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GIORDANO, L. de B.

INOUE-NAGATA, A. K.

NAGATA, T.

ÁVILA, A. C. de

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447Hortic. bras., v. 25, n. 3, jul.-set. 2007In tropical and subtropical countries,begomoviruses cause seriousproblems to tomato. The major symptomsafter begomovirus infection are yellowmosaic, mottling, rugosity and leafdistortion, resulting in reduction of thenumber of flowers, fruits, fruit weight andquality. This disease is known to betransmitted by whiteflies (Bemisia tabaci).Transmission of the virus by mechanicalinoculation is sporadically successful,hence, in nature the dissemination of thisdisease agent is supposed to hardly everoccur by mechanical means. The firstreport on the incidence of begomovirus-like disease in tomatoes in Brazil occurredINOUE-NAGATA AK; NAGATA T; ÁVILA AC; GIORDANO LB. 2007. A reliable begomovirus inoculation method for screening  Lycopersicon esculentumlines. Horticultura Brasileira 25:447-450.A reliable begomovirus inoculation method for screening  Lycopersiconesculentum linesAlice K Inoue-Nagata1; Tatsuya Nagata2; Antonio C de Ávila1; Leonardo de B Giordano11Embrapa Hortaliças, C. Postal 218, 70359-970 Brasília-DF; 2Universidade Católica de Brasília, 70790-160 Brasília-DF;alicenag@cnph.embrapa.brin the 60’s (Flores et al., 1960), and laterconfirmed as the begomovirus Tomatogolden mosaic virus (TGMV) (Matyis etal., 1975). Costa et al. (1984; 1987)described tomato infected with whitefly-transmitted geminiviruses, but theiridentification was not carried out andremains unknown. The expansion ofbegomovirus diseases on tomato wasonly observed during the last decade(Giordano et al., 1999). Thecharacterization of these begomovirusesand the breeding programs aiming tointrogress genes for begomovirusresistance on tomato plants are ongoingin various laboratories.The strategy to control this diseaseusing resistant cultivars focuses mainlyon the introgression of resistance genesinto tomato commercial cultivars. Theintroduction of the resistance usingtransgenic plants is a new approach todevelop plants resistant to diseases.However, the current transgenicstrategies introducing viral or anti-viralgenes into a plant have somedisadvantages such as the narrowresistance spectrum to specific virusspecies, and also some technical andpolitical constraints. Besides, theconventional methods of plant breedingABSTRACTDue to failures of infection after mechanical inoculation ofbegomovirus on tomato (Lycopersicon esculentum Mill.) plants, anefficient and reliable begomovirus inoculation method was developedusing the whitefly vector (Bemisia tabaci). Virus acquisition wascarried out using a ‘Tube-cage’, made of a polypropylene tube,containing one to two day-old whiteflies and a tomato plant apexinfected with a begomovirus isolate. After 48 h of acquisition accessperiod, inoculation was done using a ‘Ring-cage’, containingviruliferous whiteflies, attached to the abaxial side of the leaf ofyoung tomato plants. 48 hours after acquisition, the whiteflies wereeliminated and the inoculated plants were incubated for 28 days undergreenhouse conditions. Three viruliferous whiteflies per plant wereenough to cause infection in susceptible tomato cv. Viradoro. Thisprocedure was applied to test the resistance of tomato line 486-1(resistant line) in comparison with ‘Viradoro’ (susceptible cultivar).Dot blot hybridization confirmed the susceptibility of ‘Viradoro’ andthe resistance of the line 486-1, showing the efficiency of this methodfor screening plants for disease resistance. This method is also auseful tool in detecting the presence of virus in inoculated (lowerleaves) and in non-inoculated (upper leaves) in a given plant. Usingthis procedure, it was observed that the resistance of the line 486-1probably is expressed in the inoculated leaf without virustranslocation to the upper