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Analysis of genotypic diversity inCercospora beticola Sacc. field isolates

Analysis of genotypic diversity inCercospora beticola Sacc. field isolates Annals of Microbiology, 56 (3) 215-221 (2006) 1 2 1 1,§ 1 Maddalena MORETTI *, George KARAOGLANIDIS , Marco SARACCHI , Anna FONTANA , Gandolfina FARINA 1 2 Istituto di Patologia Vegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy; Hellenic Sugar Industry, Plant Protection Department, Sugar Factory of Platy, 59032, Platy Imathias, Greece Received 25 May 2006 / Accepted 30 June 2006 Abstract - Genetic variability and population structure of Cercospora beticola, the causal agent of Cercospora leaf spot in sugarbeet, from four sugarbeet-growing regions of Greece were investigated using growth rate, pathogenicity, and mini- and microsatellite DNA fingerprinting. Mycelial growth and pathogenicity were very diverse within and between groups, and no correlation was found between these features and the geographic origin of the isolates. High diversity was found by micro- and minisatellite fingerprinting, with an average gene diversity of 0.21, and no significant differences among populations. Among the 46 isolates, 45 different genotypes were identified, showing a high degree of genotype diversity. Analysis of the genetic profiles provided no evidence for regional patterns of variation (ΦF =0.01, P = 0.261) and the analysis of molecular variation (AMOVA) revealed that genetic variability was due mainly to ST variations within (99%) rather than between (1%) populations. Such a low level of genetic differentiation is reflected by a migration rate value Nm of 4.7. The high migration rate cannot be referred to splash dispersed conidia. To justify the absence of a regional struc- ture in these C. beticola populations, we must suppose the existence of a long-distance means of dispersal, such as seed transmission and/or man mediated transmission. Key words: Beta vulgaris, microsatellite, minisatellite, population genetic, pathogenicity. INTRODUCTION and distribution of genetic diversity within and among pop- ulations (McDonald and Linde, 2002). Methods usually Cercospora beticola Sacc., the causal agent of Cercospora leaf employed to test diversity in fungal populations include vir- spot (CLS) in sugarbeet, is one of the most destructive and ulence and molecular analyses (McDermott et al. 1989; Mil- widespread foliar diseases in the warm and humid areas groom and Fry, 1997). where this crop is cultivated (Duffus and Ruppel, 1993; Wei- Cercospora beticola is characterised by a great variabil- land and Koch, 2004). ity in pathogenicity, morphocultural and metabolic features A teleomorphic phase of this fungus has not yet been such as growth rate, mycelium colour and phytotoxin pro- identified neither in C. beticola nor in other Cercospora duction (Ruppel, 1972; Whitney and Lewellen, 1976; Moret- species monophyletic with C. beticola on the basis of rDNA ti et al., 2004). This pathogen has not yet been broadly sequences (Goodwin et al., 2001). The primary inoculum is explored by molecular techniques, and not much is known thought to be mainly constituted by conidia, produced on about its population genetic structure. Studies by Amplified stromata in crop debris (Weiland and Koch, 2004), which are Fragment Length Polymorphism (AFLP) (Weiland et al., usually ploughed under in the field. These conidia are rain, 2001), RAPD-PCR (Random Amplified Polymorphic DNA) and insect and wind dispersed over short distances (McKay and DAMD-PCR (Direct Amplification of Minisatellite-region DNA) Pool, 1918; Lawrence and Meredith, 1970). Seed contami- (Moretti et al., 2004) showed that this fungus harbours con- nation has also been reported by several authors (Ruppel, siderable genetic variation, despite the absence of a known 1986; Vereijssen et al., 2004). teleomorph. In fact, sexual populations generally have A valuable contribution to the knowledge of epidemiolo- greater genotype diversity than clonal populations (Mil- gy and, therefore, to the improvement of plant pathogen groom, 1996). management, can derive from the study of the genetic struc- Protection of sugarbeet against CLS still lies mainly on ture of a pathogen (McDonald, 1997), defined as the amount fungicide application. The development of resistant or tol- erant varieties can help to maintain crop yield under heavy disease pressure, reducing the quantities of fungicides applied. Sugarbeet breeders find it still very difficult to obtain * Corresponding author. Phone: +39 02 5031 6792; highly resistant hybrids with a root yield potential equal to Fax: +39 02 5031 6781; E-mail:maddalena.moretti@unimi.it the susceptible ones grown in the absence of C. beticola. In § Present address: Department of Biochemistry, Max Planck Insti- fact, CLS resistance is a quantitative trait, controlled by at tute for Chemical Ecology, Hans-Knöll Str. 8D-07745, Jena, Ger- least five loci, and it is characterised by a low heritability many. (Setiawan et al., 2000). 