Catiţa Plopa 1, Silvia Preda 2. Abstract

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1 TESTAREA BIOLOGICA SI SEROLOGICA A CELOR MAI IMPORTANTE VIRUSURI LA PRUN VIRAL TESTING USING BIOLOGICAL AND SEROLOGICAL ASSAY FOR MOST IMPORTANT VIRUSES TO PLUM Catiţa Plopa 1, Silvia Preda 2 1 Research Institute for Fruit Growing Pitesti, Romania 2 University of Craiova Research Station for Fruit Growing Vâlcea, Romania Abstract Establishing an accurate diagnosis in terms of viral for propagation of fruit tree is very important, it represents the most effective method of protection against viruses. Based on these considerations the primary objective of this study is to detect viruses with the highest incidence in plum by biological and ELISA serological methods, to a number of 85 samples taken from 17 varieties. Serologic testing on DAS-ELISA diagnosed 3 positive samples to Plum pox virus (PPV), 2 positives sample to Prunus necrotic ring spot virus (PNRSV) and one positive sample to Prune dwarf virus (PDV). There were not positive samples to Apple chlorotic leaf spot virus (ACLSV). The tests conducted on woody indicator plants by grafting on protect conditions and after 3-24 months assured of diagnosis for PPV, PDV, PNRSV and ACLSV viruses. The biological indicators: GF 305, Tuleu dulce and Vânăt de Italia, have shown symptoms for PNRSV for two samples. On biological indicator Vânăt de Italia and Tuleu dulce not appeared symptoms for Centenar variety tested for PPV, although the symptoms were obvious on GF 305 indicator, but viral infection was confirmed by ELISA test. Symptoms that indicate the presence of PDV occurred by Vânăt de Italia biological indicator. Cuvinte cheie: prun, virusuri, testare, seologic, biologic Keywords: plum, viruses, tested, serological, biological 1. Introduction In Romania, the most damaging viruses to plum species are represented by Plum pox potyvirus (PPV), Prune dwarf ilarvirus (PDV), Prunus necrotic ring spot ilarvirus (PNRSV) and Apple chlorotic leaf plum trichovirus spot (ACLSV). All four viruses are part of the list of quarantine organisms EPPO (EPPO / EPPO, 1990, , 2004). Németh (1994), Kegler and Hartmann (1998) and Nemchinov et.al. (1998), Waterworth and Hadidi (1998) analyzed the importance of PPV, regarding the European production of fruit. Infection with the virus can lead to yield losses ranging up to 83% and even 100% in highly susceptible varieties. Following the same authors, PDV can reduce the production with up to 50-82% and PNRSV may reduce the production if varieties are susceptible with 15-45%. Regarding the diagnosis, because sometimes distribution of viruses is irregular in the tree, visual inspection does not always detect virus requiring mandatory testing by different methods. Testing biological indicators according to established methodologies (OEPP 1983; Damsteegt et al, 1997), may lead to a diagnosis for evidence of viruses, but the reliability of the results of serological tests DAS / TAS ELISA are widely used to confirm the presence of viruses. 2. Material and methods The range used was provided from plantations RSFG Valcea and RIFG Pitesti. The varieties studied were represented by: Silvia, Diana 50, Pescăruș, d Agen, Ialomita, Stanley, Tuleu gras, Minerva, Carpatin, Centenar, Anna Spath, Mirobolan BN 4 Kr, Vinete românesti, Tita, Valcean, Sarmatic, H 9 / 24. Age of trees ranged from 5-10 years. Of each variety were evaluated by 5 trees. The choice of indicators was made according to their sensitivity (intensity of symptoms), the type of response (local and systemic infection) and incubation period (Fulton, RW, 1970). Indicators were used - plum and peach varieties susceptible Tuleu dulce, Vânăt de Italia, GF 305 (photo 1). The serological test was realized using two methods: double antibody sandwich DAS-ELISA, according to Clark and Adams (1977) using commercially available kit supplied by Bioreba and following manufacturer`s protocol and by triple antibody sandwich TAS - ELISA methods, according to Cambra et. al. (1994), using commercially available kit supplied by Sediag. Substrate p-nitro phenyl phosphate (1 mg/ml) was added to plates and reaction were reading after half and hour at room temperature. Optical densities (OD) were recorded at 405 nm and readings were done with a Bio-Rad plate reader PR

