APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUne 1993, p. 1752-1755 Vol. 59, No. 60099-2240/93/061752-04$02.00/0Copyright © 1993, American Society for Microbiology

Identification of Gremmeniella abietina Races with RandomAmplified Polymorphic DNA Markers

RICHARD C. HAMELIN,`* G. B. OUELLEiTIE,' AND LOUIS BERNIER2Laurentian Forestry Centre, lO55Rue du P.E.P. S., Sainte-Foy, Quebec GIV 4C7,1 and

Centre de Recherche en Biologie Forestiere, Universite6 Laval,Quebec GlK 7P4,2 Canada

Received 22 December 1992/Accepted 23 March 1993

Seven random amplified polymorphic DNA (RAPD) markers amplified from four oligonucleotides (10-mers)by the polymerase chain reaction were used to distinguish between the North American and European races ofGremmeniella abietina, the causal agent of Scleroderris canker of conifers. Forty-three isolates of the pathogenfrom 11 different host species originating from 11 countries, states, and provinces were tested; race designationwas consistent with results from immunogenic and soluble-protein assays. By using RAPD markers, it waspossible to identify G. abietina races by DNA amplifications directly from fruiting bodies, thus eliminating theneed to culture the fungus, as is necessary with immunogenic and soluble-protein assays. Two isolates whichhad been previously classified as intermediate were clearly identified as belonging to either one of the two racesby using RAPD markers. No interracial hybrids were detected in our survey. Patterns of amplification productsfrom the European race in North America were identical to patterns of European isolates, furthersubstantiating that this is an introduced race to the North American continent.

Scleroderris canker, caused by the ascomycete Gremme-niella abietina (Lagerb.) Morelet [= Scleroderris lagerbergiiGremmen, = Ascocalyx abietina (Lagerb.) Schlapfer-Bern-hard; anamorph, Brunchorstia pinea (Karst.) Hoehn], hasbeen recognized as a serious conifer disease in Europe formore than a century (3). It was identified in 1965 in NorthAmerica (9), where it caused considerable damage, espe-cially in nurseries and to small trees in plantations. In the1970s, a severe epidemic developed in the state of New York(12), but the symptoms associated with this new epidemicdiffered from the classical symptoms by their presence in thetops of large trees, by the general lack of stem cankers, andby the almost complete absence of the sexual stage of thefungus. It was observed that these symptoms resembled thesymptoms caused by this pathogen in Europe (5, 12).Growth rates, conidium size, and immunogenic reactionsestablished that two races of the fungus were present inNorth America and that the new race was serologicallyidentical to the race present in Europe. The new race wastherefore called the European (EU) race, while the otherrace was named the North American (NA) race (4, 5).As a result of these findings, and because the EU race was

reported to have a broader host range than the NA race (3,13), quarantines were established to contain the EU race ofthis pathogen and prevent its spread to states and provinceswhere it was not already present (1). As an alternative to theserological assay, a soluble-protein assay was developed toprovide characteristic protein banding patterns of the tworaces. This method was generally slightly faster and lesstedious than the serological assay, and it also allowed thedifferentiation between other taxa of Gremmeniella spp.(12). However, this technique still necessitated culturing thefungus (which requires approximately 1 month), and prob-lems were encountered with protein variability within raceand even within sample (2, 10, 11). For example, it wasfound that the protein banding patterns could vary with the

* Corresponding author.

age of the cultures. Also, there were difficulties with samplesthat could not be assigned to either race (9a).The presence of putative hybrids has been reported (2, 6,

10, 14, 15, 17). However, on the basis of present techniques,it was impossible to confirm or disprove the hypothesis thatthe two races hybridize in nature.To overcome these limitations, we have used the random

amplified polymorphic DNA (RAPD) technique to provide adiagnostic tool for the identification of the races of thispathogen. This diagnosis is rapid, reproducible, and usesminimal amounts of tissue, thereby allowing identification ofthe pathogen without culturing. This article describes ouruse of selected RAPD markers to differentiate between racesof G. abietina from the mycelium as well as directly fromfruiting bodies.