216 M. Moretti et al. For this reason, plant breeders should know the poten- flask was inoculated with five plugs, taken with a 5-mm cork tial for gene flow and long distance dispersal, and the mech- borer from the margins of a fungal colony growing on PDA anisms of C. beticola reproduction and genetic exchange. All and was shaken at 150 rpm, in the dark, for 10 days. The these information determine the potential of C. beticola to mycelium was harvested by filtration through filter paper circumvent sugarbeet CLS resistance and can be inferred by (Whatman paper No 3, Whatman, Maidstone, Kent, U.K.) in the knowledge of genetic structure of C. beticola populations. a Büchner funnel. For growth determination, 4 separate cul- The objective of this study was to analyse the genetic tures were filtered individually through filter paper, dried in structure of four populations of C. beticola obtained in an oven at 80 °C for 24 h and weighed. The experiment was Greece, in order to test the hypothesis that, being C. beti- repeated three times. cola mainly dispersed by conidia on a short distance, popu- lations geographically separated are also genetically differ- Measurement of pathogenicity. entiated. The analysis was conducted using a microsatellite Plant material. The experiments were conducted on Beta vul- (SAT 2) and a minisatellite primer (M 13). garis L. plants, cv. ‘Rival’, which is susceptible to C. betico- la. Plants were grown in a greenhouse (18-26 C), in plas- tic pots (18 cm diameter) containing a 2:1 mixture of peat MATERIALS AND METHODS and perlite. Each pot contained two plants. Sugarbeet seedlings were fertilised once per week with 1% N:P:K Fungal isolates. The isolates used in this study were col- (20:20:20) solution. Plants used for inoculations were 6-7 lected from sugarbeet fields during the summer of 2002. Four weeks old, at the stage of 4-5 fully expanded leaves. regions of Northern Greece (Fig. 1) were sampled and in total 46 isolates were collected, 13 from the area of Orestiada, 12 Inoculum production. Isolates for inoculum production were from Imathia, 13 from Xanthi, and 8 from Serres. grown on V-8 juice agar medium (Tuite, 1969). After 10 days All the isolates were collected as single-lesion isolates and of incubation under fluorescent light at 25 C, sporulating were transformed to single-spore isolates using the dilution colonies were rinsed with 15 ml of distilled water and the technique (Tuite, 1969). They were obtained by transferring conidial suspensions were filtered through a double-layered conidia, with the aid of a fine glass needle, to Potato Dex- sterile cheesecloth. Suspensions were adjusted to 8 x 10 -1 trose Agar (PDA, Oxoid, Unipath Ltd, Basingstoke, U.K.) conidia ml . Before inoculation, one droplet of 0.1% Tween -1 acidified with 0.5 ml l lactic acid to suppress bacterial 20 was added to each suspension. growth. The isolates were obtained from several fields in each sampling site. From each field one leaf with distinct sporu- Inoculation. Inoculation was carried out by spraying the coni- lating lesions was selected and conidia from only one lesion dial suspension until run off, with a spray atomiser. Both leaf per leaf were transferred to the culture medium to obtain one surfaces were sprayed and the potted plants were transferred isolate per field. To obtain durable stock cultures, the strains to a growth chamber and kept at 25 C and 99% RH, with a 16- were grown on PDA plates and plugs of mycelium were h photoperiod. In total 8 plants per isolate were inoculated. maintained in sterile water at room temperature. Pathogenicity recording. After the appearance of the symp- Measurement of mycelial growth. Isolates were grown toms, about 15 days after inoculation, the plants were in Potato Dextrose Broth (Difco Laboratories, Detroit, USA), removed from the growth chamber and the disease severi- in 250-ml flasks containing 50 ml of nutrient medium. Each ty was recorded on 3 randomly selected leaves per plant, FIG. 1 – Map of Greece showing the four Cercospora beticola sampling sites. Ann. Microbiol., 56 (3), 215-221 (2006) 217 according to the nine-category scale disease index of Klein- wanzlebener Saatzucht (KWS): 1 = healthy leaf, and 9 = leaf 0.45 and leaf stalk dead and dried up (Shane and Teng, 1992). The experiment was repeated twice. 0.40 DNA extraction and amplification. Cercospora beticola isolates were grown in liquid medium (DSM65: 4 g of glu- 0.35 -1 cose, 4 g of yeast extract and 10 g of malt extract l ) and incubated for one week at 25 °C on a rotary shaker (120 rpm), in the dark. Total genomic DNA was extracted from 0.30 lyophilised ground mycelium according to Kelly et al. (1998). Italian strains were used as positive control. 0.25 DNA minisatellite and microsatellite regions were ampli- fied by PCR (Polymerase Chain Reaction). Amplifications Imathia Orestiada Serres Xanthi were performed using the following reaction mixtures (50 µl Sampling sites reaction volume): 1x Taq buffer, 1.5 mM MgCl , 0.2 mM dNTPs, 0.42 µM primer, 2 U of Taq polymerase and 2 µl of FIG. 2 – Boxplot of mycelial growth of Cercospora beticola in the DNA (all PCR reagents purchased from Amersham Pharma- four sampling sites. cia). The primers used were the minisatellite marker M13 (5’- GAGGGTGGCGGTTCT-3’), and the microsatellite marker SAT2 (GTG) . For M13 the amplification programme consisted of ty (G) was calculated for each population using the meas- an initial denaturation at 94 °C for 1 min, followed by 40 ure of Stoddart and Taylor (1988). To compare populations cycles at 94 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 of different sample sizes genotypic evenness was calculat- min, with a final extension at 72 °C for 6 min. For SAT2, the ed dividing G by the number of genotypes observed (Stod- thermocycler was programmed for an initial denaturation at dart and Taylor, 1988). 