease to be endemic
(Dwinell 1999). In Mexico, the disease is widely distributed
(Guerra-Santos 1999), where it has caused considerable
losses in plantations. Pitch canker also occurs in native for-
ests in Mexico, but is apparently less damaging in these ar-
eas (J.J. Guerra-Santos, personal communication).
Limited vegetative compatibility group (VCG) diversity
suggests that the pitch canker fungus is a recent introduction
to California and that propagation is predominantly clonal
(Correll et al. 1992; Gordon et al. 1996). However, nothing
is presently known about the relationships between VCGs
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710
within the California population or the affinity of this popu-
lation with those found in other parts of the world. Insight
into these questions requires polymorphic markers, which
have proven difficult to obtain (Wikler et al. 1996). For this
reason a genomic subtraction procedure (Rosenberg et al.
1994) was used to identify polymorphic loci. Allelic differ-
ences at these loci provided a measure of the relationships
between isolates associated with different VCGs in the Cali-
fornia population of F. circinatum and a preliminary assess-
ment of the genetic distances separating this population from
those in the SE U.S.A., Japan, Mexico, and South Africa.
Materials and methods
Isolate collections
Seventy-six isolates of F. circinatum were included in this study
(Table 1). Thirty-four isolates from California and 19 from the SE
U.S.A. were from collections described previously (Correll et al.
1992; Gordon et al. 1996). The South African isolates were col-
lected from the original nursery outbreak in 1990–1991 (Viljoen et
al. 1994, 1997). Sources of the isolates from Japan and Mexico
are given in Table 1. All isolates not previously described (those
from Mexico and Japan) were identified as F. circinatum based on
microscopic morphology on carnation leaf agar, and verified as
pine pathogens by lesion development in a pathogenicity test on
Monterey pine (P. radiata), as described by Gordon et al. (1996).
Vegetative compatibility groupings
Vegetative compatibility groups (VCGs) for the California, SE
U.S.A., and South Africa isolates were determined previously
(Gordon et al. 1996; Correll et al. 1992; Viljoen et al. 1997). The
remaining isolates were assigned to VCGs using standard protocols
(Puhalla 1985).
DNA extraction and quantification
DNA was extracted as described previously (Jacobson and
Gordon 1990), with the addition of a second phenol–chloroform
extraction. The DNA was quantified by spotting 4 µL of a dilution
series onto a UV light box adjacent to DNA standards of known
concentrations, adding an equal amount of 2 µg/mL ethidium bro-
mide, and comparing the intensity of fluorescence. A fluorometer
(Hoefer Scientific Instruments) was also used to quantify the DNA
used for Southern blotting.
Marker identification and screening
To obtain polymorphic markers, a genomic subtraction proce-
dure developed by Rosenberg et al. (1994) was used. This involved
a series of DNA–DNA hybridizations to remove restriction frag-
ments common to two isolates and thereby obtaining a pool of
fragments enriched for sequences unique to one isolate. All poly-
merase chain reaction (PCR) protocols, digestions, and ligations
were performed as described by Rosenberg et al. (1994), using the
identical reagents, PCR primers, and DNA adaptors, with the ex-
ception of Taq polymerase and 10× buffer, which were obtained
from Perkin Elmer rather than Boehringer Mannheim. Our use of
the procedure is briefly described below.
Total DNA from a California isolate (PF-1) was digested with
HindIII. These restriction fragments were ligated to DNA adaptors
for PCR amplification and size-fractionated (250–1000 bp). This
representation of the original genome was termed the tracer. The
same procedure was used to obtain a representation of an isolate
from South Africa (SA0024), but with a different set of DNA adap-
tors and a slightly larger size fraction (120–1200 bp). This repre-
sentation was termed the driver. Three cycles of hybridization were
used to remove sequences common to both tracer and driver, as de-
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Can. J. Bot. Vol. 78, 2000
scribed by Rosenberg et al. (1994) and the remaining tracer frag-
ments were used as templates for amplification with tracer-specific
primers.
The fragments remaining after the third subtractive hybridiza-