leaves. Keywords: Lycopersicon esculentum, inoculation, resistance,screening, geminivirus.RESUMOUm método de inoculação de begomovírus confiável para aseleção de linhagens de Lycopersicon esculentimDevido às falhas encontradas no método de inoculação debegomovírus em plantas de tomateiro, um método eficiente econfiável de inoculação de begomovírus foi desenvolvido utilizan-do como vetor a mosca-branca (Bemisia tabaci). A aquisição viralfoi realizada em uma armadilha (‘Tube-cage’) feita a partir de umtubo de polipropileno contendo moscas-brancas de um ou dois diasde idade e o ápice de uma planta de tomate infectada com umbegomovírus. Após 48 horas de período de aquisição, procedeu-se àinoculação utilizando-se uma armadilha em forma de anel (‘Ring-cage’) contendo as moscas-brancas virulíferas. A armadilha foi co-locada na parte abaxial das folhas de plântulas de tomateiro. Após operíodo de inoculação de 48 horas, as moscas-brancas foram elimi-nadas e as plantas inoculadas foram incubadas por 28 dias em casa-de-vegetação. Utilizando-se este método três moscas-brancasvirulíferas por planta foram suficientes para causar infecção em to-mateiro susceptível cv. Viradoro. Este procedimento foi aplicadopara avaliar a resistência de tomateiro linhagem 486-1 (resistente)em comparação com ‘Viradoro’. Hibridização por dot-blot confir-mou a susceptibilidade de ‘Viradoro’ e a resistência da linhagem486-1, mostrando a utilidade deste método na seleção de plantasresistentes. Este método também permitiu a avaliação da presençado vírus nas folhas inoculadas e em folhas superiores não inocula-das, indicando que o mecanismo de resistência da linhagem 486-1poderá estar atuando nas folhas inferiores inoculadas, não havendotranslocação viral para as folhas superiores.Palavras-chave: Lycopersicon esculentum, inoculação, resistência,screening, geminivírus.(Recebido para publicação em 7 de janeiro de 2006; aceito em 25 de julho de 2007)448 Hortic. bras., v. 25, n. 3, jul.-set. 2007by inter- and intra-species crosses areviable approaches due to the availabilityof a large range of resistance genes inthe genus Lycopersicon. Some level ofresistance to Tomato yellow leaf curlvirus (TYLCV) was already reported inL. pimpinellifolium, L. cheesmani, L.hirsutum, L. peruvianum and L. chilense(Kasrawi et al., 1988; Pilowsky &Cohen, 1974; Picó et al., 1996; 1998;Scott et al., 1996).The main limitation for screeningresistant plants is related to theinoculation method. Evaluation of plantresistance to a virus is frequently basedon mechanical inoculations. However,the systemic infection of tomatobegomoviruses by mechanicalinoculation is sporadic. Therefore,mechanical inoculation is not a reliablemethod for screening tomato plants forbegomovirus resistance. The particlebombardment method could be a highlyefficient option. However, this methodrequires special equipment and is notsuitable for inoculating great numbersof plants. With this method ofinoculation it is difficult to control theinoculation pressure in each plant, animportant factor to achieve uniformityof infection. Agroinfection is analternative method, however due to thesame reasons mentioned above, it is alsonot suitable for screening plants forresistance.Inoculation of begomovirus usingwhitefly as vectors appears to be the bestmethod at this moment. Hence, itbecame important to develop a practicaland reliable method of begomovirusinoculation using whiteflies. This studyintroduces a convenient inoculationmethod, which enables the applicationof a uniform inoculation pressure to theplants.MATERIAL AND METHODSPreparation of viruliferouswhiteflies using the Tube-cageNewly emerged whitefly adults witha maximum age of 48 h were collectedusing a sucking apparatus from thecolonies established on healthypoinsettia plants. A polypropylene tubeof 50 ml (Corning) containing adetached infected tomato plant apex(isolate DFM, Tomato mottle leaf curlvirus, Inoue-Nagata