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Previous Article | Next Article 
Applied and Environmental Microbiology, October 1998, p. 4007-4014, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Polymorphisms in Phytophthora infestans:
Four Mitochondrial Haplotypes Are Detected after PCR Amplification
of DNA from Pure Cultures or from Host Lesions
Gareth W.
Griffith* and
David S.
Shaw
School of Biological Sciences, University of
Wales, Bangor, Gwynedd, LL57 2UW, Wales, United Kingdom
Received 23 February 1998/Accepted 21 July 1998
 |
ABSTRACT |
Four pairs of primers were designed for PCR amplification of known
polymorphic regions of the mitochondrial genome of Phytophthora infestans. Digestion of the amplified products with restriction enzymes allows identification of previously identified haplotypes. Product P2 cut with MspI uniquely identifies haplotypes Ib
and IIa, while types Ia and IIb are differentiated by digestion of product P4 with EcoRI. Digestion of products P1 and P3 gave
results similar to that with digestion of P4, but amplification of
these products was less robust. Thus, all four common haplotypes are identified by amplifying and digesting products P2 and P4.
Identification of haplotypes was also possible from DNA extracted
directly from small, late-blight lesions on both tomato and potato
leaves, making isolation of the fungus unnecessary. A rapid
and efficient method of monitoring changes in the pathogen population
is facilitated. These PCR primers were also useful for differentiating
other Phytophthora species.
| |
INTRODUCTION |
Populations of Phytophthora
infestans from Central Mexico are highly diverse, indicating that
this area is the center of origin of the pathogen (14, 16,
20). In contrast, populations from other parts of the world were
almost totally monomorphic for allozyme and restriction fragment
length polymorphism (RFLP) loci. Over the last 20 years, new
genotypes migrating from Mexico have become established and have
replaced the old monomorphic genotype around the world (10, 14,
16, 20, 21, 30). As some new genotypes are more pathogenic or
more resistant to curative systemic fungicides (6, 15, 23),
rapid identification of the strains is crucial so that optimal methods
of control can be implemented. The development of such identification
and detection methods is considered a high priority by the agriculture
industry (34).
Polymorphisms in mitochondrial DNA (mtDNA) of P. infestans are particularly useful for monitoring pathogen
populations; they are easily detected and, because they are
uniparentally (and probably maternally) inherited (35),
ideal for tracing lines of descent. These polymorphisms have previously
been studied by hybridization of digested total DNA with labelled,
cloned mtDNA (8, 17, 28) and digestion of isolated
mtDNA (12, 13, 24). Carter et al. (3, 4)
defined two mitochondrial types, type I and type II, by digestion of
total DNA with the frequently cutting restriction enzymes
MspI or CfoI (which produce bands of mtDNA on
a background smear of nuclear DNA upon separation). Type II differed
from type I by an insert of 1.6 kb and rearrangement of flanking
sequences. Type I was further differentiated into haplotypes Ia and Ib,
the latter possessing an additional MspI site; similarly,
type II was subdivided into haplotypes IIa and IIb, the latter
possessing an additional CfoI site. The recent sequencing of
the mitochondrial genome of P. infestans (5, 29) allows the design of primers to amplify by PCR the known polymorphic sequences of the genome.
Our objective in this study was to develop a PCR-based method for the
rapid detection of mtDNA polymorphisms in P. infestans, using small amounts of fungus from agar cultures or
infected host tissue. A secondary goal was to test whether the same
primers can be used to amplify mtDNA from other species of
Phytophthora to aid identification and diagnoses.
| |
MATERIALS AND METHODS |
Isolates of P. infestans and other
Phytophthora spp.
The cultures used in this
investigation were from the UW Bangor collection or imported to the
United Kingdom via the International Mycological Institute.
P. infestans was isolated on ryeA medium containing
antibiotics (rifamycin, ampicillin, and nystatin), as described by
Griffith et al. (22). Long-term stocks were maintained as
agar plugs in 5% dimethyl sulfoxide under liquid nitrogen
(31). Details of the origins of cultures are given in Table
1.
Isolates were grown routinely on ryeA agar at 18°C and stored under
liquid nitrogen. Cultures of other Phytophthora spp. were
from the UW Bangor collection and were cultured as described above.
Sources of host lesions.
Leaf and stem lesions of late
blight were collected from naturally infected crops in the field
(various cultivars) or were grown on detached leaflets of potato (cv.
