viruses in finland

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www.metla.f/silvaennica ISSN 0037-5330The Finnish Society o Forest Science The Finnish Forest Research Institute

SILVA FENNICASilva Fennica 43(5) research articles

Cherry leaf roll virus an Emerging Virus in Finland?

Susanne von Bargen, Elise Grubits, Risto Jalkanen and Carmen Bttner

von Bargen, S., Grubits, E., Jalkanen, R. & Bttner, C. 2009. Cherry lea roll virus an emerging

virus in Finland? Silva Fennica 43(5): 727738.

Cherry lea roll virus, CLRV, is a plant pathogen that inects a variety o deciduous treesand shrubs in temperate regions. Little is known about its occurrence at high latitudes andespecially in Finnish birch species. Still, symptoms that seemed to be associated with CLRVsuch as vein banding, lea roll and decline have been observed in birch trees throughout thecountry since the summer o 2002. Six dierent birch species, subspecies or varieties, i.e.Betula pubescens subsp.pubescens (downy birch),B. pendula (silver birch),B. nana (dwarbirch),B. pubescens var. appressa (Kiilop birch),B. pubescens subsp. czerepanovii (moun-tain birch) andB. pendula var. carelica (curly birch) originating rom all over Finland wereassessed by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR)or CLRV inection. It was shown that CLRV is widely distributed in B. pendula and B.pubescens throughout the country. Furthermore, dwar birch, mountain birch, Kiilop birchand curly birch were conrmed to be previously unkown hosts o CLRV. Genetic analysis ovirus sequence variants originating rom Finnish birch trees revealed atypical phylogeneticrelationships. In contrast to CLRV isolates rom birches growing in the United Kingdom andGermany which clustered exclusively within group A, Finnish CLRV isolates belonged eitherto group B, D or E. Thus, virus population structure in Finnish birches seems to be morevariable and host plant dependency seems not to apply or Finnish CLRV isolates.

KeywordsBetula nana, Betula pendula, Betula pubescens, IC-RT-PCR, 3 non-coding region,CLRV, phylogenetic relationshipAddressesvon Bargen, Grubits and Bttner, Humboldt-Universitt zu Berlin, Department rNutzpfanzen- und Tierwissenschaten, Fachgebiet Phytomedizin, Lentzeallee 55/57, 14195Berlin, Germany; Jalkanen, Finnish Forest Research Institute, Rovaniemi Research Unit,P.O. Box 16, FI-96301 Rovaniemi, Finland E-mail [email protected],[email protected] 23 February 2009 Revised 29 June 2009 Accepted 31 August 2009Available at http://www.metla./silvaennica/ull/s43/s435727.pd


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Silva Fennica 43(5), 2009 research articles

1 Introduction

A severe and increasing birch decline in dierentparts o Finland is observed since 2002. Treessuer rom loss o vigour, and leaves indicate

vein banding, lea roll, chlorosis and subsequentnecrosis. Reerring to these symptoms they leadto the assumption that they are caused by viruses.In a survey covering major parts o the country,symptoms on birch were especially distinct aterthe extremely dry summer o 2006, and severalbirch species are aected. Trees showed CLRV-characteristic symptoms and recently, this viruswas conrmed in Rovaniemi, northern Finland inseveralBetula pubescens Ehrh. subsp.pubescens(downy birch) trees (Jalkanen et al. 2007).

Cherry lea roll virus (CLRV) belongs tothe subgroup c o the genus Nepovirus (amilyComoviridae). The two (+)ssRNAs o the bipar-tite viral genome each contain an extraordinarylong 3 non-coding region (3 NCR, Borja et al.1995, Scott et al. 1992), which is involved inregulation o replication and translation (Dreher1999) as described or Blackcurrant reversionnepovirus (Karetnikov et al. 2006). Comparativestudies o CLRV strains rom dierent originsdone by Rebenstor et al. (2006) revealed that thesequence variability o a short stretch o the 3NCR near the 3 terminus o the virus RNA leadsto grouping o virus isolates into dierent phylo-genetic clades. This clustering can be correlatedwith serological and biological properties o virusstrains. Additionally, it was demonstrated thatvariability within this part o the virus genome islinked to the originating host plant species o thevirus isolates. CLRV inects various deciduoustrees and shrubs (Bandte and Bttner 2001) otemperate regions. It is a seed and pollen-bornevirus, which is also transmitted by mechanicalmeans, grating, root connation and was recentlyreported to be water transmissible as well (Bandteet al. 2007).

More than one th o the Finnish orests aredominated by birch species. In total, the genus

Betula represents the most common group odeciduous trees, which comprises an importantraw material in the mechanical and chemicalorest industry (Peltola 2007). The speciesBetula

pubescens subsp. pubescens (downy birch) andB. pendula Roth (silver birch) are preerably

used industrially or plywood, veneer (Luos-tarinen and Verkasalo 2000) and paper produc-tion (Viher-Aarnio and Velling 1999). Downyand silver birches are early-succession tree spe-cies abundant throughout the country in towns,

on roadsides and most common in mixed or-ests. However, north o the Arctic Circle alsoB.nana L. (dwar birch),B. pubescens var. appressa(Kallio & Y. Mkinen, Kiilop birch) and B.

pubescens subsp. czerepanovii (Orlova) Hmet-Ahti (mountain birch) are the specialisedBetulaspecies, comprising important key components othe sub-arctic ecosystem (Walker 2000, van Wijket al. 2005). The decorative wood o curly birch(B. pendula var. carelica (Mercklin) Hmet-Ahti),which itsel is an ornamental tree, is o great

economic value in Finland providing the highestprized timber used in urniture and cratwork(Velling et al. 2000).