tion were cloned as follows. An aliquot of the remaining fragments
was reamplified using the tracer primers, 23 cycles of 30 s at 94°C,
30 s at 55°C, and 3 min at 72°C, finishing with a 2-h extension at
72°C. Shrimp alkaline phosphatase was added and allowed to incu-
bate for 1 h at 37°C to dephosphorylate the dNTPs. The products
were then denatured at 99°C for 10 min and incubated at 72°C for
2 h, to eliminate poorly hybridizing fragments from the subsequent
ligation procedure. The fragments were inserted into Invitrogen’s
pCR2.1 and cloned into Escherichia coli (Invitrogen A-T cloning
kit). Individual transformed colonies were isolated, and plasmids
were purified (Bio-Rad Quantum Prep plasmid mini-prep kit).
Cloned fragments were screened, by labeling them with dig-
oxigenin (DIG), and using them in Southern hybridizations with
1.5 µg of membrane-bound HindIII digested DNA from each of
several isolates that collectively represented the worldwide dis-
tribution of F. circinatum. All Southern blotting was done using
Boehringer Mannheim’s DIG nonradioactive nucleic acid labeling
and detection system, performed according to the manufacturer’s
instructions. Eight fragments (termed loci 18, 60, 70, 74, 80, 82,
98, and 220) that revealed polymorphisms were selected for the
study, and each of these fragments was sequenced.
The M13 reverse and M13(–20) forward primers were each used
in separate reactions to sequence the fragment in both directions.
Perkin Elmer’s ABI PRISM™ Dye terminator cycle sequencing
ready reaction kit was used according to the manufacturer’s in-
structions, and the reactions were carried out in an Ericomp ther-
mocycler using 25 cycles of the following program: 12 s at 96°C,
7 s at 50°C, 4 min at 60°C. The extension products were purified
by precipitating them in 70% ethanol – 0.5 mM MgCl2. Both
strands of the fragments were sequenced using an ABI automatic
sequencer. Primers were designed to match the ends of each frag-
ment’s sequence, to amplify the homologous region in other iso-
lates and potentially capture the polymorphism.
PCR reactions using the custom primers were carried out in
25-µL volumes containing 200 µM of each dNTP, 0.5 µM of each
primer, 2.5 µL 10× reaction buffer (includes 15 mM MgCl2),
0.625 U Taq polymerase, and 50–100 ng template DNA. The am-
plification regime was 30 s at 95°C (the samples were placed in the
thermocycler when the machine came up to temperature) followed
by 30 cycles of: 30 s at 94°C, 35 s at 50°C, 3 min at 72°C, and a
final step of 10 min at 72°C after the last full cycle. Perkin Elmer
Taq and buffer were used for all reactions except for the 18, 60,
and 98 primer sets, in which HotStarTaq and buffer were used
(Qiagen), along with a 15-min initial denaturation step at 95°C.
The PCR products were run on 1% agarose gels, except for those
amplified with primer set 98 forward (F) and 98 reverse (R), which
were run on 2% agarose gels.
For four of the eight loci (18, 60, 98, and 220), polymorphisms
were detectable using PCR with the following primer sets: 18F
(TTG AAT CAC AAC TTA GAG TGG CTC) and 18R (AAG CTT
CCG CGT GCC ATT CT); 60F (TAC CCA AAG AGA CCT CCG
ATG) and 60R (AAC TTC AGG TCT TGA TCC TAA GG); 98F
(TTG GAA GCA TGA TGG TGG C) and 98R (AGT TCA AGA
CTT AAT CAC TCC TGG); and 220F (ATA TTA TCC ATC TTC
GAC TTT ACC) and 220R (GCG TTG TTG TCC CGT ATC C).
However, the largest PCR product for 60F or 60R was difficult to
amplify. Consequently, the null allele (i.e., no amplicon) was con-
firmed by using the cloned fragment (DIG labeled) representing lo-
cus 60 in Southern hybridizations with 1.5 µg of HindIII digested
DNA from the isolate in question. Similarly, Southern hybridiza-
tions were used to confirm a subset (10 of 15) of null alleles for
primer set 18F or 18R to verify that the absence of PCR products
reflected the absence of a region, rather than a refractory segment
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Wikler and Gordon
of DNA. The remaining four loci (70, 74, 80, and 82) were assayed
using nonradioactive Southern hybridizations as described above.
Genetic diversity and population structure analyses
The allele frequency at each locus was calculated for each geo-
graphic region using the program Relatedness 5.0.4 (Goodnight
Software, http://gsoft.smu.edu/GSoft.html). Total genetic diversity
for all geographic regions combined (HT) was estimated using Nei
and Chesser’s (1983) equations for calculating genetic diversity
