Biologia 68/5: 851—860, 2013 Section Botany DOI: 10.2478/s11756-013-0222-2

Genetic variation of horse chestnut and red horse chestnut and trees susceptibility to Erysiphe flexuosa and Cameraria ohridella

Lidia Irzykowska1*, Maria Werner1,JanBocianowski2,ZbigniewKarolewski1 & Dorota Fruzy˙ nska-Jó´ ´zwiak1

1Department of Phytopathology, Poznan University of Life Sciences, D˛abrowskiego 159,PL-60-594 Pozna´n, Poland; e-mail: [email protected] 2Department of Mathematical and Statistical Methods, Poznan University of Life Sciences, Wojska Polskiego 28,PL-60-637 Pozna´n, Poland

Abstract: Trees of Aesculus sp. are often present in European landscapes. Common horse chestnut and red horse chestnut are seriously damaged by the fungus Erysiphe flexuosa as well as the pest Cameraria ohridella. Both pathogen and pest have spread throughout Europe. A genetic background of trees resistance still remains unknown. The present study was undertaken to estimate genetic variation of A. hippocastanum and A.×carnea grown in urban greens and their susceptibility to powdery mildew and horse chestnut leafminer. According to obtained results both species were infected by E. flexuosa in a similar degree but there were significant differences in susceptibility of particular trees (ANOVA). C. ohridella damaged only A. hippocastanum. The correlation between age of trees and degree of infestation by pathogen and pest was not observed. The significant genetic variability between two Aesculus species was revealed by the analysis of molecular variance (AMOVA) where both the intra- and the inter-species variation were found to be significant. It was shown that 73.0% of the genetic variance was contributed by the differentiation between A. hippocastanum and A.×carnea, whereas 27.0% was partitioned within species. The medium level of genetic diversity of Aesculus spp. was determined using SRAP and RAPD analyses. The mean value of genetic similarity was equal to 0.55 for common horse chestnut and 0.98 for red horse chestnut. Among 40 polymorphic SRAP and RAPD markers 17 were associated with degree of leaf damage caused by C. ohridella. Key words: Aesculus hippocastanumi; Aesculus ×carnea; Cameraria ohridella; Erysiphe flexuosa; horse chestnut leafminer; powdery mildew; RAPD; SRAP

Introduction 2000), Slovakia (Zimmermannová-Pastirčáková & Pa- stirčák 2002), Great Britain (Ing & Spooner 2002), Trees of Aesculus sp. are often present in European Hungary (Kiss et al. 2004), Lithuania (Grigali¨unait et landscapes. The genus evolved in Eastern Asia and di- al. 2005), Estonia (Poldmaa 2006), Italy (Nali 2006), verged into major lineage in North America and Europe Spain (Campelo et al. 2007), Bulgaria (Stoykov & (Prada et al. 2011). Several species, divided among five Denchev 2008) and Turkey (Tozlu & Demirci 2010). sections, were recognized within the genus (Xiang et In Poland E. flexuosa was found for the first time in al. 1998). In Poland, Aesculus hippocastanum L. has 2002 (Pi˛atek 2002). Initial powdery mildew signs in- planted from the 17th century in parks, avenues and clude small, white patches forming on leaves and then greens and still remains one of the most attractive or- fungal colonies expand covering both leaf surfaces. Both namental trees. Additionally, the hybrid of European A. young and old leaves are usually infected. hippocastanum and American A. pavia named red horse Moreover, A. hippocastanum is damaged by pest, chestnut (Aesculus×carnea Hayne), becomes popular the horse chestnut leaf miner Cameraria ohridella as an urban tree in most of Europe (Prada et al. 2011; Deschka & Dimi´c(Lepidoptera: Gracillaridae). The Valade et al. 2009). moth has spread throughout Europe over last two Unfortunately, trees of both species are seriously decades (Johne et al. 2008; Augustin et al. 2009). The damaged by fungus Erysiphe flexuosa (Peck) U. Braun first information about its occurrence in Poland (in & S. Takam. (Ascomycota, Erysiphales) which causes Wo jslawice arboretum) was reported byLabanowski  & powdery mildew (Ale-Agha et al. 2000; Ing & Spooner Soika (1998). C. ohridella was included in the Alert 2002). This North American species recently intro- List EPPO (European and Mediterranean Plant Pro- duced to Europe were observed on chestnut trees in tection Organization) as a very serious pest of trees Germany (Ale-Agha et al. 2000), Switzerland (Bolay grown in Europe. C. ohridella develops 3–4 generation

* Corresponding author

c 2013 Institute of Botany, Slovak Academy of Sciences 852 L. Irzykowska et al.