The aim o the study was to investigate theoccurrence o CLRV in birch species and thedistribution o the virus in Finland. Furthermore,it was intended to give a rst assessment o char-acteristics o individual CLRV sequence variantsobtained rom dierent locations in Finland and

Betula species. This includes the comparison othe CLRV strains ound in Finland with otherknown virus isolates which have already beenphylogenetically characterised by Rebenstor etal. (2006).

2 Material and Methods

Seventy-seven trees exhibiting characteristicsymptoms o a virus inection (34B. pubescenssubsp. pubescens, 27B. pendula, six B. pubes-cens subsp. czerepanovii, veB. pubescens var.appressa, ourB. nana and a singleB. pendulavar. carelica) were sampled all over Finland.Two symptomatic twigs including catkins andyoung lea tissue o individual trees were col-lected between the height o 25 m o trees whichwere between 5 and 100+ years old. Twigs werewrapped in moist paper in a polyethylene bag andcooled. The samples were sent within one weekto the laboratory and were processed immediately

or kept rozen at 20 C until molecular analysis.Sampled trees were tested in duplicate by appli-


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von Bargen et al. Cherry leaf roll virus an Emerging Virus in Finland?

cation o homogenates o symptomatic leavesand buds, catkins or twig tips in a CLRV specicimmunocapture-reverse transcription-polymerasechain reaction (IC-RT-PCR) described in Jalkanenet al. (2007) in detail. CLRV particles were coated

to the wall o reaction tubes by a virus specicpolyclonal antibody (10 l o a 1:200 dilutiono 1 mg/ml IgG) which was developed in rabbitagainst an elderberry isolate o CLRV (Gentkowet al. 2007). The virus RNA becomes accessibleater washing away plant material and is copiedinto a cDNA by 200 units o the enzyme M-MuLVreverse transcriptase (Fermentas, Germany). Thegenerated cDNA is then amplied by polymer-ase chain reaction applying 2 units GoTaq fexiDNA polymerase (Promega, Wisconsin, USA)

with the supplied buer and a nal concentra-tion o 2 mM MgCl2. Specic oligonucleotidesdeveloped by Werner et al. (1997) were usedin PCR targeting the partial 3 NCR o CLRVwhich have been shown suitable to investigatethe phylogenetic relationships o virus isolateswithin the species (Rebenstor et al. 2006). PCRwas conducted in a Tgradient thermocycler (Bio-metra, Germany) with the ollowing program:4 min initial denaturation at 94 C ollowed by30 cycles o denaturation or 1 min at 94 C,annealing 45 s at 55 C and elongation or 1 minat 72 C; nal extension was or 5 min at 72 C.Amplication products o the IC-RT-PCR wereseparated by electrophoresis in 1% (w/v)-agarosegels. Nucleic acids were stained by incubation ogels in ethidiumbromide (1 g/ml TBE-buer)and visualised under uv-light (312 nm). A sampletree was scored as CLRV inected, i a specicragment o the expected size (404420 bp) wasdetected in at least one sample per tree.

Selected IC-RT-PCR generated ragmentso the CLRV 3 non-coding region that wereobtained rom three downy birches o Rovaniemi,two silver birches and one mountain birch romdierent locations in Finland (Lieksa, Vaasa,Inari) were analysed by sequencing based on thechain-termination method developed by Sangeret al. (1977). Amplied ragments were ligatedinto pBluescriptII SK(-)-vectors (Stratagene, LaJolla, USA) and transormed intoE. coli (JM109,Promega, Wisconsin, USA) using standard pro-

tocols (Sambrook et al. 1989). Plasmid-DNAwas puried with the Invisorb Spin Plasmid

Mini Kit (Invitek, Berlin, Germany) accordingto manuacturers instructions. Cloned insertswere sequenced rom both directions using aBigDye Terminator v1.1 Ready Reaction CycleSequencing Kit (Applied Biosystems, Foster City,

