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Migrational capacity of Fennoscandian populations of Venturia tremulae
Mycol. Res. 108 (1): 64–70 (January 2004). f The British Mycological Society
64
DOI: 10.1017/S0953756203008918 Printed in the United Kingdom.
Migrational capacity of Fennoscandian populations of Venturia tremulae
Risto KASANEN1*, Jarkko HANTULA1, Martti VUORINEN2, Jan STENLID3, Halvor SOLHEIM4 and Timo KURKELA1 1
Finnish Forest Research Institute, Vantaa Research Centre, P.O. Box 18, FIN-01301, Vantaa, Finland. FFRI, Suonenjoki Research Station, Suonenjoki, Finland. 3 Institute of Mycology and Forest Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden. 4 Norwegian Forest Research Institute, Aas, Norway. E-mail : risto.kasanen@metla.fi 2
Received 27 February 2003; accepted 14 November 2003.
Genetic variation in three multiallelic loci was analysed with Temperature Gradient Gel Electrophoresis in order to assess the genetic population structure of Venturia tremulae var. tremulae in order to understand the evolutionary potential of the pathogen against resistance breeding. Also the identification of the fungus was verified with molecular analysis of reference isolates. The Fst and Gst values were very low indicating no substructuring or restrictions to gene flow between Fennoscandian populations of V. tremulae. The results imply high epidemiological efficiency of the fungus and therefore continuous breeding programme should be implemented for Venturia resistance of aspen.
INTRODUCTION Wide-scale plantations of aspen (Populus tremula) and hybrid aspen (P. tremularP. tremuloides) have recently been established in Nordic and Baltic countries as the forest industry has become interested in aspen fibre. As the number of aspen stands increases, the fungal diseases will gain in both economical and ecological importance. It is even possible that damages caused by the fungal diseases increase due to the pathogens adapting to established monocultures. The shoot diseases cause visible damage which results in economical losses to the nurseries. In young plantations the shoot diseases may delay the growth of seedlings or even cause mortality. The symptoms of black shoot blight of aspen include blackened and twisted shoots (so called shepherd’s crooks) and blackspotted leaves. Two ascomycetes are capable of causing the disease: Venturia macularis (anamorph unknown) and V. tremulae var. tremulae (anamorph : Pollaccia radiosa) (Morelet 1985). Both aspen and several aspen hybrids show variation in the resistance to black shoot blight and therefore it is probable that the disease could be controlled by utilizing clonal variation of resistance (Weisgerber 1968). * Corresponding author.
However, the plantations surveyed by Weisgerber (1968) were geographically widespread and therefore variation in the virulence between local fungal populations may well have caused bias in the survey. The information about the population structure of a pathogen is important in resistance breeding of a host. Even in the screening phase, a decision must be made whether to rely on local background inoculum or whether to have inocula collected from a wider geographical area. In transporting inoculum to field trials, one must also anticipate the risk of spreading virulent genotypes of the pathogen to new areas. Differences between local populations highly depend on the migrational capacity of the fungus. The capability of Venturia sp. to produce conidia throughout the growth season (Weisgerber 1968) and the regeneration of aspen by asexual reproduction might in theory favour clonality in fungal population and therefore give epidemiological benefit of the pathogen clones virulent to the coppicing aspen clone. Unfortunately, there are no studies available on the population structure of any Venturia species causing shoot blight on Populus. Thus the evolutionary potential (McDonald & Linde 2002) and migrational capacity of Venturia species is unknown. The distribution, taxonomy and morphology of Venturia species on Populus sp. is not well known or
R. Kasanen and others
65
Table 1. Sample data. All newly obtained isolates were isolated and deposited by R.K.
n
Host
Isolation method
Collector
Eastern Finland Kesa¨lahti (1) Joutseno (2) Rautja¨rvi (3)
8 12 11
Populus tremula P. tremula P. tremula
ascospore ascospore ascospore
T. Kurkela T. Kurkela T. Kurkela
Central Finland Varpaisja¨rvi (4) Va¨a¨na¨la¨nranta (5) Lo¨ytynma¨ki (6)
11 10 10
P. tremula P. tremula P. tremula
ascospore ascospore ascospore
M. Vuorinen M. Vuorinen M. Vuorinen
9 7 7
P. tremula P. tremula P. tremula
ascospore ascospore ascospore
J. Hantula J. Hantula J. Hantula
Sweden Ra¨ttvik, Dalarna (10) Gottsunda, Uppsala (11)
10 6
P. tremula P. tremula
mycelia mycelia
J. Stenlid J. Stenlid
Norway Hof, Vestfold (12)
11
P. tremula
mycelia
H. Solheim
8
P. tremula
mycelia
T. Kurkela
ascospore not known ascospore ascospore not known not known not known spore
M. Morelet O. Servazzi M. Morelet M. Morelet O. Servazzi R. Ciferri E. Mu¨ller M. Morelet
POPULATION SAMPLES
Northern Finland Keminmaa (7) Jaatilansaari (8) Loue (9)
Russia Valamo island, Lake Ladoga (13) REFERENCE SAMPLES
Venturia tremulae var. tremulae CBS 692.85 V. tremulae var. tremulae CBS 257.38 V. tremulae var. grandidentatae CBS 695.85 V. tremulae var. populi-albae CBS 694.85 V. populina CBS 256.38 V. populina CBS 316.58 V. macularis CBS 477.61 V. macularis CBS 698.85 a
Francea, P. tremula Italy, P. tremula Canada, P. tremuloides France, P. alba Italy, P. canadiensis Italy, Populus sp. France France
Country of origin.
documented. Sivanesan (1977, 1984) treated V. macularis and V. tremulae as synonyms occurring on Populus sect. Leuce, and described Pollaccia radiosa as the anamorph. On Populus sect. Tacamahaca, the shoot blight was considered to be caused by V. populina (Dance 1961, Sivanesan 1977). However, Morelet (1985) stated that V. macularis has no known anamorph. In addition to describing morphological differences between V. tremulae and V. macularis, Morelet (1985) divided V. tremulae into three varieties: var. tremulae, var. grandidentatae, and var. populi-alba. It must also be noted that in North America, a new species has recently been described, V. inopina on Populus trichocarpa (Newcombe 2003). We have isolated Venturia species from blighted shoots of aspen in Finland. Based on the morphological key of Sivanesan (1977) and molecular analyses, we concluded that the isolates did not belong to V. macularis (syn. V. tremulae according to Sivanesan 1977, 1984) or V. populina, but to a third species (Kasanen, Hantula & Kurkela 2001). Unfortunately, we were not then aware of the paper by Morelet (1985) in which another treatment of Venturia had been proposed. Morphologically, the ascospores of our isolates (Kasanen et al. 2001) were similar to V. tremulae as described by Morelet (1985), we consider it probable
that our samples were actually V. tremulae var. tremulae, and that V. tremulae and V. macularis are not conspecific as Sivanesan (1977) proposed. However, comparison of the literature (Dance 1961, Sivanesan 1977, Morelet 1985) reveals that the ascospores of V. populina and V. tremulae varieties are highly similar and morphologically these fungi could only be identified by the morphology of the conidia. Unfortunately, according to Weisgerber’s (1968) and our observations, the conidia emerge only seldom in in vitro. In addition, none of the samples Morelet (1985) examined was from Scandinavia. The aim of this study was to assess the genetic population structure of the Venturia sp. described in Kasanen et al. (2001), with the null hypothesis that no genetic differentiation of the fungus occurs even on a more wide geographical scale. Also, the identity of the fungus was tested by molecular comparisons to the reference isolates.