2 3. Results Transmission of viruses that give systemic infection by contacting the tissue leading to two partners, was achieved in optimal conditions by double grafting and massive eyes. The rootstock Mirobalan or peach seedlings GF-503 was grafted a bud of the variety or rootstock of the plant tested and one indicator. Assortment of plum varieties and rootstocks consists of 17 reacted differently Tuleu dulce indicators, GF-305, Vanat de Italia, according to the presence of viral pathogen (Table 1). Assessments made after a reaction time of 3-24 months, the degree of infection with viruses PPV, PDV, PNRSV, ACLSV resulted that plum varieties: Diana 50, Pescăruș, Carpatin, Anna Späth, Ialomița, Agent, Mirobolan BN-4Kr, Vinete românesti, Tita, Sarmatic and H9/24 had a negative reaction (-). The plants tested were found healthy viruses. Reacted positively (+) against the virus PNRSV plum cultivars Stanley and Valcean viruses causing symptoms on indicators P.persica GF-305 and P. domestica cv. Tuleu dulce. PDV virus was found to Silvia variety, showing symptoms of the indicator Vânăt de Italia (photo 2). PPV virus manifested for Tuleu dulce and Minerva varieties, on all use indicators. For Centenar variety PPV infection not manifested than GF-305 indicator. Virus detection using DAS/TAS-ELISA technique was carried out in June-July, when the concentration of virus in plants is maximized. Results of the serological tests shown by the variability of the gradation levels in the case of infection ELISA, amount of viral protein present in each plant tested. We conducted 170 tests of varieties and rootstocks studied (2 tests/sample), highlight viruses PPV, PDV, PNRSV, ACLSV. Reacted positively to the PPV the varieties Minerva, Tuleu gras and Centenar. DAS-ELISA absorption values were 1,693; 1217 and 1398; 1124 compared with healthy control value 0,441- negative reaction (table 2). PDV virus was revealed to Silvia variety which was a DAS-ELISA value of 1.246, compared to 0,440 healthy control values (table 2). Stanley and Vâlcean plum varieties have been shown PNRSV infected samples from the extinction values of DAS-ELISA, ranged from to compared to for the negative control (table 2). ACLSV virus was not identified in the samples analyzed, as evidenced by the negative reaction resulting from testing with ELISA values below the infection. Results of virus identification obtained by DAS-ELISA were confirmed by TAS-ELISA test (table 3). Doing an analysis in terms of the degree of infection and the presence or absence of plum viruses found that out of a total of 85 samples were found positive 7 samples (figures 1 and 2), with the highest incidence being PPV (4 positive samples), two positive samples PNRSV, PDV -1 positive sample. Diagnosis performed revealed no evidence infected with ACLSV. 4. Conclusions The characterization effectuated on different indicators plants not show exactly results, and is necessary that this results to be confirmed by serological reaction. The results show that both serological methods are sensitive for these tests. References 1. Cambra, M., Asensio, M., Gorris, M.T., Pérez, E., Camarasa, E., García, J.A., Moya, J.J., López- Abella, D., Vela, C., Sanz, A., Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins. EPPO Bull. 24, Clark, M.F., Adams, M.F., Characteristics of microplate method of Enzyme linked immunosorbent assay for the detection of plant viruses. Journal of generally virology 34: Damsteegt, V.D., Waterworth, H.E., Mink, G.I., Howell, W.E, Levy L., Prunus tomentosa as a diagnostic host for detection of Plum pox virus and other Prunus viruses. Plant Disease, 81(4): ; 12 ref. 4. Kegler, H., Hartmann, W., Present status of controlling conventional strains of Plum pox virus. In: Hadidi A, Khetarpal RK, Koganezawa H, eds. Plant Virus Disease Control. St Paul, Minnesota, USA: APS Press, OEPP/EPPO, Data sheets on quarantine organisms. No 96, Plum pox virus. Bulletin OEPP/EPPO 13 (1). 6. OEPP/EPPO, Specific quarantine requirements. EPPO Technical Documents, No Paris, France: EPPO. 7. OEPP/EPPO, 1991/1992. Certification schemes. Virus-free or virus-tested fruit trees and rootstocks. Bulletin OEPP/EPPO Bulletin, 21: ; 22:

3 8. OEPP/EPPO, Standard PM 7/32 Plum pox potyvirus. Bulletin OEPP/EPPO Bulletin, 34: Nemeth, M., History and importance of Plum pox in stone-fruit production. Bulletin OEPP, 24(3): Nemchinov, L., Crescenzi, A., Hadidi, A., Piazzolla, P., Verderevskaya, T., Present status of the new cherry subgroup of Plum pox virus (PPV-C). In: Hadidi A, Khetarpal RK, Koganezawa H, eds. Plant Virus Disease Control. St Paul, Minnesota, USA: APS Press, Waterworth, H.E., Hadidi, A., Economic losses due to plant viruses. In: Hadidi A, Khetarpal RK, Koganezawa H, eds. Plant Virus Disease Control. St Paul, Minnesota, USA: APS Press, Tables and figures Table 1. Bioassay results for different viruses No crt Genotypes Samples Reaction on indicators: Tuleu dulce Vânăt de Italia GF PNRSV PPV PNRSV PPV PDV PNRSV PPV ACLSV 1. Minerva Pr. 45 P Pr. 46 P Pr. 47 P Pr. 48 P Pr. 49 P Diana 50 Pr. 50 P Pr. 52 P Pr. 53 P Pr. 54 P Pr. 55 P Tuleu gras Pr. 56 P Pr. 61 P Pr. 62 P Pr. 63 P Pr. 64 P d Agen Pr. 65 P Pr. 66 P Pr. 67 P Pr. 68 P Pr. 69 P Carpatin Pr. 70 P Pr.112R15P Pr.113R15P Pr. 114 R3P Pr. 115 R3P Centenar Pr. 12 R1P Pr. 13 R1P Pr. 15 R5P Pr. 16 R5P Pr. 80 P Anna Späth Pr. 81 P Pr. 82 P Pr. 83 P Pr. 84 P Pr. 85 P Vâlcean Pr. 86 P Pr. 87 P Pr. 88 P Pr. 89 P Pr. 90 P Vinete românesti Pr. 91 P Pr. 92 P Pr. 93 P

4 44. Pr. 94 P Pr. 95 P Stanley Pr. 96 P Pr. 97 P Pr. 98 P Pr. 99 P Pr. 100 P Pescăruş Pr. 31 P Pr. 32 P Pr. 33 P Pr. 34 P Pr. 35 P Silvia Pr. 36 P Pr. 37 P Pr. 38 P Pr. 39 P Pr. 1 P Ialomița Pr. 71 P Pr. 72 P Pr. 73 P Pr. 74 P Pr. 75 P Sarmatic Pr.76P Pr. 77 P Pr. 78 P Pr. 79 P Pr. 101 P Mirobolan 4Kr Pr. 102 P Pr. 2 P Pr. 3 P Pr. 4 P Pr. 5 P H9/24 Pr. 6 P Pr. 7 P Pr. 8 P Pr. 9 P Pr. 103 P Tita Pr. 104 P Pr. 105 P Pr. 106 P Pr. 107 P Pr. 108 P

