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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
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within the California population or the affinity of this popu- scribed by Rosenberg et al. (1994) and the remaining tracer frag-
lation with those found in other parts of the world. Insight ments were used as templates for amplification with tracer-specific
into these questions requires polymorphic markers, which primers.
have proven difficult to obtain (Wikler et al. 1996). For this The fragments remaining after the third subtractive hybridiza-
reason a genomic subtraction procedure (Rosenberg et al. tion were cloned as follows. An aliquot of the remaining fragments
1994) was used to identify polymorphic loci. Allelic differ- 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
ences at these loci provided a measure of the relationships
72°C. Shrimp alkaline phosphatase was added and allowed to incu-
between isolates associated with different VCGs in the Cali- bate for 1 h at 37°C to dephosphorylate the dNTPs. The products
fornia population of F. circinatum and a preliminary assess- were then denatured at 99°C for 10 min and incubated at 72°C for
ment of the genetic distances separating this population from 2 h, to eliminate poorly hybridizing fragments from the subsequent
those in the SE U.S.A., Japan, Mexico, and South Africa. 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
Materials and methods were purified (Bio-Rad Quantum Prep plasmid mini-prep kit).
Cloned fragments were screened, by labeling them with dig-
Isolate collections oxigenin (DIG), and using them in Southern hybridizations with
Seventy-six isolates of F. circinatum were included in this study 1.5 µg of membrane-bound HindIII digested DNA from each of
(Table 1). Thirty-four isolates from California and 19 from the SE several isolates that collectively represented the worldwide dis-
U.S.A. were from collections described previously (Correll et al. tribution of F. circinatum. All Southern blotting was done using
1992; Gordon et al. 1996). The South African isolates were col- Boehringer Mannheim’s DIG nonradioactive nucleic acid labeling
lected from the original nursery outbreak in 1990–1991 (Viljoen et and detection system, performed according to the manufacturer’s
al. 1994, 1997). Sources of the isolates from Japan and Mexico instructions. Eight fragments (termed loci 18, 60, 70, 74, 80, 82,
are given in Table 1. All isolates not previously described (those 98, and 220) that revealed polymorphisms were selected for the
from Mexico and Japan) were identified as F. circinatum based on study, and each of these fragments was sequenced.
microscopic morphology on carnation leaf agar, and verified as
pine pathogens by lesion development in a pathogenicity test on The M13 reverse and M13(–20) forward primers were each used
Monterey pine (P. radiata), as described by Gordon et al. (1996). 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-
Vegetative compatibility groupings structions, and the reactions were carried out in an Ericomp ther-
Vegetative compatibility groups (VCGs) for the California, SE mocycler using 25 cycles of the following program: 12 s at 96°C,
U.S.A., and South Africa isolates were determined previously 7 s at 50°C, 4 min at 60°C. The extension products were purified
(Gordon et al. 1996; Correll et al. 1992; Viljoen et al. 1997). The by precipitating them in 70% ethanol – 0.5 mM MgCl2. Both
remaining isolates were assigned to VCGs using standard protocols strands of the fragments were sequenced using an ABI automatic
(Puhalla 1985). sequencer. Primers were designed to match the ends of each frag-
ment’s sequence, to amplify the homologous region in other iso-
DNA extraction and quantification lates and potentially capture the polymorphism.
DNA was extracted as described previously (Jacobson and PCR reactions using the custom primers were carried out in
Gordon 1990), with the addition of a second phenol–chloroform 25-µL volumes containing 200 µM of each dNTP, 0.5 µM of each
extraction. The DNA was quantified by spotting 4 µL of a dilution primer, 2.5 µL 10× reaction buffer (includes 15 mM MgCl2),
series onto a UV light box adjacent to DNA standards of known 0.625 U Taq polymerase, and 50–100 ng template DNA. The am-
concentrations, adding an equal amount of 2 µg/mL ethidium bro- plification regime was 30 s at 95°C (the samples were placed in the
mide, and comparing the intensity of fluorescence. A fluorometer thermocycler when the machine came up to temperature) followed
(Hoefer Scientific Instruments) was also used to quantify the DNA by 30 cycles of: 30 s at 94°C, 35 s at 50°C, 3 min at 72°C, and a
used for Southern blotting. 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,
Marker identification and screening and 98 primer sets, in which HotStarTaq and buffer were used
To obtain polymorphic markers, a genomic subtraction proce- (Qiagen), along with a 15-min initial denaturation step at 95°C.
