ANNOTATED SEQUENCE RECORD
Complete nucleotide sequences of the genomes of two isolates of apple chlorotic leaf spot virus from peach (Prunus persica )in China
Feiqing Niu •Song Pan •Zujian Wu •
Dongmei Jiang •Shifang Li
Received:7September 2011/Accepted:2December 2011/Published online:26January 2012ÓSpringer-Verlag 2012
Abstract The complete nucleotide sequences of two iso-lates of apple chlorotic leaf spot virus (Z1and Z3)collected from peach in Henan Province,China,were determined.The genomes of both Z1and Z3were found to contain three open reading frames (ORFs).Sequence analysis showed that genomic sequences of Z1and Z3isolates shared 67.4%-82.9%and 67.2%-82.6%identity,respectively,with the other eight isolates of ACLSV that have been reported previously.Based on the putative amino acid sequences of the products of the three ORFs,Z1and Z3isolates showed the greatest identity to isolate PBM1(GenBank accession number AJ243438)from plum and the least identity with isolate Ta Tao5(GenBank Accession N
umber:EU223295)from peach.Considering the low level of sequence identity between Z1/Z3isolate and Ta Tao5isolate,two types of ACLSV may exist in peach.
Apple chlorotic leaf spot virus (ACLSV)is the type species of the genus Trichovirus ,family Betaflexiviridae [1].
Viruses of this species are distributed worldwide and are known to infect most pome and stone fruit tree species of the family Rosaceae ,including apple,pear,peach,plum,almond,apricot and cherry,which are of great economic value.The severity of the symptoms elicited by ACLSV depends largely on the plant species [2].ACLSV is usually asymptomatic in cultivated apple and pear trees;however,it can induce severe symptoms in stone fruit trees,including dark green sunken mottle,leaf and fruit defor-mation in peach,bark split and pseudopox in plum,bark split in cherry and graft incompatibility in apricot [3–5].The particle of ACLSV is filamentous,approximately 640-760nm in length,and contains a single-stranded positive-sense RNA surrounded by multiple copies of a 22-kDa coat protein (CP)[6].Eight complete nucleotide sequences of ACLSV have been reported from apple (P205,A4,B6and MO-5)[7,8],plum (P863and PBM1)[9,10],peach (Ta Tao5)[11]and cherry (Bal1)[12].Excluding the poly (A)tail at its 30-end,the genome of ACLSV consists of 7,474-7,561nt and has three overlap-ping open reading frames (ORFs 1,2and 3).The 216-kDa protein encoded by ORF 1is a replication-associated pr
o-tein (Rep).The 50-kDa protein encoded by ORF 2is a movement protein (MP),and CP is encoded by ORF 3[7].Although a large number of partial sequences of AC-LSV isolates are available in the GenBank database,only a few of these isolates were from peach.We determined two complete nucleotide sequences of ACLSV from peach in Henan province,China.It is the second time that complete nucleotide sequences of ACLSV isolates from peach have been reported.More important,this is the first report of complete nucleotide sequences of ACLSV isolates from China.
Twenty-four leaf samples of peach were collected from Zhengzhou,Henan Province,China.Total RNA was
Electronic supplementary material The online version of this article (doi:10.1007/s00705-011-1195-5)contains supplementary material,which is available to authorized users.
F.Niu ÁS.Pan ÁD.Jiang ÁS.Li (&)
State Key Laboratory of Biology of Plant Diseases
and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Yuanmingyuan West Road No 2,Haidian District,Beijing 100193,People’s Republic of China e-mail:sfli@ippcaas
F.Niu ÁZ.Wu ÁD.Jiang
Institute of Plant Virology,Fujian Agriculture and Forestry University,Jinshan,Fuzhou 350002,Fujian,People’s Republic of China
Arch Virol (2012)157:783–786DOI 10.1007/s00705-011-1195-5
extracted from100mg of peach leaf using TRIzol(Invit-rogen)and was used as template for the amplification of first-strand cDNA.In order to amplify the full-length genomic sequence of ACLSV,six pairs of primers were designed according to the reference sequence of ACLSV from the P863isolate(GenBank accession number NC 001409),and the sequences of all primers used in this study can be found in Supplementary Table S1.The sequences of the30-terminal region were determined using a rapid amplification of cDNA ends(RACE)kit(TaKaRa).RT-PCR was performed using procedures described previously [13].The PCR reaction conditions were as follows:94°C for5min,followed by30cycles of30s at94°C,30s at 44-52°C,1min30s at72°C,andfinal extension for 10min at72°C.PCR products were purified using a PCR purification kit(Axygen),and the resulting fragments were inserted into pMD18-T vector(TaKaRa).After    E.coli DH5a was transformed with the recombinant DNA,posi-tive clones were identified by colony PCR and sequenced using an automated
DNA sequencer(ABIPRISMTM 3730XL DNA Analyzer).The nucleotide(nt)and deduced amino acid(aa)sequences were compared with other ACLSV sequences published in GenBank using DNAMAN 6.0.A phylogenetic tree was derived using the neighbour-joining method with1,000bootstrap replicates in the Molecular Evolutionary Genetics Analysis(MEGA)soft-ware(version4.0)[14].