Table 1. The short characteristics of Aesculus hippocastanum and Aesculus×carnea trees and degree of leaf damage at the end of vegetation season (September).

Tree Aesculus species Location Age of tree Degree of leaf damage Degree of leaf damage code (years) by C. ohridella[%] by E. flexuosa [%]

1 A.×carnea 0.0

2 A.×carnea 20.0

3 A.×carnea 16.7

4 A.×carnea 13.0

5 A.×carnea square 18–25 47.0

6 A.×carnea 7.0

7 A.×carnea 0.0 27.0

8 A.×carnea 40.0

9 A.×carnea 70 43.3

10 A.×carnea 0.0

11 A.×carnea street 16–18 0.0

12 A.×carnea 0.0

13 A.×carnea park 33.3

15 A.×carnea street 90 66.7

16 A. hippocastanum park 100 38.0 0.0

17 A. hippocastanum square 70 93.0 0.0

18 A. hippocastanum 60.0 40.0

19 A. hippocastanum street 53.0 0.0

20 A. hippocastanum 48.0 0.0

21 A. hippocastanum square 50.0 0.0

22 A. hippocastanum 58.0 100.0

23 A. hippocastanum 30–35 53.0 0.0

24 A. hippocastanum 40.0 16.7

25 A. hippocastanum 70.0 43.3

26 A. hippocastanum 58.0 63.3

27 A. hippocastanum 35.0 53.3

28 A. hippocastanum park 68.0 73.0

29 A. hippocastanum 78.0 0.0

30 A. hippocastanum 78.0 0.0

31 A. hippocastanum 25 28.0 0.0

32 A. hippocastanum 23.0 47.0

33 A. hippocastanum 60–70 28.0

34 A. hippocastanum 25.0 0.0

35 A. hippocastanum 18.0 Genetic variation of horse chestnut and red horse chestnut 853

Table 1. (continued)

Tree Aesculus species Location Age of tree Degree of leaf damage Degree of leaf damage code (years) by C. ohridella[%] by E. flexuosa [%]