94 °C for 1 min, followed by 40 cycles at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 6 min. The amplification products were separated RESULTS by electrophoresis on 1.5% agarose gel in TAE buffer (Tris- acetate EDTA, Tris-HCl 40 mM and NaEDTA 1 mM). Gels were Mycelial growth and pathogenicity -1 stained with 0.5 µg ml of ethidium bromide, visualized The dry weight of the mycelia ranged from 0.25 to 0.45 g in under U.V. light and the images were acquired with Gel-Doc the Orestiada group, with a mean value of 0.35 g, from 0.25 1000 (Bio-Rad Laboratories, California, USA). to 0.47 g in the Imathia group, with a mean value of 0.37 g, from 0.27 to 0.47 g in the Xanthi group, with a mean value Data analysis. Data obtained from mycelial growth and of 0.39 g and from 0.28 to 0.42 g in the Serres group, with pathogenicity tests were subjected to an analysis of variance. a mean value of 0.36 g (Fig. 2). The diversity (P < 0.05) was Means of each isolate within sampling sites and means significant within the four populations tested, while the among sampling sites were compared using the LSD test. All means of each population were not significantly different (P the statistical analyses were performed on Mstat-C (vers. > 0.05). 2.10, Michigan State University). All the isolates caused disease symptoms on the sugar- For the molecular analysis, bands were scored for pres- beet cultivar selected for the experiment. A large variation in ence and absence. Data generated from M13 and SAT2 pathogenicity was found among the tested isolates, even primers were pooled to generate one binary matrix. The sim- within the same population (P < 0.05). The mean disease ilarity matrix was calculated using Jaccard (J) coefficient, and index of individual isolates ranged from 2.95 to 5.50 (Fig. 3) it was used to perform the Principal Components Analysis (PCA). The similarity matrix and PCAs were calculated by 0.55 NTSYSpc (vers. 2.01, Applied Biostatistics Inc.). The 46 isolates were arbitrarily subdivided into 4 groups according to their origin, and into 3 groups on the basis of 0.50 their level of pathogenicity (group I from 2.5 to 3.5, group II from 3.6 to 4.5 and group III from 4.6 to 5.5). An ANOVA 0.45 analysis was carried out to verify the significance of the dif- ferences among the genetic profiles of these groups. The test 0.40 was performed on the coordinates of the strains in the 2- dimensional PCA from molecular analysis. For the population structure analysis, the estimation of 0.35 Nei’s measure of genetic identity (Nei, 1978) and the aver- age number of migrants among populations per generation 0.30 Nm (Nei, 1972) were calculated with Popgene (vers. 1.32). The data were also subjected to analysis of molecular vari- Imathia Orestiada Serres Xanthi ance (AMOVA, Excoffier et al., 1992) with GeneAlEx (vers. Sampling sites 5.1, Peakall and Smouse, 2001), which is freely available from the Australian National University FIG. 3 – Boxplot of pathogenicity of Cercospora beticola in the four http://www.anu.edu.au/BoZo/GenAlEx/. Genotypic diversi- sampling sites. Pathogenicity (KWS) Dry weight (g) 218 M. Moretti et al. 800 bp FIG. 4 – Examples of minisatellite patterns generated by primer M13 revealing the genetic diversity between groups in Cercospora beticola. Lanes 15, 16 and 23 are strains not included in the analysis. 800 bp FIG. 5 – Examples of microsatellite patterns generated by primer SAT2 revealing the genetic diversity between groups in Cer- cospora beticola. Lanes 11, 13, 14, 15, 16 and 17 are strains not included in the analysis. and no significant differences (P > 0.05) were found among As only one genotype over 46 was found twice, genotypic the mean disease index of the four populations tested. diversity G was high in all the considered populations (Table 1). As a consequence, genotypic evenness reached the high- Fingerprinting analysis est possible value of 1 in three of the four populations. The SAT2 and M13 PCR primers generated a total of 54 bands, AMOVA showed that almost all the genetic diversity (99%) ranging from 395 to 3300 bp, 41 of which (75.9%) were was due to variation within populations and that there was polymorphic (Fig. 4 and 5, Table 1). The number of poly- a non-significant difference among populations, being ΦF ST morphic bands in each population ranged from 24 (44.4%) = 0.01 (P = 0.261) (Excoffier et al., 1992). The absence of for the isolates coming from Serres, to 37 (68.5%) for the significative differentiation among populations was confirmed ones from Orestiada. Average gene diversity (Nei, 1973) was by Nei’s measure of genetic identity (Table 2), which was rel- not significantly different (LSD test, P < 0.05) for all the atively high ranging from 0.964 between the populations groups considered, ranging from 0.154 in Imathia to 0.220 from Serres and Imathia, to 0.992 between the populations in Orestiada. from Orestiada and Xanthi. The values of the indirect esti- 100 bp ladder 100 bp ladder O1 O13 O8 S4 O12 S9 S5 X3 S7 X4 S11 X6 X5 I6 X7 I7 X12 I12 X13 A1 λ ladder λ ladder I3 A4 I8 A6 A3 A8 A7 O2 A9 O5 A10 O9 O7 S8 O10 X1 X2 X11 X9 A2 water water 100 bp ladder 100 bp ladder Ann. Microbiol., 56 (3), 215-221 (2006) 219 TABLE 1 – Gene and genotypic diversity of the 4 Greek Cercospora beticola populations Population Sample size Percentage Gene diversity Genotypes Genotypic Genotypic 1 2 of polymorphic loci (H) observed (g ) diversity (G) evenness (G/g ) obs obs Orestiada 13 68.5 0.220 (± 0.189) 13 13.0 1.000 Imathia 12 46.3 0.154 (± 0.188) 11 10.3 0.857 Serres 8 44.4 0.171 (± 0.205) 8 8.0 1.000 Xanthi 13 59.2 0.210 (± 0.201) 13 13.0 1.000 Nei (1973); Stoddart and Taylor (1988); Numbers in brackets express Standard Deviation values. TABLE 2 – Nei’s genetic identity (Nei, 1978) (above diagonal) and estimated numbers of migrants per generation Nm, (Nei, 1972) (below diag- onal) between each pair of Cercospora beticola populations Population Orestiada Imathia Serres Xanthi Orestiada - 0.966 0.987 0.992 Imathias 5.20 - 0.964 0.975 Serres 9.26 4.21 - 0.985 Xanthi 14.77 6.39 8.75 - TABLE 3 – Analysis of variance of the Cercospora