from limited and unequal sample sizes. Because all null alleles
from each locus were assumed to be identical, the diversity mea-
surements are conservative. Total genetic diversity within each re-
gion (HTR) was estimated using an adaptation of HT. For these
analyses, isolates were divided among five geographic regions:
California, SE U.S.A., Japan, Mexico, and South Africa.
Genotypic (multi-locus) diversity was determined using Shan-
non’s information statistic (MacArthur and MacArthur 1961; Shel-
don 1969; as used by Goodwin et al. 1993) and Stoddart’s
genotypic diversity statistic (Stoddart 1983; Stoddart and Taylor
1988). Sheldon’s equitability index (Sheldon 1969) as used by
Goodwin et al. (1993) was calculated to account for the influence
of sample size on the observed diversity. Variance of Stoddart’s
multi-locus diversity statistic was estimated according to the for-
mula derived by Stoddart and Taylor (1988).
Genetic distance between populations was estimated by using
two different algorithms (Paetkau et al. 1998): (i) the Cavalli-
Sforza and Edwards (1967) arc distance measurement (DARC), as
described by Swofford et al. (1996) and (ii) DLR, the genotype like-
lihood ratio distance (Paetkau et al. 1997).
Results
Eight polymorphic loci were identified, with between two
and four alleles per locus. Alternative alleles were desig-
nated by the size of the fragment(s), in base pairs, identified
at each locus (Table 1). Among the 76 isolates examined,
there were 26 unique locus/allele combinations, (i.e., haplo-
types). Seven haplotypes were found only in Mexico (locus/
allele combinations: 60/3000,1800,950; 80/1640; 80/2100;
82/6000; 82/450; 98/null; and 220/null). No haplotypes were
unique to the SE U.S.A. or to Japan, whereas both the Cali-
fornia and South Africa populations each had one unique
haplotype, 80/null and 82/900, respectively. Allele frequen-
cies and diversity statistics are shown in Table 2.
Combining all eight loci yielded 25 multi-locus geno-
types, among 76 isolates (Table 1); their distribution among
regions is illustrated in Fig. 1. Three genotypes were shared
among isolates from different regions: one between Japan,
SE U.S.A. (both Florida and Georgia), and California (geno-
type 5); a second between California and the SE U.S.A. (ge-
notype 6); and the third between Mexico and South Africa
(genotype 19). Each of these transregional genotypes was
composed of isolates from different VCGs. Additionally,
isolates representing the most common California genotype
(1) were associated with three different VCGs (Table 1). The
two most common genotypes in the SE U.S.A. accounted for
over half the isolates (57.9%) in this region; whereas no ge-
notype was represented more than twice in either Mexico
or South Africa, and only the genotype common to both
regions spanned different VCGs. Multi-locus (genotypic) di-
versity was highest in South Africa (HEM = 0.92) and Mex-
ico (HEM = 0.82) (Table 3); it was lowest in Japan (HEM =
0.00), where all isolates sampled had identical genotypes
(Table 3).
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711
Estimates of genetic distances between each pair of re-
gions are given in Table 4. The two estimates of genetic dis-
tance had a correlation coefficient of R = 0.86. By both
measures of genetic distance (DARC, DLR), Japan and SE
U.S.A. have the closest relationship (DARC = 0.37 and DLR =
0.26), followed by California and the SE U.S.A. (DARC =
0.46 and DLR = 0.96). Mexico and South Africa, overall, are
genetically more distant from the other regions. According
to DARC, Mexico is equally close to South Africa and SE
U.S.A. (0.56), but DLR ranks Mexico closer to South Africa
than to any other region (1.26).
Discussion
Correll et al. (1992) found 45 VCGs among a sample of
117 F. circinatum isolates in Florida; whereas, only 8 VCGs
were identified among 170 isolates collected throughout the
range of pitch canker in California (Gordon et al. 1996). The
greater VCG diversity in Florida was suggestive of an out-
crossing population, while the minimal VCG diversity in
California was consistent with asexual propagation (Correll
et al. 1992; Gordon et al. 1996). A subset of these isolates
was included in the present study. Surprisingly, in the SE
U.S.A. and in California, isolates in different VCGs often
shared the same genotype. For example, genotype 5 is asso-
ciated with seven VCGs, genotype 6 with five VCGs, and
genotype 1 with three VCGs.
The common genotypes among a diverse set of vegetative