Maris Piper) or tomato (cv. FMX-93) inoculated with droplets of
zoospores. Leaflets were incubated in moist chambers at 18°C for 3 to
4 days (potato) or 5 to 7 days (tomato). Some leaflet lesions were air
dried by incubation in an open tray in the laboratory for 7 days.
Extraction of DNA.
DNA was extracted by using a variation of
the method of Doyle and Doyle (9) with modifications devised
by DuTeau and Leslie (11). Briefly, this involved cutting
small slabs (ca. 1 cm2) of agar-containing mycelium from a
culture grown on ryeA medium and subjecting these to rapid freezing
twice with liquid nitrogen (poured into a microcentrifuge tube
containing the slab and allowed to evaporate; no maceration), followed
by incubation in 800 µl of modified CTAB extraction buffer (100 mM
NaCl, 100 mM Tris-HCl [pH 8.0], 1.4 M NaCl, 2% CTAB
[hexadecyltrimethylammonium bromide], 20 mM EDTA [sodium salt, pH
8.0]) for 60 min at 65°C. After removal of the agar slab with a
sterile toothpick, 600 µl of water-saturated chloroform was added and
the tube was vortex mixed for 10 s and microcentrifuged
(17,000 × g) for 10 min. Six hundred microliters of
the upper, aqueous layer was transferred to a fresh microcentrifuge tube, and 0.6 volume of isopropanol (360 µl) was added. The tubes were vortex mixed, left to stand at room temperature for 5 min, and
microcentrifuged (17,000 × g) for 10 min. The liquid
was decanted, and the DNA pellet was washed with 1 ml of 70% (vol/vol)
ethanol. Tubes were vortexed and left at 65°C for 20 min before
microcentrifugation (17,000 × g) for 10 min as before.
After the tubes had been left open at 37°C for 30 min to remove the
last traces of 70% ethanol, the DNA pellet was resuspended in 100 µl
of TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]) and stored at
20°C. Quality and concentration were checked by electrophoresis of
5 µl of the DNA solution on a 0.8% agarose gel.
DNA was extracted from late-blight lesions. Pieces of colonized leaf (5 by 5 mm), dried or fresh, were excised and ground in a microcentrifuge
tube with fine sand (ca. 50 mg) and liquid nitrogen with a disposable
polythene microcentrifuge grinder (Anachem Ltd.). Thereafter, the
extraction procedure was the same as that described above. The purified
DNA was dissolved in TE (500 µl); 1 µl was used for PCR.
PCR-RFLP procedure.
PCRs with a model PC2 PCR thermal cycler
(Techne Ltd., Cambridge, United Kingdom) were optimized to maximize
yield of the desired PCR product and reduce levels of nonspecific
products (data not shown). Amplification was as follows for all primer combinations (final concentrations): deoxynucleoside triphosphates, 200 µM each; MgCl2, 2.75 mM; oligonucleotide primer (Cruachem Ltd., Glasgow, United Kingdom; see Table 2), 0.325 mM each; ethidium bromide, 0.2 µg ml
1; bovine serum albumin, 160 µg
ml1; 1× Thermo buffer (50 mM KCl, 10 mM Tris-HCl [pH
8.3]); Taq DNA polymerase, 0.2 µl (1 U). All
reagents were supplied by Promega Ltd. (Southampton, United Kingdom)
unless stated otherwise. One to ten nanograms of total DNA was mixed
with 20 µl of a master mix of the other PCR reactants in 0.5-ml
microcentrifuge tubes (final volume, 25 µl) and overlaid with 50 µl
of mineral oil. The PCR conditions were as follows: 1 cycle of 94°C
for 90 s and 40 cycles of 94°C for 40 s, 55°C for 60 s, and 72°C for 90 s.
Three to four microliters of the amplified DNA was digested with the
following restriction enzymes in a 20-µl volume restriction digest at
37°C for a period lasting between 1 h and overnight as follows:
P1, CfoI; P2, MspI; P3, EcoRI; and P4,
EcoRI. (EcoRI had greater activity in Promega
Multi-Core buffer than in the manufacturer's recommended buffer). The
digested DNA samples were then mixed with 5 µl of gel-loading buffer
(20% Ficoll, 0.02% bromophenol blue, 0.002% xylene cyanol), and 15 µl was loaded into a slot on a 2% agarose gel (Gibco BRL Ltd.) in
1× Tris-borate-EDTA (TBE) buffer (containing 0.1 µg of ethidium
bromide ml1). The gel was run at 10 V cm1
for 60 to 90 min. Restriction patterns were visualized with a UV
transilluminator at 254 nm, and the images were recorded by a gel
documentation system (Appligene, Chester-le- Street, United Kingdom).