MATERIALS AND METHODS

Samples. A total of 43 isolates collected from 11 coniferspecies in the northeastern United States, Canada, andWestern Europe were studied (Table 1). The samples werecultured for 1 month on dialysis membranes that were placedon petri dishes containing an agar medium consisting of 25ml of Campbell's V-8 juice, 15 g of Bacto Agar, 7.5 g of maltextract, and 475 ml of water dispensed at 25 ml per petridish; the mycelium was either used immediately or lyophi-lized and stored at -20'C until needed. Samples for ampli-fications from fructifications (pycnidia and cryptopycnidia)were obtained as part of an annual survey of G. abietina(courtesy of Solange Simard, Ministere des Forets du Que-bec) or were collected from the Laurentian Park (courtesy ofGaston Laflamme, Laurentian Forestry Centre). In bothcases, the infected branches were stored at room tempera-ture in paper bags until needed.DNA extractions. DNA was extracted by a modified cetyl-

trimethylammonium bromide protocol (19). Approximately10 mg of lyophilized mycelium was ground in liquid nitrogenwith a mortar and pestle. Seven hundred microliters ofextraction buffer [700 mM NaCl, 50 mM Tris-HCl (pH 8), 10

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IDENTIFICATION OF GREMMENIELLA ABIETINA RACES 1753

TABLE 1. G. abietina isolates used in this study, the host fromwhich they were isolated, their geographic origin, and their

race identification

Isolatea Host Geographic Racespecies originb identificationc

009 Pinus resinosa Quebec (Labelle) EU032 Pinus banksiana Quebec (Labelle) NA033 P. resinosa Quebec (Labelle) EU034 P. resinosa Quebec (Labelle) EU1370P P. banksiana Quebec (Matapedia) NA1499P P. banksiana Quebec (Abitibi) INTd3459 P. resinosa Quebec (Labelle) EU3482 P. resinosa Quebec (Labelle) EU3489B P. resinosa Quebec (Champlain) EU3555 P. resinosa Quebec (Charlevoix) NA4093A P. banksiana Quebec (Portneuf) NA4094 P. resinosa Quebec (Portneuf) EU4095A P. resinosa Quebec (Portneuf) NA4096 P. resinosa Quebec (Portneuf) NA4097 Pinus sylvestris Quebec (Portneuf) NA4126B P. resinosa Quebec (Labelle) EU4128B P. resinosa Quebec (Labelle) NA4131 P. resinosa Quebec (Labelle) EU4132B P. resinosa Quebec (Labelle) EU4338 P. banksiana Quebec (Matapedia) NA4588B P. resinosa Quebec (Labelle) EU4606 P. banksiana Quebec (Labelle) NA4646 P. sylvestris Quebec (Portneuf) EU4648 P. resinosa Quebec (Portneuf) NA92-0595 P. resinosa Quebec (Labelle) EU92-0816B P. banksiana Quebec (Labelle) NAAsco-J5 Pinus pinea Italy EUC53 Pinus contorta Alberta NAC58 P. contorta Alberta NACBS134.26 Pinus paricio The Netherlands EUM10.24 Pinus cembra Switzerland EUPASE1.8 P. sylvestris Finland EUFl Larix siberica Finland EUNB810730 P. banksiana New Brunswick NAON0001 P. resinosa Ontario NAON0018 P. resinosa Ontario NAON0023 P. sylvestris Ontario NAON0025 Pinus strobus Ontario NATN493 Pinus nigra Newfoundland EUTN500 P. sylvestris Newfoundland EUUS0006 P. strobus New York NAUSOO01 Pinus ponderosa New York EUUS0015 P. sylvestris Vermont INTd

a All isolates from Quebec, except 92-0595 and 92-0816B, were part of asurvey conducted in 1991 and were formerly labelled with the prefix 91-.