compatibility groups imply that new VCGs have been gener-
ated by mutation within a clonal lineage, rather than through
outcrossing. If all eight loci, on which the genotypes are
based, were unlinked and re-assorted during meiosis, it is
highly unlikely that six SE U.S.A. VCGs would have identi-
cal genotypes (p < 0.01, based on the product of the allele
frequencies at each of the six polymorphic loci within the
SE U.S.A.). Furthermore, we have observed cross-
compatibility among some isolates from three of the most
prevalent VCGs in California (C1, C2, and C4) (unpublished
results) which share a common genotype (1). These observa-
tions further support a close relationship between these
VCGs and may indicate that isolates with identical geno-
types (based on the eight loci assayed) have lost their com-
mon VC identities through mutations at one or more vic loci
(Leslie 1993). In cases such as this, VCGs may be more sen-
sitive indicators of population diversity than molecular
markers.
Although the extent of VCG diversity provides a measure
of variability within a population, it leaves open the question
of how VCGs are related. Isolates associated with the same
VCG are not necessarily part of the same clonal lineage, be-
cause recombination can result in genotypically different or-
ganisms sharing an identical suite of alleles at all vic loci.
Thus, VCG data combined with multi-locus genotypes can
provide a more complete picture of interregional relation-
ships, as discussed below for the Japanese and California
isolates that share VCG C7 and genotype 5.
There is strong evidence linking the California, Japanese,
and SE U.S.A. populations of F. circinatum. These regions
are separated by relatively short genetic distances and share
a common genotype (5). An additional genotype (6) is
shared between California and SE U.S.A., and a VCG is
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Table 1. Collection location, source, vegetative compatibility grouping, and multi-locus genotypes for isolates of Fusarium circinatum.
Locusa
Region Isolate VCGb Locationc Sourced 18 60 70 74 80 82e 98f 220 Genotypeg
Calif. FSP 2 C1 Sunset State Beach Pinus contorta var. 510 800 380 500 400 600 950 1500 1
contorta
Calif. FSP 134 C1 Ano Nuevo P. radiata 510 800 380 500 400 600 950 1500 1
Calif. FSP 143 C1 Richmond P. muricata 510 800 380 500 400 600 950 1500 1
Calif. FSP 87 C1 Soquel P. sabiniana 510 800 380 500 400 600 950 1500 1
Calif. SL-1 C1 San Lorenzo P. radiata 510 800 380 500 400 600 950 1500 1
Calif. JC-4 C1 New Brighton P. muricata 510 800 380 500 400 600 950 1500 1
Calif. FSP 4 C2 Sea Cliff P. torreyana 510 800 380 500 400 600 950 1500 1
Calif. JC-1 C2 Sea Cliff P. muricata 510 800 380 500 400 600 950 1500 1
Calif. FSP 13 C2 Buena Vista P. radiata 510 800 380 500 400 600 950 1500 1
Calif. FSP 89 C2 San Luis Obispo P. ponderosa 510 800 380 500 400 600 950 1500 1
Calif. Z-12 C2 Scotts Valley P. radiata 510 800 380 500 400 600 950 1500 1
Calif. Z-7 C2 Aptos P. radiata 510 800 380 500 400 600 950 1500 1
Calif. FSP 5 C3 Escondido Pityophthorus sp. 510 800 380 1400 400 600 950 400 2
(twig beetle)
Calif. FSP 56 C3 Morro Bay P. radiata 510 800 380 1400 400 600 950 400 2
Calif. SK-1 C3 Santa Barbara P. radiata 510 800 380 1400 400 600 950 400 2
Calif. SK-2 C3 Santa Barbara P. radiata 510 800 380 1400 400 600 950 400 2
Calif. PF-1 C3 Torrance P. radiata 510 800 380 1400 400 600 950 400 2
Calif. PF-2 C3 Torrance P. radiata 510 800 380 1400 400 600 950 400 2
Calif. PF-3 C3 Torrance P. radiata 510 800 380 1400 400 600 950 400 2
Calif. FSP 39 C4 Watsonville P. radiata 510 800 380 500 400 600 950 1500 1
Calif. FSP 90 C4 La Selva Beach Pseudotsuga menziesii 510 800 380 500 400 600 950 1500 1
Calif. FSP 46 C4 Watsonville P. muricata 510 800 380 500 400 600 950 1500 1
Calif. Z-38 C4 Watsonville P. radiata 510 800 380 500 400 600 950 1500 1
Calif. LA 4 C5h Torrance P. radiata 1600 null null 500 null 600 950 400 4
Calif. FSP 74 C6 Rosemead P. radiata 510 3000 null 500 400 600 950 400 6
Calif. FSP 118 C6 Carmel P. radiata 510 3000 null 500 400 600 950 400 6
Calif. FSP 165 C6 Malibu P. radiata 510 3000 null 500 400 600 950 400 6
Calif. FSP 170 C6 Carmel P. radiata 510 3000 null 500 400 600 950 400 6
Calif. FSP 132 C7 Aromas P. radiata null 3000 null 500 400 600 950 400 5
Calif. FSP 45 C7 Watsonville P. radiata 510 3000 null 500 400 600 950 400 6
Calif. FSP 188 C8 San Luis Obispo P. radiata 510 3000 null 1400 400 600 950 400 3

