ALIOTO, DANIELA

Carrieri R.

Ragozzino E.

SORRENTINO, ROBERTO

Archivio della ricerca - Università degli studi di Napoli Federico II

397www.fupress.com/pm ISSN (print): 0031-9465© Firenze University Press ISSN (online): 1593-2095Phytopathologia Mediterranea!"#$%#&!'%(!#(!)*+,-$%Corresponding author: D. Alioto Fax: +39 081 2539367 E-mail: alioto@unina.itShort NotesRed valerian (Centranthus ruber L.): wild host of Cucumber mosaic virus in uncultivated areas of Campania region (Southern Italy)RAFFAELE CARRIERI, ROBERTO SORRENTINO, ESTER RAGOZZINO and DANIELA ALIOTODipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli, 80055 Portici, ItalySummary. ./!0123/4!#$$*5#$%$(!267!89:6239/!1:9/;0!"Centranthus ruber L.) showing stunting, mild leaf vein mosaic, 267<=;3>/!3/!03?6!>@!:69860!9/7!A>B620!9/7!>==903>/9:!76@>2C9;3>/!9/7!267<=;3>/!>@!;D6!/<CE62!>@!:69860(!B626!observed in Campania (Southern Italy). The presence of Cucumber mosaic virus (CMV) in the diseased plants was demonstrated by the reactions of test plants, electron microscopy, serology and RT-PCR. On the basis of highest identities and closest phylogenetic relationships, CMV from C. ruber was most closely related to CMV subgroup I FG!H>=DI0!1>0;<:9;60!B626!@<:J::67!EK!E9=LM3/>=<:9;3/4!D69:;DK!C. ruber seedlings.Key words: weed, subgroups, phylogenetic analysis.IntroductionCucumber mosaic virus (CMV) is one of the most destructive and widespread plant viruses that can infect a broad range of annual, biennal, perennial crops and weeds. Many weeds may act as alterna-tive hosts for CMV, and for this reason they are im-portant in virus epidemiology and control. The virus is transmitted in nature by more than 75 species of aphids in a non-persistent manner, and is also seed-E>2/6!3/!73N626/;!D>0;!1:9/;0GCMV is the type member of the genus Cucumovi-rus in the family of Bromoviridae, and has isometric vi-rions of approximately 30 nm diameter. The genome of CMV is divided into three positive single-stranded RNA molecules. RNA1 and RNA2 are encapsidated separately, while RNA3 is encapsidated with a sub-46/>C3=! OPF! "OPF-&(! 6/=>767! EK! ;D6! )QMD9:@! >@!OPF)!"R9:<L93;30!et al., 1992) and involved in encap-0379;3>/!"S<?<L3!et al., 1991). Some CMV strains are also associated with satellites RNAs (satRNA), which may modify the symptoms induced by the virus in D>0;!1:9/;0!"S<?<L3!et alG(!%**%T!R9:<L93;30!et al., 1992; Gallitelli, 2000; García-Arenal et al., 2000). CMV exists as a variety of strains which have been divided into two subgroups, I and II, on the basis of nucleic acid hybridization and serology >@! ;D6! =91037!12>;63/! "R9:<L93;30! et al., 1992). CMV subgroup I has been further subdivided into IA and IB on the basis of phylogenetic analyses (Gallitelli, 2000; Yu et alG(!#$$-&GCentranthus ruber (L.) DC (syn. Valeriana rubra L.), family Valerianaceae, is a plant native of Mediterra-nean area, that develops wild in many places due to the ability to adapt to poor soils. ./!0123/4!#$$*5#$%$(!;D6!#U!