ACLSV was detected in2of the24peach samples using RT-PCR.The full-length sequences of these two isolates, both of which consisted of7,552nt,excluding the poly (A)tail,have been submitted to GenBank(accession numbers JN634760and JN634761).Similar to the other eight isolates of ACLSV reported previously,the genome contains three overlapping ORFs,encoding a putative 216-kDa protein related to replication,a50.8-kDa MP,and a21.7-kDa CP,respectively.Sequence analysis demon-strated that the whole genomic nt sequences of the Z1and Z3isolates were95.1%identical.However,when aligned with the sequences of the eight other ACLSV isolates described previously,nt sequences of Z1and Z3isolates showed only67.4%-82.9%and67.2%-82.6%identity, respectively(Table1).Both of these two isolates shared the greatest identity with isolate PBM1from plum and the least identity with isolate Ta Tao5from peach,which is further confirmed by the phylogenetic tree of the complete nucleotide sequences(Fig.1a).The nt sequences of the Z1
Table1Comparison of the complete genome sequence and different genomic regions of ACLSV Z1(a)a
nd Z3(b)isolates in China to the eight ACLSV isolates reported previously
bootstrap 5
ACLSV isolate Genome50UTR ReP gene MP gene CP gene30UTR nt nt%nt nt%nt nt%aa%nt nt%aa%nt nt%aa%nt nt%
(a)
Z17552-151-5652--1383--582--190-
Z3755295.115198.7565294.796.7138396.595.758296.299.019095.8 P863755581.415194.0565580.189.3138383.383.058284.492.219083.1 PBM1754582.915194.7565281.589.5138385.387.658288.893.318391.8 TaTao5747467.415963.6564368.274.9134169.562.358271.073.614380.0 Bal1754976.514879.1566375.984.9138379.079.858280.988.617873.9 A4754879.215192.7565877.987.8137783.285.458284.591.218081.1 B6755381.115194.0564979.989.1137784.286.058287.895.919485.8 P-205755279.815192.7565878.587.6137483.085.158284.589.618780.5 MO-5756174.515279.2563473.282.4138379.479.758282.889.621581.1 (b)
Z37552-151-5652--1383--582--190-
Z1755295.115198.7565294.796.7138396.595.758296.299.019095.8 P863755581.115192.7565580.189.4138382.782.258284.593.319082.0 PBM1754582.615193.4565281.389.6138384.586.558289.394.318392.9 TaTao5747467.215962.9564368.275.3134168.662.358270.873.614379.3 Bal1754976.114877.7566375.484.4138379.179.658282.388.617874.5 A4754879.515191.4565878.487.7137782.584.558285.792.218081.7 B6755381.115192.756498089.3137783.785.858288.095.919485.8 P-205755279.615192.1565878.687.5137482.384.058284.989.618779.3 MO-5756174.515277.9563473.682.2138378.878.658283.390.721579.9 784  F.Niu et al.
and Z3isolates shared only 43%-64%and 43.4%-64%identity with other members of genus Trichovirus ,such as apricot pseudo-chlorotic leaf spot virus (APCLSV),cherry mottle leaf virus (CMLV),peach mosaic virus (PcMV)and grapevine berry inner necrosis virus (GINV)(Fig.1a).The lengths of the 50and 30untranslated regions (UTRs)were found to be 151and 190nt,respectively (Table 1).Three putative complete ORFs were identified.ORF 1,ORF 2and ORF 3extend from nt 152to 5,803,5,715to 7,097,and 6,781to 7,362,respectively,in both Z1and Z3isolates.According to the multiple alignments,the nt sequences encoding CP are the most conserved,and nt sequences encoding MP are the least conserv
ed.The aa sequences of putative Rep of Z1and Z3shared 74.9%-89.5%and 75.3%-89.6%identity,respectively,with the corresponding sequences of the eight isolates of ACLSV.When comparing the aa sequences of the 50-kDa MP,the Z1and Z3isolates showed 62.3%-87.6%and 62.3%-86.5%identity to eight other ACLSV isolates.The aa sequences of CP of both the Z1and Z3isolates shared 73.6-95.9%identity with the sequences of the eight isolates of ACLSV.The amino acids at positions 40and 75of CP,which are necessary for the infectivity of ACLSV,are Ser and Tyr,respectively,in the Z1and Z3isolates,as they are in the other ACLSV isolates except A4and P-205,which have Ala and Phe at these positions [8].