Can. J. Bot. Vol. 78, 2000

Calif. FSP 189 C8 San Luis Obispo P. radiata 510 3000 null 1400 400 600 950 400 3
Calif. FSP 200 C8 Los Osos P. radiata 510 3000 null 1400 400 600 950 400 3
Calif. FSP 62 C8 Los Osos P. radiata 510 3000 null 1400 400 600 950 400 3
© 2000 NRC Canada

Japan SA-GE 37 C7 Hisaba, Tatugou-cho P. luchuensis null 3000 null 500 400 600 950 400 5
Japan KU-TI-5 C7 Kujirahama, Tatsugo-cho P. luchuensis null 3000 null 500 400 600 950 400 5
Japan SAK-1 C7 Sakihara, Naze-city P. luchuensis null 3000 null 500 400 600 950 400 5
Japan JU-KU-1 C7 Kujirahama, Tatsugo-cho P. luchuensis null 3000 null 500 400 600 950 400 5
Japan TA-BW-11 C7 Oogachi, Tatusgou-cho P. luchuensis null 3000 null 500 400 600 950 400 5
SE U.S.A. FK867 SE 4 Georgia P. taeda 510 800 380 1400 400 600 1400/2000 400 9
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SE U.S.A. FK 313 SE 8 Texas P. taeda null 800 380 1400 400 600 950 400 10
SE U.S.A. FK165 SE 44 Georgia P. elliottii 1600 800 380 500 400 600 1400/2000 1500 11
SE U.S.A. D115 SE 43 Alabama P. virginiana null 3000 380 500 400 600 950 400 12
SE U.S.A. FL102 SE24 Franklin Co., Fla. P. elliottii 510 null 380 500 400 600 1400/2000 400 7
SE U.S.A. FL103 SE32 Franklin Co., Fla. P. elliottii 510 null 380 500 400 600 1400/2000 400 7
SE U.S.A. FL15 SE7 Alacia Co., Fla. P. elliottii 510 800 380 500 400 600 1400/2000 400 8
SE U.S.A. FL 105 SE28 Franklin Co., Fla. P. elliottii 510 800 380 500 400 600 1400/2000 400 8
SE U.S.A. FL 1 SE2 Alacia Co., Fla. P. elliottii 510 3000 null 500 400 600 950 400 6
SE U.S.A. FL 3 SE9 Alacia Co., Fla. P. elliottii 510 3000 null 500 400 600 950 400 6
SE U.S.A. FL 19 SE10 Alacia Co., Fla. P. elliottii 510 3000 null 500 400 600 950 400 6
SE U.S.A. FL 58 SE2 Volusia Co. Break7, Fla. P. elliottii 510 3000 null 500 400 600 950 400 6
SE U.S.A. FL 52 SE21 Volusia Co. Break7, Fla. P. elliottii null 3000 null 500 400 600 950 400 5
SE U.S.A. FK870 SE 42 Georgia P. elliottii null 3000 null 500 400 600 950 400 5
SE U.S.A. FL11 SE5 Alacia Co., Fla. P. elliottii null 3000 null 500 400 600 950 400 5
SE U.S.A. FL 4 SE1 Alacia Co., Fla. P. elliottii null 3000 null 500 400 600 950 400 5
SE U.S.A. FL 10 SE1 Alacia Co., Fla. P. elliottii null 3000 null 500 400 600 950 400 5
SE U.S.A. FL 88 SE22 Volusia Co. Maytown, Fla. P. elliottii null 3000 null 500 400 600 950 400 5
SE U.S.A. FL72 SE6 Volusia Co. Maytown, Fla. P. elliottii null 3000 null 500 400 600 950 400 5
S. Africa SA 0024 SA10 Nursery, Eastern Transvaal P. patula 1600 null 380 6300 400 900 1400/2000 1500 14
S. Africa SA0006 SA 15 Nursery, Eastern Transvaal P. patula 1600 800 380 1400 400 600 1400/2000 400 19
S. Africa SA 0035 SA 5 Nursery, Eastern Transvaal P. patula 1600 800 380 6300 400 600 950 1500 15
S. Africa SA 0046 SA 6 Nursery, Eastern Transvaal P. patula 1600 800 380 6300 400 600 950 400 16
S. Africa SA 0153 SA 23 Nursery, Eastern Transvaal P. patula 510 800 380 1400 400 600 1400/2000 1500 17
S. Africa SA 0164 SA 22 Nursery, Eastern Transvaal P. patula 510 800 380 1400 400 600 950 1500 18
S. Africa SA 0009 SA18 Nursery, Eastern Transvaal P. patula 510 null 380 1400 400 600 1400/2000 1500 13
S. Africa SA 0026 SA18 Nursery, Eastern Transvaal P. patula 510 null 380 1400 400 600 1400/2000 1500 13
Mexico T1 M1 Northeastern Michoacan P. teocote 1600 3000 null 6300 400 6000 1400/2000 400 20
Mexico T5 M1 Northeastern Michoacan P. teocote 1600 3000 null 6300 400 6000 1400/2000 400 20
Mexico A3 M4 Eastern Mexico P. patula 1600 800 null 6300 400 600 1400/2000 400 21
Mexico A5 M4 Eastern Mexico P. patula 1600 800 null 6300 400 600 1400/2000 400 21
Mexico G1 M5 Eastern Mexico P. greggii 1600 800 380 1400 400 600 1400/2000 400 19
Mexico E2 M5 Eastern Mexico P. greggii 1600 800 380 1400 400 600 1400/2000 400 19
Mexico JAL-3 M6 Jalisco P. douglasiana 510 3000 null 6300 1640 600 950 400 22
Mexico HGO-5 M7 Hildalgo P. patula 1600 3000, 1800, 950 380 6300 2100 450 null null 23
Mexico T3 M2 Northeastern Michoacan P. teocote 510 3000 null 6300 1640 600 1400/2000 400 24
Mexico LJ M3 North-central Michoacan P. leiophylla 1600 3000 null 6300 1640 600 950 400 25
No. of single locus haplotypes 3 4 2 3 4 4 3 3
Note: Alleles in italics represent unique, reginal haplotypes. Isolates from Japan were kindly provided by Y. Sato and M. Muramoto. Sources for the isolates from Mexico are as follows: T1, T5, T3,
and LJ were collected by the first author; A3, A5, G1, and E2 were collected by M. Wingfield; and JAL-3 and HGO-5 were collected by J. Guerra-Santos.
a
The alleles of each locus are designated as the fragment length, in base pairs.
© 2000 NRC Canada

