Browsing by Author "Zwane, Zanele Rebecca."

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Distribution, epidemiology and management strategies for Avocado sunblotch disease in South Africa.
(2017) Zwane, Zanele Rebecca.; Jooste, Anna Elizabeth Catharina.; Gubba, Augustine.
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 (1985) primer pair that resulted in a 247 bp product and secondly the Jooste (unpublished) primer pair that generates a 99 bp product. Of all the methods tested, RNA extraction with the cellulose column chromatography and amplification using the Jooste (unpublished) primer pair was the most sensitive and reliable for large scale ASBVd indexing.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.
A study on avocado sunblotch viroid (asbvd) with a focus on symptomless carrier trees.
Zwane, Zanele Rebecca.; Jooste, Anna Elizabeth Catharina.
The avocado (Persea americana Mill.) is an important subtropical fruit worldwide (Blakey et al, 2014). South Africa is among the top five avocado exporters and the exports are primarily aimed at the European market (DAFF, 2019). According to the profile of the South African avocado market value chain by DAFF (2019), approximately 54% of total avocados produced in South Africa are exported, 14% are sold through the National Fresh Produce Markets (NFPMs), 12% are sold to the informal markets (bakkies and hawkers), 10% are processed, while the remaining 9% are delivered directly to retailers. Avocado sunblotch disease (ASBD), caused by avocado sunblotch viroid (ASBVd), is one of the smallest pathogens inciting economical losses in the avocado crop worldwide (Palukaitis et al. 1979). The infected trees can either express ASBD symptoms phenotypically or can show no symptoms or signs of infection but still be carriers of ASBVd; these trees are known as symptomless carrier trees (Acheampong et al., 2008). Characteristic ASBD symptoms include the appearance of irregular, sunken areas of white, yellow, or reddish colour in infected fruit; white/yellow sunken streaks on green stems and variegated or bleached symptoms on the leaves (Vallejo-Perez et al., 2014). The ASBVd symptomless carrier trees have been described as the primary sources of infection by spreading the disease through budding and grafting practices thus playing an essential role in the epidemiology of ASBVd (Saucedo Carabez et al., 2019). Therefore, the main aim of the study was to investigate ASBVd in avocado symptomless carrier trees. The specific objectives of the current study were to (1) identify and monitor ASBVd-infected trees from the flowering stage until harvest for any visible physical indications of the disease compared with healthy trees, (2) determine the maturity of ASBVd - infected and healthy fruits over time from early development stages until harvest, (3) determine the productivity of ASBVd - infected fruits of the symptomless carrier trees to healthy ones by counting the number of fruit per tree, (4) investigate direct ripening, storage effects and internal quality of ASBVd - infected fruits, (5) investigate honeybees as possible ASBVd vectors for tree-to-tree transmission during pollination, (6) determine the occurrence of root graft transmission of ASBVd from root systems of infected trees to healthy trees, (7) investigate the impact of cutting tools on the plant to plant transmission, (8) study the movement of ASBVd from initially infected cells to the rest of the tree through the grafting of infected scions on healthy rootstocks and (9) investigate t 85 he genetic differences between ASBVd variants within a clonal symptomless carrier ‘Fuerte’ tree population. Differences were observed in the orchard between infected and healthy trees; medium and highly infected trees excessively produced flowers, lost leaves during flowering and ultimately bore few to no fruit at the end of the season. The dry matter content results showed that ASBVd did not affect the maturity of the fruit; fruit from infected and healthy trees matured at the same time. Yield measurements indicated that medium and highly infected trees produced between 83% and 96% lower yields compared to healthy trees. Postharvest studies showed that medium and highly infected fruit significantly lost firmness and developed colour more rapidly than healthy fruit. Infected fruit also developed external rots and shrivels for non-stored fruit, however, these injuries were reduced for fruit stored at 5°C for 28 days. Therefore, flower overbearing with the shedding of leaves and lower yields can be used as an indication of ASBVd infection in ‘Hass’ orchards; however, confirmation with molecular testing is required. These observations can be incorporated as an ASBD management strategy in 'Hass' orchards. Graft transmission (top-work) was demonstrated by grafting 30 healthy and 30 infected ‘Hass’ scions onto a healthy ‘Bounty’ rootstock. There was a 53% infection success rate for the infected symptomless carrier trees compared to the 43.3% for the healthy trees. The statistical analysis showed that there were no significant differences between grafting the healthy or infected scions to the rootstocks (p≤0.05), implying that ASBVd spread through the infected scions is unavoidable unless viroid-free scions are used. Two separate root graft experiments were conducted, one in the tunnels where a single infected tree was planted in the 10L plastic bag to force root contact with four healthy trees; the healthy trees outcompeted the infected trees in all six experiments and they died. In the field, the healthy young ‘Fuerte’ trees were planted one meter closer to the old infected orchard trees with confirmed ASBVd positive roots to force root contact. Eventually, the trees died from the lack of sunlight and water stress and the experiment was discontinued. Seventy-five ‘Fuerte’ clonal trees were used in the mechanical transmission experiment using pruning shears (secateurs). The trees were infected by cutting the infected branch and then using the same shear to prune the healthy trees. The shears were treated with four different percentages of sodium hypochlorite (3.5% M/V) and the untreated shears were used as controls. ASBVd was not successfully transmitted 118 mechanically using pruning shears, however, when the stability of ASBVd was tested on different surfaces, it showed to survive up to 24 hours. Therefore, if equipment used on an infected tree is used for the next tree, there is a chance that ASBVd will be transmitted to the next tree and lead to ASBVd spread in an orchard. Pollen and bees were sampled from the beehives at four different sites in KwaZulu-Natal. ASBVd was successfully detected in the pollen from all four site sites, however, only detected in the bees from three sites The samples were sequenced and the blast results confirmed the sequences to be ASBVd and the phylogenetic analysis gave a 93% identity between the detected sequences and the existing ASBVd variants retrieved from the GenBank® database. From the current study, it was confirmed that graft transmission is the most prevalent mode of transmission for ASBVd and that honey bees carry pollen from infected trees to healthy trees in the field and can play an important role in pollen transmission. A total of 103 positive symptomless carrier trees were detected with a real-time qRT132 PCR assay from a population of 453 young ‘Fuerte’ trees. Results showed that 22% of this population was infected with ASBVd without showing signs of infection. In a further investigation, complete ASBVd genomes were obtained by using a conventional PCR with primer sequences that yielded a 250 bp product, here only 76 samples tested positive for ASBVd, the remaining 27 samples tested negative. The 76 samples were sequenced and the sequences obtained had lengths that variedbetween 248 and 253 nucleotides. From the original 76 sequences, 42 ASBVd variants were identified and several sequences were repeatedly detected in the population. The variants were deposited to the NCBI GenBank® and were assigned ON135462 to ON135503. The phylogenetic analysis of the variants obtained from this study showed a high sequence identity of 97% with the reference ASBVd variants obtained from GenBank®. The current study is crucial for the development of accurate detection techniques for ASBVd and contributes to the ASBVd action plan goals that promote the removal of all infected material from avocado orchards to prevent the further spread of the viroid in avocado orchards.