dure developed by Rosenberg et al. (1994) was used. This involved The PCR products were run on 1% agarose gels, except for those
a series of DNA–DNA hybridizations to remove restriction frag- amplified with primer set 98 forward (F) and 98 reverse (R), which
ments common to two isolates and thereby obtaining a pool of were run on 2% agarose gels.
fragments enriched for sequences unique to one isolate. All poly- For four of the eight loci (18, 60, 98, and 220), polymorphisms
merase chain reaction (PCR) protocols, digestions, and ligations were detectable using PCR with the following primer sets: 18F
were performed as described by Rosenberg et al. (1994), using the (TTG AAT CAC AAC TTA GAG TGG CTC) and 18R (AAG CTT
identical reagents, PCR primers, and DNA adaptors, with the ex- CCG CGT GCC ATT CT); 60F (TAC CCA AAG AGA CCT CCG
ception of Taq polymerase and 10× buffer, which were obtained ATG) and 60R (AAC TTC AGG TCT TGA TCC TAA GG); 98F
from Perkin Elmer rather than Boehringer Mannheim. Our use of (TTG GAA GCA TGA TGG TGG C) and 98R (AGT TCA AGA
the procedure is briefly described below. CTT AAT CAC TCC TGG); and 220F (ATA TTA TCC ATC TTC
Total DNA from a California isolate (PF-1) was digested with GAC TTT ACC) and 220R (GCG TTG TTG TCC CGT ATC C).
HindIII. These restriction fragments were ligated to DNA adaptors However, the largest PCR product for 60F or 60R was difficult to
for PCR amplification and size-fractionated (250–1000 bp). This amplify. Consequently, the null allele (i.e., no amplicon) was con-
representation of the original genome was termed the tracer. The firmed by using the cloned fragment (DIG labeled) representing lo-
same procedure was used to obtain a representation of an isolate cus 60 in Southern hybridizations with 1.5 µg of HindIII digested
from South Africa (SA0024), but with a different set of DNA adap- DNA from the isolate in question. Similarly, Southern hybridiza-
tors and a slightly larger size fraction (120–1200 bp). This repre- tions were used to confirm a subset (10 of 15) of null alleles for
sentation was termed the driver. Three cycles of hybridization were primer set 18F or 18R to verify that the absence of PCR products
used to remove sequences common to both tracer and driver, as de- reflected the absence of a region, rather than a refractory segment
© 2000 NRC Canada
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of DNA. The remaining four loci (70, 74, 80, and 82) were assayed Estimates of genetic distances between each pair of re-
using nonradioactive Southern hybridizations as described above. gions are given in Table 4. The two estimates of genetic dis-
tance had a correlation coefficient of R = 0.86. By both
Genetic diversity and population structure analyses measures of genetic distance (DARC, DLR), Japan and SE
The allele frequency at each locus was calculated for each geo- U.S.A. have the closest relationship (DARC = 0.37 and DLR =
graphic region using the program Relatedness 5.0.4 (Goodnight 0.26), followed by California and the SE U.S.A. (DARC =
Software, http://gsoft.smu.edu/GSoft.html). Total genetic diversity 0.46 and DLR = 0.96). Mexico and South Africa, overall, are
for all geographic regions combined (HT) was estimated using Nei
genetically more distant from the other regions. According
and Chesser’s (1983) equations for calculating genetic diversity
from limited and unequal sample sizes. Because all null alleles to DARC, Mexico is equally close to South Africa and SE
from each locus were assumed to be identical, the diversity mea- U.S.A. (0.56), but DLR ranks Mexico closer to South Africa
surements are conservative. Total genetic diversity within each re- than to any other region (1.26).