b For samples from Quebec, the county of origin is given in parentheses.c Race identification by a soluble-protein (10) or serology (6) assay. INT,

intermediate reaction.d Isolates 1499p and US0015 were assigned to the NA and EU races,

respectively, by using RAPD markers.

mM EDTA, 1% (1)-2-mercaptoethanol, 1% cetyl-trimethyl-ammonium bromide] was added to each mycelium sample,and the mixtures were incubated at 65°C for 1 h. Themixtures were emulsified by adding an equal volume ofchloroform-isoamyl alcohol (24:1), vortexing, and centrifug-ing for 5 min at 12,000 x g. The upper phases wereprecipitated with 75 ,ul of ammonium acetate (7.5 mM) and600 RI of cold absolute ethanol and centrifuged for 5 min at10,000 x g. The resulting pellets were washed with 70%ethanol and then dried and resuspended in 50 ,u1 of TE8 (10mM Tris-HCl [pH 8], 1 mM EDTA). DNAs were stored at4°C until needed. A 1:100 dilution of each extraction wasused for the amplifications.For extractions from fruiting bodies, one pycnidium or

cryptopycnidium was ground with a disposable polypropy-

lene pellet pestle in a 1.5-ml Eppendorf tube containing 50 ,ulof extraction buffer and approximately 10 mg of diatoma-ceous earth (Sigma Chemical Co., St. Louis, Mo.); 350 p.l ofextraction buffer was added, and the extraction was per-formed as already described. A 1:10 dilution was used forthese amplifications.DNA amplifications. Amplifications, modified from the

method of Williams et al. (18), were performed in volumes of25 ,ul containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2mM MgCl2, 0.0001% gelatin, 100 p.M each deoxynucleosidetriphosphate (Pharmacia, Uppsala, Sweden), 0.2 p.M oligo-nucleotides (10-mer Kit A; Operon Technologies, Alameda,Calif.), 2 ,ul (approximately 10 ng) of genomic DNA, and 0.5U of Taq DNA polymerase (Boehringer Mannheim Bio-chemica, Mannheim, Germany) overlaid with a drop ofmineral oil. Amplifications were performed in a DNA ther-mal cycler (model 480; Perkin-Elmer Cetus, Norwalk,Conn.) programmed for a denaturation step at 94°C for 3min, followed by 1 cycle at 35°C for 4 min and 72°C for 2 min,and then 45 cycles of 94°C for 1 min, 35°C for 1 min, and 72°Cfor 2 min. The amplifications ended with a 10-min extensionat 72°C. Amplification products were separated by electro-phoresis on 1.2 or 1.5% agarose gels by using TAE buffer (40mM Tris-acetate-1 mM EDTA [pH 8.0]) and visualized byUV fluorescence after ethidium bromide staining.

Data analysis. We selected five isolates (NA isolates 4093,4338, and 4648; EU isolates 3459 and 4131) to screen a totalof 20 oligonucleotides for RAPD polymorphisms. Oligonu-cleotides that produced reproducible polymorphisms inter-racially but not intraracially were then retained to screen theremainder of our collection. Diagnostic polymorphisms wereidentified by the name of the oligonucleotide and the size ofthe DNA amplification product. Amplification of polymor-phic primer-template combinations were repeated at leasttwice to ensure reproducibility.

RESULTS

Four primers (OPA3, OPA4, OPA7, and OPA11) pro-duced seven amplification products that were monomorphicintraracially but polymorphic interracially. Amplificationswith primer OPA3 resulted in the production of a 1,700-bpDNA fragment in all of the NA samples tested but in none ofthe EU isolates. However, amplifications with the sameprimer produced a DNA fragment (750 bp) in all EU isolateswhich was absent in NA isolates (Fig. 1A; Table 2). Simi-larly, amplifications with primer OPA4 resulted in a 2,100-bpproduct in all NA isolates but in none of the EU isolates(Table 2). Two other primers allowed separation of the EUand NA races: amplifications with primer OPAll produced a1,200-bp fragment only in NA isolates and a 2,300-bp DNAfragment only in EU isolates (Fig. 1B; Table 2), and OPA7produced two DNA fragments (1,800 and 850 bp) that werepresent in EU isolates but absent in NA isolates (Fig. 2;Table 2). The remainder of the primers tested producedpolymorphisms both inter- and intraracially and were there-fore not considered useful for diagnosis purposes. However,such primers would be useful to study intraracial variabilityand population structure.Although some primers were essentially monomorphic