36 A. hippocastanum 93.0

37 A. hippocastanum 95.0

38 A. hippocastanum street 16–20 93.0 0.0

39 A. hippocastanum 73.0

40 A. hippocastanum 55.0

41 A. hippocastanum square 60 3.0

per year in Central Europe. The moth overwinters as Material and methods pupae in dry dropped leaves. Damages caused by lar- Plant material val feeding inside leaves are spectacular. Leaves brown- The study was carried out in Pozna´n (Western Poland) in ing and premature defoliation is typical for seriously urban parks, streets and greens. Based on observations per- damaged trees. What it worse, pest causes not only formed during 2008-2011 years a set of 40 Aesculus trees aesthetic damages. Repeated defoliation has been re- was chosen (Werner et al. 2012) for molecular study and ported to cause decrease in radial growth of trees and estimation of susceptibility to E. flexuosa and C. ohridella. a progressive decline due to reduced production of pho- Short characteristics of Aesculus trees were given in Table 1. tosynthates. Moreover, the allocation of photosynthates to seeds is strongly reduced in infested trees (Salleo et Estimation of damage caused by E. flexuosa and C. ohridella The assessment of leaf damage was conducted from May al. 2003). to September in 2011. Tree infestation rate by E. flexuosa Interestingly, C. ohridella damaged mainly A. hip- was determined in two repetitions according to the method pocastanum while other species such as A. pavia and A. described earlier (Werner et al. 2012). The degree of leaf flava were infested only occasionally (Straw & Tilbury damages caused by C. ohridella was also evaluated. Ten 2006; Ferracini et al. 2010). A genetic background of leaves from each tree were selected randomly and estimated trees resistance to E. flexuosa and C. ohridella is prob- according to the 5-point scale given by Baranowski et al. ably complex and still remains unknown. Knowledge of (2004). inter- and intraspecies genetic variation of species be- The mean damage degree of leaves (P )causedby longing to Aesculus genus is limited and link between E. flexuosa or C. ohridella was calculated according to Townsend and Heuberger’s formula (Puntener 1992): genetic variability of Aesculus species and trees resis- tance to pest and pathogen attack was not found so
(nv) far. Genetic diversity of A. flava and A. glabra was P × (%) = NV 100 examined with multilocus VNTR (variable number of tandem repeats) probes (Lim et al. 2002). Thomas et where v – number of leaves per category, V – total number al. (2008) found a high genetic variation of A. flava, of leaves screened, n – degree of leaf damages according to A. sylvatica, A. pavia and hybrids between them us- the scale, N – the highest degree of damage. ing microsatellites and ISSR markers (inter simple se- quence repeat). Prada et al. (2011) applied AFLP (am- Molecular marker analyses plified fragment length polymorphism) genotyping to DNA was isolated from leaves buds of particular trees. Fresh investigate variability of A. hippocastanum. In this material was surface-disinfected for 2 min. in 1% sodium study a SRAP (sequence-related amplified polymor- hypochlorite. Then tissues were crushed with liquid nitro- gen. DNA was extracted and purified using a DNeasy Mini phism) method was adopted to study the genetic vari- × Kit (QIAGEN Inc., Hilden, Germany) according to the man- ability of A. hippocastanum and A. carnea. ufacturer’s recommendations. The present study was undertaken to estimate ge- The RAPD-PCR and SRAP-PCR reactions were car- netic variation of A. hippocastanum and A.×carnea ried out using a Taq PCR Core Kit (QIAGEN, Inc., Hilden, grown in urban greens and their susceptibility to pow- Germany). The reaction mixture contained 10 ng of DNA dery mildew and horse chestnut leafminer. The prin- from young leaves, 200 µMofeachdNTP,1µMprimerand cipal aims of this study were: (1) to determine the 0.5 U of Taq DNA polymerase in 1× reaction buffer with inter- and intraspecies genetic diversity of trees using 2.5 mM magnesium chloride. A total of 22 PCR primers SRAP and RAPD (randomly amplified polymorphic including 16 RAPD primers: OPA-01 – OPA-12, OPA-18, OPC-02, OPL-11 – OPL-12 (Qiagen Operon, Cologne, Ger- DNA) methods (2) to evaluate and compare the tree many) and 6 SRAP primer pairs (mew-emw: AT-CT, CG- infestation/damage rate caused by E. flexuosa and C. CG, GA-TG, TA-AC, TC-GA, TG-GT) were used to screen ohridella and (3) to find molecular markers associated the set of trees for polymorphism (Table 3). Finally, 5 RAPD with resistance A. hippocastanum and A.×carnea to primers (OPA-02, OPA-04, OPA-08, OPA-18, OPC-02) and powdery mildew and/or horse chestnut leafminer. 3 SRAP primer pairs (mew-emw: TA-AC, AT-CT, TG-GT) 854 L. Irzykowska et al.

Table 2. Mean squares from two-way analysis of variance (ANOVA) for the degree of leaf damage and degree of leaf infection of A. hippocastanum and A.×carnea.

Source of variation Number of degrees of freedom Degree of leaf damage Degree of leaf infection

Trees 39 2,756.81*** 1,239.74*** Months 3 16,291.33*** 5,762.73*** Trees × months interaction 117 396.56*** 344.69*** Residual 160 32.73 3.19

*** P < 0.001

Table 3. Sequences of primers tested and the number of polymorphic markers.