PCR amplification of longer fragments of the mitochondrial genome was
achieved by a modification of the long PCR technique of Barnes
(2) as follows (final concentrations): deoxynucleoside triphosphates, 325 µM each; 0.325 µM (each) oligonucleotide primer; ethidium bromide, 0.2 µg ml1; 1× long PCR buffer (50 mM Tris-HCl [pH 9.2 at 25°C], 16 mM
(NH4)2SO4, 1.75 mM
MgCl2). A mixture of Taq DNA polymerase
(Promega) and Tli polymerase (250 U of Taq, 1 U
of Tli polymerase) was made, and 0.2 µl (i.e., 1 U of
Taq plus 0.004 U of Tli polymerase) was added to
each reaction in thin-walled 0.5-ml tubes. PCR cycling conditions were
as follows: 1 cycle of 95°C for 1 s, then 94°C for 20 s;
20 cycles of 95°C for 1 s, 94°C for 10 s, 54°C for
60 s, and 67.5°C for 5 min; 8 cycles of 95°C for 1 s,
94°C for 10 s, and 67.5°C for 6 min.
| |
RESULTS |
Design of primers for analysis of mitochondrial haplotypes.
The complete mitochondrial genome of P. infestans
(Ib mtDNA haplotype; 37,957 bp) has been sequenced, and parts
of it have been published (5, 29; also
http://megasun.bch.umontreal.ca/People/lang/species/phyti/phyti.html). Restriction site characteristics of the four haplotypes identified by
Carter et al. (4; referred to here as Carter
haplotypes) were located within the complete mitochondrial genome
sequence (kindly supplied prior to full publication by B. F. Lang). Based on this sequence, pairs of oligonucleotide primers (listed
in Table 2) were designed to amplify four
regions of the mitochondrial genome in which polymorphisms were known
to occur (Fig. 1). P2 is the site of the
Ia/Ib polymorphism, while P1, P3, and P4 are sites of
polymorphism between haplotype Ia/Ib and haplotype IIa/IIb. The primers were predicted to produce amplification
products of 1,118 (P1), 1,070 (P2), 1,308 (P3), and 964 bp (P4).
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FIG. 1.
Diagrammatic representation of the mitochondrial genome
of P. infestans illustrating the location of the
various amplification products (shaded). Sequence locations are
according to the provisional sequence supplied by B. F. Lang. The
position of a ca.-1.6-kb insert present in haplotype II isolates
(Cornell haplotype B) is indicated. The darker shading indicates the
extent of the longer F4-R2 PCR product. Sequence positions
correspond to those published by Paquin et al. (29)
(available at
http://megasun.bch.umontreal.ca/People/lang/species/phyti/phytimtDNA.html).
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Once optimized, amplification of all four templates from relatively
crudely extracted DNA from agar cultures was robust and generated a
single band of the predicted size. The template DNA for P4 could be
diluted to 150 pg and still provide suitable amplification (data
not shown). Similar minimum quantities of template were required
for amplification of the other products.
Restriction enzyme digestion of the four PCR products generally
revealed the polymorphisms predicted by Carter et al. (4) (Fig. 2 and
3). A CfoI site on the P1
product was present in haplotypes Ia and Ib, while in both the P3 and
P4 products, additional EcoRI sites were present in Ia and
Ib haplotypes but not in types IIa and IIb. Digestion of the P1, P3,
and P4 products provided the same information, but the P4 primers were
used routinely because they amplified more reproducibly and produced a
more distinctive restriction pattern. As predicted (4),
an MspI digest of P2 yielded two fragments for haplotype Ia
and IIb isolates and three fragments for haplotype Ib; however,
haplotype IIa isolates unexpectedly also yielded three fragments due to
an MspI site not identified previously due to the small size
of the additional fragment (203 bp; Fig. 3). The reliability of the
assay was confirmed with a double blind test in which a number of
isolates, previously genotyped by the method of Carter et al.
(4), were correctly identified.
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FIG. 2.
Restriction enzyme digestions of PCR products amplified
from P. infestans with primer pairs F1-R1 (cut with
CfoI), F2-R2 (cut with MspI), F3-R3 (cut with
EcoRI), and F4-R4 (cut with EcoRI).