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. Discussion
Genotypic (multi-locus) diversity was determined using Shan-
non’s information statistic (MacArthur and MacArthur 1961; Shel- Correll et al. (1992) found 45 VCGs among a sample of
don 1969; as used by Goodwin et al. 1993) and Stoddart’s 117 F. circinatum isolates in Florida; whereas, only 8 VCGs
genotypic diversity statistic (Stoddart 1983; Stoddart and Taylor were identified among 170 isolates collected throughout the
1988). Sheldon’s equitability index (Sheldon 1969) as used by range of pitch canker in California (Gordon et al. 1996). The
Goodwin et al. (1993) was calculated to account for the influence greater VCG diversity in Florida was suggestive of an out-
of sample size on the observed diversity. Variance of Stoddart’s crossing population, while the minimal VCG diversity in
multi-locus diversity statistic was estimated according to the for-
California was consistent with asexual propagation (Correll
mula derived by Stoddart and Taylor (1988).
Genetic distance between populations was estimated by using
et al. 1992; Gordon et al. 1996). A subset of these isolates
two different algorithms (Paetkau et al. 1998): (i) the Cavalli- was included in the present study. Surprisingly, in the SE
Sforza and Edwards (1967) arc distance measurement (DARC), as U.S.A. and in California, isolates in different VCGs often
described by Swofford et al. (1996) and (ii) DLR, the genotype like- shared the same genotype. For example, genotype 5 is asso-
lihood ratio distance (Paetkau et al. 1997). ciated with seven VCGs, genotype 6 with five VCGs, and
genotype 1 with three VCGs.
The common genotypes among a diverse set of vegetative
Results
compatibility groups imply that new VCGs have been gener-
Eight polymorphic loci were identified, with between two ated by mutation within a clonal lineage, rather than through
and four alleles per locus. Alternative alleles were desig- outcrossing. If all eight loci, on which the genotypes are
nated by the size of the fragment(s), in base pairs, identified based, were unlinked and re-assorted during meiosis, it is
at each locus (Table 1). Among the 76 isolates examined, highly unlikely that six SE U.S.A. VCGs would have identi-
there were 26 unique locus/allele combinations, (i.e., haplo- cal genotypes (p < 0.01, based on the product of the allele
types). Seven haplotypes were found only in Mexico (locus/ frequencies at each of the six polymorphic loci within the
allele combinations: 60/3000,1800,950; 80/1640; 80/2100; SE U.S.A.). Furthermore, we have observed cross-
82/6000; 82/450; 98/null; and 220/null). No haplotypes were compatibility among some isolates from three of the most
unique to the SE U.S.A. or to Japan, whereas both the Cali- prevalent VCGs in California (C1, C2, and C4) (unpublished
fornia and South Africa populations each had one unique results) which share a common genotype (1). These observa-
haplotype, 80/null and 82/900, respectively. Allele frequen- tions further support a close relationship between these
cies and diversity statistics are shown in Table 2. VCGs and may indicate that isolates with identical geno-
Combining all eight loci yielded 25 multi-locus geno- types (based on the eight loci assayed) have lost their com-
types, among 76 isolates (Table 1); their distribution among mon VC identities through mutations at one or more vic loci
regions is illustrated in Fig. 1. Three genotypes were shared (Leslie 1993). In cases such as this, VCGs may be more sen-
among isolates from different regions: one between Japan, sitive indicators of population diversity than molecular
SE U.S.A. (both Florida and Georgia), and California (geno- markers.
type 5); a second between California and the SE U.S.A. (ge- Although the extent of VCG diversity provides a measure
notype 6); and the third between Mexico and South Africa of variability within a population, it leaves open the question
(genotype 19). Each of these transregional genotypes was of how VCGs are related. Isolates associated with the same
composed of isolates from different VCGs. Additionally, VCG are not necessarily part of the same clonal lineage, be-
isolates representing the most common California genotype cause recombination can result in genotypically different or-
(1) were associated with three different VCGs (Table 1). The ganisms sharing an identical suite of alleles at all vic loci.
two most common genotypes in the SE U.S.A. accounted for Thus, VCG data combined with multi-locus genotypes can
over half the isolates (57.9%) in this region; whereas no ge- provide a more complete picture of interregional relation-
notype was represented more than twice in either Mexico ships, as discussed below for the Japanese and California
or South Africa, and only the genotype common to both isolates that share VCG C7 and genotype 5.
