- Title
- Development of a UV-tolerant strain of the South African isolate of Cryptophlebia leucotreta granulovirus for use as an enhanced biopesticide for Thaumatotibia leucotreta control on citrus
- Creator
- Mwanza, Patrick
- Subject
- Baculoviruses -- South Africa
- Subject
- Ultraviolet astronomy
- Date Issued
- 2020
- Date
- 2020
- Type
- Thesis
- Type
- Doctoral
- Type
- PhD
- Identifier
- http://hdl.handle.net/10948/48533
- Identifier
- vital:40885
- Description
- Baculoviruses are pathogenic to insects in the orders Diptera, Hymenoptera, and Lepidoptera. As a result of this natural relationship with insects they provide an environmentally friendly method to combat crop and forest pests. As such, a number of baculoviruses have been formulated into biopesticides. The use of baculovirus biopesticides is gaining popularity as the use of chemical pesticides has come under stringent regulatory conditions imposed by governments and continental blocks such as the European Union. Baculoviruses have a narrow host range and therefore do not harm non-pests or humans who consume the crops. One such baculovirus is Cryptophlebia leucotreta granulovirus (CrleGV), which is pathogenic to the citrus pest Thaumatotibia leucotreta, commonly referred to as the false codling moth (FCM). CrleGV has an occlusion body (OB) that encloses a single virion. Several CrleGV biopesticides have been registered in South Africa for use on citrus, avocadoes, macadamias, grapes and other crops by two commercial producers, River Bioscience (SA) and Andermatt (Switzerland). These biopesticides are used as part of the FCM integrated pest management (IPM) programme, a multifacetted approach to controlling FCM. However, baculoviruses are susceptible to the ultraviolet (UV) radiation component of sunlight and lose their activity within hours to a few days, after exposure to UV. Several substances have been tested as UV protectants to improve the persistence of baculovirus biopesticides in the field. These include optical brighteners, UV absorbers and anti-oxidants. While very promising in the laboratory, UV-protectants have not been as successful in the field. A few published reports have reported, that UV-tolerant baculoviruses could be isolated from a population by repeatedly exposing and re-exposing the virus to UV irradiation with a propagation step in insect host fourth or fifth instars between each exposure cycle. In this study, the South African isolate of Cryptophlebia leucotreta (CrleGV-SA) was exposed to UV irradiation for 5 exposure cycles in a Q-Sun Xe-3 HC test chamber (Qlab, USA) with parameters set to mimic a typical summer day in the Sundays River Valley, Eastern Cape Province, in South Africa. In between exposures the virus survivors were allowed to multiply in FCM fifth instars. Surface dose bioassays were also conducted to determine the LC50 of the virus after each exposure cycle. Samples from exposure cycle 1 and cycle 5 (UV-tolerant) irradiated for 72 h were prepared for Next Generation Sequencing (NGS) of DNA. The resultant sequence data were analysed using the Geneious R11 software (New Zealand) and compared with the unexposed CrleGV-SA sequence. In-silico restriction enzyme analsysis (REN) with several enzymes was also carried on both the cycle 1 and cycle 5 exposed samples and the resulting digestion patterns were compared with the original CrleGV-SA digestion patterns. The same samples were also analysed by transmission electron microscopy (TEM) and Attenuated Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) to evaluate the effect of UV irradiation on the structure of the CrleGV-SA OB. In addition, three UV protectants, lignin sulphate (Sappi, SA), BREAK-THRU®OE446 (OE446) (Evonik Industries, Germany) and Uvinul Easy (BASF, Germany) were prepared with CrleGV-SA to give final protectant concentrations of 0.09 %, 0.9 % and 9 %. The protectant-virus suspensions were exposed to UV for 24 h in the Q-Sun test chamber and bioassays conducted to determine the protective effect of each protectant concentration. The most successful protectants were then combined with the UV-tolerant CrleGV-SA and exposed to UV for 24 h in the Q-Sun test chamber and surface dose bioassays conducted afterwards. Samples exposed to UV in cycle 5 had lower LC50 values compared to samples in the early cycles. With each re-exposure cycle the LC50 values moved closer to that of the unexposed control. The LC50 of virus samples decreased from 2.89 x 108 OBs/ml after 24 h UV-exposure in cycle 1 to 2.16 x 105 OBs/ml after the same duration of exposure in cycle 5; and from 2.11 x 109 OBs/ml in cycle 1 after 72 h UV-exposure to 1.73 x 106 OBs/ml after the same duration of exposure. This represented a 1338-fold difference and a 1220-fold difference, respectively. When the UV-tolerant samples were sequenced seven SNPs were identified in cycle 1, which were thought to help establish UV tolerance, while a further seven SNPs were identified in cycle 5 samples; these were thought to further establish and maintain the UV-tolerance. Additionally, REN analysis with EcoR1 for both test samples yielded digestion patterns that were different from those of the original CrleGV-SA. TEM data showed that UV damages the virion as well as the crystalline structure of the OB. This is the first time visual evidence for UV damage to baculoviruses has been published. Comparison of cycle 1 and cycle 5 UV exposed OBs revealed that the cycle 5 OBs were significantly larger than the cycle 1 OBs (P<0.05). In addition, several peaks in the fingerprint region were shown to have either appeared or disapeered from the ATR-FTIR spectra after UV irradiation. However, there was no difference in the spectra of the Cycle 1 and Cycle 5 virus samples. The tests with potential UV-protectants revealed that the 0.9 % lignin, 9 % OE446 and 9 % Uvinul Easy were the most effective in protecting the virus from UV. However, there was no significant difference in their protection of UV tolerant CrleGV-SA and wild type CrleGV-SA. Going forward, it is recommended that the 0.9 % lignin, 9 % OE446 and 9 % Uvinul Easy combinations be explored further in future studies, particulary in the field. This study therefore forms an important foundation for the development of UV-tolerant baculovirus that will last longer in the field.
- Format
- XXvii, 191 leaves
- Format
- Publisher
- Nelson Mandela University
- Publisher
- Faculty of Science
- Language
- English
- Rights
- Nelson Mandela University
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NMU School of BioMolecular and Chemical Sciences
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| View Details Download | SOURCE1 | Mwanza, P 209201009 Thesis April 2020.pdf | 1 MB | Adobe Acrobat PDF | View Details Download |