Amplifications were conducted with DNA from four isolates representing
each of the four Carter (4) mitochondrial DNA haplotypes,
namely Ia, 96.70; Ib, K1067; IIa, E14c2; and IIb, Ca65. Lanes marked M
contain 100-bp ladders (Gibco BRL Ltd.).
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FIG. 3.
Diagrammatic representation of the sizes of restriction
fragments generated by digestion of the four PCR products from
P. infestans. Restriction sites are indicated with an
arrow, and the presence or absence of these sites is indicated by + or .
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The four PCR products were tested for new RFLPs by digestion with
a further eight enzymes which cut the mtDNA frequently
(AluI, DraI, EcoRI, EcoRV,
HaeIII, HindIII, RsaI, and
Sau3AI). However, among 12 geographically diverse isolates,
which included all four Carter haplotypes, no further polymorphisms
were detected, and all restriction fragments were as predicted from the
sequence data.
Amplification of larger portions of the mtDNA was achieved with
other combinations of the eight primers described in Table 2 and the
modified long PCR amplification conditions of Barnes (2).
Amplification with primers F4 and R2 yielded a PCR product, P2/4,
5,359 bp in length (including both the P2 and P4 products as well as
the intervening region; Fig. 1). Digestion of P2/4 from six
isolates, including all four haplotypes, with eight enzymes known to
cut the predicted product at several positions (Fig. 4) did not reveal additional
polymorphisms. This long PCR procedure was also successful in
amplifying the 7,400-bp fragment of mtDNA between primers F3 and R4
(on either side of the 1.6-kb insert in haplotypes IIa and IIb) from a
haplotype Ia isolate, but several smaller, nonspecific products were
also amplified, and no restriction analysis was conducted (data not
shown).
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FIG. 4.
Long PCR amplification with F4-R2 primers of isolates
Cer002t (haplotype Ia, lanes 1); GF266 (haplotype IIa, lanes 2); 1.T1
(haplotype Ib, lanes 3); and Tail (haplotype Ib, lanes 4) digested with
HindIII, MspI, and RsaI. Lane M,
1-kb ladder (Gibco BRL Ltd.).
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Isolates from 17 countries from both potato and tomato were screened by
amplifying and digesting P2 (MspI digest; Fig.
5) and P4 (EcoRI digest). The
presence of ethidium bromide in the PCR mixture allowed amplification
to be verified by examining the PCR tubes with a UV transilluminator,
making preliminary electrophoresis unnecessary. Products were digested
most conveniently with microtiter plates.
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FIG. 5.
F2-R2 PCR products amplified from various Russian
isolates of P. infestans and digested with
MspI. Further details about these isolates are given in
Table 1. Lane M, 1-kb ladder (Gibco BRL Ltd.).
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Haplotype IIb was found only in California and Canada
(Vancouver). These isolates were mating types A1 and A2. Haplotypes Ia and IIa were both found in most countries. Haplotype Ia was most
often associated with A1, and haplotype IIa was most often associated
with A2 mating type. The Ib haplotype was detected in five countries
(16 isolates in total) and tended to be from older collections.
Direct analysis of mitochondrial haplotype from late-blight
lesions.
DNA was extracted from late-blight lesions on potato
leaflets and was amplified with all four pairs of primers to yield
amounts of product comparable to those amplified with DNA from pure
cultures. A background smear or faint additional bands were frequently
observed but did not obscure digestion products. P2 and P4 were
reliably amplified from DNA from fresh lesions and from air-dried
lesions of both tomato and potato. Four samples of air-dried lesions on tomato leaflets sent to Wales from Tanzania were found to be of the same haplotype (Ib) as a pure culture established in Tanzania (TNZW1; Table 1) from the same crop.
Amplification of mtDNA from other Phytophthora
spp.
The sequences of mitochondrial genomes in
Phytophthora spp. are believed to be moderately conserved
(12, 29), meaning that the primers described above could be
used to identify and characterize mtDNA of other species.
We tested DNA from nine Phytophthora spp. with the
mitochondrial primers described above. At an annealing temperature
of 55°C (optimal for P. infestans), amplification was
generally poor for all primers. However, when a lower annealing
temperature (53.5°C) was used, amplification was successful in the
majority of cases as shown in Table 3.
The P1 and P3 primers were the most suitable for the reliable
amplification of mtDNA from these other species. The amount of P2
and P4 products were generally less than the amount from P. infestans.
There was some variation in the sizes of the undigested PCR products
from the different species (data not shown), but more distinctive
differences were seen following endonuclease digestion (Fig.
6). There were frequently similarities
between the restriction patterns obtained from different species.
