Apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) : detection and isolate characterization in South African pome fruit

Gagiano, Magdalena Christiena (2015-03)

Thesis (MScAgric)--Stellenbosch University, 2015.

Thesis

Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) are known to infect pome fruit in all pome fruit producing regions of the world. In this study, a comparison between double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) for ASGV detection in pome fruit was performed. A total of 15 ASGV positive orchard leaf samples were detected using RT-PCR whilst DAS-ELISA could only detect 13. RT-PCR was found to be at least 126 fold more sensitive than DAS-ELISA. In an assessment of the genetic variation of ASGV in South Africa, the coat protein (CP) gene of isolates was sequenced, aligned and phylogenetically analysed with ASGV CP gene sequences from GenBank. Parsimony analysis identified groups of isolates, but could not resolve the relationships between them. In order to obtain better resolution, whole genome sequences of international ASGV and Citrus tatter leaf virus (CTLV) isolates were aligned with ASGV and CTLV CP gene sequences and phylogenetically analysed with parsimony. South African ASGV isolates grouped into three clades and showed multiple origins and no geographical trend. In an assessment of the genetic variation of ASPV in South Africa, the CP gene sequences of infected samples were aligned with international CP gene sequences obtained from GenBank and phylogenetically analysed using parsimony. Results from the analysis using parsimony revealed low CI and RI values indicating homoplasy in the CP gene data. To address the homoplasy, two additional analyses were performed in which the gene sequences were converted to amino acid sequences and in which the third position of the codon was excluded from the alignment. Both of these approaches resulted in a reduction in homoplasy. In an attempt to further increase the resolution of the phylogeny, the phylogenetic analysis was repeated using maximum likelihood. In the first codon unpartitioned analysis a tree with low support was retrieved followed by, as with the parsimony analysis, an analysis performed on the data translated to amino acid sequences, which showed better resolution and higher clade support. The tree with the highest resolution and clade support was retrieved by codon partitioning into first, second and third positions. South African ASGV isolates grouped into five clades and showed multiple origins and no geographical trend. This study is the first in which ASGV and ASPV have been detected using RT-PCR in South Africa. Dual infections of ASGV and ASPV were recorded in 24.7% of samples analysed. This is the first report of South African pear trees exhibiting symptoms of pear stony pit and fruit deformation associated with ASPV infection.

Apple stem grooving virus (ASGV) en Apple stem pitting virus (ASPV) kom wêreldwyd voor waar kernvrugte geproduseer word. In hierdie studie is ‘n vergelyking tussen die opsporingsgrense van “double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA)” en “reverse transcriptase polymerase chain reaction (RT-PCR)” vir ASGV bepaal. RT-PCR kon ASGV opspoor in 15 ASGV geïnfekteerde kernvrug isolate terwyl die DAS-ELISA slegs ASGV kon opspoor in 13 van die isolate. Daar is gevind dat RT-PCR met ‘n minimum van 126 keer meer sensitief is as DAS-ELISA. In ‘n ondersoek na die genetiese variasie van ASGV in Suid Afrika is die nukleotiedvolgorde van die mantelproteïngeen van isolate bepaal, in lyn geplaas- en filogeneties geanaliseer met volgordes wat van GenBank verkry is. ‘n Parsimony analise het isolaat groepe geïdentifiseer, maar kon nie die verhouding tussen hulle oplos nie. In ’n poging om beter resolusie te verkry is ASGV en “Citrus tatter leaf virus (CTLV)” heel genoom volgordes in lyn geplaas met die mantelproteïn volgordes en filogeneties geanaliseer met parsimony. Suid-Afrikaanse isolate groepeer in drie klades van veelvuldige oorspronge wat dus geen geografiese tendens aandui nie. In ’n ondersoek na die genetiese variasie van ASPV in Suid-Afrika is die nukleotied volgorde van die mantelproteïngeen van isolate bepaal, in lyn geplaas- en filogeneties geanaliseer met ASPV volgordes wat van GenBank verkry is. ‘n Parsimony analise het isolaat groepe geïdentifiseer maar kon nie die verhouding tussen hulle oplos nie. Lae CI en RI waardes het aangedui dat daar homoplasie in die data van die mantelproteïngeen is. Die resultate van twee addisionele analises, waarin geenvolgordes onderskeidelik omgeskakel is na aminosuurvolgordes en die derde posisie van die kodon uitgesluit is in die volgorde, dui ‘n afname in homoplasie aan. In ’n poging om die resolusie van die filogenie te verbeter is die filogenetiese analise herhaal deur “maximum likelihood” te gebruik. In die eerste analise, waar die kodon nie verdeel is nie, is ’n boom met lae ondersteuning opgespoor. Dit is opgevolg met, net soos by parsimony, ’n analise van data wat omgeskakel is na aminosuurvolgorde. ’n Boom met hoër resolusie en waarvan die klades beter ondersteun is, is verkry. Die boom met die hoogste resolusie en klade ondersteuning is opgespoor deur die verdeling van die kodon in eerste, tweede en derde posisies. Suid-Afrikaanse isolate groepeer in vyf klades van veelvuldige oorspronge wat dus geen geografiese tendens aandui nie. Hierdie is die eerste studie waarin die teenwoordigheid ASGV en ASPV met behulp van RT-PCR in Suid-Afrika bepaal is. Dubbel-infeksie van ASGV en ASPV is verkry in 24.7% van monsters wat in die studie geanaliseer is. Hierdie is die eerste verslag op rekord van Suid-Afrikaanse pere wat simptome van peerverpitting en vrugvervorming toon wat geassosieer is met ASPV infeksie.

ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) are known to infect pome fruit in all pome fruit producing regions of the world. In this study, a comparison between double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) for ASGV detection in pome fruit was performed. A total of 15 ASGV positive orchard leaf samples were detected using RT-PCR whilst DAS-ELISA could only detect 13. RT-PCR was found to be at least 126 fold more sensitive than DAS-ELISA. In an assessment of the genetic variation of ASGV in South Africa, the coat protein (CP) gene of isolates was sequenced, aligned and phylogenetically analysed with ASGV CP gene sequences from GenBank. Parsimony analysis identified groups of isolates, but could not resolve the relationships between them. In order to obtain better resolution, whole genome sequences of international ASGV and Citrus tatter leaf virus (CTLV) isolates were aligned with ASGV and CTLV CP gene sequences and phylogenetically analysed with parsimony. South African ASGV isolates grouped into three clades and showed multiple origins and no geographical trend. In an assessment of the genetic variation of ASPV in South Africa, the CP gene sequences of infected samples were aligned with international CP gene sequences obtained from GenBank and phylogenetically analysed using parsimony. Results from the analysis using parsimony revealed low CI and RI values indicating homoplasy in the CP gene data. To address the homoplasy, two additional analyses were performed in which the gene sequences were converted to amino acid sequences and in which the third position of the codon was excluded from the alignment. Both of these approaches resulted in a reduction in homoplasy. In an attempt to further increase the resolution of the phylogeny, the phylogenetic analysis was repeated using maximum likelihood. In the first codon unpartitioned analysis a tree with low support was retrieved followed by, as with the parsimony analysis, an analysis performed on the data translated to amino acid sequences, which showed better resolution and higher clade support. The tree with the highest resolution and clade support was retrieved by codon partitioning into first, second and third positions. South African ASGV isolates grouped into five clades and showed multiple origins and no geographical trend. This study is the first in which ASGV and ASPV have been detected using RT-PCR in South Africa. Dual infections of ASGV and ASPV were recorded in 24.7% of samples analysed. This is the first report of South African pear trees exhibiting symptoms of pear stony pit and fruit deformation associated with ASPV infection.

AFRIKAANSE OPSOMMING: Apple stem grooving virus (ASGV) en Apple stem pitting virus (ASPV) kom wêreldwyd voor waar kernvrugte geproduseer word. In hierdie studie is ‘n vergelyking tussen die opsporingsgrense van “double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA)” en “reverse transcriptase polymerase chain reaction (RT-PCR)” vir ASGV bepaal. RT-PCR kon ASGV opspoor in 15 ASGV geïnfekteerde kernvrug isolate terwyl die DAS-ELISA slegs ASGV kon opspoor in 13 van die isolate. Daar is gevind dat RT-PCR met ‘n minimum van 126 keer meer sensitief is as DAS-ELISA. In ‘n ondersoek na die genetiese variasie van ASGV in Suid Afrika is die nukleotiedvolgorde van die mantelproteïngeen van isolate bepaal, in lyn geplaas- en filogeneties geanaliseer met volgordes wat van GenBank verkry is. ‘n Parsimony analise het isolaat groepe geïdentifiseer, maar kon nie die verhouding tussen hulle oplos nie. In ’n poging om beter resolusie te verkry is ASGV en “Citrus tatter leaf virus (CTLV)” heel genoom volgordes in lyn geplaas met die mantelproteïn volgordes en filogeneties geanaliseer met parsimony. Suid-Afrikaanse isolate groepeer in drie klades van veelvuldige oorspronge wat dus geen geografiese tendens aandui nie. In ’n ondersoek na die genetiese variasie van ASPV in Suid-Afrika is die nukleotied volgorde van die mantelproteïngeen van isolate bepaal, in lyn geplaas- en filogeneties geanaliseer met ASPV volgordes wat van GenBank verkry is. ‘n Parsimony analise het isolaat groepe geïdentifiseer maar kon nie die verhouding tussen hulle oplos nie. Lae CI en RI waardes het aangedui dat daar homoplasie in die data van die mantelproteïngeen is. Die resultate van twee addisionele analises, waarin geenvolgordes onderskeidelik omgeskakel is na aminosuurvolgordes en die derde posisie van die kodon uitgesluit is in die volgorde, dui ‘n afname in homoplasie aan. In ’n poging om die resolusie van die filogenie te verbeter is die filogenetiese analise herhaal deur “maximum likelihood” te gebruik. In die eerste analise, waar die kodon nie verdeel is nie, is ’n boom met lae ondersteuning opgespoor. Dit is opgevolg met, net soos by parsimony, ’n analise van data wat omgeskakel is na aminosuurvolgorde. ’n Boom met hoër resolusie en waarvan die klades beter ondersteun is, is verkry. Die boom met die hoogste resolusie en klade ondersteuning is opgespoor deur die verdeling van die kodon in eerste, tweede en derde posisies. Suid-Afrikaanse isolate groepeer in vyf klades van veelvuldige oorspronge wat dus geen geografiese tendens aandui nie. Hierdie is die eerste studie waarin die teenwoordigheid ASGV en ASPV met behulp van RT-PCR in Suid-Afrika bepaal is. Dubbel-infeksie van ASGV en ASPV is verkry in 24.7% van monsters wat in die studie geanaliseer is. Hierdie is die eerste verslag op rekord van Suid-Afrikaanse pere wat simptome van peerverpitting en vrugvervorming toon wat geassosieer is met ASPV infeksie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96727
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