intraracially, others showed intraracial polymorphisms. Forexample, amplifications with primer OPA7 produced twogenotypes in the EU race from North America, but eightgenotypes were found in the NA race (Fig. 2). Nei's measureof gene diversity (8) (the probability that two individuals

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1754 HAMELIN ET AL.

A

2.01.6

1.0

0.5

B

2.01.6

1.0

0.5

~I

EU NA

FIG. 1. Amplification products of G. abietina DNA from NA andEU races by using oligonucleotide primers OPA3 (A) and OPAll(B). Arrows indicate diagnostic DNA fragments that are monomor-phic intraracially but polymorphic interracially.

sampled at random differ in type) was 0.167 in EU isolatesfrom North America and 0.894 in NA isolates.

Patterns of amplification from pycnidia and cryptopyc-nidia were clear and consistent with amplifications frommycelial DNA (Fig. 3).

All of our race assignments were consistent with thosepreviously given by using the soluble-protein assay. How-ever, two samples, 1499p and US0015, which until nowcould not be assigned to a race, were clearly identified asbelonging to the NA and EU races, respectively (Fig. 1). Noapparent hybrids were detected in our collections.

TABLE 2. RAPD markers diagnostic of NA and EU racesof G. abietina

Primer' Length of fragment Race(bp) identified

OPA3 1,700 NAOPA3 750 EUOPA4 2,100 NAOPA7 1,800 EUOPA7 850 EUOPAll 1,200 NAOPAll 2,300 EU

' Primers were 10-mers obtained from Operon Technologies. Primer se-quences are as follows: OPA3, 5'-AGTCAGCCAC-3'; OPA4, 5'-AATCGGCTG-3'; OPA7, 5'-GAAACGGGTG-3'; OPAll, 5'-CAATCGCCGT-3'.

2.01.6

1.0

0.5

-z (A 3 >i 0" l 2 S wj O $

NA EU

FIG. 2. Amplification products of G. abietina DNA from NA andEU races by using oligonucleotide primer OPA7. Arrows indicatediagnostic DNA fragments that are monomorphic intraracially butpolymorphic interracially.

DISCUSSION

The results clearly indicate the potential for using DNAamplification techniques to quickly assign G. abietina toeither of its two most important races found in NorthAmerica. This technique offers several advantages over theconventional diagnostic tools, serological and soluble-pro-tein assays. The RAPD technique is quick, reliable, insensi-tive to the age of the tissue sampled, and requires onlyminute amounts of material for identification. Since DNAcan be extracted directly from fructifications, it is nowpossible to identify races of G. abietina without having toculture the fungus. This will greatly facilitate surveys of thefrequency and distribution of these two races in NorthAmerica and allow unequivocal identification of interracialhybrids.Our results support findings from previous surveys based

on serological and soluble protein assays concerning theprobable common origin of the EU race present in NorthAmerica and the race present in Europe (4, 5). Even thoughisolates originated from different countries and continents,the patterns of amplification products were consistent in-traracially for the EU race for most of the primers studiedbut clearly different from the NA race.

In recent surveys of Canada and the United States, both

EU o2.01.6

NA

0.5

<- NA

EU

.- 00 1 00 01O CO\ 01 0' 0n

All A3FIG. 3. Amplification products of G. abietina DNA extracted

directly from fruiting bodies. Isolate 816 (92-0816B) belongs to theNA race and was extracted from pycnidia, and isolate 595 (92-0595)belongs to the EU race and was extracted from cryptopycnidia.