Primers P2 and P4, routinely used for P. infestans,
either failed to generate PCR product or gave a distinctive restriction
pattern after digestion with the other species; on two occasions
(data not shown), isolates from foliage blight of tomato from Taiwan
produced restriction patterns quite different from those of
P. infestans. These were subsequently identified as
Phytophthora capsici on the basis of colony and sporangial morphology.
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FIG. 6.
Amplification of DNA from various
Phytophthora species with the P3 primers (at an annealing
temperature of 53.5°C) followed by digestion with DraI.
Lanes: 1, P. capsici (P. cap) (P139);
2, P. citrophthora (P. cit)
(IMI362668); 3, P. colocasiae (P. col)
IMI 368918); 4, P. drechsleri (P. dre)
(P533); 5, P. fragariae var. fragariae
(P. fra) (FVF24); 6, P. nicotianae
(P. nic) (Nic1); 7, P. infestans
(P. inf) (86.126.1a). Lanes M, contain a 100-bp ladder
(Gibco BRL Ltd.). DNA was kindly supplied by David Cooke, SCRI, Dundee,
Scotland.
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DISCUSSION |
Isolation of P. infestans into pure culture
followed by the production of sufficient mycelium (typically >1 g) for
Southern analysis or purification of mitochondrial DNA is a process
which requires several weeks to complete. Methods for which only small (micrograms) quantities of pure P. infestans DNA are
needed and methods not requiring the isolation of fungus into pure
culture permit rapid and accurate monitoring of populations, which
could help determine the most appropriate control strategy. For
example, haplotype Ib has never become highly resistant to one of the
most useful systemic and curative fungicides, metalaxyl (1,
6).
Restriction enzyme digestion of PCR products for RFLP analysis
facilitates monitoring of genetic changes among field populations of
P. infestans. We have shown that the screening process
can be simplified by the direct analysis of genotypes from fresh or dried blight lesions and by the use of microtiter plates for digestion. One advantage of our method is that it includes both positive and
negative controls; differences in the size of a positive product rather
than a simple product/no product result are scored. By ribosomal DNA
(rDNA)-based detection methods (32, 34) results could be
false negative if a PCR fails due to sample contamination. A positive
control for restriction digestion is also included, as one
MspI site (P2) and one EcoRI site (P4) are
present in all haplotypes; digestion failures are identified by uncut
product.
Digestion of products P2 and P4, with MspI and
EcoRI respectively, permits the unambiguous identification
of the four Carter mitochondrial haplotypes. The simplicity of
the process is such that 50 to 100 samples can be processed
(i.e., DNA extraction, PCR amplification, restriction digestion, and
gel electrophoresis) by a single person in less than 2 working days.
This method might be further simplified by substituting a DNA
extraction from late-blight lesions with NaOH (32, 34).
Some correlation has been made between the Carter haplotypes and the
six mitochondrial haplotypes (A to F) designated by Goodwin (17), and it is known that the haplotype Ib isolates also
have a nuclear DNA fingerprint (with probe RG57; 18)
identical to the US-1 clonal lineage (6). Screening of
isolates known to contain Goodwin's haplotypes C to F (17)
may reveal additional polymorphism within the PCR products described
here.
In this study, haplotype Ib was detected in collections from most
countries. It is now clear that this haplotype is almost always
associated with A1 mating type, isozyme genotypes Gpi-1 86/100 and Pep-1 92/100 and multilocus RG57 fingerprint
US-1. The four isolates from Taiwan were, as expected, haplotype Ib, as
Taiwan is one of few countries in which this multilocus genotypes has
been the only one detected (25). All older collections from Russia, Europe, and the eastern United States were only of this genotype (21); more recent collections show that this
apparent clone has been or is being replaced by genotypes of haplotypes Ia or IIa, different isozyme alleles, and different RG57 fingerprints. It appears that the old genotype did not recombine with the new, highly
variable genotypes which probably migrated from Mexico in the 1970s
(21). An exception was detected in an English isolate from
1995 which was haplotype Ib but had a fingerprint quite different from
US-1 (6); another isolate from Brittany in 1996 was
Gpi-1 86/100 Pep-1 92/100 and haplotype Ib but again had a
non-US-1 fingerprint (26).