USA) ater standard protocol with vector specicprimers and an ABI PRISM 310 Genetic Analyzer(Applied Biosystems, Foster City, USA). Obtainedsequences were assembled in the sequence align-ment editor sotware BioEdit 7.0.5.2 (Hall 1999).They were subjected to an online database searchusing BLAST (Basic Local Alignment SearchTool, Altschul et al. 1997) at the National Centeror Biotechnology Inormation (NCBI) to retrieverelated sequences with signicant scores. CLRVsequences obtained rom Finnish birches were

aligned and compared with corresponding nucle-otide ragments extracted rom the NCBI data-base (Table 1) applying the computer programClustalX 1.83 (Thompson et al. 1997). Aterremoval o primer sequences pairwise sequenceidentities were calculated rom resulting sequenceragments. Comparison o the pairwise sequenceidentities o the group o CLRV sequence vari-ants originating rom Finland with diversities oa second group o birches rom other geographicorigins were done by calculation o mean valuesand standard deviation o the two groups. Phy-logenetic trees were inerred rom the sequencealignment using neighbour-joining, maximum-likelihood, and maximum-parsimony methodsincorporated in the BioEdit sotware package inorder to apply dierent algorithms. To test or sta-tistical robustness o data, the neighbour-joiningtree was calculated with ClustalX 1.83 includingbootstrap analyses with 1000 repetitions; otherparameters were let in deault settings.

3 Results

The sampled trees rom southern, central andnorthern Finland revealed numerous CLRV inec-tions. Results conrm that CLRV is widely dis-tributed throughout Finland (Fig. 1), even northo Rovaniemi and the Arctic Circle up to thenorthern and alpine tree line. Tree samples origi-

nated rom rural areas, i.e. rom alleys, parks(churchyard, schoolyard) and along roadsides in


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Table 1. CLRV isolates rom the database included in the phylogenetic analyses.

Accession no. a) CLRV isolate Host plant Geographic origin Phylogeneticgroupb)

AB168098 chinese chive Allium tuberosum Rottl. Japan C

ex Spreng.AB168099 Rumex AGBC Rumex acetosella L. Japan CAB168100 Rumex acetosella-21 Rumex acetosella L. Japan CAJ877118 E120 Betula pendula Roth Berlin, Germany AAJ877119 E499s Betula pendula Roth Berlin, Germany AAJ877120 E896s Betula pendula Roth Berlin, Germany AAJ877121 E696s Betula pendula Roth Hamburg, Germany AAJ877122 E111 Betula pendula Roth Hamburg, Germany AAJ877123 E806 Betula pendula Roth United Kingdom AAJ877124 E1469 Betula pendula Roth United Kingdom AAJ877125 E836s Betula nigra L. Lower Saxony, Germany AAJ877126 GAY Juglans regia L. United Kingdom D1AJ877127 E327 Prunus avium L. North-Rhine-Westalia, Germany A

AJ877128 E803 Prunus avium L. United Kingdom AAJ877129 E1472 Prunus avium L. United Kingdom AAJ877130 E676s Sambucus nigra L. Schleswig-Holstein, Germany BAJ877131 E485 Sambucus nigra L. Mecklenburg-Western Pomerania, E

GermanyAJ877132 E603 Sambucus nigra L. Brandenburg, Germany EAJ877133 E583 Sambucus nigra L. Brandenburg, Germany EAJ877134 E119s Sambucus nigra L. Berlin, Germany EAJ877135 E622s Sambucus nigra L. Berlin, Germany EAJ877136 E839s Sambucus nigra L. Berlin, Germany EAJ877137 E541s Sambucus nigra L. Berlin, Germany EAJ877138 E443 Sambucus nigra L. Berlin, Germany E

AJ877139 E441 Sambucus nigra L. Saxony-Anhalt, Germany AAJ877140 E950s Sambucus nigra L. Saxony-Anhalt, Germany EAJ877141 E568 Sambucus nigra L. North-Rhine-Westalia, Germany EAJ877142 E576 Sambucus nigra L. North-Rhine-Westalia, Germany EAJ877143 E492 Sambucus nigra L. Hungary EAJ877144 PV-0276 Sambucus nigra L. North-Rhine-Westalia, Germany EAJ877145 E804 Sambucus canadensis L. United States o America EAJ877146 E326 Juglans regia L. North-Rhine-Westalia, Germany D1AJ877147 E648 Juglans regia L. France D1AJ877148 4WJUG Juglans regia L. United Kingdom D1AJ877149 E800 Juglans regia L. United Kingdom D1AJ877150 E156 Juglans regia L. Hungary D2AJ877151 CTIFL Juglans regia L. France D1

AJ877152 Ludmila Juglans regia L. Slovakia D2AJ877153 E697s Sorbus aucuparia L. Hamburg, Germany BAJ877154 E695s Sorbus aucuparia L. Schleswig-Holstein, Germany BAJ877155 E693 Sorbus aucuparia L. Baden-Wrttemberg, Germany EAJ877156 E141s Carpinus betulus L. North-Rhine Westalia, Germany EAJ877157 E575s Aegopodium podagraria L. North-Rhine Westalia, Germany BAJ877159 E113 Fagus sylvatica L. North-Rhine Westalia, Germany AAJ877160 E801 Ulmus americana L. United States o America AAJ877161 E797 Cornus forida L. United States o America AAJ877162 E802 Rubus idaeus L. New Zealand CAJ877163 E805 Rubus ruticosus L. United Kingdom AAJ877164 E1636 Vitis viniera L. Rhineland-Palatinate, Germany A

AJ877165 E395 Rheum rhabarbarum L. North-Rhine Westalia, Germany BAJ888533 E678s Fraxinus excelsiorL. Bavaria, Germany B


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town centres but also rom natural stands as orinstance the samples collected in Inari. Alto-gether, CLRV inection was proved in 44 out o77 o the symptomatic trees, thereo 20 downybirches and 14 silver birches (Table 2).