Primer code Sequence 5’ to 3’ Polymorphic Polymorphic band size (bp) band number

OPA-01 CAGGCCCTTC 0 – OPA-02 TGCCGAGCTG 9 a-340, b-630, c-760, d-950, e-1350, f-1440, g-1510, h-1800, i-2000 OPA-03 AGTCAGCCAC 0 – OPA-04 AATCGGGCTG 7 a-560, b-580, c-910, d-980, e-1020, f-1150, g-1360 OPA-08 GTGACGTAGG 6 a-330, b-405, c-440, d-890, e-995, f-2400 OPA-09 GGGTAACGCC 0 – OPA-10 GTGATCGCAG 0 – OPA-11 CAATCGCCGT 0 not reproducible OPA-12 TCGGCGATAG 0 – OPA-18 AGGTGACCGT 3 a-485, b-600, c-900 OPC-02 GTGAGGCGTC 4 a-480, b-570, c-845, d-890 OPL-11 ACGATGAGCC 0 – OPL-12 GGGCGGTACT 0 – mew-CG TGAGTCCAAACCGGCG 0– emw-CG GACTGCGTACGAATTCG mew-GA TGAGTCCAAACCGGGA 0– emw-TG GACTGCGTACGAATTTG mew-TC TGAGTCCAAACCGGTC 0– emw-GA GACTGCGTACGAATTGA mew-TA TGAGTCCAAACCGGTA 5 a-200, b-550, c-690, d-705, e-1500 emw-AC GACTGCGTACGAATTAC mew-AT TGAGTCCAAACCGGAT 3 a-480, b-995, c-1150 emw-CT GACTGCGTACGAATTCT mew-TG TGAGTCCAAACCGGTG 3 a-230, b-295, c-730 emw-GT GACTGCGTACGAATTGT

gave polymorphic bands. Amplification was carried out in a of RAPD or SRAP products of the same length. The coef- BIO-RAD C1000 Touch Thermal Cycler (Whatman Biome- ficients of genetic similarity (GS) of the investigated trees tra, Goettingen, Germany) as described earlier (Li & Quiros were calculated using the following formula (Nei & Li 1979): 2001; Irzykowska et al. 2012a). PCR was repeated twice to check their reproducibility. GSij =2Nij /(Ni + Nj ), The PCR products were separated by electrophoresis in 1.5% agarose gels with 1× TBE buffer (89 mM Tris- where Nij is the number of alleles present at i-th and j-th borate and 2 mM EDTA, pH 8.0) and visualised under UV trees, Ni – the number of alleles present at the i-th tree, Nj – light following ethidium bromide staining. A Gene RulerTM the number of alleles present at the j-th tree, i, j =1,2,... 100 bp DNA Ladder Plus (Fermentas GmbH, St. Leon-Rot, , 40. Basing on calculated coefficients trees were grouped Germany) was used as a molecular size standard for PCR hierarchically using the unweighted pair group method of products. arithmetic means (UPGMA) and the relationship among them was presented in the form of a dendrogram. The association between molecular markers and degree Statistical analysis of leaf damage or infection was estimated using regression A two-way analysis of variance (ANOVA) was carried out to analysis (Bocianowski et al. 2011). The marker observations determine the effects of trees, months and trees × months were tested as independent variables and considered in in- interaction on the variability of degree of leaf damage and dividual models. The critical significance level amounting degree of leaf infection. The influence of tree age and tree to 0.001, resulting from the Bonferroni correction (Province species on observed traits were tested by contrast analyses. 1999), was used. The Bonferroni correction is a method used The comparison of each band profile for each primer was to counteract the problem of multiple comparison. In total done on the basis of the presence (1) versus absence (0) number of all markers is equal to 40, than the global signif- Genetic variation of horse chestnut and red horse chestnut 855

Fig. 1. Example of RAPD patterns of A.×carnea (lanes 1–15) and A. hippocastanum (lanes 16–41) amplified with primer OPC-04. Gene Ruler TM 100 bp DNA Ladder Plus (Fermentas) was used as a molecular size standard (lane M), * – polymorphic DNA fragments. icant level needed to be adjusted α = 0.05/40 = 0.00125. that two alleles drawn at random from a subpopulation dif- Moreover, analysis of molecular variance analysis fer in state (which, for a two-allele system, will always be (AMOVA) was used to compute the distribution of genetic 2piqi,wherepi is the observed allelic frequency in subpop- variability among and within species. AMOVA was per- ulation i), averaged over subpopulations. formed using the program GenAlEx 6.5 (Peakall & Smouse 2012). Significance levels for variance component estimates Results were computed using 1000 permutations. FST was used as a measure of population genetic structure and calculated by formula: FST =(HT – HS)/HT,whereHT is the proba- Significant differentiation of A. hippocastanum and bility that two alleles drawn at random (with replacement) A.×carnea trees in susceptibility to E. flexuosa and C. from the entire population differ in state (i.e., the probabil- ohridella was found. The results of conducted two-way ity with no population structure), and HS is the probability ANOVA indicate a very strong influence (P < 0.001) 856 L. Irzykowska et al.