MATERIALS AND METHODS Fungal material Nine Finnish populations of Venturia were collected in June 1999 (Table 1). In the sampling, three local
Population structure of Venturia tremulae
66
Table 2. The distribution of allelic variation in marker loci. Population names and locations are given in Table 1. Alleles VII, IX and XI in loci CT1500, CTMS and Vscar3 are null alleles, respectively. pop1 8
pop2 12
pop3 11
pop4 11
pop5 10
pop6 10
pop7 9
pop8 7
pop9 7
pop10 10
pop11 6
pop12 11
pop13 8
Locus CT1500 Alleles I II III IV V VI VII
0.750 0.000 0.000 0.250 0.000 0.000 0.000
0.583 0.083 0.000 0.167 0.000 0.000 0.167
0.364 0.091 0.000 0.455 0.000 0.000 0.091
0.636 0.000 0.273 0.091 0.000 0.000 0.000
0.700 0.000 0.100 0.100 0.000 0.000 0.100
0.400 0.100 0.100 0.300 0.000 0.100 0.000
0.667 0.000 0.111 0.222 0.000 0.000 0.000
0.571 0.000 0.143 0.143 0.143 0.000 0.000
0.571 0.286 0.000 0.143 0.000 0.000 0.000
0.500 0.000 0.100 0.400 0.000 0.000 0.000
0.667 0.000 0.167 0.167 0.000 0.000 0.000
0.818 0.000 0.000 0.182 0.000 0.000 0.000
0.375 0.125 0.250 0.125 0.000 0.000 0.125
Locus CTMS Alleles I II III IV V VI VII VIII IX
0.000 0.000 0.250 0.625 0.125 0.000 0.000 0.000 0.000
0.000 0.083 0.083 0.583 0.083 0.000 0.000 0.000 0.167
0.000 0.000 0.000 0.091 0.182 0.091 0.091 0.455 0.091
0.091 0.091 0.182 0.636 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.000 0.800 0.100 0.100 0.000 0.000 0.000
0.000 0.100 0.400 0.500 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.222 0.444 0.000 0.333 0.000 0.000 0.000
0.000 0.000 0.286 0.714 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.286 0.714 0.000 0.000 0.000 0.000 0.000
0.000 0.100 0.200 0.600 0.000 0.100 0.000 0.000 0.000
0.000 0.333 0.000 0.500 0.000 0.167 0.000 0.000 0.000
0.000 0.091 0.273 0.364 0.091 0.091 0.091 0.000 0.000
0.000 0.000 0.125 0.750 0.000 0.125 0.000 0.000 0.000
0.000 0.000 0.000 0.000 0.250 0.375 0.125 0.250 0.000 0.000 0.000
0.000 0.083 0.000 0.333 0.000 0.083 0.250 0.083 0.083 0.000 0.083
0.000 0.091 0.273 0.182 0.182 0.091 0.091 0.091 0.000 0.000 0.000
0.000 0.000 0.000 0.091 0.091 0.000 0.273 0.000 0.545 0.000 0.000
0.000 0.000 0.000 0.100 0.000 0.100 0.500 0.200 0.100 0.000 0.000
0.000 0.000 0.000 0.000 0.300 0.100 0.200 0.200 0.100 0.000 0.100
0.000 0.000 0.000 0.444 0.000 0.000 0.333 0.111 0.111 0.000 0.000
0.000 0.000 0.000 0.000 0.000 0.000 0.286 0.571 0.143 0.000 0.000
0.000 0.000 0.000 0.143 0.286 0.143 0.143 0.286 0.000 0.000 0.000
0.000 0.000 0.000 0.200 0.000 0.200 0.200 0.200 0.100 0.100 0.000
0.333 0.000 0.000 0.000 0.167 0.167 0.333 0.000 0.000 0.000 0.000
0.000 0.000 0.000 0.182 0.000 0.182 0.364 0.182 0.091 0.000 0.000
0.000 0.000 0.000 0.000 0.125 0.125 0.500 0.125 0.125 0.000 0.000
Populations
Locus Vscar3 Alleles
n
I II III IV V VI VII VIII IX X XI
populations separated by approx. 100 km were collected from eastern, southern and northern Finland. The distance between regions was more than 250 km. Single ascospores were isolated from each shoot using the following procedure. The twisted tip of the shoot was cut apart, placed on 1.5 % water agar and incubated at room temperature for 1–3 d. The plates were inspected daily under a microscope for spore release. As sporulation was observed, 3–5 Venturia ascospores per plate were isolated with a sterile modified pasteur pipette and further cultured on 2% malt agar. When growth of the mycelia was observed, one single-spore colony per plate was randomly selected and subcultured on 1.5 % malt agar covered with cellophane membrane. The populations from Norway, Sweden and Russia were collected in 2000 and 2001 (Table 1). The distance between the Norwegian population and the nearest Swedish population was more than 270 km, and the Russian population was more than 200 km from the nearest Finnish populations. Because the collection dates varied between June and September, ascospore release had in most cases already taken place or samples represented the current season infection. Mycelial cultures of the fungus were isolated from dead shoots as follows. Fresh infection sites (recently blackened spots that could be caused by conidia or ascospores) were
selected and cut from the shoot material. The shoot pieces were surface-sterilized by rinsing in 75% ethanol, 4 % sodium hypochloride and sterile water (10 s each) and placed on 2% malt agar. Reference cultures of different Venturia species and varieties were obtained from the Centraalbureau voor Schimmelcultures (CBS, Utrecht) (Table 1). DNA isolations DNA isolations were made as described in Vainio, Korhonen & Hantula (1998). The fungal tissue was homogenized in 1.5 ml disposable plastic tubes by grinding with glass rod and quartz sand. Briefly, the DNA isolation procedure included two phenol-chlorophorm (1 : 1) extractions, chloroform-phenol-isoamyl alcohol extractions, precipitation with polyethylene glycol (PEG) and drying. The DNA was resuspended into 10 mM Tris HCl buffer containing 1 mM EDTA. Marker design For markers CT1500 and Vscar3, single bands were chosen from RAMS fingerprints amplified with primers DVD(CT)7C and DHB(CGA)5 respectively (Hantula, Dusabenyagasani & Hamelin 1996). The amplification of RAMS fingerprints was performed as described in
R. Kasanen and others Hantula et al. (1996) and markers were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The clones were sequenced with LI-COR IR2 automated sequencer according to the manufacturer’s recommendations. Based on the sequences obtained two pairs of primers were designed in order to amplify regions of 1500 and 900 bp from the sequence of the original RAMS markers, respectively. A GC-clamp of 40 bp was added to forward primer sequences to allow optimal resolution in Temperature Gradient Gel Electrophoresis (TGGE). The primer sequences were: (1) CT1500 GC : 5k CCC CCG CCG CGC GCC GCG CGG CGG (2) CCG GCC GCA CGC GCC GGC TTT TCT CAT AGG CCC CTC ; CT1500R : 5k CTT CAT GCT GAA CCC ACC TC ; (3) Vscar3R : 5k CGA AGG CTG GGA GTA CTT TG ; and (4) Vscar3 FGC : 5k CCC CCG CCG CGC GCC GCG CGG CGG CCG GCC GCA CGC GCC TTA TGA AGG CGG AGG TGA AC. For marker CTMS, based on the DVD(CT)7C fingerprint, the sequence analysis revealed a ten-fold repeat of CT (microsatellite motif ) at a binding site for the RAMS primer. To obtain sequence information of the flanking region of the microsatellite the following procedure was used. A pair of primers was designed near the microsatellite so that the reverse primer was located upstream of the forward primer. Total DNA of Venturia sample was digested with CfoI restriction enzyme (Boehringer-Mannheim) according to manufacturers recommendations. The restriction fragments were ligated with T4 ligase (Fermentas) to produce circular DNA, which was used as a template in the following PCR which resulted in a 500 bp amplification product. The sequence analysis of the product confirmed that the