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
- Authors: Mwanza, Patrick
- Date: 2020
- Subjects: Baculoviruses -- South Africa , Ultraviolet astronomy
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/48533 , 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.
- Full Text:
- Date Issued: 2020
- Authors: Mwanza, Patrick
- Date: 2020
- Subjects: Baculoviruses -- South Africa , Ultraviolet astronomy
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/48533 , 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.
- Full Text:
- Date Issued: 2020
Production of Cydia pomonella granulovirus (CpGV) in a heteralogous host, Thaumatotibia Leucotreta (Meyrick) (False codling moth)
- Authors: Chambers, Craig Brian
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Codling moth -- South Africa , Apples -- Diseases and pests -- South Africa , Codling moth -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Biological pest control agents -- South Africa , Baculoviruses -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5935 , http://hdl.handle.net/10962/d1017906
- Description: Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.
- Full Text:
- Date Issued: 2015
- Authors: Chambers, Craig Brian
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Codling moth -- South Africa , Apples -- Diseases and pests -- South Africa , Codling moth -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Biological pest control agents -- South Africa , Baculoviruses -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5935 , http://hdl.handle.net/10962/d1017906
- Description: Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.
- Full Text:
- Date Issued: 2015
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