Department of Plant Pathology

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Browsing Department of Plant Pathology by browse.metadata.advisor "Bellstedt, D. U."

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    Apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) : detection and isolate characterization in South African pome fruit
    (Stellenbosch : Stellenbosch University, 2015-03) Gagiano, Magdalena Christiena; Bellstedt, D. U.; Mostert, Lizel; Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology.
    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.
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    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
    (Stellenbosch : Stellenbosch University, 2014-12) Malan, Sophia Susanna; Bellstedt, D. U.; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    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.