>@!267!89:6239/!1:9/;0!0D>B3/4!0;</;3/4(!C3:7!863/!C>093=!9/7!C92L67!26-7<=;3>/!3/!03?6!>@!:69860!9/7!A>B620!"V34<260!%!9/7!2), and occasional deformation and reduction of the number of leaves, were observed on the volcanic soils of Vesuvius in Campania region (Southern Ita-:K&(!)5-!L3:>C6;260!9B9K!@2>C!=2>1167!J6:70G!In order to identify the virus responsible of these symptoms, leaves from twenty symptomatic and ten asymptomatic plants of C. ruber were collected in the 9E>86!C6/;3>/67!9269!B3;D3/!9/!9269!>@!%!LC2. Sub-Phytopathologia Mediterranea398R. Carrieri et al.06W<6/;:K(!J86!><;!>@! ;D6! ;B6/;K! 0KC1;>C9;3=! 09C-ples were ground in cold 0.01 M potassium phosphate E<N62! 1X! +5+G#! 9/7! C6=D9/3=9::K! 3/>=<:9;67! >/;>!;D266!;60;!1:9/;0!>@!69=D!>@!%-!;60;!016=360(!12683><0:K!dusted with carborundum powder. The test species were: Chenopodiaceae (Chenopodium quinoa Willd., C. amaranticolor Coste et Reyn, C. murale L.), Cucurbita-ceae (Cucumis sativus L., C. melo L., Cucurbita pepo L.), Lamiaceae (Ocimum basilicum L.), Leguminosae (Vigna sinensis Endl.), Solanaceae (Nicotiana tabacum L. “Sam-sun”, N. glutinosa L., N. rustica L., N. occidentalis XGMYGWheeler, N. benthamiana L., Lycopersicon esculentum ZG&G!R:9/;0!B626!L61;! 3/!9!4266/D><06!"#$5##[\&!9/7!>E062867!793:K!@>2!0KC1;>C!6]126003>/!@>2!-!B66L0GMechanical sap inoculations revealed successful transmission of the virus from red valerian to all the inoculated test plants of each species, except O. ba-silicum. Virus induced chlorotic-necrotic local lesions developed on C. quinoa, C. murale, C. amaranticolor and V. sinensis(!+5%$!79K0!1>0;!3/>=<:9;3>/!"713&G!^D6!Figure 1. Vein clearing and mosaic on the leaves of Cen-tranthus ruber naturally infected with Cucumber mosaic virus (CMV).!Figure 2. Size reduction of Centranthus ruber! A>B62! 9@-@6=;67!EK!\Y_!":6@;&!=>C19267!;>!9!A>B62!@2>C!9!D69:;DK!plant (right).399Vol. 51, No. 2, August, 2012Centranthus ruber L.: wild host of Cucumber mosaic virusvirus also induced systemic mosaic on N. tabacum, N. benthamiana, N. occidentalis, N. glutinosa, N. rustica and L. esculentum(! %-5%`!713G!a<23/4!0123/4! "#$5#'!dpi), L. esculentum inoculated plants also exhibited fern leaf symptoms. Cucumis sativus , C. melo and C. pepo showed chlorotic mottle and malformations on younger leaves. The host range and symptoms in-duced on indicator plants were typical of those in-duced by CMV.The indications given by biological assays were =>/J2C67!EK! ;D6!6:6=;2>/!C3=2>0=>1K!>E06289;3>/0!of leaf dips of symptomatic C. ruber plants, mounted 3/!%U!9W<6><0!<29/K:!9=6;9;6!9/7!>E062867!B3;D!9!Philips EM 208 electron microscope. These observa-tions revealed the consistent presence of isometric particles approximately 30 nm in diameter, typical of Cucumovirus particles.Subsequently, the thirty C. ruber samples, twenty symptomatic and ten asymptomatic, were analysed by DAS-ELISA, using commercially produced anti-serum (Loewe-Phytodiagnostica Biochemical, Sau-erlach, Germany), against CMV, according to manu-facturer’s instructions. Only the symptomatic plants B626!1>03;386!;>!