microsatellite and the flanking region had been cloned. A new primer pair was designed to amplify the microsatellite region and a GC-clamp of 40 bp was added to forward primer sequences to allow optimal resolution in TGGE. The primer sequences were : CTMS-R3, 5k TGG TGT TGA TAC CTG TTT TTA CT ; and CTMS-GC, 5k CCC CCG CCG CGC GCC GCG CGG CGG CGG GCC GCA CGC GCC TGC GTG CAA GTA CAA TCC AT. On agarose gels, CTMS marker was seen as a single band, but after TGGE run additional faint bands were seen. The most intense band was scored as the marker. In TGGE the samples are run in polyacrylamide gel containing a constant concentration of urea. As the temperature is increased gradually and uniformly during the electrophoresis, the result is a linear temperature gradient over the length of the electrophoresis run. Thus a denaturing gradient is formed by the constant concentration of urea in combination with temporal temperature change. The temperature gradients, ramping rates of temperature and run voltages used for markers CT1500, CTMS and Vscar3 were 40–65 xC, 1.3 x hx1, 150 V ; 54–59 x, 1.3 x hx1, 130 V, and 53–63 x, 2.5 x hx1, 170 V, respectively. All markers were analysed with DCode electrophoresis apparatus, in 6 % polyacrylamide gels containing 6 M urea. The running conditions for markers were optimized according to
67
M
1
2
3
4
5
6
7
8
M
Fig. 1. Temperature gradient gel electrophoresis of alleles in locus CT1500 marker. Lane M, standard sample (note that due to denaturing process the standard is applicable only in order to detect lane distortion) ; lane 1, allele found only in the reference isolate CBS 695.85 ; lanes 2–3, isolates from northern and eastern Finland having the same allele (I); and lanes 4–8 showing alleles II–VI, respectively.
1
2
3
4
5
6
7
8
9
10
Fig. 2. An example of TGGE screening of alleles of locus CTMS. Lane 1, an isolate from population 7, northern Finland (allele VI) ; lane 2, an isolate from population 12, Sweden (allele VII) ; lane 3, an isolate from population 7 (allele VI) ; lanes 4 and 5, isolates from population 4, central Finland (alleles I and II, respectively) ; lanes 6 and 7, isolates from populations 12, Sweden and 6, central Finland, share the same allele (III) ; lanes 8 and 9, isolates from populations 9 (northern Finland) and 4 (central Finland) share the same allele (IV) ; lane 10, a isolate from population 9 (allele V). The most intense band on each lane was scored.
manufacturers recommendations (Bio-Rad, Hercules, CA). Because of occasional lane distortion, some alleles which had only small differences in their mobility were difficult to score over the width of the gel. In such cases samples were run several times on adjacent lanes. Also a mixture of PCR products of different alleles was made and used as a standard (Fig. 3). Analysis of genetic diversity To assess the genetic diversity within populations, we calculated Nei’s unbiased gene diversities (Nei 1987). In order to study genetic differentiation among populations, we calculated the Fst analogue defined by
Population structure of Venturia tremulae
1
2
3
4
5
68
6
7
8
9
10
11
12
13
14
Fig. 3. An example of TGGE screening of alleles of locus Vscar3. Lanes 8, 11 and 13, allelic ladders composed of a mixture of different PCR products used as an internal standard in screening because of occasional lane distortions during the electrophoresis. Lanes 1 and 2, isolates from populations 6 and 4 (allele V); lanes 3 and 4, isolates from population 4 (allele VII) ; lane 5, an isolate from population 11 (allele VI) ; lane 6, an isolate from population 12 (allele X) ; lane 7, CBS 692.85 (allele IV) ; lanes 9 and 10, CBS 257.38 and CBS 695.85 (allele VIII) ; lane 12, CBS 694.85 (allele V); and lane 14, isolate from population 1 (allele VI).
Weir & Cockerham (1986) and Gst value (Nei 1987). For all the calculations, the populations were grouped in two ways ; either all 13 populations were considered separate, or they were divided to six populations based on geographic locations ; Russia, Sweden, Norway, northern, central and southern Finland (Table 1). To compare the genetic diversity (Nei 1987) between different loci over all samples, the samples were assigned to one population for the analysis. All calculations were made with FSTAT software (Goudet 2000). As FSTAT has been originally designed for diploid data sets, haploid fungal genotypes were encoded as diploid homozygotes as suggested by Goudet (2000). RESULTS Amplification of markers Among the 120 isolates analysed six, eight and ten alleles were observed in CT1500 (Fig. 1), CTMS (Fig. 2) and Vscar3 (Fig. 3) loci, respectively (Table 2). In addition five, three and one isolate out of the 120 had a null allele in these loci, respectively. The null alleles were considered as additional alleles in the analysis. The amplification of all markers was successful from the reference isolate of Venturia tremulae var. tremulae (CBS 692.85). However, only marker Vscar3 was amplified from the other V. tremulae var. tremulae reference isolate (CBS 257.38). Also all three markers were amplifiable from reference isolate of V. tremulae var. populi-albae (CBS 694.85) and two markers (CT1500 and Vscar3) could be amplified from V. tremulae var. grandidentatae (CBS 695.85) (Fig. 3). The PCR amplification from the DNA of V. populina or V. macularis isolates produced no PCR products (Table 3). Distribution of genetic diversity The genetic diversities between different loci over all samples were of the same magnitude (0.605, 0.658 and
Table 3. Alleles of marker loci amplified from reference samples.
Venturia tremulae var. tremulae CBS 692.85 V. tremulae var. tremulae CBS 257.38 V. tremulae var. grandidentatae CBS 695.85 V. tremulae var. populi-albae CBS 694.85 V. populina CBS 256.38 V. populina CBS 316.58 V. macularis CBS 477.61 V. macularis CBS 698.85
CT1500
CTMS
Vscar3
I
VI
IV
–
–
VII
Additional allele
–
VIII
II
IV
V
–
–
–
–
–
–
–
–
–
–
–
–
0.845 for CT1500, CTMS and Vscar3, respectively), but the genetic diversities within individual populations were highly variable. The highest value 0.911 was calculated for Vscar3 locus in population 10 (Ra¨ttvik, Sweden) and the lowest 0.327 for CT1500 locus in the Norwegian population 12 (Table 4). The Fst and Gst values were close to zero and therefore practically no genetic differentiation occurred between V. tremulae populations (Table 5). Totally, we observed 61 haplotypes, of which 40 were unique, i.e. observed only once (Fig. 4). The expected frequencies were calculated for haplotypes that were observed five times or more, by multiplying the observed frequencies of each allele/ locus. In the dataset, we observed three common haplotypes with frequencies of 5, 9 and 14. The expected frequencies were 4.5, 6.4 and 10.6, respectively, with no statistical difference to observed frequencies (Chi-square ; P>0.1).
