Distribution, epidemiology and management strategies for Avocado sunblotch disease in South Africa.
Zwane, Zanele Rebecca.
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Avocado (Persia americana Mill.) is an economical important subtropical fruit worldwide. In the republic of South Africa (RSA) avocado contributes approximately 29% to the total gross value of subtropical fruits. Avocado sunblotch disease caused by Avocado sunblotch viroid (ASBVd) is an important disease that affects yield and quality in avocado production worldwide. Typical symptoms are found on leaves, fruit and bark of the tree. However, some trees do not display any visible symptoms and these are termed symptomless carrier trees. The most important control measure for Sunblotch disease is careful selection of pathogen-free bud wood and seed that are used for propagation which is achieved through indexing. The objectives of the current study were to (1) validate the sensitivity of ASBVd detection techniques used for indexing, (2) study the distribution of ASBVd in a single infected tree and (3) conduct an online- and field survey on commercial farms for sunblotch disease incidence, management and strain variations in the Limpopo and Mpumalanga provinces, RSA. To validate the sensitivity of ASBVd detection techniques, an ASBVd infected tree with typical ASBVd symptoms on the leaves and stem was selected from the glasshouse at the Agricultural Research Council- Institute for Tropical and Subtropical Crops (ARC-TSC). A single ASBV infected leaf was selected as a positive control and mixed with 9, 19, 29, 39, 18 and 49 healthy avocado leaves, respectively. RNA was extracted from the leaves using two methods, a small-scale cetyltrimethylammonium bromide (CTAB) - based RNA extraction method that was compared to a large-scale cellulose column chromatography extraction method. From each method, cDNA was amplified using a fluorescent-based one-step real-time RT-PCR reaction, in a Rotor Gene Q instrument. Two primer sets were compared in separate reactions, firstly the Bar- Joseph et al. (1985) primer pair that resulted in a 247 bp product and secondly the Jooste (unpublished) primer pair that generates a 99 bp product. Of all the methods tested, RNA extraction with the cellulose column chromatography and amplification using the Jooste (unpublished) primer pair was the most sensitive and reliable for large scale ASBVd indexing. Further, cDNA was amplified using a two-step conventional RT- PCR. Two primer pairs were compared in a conventional RT-PCR: first the Bar- Joseph et al. (1985) primer pair resulting in a 247 bp product and secondly the published primer pair from Luttig and Manicom (1999) with a 250 bp product. The products were visualised on a 1.5% agarose gel, stained with ethidium bromide. The most sensitive results were obtained using the Bar- Joseph et al. (1985) primer set from RNA extracted using both the CTAB and Cellulose column chromatography extraction methods. In this study the sensitivity and reliability of a large scale indexing method for ASBVd was validated. The distribution of ASBVd in a single infected tree was studied from avocado trees collected at three nurseries in the Limpopo province that included symptom bearing trees and known ASBVd positive symptomless carrier trees. Branches of the same tree were sampled separately collected (young and old) with fruits being included when present. Further, ASBVd distribution within a single infected fruit between the green skin (healthy part) and yellow skin (symptom bearing) was investigated. RNA was extracted using the large-scale cellulose column chromatography method and amplified in a fluorescent based one-step real time RT-PCR reaction in the Rotor Gene Q instrument using the Jooste (unpublished) primer pair. In this study, an even distribution of ASBVd between the branches of the symptomless trees and symptomless fruits was demonstrated. An uneven distribution of ASBVd in symptom bearing trees was observed. These findings will improve the sampling method thus increase the reliability of ASBVd indexing. This will also lead to improved management of Sunblotch disease in RSA. Two surveys were conducted during this study, firstly an online survey which was created using Google sheets and submitted to the South African Subtropical Growers’ Association (Subtrop) website. The survey was conducted to determine the knowledge farmers have about Sunblotch disease. From the responses it was discovered that not all avocado growers are familiar with Sunblotch disease symptoms; some farmers do not take precautions with their cutting tools and removal of infected trees from the field, which could pose a serious threat in disease spread. A field survey was conducted to determine the spread of Sunblotch disease and to determine the commonly occurring ASBVd variants in RSA. A total of 310 trees were randomly sampled from 20 commercial farms in the Limpopo and Mpumalanga provinces. RNA was extracted using a large-scale cellulose column chromatography method, samples were indexed using a fluorescent based one-step real-time RT-PCR reaction in a Rotor Gene Q instrument using the Jooste (unpublished) primer pair that generates a 99 bp product. Only 27 (10.3%) of trees tested positive for ASBVd, four of which manifested symptoms and the rest were symptomless carrier trees. All positive samples were further amplified using a two- step conventional RT- PCR using the Bar- Joseph et al. (1985) primer pair and PCR products were sent for sequencing. The evolutionary history was inferred using the Neighbor-Joining method. Sequences detected in the current study aligned with other ASBVd sequences already deposited in the GenBank® database with a 98% identity. Different ASBVd variants were detected which were the result of minor changes in the sequence nucleotides.