5 Table 2. Detection of PPV, PDV, PNRSV and ACLSV using DAS-ELISA serological test No. Genotypes Samples ELISA level for viruses tested: crt. PPV Semnification PDV Semnification PNRSV Semnification ACLSV Semnification 1. Minerva Pr. 45 P11 1,693 positive 0,486 negative 0,389 negative 0,485 negative 2. Pr. 46 P35 0,416 negative 0,437 negative 0,385 negative 0,450 negative 3. Pr. 47 P13 0,416 negative 0,460 negative 0,414 negative 0,474 negative 4. Pr. 48 P16 0,403 negative 0,436 negative 0,395 negative 0,459 negative 5. Pr. 49 P18 0,399 negative 0,446 negative 0,401 negative 0,474 negative 6. Diana 50 Pr. 50 P17 0,050 negative 0,428 negative 0,393 negative 0,449 negative 7. Pr. 52 P10 0,425 negative 0,458 negative 0,401 negative 0,493 negative 8. Pr. 53 P12 0,411 negative 0,450 negative 0,402 negative 0,456 negative 9. Pr. 54 P8 0,444 negative 0,442 negative 0,418 negative 0,484 negative 10. Pr. 55 P1 0,422 negative 0,446 negative 0,394 negative 0,464 negative 11. Tuleu gras Pr. 56 P2 1,217 positive 0,487 negative 0,407 negative 0,490 negative 12. Pr. 61 P14 0,404 negative 0,452 negative 0,393 negative 0,451 negative 13. Pr. 62 P4 1,398 positive 0,456 negative 0,389 negative 0,484 negative 14. Pr. 63 P31 0,430 negative 0,463 negative 0,388 negative 0,476 negative 15. Pr. 64 P33 0,417 negative 0,446 negative 0,410 negative 0,480 negative 16. Agen Pr. 65 P32 0,060 negative 0,447 negative 0,501 negative 0,481 negative 17. Pr. 66 P24 0,423 negative 0,484 negative 0,474 negative 0,491 negative 18. Pr. 67 P21 0,417 negative 0,454 negative 0,497 negative 0,483 negative 19. Pr. 68 P34 0,411 negative 0,448 negative 0,473 negative 0,489 negative 20. Pr. 69 P27 0,405 negative 0,449 negative 0,484 negative 0,492 negative 21. Carpatin Pr. 70 P23 0,023 negative 0,470 negative 0,449 negative 0,488 negative 22. Pr.112R15P15 0,421 negative 0,464 negative 0,495 negative 0,468 negative 23. Pr.113R15P15 0,387 negative 0,466 negative 0,476 negative 0,498 negative 24. Pr.114R3P21 I 0,409 negative 0,464 negative 0,492 negative 0,479 negative 25. Pr.115R3P21 I 0,395 negative 0,446 negative 0,475 negative 0,505 negative 26. Centenar Pr. 12 R1P1 1,124 positive 0,459 negative 0,484 negative 0,494 negative 27. Pr. 13 R1P1 0,401 negative 0,447 negative 0,410 negative 0,489 negative 28. Pr. 15 R5P3 0,386 negative 0,441 negative 0,496 negative 0,503 negative 29. Pr. 16 R5P3 0,407 negative 0,451 negative 0,478 negative 0,480 negative 30. Pr. 80 P22 0,389 negative 0,447 negative 0,499 negative 0,460 negative 31. Anna Späth Pr. 81 P4 0,051 negative 0,435 negative 0,476 negative 0,490 negative 32. Pr. 82 P12 0,405 negative 0,469 negative 0,481 negative 0,488 negative 33. Pr. 83 P7 0,391 negative 0,458 negative 0,428 negative 0,475 negative 34. Pr. 84 P9 0,394 negative 0,434 negative 0,532 negative 0,462 negative 35. Pr. 85 P3 0,391 negative 0,456 negative 0,481 negative 0,479 negative 76

6 36. Vâlcean Pr. 86 P2 0,066 negative 0,445 negative 1,308 positive 0,465 negative 37. Pr. 87 P1 0,404 negative 0,443 negative 0,477 negative 0,484 negative 38. Pr. 88 P6 0,412 negative 0,471 negative 0,483 negative 0,493 negative 39. Pr. 89 P5 0,398 negative 0,463 negative 0,432 negative 0,473 negative 40. Pr. 90 P14 0,393 negative 0,459 negative 0,494 negative 0,487 negative 41. Vinete românesti Pr. 91 P10 0,068 negative 0,461 negative 0,532 negative 0,480 negative 42. Pr. 92 P32 0,390 negative 0,434 negative 0,481 negative 0,471 negative 43. Pr. 93 P20 0,489 negative 0,465 negative 0,483 negative 0,485 negative 44. Pr. 94 P35 0,416 negative 0,446 negative 0,477 negative 0,507 negative 45. Pr. 95 P34 0,440 negative 0,478 negative 0,483 negative 0,496 negative 46. Stanley Pr. 96 P31 0,025 negative 0,498 negative 1,325 positive 0,501 negative 47. Pr. 97 P11 0,459 negative 0,455 negative 0,494 negative 0,464 negative 48. Pr. 98 P30 0,452 negative 0,400 negative 0,481 negative 0,487 negative 49. Pr. 99 P14 0,437 negative 0,420 negative 0,493 negative 0,500 negative 50. Pr. 100 P19 0,455 negative 0,383 negative 0,483 negative 0,483 negative 51. Pescăruş Pr. 31 P14 0,023 negative 0,411 negative 0,489 negative 0,489 negative 52. Pr. 32 P4 0,430 negative 0,386 negative 0,434 negative 0,492 negative 53. Pr. 33 P31 0,425 negative 0,395 negative 0,520 negative 0,488 negative 54. Pr. 34 P33 0,453 negative 0,382 negative 0,486 negative 0,468 negative 55. Pr. 35 P32 0,446 negative 0,406 negative 0,507 negative 0,498 negative 56. Silvia Pr. 36 P24 0,027 negative 1,246 positive 0,496 negative 0,479 negative 57. Pr. 37 P21 0,429 negative 0,411 negative 0,513 negative 0,505 negative 58. Pr. 38 P34 0,410 negative 0,383 negative 0,517 negative 0,494 negative 59. Pr. 39 P27 0,423 negative 0,390 negative 0,527 negative 0,489 negative 60. Pr. 1 P23 0,453 negative 0,372 negative 0,511 negative 0,503 negative 61. Ialomița Pr. 71 P25 0,027 negative 0,408 negative 0,518 negative 0,479 negative 62. Pr. 72 P19 0,393 negative 0,386 negative 0,462 negative 0,480 negative 63. Pr. 73 P22 0,068 negative 0,390 negative 0,538 negative 0,460 negative 64. Pr. 74 P20 0,390 negative 0,384 negative 0,524 negative 0,490 negative 65. Pr. 75 P26 0,489 negative 0,381 negative 0,510 negative 0,488 negative 66. Sarmatic Pr. 76 P28 0,053 negative 0,382 negative 0,513 negative 0,475 negative 67. Pr. 77 P29 0,404 negative 0,415 negative 0,517 negative 0,462 negative 68. Pr. 78 P30 0,412 negative 0,391 negative 0,527 negative 0,479 negative 69. Pr. 79 P5 0,398 negative 0,403 negative 0,511 negative 0,465 negative 70. Pr. 101 P22 0,393 negative 0,387 negative 0,518 negative 0,484 negative 71. Mirobolan 4Kr Pr. 102 P4 0,061 negative 0,394 negative 0,462 negative 0,493 negative 72. Pr. 2 P12 0,390 negative 0,403 negative 0,538 negative 0,473 negative 73. Pr. 3 P7 0,489 negative 0,389 negative 0,524 negative 0,487 negative 74. Pr. 4 P9 0,416 negative 0,389 negative 0,510 negative 0,480 negative 75. Pr. 5 P3 0,440 negative 0,385 negative 0,456 negative 0,471 negative 77