R. Kasanen and others
69
Table 4. Nei’s (1987) unbiased gene diversity per locus and population. Population names and locations are given in Table 1. Population Locus
1
2
3
4
5
6
7
8
9
10
11
12
13
CT1500 CTMS Vscar3
0.429 0.607 0.821
0.652 0.667 0.864
0.709 0.800 0.909
0.564 0.600 0.673
0.533 0.378 0.756
0.800 0.644 0.889
0.556 0.722 0.750
0.714 0.476 0.667
0.667 0.476 0.905
0.644 0.644 0.911
0.600 0.733 0.867
0.327 0.836 0.836
0.857 0.464 0.786
Table 5. The genetic differentiation among Venturia tremulae populations. Grouping of isolates to 6 populations (regions) Locus
Fst
CT1500 x0.003 CTMS 0.008 Vscar3 0.002 Over all loci 0.002
Grouping of isolates to 13 populations (sites)
Gst
Fst
Gst
0.000 x0.005 x0.008 x0.005
x0.016 0.047 0.032 0.023
x0.022 0.036 0.030 0.017
DISCUSSION The successful amplification of the markers from V. tremulae varieties that the fungus studied belongs to V. tremulae var. tremulae, and not an undescribed species as concluded previously (Kasanen et al. 2001). Ascospore morphology and host species (P. tremula) are congruent with such an identification. All markers were amplified from V. tremulae var. populi-albae (CBS 694.85) and two loci (CT1500 and Vscar3) could be amplified from V. tremulae var. grandidentatae. Thus, the markers were species-specific, but not applicable for the identification of the infraspecific taxa described by Morelet (1985). The genetic differentiation between the populations of V. tremulae var. tremulae was practically absent, and in our analysis the dataset resembled a single population. The amount of gene flow between populations must be high and continuous to prevent any population subdivision despite considerable geographical distances and barriers (the Baltic Sea between Finland and Sweden). Neither did the results of a previous study with RAMS markers show evidence of genetic differentiation in Venturia tremulae (Kasanen et al. 2001). Interestingly, Tenzer et al. (1999) found that the degree of genetic differentiation (Gst average of 0.07) was low also among the European populations of the causative agent of apple scab, V. inaequalis. Therefore, it can be concluded that the genetic population structure of V. inaequalis and V. tremulae has similarities despite differences in their host distribution. Other common ascomycetes associated with trees such as Nectria fuckeliana infecting bark injuries of Norway spruce, showed some although limited differentiation among populations between Sweden and Lithuania (Vasiliauskas & Stenlid 1997).
The Finnish isolates were collected from ascospores of the fungus but other isolates were cultured from plant tissue where both ascospores or conidia could have caused infection. Weisgerber (1968) suggested that the conidia would be the main cause of infection during the summer. As the ascospores are products of meiosis and the conidia arise asexually (clonally), it could be suspected that the mycelial isolates were less polymorphic due to multiple sampling of the same conidial genotype (Paavolainen et al. 2001). This was not seen in our analysis ; one third of the isolates had unique haplotypes and the frequencies of the most common haplotypes were not statistically different from their expected frequencies. It should also be noted that clones would most probably be local and therefore their repeated sampling would only have increased Fst and Gst values (Paavolainen et al. 2001). In addition, the genetic diversities within individual populations were comparable and the highest values were from population samples collected as mycelial isolates (Table 4). This implies that the role of ascospores in infections could be more important than Weisgerber (1968) suggested. That would also be in accordance with our observations on spore disperal, showing ascospores being released even in the end of July (T.K., unpubl.). Two of the markers used in this study (CT1500 and Vscar3) produced easily scorable marker alleles, i.e. single bands. In contrast, the analysis of CTMS marker in the TGGE resulted in additional faint bands (Fig. 2). This was probably due to microsatellite motif within the marker, as ladderlike banding patterns are frequently observed in the analysis of microsatellite markers (Tenzer et al. 1999, Kasanen, Kaitera & Hantula 2000). From a technical point of view, it is noteworthy that the analysis of microsatellite locus CTMS, after laborous set-up of electrophoresis conditions, did not reveal more variation than the other markers that contained no microsatellite-like motifs but were technically easier to analyse. Our analysis of the distribution of genetic variation suggests considerable gene flow between populations of V. tremulae var. tremulae over large distances, implying that the fungus is an epidemiologically efficient pathogen. The observed genetic variation within populations and haplotype diversity suggests that the sexual lifecycle is important in the dissemination of the fungus. Consequently, the population structure and life-cycle
Population structure of Venturia tremulae
70
Fig. 4. The frequency distribution of haplotypes. For example, 40 haplotypes were observed once, seven were observed twice, etc.
suggest a high evolutionary potential, as described by McDonald & Linde (2002), and thus resistant aspen clones selected will avoid pathogen infections only for a limited time. Therefore, a continuous breeding programme should be implemented for Venturia resistance of aspen.
ACKNOWLEDGEMENTS We thank Antti Komulainen for skillful and precise laboratory work, and Osmo Korhonen for collecting aspen shoots from the field. The Centraalbureau voor Schimmelcultures (CBS) is acknowledged for supplying the V. tremulae strains free of charge. The study was financially supported by the Metsa¨liitto Group.
REFERENCES Dance, B. W. (1961) Leaf and shoot blight of poplars (section Tacamahaca Spach.) caused by Venturia populina (Vuill.) Fabric. Canadian Journal of Botany 39: 875–890. Goudet, J. (2000) FSTAT, a program to estimate and test gene diversities and fixation indices. Version 2.9.3 ; http://www.unil.ch/izea/ softwares/fstat.html. Hantula, J., Dusabenyagasani, M. & Hamelin, R. C. (1996) Random amplified microsatellites (RAMS) – a novel method for characterizing genetic variation within fungi. European Journal of Forest Pathology 26: 159–166. Kasanen, R., Kaitera, J. & Hantula, J. (2000) The genetic composition of Peridermium pini and Cronartium flaccidum cankers on Scots pine as revealed by two multi-allelic loci. Forest Pathology 30: 221–230. Kasanen, R., Hantula, J. & Kurkela, T. (2001) The occurrence of an undescribed species of Venturia in blighted shoots of Populus tremula. Mycological Research 108: 338–343.