\Y_(!=>/J2C3/4!;D6!900>=39;3>/!>@!CMV with the diseased plants and indicating that the virus is probably not latent in C. ruber. H>=DI0!1>0;<:9;60!B626!@<:J::67!EK!E9=L!3/>=<:9;-ing C. quinoa, V. sinensis, C. pepo and N. glutinosa sap to healthy C. ruber seedlings (three plants for each in-oculation). Inoculated plants reacted showing stunt-ing, mosaic and reduction in leaf size. In the case of C. quinoa and V. sinensis(!E9=L! 3/>=<:9;3>/!B90!162-formed after three monolesion passages. The pres-6/=6!>@!\Y_!3/!;D6!E9=LM3/>=<:9;67!0KC1;>C9;3=!C. ruber!1:9/;0!B90!=>/J2C67!EK!aFSMbZ.SFG!In order to verify the transmissibility of CMV by C. ruber seeds, 165 plants derived from seed from two diseased plants were tested by DAS-ELISA, with negative results, suggesting that CMV is not seed-transmitted in this host.For RT-PCR, total nucleic acids were extracted from the leaves (100 mg) of three symptomatic and one asymptomatic C. ruber plants using the silica capture method (Foissac et al. (2000). An aliquot "#! cZ&!B90! <067! 90! ;6C1:9;6! @>2! 2686206! ;29/0=231-tion with the enzyme Superscript III (Invitrogen, Carlsbad, CA, USA), following manufacturer’s in-0;2<=;3>/0G! ./=<E9;3>/!C3];<260! @>2! R\O! 9C1:3J=9-;3>/0!=>/;93/67!#!cZ!>@!=aPF(!%G'!CY!Y4\:2, 200 cY!69=D!7P^R(!-$$!/Y!@>2!69=D!123C62!9/7!#G'!d!of Taq DNA polymerase (Sigma-Aldrich, St Louis, MI, USA). Primers used were CMV3F1 and CMV3R (Davino et alG(!#$$'&(!016=3J=!@>2!9!=>/062867!2643>/!of CMV coat protein gene (CP), and primers M1 and Y#(!016=3J=!@>2!9!2643>/!=>73/4!@>2!\Y_!C>86C6/;!protein (MP) (Lin et alG(!#$$-&G!F::!26946/;0!B626!=>C-E3/67!9/7!C93/;93/67!9;!*'[\!@>2!)!C3/G!^D32;KM634D;!=K=:60!>@!R\O!B626!162@>2C67!EK!D69;3/4!9;!*'[\!@>2!)$!06=(!'$[\!"123C620!\Y_)V%!9/7!\Y_)O&!>2!''[\!"Y%!9/7!Y#&!@>2!%!C3/(!9/7!+#[\!@>2!%!C3/(!@>::>B67!EK!J/9:!1623>7!9;! +#[\! @>2! %$!C3/G!F0! ;D6!1>03;386!control, a CMV isolate from the authors’ collection B90!<067G!R\O!9C1:3=>/0!>@!;D6!6]16=;67!03?6!"-)`!E1! 9/7! e-#! E1(! 26016=;386:K&! B626! 1<23J67! <03/4!R2>C649!1<23J=9;3>/!L3;!"f3?927g!S_!46:!9/7!R\O!Clean-UpSystem, Madison, WI, USA) and directly sequenced (BMR Genomics, Padova, Italy) in both 7326=;3>/0!<03/4!;D6!016=3J=!123C620G!^D6!06W<6/=60!obtained were analysed using the BLASTn program with default parameters (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990). Using primer pairs for the CP and MP regions of \Y_!46/>C6(! ;D6!6]16=;67! 03?6!9C1:3=>/0!>@! -)`!E1! 9/7! e-#! E1(! 26016=;386:K(!B626! 9C1:3J67! @2>C!;D6! 0KC1;>C9;3=! 09C1:60! 46/>C6G! X>B6862(! />!9C1:3J=9;3>/!B90!>E;93/67!@2>C!;D6!90KC1;>C9;3=!plant of C. ruber used as the negative control, which previously resulted negative to CMV by ELISA. The analyses of the movement protein sequences of the three C. ruber!30>:9;60(!26869:67!9!%$$U!376/-;3;K! ;>! 69=D! >;D62! 9/7! **U! B3;D! \Y_! 06W<6/=60!1<E:30D67! 3/! ;D6!h6/i9/L! 79;9E906! "http://blast.ncbi.nlm.nih.gov/). This sequence was submitted ;>!h6/i9/L!79;9E906!