Fig. 2. Dendrogram of A.×carnea (1–13, 15) and A. hippocastanum (16–41) based on DNA polymorphism analyses by SRAP and RAPD methods. Trees were grouped hierarchically using the unweighted pair group method of arithmetic means (UPGMA).

of tree species, terms and trees × terms interaction on ucts were obtained, from 3 to 9 per primer (Table 3). the degree of leaf damage (Table 2). No correlation was The size of PCR products ranged from 0.2 to 2.4 kb found between age of trees and degree of leaf infestation (Fig. 1). The relationship among trees was presented on by pest or fungus (P = 0.087 and P = 0.875, respec- the dendrogram (Fig. 2). Species differed in about 60% tively). in analysed genome parts. The highest genetic similar- Both Aesculus species were infected by E. flexu- ity (equal 1) in screened genome regions was revealed osa. In September the leaf area covered by mycelial between lots of pairs of A. ×carnea trees, whereas the patches reached even more than 50% in case of some lowest genetic similarity (equal 0.202) was found for a trees (Table 1). The degree of leaf infection was not sig- pair 10-17 (Table 4). The mean value of genetic simi- nificantly different for common and red horse chestnuts larity was equal to 0.55 for common horse chestnut and (P = 0.459; 8.1 and 6.5, respectively, where LSD0.05 0.98 for red horse chestnut. = 4.22). On infected leaves conidia were formed abun- The significant differentiation (FST = 0.726; P = dantly. Moreover, from July to September many chas- 0.010) between two Aesculus species was further sup- mothecia (the sexual fruiting bodies also called cleis- ported by the AMOVA results (Table 5). The intra- tothecia in earlier works) appeared, mostly on the lower and the inter-species variability were found to be leaf surface. significant, with 73.0% of the genetic variance con- A. hippocastanum and A.×carnea significantly dif- tributed by the differentiation between A. hippocas- fered in degree of leaf damage caused by leaf miner (P < tanum and A.×carnea, whereas 27.0% was partitioned 0.001, 30.8 and 0.0, respectively). C. ohridella infested within species. only A. hippocastanum trees. Damages caused by larval Among 40 polymorphic SRAP and RAPD mark- feeding inside leaves were growing gradually from May ers, 17 DNA fragments were associated with degree of to September when reached even 95% of leaf sheet. leaf damage by C. ohridella (Table 6). Four of them The level of inter- and intraspecies genetic diver- were present in the genome of strongly damaged trees. sity of Aesculus spp. was determined using SRAP and Markers associated with degree of leaf infection by E. RAPD analyses. Finally 40 polymorphic PCR prod- flexuosa were not found. Genetic variation of horse chestnut and red horse chestnut 857

Table 4. Coefficients of genetic similarity among trees from Aesculus hippocastanum and Aesculus×carnea species.