beticola isolates coordinates by principal components analysis of M13 and SAT2 fingerprinting Dimension 1 (68.2%) Dimension 2 (6.9%) Source of variation F P F P Geographic origin 1.50 0.229 2.08 0.117 Virulence 0.50 0.615 1.53 0.227 Fisher F mates of migration Nm between each pair of populations are reported in Table 2. No significant negative correlation between the logarithm of geographic distance and the log- arithm of Nm index could be detected. The average Nm value for all the isolates was 4.7. The PCA of the matrix obtained by M13 and SAT2 PCR DNA amplification reported in Fig. 6 shows that 68.2% of the total genetic variability was distributed on dimension 1, and only 6.9% on dimension 2. The ANOVA of the coordinates of each isolate on these two dimensions showed that the groups of isolates constituted on the basis of the geograph- ic origin or pathogenicity were never significantly different (P > 0.05) from each other (Table 3). So, the PCA failed to find in the geographic origin the principal component for the genetic variability, and there was no correlation between genetic fingerprinting and pathogenicity. Dim – 1 (68.2%) DISCUSSION FIG. 6 – PCA of the Cercospora beticola isolate fingerprintings obtained by mini and microsatellite amplification of DNA. The results of this research establish that the naturally occur- ring populations of C. beticola studied are significantly vari- able phenotipically for mycelial growth and pathogenicity, as diversity among isolates on a genetic basis, as the examined previously reported by other authors (Ruppel, 1972; Whit- isolates of C. beticola were characterised by high gene and ney and Lewellen, 1976). genotype diversity. Similar results were reported by sever- The fingerprinting techniques used confirmed the high al authors, using RAPD, AFLP and DAMD-PCR, both in C. beti- Dim – 2 (6.9%) 220 M. Moretti et al. cola, (Weiland et al., 2001; Moretti et al., 2004) and in other However, we found no evidence for population subdivision over spatial scales of hundreds of kilometres. Furthermore, supposed asexually reproducing pathogens (Salamati et al., 2000; Goodwin et al., 2003; Van der Waals et al., 2004). the eventual teleomorph of C. beticola should be identified High level of genotypic diversity was also found in Cer- in the genus Mycosphaerella (Goodwin et al., 2001), but it cospora kikuchii by Cai and Schneider (2005). They assayed is doubtful that ascospores could maintain this low level of population structure of this fungus by vegetative compati- regional genetic differentiation, since it remains questionable bility tests, and they found a high level of diversity, with 56 whether ascospores could be disseminated by the wind in strains distributed in 46 VCGs. large distances. A fungus which reproduces exclusively asexually, as C. To justify the absence of a regional structure in C. beti- beticola is thought to do, is supposed to display low geno- cola populations, we must suppose the existence of a long- type diversity among a limited number of clonal lineages (Mil- distance means of dispersal. Such means could be either groom, 1996). The high level of diversity observed in the C. seed transmission, which has been demonstrated by Verei- beticola tested isolates suggests the existence of a means jssen and co-workers (2004), and/or man mediated trans- to promote genome exchange. Such means could be hyphal mission. As a matter of fact in Greece sugar beet seeds are anastomosis, followed by parasexual recombination or an produced in a restricted area far away from commercial elusive mating system (Weiland and Koch, 2004). Parasex- sugar beet fields, and then distributed to the growers all over ual cycle may represent a tool to maintain genetic diversity the country. Furthermore, the same harvest machineries in natural populations in the absence of sexual recombina- are involved in the harvest process in all the regions of tion (Sanders, 1999); the prerequisite for parasexual recom- sugar beet cultivation so they could contribute to the bination is the possibility to exchange nuclei by hyphal anas- pathogen dispersal by carrying plant debris with conidia and tomosis, and, in fact, the possibility of heterokaryon forma- stromata from one region to another. Okori and co-workers tion between strains has been demonstrated in C. kikuchii (2004) suggested that the absence of genetic differentiation by Cai and Schneider (2005). Work is in progress to assess in populations of C. sorghi could be related to the role of the this feature also in C. beticola. wild sorghum plants in gene flow. Regarding C. beticola, fur- Otherwise we should consider the hypothesis that the ther studies should be done to understand the eventual role pathogen undergoes cycles of sexual reproduction that have of the beet progenitor of sugar beet, Beta vulgaris ssp. mar- remained unobserved (Goodwin et al., 2003), but inferences itima, in Greece as a possible cause of gene flow between on recombination can only be done by testing linkage dise- sugarbeet fields. quilibrium between loci (Milgroom, 1996). This kind of analy- In conclusion, high level of diversity in Cercospora spp. sis requires a larger samples size and more specific amplifi- seems to be a rule, more than an exception. The mechanisms cation strategies. Further work will be done in this direction by which this fungus produces and maintains its variability to assess this important feature of C. beticola biology. are still largely unknown, so they need to be further inves- No correlation was noted between pathogenicity and tigated. 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Analysis of genotypic diversity inCercospora beticola Sacc. field isolates

