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Detection of Citrus Tristeza Virus by Print Capture and Squash Capture-PCR in Plant Tissues and Single Aphids

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Detection of Citrus Tristeza Virus by Print Capture and Squash Capture-PCR in Plant Tissues and Single Aphids Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus Detection of Citrus Tristeza Virus by Print Capture and Squash Capture-PCR in Plant Tissues and Single Aphids M. Cambra, A. Olmos, M. T. Gorris, C. Marroquín, O. Esteban, S. M. Garnsey, R. Llauger, L. Batista, I. Peña, and A. Hermoso de Mendoza ABSTRACT. Two sensitive methods using immobilized targets on membranes (hemi-nested- PCR and nested-PCR in a single closed tube) were applied successfully for amplification of citrus tristeza virus (CTV) targets from plant material (print capture) and aphids (squash capture). Amplification was obtained from different aphid species regardless of transmission efficiency showing that CTV is acquired by different aphid species. The use of preprinted or presquashed targets offers a new and sensitive diagnostic tool for CTV detection for many applications includ- ing epidemiological studies. Index words. Citrus tristeza virus, detection, aphids, Toxoptera citricida, Aphis gossypii, Aphis nerii, Hyalopterus pruni, squash capture-PCR, print capture-PCR, hemi-nested-PCR, nested-PCR. Citrus tristeza virus (CTV) is one The use of certified pathogen-free of the most detrimental virus diseas- plants and epidemiological studies es of citrus (2), especially where CTV- to track the incidence and spread of sensitive rootstocks are used. CTV is CTV in the field are the two strate- vectored by a number of aphid spe- gies to manage the tristeza problem. cies (Homoptera, Aphididae) in a Accurate epidemiological data al- noncirculative, semi-persistent man- lows the proper recommendation to ner (19). Seven aphid species have be given to a grower on what to do to been determined as CTV vectors in control CTV such as the use of se- different geographical areas (13). lected varieties grafted on tristeza- Although the transmission effi- tolerant rootstocks produced under ciency of CTV by Toxoptera citricida a certification scheme. is high (23), Aphis gossypii has also Sensitive and reliable detection proved to be an efficient vector of of CTV in plants and vectors is es- the virus in California, Israel and sential for suitable control of the vi- Spain (1, 6, 11, 13). High popula- rus and better understanding of the tions of A. gossypii in some Spanish relationships between CTV and its areas were responsible for spread of insect vectors. ELISA has been suc- the tristeza disease (6). The non-ef- cessfully applied for the detection of ficient vectors, Toxoptera aurantii CTV in plant material (12) and in and Aphis spiraecola (13), along aphids (7, 8). Nevertheless, in the with uncontrolled movement of in- latter case, positive reactions were fected plant materials, were impli- obtained only when extracts were cated in the CTV-induced death of prepared from multiple aphids (more about 20 million trees in Spain from than 20). The low titer of semipersis- 1930-35 to 1989. The recent CTV ep- tent transmitted viruses in aphids idemic from 1989 to present is due make their serological detection in to spread by A. gossypii and has single individuals difficult. caused the decline and death of an Recently, detection of CTV tar- additional 20 million trees. Other gets in a single aphid has been ob- aphids such as Aphis craccivora, tained using conventional reverse- Uroleucon jaceae, and Myzus persi- transcription polymerase chain re- cae have been described in India as action (RT-PCR) (14) and a variation CTV vectors (20, 21). called print-, spot-, or squash-cap- 42 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus 43 ture PCR (PC- or SC-PCR) (10). field were collected in individual These methods use sensitive and plastic bags and ground in PBS, pH simple procedures developed for the 7.2 to 7.4, supplemented with 0.2% preparation of plant and insect sam- DIECA (1/20, w/v). Sections of these ples before PCR amplification with- plant materials were also directly out the need of extract preparation blotted on Whatman 3MM paper (17, 18). This method essentially us- (18). Ten imprints from each plant es imprinted sections of plant ma- were used (two imprints per stem terial or squashed aphids on What- section) on about 1 cm2 paper, using man 3MM paper (10). The immobi- both sides of the paper when need- lized or captured targets are re- ed. The printed papers prepared in leased from the blotted paper with a Valencia were stored at 4°C in plas- simple Triton treatment (18). tic bags for as long as 1 mo before In this study, the PC- or SC-PCR being processed. The prints made in method, previously applied to other Cuba or the Dominican Republic were RNA and DNA viruses (9, 10, 15, 17, sent to