As an example, CLRV could be detected inall birch species growing in the municipality oInari (Lapland); the number o the sampled dwarbirches (4), mountain birches (6) and Kiilopbirches (5) included in the assay was limited;still, CLRV detection was successul at least intwo trees per species. Additionally, the one curlybirch sampled rom a garden in Rovaniemi wasCLRV positive. This is the rst time that thesespecies have been conrmed to be hosts or thevirus. Additionally, our CLRV inected saplings twoB. pendula and twoB. pubescens origi-nated rom a 100-year-old seed-production standin Stken, Kittil. So ar CLRV has never been

recorded north o the Arctic Circle or inB. pubes-cens andB. pendula trees in the entire country.

Accession no. a) CLRV isolate Host plant Geographic origin Phylogeneticgroupb)

AJ888534 E698s Fraxinus excelsiorL. Rhineland-Palatinate, Germany BEF182751 Edremit 2 Juglans regia L. Turkey -S84124 I2-RNA1 Betula pendula Roth United Kingdom A

S84125 I2-RNA2 Betula pendula Roth United Kingdom AS84126 R25 Rheumrhabarbarum L. United Kingdom BU24694 W8-RNA2 Juglans regia L. United States o America D1X99828 PetHH1 Petunia hybrida Hamburg, Germany -Z344265 W8-RNA1 Juglans regia L. United States o America D1

a) Database accession via National Center or Biotechnology Inormation (NCBI)b) According to Rebenstor et al. 2006

Table 2. Detection o CLRV by IC-RT-PCR in sympto-matic birch species in Finland.

Species Sampled, CLRVno. positive,

no.

B. pubescens subsp.pubescens 34 20B. pendula 27 14B. pubescens subsp. czerepanovii 6 2B. pubescens var. appressa 5 5B. nana 4 2B. pendula var. carelica 1 1Total 77 44

Fig. 1. Location o sampled trees expressing virus-likesymptoms in Finland. Species are indicated by theollowing symbols:Betula pubescens subsp.pubes-cens (), B. pendula (), B. pubescens subsp.czerepanovii (),B. pubescens var. appressa (),B. nana (), and B. pendula var. carelica ().CLRV inected trees conrmed by IC-RT-PCR are

indicated by dark symbols. Small symbols repre-sent one individual tree, large symbols 45 trees.

Rovaniemi

Lieksa

Vaasa

Inari

Kittil

161 km


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CLRV inection o three downy, two silverbirches and one mountain birch was conrmed bysequencing (Table 3). CLRV sequence ragmentso the 3 NCR received rom Finnish birches havebeen submitted to the sequence database main-tained by the European Molecular Biology Labo-ratory (EMBL) and are available by the accessionnumbers AM981029AM981037. These are therst partial sequences available or CLRV obtainedrom Finnish host plants. The samples originatedrom various locations in Finland, i.e. Rovaniemiand Inari (North), Lieksa (East) and Vaasa (West)(Fig. 1), and display size dierences o the partial3 NCR between 404 and 412 bp (Fig. 2) as wellas sequence variability. Phylogenetic analyses oCLRV sequences applying maximum-likelihoodor maximum parsimony algorithms led to simi-lar grouping and conrmed the neighbour-join-ing tree (Fig. 3). Sequence ragments o CLRVobtained rom Finnish birches shared highestsequence identities to CLRV isolates character-ised previously belonging to phylogenetic groupB, D1 or E. Two identical sequences derived romdierent sample trees (EG22 and EG31) cluster-ing within phylogenetic group D1 were 363 bplong excluding primer sequences. This is dueto a deletion o eight consecutive nucleotides atposition 132140 within the alignment (data notshown). Interestingly, this deletion is also presentin other CLRV isolates previously characterisedas members o group D1 (accessions AJ877126,

AJ877146, AJ877147, AJ877148, AJ877149,AJ877151, U24694, Z34265). Seven o the inves-

Table 3. Characteristics o CLRV isolates obtained rom various Finnish birch trees.

Accession no. Species, tree no., tissue Origin, sampling date CLRV isolate, Fragment Phylo-clone length a), genetic

bp group b)

AM981029 B. pubescens subsp.pubescens, 1, lea Rovaniemi, May 2007 E2484, EG1 412 BAM981030 B. pubescens subsp.pubescens, 3, lea Rovaniemi, May 2007 E2485, EG3 412 EAM981031 E2485, EG12 412 BAM981032 B. pubescens subsp.pubescens, 20, lea Rovaniemi, July 2007 E2501, EG9 412 EAM981033 E2501, EG10 412 EAM981034 B. pendula, 35, lea Lieksa, July 2007 E2532, EG22 404 D1AM981035 B. pendula, 55, twig tip Vaasa, July 2007 E2558, EG28 412 EAM981036 B. pubescens subsp. czerepanovii, 98, bud Inari, July 2007 E2621, EG31 404 D1AM981037 E2621, EG32 412 B

a) IC-RT-PCR product including primer sequencesb) According to Rebenstor et al. (2006)

Fig. 2. CLRV specic ragments o the partial 3 non-coding region (412 bp) amplied by IC-RT-PCRrom dierent birch trees. Sample names areaccording to Table 3. As the negative control is

water instead o nucleic acid template applied inPCR. The marker M is the 1 kb Ladder, Fermen-tas, Germany and sizes in basepairs (bp) o somereerence ragments are indicated on the right. Theasterisks indicate the shorter ragments o CLRVsequence variants o 404 bp.