et al., 2006) wasattached at the end of the suckingapparatus and the whiteflies werereleased inside the tube. This new Tube-cage was then covered with a whitefly-proof nylon screen and tightly screw-capped (Figure 1).Tomato inoculation using theRing-cageThree, five or ten viruliferouswhiteflies previously prepared using theTube-cage method (for virusacquisition) were transferred into thenewly developed leaf-cages (Figure 2)settled onto the abaxial side of tomatoseedlings leaves cv. Viradoro. Tomatoplants at four true-leaf staged were usedfor the inoculation. The whiteflies wereplaced in contact with the leaves of theplants to be evaluated for diseasereaction using a Ring-cage. Theinoculation access period was 48 h. Thelimited space inside the cage forces theinsect to feed on the plants to be tested,increasing the efficiency of virustransmission. After 48 h of incubationat room temperature, the whiteflies wereeliminated by spraying the insecticideConfidor (imidacloprid, Bayer). Theinoculated plants were incubated in thegreenhouse up to one month forsymptom development. Leaf disks werecollected from the inoculated plants toconfirm virus infection and tested by dotblot hybridization according to Santoset al. (2004).RESULTS AND DISCUSSIONNumber of whiteflies used in theinoculation procedureForty-eight hours after inoculationthe mortality of the whiteflies was low(averaging 10% for both ten and fivewhitefly inoculation cohorts, and 16.7%for three whitefly inoculation cohort).All the plants inoculated with ten (6/6)and three whiteflies (6/6) were infectedwithin one month. Only one out of sixplants was not infected when fiveviruliferous whiteflies were used. Sinceall six plants inoculated with threeviruliferous whiteflies were infectedwith begomovirus, the use of threewhiteflies per plant for inoculation wasdemonstrated to be appropriate for mostpurposes.The application of the method forthe pre-screening testIn order to test the usefulness of thismethod for screening, two tomato lineswere tested, ‘Viradoro’, the susceptiblecultivar, and the tomato line 468-1,previously selected for resistance to thevirus (Santana et al., 1998; 2001). Tenplants of ‘468-1’ and eight of ‘Viradoro’were inoculated using three whiteflies.The development of symptom wasobserved and recorded weekly. Thenumber of infected plants of ‘Viradoro’and ‘468-1’, based on symptomdevelopment and dot-blot hybridization,are presented in Table 1. Based uponsymptom development, tomato plants´Viradoro’ showed a high infection rate(7/8). On the other hand, tomato line´468-1’ showed only one plant withsymptom. To confirm the virusinfection, dot blot hybridization wasemployed using the leaf materialscollected 28 days after inoculation.Seven out of eight plants of ´Viradoro’Table 1. Symptom expression and infection of begomovirus on inoculated tomato plants(Expressão de sintomas e infecção de begomovírus em plantas de tomate inoculadas). Brasília,Embrapa Hortaliças, 2005.1Number of plants with symptoms (before the slash) out of the total (after the slash); 2Numberof plants positively detected (before the slash) out of the total (after the slash) detected bydot blot hybridization (1Número de plantas com sintomas (antes da barra) em relação aototal (após a barra); 2Número de plantas detectadas positivamente (antes da barra) em rela-ção ao total (após a barra) detectados por hibridização “dot blot”).AK Inoue-Nagata et al.449Hortic. bras., v. 25, n. 3, jul.