2 1.00 3 1.00 1.00 4 1.00 1.00 1.00 5 0.91 0.91 0.91 0.91 6 1.00 1.00 1.00 1.00 0.91 7 1.00 1.00 1.00 1.00 0.91 1.00 8 1.00 1.00 1.00 1.00 0.91 1.00 1.00 9 1.00 1.00 1.00 1.00 0.91 1.00 1.00 1.00 10 0.96 0.96 0.96 0.96 0.91 0.96 0.96 0.96 0.96 11 1.00 1.00 1.00 1.00 0.91 1.00 1.00 1.00 1.00 0.96 12 1.00 1.00 1.00 1.00 0.91 1.00 1.00 1.00 1.00 0.96 1.00 13 1.00 1.00 1.00 1.00 0.91 1.00 1.00 1.00 1.00 0.96 1.00 1.00 15 1.00 1.00 1.00 1.00 0.91 1.00 1.00 1.00 1.00 0.96 1.00 1.00 1.00 16 0.41 0.41 0.41 0.41 0.46 0.41 0.41 0.41 0.41 0.38 0.41 0.41 0.41 0.41 17 0.25 0.25 0.25 0.25 0.22 0.25 0.25 0.25 0.25 0.21 0.25 0.25 0.25 0.25 0.62 18 0.40 0.40 0.40 0.40 0.39 0.40 0.40 0.40 0.40 0.37 0.40 0.40 0.40 0.40 0.76 0.74 19 0.49 0.49 0.49 0.49 0.49 0.49 0.49 0.49 0.49 0.46 0.49 0.49 0.49 0.49 0.73 0.58 0.67 20 0.41 0.41 0.41 0.41 0.40 0.41 0.41 0.41 0.41 0.38 0.41 0.41 0.41 0.41 0.64 0.69 0.71 0.73 21 0.34 0.34 0.34 0.34 0.32 0.34 0.34 0.34 0.34 0.31 0.34 0.34 0.34 0.34 0.73 0.52 0.61 0.63 0.60 22 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.49 0.51 0.51 0.51 0.51 0.64 0.55 0.65 0.67 0.71 0.53 23 0.39 0.39 0.39 0.39 0.43 0.39 0.39 0.39 0.39 0.36 0.39 0.39 0.39 0.39 0.80 0.71 0.72 0.63 0.67 0.75 24 0.47 0.47 0.47 0.47 0.51 0.47 0.47 0.47 0.47 0.44 0.47 0.47 0.47 0.47 0.69 0.48 0.74 0.82 0.69 0.71 25 0.41 0.41 0.41 0.41 0.40 0.41 0.41 0.41 0.41 0.38 0.41 0.41 0.41 0.41 0.71 0.69 0.76 0.73 0.71 0.67 26 0.51 0.51 0.51 0.51 0.46 0.51 0.51 0.51 0.51 0.49 0.51 0.51 0.51 0.51 0.69 0.61 0.74 0.76 0.63 0.71 27 0.36 0.36 0.36 0.36 0.40 0.36 0.36 0.36 0.36 0.38 0.36 0.36 0.36 0.36 0.79 0.48 0.76 0.53 0.57 0.60 28 0.47 0.47 0.47 0.47 0.46 0.47 0.47 0.47 0.47 0.44 0.47 0.47 0.47 0.47 0.75 0.67 0.84 0.76 0.75 0.71 29 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.54 0.51 0.51 0.51 0.51 0.64 0.48 0.71 0.73 0.64 0.53 30 0.49 0.49 0.49 0.49 0.43 0.49 0.49 0.49 0.49 0.46 0.49 0.49 0.49 0.49 0.73 0.58 0.78 0.75 0.73 0.69 31 0.38 0.38 0.38 0.38 0.42 0.38 0.38 0.38 0.38 0.35 0.38 0.38 0.38 0.38 0.65 0.63 0.81 0.61 0.58 0.55 32 0.40 0.40 0.40 0.40 0.39 0.40 0.40 0.40 0.40 0.37 0.40 0.40 0.40 0.40 0.76 0.60 0.69 0.65 0.69 0.77 33 0.44 0.44 0.44 0.44 0.43 0.44 0.44 0.44 0.44 0.41 0.44 0.44 0.44 0.44 0.67 0.45 0.67 0.81 0.60 0.63 34 0.30 0.30 0.30 0.30 0.28 0.30 0.30 0.30 0.30 0.26 0.30 0.30 0.30 0.30 0.62 0.80 0.80 0.52 0.76 0.58 35 0.37 0.37 0.37 0.37 0.35 0.37 0.37 0.37 0.37 0.39 0.37 0.37 0.37 0.37 0.67 0.57 0.67 0.76 0.67 0.55 36 0.42 0.42 0.42 0.42 0.41 0.42 0.42 0.42 0.42 0.39 0.42 0.42 0.42 0.42 0.74 0.71 0.73 0.76 0.67 0.55 37 0.47 0.47 0.47 0.47 0.46 0.47 0.47 0.47 0.47 0.44 0.47 0.47 0.47 0.47 0.69 0.48 0.74 0.65 0.56 0.71 38 0.39 0.39 0.39 0.39 0.38 0.39 0.39 0.39 0.39 0.41 0.39 0.39 0.39 0.39 0.67 0.58 0.72 0.63 0.67 0.69 39 0.42 0.42 0.42 0.42 0.46 0.42 0.42 0.42 0.42 0.39 0.42 0.42 0.42 0.42 0.69 0.73 0.74 0.71 0.63 0.59 40 0.43 0.43 0.43 0.43 0.38 0.43 0.43 0.43 0.43 0.41 0.43 0.43 0.43 0.43 0.69 0.78 0.83 0.76 0.69 0.70 41 0.48 0.48 0.48 0.48 0.47 0.48 0.48 0.48 0.48 0.45 0.48 0.48 0.48 0.48 0.71 0.69 0.86 0.73 0.77 0.61 1 2 3 4 5 6 7 8 9 1011121315161718192021