7 76. H9/24 Pr. 6 P2 0,022 negative 0,414 negative 0,500 negative 0,485 negative 77. Pr. 7 P1 0,405 negative 0,395 negative 0,498 negative 0,507 negative 78. Pr. 8 P6 0,391 negative 0,401 negative 0,488 negative 0,496 negative 79. Pr. 9 P5 0,394 negative 0,393 negative 0,456 negative 0,501 negative 80. Pr. 103 P14 0,391 negative 0,401 negative 0,500 negative 0,464 negative 81. Tita Pr. 104 P10 0,027 negative 0,402 negative 0,498 negative 0,487 negative 82. Pr. 105 P32 0,404 negative 0,418 negative 0,488 negative 0,500 negative 83. Pr. 106 P20 0,412 negative 0,394 negative 0,543 negative 0,464 negative 84. Pr. 107 P27 0,398 negative 0,407 negative 0,503 negative 0,488 negative 85. Pr. 108 P28 0,393 negative 0,393 negative 0,523 negative 0,475 negative 86. Positive control >3,014 positive >3,000 positive 3,030 positive >3,000 positive 87. Negative control 0,441 negative 0,440 negative 0,487 negative 0,570 negative 78

8 Table 3. Viruses identified and detection using different methods for tested Specification No of samples/no Method/Virus identification positives samples Silvia 5/1 a 2 /PDV;b/PDV;c/PDV Diana 50 5/0 - Pescăruș 5/0 - Agen 5/0 - Ialomița 5/0 - Stanley 5/1 a 1;2;3 /PNRSV;b/PNRSV;c/PNRSV Tuleu gras 5/2 a 1;2;3 /PPV;b/PPV;c/PPV Minerva 5/1 - Carpatin 5/0 - Centenar 5/1 a 3 /PPV;b/PPV;c/PPV Anna Spath 5/0 - Mirobolan BN 4 Kr 5/0 - Vinete românesti 5/0 - Tita 5/0 - Vâlcean 5/1 a 1;2;3 /PNRSV;b/PNRSV;c/PNRSV Sarmatic 5/0 - H 9/24 5/0 - a) virus identification by bioassay 1) Tuleu dulce 2) Vânăt de Italia 3) GF 305 b) virus identification by DAS-ELISA c) virus identification by TAS-ELISA Photo 1. Biological testing on GF 305 indicator Photo 2. Prune dwarf virus identification to Silvia variety 79

9 Fig. 1. The percentage of positive samples detected Fig. 2. Share viruses in positive samples 80

10