E2484,

EG1

E2485,

EG3

E2485,

EG12

E2501,

EG9

E2501,

EG10

E2532,

EG22

E2558,

EG28

E2621,

EG31

E2621,

EG32

negativecontrol

M

-1000 bp

-2000 bp

-250 bp

-500 bp* *


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Fig. 3. Phylogenetic tree inerred rom the nucleotide sequence alignment (386 bp) o the 3 non-coding regionsoCherry lea roll virus isolates generated with ClustalX 1.83 applying the neighbour-joining algorithm.Bootstrap analysis was perormed with 1000 repetitions and values above 800 are indicated at branch nodes.The scale bar o 0.02 represents 2 nucleotide substitutions per 100 nucleotides o the aligned sequences.Sequences obtained rom the NCBI database are indicated by their accession numbers according to Table1. CLRV sequences obtained romBetula spp. in Finland (Table 3) are indicated with arrowheads, isolatesoriginating romBetula spp. in Germany and United Kingdom are pinpointed with asterisks. Phylogenetic

groups (A to E) according to Rebenstor et al. (2006) are indicated by bold lines on the right.


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tigated sequences were 371 bp in length exclud-ing primer sequences. Four clones (EG3, EG9,EG10 and EG28) revealed identical sequencesand showed highest sequence identities to CLRVisolates determined as phylogenetic group E (Fig.3); three Finnish CLRV sequence variants (clones,EG1, EG12 and EG32) related to phylogeneticgroup B isolates and showed sequence variabilityo max. 2.2%. It was ound that a single birchtree harbour dierent CLRV sequence variants.In detail, one individual clone romB. pubescenssubsp.pubescens (CLRV-E2485, EG3) relates togroup E, while the second (CLRV-E2485, EG12)represents group B; sequences diered in 30 posi-tions o 371 aligned nucleotides (identity score o0.919). Two dierent CLRV clones sequencedromB. pubescens subsp. czerepanovii belongedeither to group B (CLRV-E2621, EG32) or D1(CLRV-E2621, EG31) exhibiting a slightly lowersequence identity o 0.913. A higher variabil-ity o sequence ragments obtained rom CLRVinected birches in Finland was substantiated bycomparison o the group o nine Finnish sequencevariants (Table 3) with a subgroup o ten CLRVisolates originating romBetula sp. in the UnitedKingdom and Germany available in the database(accessions AJ877118, AJ877119, AJ877120,AJ877121, AJ877122, AJ877123, AJ877124,AJ877125, S84124, S84125). The group oFinnish CLRV strains revealed a lower meanintra-group sequence identity o 0.93 within theanalysed region o the 3 NCR (Table 4). Lowest

identity scores o 0.892 were determined or theFinnish sequence variants relating to phylogenetic

group D1 (clones EG31 and EG22) and E (EG3,EG9, EG10 and EG28). Birch CLRV isolatesrom dierent locations in the UK and Germany,all clustering within phylogenetic clade A, showat least mean identities o 0.968 with a minimumsequence identity o 0.930. Mean inter-groupsequence identities o these two CLRV groupsoriginating either rom Finland or rom otherEuropean countries was even lower and rangedbetween 0.8480.893.

4 Discussion

CLRV has been conrmed in symptomatic birchtrees rom several places in Finland by molecularmeans, revealing that the virus is widely distrib-uted in the country and also aects at least sixbirch species native to Fennoscandia. The mainroute o CLRV dispersal in birch in natural habi-tats is assumed to be pollen and seed transmission,which has been studied in detail beore (Cooper1976, 1979, Cooper et al. 1984). Hybridisationand cytoplasmic introgression occur requentlywithin the genus Betula and hybrids o silver,downy, and dwar birch are reported rom Scan-dinavia as well (Thrsson et al. 2001, Palme et al.2004). Thus, interspecic ertilisation by CLRVinected pollen may contribute to virus disper-sal within this genus. However, pollen mediatedvertical transmission as an exclusive mode o

CLRV dissemination cannot suciently explainthe sudden appearance o symptoms in CLRV

Table 4. Sequence identities o CLRV sequences ater pairwise comparison received rom theBetula spp. grouprom Finland and the group rom United Kingdom or Germany.

Parameter n a) Mean (S.D.) Min. Max.