b
Vegetative compatibility group.
c
Most proximate location of the collection site given: town, county, or state.
d
The tree (Pinus spp. or Pseudotsuga menziesii) or insect (Pityophthorus sp.) from which the fungus was isolated.
e
Allele 600 refers to a fragment of this size (in bp) which was always present, although additional fragments would occasionally hybridize to the probe.
f
Allele 950 refers to an amplicon of this size (or hybridization to a band of this size), although additional fragments occasionally hybridized or amplified. Allele 1400/2000 refers to isolates where no
950-bp band amplified or hybridized.
g
Each number corresponds with a unique combination of alleles at eight polmorphic loci (= multi-locus genotype).

713
h
This is the only known isolate of this VCG.
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714 Can. J. Bot. Vol. 78, 2000

Table 2. Allele frequencies and estimates of genetic diversity in populations of Fusarium

circinatum within each region.
Allele frequencya = (1 – Σpi2 )
Locus California SE U.S.A. Japan South Africa Mexico Total
18 0.12 0.55 0.00 0.50 0.32 0.65
60 0.46 0.52 0.00 0.47 0.59 0.60
70 0.44 0.49 0.00 0.00 0.42 0.50
74 0.44 0.19 0.00 0.47 0.32 0.62
80 0.06 0.00 0.00 0.00 0.54 0.16
82 0.00 0.00 0.00 0.22 0.46 0.16
98 0.00 0.43 0.00 0.47 0.46 0.50
220 0.50 0.10 0.00 0.38 0.18 0.37
HTRb 0.26 0.30 0.00 0.36 0.46 HTc = 0.45
a
Allele frequency, where pi is the frequency of the ith allele (Nei 1973); calculated using the program
Relatedness 5.0.4 (Goodnight Software, http:/gsoft.smu.edu/GSoft.html).
b
HTR is the mean regional genetic diversity adjusted for sample size such that HTR = [(n)/(n – 1)](1 – Σpi2),
where n is the sample size; adapted from HT (Nei and Chesser 1983).
c
HT is the total genetic diversity, adjusted for sample size, such that HT = [(ñ)/(ñ – 1)](1 – Σpi2), where ñ is
the harmonic mean of the regional sample sizes (Nei and Chesser 1993).