McDonald, B. A. & Linde, C. (2002) Pathogen population genetics, evolutionary potential and durable resistance. Annual Review of Phytopathology 40: 349–379. Morelet, M. (1985) Les Venturia des peupliers de la section Leuce I. – Taxonomie. Cryptogamie, Mycologie 6: 101–117. Nei, M. (1987) Molecular Evolutionary Genetics. Columbia University Press, New York. Newcombe, G. (2003) Native Venturia inopina sp. nov., specific to Populus trichocarpa and its hybrids. Mycological Research 107: 108–116. Paavolainen, L., Kurkela, T., Suhonen, J. & Hantula, J. (2001) The genetic population structure of Pyrenopeziza betulicola the causative agent of birch leaf spot disease. Mycological Research 93: 258–264. Sivanesan, A. (1977) The taxonomy and pathology of Venturia species. Bibliotheca Mycologica 59: 1–139. Sivanesan, A. (1984) The Bitunicate Ascomycetes and their Anamorphs. J. Cramer, Vaduz. Tenzer, I., Ivanissevich, S. D., Morgante, M. & Gessler, C. (1999) Identification of microsatellite markers and their application to population genetics of Venturia inaequalis. Phytopathology 89: 748–753. Vainio, E. J., Korhonen, K. & Hantula, J. (1998) Genetic variation in Phlebiopsis gigantea as determined with random amplified microsatellite (RAMS) markers. Mycological Research 102: 187–192. Vasiliauskas, R. & Stenlid, J. (1997) Population structure and genetic variation in Nectria fuckeliana. Canadian Journal of Botany 75: 1707–1713. Weisgerber, H. (1968) Die Bedeutung der Triebspitzenkrankheit an Pappeln der Sektion Leuce Duby II. Holzzucht 22: 38–44. Weir, B. S. & Cockerham, C. C. (1984) Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370.
Corresponding Editor: H. T. Lumbsch
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DOI: 10.1017/S0953756203008918 Printed in the United Kingdom.
Migrational capacity of Fennoscandian populations of Venturia tremulae
Risto KASANEN1*, Jarkko HANTULA1, Martti VUORINEN2, Jan STENLID3, Halvor SOLHEIM4 and Timo KURKELA1 1
Finnish Forest Research Institute, Vantaa Research Centre, P.O. Box 18, FIN-01301, Vantaa, Finland. FFRI, Suonenjoki Research Station, Suonenjoki, Finland. 3 Institute of Mycology and Forest Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden. 4 Norwegian Forest Research Institute, Aas, Norway. E-mail : risto.kasanen@metla.fi 2
Received 27 February 2003; accepted 14 November 2003.
Genetic variation in three multiallelic loci was analysed with Temperature Gradient Gel Electrophoresis in order to assess the genetic population structure of Venturia tremulae var. tremulae in order to understand the evolutionary potential of the pathogen against resistance breeding. Also the identification of the fungus was verified with molecular analysis of reference isolates. The Fst and Gst values were very low indicating no substructuring or restrictions to gene flow between Fennoscandian populations of V. tremulae. The results imply high epidemiological efficiency of the fungus and therefore continuous breeding programme should be implemented for Venturia resistance of aspen.
INTRODUCTION Wide-scale plantations of aspen (Populus tremula) and hybrid aspen (P. tremularP. tremuloides) have recently been established in Nordic and Baltic countries as the forest industry has become interested in aspen fibre. As the number of aspen stands increases, the fungal diseases will gain in both economical and ecological importance. It is even possible that damages caused by the fungal diseases increase due to the pathogens adapting to established monocultures. The shoot diseases cause visible damage which results in economical losses to the nurseries. In young plantations the shoot diseases may delay the growth of seedlings or even cause mortality. The symptoms of black shoot blight of aspen include blackened and twisted shoots (so called shepherd’s crooks) and blackspotted leaves. Two ascomycetes are capable of causing the disease: Venturia macularis (anamorph unknown) and V. tremulae var. tremulae (anamorph : Pollaccia radiosa) (Morelet 1985). Both aspen and several aspen hybrids show variation in the resistance to black shoot blight and therefore it is probable that the disease could be controlled by utilizing clonal variation of resistance (Weisgerber 1968). * Corresponding author.
However, the plantations surveyed by Weisgerber (1968) were geographically widespread and therefore variation in the virulence between local fungal populations may well have caused bias in the survey. The information about the population structure of a pathogen is important in resistance breeding of a host. Even in the screening phase, a decision must be made whether to rely on local background inoculum or whether to have inocula collected from a wider geographical area. In transporting inoculum to field trials, one must also anticipate the risk of spreading virulent genotypes of the pathogen to new areas. Differences between local populations highly depend on the migrational capacity of the fungus. The capability of Venturia sp. to produce conidia throughout the growth season (Weisgerber 1968) and the regeneration of aspen by asexual reproduction might in theory favour clonality in fungal population and therefore give epidemiological benefit of the pathogen clones virulent to the coppicing aspen clone. Unfortunately, there are no studies available on the population structure of any Venturia species causing shoot blight on Populus. Thus the evolutionary potential (McDonald & Linde 2002) and migrational capacity of Venturia species is unknown. The distribution, taxonomy and morphology of Venturia species on Populus sp. is not well known or
R. Kasanen and others
65
Table 1. Sample data. All newly obtained isolates were isolated and deposited by R.K.
n
Host
Isolation method
Collector
Eastern Finland Kesa¨lahti (1) Joutseno (2) Rautja¨rvi (3)
8 12 11
Populus tremula P. tremula P. tremula
ascospore ascospore ascospore
T. Kurkela T. Kurkela T. Kurkela
Central Finland Varpaisja¨rvi (4) Va¨a¨na¨la¨nranta (5) Lo¨ytynma¨ki (6)
11 10 10
P. tremula P. tremula P. tremula
ascospore ascospore ascospore
M. Vuorinen M. Vuorinen M. Vuorinen
9 7 7
P. tremula P. tremula P. tremula
ascospore ascospore ascospore
J. Hantula J. Hantula J. Hantula
Sweden Ra¨ttvik, Dalarna (10) Gottsunda, Uppsala (11)
10 6
P. tremula P. tremula
mycelia mycelia
J. Stenlid J. Stenlid
Norway Hof, Vestfold (12)
11
P. tremula
mycelia
H. Solheim
8
P. tremula
mycelia
T. Kurkela
ascospore not known ascospore ascospore not known not known not known spore
M. Morelet O. Servazzi M. Morelet M. Morelet O. Servazzi R. Ciferri E. Mu¨ller M. Morelet
POPULATION SAMPLES
Northern Finland Keminmaa (7) Jaatilansaari (8) Loue (9)
Russia Valamo island, Lake Ladoga (13) REFERENCE SAMPLES
Venturia tremulae var. tremulae CBS 692.85 V. tremulae var. tremulae CBS 257.38 V. tremulae var. grandidentatae CBS 695.85 V. tremulae var. populi-albae CBS 694.85 V. populina CBS 256.38 V. populina CBS 316.58 V. macularis CBS 477.61 V. macularis CBS 698.85 a
Francea, P. tremula Italy, P. tremula Canada, P. tremuloides France, P. alba Italy, P. canadiensis Italy, Populus sp. France France
Country of origin.