Valencia by international 18), are coupled to the new tech- courier at ambient temperature niques of hemi-nested-PCR and and stored at 4°C until processed. nested-PCR in a single closed tube, To estimate the limits of sensitiv- allowing sensitive amplification of ity of the print-capture method, an CTV targets from infected plants or experiment was performed imprint- aphid tissues directly from the blots ing the paper with a sectioned stem on paper without any need of nucle- collected from a sweet orange tree ic acid extraction or immunocapture infected isolate T-308 in Moncada phase. along with imprints of zero, four, nine, 14 or 19 imprints from a MATERIAL AND METHODS healthy tree. Preparation of aphids squash- Virus sources and plant mate- es. Aphids collected from field trees rial preparation. Twenty three or fed on CTV-infected hosts under characterized Spanish CTV isolates experimental conditions were indi- from the IVIA collection, main- vidually squashed on Whatman tained in an insect-proof screen- 3MM paper. The round bottom of house in sweet orange grafted on Eppendorf tubes was used like a Troyer citrange in Moncada (Valen- pestle to crush each aphid (17). cia), and the Cuban CTV isolate, Seventy-nine T. citricida apterae Cu50, from the IICF collection were individually collected and maintained in sweet orange in a squashed in June 1996 from an es- greenhouse in La Habana, were tablished colony on a CTV-infected used in this study. In addition, sweet orange tree in the Dominican shoots from sweet orange trees in- Republic. Imprints of sections of fected with CTV isolate I.7 from shoots from which the aphids were CTV epidemiology plots at IVIA, captured were simultaneously test- Moncada and naturally-infected trees ed. Healthy controls were prepared from the Dominican Republic were by crushing single T. citricida fed on used. In addition, 12 Washington a CTV-free sweet orange tree. The Navel sweet orange trees were sur- paper membranes were mailed to veyed (five stems/tree) in Moncada Valencia at room temperature and in November. CTV negative controls stored at 4°C until used. The analy- from different citrus species were sis of the squashed aphids was per- kindly provided by Dr. L. Navarro formed in three different periods: from the pathogen-free germplasm September 1996; April 1997; and collection maintained at IVIA. April 1998. Samples from healthy and CTV- Individual T. citricida and A. gos- infected plants from the collection or sypii were collected from a colony on 44 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus an infected sweet orange tree (iso- ly some tubes were coated with late Cu50) and a non-infected sweet plum pox virus (PPV) specific MCA orange tree in Cuba. The squashed 5B-IVIA (3) at 1 µg/ml, with carbon- aphids were sent to IVIA by conven- ate buffer alone, and other tubes re- tional mail and analyzed about 1 mo ceived no treatment. later. Amplification was made by con- A. gossypii grown on cotton in a ventional one-step RT-PCR using growth chamber were individually CTV-specific primers: PT1: 5’ GGT transferred to detached young TCA CGC ATA CGT TAA GCC TCA shoots of a CTV infected Washington CTT and PT2: 5’ TAT CAC TAG Navel tree (I.7) maintained in water. ACA ATA ACC GGA TGG GTA. The young growing shoots with Briefly, 25 µl of RT-PCR mix (10 mM aphids were caged in 3 × 8 cm plastic Tris-HCl pH 8.8, 50 mM KCl, 1.5 µ cylinders. After a 5 h virus acquisi- mM MgCl2, 0.3% Triton X100, 1 M tion feeding period, the aphids were of each primer, 250 µM dNTPs, collected with a brush, placed on the 0.25U AMV and 0.5U TaqPol) was membrane and squashed. The same added to each tube. cDNA synthesis operation was performed with Aphis was performed at 42°C for 45 min nerii and Hyalopterus pruni, previ- followed by a denaturation step of ously captured in Nerium oleander 94°C for 2 min. The amplification and Prunus armeniaca, respectively. process consisted of 40 cycles of Some individuals of each of the three 92°C for 30 sec, 60°C for 30 sec and aphid species were fed for 5 h on 72°C for 1 min. Ten µl of PCR prod- young shoots of a healthy sweet or- ucts were analysed by electrophore- ange seedling cv. Pineapple as nega- sis on 3% agarose gels, stained by tive controls. ethidium bromide and visualized Apterae of T. citricida (from Cu- under UV transilluminator. In some ba) and A. gossypii (from Spain) cases, the preliminary capture were fed on sweet orange CTV in- phase of IC-PCR was also used to fected (Cu50 and T-397, respective- prepare samples before amplifica- ly) shoots for several acquisition tion using the hemi-nested-PCR or times ranging from 15 min to 48 h. nested-PCR in a single closed-tube Ten individuals per acquisition peri- procedures. od were tested. T. citricida were Print and Squash capture squashed and the blots mailed to IV- preparation of samples before IA in Valencia; whereas A.
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