</762! ;D6!9==6003>/!/<CE62!JF951999.For phylogenetic analysis, the coat protein se-quences of the CMV isolated from red valerian were compared to the corresponding sequences of 20 CMV 016=360! 26;236867! @2>C! h6/i9/L! 79;9E906! 6/;2360j!seven belonging to subgroup IA (Accession numbers a%$')e(!d##e#%(!Fk'%%**$(!a%$')*(!a$$)e`(!a%#-**(!Fk#+`-e%&(! 03]! ;>! 0<E42><1! .i! "F==6003>/! /<CE620!Fi$$e+++(!Fi$-##*-(!d#$#%*(!a#e+e$(!l%`*#`&!9/7!seven  to subgroup II (Accession numbers AF063610, Fk)$-)*e(! FV%*e%$)(! Z%'))`(! Fk)$--$)(! FV%#+*+`(!Y#%-`-&GThe coat protein sequences obtained in this study and corresponding to the isolates previously cited, D97!%$$U!376/;3;K!;>!69=D!>;D62!"h6/i9/L!9==6003>/!P[!JF802035) and showed an average identity of 96, Phytopathologia Mediterranea-$$R. Carrieri et al.*#! 9/7! e%U!B3;D! ;D>06! >@! \Y_! 0;293/0! E6:>/43/4!to subgroups IA, IB and II, respectively. A phyloge-netic tree was inferred using the program MEGA v. 3.1 (Kumar et al.,! #$$-&!B3;D! ;D6!/634DE><2Mm>3/3/4!method, and the statistical reliability of the branch-es was evaluated by performing bootstrap analysis (1,000 replicas). The results showed that CMV iso-lated from C. ruber formed a monophyletic group 0<11>2;67!EK!%$$U!E>>;0;291!9/7!91169267!=:>06:K!26:9;67!;>!Fk#+`-e%!"0<E42><1!.F&!"/<=:6>;376!376/-;3;K! >@! *`U&! "V34<26! )&G! Y>26! 730;9/;! 26:9;3>/0D310!were observed for subgroups IB and II. In particular, ;D6!;266!=>C123067!;D266!C9m>2!=:9760!@>2C67!EK!;D6!CMV isolates belonging to the three subgroups. Figure 3. Unrooted phylogenetic tree calculated from a conserved region of the coat protein gene (CP) of CMV species belonging to the three subgroups IA, IB and II. CMV-RED V is the sequence obtained by the authors.! D10538 - CMV IA U22821 - CMV IA AJ511990 - CMV IA D10539 - CMV IA D00385 - CMV IA D12499 - CMV IA AJ276481 - CMV IA CMV - RED V D49496 - CMV IB AB008777 - CMV IB AB042294 - CMV IB U20219 - CMV IB D28780 - CMV IB Y16926 - CMV IB AF063610 - CMV II AJ304398 - CMV II AF198103 - CMV II L15336 - CMV II AJ304403 - CMV II AF127976 - CMV II M21464 - CMV II878025478910010010064934383656852789763-$%Vol. 51, No. 2, August, 2012Centranthus ruber L.: wild host of Cucumber mosaic virusThe studies at the biological, serological and mo-lecular level show that the C. ruber disease is caused by an isolate of CMV. Based on the analysis of coat protein gene sequence, this isolate belongs to sub-group IA. A previous study by Crescenzi et al. (1993) in Campania investigated CMV strains, and the role played by so-called arable weeds (within a crop or 3/!92690!97m9=6/;!;>!=2>1!1:9/;0&!3/!61376C3>:>4K!>@!the virus. They showed the occurrence of CMV with greater frequency of strains of subgroup I (formerly f^!0;293/0&!3/!#$!73N626/;!016=360!