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Publisher
Springer Journals
Copyright
Copyright © 2006 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/BF03175008
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Abstract

Annals of Microbiology, 56 (3) 215-221 (2006) 1 2 1 1,§ 1 Maddalena MORETTI *, George KARAOGLANIDIS , Marco SARACCHI , Anna FONTANA , Gandolfina FARINA 1 2 Istituto di Patologia Vegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy; Hellenic Sugar Industry, Plant Protection Department, Sugar Factory of Platy, 59032, Platy Imathias, Greece Received 25 May 2006 / Accepted 30 June 2006 Abstract - Genetic variability and population structure of Cercospora beticola, the causal agent of Cercospora leaf spot in sugarbeet, from four sugarbeet-growing regions of Greece were investigated using growth rate, pathogenicity, and mini- and microsatellite DNA fingerprinting. Mycelial growth and pathogenicity were very diverse within and between groups, and no correlation was found between these features and the geographic origin of the isolates. High diversity was found by micro- and minisatellite fingerprinting, with an average gene diversity of 0.21, and no significant differences among populations. Among the 46 isolates, 45 different genotypes were identified, showing a high degree of genotype diversity. Analysis of the genetic profiles provided no evidence for regional patterns of variation (ΦF =0.01, P = 0.261) and the analysis of molecular variation (AMOVA) revealed that genetic variability was due mainly to ST variations within (99%) rather than between (1%) populations. Such a low level of genetic differentiation is reflected by a migration rate value Nm of 4.7. The high migration rate cannot be referred to splash dispersed conidia. To justify the absence of a regional struc- ture in these C. beticola populations, we must suppose the existence of a long-distance means of dispersal, such as seed transmission and/or man mediated transmission. Key words: Beta vulgaris, microsatellite, minisatellite, population genetic, pathogenicity. INTRODUCTION and distribution of genetic diversity within and among pop- ulations (McDonald and Linde, 2002). Methods usually Cercospora beticola Sacc., the causal agent of Cercospora leaf employed to test diversity in fungal populations include vir- spot (CLS) in sugarbeet, is one of the most destructive and ulence and molecular analyses (McDermott et al. 1989; Mil- widespread foliar diseases in the warm and humid areas groom and Fry, 1997). where this crop is cultivated (Duffus and Ruppel, 1993; Wei- Cercospora beticola is characterised by a great variabil- land and Koch, 2004). ity in pathogenicity, morphocultural and metabolic features A teleomorphic phase of this fungus has not yet been such as growth rate, mycelium colour and phytotoxin pro- identified neither in C. beticola nor in other Cercospora duction (Ruppel, 1972; Whitney and Lewellen, 1976; Moret- species monophyletic with C. beticola on the basis of rDNA ti et al., 2004). This pathogen has not yet been broadly sequences (Goodwin et al., 2001). The primary inoculum is explored by molecular techniques, and not much is known thought to be mainly constituted by conidia, produced on about its population genetic structure. Studies by Amplified stromata in crop debris (Weiland and Koch, 2004), which are Fragment Length Polymorphism (AFLP) (Weiland et al., usually ploughed under in the field. These conidia are rain, 2001), RAPD-PCR (Random Amplified Polymorphic DNA) and insect and wind dispersed over short distances (McKay and DAMD-PCR (Direct Amplification of Minisatellite-region DNA) Pool, 1918; Lawrence and Meredith, 1970). Seed contami- (Moretti et al., 2004) showed that this fungus harbours con- nation has also been reported by several authors (Ruppel, siderable genetic variation, despite the absence of a known 1986; Vereijssen et al., 2004). teleomorph. In fact, sexual populations generally have A valuable contribution to the knowledge of epidemiolo- greater genotype diversity than clonal populations (Mil- gy and, therefore, to the improvement of plant pathogen groom, 1996). management, can derive from the study of the genetic struc- Protection of sugarbeet against CLS still lies mainly on ture of a pathogen (McDonald, 1997), defined as the amount fungicide application. The development of resistant or tol- erant varieties can help to maintain crop yield under heavy disease pressure, reducing the quantities of fungicides applied. Sugarbeet breeders find it still very difficult to obtain * Corresponding author. Phone: +39 02 5031 6792; highly resistant hybrids with a root yield potential equal to Fax: +39 02 5031 6781; E-mail:maddalena.moretti@unimi.it the susceptible ones grown in the absence of C. beticola. In § Present address: Department of Biochemistry, Max Planck Insti- fact, CLS resistance is a quantitative trait, controlled by at tute for Chemical Ecology, Hans-Knöll Str. 8D-07745, Jena, Ger- least five loci, and it is characterised by a low heritability many. (Setiawan et al., 2000). 