regions spanned different VCGs. Multi-locus (genotypic) di- There is strong evidence linking the California, Japanese,
versity was highest in South Africa (HEM = 0.92) and Mex- and SE U.S.A. populations of F. circinatum. These regions
ico (HEM = 0.82) (Table 3); it was lowest in Japan (HEM = are separated by relatively short genetic distances and share
0.00), where all isolates sampled had identical genotypes a common genotype (5). An additional genotype (6) is
(Table 3). shared between California and SE U.S.A., and a VCG is
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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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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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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 the SE U.S.A. long before it was known in California or Ja-
these two genotypes lurk somewhere in Mexico, their high pan, it is most likely that the path of spread was westward.
frequency of occurrence in the SE U.S.A. (37% and 21%, Vegetative compatibility between the Japanese and Califor-
respectively) implicates the latter is the likely founding nia isolates of the same genotype suggests both populations
source of the California and Japanese introductions of iden- trace their origins to the SE U.S.A. Because pitch canker
tical genotypes. Because the disease was well established in was reported in Japan after it was reported in California, it is
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Table 4. Genetic distance, DARCa, and DLRb, of Fusarium circinatum between each pair of regions.
possible the pathogen in Japan was introduced indirectly via supported by an allele (locus 74: allele 6300) that is present
California, but it may also have come directly from the SE at a high frequency in both populations, but found in no
U.S.A. other region (Table 1). These two populations also share a
Although our small sample precludes a definite conclu- common genotype (19). Despite the possibility of outcross-
sion, the total lack of diversity in the Japanese collection ing in both of these geographic regions, it is more likely that
strongly implies the population arose from a recent introduc- the shared genotype is representative of a clonal line, rather
tion, rather than being endemic to that country. Furthermore, than a coincidental, identical reassortment. The high intrare-
that F. circinatum in Japan is identical to isolates from North gional diversity within both Mexico and South Africa, indi-
America, in both genotype and vegetative compatibility, in- cates that a random match at all eight loci would be unlikely
dicates that the fungus entered the country from the outside, (p = 0.02, if the probability is based on the average allele
rather than originating locally, and did not host-jump from frequency at each locus in both populations).
F. subglutinans infected sugarcane grown on the southern is- Clearly the data are not definitive on the origin of pitch
lands of Japan (Kobayashi and Muramoto 1989). canker in South Africa, given the moderately short genetic
The South African population includes a large number of distance between the South Africa and California popula-
compatibility groups (Viljoen et al. 1997), and, like the tions (DARC = 0.48 and DLR = 1.63). Furthermore, because
Mexican population, no isolates from different VCGs shared the markers were chosen from a pool of fragments that was
the same genotype. These high levels of genotypic and VCG biased for differences between a California isolate and a
diversity are suggestive of an outcrossing population. Fur- South Africa isolate, the genetic distance between these two
thermore, some evidence, discussed below, indicates the populations may be exaggerated, relative to other compari-
South African population may have been derived from the sons. Whereas it is possible that South Africa received its
Mexican population. Therefore, South Africa may have di- isolates from multiple introductions (perhaps Mexico and
rectly inherited some of its genetic diversity from Mexico California), it would be highly coincidental if the original
(HTR is higher in South Africa than in any other region ex- 1990–1991 pitch canker outbreak at a single nursery (where
cept Mexico). all isolates used in this study are from) had multiple sources.
Because the pitch canker infestation in South Africa origi- The apparent association between California and South Af-
nated in a single nursery where it killed millions of seed- rica can be explained, in part, by the high frequency of allele
lings, it is quite possible the original source of inoculum was 1500 at locus 220 in both geographic regions. The absence
in the seeds used to plant the nursery. Though South Africa of this allele in Mexico may only be an artifact of our lim-
has restrictions on imported Mexican pine seed, it is con- ited sample of isolates from this area. The prevalence of this
ceivable the pitch canker pathogen in this P. patula nursery allele, as well as allele 400 at locus 80 and allele 600 at lo-
entered South Africa on seed collected in Mexico, where cus 82, in both South Africa and California could also be the
P. patula is native (Wingfield et al. 1999). This connection is result of a founder effect common to both populations.
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The high level of genetic diversity in Mexico implies that Cavalli-Sforza, L.L., and Edwards, A.W.F. 1967. Phylogenetic
the fungus has been in that region longer than the recent first analysis: Models and estimation procedures. Am. J. Hum.
report would suggest (Guerra-Santos and Cibrian-Tovar Genet. 19: 233–257.