Subgroup sequence identity

CLRV, United Kingdom, Germanyc) 10 0.968 ( 0.016) 0.930 1b)

CLRV, Finlandd) 9 0.930 ( 0.042) 0.892 1

Sequence identity ater pairwise comparison o sequences between subgroups

CLRV romBetula spp. 19 0.875 ( 0.012) 0.848 0.893

a) Sequences included in the pairwise comparisonb) Identical sequencesc) Isolates originating romBetula spp. in the United Kingdom and Germany are indicated in Table 1.d) See Table 3


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inected birch trees or the wide distribution allover the country. As birch pollen is anemophilous,one would expect virus-induced symptoms rston wind exposed sites and sides o the canopy.This seems not to be the case, because sampled

trees rom sheltered sites within orest standswere also ound to be aected by the disease.Vertical transmission by seed may also be ham-pered by lower germination rates and less viableCLRV inected birch seedlings (Cooper et al.1984). Still, in our investigations we ound CLRVinected silver and pubescent seedlings in a seedproduction stand in Kittil in northern Finland.Thus, contaminated seed could be a possible routeo CLRV dispersal into planted birch populations,which exist or instance as alleys in Finnish town

centres. However, most alleys and the sympto-matic town birches are rather old, 40 to 80 years,suggesting that contaminated seed is not involvedin the rapid spread o CLRV unless the trees areinected without symptoms.

Sequence comparisons revealed an unusual phy-logenetic relationship o Finnish CLRV isolates.Until now, phylogenetically characterised CLRVisolates o birch trees rom the United Kingdomand Germany exclusively cluster within clade A(Rebenstor et al. 2006). It was shown that CLRVisolates could be genetically dierentiated accord-ing to the originating host plant species. From thisnding the authors concluded that co-evolution oCLRV with the host plant species is an importantactor responsible or the quick genetic adapta-tion o the virus population to the host species.In our study we ound that CLRV sequence vari-ants rom Finnish birch species did not clusterin group A with previously characterised CLRVisolates originating rom birch. For instance twoFinnish CLRV sequence variants rom silverbirch and mountain birch (CLRV-E2532, EG22,CLRV-E2621, EG31) clustered within phyloge-netic group D. This is remarkable, because in thisclade CLRV isolates originating only rom walnuttrees were established to date and thereore the co-evolutional mechanisms postulated by Rebenstoret al. (2006) seem not to apply to CLRV oundin Finnish birches. Instead our ndings suggestthat a more diverse CLRV population is presentin Finnish birches, which may also explain the

wide occurrence and the aggressive spreadingo the virus in Finland in a very short time. This

may be due to new introduction o CLRV into theregion. However, this cannot be secured becauseo the ew individuals characterised so ar. Themajority o nine sequenced clones was ound torelate to group B or represent E variants. Both

phylogenetic clades comprise CLRV isolates oa wider range o host plant species includingFraxinus excelsiorL., Sorbus aucuparia L. andSambucus sp. (Rebenstor et al. 2006), whichare native to southern Finnish ecosystems, too(Mikk and Mander 1995, Simola 2006). This mayindicate that CLRV strains o these phylogeneticgroups are not restricted in host range and areable to inect various plant genera. Thus, CLRVinections in Finland are most likely not restrictedto the genusBetula, but the virus may also inect

other trees and shrubs o the Finnish fora.On the contrary, birches may have recently

acquired CLRV rom other host plants, and thevirus population is not adapted to specic host plantspecies yet. This is substantiated by a single reportrom Cooper and Edwards (1980), who mentioneda CLRV isolate that was obtained in Finland romSambucus racemosa L. This would imply otherroutes o CLRV transmission than by pollen orseed to be important in Finland. Interspecicviral spread might be acilitated by mechanicalinoculation, either with the assistance o suitableinsect vectors (Werner et al. 1997) or through virusentrance into roots via CLRV contaminated soiland water in conjunction with wounding. As it isstill unknown, whether insects, mites or nematodesare involved in the transmission o this virus, thecontributing agents o CLRV dissemination in theFinnish environment have to be investigated inthe uture. In particular, global warming may leadto a changed ecosystem, which alters the plantsphysiological condition. According to Garret et al.(2006) this may contribute to higher susceptibilityo plants causing increased symptom developmentater virus inection or enhancing virus replicationcapabilities within the host plant. Climate changepredictions or Finland conducted by Jylh et al.(2004) estimate that annual mean temperatures aswell as precipitation will raise within the next teenyears, including signicantly aected seasonaltemperatures. Plant species sensitive to elevatedtemperatures may respond to a warmer climate

with loss o virus tolerance, thus acilitating virusreplication in the host plant.


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Also water transmission o CLRV has to betaken into serious consideration (Bandte et al.2007). Earlier thawing o ice and ground rost incombination with an earlier start o the vegetationperiod may stress plants to loose their resistance

to pathogens. Although the response to warmingis generally understood, concurrent changes inother climatic actors may aect plantpathogeninteractions, leading to a shit in disease distribu-tion as outlined by Garret et al. (2006).