216 M. Moretti et al. For this reason, plant breeders should know the poten- flask was inoculated with five plugs, taken with a 5-mm cork tial for gene flow and long distance dispersal, and the mech- borer from the margins of a fungal colony growing on PDA anisms of C. beticola reproduction and genetic exchange. All and was shaken at 150 rpm, in the dark, for 10 days. The these information determine the potential of C. beticola to mycelium was harvested by filtration through filter paper circumvent sugarbeet CLS resistance and can be inferred by (Whatman paper No 3, Whatman, Maidstone, Kent, U.K.) in the knowledge of genetic structure of C. beticola populations. a Büchner funnel. For growth determination, 4 separate cul- The objective of this study was to analyse the genetic tures were filtered individually through filter paper, dried in structure of four populations of C. beticola obtained in an oven at 80 °C for 24 h and weighed. The experiment was Greece, in order to test the hypothesis that, being C. beti- repeated three times. cola mainly dispersed by conidia on a short distance, popu- lations geographically separated are also genetically differ- Measurement of pathogenicity. entiated. The analysis was conducted using a microsatellite Plant material. The experiments were conducted on Beta vul- (SAT 2) and a minisatellite primer (M 13). garis L. plants, cv. ‘Rival’, which is susceptible to C. betico- la. Plants were grown in a greenhouse (18-26 C), in plas- tic pots (18 cm diameter) containing a 2:1 mixture of peat MATERIALS AND METHODS and perlite. Each pot contained two plants. Sugarbeet seedlings were fertilised once per week with 1% N:P:K Fungal isolates. The isolates used in this study were col- (20:20:20) solution. Plants used for inoculations were 6-7 lected from sugarbeet fields during the summer of 2002. Four weeks old, at the stage of 4-5 fully expanded leaves. regions of Northern Greece (Fig. 1) were sampled and in total 46 isolates were collected, 13 from the area of Orestiada, 12 Inoculum production. Isolates for inoculum production were from Imathia, 13 from Xanthi, and 8 from Serres. grown on V-8 juice agar medium (Tuite, 1969). After 10 days All the isolates were collected as single-lesion isolates and of incubation under fluorescent light at 25 C, sporulating were transformed to single-spore isolates using the dilution colonies were rinsed with 15 ml of distilled water and the technique (Tuite, 1969). They were obtained by transferring conidial suspensions were filtered through a double-layered conidia, with the aid of a fine glass needle, to Potato Dex- sterile cheesecloth. Suspensions were adjusted to 8 x 10 -1 trose Agar (PDA, Oxoid, Unipath Ltd, Basingstoke, U.K.) conidia ml . Before inoculation, one droplet of 0.1% Tween -1 acidified with 0.5 ml l lactic acid to suppress bacterial 20 was added to each suspension. growth. The isolates were obtained from several fields in each sampling site. From each field one leaf with distinct sporu- Inoculation. Inoculation was carried out by spraying the coni- lating lesions was selected and conidia from only one lesion dial suspension until run off, with a spray atomiser. Both leaf per leaf were transferred to the culture medium to obtain one surfaces were sprayed and the potted plants were transferred isolate per field. To obtain durable stock cultures, the strains to a growth chamber and kept at 25 C and 99% RH, with a 16- were grown on PDA plates and plugs of mycelium were h photoperiod. In total 8 plants per isolate were inoculated. maintained in sterile water at room temperature. Pathogenicity recording. After the appearance of the symp- Measurement of mycelial growth. Isolates were grown toms, about 15 days after inoculation, the plants were in Potato Dextrose Broth (Difco Laboratories, Detroit, USA), removed from the growth chamber and the disease severi- in 250-ml flasks containing 50 ml of nutrient medium. Each ty was recorded on 3 randomly selected leaves per plant, FIG. 1 – Map of Greece showing the four Cercospora beticola sampling sites. Ann. Microbiol., 56 (3), 215-221 (2006) 217 according to the nine-category scale disease index of Klein- wanzlebener Saatzucht (KWS): 1 = healthy leaf, and 9 = leaf 0.45 and leaf stalk dead and dried up (Shane and Teng, 1992). The experiment was repeated twice. 0.40 DNA extraction and amplification. Cercospora beticola isolates were grown in liquid medium (DSM65: 4 g of glu- 0.35 -1 cose, 4 g of yeast extract and 10 g of malt extract l ) and incubated for one week at 25 °C on a rotary shaker (120 rpm), in the dark. Total genomic DNA was extracted from 0.30 lyophilised ground mycelium according to Kelly et al. (1998). Italian strains were used as positive control. 0.25 DNA minisatellite and microsatellite regions were ampli- fied by PCR (Polymerase Chain Reaction). Amplifications Imathia Orestiada Serres Xanthi were performed using the following reaction mixtures (50 µl Sampling sites reaction volume): 1x Taq buffer, 1.5 mM MgCl , 0.2 mM dNTPs, 0.42 µM primer, 2 U of Taq polymerase and 2 µl of FIG. 2 – Boxplot of mycelial growth of Cercospora beticola in the DNA (all PCR reagents purchased from Amersham Pharma- four sampling sites. cia). The primers used were the minisatellite marker M13 (5’- GAGGGTGGCGGTTCT-3’), and the microsatellite marker SAT2 (GTG) . For M13 the amplification programme consisted of ty (G) was calculated for each population using the meas- an initial denaturation at 94 °C for 1 min, followed by 40 ure of Stoddart and Taylor (1988). To compare populations cycles at 94 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 of different sample sizes genotypic evenness was calculat- min, with a final extension at 72 °C for 6 min. For SAT2, the ed dividing G by the number of genotypes observed (Stod- thermocycler was programmed for an initial denaturation at dart and Taylor, 1988). 