In some countries only a single haplotype was detected (e.g.,
Costa Rica, 18 isolates all haplotype Ia; Bolivia, 10 isolates all
haplotype IIa; data not shown), while in others (e.g., Russia, United Kingdom; Table 1) two or three haplotypes were detected. Lack of
diversity may be due to the limited number of samples collected or
sites sampled and/or to restricted sampling dates. In Western Europe,
recent collections are predominantly of haplotype Ia, with much lower
frequencies of IIa (6, 26). In this study, haplotype IIb was
detected only in California and British Columbia (Vancouver), but it
has since been detected in one isolate from The Netherlands
(11a).
The amplification procedure described here works well with crudely
prepared template DNA from both pure cultures and late-blight lesions, and is sensitive to as little as 150 pg of template DNA, which
is equivalent to 100 to 200 nuclei of P. infestans
(33). A PCR-based diagnostic assay devised by Trout et al.
(34) to detect symptomless early infection detected as
little as 10 pg of DNA of P. infestans but did not
permit differentiation of genetically different isolates. The increased
sensitivity of the latter assay is probably due to the higher copy
number of the rDNA target sequences and the short 456-bp product which
was generated. Like the method used in the present study, the rapid
cellulose acetate electrophoresis method of Goodwin et al.
(19) for isozyme analysis also does not require isolation of
the fungus into pure culture. However, incubation of lesions may be
required to induce development of sporangia. Our PCR-RFLP method
has been tested successfully on small numbers of sporangia
(26b).
The results shown in Fig. 4 demonstrate that modified long PCR
amplification can be used as a preliminary search for restriction polymorphisms. The advantage of amplifying a longer fragment lies in
the increased probability of locating polymorphic sites. Unfortunately, with the combinations of restriction enzymes and DNA samples tested here, polymorphisms were not detected. However, a similar method could be used to localize the mutations and/or insertions
characteristic of mitochondrial haplotypes C to F described by Goodwin
(17). We have also used a similar long PCR approach to
amplify the 6-kb sequence of the InterGenic Spacer region (IGS) of the
rDNA locus and digestion with RsaI to detect polymorphisms
(data not shown).
Over the past decade, RFLPs have been exploited to analyze the
mitochondrial genomes of various Phytophthora species (e.g., references 7, 8, 12, 13, 28). However, in all cases it was necessary to purify mtDNA by a time-consuming method of cesium chloride centrifugation. In the present study, PCR primers, originally designed to amplify mtDNA from P. infestans, were found to be effective for other
Phytophthora species. In particular, P1 and P3 permitted
amplification across a wide range of species. Although these primers
were tested on only one isolate of each species here, it is anticipated
that they will be of general use for diagnostic assays.
Our aim in this study was to provide a simple, robust system whereby
mtDNA haplotypes could be characterized according to a unified
system which was easily comparable between laboratories. The simplicity
of the method described combined with the wide availability of
facilities for conducting PCR in most laboratories should mean
that this and similar fingerprinting techniques can be used by those
with little previous experience of molecular biology. Informal
communication has already resulted in this method being used in a
number of laboratories in several countries studying populations of
P. infestans. We would be happy to comply with requests
for moderate quantities of these primer pairs (along with a more
detailed protocol).
| |
ACKNOWLEDGMENTS |
The financial support of the United Kingdom Government Overseas
Development Administration (Department for International Development) is gratefully acknowledged.
Maintenance and culture of exotic isolates were conducted under the
conditions of MAFF license no. PHF 1571/1156/84. We are grateful to the
following people for supplying DNA or cultures of P. infestans and other species: Greg Forbes (CIP, Peru); Yuri Dyakov,
Svetlana Bagirova, and Julia Maleeva (Moscow State University), Lowell Black and Yi-Hsiou Huang (AVRDC, Tainan, Taiwan), Remi Non-Wondim (AVRDC, Tanzania), K. M. Zhang, (Hainan, China), Vera Sanchez (CATIE, Costa Rica), and David Cooke (SCRI, Dundee, Scotland). We are grateful to Geoffrey Hall of the International Mycological Institute for assistance with importation of isolates, and we are
indebted to B. F. Lang of the University of Montreal for access to
unpublished sequence data for the P. infestans
mitochondrial genome. Also, we thank the following persons for useful
discussions: Jenny Day, Sue Whittaker, Nick Pipe, Richard Shattock, and
David Cooke.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biological Sciences, University of Wales, Aberystwyth, Ceredigion
SY23 3DA, Wales, United Kingdom. Phone: 44-1970-622325. Fax: 44-1970-622350. E-mail: GWG{at}aber.ac.uk.
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Applied and Environmental Microbiology, October 1998, p. 4007-4014, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