1991). Though the Mexican region includes isolates from a Correll, J.C., Gordon, T.R., and McCain, A.H. 1992. Examination
broader geographic area than the regions to which it is being of genetic diversity in California and Florida populations of the
compared, lumping the SE U.S.A. and California popula- pitch canker fungus Fusarium subglutinans f. sp. pini. Phyto-
tions (0.31) still does not match the genetic diversity exhib- pathology, 82: 415–420.
ited in Mexico (HTR = 0.46). Even correcting for repetitive Dwinell, L.D. 1999. Global distribution of the pitch canker fungus.
sampling of identical VCGs within confined localities in In Current and potential impacts of pitch canker in radiata pine.
Proceedings of IMPACT Monterey workshop, November 30 –
California and the SE U.S.A. does not raise the genetic di-
December 3, 1998, Monterey, Calif. Edited by M.E. Devey, A.C.
versity (0.33) to the level found in Mexico. Concomitantly,
Matheson, and T.R. Gordon. CSIRO, Australia. pp. 54–57.
in Mexico the disease is quite widespread (Guerra-Santos
Dwinell, L.D., Barrows-Broaddus, J.B., and Kuhlman, E.G. 1985.
and Cibrian-Tovar 1991), but only reaches epidemic propor- Pitch canker: a disease complex of southern pines. Plant Dis. 69:
tions in off-site plantings, supportive of the notion that the 270–276.
fungus may be an endemic and balanced member of the na- Goodwin, S.B., Saghai Maroof, M.A., Allard, R.W., and Webster,
tive forest communities in that region. If this fungal patho- R.K. 1993. Isozyme variation within and among populations of
gen indeed originated in Mexico, it would follow the trend Rhynchosporium secalis in Europe, Australia and the United
found in other devastating plant diseases that appear to have States. Mycol. Res. 97: 49–58.
originated in their host’s center of diversity as benign co- Gordon, T.R., Storer, A.J., and Okamoto, D. 1996. Population
inhabitants, and only cause epidemics outside their natural structure of the pitch canker pathogen, Fusarium subglutinans
communities (Schmidt 1978; Harrington and Wingfield f.sp. pini, in California. Mycol. Res. 100: 850–854.
1998). Two classic examples of this phenomenon are Guerra-Santos, J.J. 1999. Pitch canker in Monterey pine in Mexico.
Seiridium canker of Monterey Cypress (Cupressus macro- In Current and Potential Impacts of Pitch Canker in Radiata
carpa) and Dothistroma needle blight of Monterey pine. Pine. Proceedings of IMPACT Monterey Workshop, Monterey,
Both diseases are virtually undetectable within their host’s Calif., 30 November – 3 December 1998. Edited by M.E. Devey,
native geographic range, but become aggressive pathogens A.C. Matheson, and T.R. Gordon. CSIRO, Australia. pp. 58–61.
outside that range (Sinclair et al. 1987). Harrington, T.C., and Wingfield, M.J. 1998. Diseases and the ecol-
Though it seems most likely that the pitch canker fungus ogy of indigenous and exotic pines. In Ecology and bio-
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tensive surveying and sampling in northern Mexico might DNA as an indicator of relationships between populations of Fu-
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script. We also are grateful to M. Wingfield, Y. Sato, M. Strobeck,C. 1997. An empirical evaluation of genetic distance
Muramoto, and J. Guerra-Santos for donating isolates from statistics using microsatellite data from bear (Ursidae) popula-
their collections, and to D. Cibrian-Tovar for assistance lo- tions. Genetics, 147: 1943–1957.
cating pitch canker in Mexico. Funding provided in part Paetkau, D., Shields, G.F., and Strobeck, C. 1998. Gene flow be-
tween insular, coastal and interior populations of brown bears in
by U.S. Department of Agriculture – National Research Ini-
Alaska. Mol. Ecol. 7: 1283–1292.
tiative grant 96-35302-3821 and an Environmental Protec-
Puhalla, J.E. 1985. Classification of strains of Fusarium oxysporum
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