CLRV inection may aect the local woodindustry because o its abundance in the economi-cally important birch species B. pubescens and

B. pendula. The virus infuence on machiningquality is not yet known, but its appearance inorest trees may avour other predisposing and

inciting environmental actors o decline, whichmay entail reduced tree vitality as well as woodquality. Reerring to the rate at which symptomshave spread within the last ew years, CLRVmight become a serious problem in northernbirch orest ecosystems. In combination with theexpected climatic changes the virus may contrib-ute to impair key species o the delicate sub-arcticplant communities likeBetula nana. In order toprevent urther distribution o CLRV, its earlydetection in birch trees as well as investigationo other possible host plants o the Finnish forais necessary.

Finally, the etiology o the disease aectingat least six Finnish birch species has to be thor-oughly investigated in the uture. We could detectCLRV in only 57% o the symptomatic birch treeswhich may be explained by the applied methodor virus detection including an immunocapturestep. The polyclonal antibody which was devel-oped against an elderberry isolate o CLRV maynot recognise all strains o the virus, because itis known that the species consists o dierentserotypes (Rowhani and Mircetich 1988, Jones etal. 1990, Rebenstor et al. 2006). Furthermore, itcannot be excluded that other viruses are involvedin the disease aecting birches (Nienhaus andCastello 1989). Although the symptoms in birchare characteristic or an inection with CLRV(Schmelzer 1972, Cooper and Atkinson 1975),Kochs postulates remain to be ullled in orderto prove CLRV as causal agent o the disease

observed in birch species in Finland.Concluding, detailed studies are needed, espe-

cially concerning mode o CLRV transmissionwithin birch species as well as virus dispersalwithin the Finnish environment including otherpossible hosts, because CLRV has emerged in aew years time in all Finnish regions and occurs in

all birch species investigated to date. Additionally,ecological and economic impact o the emerg-ing virus disease has to be estimated in the nearuture. It is o particular interest to determine thevariability o CLRV sequences and monitor thedevelopment o CLRV virus populations oundin Finnish plant species, because they dier con-siderably rom previous ndings and may enablethe investigation o genetic adaptation processeso CLRV to dierent woody hosts.

Acknowledgements

We thank the DFG (Deutsche Forschungsgemein-schat) or unding by the project Bu 890/8-1 andBu 890/8-2 and acknowledge the contribution oR. Junge to this work through skilul technicalassistance.

References

Altschul, S.F., Madden, T.L., Scher, A.A., Zhang,J., Zhang, Z., Miller, W. & Lipman, D.J. 1997.Gapped BLAST and PSI-BLAST: a new genera-tion o protein database search programs. NucleicAcids Research 25: 33893402.

Bandte, M. & Bttner, C. 2001. A review o an impor-tant virus o deciduous trees cherry lea roll virus:occurrence, transmission and diagnosis. Pfanzen-schutzberichte 59: 119.

, Echevarria Laza, H.J., Paschek, U., Ulrichs,Ch., Pestemer, W., Schwarz, D. & Bttner, C.2007. Transmission o plant pathogenic virusesby water. In: Fischer, G., Magnitskiy, S., Flrez,L.E., Miranda, D. & Medina, A. (eds.). SociedadColumbiana de Ciencias Horticolas. 2do CongresoColombiano de Horticultura, Bogot 1214 Sep-tember. p. 3143.

Borja, M., Snchez, F., Rowhani, A., Bruening, G. &

Ponz, F. 1995. Long, nearly identical untranslatedsequences at the 3terminal regions o the genomic


11/12

737


RNAs o Cherry lea roll virus (walnut strain).Virus Genes 10(3): 245252.

Cooper, J.I. 1976. The possible epidemiological signi-cance o pollen and seed transmission in the cherrylea roll virus/Betula spp. complex. Mitteilungen

der Biologischen Bundesanstalt r Land- undForstwirtschat Berlin-Dahlem 170: 1727.

1979. Virus diseases o trees and shrubs. Institute oTerrestrial Ecology, Cambrian News Aberystwyth,Cambridge. 229 p.

& Atkinson, M.A. 1976. Cherry lea roll virus, acause o chlorotic symptoms in Betula spp. in theUnited Kingdom. Forestry 48: 193203.

& Edwards, M.L. 1980. Cherry lea roll virus inJuglans regia in the United Kingdom. Forestry 53:4150.

, Massalski, P.R. & Edward, M.L. 1984. Cherrylea roll virus in the emale gametophyte and itsrelevance to vertical virus transmission. Annals oApplied Biology 105: 5564.

Dreher, T.W.1999. Functions o the 3-untranslatedregions o positive strand viral genomes. AnnualReview o Phytopathology 37: 151174.

Garret, K.A., Dendy, S.P., Frank, E.E., Rouse, M.N. &Travers, S.E. 2006. Climate change eects on plantdisease: genomes to ecosystems. Annual Reviewo Phytopathology 44: 489509.

Gentkow, J., von Bargen, S., Petrik, K. & Bttner, C.2007. Mglichkeiten zur Detektion des Kirschen-blattrollvirus (CLRV) in Gehlzen durch serolo-gische und molekulare Methoden. In: Dujesieken,D. & Kockerbeck, P. (eds.). Yearbook o Arbo-riculture. Haymarket Media, Braunschweig, p.279284.