94 °C for 1 min, followed by 40 cycles at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 6 min. The amplification products were separated RESULTS by electrophoresis on 1.5% agarose gel in TAE buffer (Tris- acetate EDTA, Tris-HCl 40 mM and NaEDTA 1 mM). Gels were Mycelial growth and pathogenicity -1 stained with 0.5 µg ml of ethidium bromide, visualized The dry weight of the mycelia ranged from 0.25 to 0.45 g in under U.V. light and the images were acquired with Gel-Doc the Orestiada group, with a mean value of 0.35 g, from 0.25 1000 (Bio-Rad Laboratories, California, USA). to 0.47 g in the Imathia group, with a mean value of 0.37 g, from 0.27 to 0.47 g in the Xanthi group, with a mean value Data analysis. Data obtained from mycelial growth and of 0.39 g and from 0.28 to 0.42 g in the Serres group, with pathogenicity tests were subjected to an analysis of variance. a mean value of 0.36 g (Fig. 2). The diversity (P < 0.05) was Means of each isolate within sampling sites and means significant within the four populations tested, while the among sampling sites were compared using the LSD test. All means of each population were not significantly different (P the statistical analyses were performed on Mstat-C (vers. > 0.05). 2.10, Michigan State University). All the isolates caused disease symptoms on the sugar- For the molecular analysis, bands were scored for pres- beet cultivar selected for the experiment. A large variation in ence and absence. Data generated from M13 and SAT2 pathogenicity was found among the tested isolates, even primers were pooled to generate one binary matrix. The sim- within the same population (P < 0.05). The mean disease ilarity matrix was calculated using Jaccard (J) coefficient, and index of individual isolates ranged from 2.95 to 5.50 (Fig. 3) it was used to perform the Principal Components Analysis (PCA). The similarity matrix and PCAs were calculated by 0.55 NTSYSpc (vers. 2.01, Applied Biostatistics Inc.). The 46 isolates were arbitrarily subdivided into 4 groups according to their origin, and into 3 groups on the basis of 0.50 their level of pathogenicity (group I from 2.5 to 3.5, group II from 3.6 to 4.5 and group III from 4.6 to 5.5). An ANOVA 0.45 analysis was carried out to verify the significance of the dif- ferences among the genetic profiles of these groups. The test 0.40 was performed on the coordinates of the strains in the 2- dimensional PCA from molecular analysis. For the population structure analysis, the estimation of 0.35 Nei’s measure of genetic identity (Nei, 1978) and the aver- age number of migrants among populations per generation 0.30 Nm (Nei, 1972) were calculated with Popgene (vers. 1.32). The data were also subjected to analysis of molecular vari- Imathia Orestiada Serres Xanthi ance (AMOVA, Excoffier et al., 1992) with GeneAlEx (vers. Sampling sites 5.1, Peakall and Smouse, 2001), which is freely available from the Australian National University FIG. 3 – Boxplot of pathogenicity of Cercospora beticola in the four http://www.anu.edu.au/BoZo/GenAlEx/. Genotypic diversi- sampling sites. Pathogenicity (KWS) Dry weight (g) 218 M. Moretti et al. 800 bp FIG. 4 – Examples of minisatellite patterns generated by primer M13 revealing the genetic diversity between groups in Cercospora beticola. Lanes 15, 16 and 23 are strains not included in the analysis. 800 bp FIG. 5 – Examples of microsatellite patterns generated by primer SAT2 revealing the genetic diversity between groups in Cer- cospora beticola. Lanes 11, 13, 14, 15, 16 and 17 are strains not included in the analysis. and no significant differences (P > 0.05) were found among As only one genotype over 46 was found twice, genotypic the mean disease index of the four populations tested. diversity G was high in all the considered populations (Table 1). As a consequence, genotypic evenness reached the high- Fingerprinting analysis est possible value of 1 in three of the four populations. The SAT2 and M13 PCR primers generated a total of 54 bands, AMOVA showed that almost all the genetic diversity (99%) ranging from 395 to 3300 bp, 41 of which (75.9%) were was due to variation within populations and that there was polymorphic (Fig. 4 and 5, Table 1). The number of poly- a non-significant difference among populations, being ΦF ST morphic bands in each population ranged from 24 (44.4%) = 0.01 (P = 0.261) (Excoffier et al., 1992). The absence of for the isolates coming from Serres, to 37 (68.5%) for the significative differentiation among populations was confirmed ones from Orestiada. Average gene diversity (Nei, 1973) was by Nei’s measure of genetic identity (Table 2), which was rel- not significantly different (LSD test, P < 0.05) for all the atively high ranging from 0.964 between the populations groups considered, ranging from 0.154 in Imathia to 0.220 from Serres and Imathia, to 0.992 between the populations in Orestiada. from Orestiada and Xanthi. The values of the indirect esti- 100 bp ladder 100 bp ladder O1 O13 O8 S4 O12 S9 S5 X3 S7 X4 S11 X6 X5 I6 X7 I7 X12 I12 X13 A1 λ ladder λ ladder I3 A4 I8 A6 A3 A8 A7 O2 A9 O5 A10 O9 O7 S8 O10 X1 X2 X11 X9 A2 water water 100 bp ladder 100 bp ladder Ann. Microbiol., 56 (3), 215-221 (2006) 219 TABLE 1 – Gene and genotypic diversity of the 4 Greek Cercospora beticola populations Population Sample size Percentage Gene diversity Genotypes Genotypic Genotypic 1 2 of polymorphic loci (H) observed (g ) diversity (G) evenness (G/g ) obs obs Orestiada 13 68.5 0.220 (± 0.189) 13 13.0 1.000 Imathia 12 46.3 0.154 (± 0.188) 11 10.3 0.857 Serres 8 44.4 0.171 (± 0.205) 8 8.0 1.000 Xanthi 13 59.2 0.210 (± 0.201) 13 13.0 1.000 Nei (1973); Stoddart and Taylor (1988); Numbers in brackets express Standard Deviation values. TABLE 2 – Nei’s genetic identity (Nei, 1978) (above diagonal) and estimated numbers of migrants per generation Nm, (Nei, 1972) (below diag- onal) between each pair of Cercospora beticola populations Population Orestiada Imathia Serres Xanthi Orestiada - 0.966 0.987 0.992 Imathias 5.20 - 0.964 0.975 Serres 9.26 4.21 - 0.985 Xanthi 14.77 6.39 8.75 - TABLE 3 – Analysis of variance of the Cercospora beticola isolates coordinates by principal components analysis of M13 and SAT2 fingerprinting Dimension 1 (68.2%) Dimension 2 (6.9%) Source of variation F P F P Geographic origin 1.50 0.229 2.08 0.117 Virulence 0.50 0.615 1.53 0.227 Fisher F mates of migration Nm between each pair of populations are reported in Table 2. No significant negative correlation between the logarithm of geographic distance and the log- arithm of Nm index could be detected. The average Nm value for all the isolates was 4.7. The PCA of the matrix obtained by M13 and SAT2 PCR DNA amplification reported in Fig. 6 shows that 68.2% of the total genetic variability was distributed on dimension 1, and only 6.9% on dimension 2. The ANOVA of the coordinates of each isolate on these two dimensions showed that the groups of isolates constituted on the basis of the geograph- ic origin or pathogenicity were never significantly different (P > 0.05) from each other (Table 3). So, the PCA failed to find in the geographic origin the principal component for the genetic variability, and there was no correlation between genetic fingerprinting and pathogenicity. Dim – 1 (68.2%) DISCUSSION FIG. 6 – PCA of the Cercospora beticola isolate fingerprintings obtained by mini and microsatellite amplification of DNA. The results of this research establish that the naturally occur- ring populations of C. beticola studied are significantly vari- able phenotipically for mycelial growth and pathogenicity, as diversity among isolates on a genetic basis, as the examined previously reported by other authors (Ruppel, 1972; Whit- isolates of C. beticola were characterised by high gene and ney and Lewellen, 1976). genotype diversity. Similar results were reported by sever- The fingerprinting techniques used confirmed the high al authors, using RAPD, AFLP and DAMD-PCR, both in C. beti- Dim – 2 (6.9%) 220 M. Moretti et al. cola, (Weiland et al., 2001; Moretti et al., 2004) and in other However, we found no evidence for population subdivision over spatial scales of hundreds of kilometres. Furthermore, supposed asexually reproducing pathogens (Salamati et al., 2000; Goodwin et al., 2003; Van der Waals et al., 2004). the eventual teleomorph of C. beticola should be identified High level of genotypic diversity was also found in Cer- in the genus Mycosphaerella (Goodwin et al., 2001), but it cospora kikuchii by Cai and Schneider (2005). They assayed is doubtful that ascospores could maintain this low level of population structure of this fungus by vegetative compati- regional genetic differentiation, since it remains questionable bility tests, and they found a high level of diversity, with 56 whether ascospores could be disseminated by the wind in strains distributed in 46 VCGs. large distances. A fungus which reproduces exclusively asexually, as C. To justify the absence of a regional structure in C. beti- beticola is thought to do, is supposed to display low geno- cola populations, we must suppose the existence of a long- type diversity among a limited number of clonal lineages (Mil- distance means of dispersal. Such means could be either groom, 1996). The high level of diversity observed in the C. seed transmission, which has been demonstrated by Verei- beticola tested isolates suggests the existence of a means jssen and co-workers (2004), and/or man mediated trans- to promote genome exchange. Such means could be hyphal mission. As a matter of fact in Greece sugar beet seeds are anastomosis, followed by parasexual recombination or an produced in a restricted area far away from commercial elusive mating system (Weiland and Koch, 2004). Parasex- sugar beet fields, and then distributed to the growers all over ual cycle may represent a tool to maintain genetic diversity the country. Furthermore, the same harvest machineries in natural populations in the absence of sexual recombina- are involved in the harvest process in all the regions of tion (Sanders, 1999); the prerequisite for parasexual recom- sugar beet cultivation so they could contribute to the bination is the possibility to exchange nuclei by hyphal anas- pathogen dispersal by carrying plant debris with conidia and tomosis, and, in fact, the possibility of heterokaryon forma- stromata from one region to another. Okori and co-workers tion between strains has been demonstrated in C. kikuchii (2004) suggested that the absence of genetic differentiation by Cai and Schneider (2005). Work is in progress to assess in populations of C. sorghi could be related to the role of the this feature also in C. beticola. wild sorghum plants in gene flow. Regarding C. beticola, fur- Otherwise we should consider the hypothesis that the ther studies should be done to understand the eventual role pathogen undergoes cycles of sexual reproduction that have of the beet progenitor of sugar beet, Beta vulgaris ssp. mar- remained unobserved (Goodwin et al., 2003), but inferences itima, in Greece as a possible cause of gene flow between on recombination can only be done by testing linkage dise- sugarbeet fields. quilibrium between loci (Milgroom, 1996). This kind of analy- In conclusion, high level of diversity in Cercospora spp. sis requires a larger samples size and more specific amplifi- seems to be a rule, more than an exception. The mechanisms cation strategies. Further work will be done in this direction by which this fungus produces and maintains its variability to assess this important feature of C. beticola biology. are still largely unknown, so they need to be further inves- No correlation was noted between pathogenicity and tigated. 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