23 0.60 24 0.69 0.71 25 0.64 0.73 0.75 26 0.63 0.71 0.83 0.81 27 0.57 0.67 0.69 0.57 0.63 28 0.75 0.76 0.83 0.81 0.78 0.75 29 0.71 0.67 0.81 0.79 0.81 0.64 0.81 30 0.60 0.69 0.76 0.80 0.88 0.67 0.82 0.73 31 0.65 0.61 0.74 0.77 0.74 0.65 0.74 0.65 0.73 32 0.62 0.65 0.67 0.62 0.73 0.62 0.67 0.55 0.71 0.69 33 0.67 0.50 0.82 0.67 0.76 0.60 0.71 0.67 0.69 0.73 0.71 34 0.62 0.71 0.61 0.62 0.67 0.69 0.73 0.55 0.65 0.63 0.67 0.58 35 0.67 0.69 0.71 0.74 0.65 0.67 0.77 0.74 0.62 0.53 0.50 0.62 0.57 36 0.67 0.62 0.71 0.89 0.77 0.59 0.77 0.74 0.76 0.73 0.64 0.69 0.57 0.69 37 0.56 0.71 0.78 0.63 0.72 0.69 0.67 0.63 0.65 0.69 0.67 0.71 0.61 0.58 0.52 38 0.60 0.69 0.76 0.67 0.71 0.80 0.82 0.67 0.75 0.67 0.65 0.63 0.71 0.76 0.62 0.71 39 0.69 0.76 0.72 0.56 0.67 0.63 0.78 0.63 0.59 0.69 0.73 0.65 0.73 0.65 0.58 0.67 0.71 40 0.63 0.70 0.72 0.80 0.77 0.57 0.82 0.69 0.76 0.74 0.67 0.65 0.67 0.71 0.71 0.72 0.76 0.77 41 0.77 0.73 0.80 0.84 0.80 0.71 0.91 0.84 0.79 0.82 0.69 0.67 0.75 0.73 0.80 0.69 0.73 0.74 0.79 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Discussion trees to E. flexuosa and C. ohridella were found. Both species of Aesculus were infected by E. flexuosa.This The epidemic occurrence of powdery mildew and horse concurs with other reports focused on Aesculus suscep- chestnut leafminer constitutes a serious problem espe- tibility to powdery mildew (Milevoj 2004; Werner et al. cially in urban areas where Aesculus trees are very com- 2009). Significant variation in susceptibility to E. flex- mon in parks, streets and private gardens. In present uosa between particular trees was revealed. In many work significant differences in susceptibility of Aesculus places infected trees were situated next to the healthy 858 L. Irzykowska et al.

Table 5. Summary of analysis of molecular variance (AMOVA) for comparison between and within two Aesculus species.

Source of variation Degrees of freedom Sum of squares Mean squares Estimated variance Percentage of variation

Between species 1 167.775 167.775 9.032 73.0 Within species 38 129.275 3.402 3.402 27.0 Total 39 297.050 12.433

* Probability of obtaining a larger component estimate. Number of permutations = 1000

Table 6. Molecular markers associated with degree of Aesculus hippocastanum leaves damage by Cameraria ohridella.