-set. 2007showed positive signals and two out often tomato plants of the line ´468-1’showed positive infection (data notshown). The result indicated that theprocedure could differentiate distinctlevels of virus susceptibility (resistance)occurring in tomato varieties.The virus distribution and thedevelopment of the infection process intomato plants can define the mechanismof resistance. One type of resistance, forinstance, is related to long distance virusmigration in plants. The virus canreplicate in the inoculated leaf but is notable to translocate in the whole plant.Since this method allows thedifferentiation of the inoculated leaffrom the non-inoculated ones on a singleplant basis, the inoculated leaf and onetop leaf were collected separately forboth tomato germplasm during a 28-dayperiod. Four plants of each line wereseparated and the presence of virus wasevaluated at 7, 14, 21, and 28 days afterinoculation by dot blot hybridization(Figure 3). The virus was detected in theinoculated and in the non-inoculatedleaves (top) of ´ Viradoro’ within 14 daysafter inoculation (a.i.) (Figure 3). Thevirus could also be detected in theseleaves up to 28 days a.i. No positivesignals were obtained from samples ofthe inoculated and of the top leaves,when tomato ´468-1’ was used as testplants. These results suggested that theresistance involved in tomato ´ 468-1’ isnot related to the inhibition of long-distance migration of the virus, but mostprobably with cell-to-cell movement orwithin even in earlier stages of virusinfection. For the Ty-1 gene (toleranceto Tomato yellow leaf curl virus –TYLCV) the resistance, at low titerinoculum, is expressed through asignificant reduction of the viralaccumulation in the inoculated tissue,and at high titer inoculum through asignificant limitation of the long-distance translocation of the virus(Michelson et al., 1994). However forthe same virus, a different tomato lineneither developed disease symptoms noraccumulated high virus titer (Friedmannet al., 1998).The use of polypropylene (PP) tubecage for virus acquisition and leaf cagefor inoculation provided importantA reliable begomovirus inoculation method for screening  Lycopersicon esculentum linesFigure 2. Ring-cage. The leaf is sandwiched between big and small rings of plastic tube andwhiteflies were introduced through the small hole by using sucking-blowing apparatusassembled with a narrow silicon tube and yellow tip with wide hole by cutting the top and apiece of screen between them. After the transferrence of the whiteflies, the small hole wassealed with a piece of cotton (“Ring-cage”. A folha é colocada entre anéis largos e estreitosde tubos plásticos e moscas-brancas foram introduzidas através de um pequeno orifíciousando um aparato sugador, montado com um tubo de silicone estreito com extremidade deponteira amarela com a ponta alargada por corte e uma barreira de tela entre eles. Após atransferência das moscas-brancas, o pequeno orifício é selado com de algodão). EmbrapaHortaliças, 2005.Figure 1. Tube-cage with infected leaf material and whitefly (“Tube-cage” com materialfoliar infectado e mosca-branca). Brasília, Embrapa Hortaliças, 2005.450 Hortic. bras., v. 25, n. 3, jul.-set. 2007Figure 3. Comparison of susceptibility between ´ Viradoro’ (lanes 1-4) and ´ 468-1’ (lanes 6-9)to begomovirus infection by dot blot hybridization. Infection on tomato ´ Viradoro’ (lanes 1-3)became clear from 14 days after inoculation. No positive signals were found in tomato ´ 468-1’and one tomato plant ´Viradoro’ (lane 4). Weak reactions were considered as backgroundreaction. Lane 5 = non-inoculated tomato ´ Viradoro’. 7d, 14d, 21d and 28d = 7, 14, 21, 28 daysafter inoculation (Comparação de susceptibilidade entre ‘Viradoro’ (colunas 1-4) e ‘468-1’(colunas 6-9) a infecção por begomovírus por hibridização “dot blot”. Infecção de tomate‘Viradoro’ (colunas 1-3) tornou-se clara a partir de 14 dias após a inoculação. Nenhum sinalpositivo foi encontrado nos tomates ‘468-1’ e em um tomate ‘Viradoro’ (coluna 4). Reaçõesfracas foram consideradas como negativas. Coluna 5 = tomate ‘Viradoro’ não inoculado. 