Preferential acquisition and inoculation of PVY NTN over PVY O in potato by the green peach aphid Myzus persicae (Sulzer)

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1 Journl of Generl Virology (2016), 97, DOI /jgv Short Communiction Correspondence S. M. Gry Preferentil cquisition nd inocultion of PVY NTN over PVY O in potto by the green pech phid Myzus persice (Sulzer) J. E. Crroll, 1 D. M. Smith 2 nd S. M. Gry 3 1 New York Stte IPM Progrm nd Section of Plnt Pthology nd Plnt Microbe Biology, Cornell University, 630 W. North St., Genev, NY 14456, USA 2 Section of Plnt Pthology nd Plnt-Microbe Biology, School of Plnt Sciences, Cornell University, Ithc, NY 14853, USA 3 USDA ARS, Robert W. Holley Center for Agriculture nd Helth nd Section of Plnt Pthology nd Plnt-Microbe Biology, School of Plnt Sciences, Cornell University, Ithc, NY 14853, USA Received 2 September 2015 Accepted 15 December 2015 In the pst decde, the incidence nd distribution of the recombinnt, tuber necrotic strin of Potto virus Y (PVY NTN ) hs been incresing in the US seed potto crop while the ordinry strin (PVY O ) hs been decresing. The trnsmission efficiency of both strins ws determined from two potto cultivrs when cquired sequentilly by the sme phid or when cquired by seprte phids nd inoculted to the sme plnt. PVY NTN ws trnsmitted more efficiently thn PVY O nd the order of cquisition or inocultion did not ffect the preferentil trnsmission of PVY NTN. When recipient plnt becme infected with both strins, PVY NTN mintined higher titre thn PVY O nd would fcilitte the cquisition of PVY NTN. Furthermore, the cquisition nd trnsmission of PVY NTN over PVY O ws enhnced in the potto cultivr tht expressed strinspecific Ny-like resistnce gene tht confers prtil resistnce to PVY O. Potto virus Y (PVY) is currently the most economiclly importnt disese ffecting seed potto production in the US nd is the primry reson seed lots fil recertifiction (Frost et l., 2013). Further excerbting the sitution is the emergence of recombinnt PVY strins (e.g. PVY NTN ) tht induce tuber necrosis nd ttenuted folir symptoms in mny populr North Americn potto cultivrs (Gry et l., 2010). These plnts often escpe visul detection during field inspections, wheres most isoltes of the ordinry strin (PVY O ) cuse distinctive folir mosic, but do not induce tuber necrosis. In the pst ten yers, PVY NTN hs become widespred nd incidence is incresing in US seed potto production res, though it still only ccounts for bout 20 % of the totl PVY incidence (Gry et l., 2010 nd unpublished). In contrst, PVY NTN now predomintes in mny seed potto production regions in Europe (Rollnd et l., 2008) nd is common in most potto production res worldwide (Ali et l., 2013, 2010; Bouhchem et l., 2008; Rmírez-Rodríguez et l., 2009; Wng et l., 2012). While less definitive folir symptoms induced by PVY NTN hve contributed to its emergence in US seed stocks, other fctors my be involved. Multiple studies report tht PVY NTN is more efficiently trnsmitted by phid vectors thn other strins (Cervntes & Alvrez, 2011; Srinivsn et l., 2012), lthough contrsting findings hve lso been published (Dvis et l., 2005; Verbeek et l., 2010). In mixed strin infections, PVY NTN seems to outcompete other PVY strins in potto, which my contribute to enhnced trnsmission efficiency (Cervntes & Alvrez, 2011; Syller & Grup, 2014). During surveys of US seed pottoes (Gry et l., 2010), PVY NTN ws often found in the sme field nd occsionlly in the sme plnt with PVY O. The experiments reported here investigted the cquisition nd inocultion efficiencies of PVY NTN nd PVY O when they were (1) cquired sequentilly by the sme phid nd then inoculted to helthy recipient plnt or (2) cquired individully by seprte phids nd then sequentilly inoculted to the sme helthy recipient potto plnt. All potto (S. tuberosum L.) plnts were grown in Cornell Mix (Boodley & Sheldrke, 1977) nd mintined in greenhouse screened to exclude phids. Helthy recipient potto plnts were grown from true seed of the cultivr Red Mri. Seedlings were used t the 6 8 lef stge. Plnts of the cultivrs, Pike nd Superior, were mechniclly inoculted with PVY O or PVY NTN, mintined vi shoot cuttings rooted in vermiculite nd used s the sources of virus for the subsequent phid trnsmission experiments. The PVY O isolte Oz ws described previously (Krsev et l., 2011); the PVY NTN isolte ME ws obtined during ntionl survey of lte genertion seed potto lots (Gry et l., 2010) G 2015 Downloded from by Printed in Gret Britin 797