Molecular marker Estimates of P value Percentage variation regression coefficients accounted

OPA-02 a 340; OPA-02 b 630; OPA-04 a 560; OPA-04 d 980; –30.79 <0.001 63.3 OPA-08 c 440; SRAP AT-CT b 995; SRAP TG-GT a 230; OPA-18 a 485; OPC-02 a 480; OPC-02 b 570 OPA-04 c 910 30.79 <0.001 63.3 OPA-08 d 890 –29.65 <0.001 56.3 OPA-02 d 950 –28.59 <0.001 49.8 OPA-02 h 1800 25.42 <0.001 44.7 OPA-02 e 1350 24.20 <0.001 41.1 OPA-04 f 1150 23.47 <0.001 39.4 OPA-02 c 760 –20.97 <0.001 30.9

ones. The mean degree of leaf damage caused by E. sess the amount and distribution of genetic variation flexuosa was similar for common horse chestnuts (17%) within and among species. In this study we assessed and red horse chestnuts (22%). It was possible to find A. the genetic diversity within and between two Aescu- hippocastanum and A. ×carnea trees resistant to pow- lus species using well known RAPD method and rarely dery mildew and damaged by C. ohridella toalesser applied SRAP method. RAPD method is useful in de- extent (or resistant in case of red horse chestnut) and tection of DNA polymorphisms without prior knowl- such specimens selected as the maternal trees could be edge of the genome sequence and was widely applied to used in grafting process in urban greens. the plant genome study (Irzykowska et al. 2002; Nanda First symptoms of foliage damage by C. ohridella et al. 2004; Sesli & Ye˘geno˘glu 2009; Samantaray et were appeared earlier on A. hippocastanum trees grow- al. 2011). In our study RAPD analyses also were ef- ing in parks. Probably sweeping of leaves in the streets ficient and from 6 to 9 polymorphic DNA fragments reduces population of moth temporarily but popula- per primer were amplified. Moreover, we proved a use- tion rebuilds quickly despite control efforts (Gilbert fulness of a novel SRAP method to successfully scan et al. 2003). The potential population reservoirs are the genomic DNA of Aesculus sp. SRAP is considered localized in parks and gardens where pupae overwin- to be more powerful techniques than RAPD for reveal- ter inside fallen leaves and in spring moths disperse ing genetic diversity. SRAP markers should be tightly also toward trees growing along streets (Augustin et al. linked to actual genes, and generate a fingerprint of 2009). Red horse chestnut was not infested by horse the coding sequences (Jones et al. 2009; Irzykowska et chestnut leafminer. This concurs with earlier observa- al. 2012b). SRAP preferentially amplifies open reading tions in Pozna´n urban area (Werner et al. 2009). C. frames (ORFs) and allows easy isolation of bands for se- ohridella was found on A. turbinata, A. flava or A. quencing. Up to now, SRAPs have been applied in very pavia but not on the red horse chestnut (Freise & Heit- few studies of plant species (Li & Quiros 2001; Budak land 2004). However, Freise et al. (2003) claimed that et al. 2004; Gulsen et al. 2007; Han et al. 2008; Uzun et females of C. ohridella, do not show preference to A. al. 2010). To our knowledge up to now SRAP methods hippocastanum, but mortality of larvae on A. ×carnea have not been applied to study of Aesculus genus. is much more higher than on common horse chestnut. Based on genetic similarity coefficients all trees It would be interesting to elucidate why moth cannot analysed together were divided into two main clusters develop on leaves of red horse chestnut. Probably in on the dendrogram at 60% similarity level. It means leaves are present some substances harmful for larval that A. hippocastanum differed from A.×carnea in 40% stage of C. ohridella. The important findings of Kukula- in analysed genome parts. Considering that A. ×carnea Mlynarczyk et al. (2006) shed light on the possible de- is a hybrid of A. hippocastanum and A. pavia (Thomas fensive role of saponins against leafminer. Extensive et al. 2008) obtained results are justifiable. According to studies are necessary to elucidate which parts of Aescu- AMOVA results the majority of variation revealed was lus genome determine biosynthesis of compounds mak- partitioned between species. Moreover, common horse ing some species unattractive or even toxic for the pest. chestnut was much more differentiated than red horse Many molecular tools have been applied to as- chestnut. In case of A. hippocastanum probably it is Genetic variation of horse chestnut and red horse chestnut 859 due to generative reproduction when crossing-over oc- leafminer Cameraria ohridella: a tale of two cities. Entomol. curs. On the contrary, A. ×carnea forms less seeds and Exp. Appl. 107: 25–37. Erysiphe is reproduced mainly in vegetative manner hence the Grigali¨unait B., Meškauskien V. & Matelis A. 2005. flexuosa on Aesculus hippocastanum in Lithuania. 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