7d,14d, 21d e 28d = 7, 14, 21, 28 dias após a inoculação). Embrapa Hortaliças, 2005.AK Inoue-Nagata et al.FRIEDMANN M; LAPIDOT M; COHEN S;PILOWSKY M. 1998. A novel source ofresistance  to Tomato Yellow Leaf Curl virusexhibiting a symptomless reaction to viralinfection. Journal of the American Society forHorticultural Science 123: 1004–1007.GIORDANO LB; BEZERRA IC; FERREIRAPTO; BORGES NETO CR. 1999. Breedingtomatoes for resistance to whitefly-transmitedGeminivirus with bipartite genome in Brazil.Acta Horticulturae 487: 357-360.INOUE-NAGATA AK; MARTIN D; BOITEUXLS; GIORDANO LB; BEZERRA IC; ÁVILAAC. Evidence for new viral species emergencevia recombination among isolates of the Braziliantomato-infecting Begomovirus complex. Pesqui-sa Agropecuária Brasileira, no prelo.KASRAWI MA; SUWWAN MA; MANSOUR A.1988. Sources of resistance to tomato yellowleaf curl virus in Lycopersicon species.Euphytica 37: 61-64.MATYIS JC; SILVA DM; OLIVEIRA AR; COS-TA AS. 1975. Purificação e morfologia do ví-rus do mosaico dourado do tomateiro. SummaPhytopathologica 1: 267-275.MICHELSON I; ZAMIR D; CHOSNEK H. 1994.Accumulation and translocation of TomatoYellow Leaf Curl Virus (TYLCV) in aLycopersicon esculentum breeding linecontaining the L. chilense TYLCV tolerancegene Ty-1. Phytopathology 34: 928-933.PICÓ B; JOSÉ DÍEZ MA; NUEZ F. 1996. Viraldiseases causing the greatest economic lossesto the tomato crop. II. The tomato yellow leafcurl virus – a review. Scientia Horticulturae67: 151-196.PICÓ B; JOSÉ DÍEZ MA; NUEZ F. 1998.Evaluation of whitefly-mediated inoculationtechniques to screen Lycopersicon esculentumand wild relatives for resistance to Tomatoyellow leaf curl virus. Euphytica 101: 259-271.PILOWSKY M; COHEN S. 1974. Inheritance ofresistance to tomato yellow leaf curl virus.Phytopathology 64: 632-635.SANTANA FM, RIBEIRO SG; MOREIRA JUNIORD; GIORDANO LB. 1998. Evaluation oftomatoes with resistance to a bipartite begomovirusfrom the Federal District and Pernambuco. inCongresso Latino Americano e do Caribe sobreMosca-branca e Geminivírus, Recife, PE.SANTANA FM; RIBEIRO SG; MOITA AW;MOREIRA JUNIOR DJ; GIORDANO LB.2001. Sources of resistance in Lycopersicon spp.to a bipartite whitefly-transmitted geminivirusfrom Brazil. Euphytica 122: 45-51.SANTOS CDG; AVILA AC; INOUE-NAGATAAK; RESENDE RO. 2004. Plant species hostsof begomovirus obtained from tomato in Goiásand Federal District of Brazil. Fitopatologia.Brasileira 29: 450-455.SCOTT JW; STEVENS MR; BARTEN JHM;THOME CR; POLSTON JE; SCHUSTER DJ;SERRA CA. 1996. Introgression of resistanceto whitefly-transmitted begomoviruses fromLycopersicon chilense to tomato. In: DGERLING RT. Mayer (Ed.). Bemisia: 1995,taxonomy, biology, damage, control andmanagement. Andover: Intercept, p.357-377.alternative methods for theimprovement of whitefly mediatedinoculation during the screeningprocedure. The PP tube cage withinfected leaves was a very effectivemethod for obtaining viruliferouswhiteflies. The leaf cage allows theutilization of only three viruliferouswhiteflies per plant for proceeding withthe inoculation process. It is alsoworthwhile to observe that the protocoltakes a very short time to prepare theviruliferous whiteflies. In addition, thisprocedure makes inoculation pressuremuch more uniform and the results morereliable. This inoculation procedureproved to be an efficient and easymethod, and demanding a smallnumbers of whiteflies individuals.Furthermore, it turned out to be a goodtool to study the mechanism ofbegomovirus resistance on tomato..ACKNOWLEDGEMENTSWe are grateful to Élcio Vieira daSilva Lopes (in memorian) for histechnical assistance.REFERENCESCOSTA AS. 1974. Moléstias do tomateiro trans-mitidos por mosca branca Bemisia tabaci.Fitopatologia (Lima) 9: 47.COSTA CL; CHOUDHURY MM; KITAJIMA EW;SILVA NR. 1987. Doença do tipo “curl” consta-tada em tomatais da região de Petrolina, emPernambuco. Fitopatologia Brasileira 12: 155.FLORES E; SILBERSCHMIDT K; KRAMER M.1960. Observações de “clorose infecciosa” dasmalváceas em tomateiros do campo. O Bioló-gico 26: 65-69.

A reliable begomovírus inoculation method for screening Lycopersicon esculentum lines.

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