2 J. E. Crroll, D. M. Smith nd S. M. Gry The green pech phid, Myzus persice (Sulzer), clone used in trnsmission tests hd been mintined on cged Physlis floridn (Rydb.) plnts in plnt growth room t our lbortory for pproximtely 15 yers (Gry, 2008). Aphids, strved for t lest 1 h, were plced on detched PVY O - or PVY NTN -infected source leves nd observed for probing behviour during 1 3 min cquisition ccess period (AAP). Individul phids were trnsferred to the youngest fully developed lef on helthy Red Mri seedling nd the plnt cged for n inocultion ccess period (IAP) of t lest 2 h. The sequentil cquisition experiments, designted O/NTN nd NTN/O, used single M. persice given n AAP on lef infected with one strin nd then moved immeditely to lef infected with the other strin for the second AAP before being plced on the recipient plnt (Fig. 1). The sequentil inocultion experiments, designted O+NTN nd NTN+O, used two phids per plnt ech given n AAP on single strin. There ws n pproximtely min lg between plcing the first nd second phids on the recipient plnt (Fig. 1). Control tretments consisted of uninoculted plnts nd plnts inoculted with single phids tht hd been given n AAP on PVY O - or PVY NTN -infected leves. The experiment ws repeted six times, twice using Pike source tissue nd four times using Superior source tissue. In ech experiment 20 to 22 recipient plnts were used per tretment. Following the IAP, the plnts were treted with mixture of pymetrozine, Endevour 50WG, t 1.6 g (93 m 2 ) 21 [0.058 oz (1000 ft 2 ) 21 ] nd bifenthrin, Tlstr P, t 15 ml (93 m 2 ) 21 [0.5 fl oz (1000 ft 2 ) 21 ] to kill ll phids before being moved to the greenhouse. Inoculted plnts were observed for symptom development nd tested t 35 to 42 dys post-inocultion (dys p.i.) by TAS-ELISA (Agdi) using monoclonl ntibody (4C3) tht recognizes ll strins of PVY or monoclonl ntibodies specific for PVY O/C serotypes (Mb2) or for PVY N serotypes (1F5) (Blduf et l., 2006). Source leves nd subset of plnts infected with PVY O nd/or PVY NTN were lso tested by immunocpture multiplex reverse trnscription (RT)- PCR (Lorenzen et l., 2006). O/NTN One phid NTN/O One phid O+NTN Two phids NTN+O Two phids Recipient plnt Recipient plnt Recipient plnt Recipient plnt Fig. 1. Schemtic of the sequentil cquisition tretments O/NTN nd NTN/O where one phid ws given n cquisition ccess period (AAP) sequentilly on source tissue infected with ech strin (PVY O nd PVY NTN ) before being plced on recipient plnt nd of the sequentil inocultion tretments O+NTN nd NTN+O in which two phids, ech given n AAP on tissue infected with one of the virus strins, were used to sequentilly inoculte single recipient plnt. Downloded from by 798 Journl of Generl Virology 97

3 Preferentil trnsmission of PVT NTN over PVT O Sttisticl nlyses of experiments were conducted using effect likelihood rtios nd odds rtios determined by fitting dt to nominl logistic model, contingency nlysis, distribution probbility nlysis nd chi-squre tests. Experiment ws not significnt nd experimentl results were combined for nlyses shown. All nlyses were done with JMP Pro version (SAS Institute) or QuickClcs (GrphPd Softwre). When using infected Pike leves s the source of virus, the trnsmission efficiency of PVY NTN ws similr cross ll tretments described in Fig. 1 (x , P ). The number of plnts infected with PVY NTN ws similr whether the AAP ws on PVY