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IDENTIFICATION OF GREMMENIELLA ABIETINA RACES 1755

races were found in New York, Vermont, New Hampshire,Maine, Quebec, Ontario, and New Brunswick, while onlythe EU race was present in Newfoundland and only the NArace was present in Michigan, Minnesota, and Wisconsin (7,15, 16). However, the serology assay did not provide infor-mation concerning intraracial variability. It is interesting tonote, as was reported for protein profiles (10), that very lowlevels of polymorphism were detected among representa-tives of the EU race in North America but some primersrevealed high levels of intraracial polymorphism in the NArace (e.g., OPA7; Fig. 2). Although more data need to begathered to compare genetic diversity in the European andNorth American populations of the EU race, the preliminaryresults reported here are consistent with the hypothesis of afairly recent introduction of the EU race to North America(5) and the resulting founder effect (i.e., colonization by fewindividuals) and reduction of genetic variability.

Putative interracial hybrids showing symptoms character-istic of both races have been reported previously by usingserological and soluble-protein assays (2, 6, 10, 14, 15, 17).For example, one isolate found in the upper crown of a tree(typical of the EU race) also produced abundant apothecia(typical of the NA race) (15). In our limited collection, twoisolates classified as intermediate by using either the serol-ogy (6) or soluble-protein assay (9a) were clearly assigned toa race by each of our seven RAPD markers. Since we choseour markers for their intraracial monomorphism, true hy-brids should be easily detected with our assay, resulting inthe presence of some markers diagnostic of both races in asingle isolate. Nevertheless, a more exhaustive survey of theG. abietina populations with RAPD markers, especially inareas of heavy infection with both races, might detect thepresence of interracial hybrids.A significant improvement reported here is the possibility

to assign races directly from fruiting bodies, thereby elimi-nating the tedious step of culturing the fungus prior to theassay. Furthermore, amplifications of DNA extracted fromeither pycnidia or cryptopycnidia yielded the same patternsof amplified DNA fragments. This could be important sincedifferent types of fruiting bodies are often present on differ-ent samples.The technique described here can be further refined by

sequencing diagnostic DNA fragments and synthesizingrace-specific primers which would amplify a single diagnos-tic DNA fragment (sequence-tagged sites) instead of com-plex patterns of amplification products. This improvementwould allow identification of G. abietina races from anyinfected tissue even prior to sporulation and severe epidemicdevelopment.

ACKNOWLEDGMENTS

We acknowledge the help of Nicole Lecours and Robert Blais(Laurentian Forestry Centre) for providing and culturing the iso-lates, of Vincent Quintin for technical assistance, of Claude Moffetfor photography (Laurentian Forestry Centre), of Ken Dewar forreviewing the manuscript, and of Gaston Laflamme (LaurentianForestry Centre) for insightful discussions.This work was made possible by grant OGPIN003 from the

National Science and Engineering Research Council of Canada.

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6. Dorworth, C. E., D. P. Webb, J. Krywienczyk, and D. McNa-mara. 1982. Comparisons between serologic and gas chromato-graphic techniques for characterization of Gremmeniella abiet-ina and related species. Eur. J. For. Pathol. 12:209-217.

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9. Ohman, J. H. 1966. Scleroderris lagerbergii Gremmen: thecause of dieback and mortality of red and jack pines in upperMichigan plantations. Plant Dis. Rep. 50:402-405.

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1988. Investigations on new means of identifying races ofAscocalix abietina, p. 73-79. In E. Donaubauer and B. R.Stephan (ed.), Recent research on Scleroderris canker of coni-fers. Mitteilungen der forstlichen Bundesversuchsanstalt Wienno. 162. Osterreichischer Agrarverlag, Vienna.

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12. Setliff, E. C., J. A. Sullivan, and J. H. Thompson. 1975.Scleroderris lagerbergii in large red and Scots pine trees in NewYork. Plant Dis. Rep. 59:380-381.

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