documented. Sivanesan (1977, 1984) treated V. macularis and V. tremulae as synonyms occurring on Populus sect. Leuce, and described Pollaccia radiosa as the anamorph. On Populus sect. Tacamahaca, the shoot blight was considered to be caused by V. populina (Dance 1961, Sivanesan 1977). However, Morelet (1985) stated that V. macularis has no known anamorph. In addition to describing morphological differences between V. tremulae and V. macularis, Morelet (1985) divided V. tremulae into three varieties: var. tremulae, var. grandidentatae, and var. populi-alba. It must also be noted that in North America, a new species has recently been described, V. inopina on Populus trichocarpa (Newcombe 2003). We have isolated Venturia species from blighted shoots of aspen in Finland. Based on the morphological key of Sivanesan (1977) and molecular analyses, we concluded that the isolates did not belong to V. macularis (syn. V. tremulae according to Sivanesan 1977, 1984) or V. populina, but to a third species (Kasanen, Hantula & Kurkela 2001). Unfortunately, we were not then aware of the paper by Morelet (1985) in which another treatment of Venturia had been proposed. Morphologically, the ascospores of our isolates (Kasanen et al. 2001) were similar to V. tremulae as described by Morelet (1985), we consider it probable
that our samples were actually V. tremulae var. tremulae, and that V. tremulae and V. macularis are not conspecific as Sivanesan (1977) proposed. However, comparison of the literature (Dance 1961, Sivanesan 1977, Morelet 1985) reveals that the ascospores of V. populina and V. tremulae varieties are highly similar and morphologically these fungi could only be identified by the morphology of the conidia. Unfortunately, according to Weisgerber’s (1968) and our observations, the conidia emerge only seldom in in vitro. In addition, none of the samples Morelet (1985) examined was from Scandinavia. The aim of this study was to assess the genetic population structure of the Venturia sp. described in Kasanen et al. (2001), with the null hypothesis that no genetic differentiation of the fungus occurs even on a more wide geographical scale. Also, the identity of the fungus was tested by molecular comparisons to the reference isolates.
MATERIALS AND METHODS Fungal material Nine Finnish populations of Venturia were collected in June 1999 (Table 1). In the sampling, three local
Population structure of Venturia tremulae
66
Table 2. The distribution of allelic variation in marker loci. Population names and locations are given in Table 1. Alleles VII, IX and XI in loci CT1500, CTMS and Vscar3 are null alleles, respectively. pop1 8
pop2 12
pop3 11
pop4 11
pop5 10
pop6 10
pop7 9
pop8 7
pop9 7
pop10 10
pop11 6
pop12 11
pop13 8
Locus CT1500 Alleles I II III IV V VI VII
0.750 0.000 0.000 0.250 0.000 0.000 0.000
0.583 0.083 0.000 0.167 0.000 0.000 0.167
0.364 0.091 0.000 0.455 0.000 0.000 0.091
0.636 0.000 0.273 0.091 0.000 0.000 0.000
0.700 0.000 0.100 0.100 0.000 0.000 0.100
0.400 0.100 0.100 0.300 0.000 0.100 0.000
0.667 0.000 0.111 0.222 0.000 0.000 0.000
0.571 0.000 0.143 0.143 0.143 0.000 0.000
0.571 0.286 0.000 0.143 0.000 0.000 0.000
0.500 0.000 0.100 0.400 0.000 0.000 0.000
0.667 0.000 0.167 0.167 0.000 0.000 0.000
0.818 0.000 0.000 0.182 0.000 0.000 0.000
0.375 0.125 0.250 0.125 0.000 0.000 0.125
Locus CTMS Alleles I II III IV V VI VII VIII IX
0.000 0.000 0.250 0.625 0.125 0.000 0.000 0.000 0.000
0.000 0.083 0.083 0.583 0.083 0.000 0.000 0.000 0.167
0.000 0.000 0.000 0.091 0.182 0.091 0.091 0.455 0.091
0.091 0.091 0.182 0.636 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.000 0.800 0.100 0.100 0.000 0.000 0.000
0.000 0.100 0.400 0.500 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.222 0.444 0.000 0.333 0.000 0.000 0.000
0.000 0.000 0.286 0.714 0.000 0.000 0.000 0.000 0.000
0.000 0.000 0.286 0.714 0.000 0.000 0.000 0.000 0.000
0.000 0.100 0.200 0.600 0.000 0.100 0.000 0.000 0.000
0.000 0.333 0.000 0.500 0.000 0.167 0.000 0.000 0.000
0.000 0.091 0.273 0.364 0.091 0.091 0.091 0.000 0.000
0.000 0.000 0.125 0.750 0.000 0.125 0.000 0.000 0.000
0.000 0.000 0.000 0.000 0.250 0.375 0.125 0.250 0.000 0.000 0.000
0.000 0.083 0.000 0.333 0.000 0.083 0.250 0.083 0.083 0.000 0.083
0.000 0.091 0.273 0.182 0.182 0.091 0.091 0.091 0.000 0.000 0.000
0.000 0.000 0.000 0.091 0.091 0.000 0.273 0.000 0.545 0.000 0.000
0.000 0.000 0.000 0.100 0.000 0.100 0.500 0.200 0.100 0.000 0.000
0.000 0.000 0.000 0.000 0.300 0.100 0.200 0.200 0.100 0.000 0.100
0.000 0.000 0.000 0.444 0.000 0.000 0.333 0.111 0.111 0.000 0.000
0.000 0.000 0.000 0.000 0.000 0.000 0.286 0.571 0.143 0.000 0.000
0.000 0.000 0.000 0.143 0.286 0.143 0.143 0.286 0.000 0.000 0.000
0.000 0.000 0.000 0.200 0.000 0.200 0.200 0.200 0.100 0.100 0.000
0.333 0.000 0.000 0.000 0.167 0.167 0.333 0.000 0.000 0.000 0.000
0.000 0.000 0.000 0.182 0.000 0.182 0.364 0.182 0.091 0.000 0.000
0.000 0.000 0.000 0.000 0.125 0.125 0.500 0.125 0.125 0.000 0.000
Populations
Locus Vscar3 Alleles
n
I II III IV V VI VII VIII IX X XI
populations separated by approx. 100 km were collected from eastern, southern and northern Finland. The distance between regions was more than 250 km. Single ascospores were isolated from each shoot using the following procedure. The twisted tip of the shoot was cut apart, placed on 1.5 % water agar and incubated at room temperature for 1–3 d. The plates were inspected daily under a microscope for spore release. As sporulation was observed, 3–5 Venturia ascospores per plate were isolated with a sterile modified pasteur pipette and further cultured on 2% malt agar. When growth of the mycelia was observed, one single-spore colony per plate was randomly selected and subcultured on 1.5 % malt agar covered with cellophane membrane. The populations from Norway, Sweden and Russia were collected in 2000 and 2001 (Table 1). The distance between the Norwegian population and the nearest Swedish population was more than 270 km, and the Russian population was more than 200 km from the nearest Finnish populations. Because the collection dates varied between June and September, ascospore release had in most cases already taken place or samples represented the current season infection. Mycelial cultures of the fungus were isolated from dead shoots as follows. Fresh infection sites (recently blackened spots that could be caused by conidia or ascospores) were
selected and cut from the shoot material. The shoot pieces were surface-sterilized by rinsing in 75% ethanol, 4 % sodium hypochloride and sterile water (10 s each) and placed on 2% malt agar. Reference cultures of different Venturia species and varieties were obtained from the Centraalbureau voor Schimmelcultures (CBS, Utrecht) (Table 1). DNA isolations DNA isolations were made as described in Vainio, Korhonen & Hantula (1998). The fungal tissue was homogenized in 1.5 ml disposable plastic tubes by grinding with glass rod and quartz sand. Briefly, the DNA isolation procedure included two phenol-chlorophorm (1 : 1) extractions, chloroform-phenol-isoamyl alcohol extractions, precipitation with polyethylene glycol (PEG) and drying. The DNA was resuspended into 10 mM Tris HCl buffer containing 1 mM EDTA. Marker design For markers CT1500 and Vscar3, single bands were chosen from RAMS fingerprints amplified with primers DVD(CT)7C and DHB(CGA)5 respectively (Hantula, Dusabenyagasani & Hamelin 1996). The amplification of RAMS fingerprints was performed as described in