Fig. 1. Genotype distribution among five regions. Isolates associated with the same genotype have identical alleles at all eight loci. The
number above each bar indicates the number of different VCGs associated with each genotype.

common to California and Japan. Though it is possible that
these two genotypes lurk somewhere in Mexico, their high
frequency of occurrence in the SE U.S.A. (37% and 21%,
respectively) implicates the latter is the likely founding
source of the California and Japanese introductions of iden-
tical genotypes. Because the disease was well established in
the SE U.S.A. long before it was known in California or Ja-
pan, it is most likely that the path of spread was westward.
Vegetative compatibility between the Japanese and Califor-
nia isolates of the same genotype suggests both populations
trace their origins to the SE U.S.A. Because pitch canker
was reported in Japan after it was reported in California, it is

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Table 3. Genotype (multi-locus) diversity of Fusarium circinatum populations within regions.

Multi-locus diversity
Location (HM)a HM adjusted for sample size (HEM)b Stoddart’s genotypic diversity (GO)c
California 1.42 0.40 3.32 ± 0.39
SE U.S.A. 1.79 0.61 4.69 ± 1.70
Japan 0.00 0.00 1.00 ± 0.00
South Africa 1.91 0.92 6.40 ± 2.46
Mexico 1.89 0.82 6.25 ± 1.46
a
Shannon’s information statistic, HM = –Σgilngi; gi = the frequency of the ith genotype (denoted by Sheldon
(1969) as H and by Goodwin et al. (1993) as M).
b
Sheldon’s index, HEM = HM/HMAX; where HMAX = lnS, and S = sample size (Sheldon 1969; referred to by
Goodwin et al. (1993) as M$ ).
c
GO = 1/Σgi2 and variance ≈ 4/N(GO2)(GO2Σgi3 – 1) (Stoddart 1983; Stoddart and Taylor 1988).

Table 4. Genetic distance, DARCa, and DLRb, of Fusarium circinatum between each pair of regions.

California SE U.S.A. Japan South Africa Mexico

California — 0.46 0.49 0.48 0.60
SE U.S.A. 0.96 — 0.37 0.57 0.56
Japan 1.92 0.26 — 0.78 0.64
South Africa 1.63 2.23 3.31 — 0.56
Mexico 2.99 1.81 1.95 1.26 —
a
Values for DARC, listed above the diagonal, were estimated using the equation DARC = [(l/L)Σ(2Θ/Π)2]0.5,
where Θ = cos–1 Σ(xiyi)0.5 and L is the number of loci (Cavalli-Sforza and Edwards 1967 as described by
Swofford et al. 1996). The upper value is 1.0.
b
Values for DLR, listed below the diaganol, were computed using the assignment calculator from
http://www.biology.ualberta.ca/jbrzusto/Doh.html (Paetkau et al. 1997).