NTN -infected tissue (18.6 %, n543), on PVY NTN nd then PVY O (16.7 %, n542), on PVY O nd then on PVY NTN (19.5 %, n541) or when two phids, ech given n AAP on one virus, were plced on the recipient plnt in either order (PVY NTN first, 14.0 %, n543; PVY O first, 23.3 %, n543). In contrst to the mild mosic symptoms observed on Pike plnts infected with PVY NTN, infection with PVY O led to vein necrosis nd chlorotic/necrotic spots on leves. PVY O ws not trnsmitted from Pike source tissue in ny of the experiments. The vriety Pike my possess one or more of the poorly chrcterized Ny resistnce genes tht confer incomplete resistnce to PVY O, mnifested s hypersensitive response nd restriction in virus movement (Rowley et l., 2015). This could hve prevented PVY O cquisition nd trnsmission. Ny resistnce is often strin specific, temperture sensitive nd ineffective ginst the recombinnt strins of PVY, e.g. PVY NTN. Ny-like resistnce to PVY O is common in North Americn cultivrs, nd reduced spred of PVY O by phids nd through tubers from those cultivrs could be contributing to the shift in PVY strin predominnce from PVY O to PVY NTN nd other recombinnt PVY strins. The Superior plnts infected with PVY O or PVY NTN expressed typicl mosic symptoms without vein or lef necrosis. When leves from PVY-infected Superior plnts were used s the virus source, the number of PVY-infected recipient plnts ws significntly different cross tretments (x , P ) (Tble 1). PVY NTN trnsmission efficiency ws higher, 28.1 %, thn PVY O, 17.5 %. When single phid ws fed sequentilly on PVY O then PVY NTN, or the reverse order, the trnsmission efficiencies were 19.5 % (O/NTN) nd 22.2 % (NTN/O), respectively. The highest trnsmission efficiencies, 35.4 % nd 36.6 %, were in the sequentil inocultion tretments, O+NTN nd NTN+O, respectively. These higher PVY trnsmission efficiencies were perhps expected, since these plnts were inoculted by two potentilly viruliferous phids. The trnsmission efficiencies for PVY O in tretments O+NTN (15.9 %) nd NTN+O (19.5 %) were not significntly different (x , P ) from the PVY O control (17.5 %) (Tble 1). Similrly, PVY NTN trnsmission efficiency ws 24.4 % in both sequentil inocultion tretments nd not significntly different (x , P ) from the PVY NTN control (28.1 %) (Tble 1). These results suggest tht while multiple viruliferous phids lnding on the plnt will increse the probbility of infection, the probbility of either PVY O or PVY NTN being inoculted does not chnge, i.e. overll inocultion efficiency by multiple phids crrying different virus strins is dditive reltive to single phids inoculting the plnt. Aphids sequentilly cquiring both virus strins in tretments (O/NTN or NTN/O) ctully inoculted successful mixed infection in only one out of 163 plnts (0.6 %) (Tble 1). Although rre, these results show single phid is cpble of probing sequentilly on two plnts, ech infected with different strin, nd trnsmitting both strins to single plnt. Therefore, n phid need not cquire both strins from one source plnt in order to trnsmit multiple viruses. In fct, cquisition nd subsequent trnsmission of both virus strins from mixedinfected plnt is not generlly efficient (Srinivsn et l., 2012; Syller & Grup, 2014) nd mixed-infected plnts re not bundnt in the field (Gry et l., 2010). Our results suggest tht mixed-infected plnts would more likely be generted in the field vi inocultion by multiple phids tht hve fed on plnts infected with different strins thn by single phids probing multiple plnts singly Tble 1. Number of Red Mri seedlings infected with PVY, PVY O, PVY NTN or mixed-infected (Mix) in the four experiments, using Superior s source tissue Mixed-infected plnts re counted lso s both O-infected nd NTN-infected. Letters next to the numbers indicte significnt differences in the odds rtios for uninfected plnts cross tretments within ech Totl column. Sequentil tretment Plnts (n) Totl PVY % PVY Totl PVY O % PVY O Totl PVY NTN % PVY NTN Totl Mix % Mix O control b 17.5 O/NTN b 1.2 NTN/O b NTN control bc O+NTN bc b bc 4.9 NTN+O c b c Downloded from by 799