Z1and Z3isolates of ACLSV from peach showed the most sequence identity to a PBM1isolate from plum in Germany,not only for the whole genome sequence but also for the Rep,MP,50UTR,and 30UTR sequences.Among the four isolates (B6,A4,P-205,MO-5)from apple,the B6isolate shared the greatest similarity with the Z1and Z3isolates.Except for the 30UTR sequence,the Z1and Z3isolates showed the least identity (70.8%-71.0%)to the Ta Tao5isolate from peach in the USA,especially for the CP gene sequences,which are relatively conserved compared with other ORFs of the genome of ACLSV.In addition,the complete CP gene sequences of forty ACLSV isolates we obtained from peach,including the Z1and Z3isolates (accession numbers JN848971-JN849010)and six isolates reported previously fr
om peach,(HBP,PE,Ta Tao5,Alberta,In-peach,Peach)were analyzed,and the results of phylogenetic analysis showed that the Z1and Z3isolates were grouped into a cluster and the Ta Tao5isolate was grouped into another cluster (Fig.1b).Therefore,based on the phylogenetic analysis result of complete
genome
Fig.1a Phylogenetic analysis of ACLSV sequences from different isolates,including Z1and Z3from peach in China,P205,A4,B6and MO-5from apple,P863and PBM1from plum,Bal1from cherry,and Ta Tao5from peach in the USA.Four members of the genus Trichovirus (APCLSV,CMLV,PcMV,GINV)were also used to constructed the phylogenetic tree.b Phylogenetic analysis of different ACLSV isolates from peach based on the nucleotide sequences of CP gene,including 40isolates we obtained earlier from China (accession numbers JN848971-JN849010),HBP and PE from China,Ta Tao5from the USA,and Alberta,In-peach and Peach from India.The phylogenetic trees were both constructed by the neighbor-joining method with 1000bootstrap replicates using MEGA software (version 4.0)
Apple chlorotic leaf spot virus from peach in China 785
sequence and CP gene sequence,we proposed that two types of ACLSV isolates may exist in peach,the Z1type and the Ta Tao5type.
Acknowledgments This work was supported by grants from the Earmarked Fund for China Agriculture Research System(CARS-31), the National Basic Research and Development Program of China(973 Program)(No.2009CB119200),the National Natural Science Foun-dation of China(No.31171819).The authors are also grateful to the unknown reviewers for critical revision and help with English lan-guage editing of this manuscript.
References
1.Carstens EB(2010)Ratification vote on taxonomic proposals to
the international committee on taxonomy of virus(2009).Arch Virol155:133–146
2.Nemeth M(1986)Viruses,mycoplasma and rickettsia diseases of
fruit trees.Martinus Nijhoff Publishers,The Netherlands and Akade´miai Kiado´,Hungary
3.Dunez J,Marenaud G,Delbos RP,Lansac M(1972)Variability
of symptoms induced by the Apple chlorotic leaf spot vius (CLSV).A type of CLSV probably responsible for bark split disease of prune trees.Plant Dis Rep56:293–295
4.Desvignes JC,Boye´R(1989)Different diseases caused by the
Chlorotic leaf spot virus on the fruit trees.Acta Hort235:31–38 5.Jelkmann W,Kunze L(1995)Plum pseudopox in German prune
after infection with an isolate of Apple chlorotic leafspot virus causing plum line pattern.Acta Hort386:1
22–125
6.Yoshikawa N,Takahashi T(1988)Properties of RNAs and pro-
teins of Apple stem grooving and Apple chlorotic leaf spot viruses.J Gen Virol88:2611–2618
7.Sato K,Yoshikawa N,Takahashi T(1993)Complete nucleotide
sequence of the genome of an apple isolate of Apple chlorotic leaf spot virus.J Gen Virol74:1927–1931
8.Yaegashi H,Isogai M,Tajima H,Sano T,Yoshikawa N(2007)
Combinations of two amino acids(Ala40and Phe75or Ser40and Tyr75)in the coat protein of Apple chlorotic leaf spot virus are crucial for infectivity.J Gen Virol88:2611–2618
9.German S,Candresse T,Lanneau M,Huet JC,Pernollet JC,
Dunez J(1990)Nucleotide sequence and genomic organization of Apple chlorotic leaf spot closterovirus.Virology179:104–112 10.Jelkmann W(1996)The nucleotide sequence of a strain of Apple
chlorotic leaf spot virus(ACLSV)responsible for plum pseudo-pox and its relation to an apple and plum
bark split strain.
Phylopathol86:101
11.Marini DB,Gibson PG,Scott SW(2008)The complete nucleo-
tide sequence of an isolate of Apple chlorotic leaf spot virus from peach(Prunus persica(L.)Batch).Arch Virol153:1003–1005 12.German S,Delbos RP,Candresse T,Lanneau M,Dunez J(1997)
Complete nucleotide sequence of the genome of a severe cherry isolate of Apple chlorotic leaf spot tricho virus(ACLSV).Arch Virol142:833–841
13.He Z,Jiang DM,Liu AQ,Sang LW,Li WF,Li SF(2011)The
complete sequence of Cymbidium mosaic virus from Vanilla fragrans in Hainan,China.Virus genes42:440–443
14.Tamura K,Dudley J,Nei M,Kumar S(2007)MEGA4:Molec-
ular Evolutionary Genetics Analysis(MEGA)software version
4.0.Mol Biol Evol24:1596–1599
786  F.Niu et al.

版权声明：本站内容均来自互联网，仅供演示用，请勿用于商业和其他非法用途。如果侵犯了您的权益请与我们联系QQ:729038198，我们将在24小时内删除。