R. Kasanen and others Hantula et al. (1996) and markers were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The clones were sequenced with LI-COR IR2 automated sequencer according to the manufacturer’s recommendations. Based on the sequences obtained two pairs of primers were designed in order to amplify regions of 1500 and 900 bp from the sequence of the original RAMS markers, respectively. A GC-clamp of 40 bp was added to forward primer sequences to allow optimal resolution in Temperature Gradient Gel Electrophoresis (TGGE). The primer sequences were: (1) CT1500 GC : 5k CCC CCG CCG CGC GCC GCG CGG CGG (2) CCG GCC GCA CGC GCC GGC TTT TCT CAT AGG CCC CTC ; CT1500R : 5k CTT CAT GCT GAA CCC ACC TC ; (3) Vscar3R : 5k CGA AGG CTG GGA GTA CTT TG ; and (4) Vscar3 FGC : 5k CCC CCG CCG CGC GCC GCG CGG CGG CCG GCC GCA CGC GCC TTA TGA AGG CGG AGG TGA AC. For marker CTMS, based on the DVD(CT)7C fingerprint, the sequence analysis revealed a ten-fold repeat of CT (microsatellite motif ) at a binding site for the RAMS primer. To obtain sequence information of the flanking region of the microsatellite the following procedure was used. A pair of primers was designed near the microsatellite so that the reverse primer was located upstream of the forward primer. Total DNA of Venturia sample was digested with CfoI restriction enzyme (Boehringer-Mannheim) according to manufacturers recommendations. The restriction fragments were ligated with T4 ligase (Fermentas) to produce circular DNA, which was used as a template in the following PCR which resulted in a 500 bp amplification product. The sequence analysis of the product confirmed that the microsatellite and the flanking region had been cloned. A new primer pair was designed to amplify the microsatellite region and a GC-clamp of 40 bp was added to forward primer sequences to allow optimal resolution in TGGE. The primer sequences were : CTMS-R3, 5k TGG TGT TGA TAC CTG TTT TTA CT ; and CTMS-GC, 5k CCC CCG CCG CGC GCC GCG CGG CGG CGG GCC GCA CGC GCC TGC GTG CAA GTA CAA TCC AT. On agarose gels, CTMS marker was seen as a single band, but after TGGE run additional faint bands were seen. The most intense band was scored as the marker. In TGGE the samples are run in polyacrylamide gel containing a constant concentration of urea. As the temperature is increased gradually and uniformly during the electrophoresis, the result is a linear temperature gradient over the length of the electrophoresis run. Thus a denaturing gradient is formed by the constant concentration of urea in combination with temporal temperature change. The temperature gradients, ramping rates of temperature and run voltages used for markers CT1500, CTMS and Vscar3 were 40–65 xC, 1.3 x hx1, 150 V ; 54–59 x, 1.3 x hx1, 130 V, and 53–63 x, 2.5 x hx1, 170 V, respectively. All markers were analysed with DCode electrophoresis apparatus, in 6 % polyacrylamide gels containing 6 M urea. The running conditions for markers were optimized according to
67
M
1
2
3
4
5
6
7
8
M
Fig. 1. Temperature gradient gel electrophoresis of alleles in locus CT1500 marker. Lane M, standard sample (note that due to denaturing process the standard is applicable only in order to detect lane distortion) ; lane 1, allele found only in the reference isolate CBS 695.85 ; lanes 2–3, isolates from northern and eastern Finland having the same allele (I); and lanes 4–8 showing alleles II–VI, respectively.
1
2
3
4
5
6
7
8
9
10
Fig. 2. An example of TGGE screening of alleles of locus CTMS. Lane 1, an isolate from population 7, northern Finland (allele VI) ; lane 2, an isolate from population 12, Sweden (allele VII) ; lane 3, an isolate from population 7 (allele VI) ; lanes 4 and 5, isolates from population 4, central Finland (alleles I and II, respectively) ; lanes 6 and 7, isolates from populations 12, Sweden and 6, central Finland, share the same allele (III) ; lanes 8 and 9, isolates from populations 9 (northern Finland) and 4 (central Finland) share the same allele (IV) ; lane 10, a isolate from population 9 (allele V). The most intense band on each lane was scored.
manufacturers recommendations (Bio-Rad, Hercules, CA). Because of occasional lane distortion, some alleles which had only small differences in their mobility were difficult to score over the width of the gel. In such cases samples were run several times on adjacent lanes. Also a mixture of PCR products of different alleles was made and used as a standard (Fig. 3). Analysis of genetic diversity To assess the genetic diversity within populations, we calculated Nei’s unbiased gene diversities (Nei 1987). In order to study genetic differentiation among populations, we calculated the Fst analogue defined by
Population structure of Venturia tremulae
1
2
3
4
5
68
6
7
8
9
10
11
12
13
14
Fig. 3. An example of TGGE screening of alleles of locus Vscar3. Lanes 8, 11 and 13, allelic ladders composed of a mixture of different PCR products used as an internal standard in screening because of occasional lane distortions during the electrophoresis. Lanes 1 and 2, isolates from populations 6 and 4 (allele V); lanes 3 and 4, isolates from population 4 (allele VII) ; lane 5, an isolate from population 11 (allele VI) ; lane 6, an isolate from population 12 (allele X) ; lane 7, CBS 692.85 (allele IV) ; lanes 9 and 10, CBS 257.38 and CBS 695.85 (allele VIII) ; lane 12, CBS 694.85 (allele V); and lane 14, isolate from population 1 (allele VI).
Weir & Cockerham (1986) and Gst value (Nei 1987). For all the calculations, the populations were grouped in two ways ; either all 13 populations were considered separate, or they were divided to six populations based on geographic locations ; Russia, Sweden, Norway, northern, central and southern Finland (Table 1). To compare the genetic diversity (Nei 1987) between different loci over all samples, the samples were assigned to one population for the analysis. All calculations were made with FSTAT software (Goudet 2000). As FSTAT has been originally designed for diploid data sets, haploid fungal genotypes were encoded as diploid homozygotes as suggested by Goudet (2000). RESULTS Amplification of markers Among the 120 isolates analysed six, eight and ten alleles were observed in CT1500 (Fig. 1), CTMS (Fig. 2) and Vscar3 (Fig. 3) loci, respectively (Table 2). In addition five, three and one isolate out of the 120 had a null allele in these loci, respectively. The null alleles were considered as additional alleles in the analysis. The amplification of all markers was successful from the reference isolate of Venturia tremulae var. tremulae (CBS 692.85). However, only marker Vscar3 was amplified from the other V. tremulae var. tremulae reference isolate (CBS 257.38). Also all three markers were amplifiable from reference isolate of V. tremulae var. populi-albae (CBS 694.85) and two markers (CT1500 and Vscar3) could be amplified from V. tremulae var. grandidentatae (CBS 695.85) (Fig. 3). The PCR amplification from the DNA of V. populina or V. macularis isolates produced no PCR products (Table 3). Distribution of genetic diversity The genetic diversities between different loci over all samples were of the same magnitude (0.605, 0.658 and
Table 3. Alleles of marker loci amplified from reference samples.