4 J. E. Crroll, D. M. Smith nd S. M. Gry infected with different strins. In the sequentil inocultion tretments, four of 29 nd six of 30 plnts becme infected with both viruses in the O+NTN nd NTN+O tretments, respectively (Tble 1). When single phids were given sequentil AAPs on both strins of the virus there were significnt differences in the numbers of PVY O -, PVY NTN - or mixed-infected plnts in ech of the two tretments (O/NTN, x , P ; NTN/O, x , P ) (Fig. 2). PVY NTN ws trnsmitted 3 4 times more efficiently thn PVY O nd only one plnt becme infected with both viruses (Fig. 2). The order in which the strin ws () Number of infected plnts (b) Number of infected plnts b b O/NTN NTN/O Sequentil virus cquistion tretment b PVY-O PVY-NTN Mixture O+NTN Sequentil virus cquistion tretment b c NTN+O PVY-O PVY-NTN Mixture Fig. 2. The distribution of Red Mri seedlings infected with PVY O, PVY NTN or both strins (Mixture) within the sequentil trnsmission tretments using Superior s source tissue. Letters bove the brs indicte significnt differences in the distribution of infected plnts. () Sequentil cquisition in which one phid given n AAP sequentilly on ech strin, the first listed strin first, ws plced on recipient plnt. (b) Sequentil inocultion in which two phids ech given n AAP on different strin, were plced on recipient plnt, the first strin listed first. cquired by the sme phid hd no significnt effect on the numbers of plnts infected with either PVY NTN (x , P ) or PVY O (x , P ), suggesting tht PVY NTN cn displce PVY O when PVY O is cquired first, but the reverse does not hppen. Alterntively, both virus strins my bind the phid stylets eqully, but PVY NTN is preferentilly relesed when the phid slivtes. Fsting prior to the first AAP nd not the second AAP my lso hve impcted virus strin cquisition or trnsmission by ffecting phid behviour or ltering the complement of inhibitors or helper components in the stylet. However, Syller & Grup (2014) lso reported tht the cquisition order of PVY O nd PVY NTN did not ffect trnsmission efficiency, but in their experiments PVY NTN nd PVY O were trnsmitted with similr efficiencies, suggesting fsting is not likely fctor. PVY NTN infected more recipient plnts in the sequentil inocultion tretments (O+NTN, NTN+O) (Tble 1, Fig. 2b), lthough differences in the number of plnts infected with ech strin or strin mixture were only significnt in the O+NTN tretment (x , P ). The mechnism for this outcome is unknown, but it is unlikely both phids would inoculte the sme or closely ssocited cells. Therefore, ny synergistic or competitive effects of both strins would occur fter mny rounds of repliction nd cell-to-cell or systemic movement. The order of inocultion nd perhps the time intervl between inocultions likely influence the outcome. Perhps PVY NTN hs stronger silencing suppressor (Ksschu & Crrington, 1998) thn PVY, which could fcilitte superinfection of PVY O in PVY NTN -infected plnt with suppressed plnt defences, lthough PVY NTN ppers to win over PVY O more of the time. Interestingly, in the 11 mixed-infected plnts from ll tretments (Tble 1), PVY NTN lwys outcompeted PVY O. At 35 to 42 dys p.i., the verge ELISA bsorbnce vlues for PVY NTN were similr in mixedinfected (1.952, n511) nd singly infected (2.304, n528) plnts, while the verge ELISA bsorbnce vlues for PVY O were (n511) nd (n517) in mixedinfected nd singly infected plnts, respectively. At 98 to 140 dys p.i., nine surviving mixed-infected plnts were retested with TAS-ELISA, multiplex RT-PCR nd by two biossys: bck-inocultion to systemic host, Nicotin tbcum, nd to locl lesion host, Chenopodium mrnticolor, Coste & A. Reyn. Seven of the nine plnts filed to test positive for PVY O ; the other two plnts gve slightly bove bckground levels for PVY O with TAS-ELISA nd RT-PCR, but PVY O ws not detected by the plnt biossys. Aphids were llowed n AAP on leves from these two plnts nd 20 phids from ech source lef were trnsferred to single recipient plnt. PVY O ws trnsmitted from one of the two mixed infected plnts. Finlly, tubers collected from the mixed-infected plnts were sprouted nd the sprouts tested for PVY by TAS-ELISA nd RT- PCR; only PVY NTN ws detected in the developing sprouts. Syller & Grup (2014) reported tht PVY NTN reched higher titres thn PVY O in mixed-infected potto plnts 800 Downloded from by Journl of Generl Virology 97