possible the pathogen in Japan was introduced indirectly via
California, but it may also have come directly from the SE
U.S.A.
Although our small sample precludes a definite conclu-
sion, the total lack of diversity in the Japanese collection
strongly implies the population arose from a recent introduc-
tion, rather than being endemic to that country. Furthermore,
that F. circinatum in Japan is identical to isolates from North
America, in both genotype and vegetative compatibility, in-
dicates that the fungus entered the country from the outside,
rather than originating locally, and did not host-jump from
F. subglutinans infected sugarcane grown on the southern is-
lands of Japan (Kobayashi and Muramoto 1989).
The South African population includes a large number of
compatibility groups (Viljoen et al. 1997), and, like the
Mexican population, no isolates from different VCGs shared
the same genotype. These high levels of genotypic and VCG
diversity are suggestive of an outcrossing population. Fur-
thermore, some evidence, discussed below, indicates the
South African population may have been derived from the
Mexican population. Therefore, South Africa may have di-
rectly inherited some of its genetic diversity from Mexico
(HTR is higher in South Africa than in any other region ex-
cept Mexico).
Because the pitch canker infestation in South Africa origi-
nated in a single nursery where it killed millions of seed-
lings, it is quite possible the original source of inoculum was
in the seeds used to plant the nursery. Though South Africa
has restrictions on imported Mexican pine seed, it is con-
ceivable the pitch canker pathogen in this P. patula nursery
entered South Africa on seed collected in Mexico, where
P. patula is native (Wingfield et al. 1999). This connection is
supported by an allele (locus 74: allele 6300) that is present
at a high frequency in both populations, but found in no
other region (Table 1). These two populations also share a
common genotype (19). Despite the possibility of outcross-
ing in both of these geographic regions, it is more likely that
the shared genotype is representative of a clonal line, rather
than a coincidental, identical reassortment. The high intrare-
gional diversity within both Mexico and South Africa, indi-
cates that a random match at all eight loci would be unlikely
(p = 0.02, if the probability is based on the average allele
frequency at each locus in both populations).
Clearly the data are not definitive on the origin of pitch
canker in South Africa, given the moderately short genetic
distance between the South Africa and California popula-
tions (DARC = 0.48 and DLR = 1.63). Furthermore, because
the markers were chosen from a pool of fragments that was
biased for differences between a California isolate and a
South Africa isolate, the genetic distance between these two
populations may be exaggerated, relative to other compari-
sons. Whereas it is possible that South Africa received its
isolates from multiple introductions (perhaps Mexico and
California), it would be highly coincidental if the original
1990–1991 pitch canker outbreak at a single nursery (where
all isolates used in this study are from) had multiple sources.
The apparent association between California and South Af-
rica can be explained, in part, by the high frequency of allele
1500 at locus 220 in both geographic regions. The absence
of this allele in Mexico may only be an artifact of our lim-
ited sample of isolates from this area. The prevalence of this
allele, as well as allele 400 at locus 80 and allele 600 at lo-
cus 82, in both South Africa and California could also be the
result of a founder effect common to both populations.

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716
The high level of genetic diversity in Mexico implies that
the fungus has been in that region longer than the recent first
report would suggest (Guerra-Santos and Cibrian-Tovar
1991). Though the Mexican region includes isolates from a
broader geographic area than the regions to which it is being
compared, lumping the SE U.S.A. and California popula-
tions (0.31) still does not match the genetic diversity exhib-
ited in Mexico (HTR = 0.46). Even correcting for repetitive
sampling of identical VCGs within confined localities in
California and the SE U.S.A. does not raise the genetic di-
versity (0.33) to the level found in Mexico. Concomitantly,
in Mexico the disease is quite widespread (Guerra-Santos
and Cibrian-Tovar 1991), but only reaches epidemic propor-
tions in off-site plantings, supportive of the notion that the
fungus may be an endemic and balanced member of the na-
tive forest communities in that region. If this fungal patho-
gen indeed originated in Mexico, it would follow the trend
found in other devastating plant diseases that appear to have
originated in their host’s center of diversity as benign co-
inhabitants, and only cause epidemics outside their natural
communities (Schmidt 1978; Harrington and Wingfield
1998). Two classic examples of this phenomenon are
Seiridium canker of Monterey Cypress (Cupressus macro-
carpa) and Dothistroma needle blight of Monterey pine.
Both diseases are virtually undetectable within their host’s
native geographic range, but become aggressive pathogens
outside that range (Sinclair et al. 1987).
Though it seems most likely that the pitch canker fungus
has been recently introduced in Japan, California, and South
Africa, and is endemic in Mexico, the history of the disease
in the SE U.S.A. is less clear. The moderately short genetic
distance between Mexico and the SE U.S.A. suggests a rela-
tionship between the isolates in these two regions. More ex-
tensive surveying and sampling in northern Mexico might
help establish if the SE U.S.A. population is another disjunct
population, also derived from Mexico, or if it is part of a
more continuous metapopulation that spans central Mexico
and the SE U.S.A.
Acknowledgements
Thanks to M. Neff for guidance in performing the ge-
nomic subtraction procedure, to J. Rine for providing labora-
tory space and resources for this procedure and for DNA
sequencing, to J. Correll for nits of the SE U.S.A. isolates,
and to M. Ishikawa and P. Tran for lab assistance. Thanks
also to S. Clark, M. Garbelotto, J. Petersen, E. Bonello, J.
Taylor, and M. Rademacher for helpful reviews of the manu-
script. We also are grateful to M. Wingfield, Y. Sato, M.
Muramoto, and J. Guerra-Santos for donating isolates from
their collections, and to D. Cibrian-Tovar for assistance lo-
cating pitch canker in Mexico. Funding provided in part
by U.S. Department of Agriculture – National Research Ini-
tiative grant 96-35302-3821 and an Environmental Protec-
tion Agency STAR graduate fellowship to K.W.
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