Venturia tremulae var. tremulae CBS 692.85 V. tremulae var. tremulae CBS 257.38 V. tremulae var. grandidentatae CBS 695.85 V. tremulae var. populi-albae CBS 694.85 V. populina CBS 256.38 V. populina CBS 316.58 V. macularis CBS 477.61 V. macularis CBS 698.85
CT1500
CTMS
Vscar3
I
VI
IV
–
–
VII
Additional allele
–
VIII
II
IV
V
–
–
–
–
–
–
–
–
–
–
–
–
0.845 for CT1500, CTMS and Vscar3, respectively), but the genetic diversities within individual populations were highly variable. The highest value 0.911 was calculated for Vscar3 locus in population 10 (Ra¨ttvik, Sweden) and the lowest 0.327 for CT1500 locus in the Norwegian population 12 (Table 4). The Fst and Gst values were close to zero and therefore practically no genetic differentiation occurred between V. tremulae populations (Table 5). Totally, we observed 61 haplotypes, of which 40 were unique, i.e. observed only once (Fig. 4). The expected frequencies were calculated for haplotypes that were observed five times or more, by multiplying the observed frequencies of each allele/ locus. In the dataset, we observed three common haplotypes with frequencies of 5, 9 and 14. The expected frequencies were 4.5, 6.4 and 10.6, respectively, with no statistical difference to observed frequencies (Chi-square ; P>0.1).
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Table 4. Nei’s (1987) unbiased gene diversity per locus and population. Population names and locations are given in Table 1. Population Locus
1
2
3
4
5
6
7
8
9
10
11
12
13
CT1500 CTMS Vscar3
0.429 0.607 0.821
0.652 0.667 0.864
0.709 0.800 0.909
0.564 0.600 0.673
0.533 0.378 0.756
0.800 0.644 0.889
0.556 0.722 0.750
0.714 0.476 0.667
0.667 0.476 0.905
0.644 0.644 0.911
0.600 0.733 0.867
0.327 0.836 0.836
0.857 0.464 0.786
Table 5. The genetic differentiation among Venturia tremulae populations. Grouping of isolates to 6 populations (regions) Locus
Fst
CT1500 x0.003 CTMS 0.008 Vscar3 0.002 Over all loci 0.002
Grouping of isolates to 13 populations (sites)
Gst
Fst
Gst
0.000 x0.005 x0.008 x0.005
x0.016 0.047 0.032 0.023
x0.022 0.036 0.030 0.017
DISCUSSION The successful amplification of the markers from V. tremulae varieties that the fungus studied belongs to V. tremulae var. tremulae, and not an undescribed species as concluded previously (Kasanen et al. 2001). Ascospore morphology and host species (P. tremula) are congruent with such an identification. All markers were amplified from V. tremulae var. populi-albae (CBS 694.85) and two loci (CT1500 and Vscar3) could be amplified from V. tremulae var. grandidentatae. Thus, the markers were species-specific, but not applicable for the identification of the infraspecific taxa described by Morelet (1985). The genetic differentiation between the populations of V. tremulae var. tremulae was practically absent, and in our analysis the dataset resembled a single population. The amount of gene flow between populations must be high and continuous to prevent any population subdivision despite considerable geographical distances and barriers (the Baltic Sea between Finland and Sweden). Neither did the results of a previous study with RAMS markers show evidence of genetic differentiation in Venturia tremulae (Kasanen et al. 2001). Interestingly, Tenzer et al. (1999) found that the degree of genetic differentiation (Gst average of 0.07) was low also among the European populations of the causative agent of apple scab, V. inaequalis. Therefore, it can be concluded that the genetic population structure of V. inaequalis and V. tremulae has similarities despite differences in their host distribution. Other common ascomycetes associated with trees such as Nectria fuckeliana infecting bark injuries of Norway spruce, showed some although limited differentiation among populations between Sweden and Lithuania (Vasiliauskas & Stenlid 1997).
The Finnish isolates were collected from ascospores of the fungus but other isolates were cultured from plant tissue where both ascospores or conidia could have caused infection. Weisgerber (1968) suggested that the conidia would be the main cause of infection during the summer. As the ascospores are products of meiosis and the conidia arise asexually (clonally), it could be suspected that the mycelial isolates were less polymorphic due to multiple sampling of the same conidial genotype (Paavolainen et al. 2001). This was not seen in our analysis ; one third of the isolates had unique haplotypes and the frequencies of the most common haplotypes were not statistically different from their expected frequencies. It should also be noted that clones would most probably be local and therefore their repeated sampling would only have increased Fst and Gst values (Paavolainen et al. 2001). In addition, the genetic diversities within individual populations were comparable and the highest values were from population samples collected as mycelial isolates (Table 4). This implies that the role of ascospores in infections could be more important than Weisgerber (1968) suggested. That would also be in accordance with our observations on spore disperal, showing ascospores being released even in the end of July (T.K., unpubl.). Two of the markers used in this study (CT1500 and Vscar3) produced easily scorable marker alleles, i.e. single bands. In contrast, the analysis of CTMS marker in the TGGE resulted in additional faint bands (Fig. 2). This was probably due to microsatellite motif within the marker, as ladderlike banding patterns are frequently observed in the analysis of microsatellite markers (Tenzer et al. 1999, Kasanen, Kaitera & Hantula 2000). From a technical point of view, it is noteworthy that the analysis of microsatellite locus CTMS, after laborous set-up of electrophoresis conditions, did not reveal more variation than the other markers that contained no microsatellite-like motifs but were technically easier to analyse. Our analysis of the distribution of genetic variation suggests considerable gene flow between populations of V. tremulae var. tremulae over large distances, implying that the fungus is an epidemiologically efficient pathogen. The observed genetic variation within populations and haplotype diversity suggests that the sexual lifecycle is important in the dissemination of the fungus. Consequently, the population structure and life-cycle
Population structure of Venturia tremulae
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Fig. 4. The frequency distribution of haplotypes. For example, 40 haplotypes were observed once, seven were observed twice, etc.
suggest a high evolutionary potential, as described by McDonald & Linde (2002), and thus resistant aspen clones selected will avoid pathogen infections only for a limited time. Therefore, a continuous breeding programme should be implemented for Venturia resistance of aspen.
ACKNOWLEDGEMENTS We thank Antti Komulainen for skillful and precise laboratory work, and Osmo Korhonen for collecting aspen shoots from the field. The Centraalbureau voor Schimmelcultures (CBS) is acknowledged for supplying the V. tremulae strains free of charge. The study was financially supported by the Metsa¨liitto Group.
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