Hall, T.A. 1999. BioEdit: a user-riendly biologicalsequence alignment editor and analysis programor Windows 95/98/NT. Nucleic Acids SymposiumSeries 41: 9598.

Jalkanen, R., Bttner, C. & von Bargen, S. 2007. Cherrylea roll virus CLRV, abundant on Betula pubescensin Finland. Silva Fennica 41: 755762.

Jones, A.T., Koenig, R., Lesemann, D.-E., Hamacher,J., Nienhaus, F. & Winter, S. 1990. Serologicalcomparison o isolates o cherry learoll virus romdiseased beech, and birch trees in a orest declinearea in Germany with other isolates o the virus.Journal o Phytopathology 129: 339344.

Jylh, K., Tuomenvirta, H. & Ruosteenoja, K. 2004.

Climate change projections or Finland duringthe 21st century. Boreal Environment Research

9: 127152.Karetnikov, A., Kernen, M. & Lehto, K. 2006. Role

o the RNA2 3 non-translated region o Blackcur-rant reversion nepovirus in translational regulation.Virology 354: 178191.

Luostarinen, K. & Verkasalo, E. 2000. Birch as sawntimber and in mechanical urther processing inFinland. A literature study. Silva Fennica Mono-graphs 1. 40 p.

Mikk, M. & Mander, . 1995. Species diversity oorest islands in agricultural landscapes o southernFinland, Estonia and Lithuania. Landscape andUrban Planning 31: 153169.

Nienhaus, F. & Castello, J.D. 1989. Viruses in oresttrees. Annual Review o Phytopathology 27: 165186.

Palme, A.E., Su, Q., Palsson, S. & Lascoux, M. 2004.Extensive sharing o chloroplast haplotypes amongEuropean birches indicates hybridization amongBetula pendula, B. pubescens and B. nana. Molecu-lar Ecology 13: 167178.

Peltola, A. (ed.). 2007. Finnish Statistical Yearbook oForestry. Metla, Helsinki. 436 p.

Rebenstor, K., Candresse, T., Dulucq, M.J., Bttner,C. & Obermeier, C. 2006. Host species-dependentpopulation structure o a pollen-borne plant virus,Cherry lea roll virus. Journal o Virology 80:24532462.

Rowhani, A. & Mircetich, S.M. 1988. Pathogenicity onwalnut and serological comparisons o cherry lea-roll virus strains. Phytopathology 78: 817820.

Sambrook, J., Fritsch, E.F. & Maniatis, T. 1989. Molec-ular cloning a laboratory manual. Cold SpringHarbour, New York.

Sanger, F., Nicklen, S. & Coulsen, A.R. 1977. DNAsequencing with chain-terminating inhibitors. Pro-ceedings o the National Academy o SciencesUSA 74: 54635467.

Schmelzer, K. 1972. Das Kirschenblattrollvirus aus derBirke (Betula pendula Roth.). Zentralblatt r Bak-teriologie, Parasitenkunde, Inektionskrankheitenund Hygiene, II. Abteilung 127: 1012.

Scott, N.W., Cooper, J.I., Liu, Y.Y. & Hellen, C.U.T.1992. A 1.5 kb sequence homology in 3-terminalregions o RNA-1 and RNA-2 o a birch isolate ocherry lea roll nepovirus is also present, in part,in a rhubarb isolate. Journal o General Virology73: 481485.

Simola, H. 2006. Cultural land use history in Finland.The Finnish Environment 23: 163172.


12/12

738


Thompson, J.D., Gibson, T.J., Plewniak, F., Jean-mougin, F. & Higgins, D.G. 1997. The ClustalXwindows interace: fexible strategies or multiplesequence alignment aided by quality analysis tools.Nucleic Acids Research 25: 48764882.

Thrsson, .T., Salmela, E., & Anamthawat-Jnsson,K. 2001. Morphological, cytogenetic, and molec-ular evidence or introgressive hybridization inbirch. Journal o Heredity 92: 404408.

Velling, P., Viher-Aarnio, A., Hagqvist, R. & Lehto,J. 2000. Valuable wood as a result o abnormalcambial activity the case o Betula pendula var.carelica. In: Savidge, R.A., Barnett, J.R. & Napier,R. (eds.). Cell and molecular biology o woodormation. BIOS Scientic Publishers Limited,Oxord. p. 377386.

Viher-Aarnio, A. & Velling, P. 1999. Growth and stemquality o mature birches in a combined species andprogeny trial. Silva Fennica 33: 225234.

Walker, D.A. 2000. Hierarchical subdivision o Arctictundra based on vegetation response to climate,parent material and topography. Global ChangeBiology 6: 1934.

Werner, R., Mhlbach, H.P. & Bttner, C. 1997. Detec-

tion o cherry lea roll nepovirus (CLRV) in birch,beech and petunia by immunocapture RT-PCRusing a conserved primer pair. European Journalo Forest Pathology 27: 309318.

van Wijk, M.T., Williams, M. & Shaver, G.R. 2005.Tight coupling between lea area index and oliageN content in arctic plant communities. Oecologia142: 421427.

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