5 Preferentil trnsmission of PVT NTN over PVT O nd tht PVY O ws often undetected by ELISA by 35 dys p.i., but detected by bck inocultion to tobcco nd by phid trnsmission. Agin, PVY NTN my hve stronger silencing suppressor giving it competitive edge or perhps PVY O is more efficiently trgeted by the silencing mchinery. Alterntively, the RNA of PVY O might be trnsencpsidted by the PVY NTN cot protein, directly ffecting the ELISA, immunocpture multiplex RT-PCR results nd the trnsmission by phids, or the PVY NTN RNA my outcompete the PVY O RNA for both cot proteins leving the PVY O RNA more vulnerble to cellulr degrdtion. Clerly, PVY O survives in some plnts t low levels, but PVY NTN hs distinct dvntge of being cquired nd trnsmitted from these mixed-infected plnts. PVY NTN trnsmission efficiency by M. persice s mesured by counting infected plnts 35 to 42 dys p.i. is higher thn PVY O trnsmission from mixed infections (Srinivsn et l., 2012; Syller & Grup, 2014) despite the use of different clones of M. persice, different isoltes of the virus strins nd different host plnt species (both s source nd recipient). Also, PVY NTN titre in mixed-infected recipient plnts is usully higher thn PVY O titre (Srinivsn et l., 2012; Syller & Grup, 2014 nd this study). Our results here suggest PVY NTN will lso hve competitive dvntge over PVY O s result of sequentil cquisition or inocultion. It is likely tht these re consistent phenomen cross most isoltes within these strins. Whether this is true for other phid vector species remins to be determined. However, wht is unknown is the contribution of ech step in this process, i.e. do both strins differ in cquisition nd inocultion efficiency or is the differentil trnsmission primrily result of virus plnt interctions tht llow PVY NTN to outcompete PVY O in greter number of instnces? Perhps phids ctully ply minor or no role in differentil trnsmission, since we do not know if both strins re eqully cquired (bound) on the phid stylet or eqully inoculted (relesed) into the recipient plnt with equl efficiency. Our results suggest tht phids do ply role nd tht PVY NTN is either cquired or relesed more efficiently. Nonetheless, becuse trnsmission efficiency ws mesured s the number of plnts tht become infected 35 to 42 dys p.i., it my be tht both viruses re eqully inoculted into plnts by the phid nd the resulting finl number of plnts infected t 35 to 42 dys p.i. is strictly function of plnt virus interctions nd competitions. A creful dissection of mechnisms will require dditionl crefully plnned experiments. Regrdless of the mechnisms, it is pprent tht the end result is higher likelihood of PVY NTN -infected plnts over PVY O -infected plnts vi phid trnsmission, nd perhps tuber trnsmission. This my, in prt, help explin the observed shift in PVY strin distribution nd incidence in the US seed potto crop over the pst decde. Acknowledgements The uthors cknowledge nd thnk Alex Wilson, Sm Hodgson nd Kspr Kuhne for their technicl support nd ssistnce during the course of these experiments; Json Ingrm, USDA-ARS reserch technicin, for ssistnce with mnging plnts in the greenhouse; Yu-hsun Lin, Postdoctorl Reserch Associte, USDA-ARS, for testing tuber-grown plnts for presence of virus mixtures nd Erik Mudrk, reserch support specilist Sttisticl Consulting Unit, for guidnce with sttisticl nlyses. 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