>@!B6670(!76C>/-strating an important role of these weeds as survival hosts for this virus. The present study also indicates that red valerian plants may play a role in the epide-miology of CMV, allowing the virus to overwinter during the unfavourable season and be reintroduced by aphids on agricultural crops during the growing season. Although C. ruber is listed as natural host of CMV EK!0>C6!9<;D>20!"H:3/L>B0L3!9/7!d0=729B63;(!%*`eT!X>289;D(! %*e$&(! ;>! ><2! L/>B:6746! ;D30! 30! J20;! 76-scription of CMV symptoms on this host in nature.Literature citedAltschul S.F., W. Gish, W. Miller, E.W. Myers and D.J. Lipman, 1990. Basic local alignment   search tool. Journal of Molecu-lar Biology!#%'(!-$)5-%$GCrescenzi A., L. Barbarossa, D. Gallitelli and G.P. Martelli, 1993. Cucumber mosaic cucumovirus populations in Italy under natural epidemic conditions and after a satellite-mediated protection test. Plant Disease!++(!#e5))G!Davino S., M.G. Bellardi, M. Di Bella, M. Davino and A. Bert-accini, 2005. Characterization of a      Cucumber mosaic vi-rus isolate infecting Mandevilla sanderi!"X6C0:G&!f>>70>/G!Phytopathologia Mediterranea --(!##$5##'G!!Foissac X., L. Svanella-Dumas, P. Gentit, M.J. Dulucq and T. Candresse, 2001. Polyvalent detection of fruit tree Tricho, Capillo and Foveaviruses by Nested RT-PCR using degen-erate and inosine containing primers (PDO RT-PCR). Acta X>2;3=>:;<296!".SXS&!''$(!)+5--GGallitelli D., 2000. The ecology of cucumber mosaic virus and sustainable agriculture. Virus Research!+%(!*5#%GGarcía-Arenal F., F. Escriu, M.A. Aranda, J.L. Alonso-Prados, J.M. Malpica and A. Fraile, 2000. Molecular epidemiology of Cucumber mosaic virus and its satellite RNA. Virus Re-search!+%(!%5eGX>289;D!kG(!%*e$G!_32<060!>@! :6;;<=6!..G!X>0;!29/460!>@! :6;;<=6!mosaic virus and Cucumber mosaic virus. Acta Agronomica Academiae Scientiarum Hungaricae!#*(!)))5)'#G!H:3/L>B0L3!YG! 9/7!XGFG!d0=729B63;(! %*`eG!a90!h<2L/6C>-093L!')5'+!./j!!"#$%&'()*+,'-.&./'*+00(!"YG!H:3/L>B0L3(!67G&!FL976C39!_62:94(!i62:3/(!-`$!11GH<C92!SG(!HG! 9^C<29!9/7!YG!P63(!#$$-G!YbhF)j! ./;6429;67!software for Molecular Evolutionary Genetics Analy-sis and sequence alignment. 1-'*2$/3+ '$+ 1'.'$4.-5#6'(3 5, %'$5%`)GZ3/!XG(!ZG!O<E3>(!FGiG!SCK;D6!9/7!iGfG!V9:L(!#$$-G!Y>:6=<:92!population genetics of Cucumber mosaic virus evidence for @></762! 6N6=;0! 9/7! 26900>2;C6/;G! Journal of Virology 78, ````5``+'GR9:<L93;30!RG(!YGkG!O>>003/=L(!OGhG!a36;?46/!9/7!OG.GiG!V29/=L3(!1992. Cucumber mosaic virus. Advances in Virus Research!-%(!#e%n)-eGS<?<L3!YG(!SG!H<B9;9(!\G!Y90<;9(!YG!P3;;9!9/7! G^! 9^L9/9C3(!1991. Functional analysis of deletion mutants of Cucumber mosaic virus RNA3 using an in vitro transcription system. Virology!%e)(!%$`5%%)Gl<!\G(! kG!f<!9/7!oG!pD><(!#$$-G!a6;6=;3>/!9/7!0<E42><13/4!of Cucumber mosaic virus isolates by TAS-ELISA and im-munocapture RT-PCR. Journal of Virological Methods 123, %''5%`%GAccepted for publication: November 21, 2011

Red valerian (Centranthus ruber L.): wild host of Cucumber mosaic virus in uncultivated areas of Campania region (Southern Italy)

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Archivio della ricerca - Università degli studi di Napoli Federico II