ELISA detection of Apple chlorotic leafspot virus (ACLSV) and Apple mosaic virus (ApMV) in comparison to RT-PCR detection and the determination of genetic variation of these virus species in South Africa

Thesis (MScAgric)--Stellenbosch University, 2014.

ENGLISH ABSTRACT: Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV) are responsible for reduced yield in the South African deciduous fruit industry. These two diseases are regulated by the South African Deciduous Fruit Plant Certification Scheme whereby no trees infected with these viruses are permitted for plantings. Currently the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) is prescribed as the test method for routine detection of these viruses in plant material. In the first part of this study, detection limits of DAS-ELISA and reverse transcriptase polymerase chain reaction (RT-PCR) for ApMV and ACLSV were compared. The RT-PCR was found to be 70.5 and 71 fold more sensitive than DAS-ELISA in the detection of ACLSV and ApMV, respectively. No ApMV isolates were detected by DAS-ELISA in pears, but ten isolates were detected by RT-PCR. This is of major concern as ApMV tests are not prescribed by the South African Deciduous Fruit Certification Scheme for pears, as it was not considered a host of ApMV and no ApMV symptoms have been observed. In the second part of this study, the genetic variation of ApMV and ACLSV isolates from South Africa was investigated. Extracted RNA was used for RT-PCR of the coat protein genes which were then sequenced. Phylogenetic trees were constructed using these sequences as well as reference sequences from GenBank. Phylogenetic analysis revealed that South African isolates of ACLSV were similar to isolates from the rest of the world, grouping into 3 of 4 possible clades, and that the majority of isolates are not restricted to a particular fruit group. This indicates that cross-infection between pome- and stonefruit is possible. Certain isolates of ApMV were detected by RT-PCR, but not detected by DAS-ELISA in apples and peach. It is also concluded that ACLSV was imported from various regions of the world, since similarity with a number of different overseas isolates was found. Phylogenetic analysis of the coat protein gene sequences of ApMV isolates indicated that two major groups occur in South Africa. Phylogenetic analysis also revealed that South African isolates in individual clades are not restricted to a single fruit group, which indicates the risk of cross-infection.

AFRIKAANSE OPSOMMING: Apple chlorotic leaf spot virus (ACLSV) en Apple mosaic virus (ApMV) is verantwoordlelik vir verlaagde opbrengs in die Suid-Afrikaanse sagtevrugte industrie. Dit word gereguleer deur die Suid-Afrikaanse Sagtevrugte Plant Sertifiseringsskema waar geen aanplantings wat met hierdie virusse besmet is, toegelaat word nie. Tans word DAS-ELISA voorgeskryf as die toetsmetode waarmee roetine opsporing van hierdie viruses in plant material moet geskied. In die eerste deel van hierdie studie, is die opsporingsdrempelgrense van DAS-ELISA en RT-PKR vir ApMV en ACLSV bepaal. Die RT-PKR tegniek was 70.5 en 71 keer meer sensitief as DAS-ELISA in die opsporing van ACLSV en ApMV onderskeidelik. Geen ApMV isolate is by pere deur DAS-ELISA opgespoor nie, terwyl 10 sulke isolate deur RT-PKR opgespoor is. Dit is ’n groot bekommernis, aangesien ApMV tans nie voorgeskryf word vir toetsing by pere deur die Suid-Afrikaanse Sagtevrugte Plant Verbeteringsskema nie, aangesien peer nie as gasheer van ApMV beskou word nie. In die tweede gedeelte van hierdie studie, is die genetiese variasie van Suid-Afrikaanse ApMV en ACLSV isolate ondersoek. RNA wat geїsoleer is, is vir RT-PKR van die mantelproteïen geen gebruik en die nukleotied volgorde is vervolgens bepaal. Filogenetiese bome is getrek vanaf hierdie Suid-Afrikaanse nukleotied volgordes asook verwysings isolate in GenBank. Die filogenetiese analises het getoon dat Suid-Afrikaanse ACLSV isolate in 3 van 4 moontlike klades groepeer en dat die meerderheid van Suid-Afrikaanse ACLSV isolate nie beperk is tot bepaalde vruggroepe nie. Dit dui daarop dat kruis-infeksie tussen kern- en steenvrugte ’n moontlikheid is. Sekere isolate in opgespoor in appel en perske, maar dit is nie opgespoor deur DAS-ELISA nie. Uit die resultate kon ook afgelei word dat ACLSV vanaf verskeie lande ingevoer is, aangesien sterk ooreenkomste met ’n aantal verskillende buitelandse isolate gevind is. Filogenetiese analises van die mantelproteïen volgorde van ApMV isolate het aangetoon dat twee hoof groepe in Suid Afrika voorkom. Uit die ontledings word afgelei dat ApMV isolate binne individuele klades ook nie beperk is tot ’n bepaalde vruggroep nie wat die moontlike risiko van kruis-besmetting aandui.

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