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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:
- 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:
Studies on existing and new isolates of Cryptophlebia leucotreta granulovirus (CrleGV) on Thaumatotibia leucotreta populations from a range of geographic regions in South Africa
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2012
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Biological control , Cryptophlebia leucotreta -- Life cycles , Baculoviruses , Lepidoptera -- Biological control , Tortricidae -- Biological control , Microbial insecticides , Pests -- Integrated control
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5778 , http://hdl.handle.net/10962/d1005466
- Description: Baculoviruses are arthropod-specific DNA viruses that are highly virulent to most lepidopteran insects. Their host specificity and compatibility with IPM programmes has enabled their usage as safe microbial insecticides (biopesticides). Two baculovirus-based biopesticides, Cryptogran and Cryptex, which have been formulated with Cryptophlebia leucotreta granulovirus (CrleGV) have been registered for the control of false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa and have been successfully incorporated into IPM programmes. However, several studies have indicated that insects can develop resistance to baculovirus-based biopesticide as was shown with field populations of codling moth (CM), Cydia pomonella (L.), which developed resistance to the biopesticide Cydia pomonella granulovirus (CpGV-M) in Europe. Other studies have shown that, under laboratory conditions, FCM populations differ in their susceptibility to Cryptogran and Cryptex. In order to investigate difference in susceptibility as well as protect against any future resistance by FCM to Cryptogran and Cryptex, a search for novel CrleGV-SA isolates from diseased insects from different geographic regions in South Africa was performed. Six geographic populations (Addo, Citrusdal, Marble Hall, Nelspruit, Baths and Mixed colonies) of FCM were established and maintained in the laboratory. Studies on the comparative biological performance based on pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and duration of life cycle of the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies revealed a low biological performance for the Citrusdal colony. This was attributed to the fact that FCM populations found in the Citrusdal area are not indigenous and may have been introduced from a very limited gene pool from another region. When insects from five colonies, excluding the Baths colony, were subjected to stress by overcrowding , a latent baculovirus resident in the Addo, Nelspruit, Citrusdal, Marble Hall and Mixed colonies was brought into an overt lethal state. Transmission electron micrographs revealed the presence of GV occlusion bodies (OBs) in diseased insects. DNA profiles obtained by single restriction endonuclease analysis of viral genomic DNA using BamH 1, Sa/1, Xba1 , Pst1, Xh01 , Kpn1, Hindlll and EcoR1 revealed five CrleGV-SA isolates latent within the insect populations. The new isolates were named CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl, CrleGVSA Nels and CrleGV-SA Mix isolates. The novelty of the five CrleGV-SA isolates was confirmed by the presence of unique submolar bands, indicating that each isolate was genetically different. PCR amplification and sequencing of the granulin and egt genes from the five isolates revealed several single nucleotide polymorph isms (SNPs) which, in some cases, resulted in amino acid substitutions. DNA profiles from RFLPs, as well as phylogenetic analysis based on granulin and egt sequencing showed the presence of two CrleGV-SA genome types for the CrleGV-SA isolate. Cryptex and CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl and CrleGV-SA Mix were placed as members of Group one CrleGV-SA, and Cryptogran and CrleGV-SA Nels isolate were placed into Group two CrleGV-SA. In droplet feeding bioassays, the median survival time (STso) for neonate larvae inoculated with Group one and two CrleGV-SA were determined to range from 80 - 88 hours (3.33 - 3.67 days), for all five colonies. LDso values for Group one and two CrleGV-SA against neonates from the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies varied between some populations and ranged from 0.80 - 3.12 OBs per larva, indicating some level of variation in host susceptibility. This is the first study reporting the existence of genetically distinct CrleGV baculovirus isolates infecting FCM in different geographical areas of South Africa. The results of this study have broad-ranging implications for our understanding of baculovirus-host interactions and for the application of baculovirus basedbiopesticides.
- Full Text:
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2012
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Biological control , Cryptophlebia leucotreta -- Life cycles , Baculoviruses , Lepidoptera -- Biological control , Tortricidae -- Biological control , Microbial insecticides , Pests -- Integrated control
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5778 , http://hdl.handle.net/10962/d1005466
- Description: Baculoviruses are arthropod-specific DNA viruses that are highly virulent to most lepidopteran insects. Their host specificity and compatibility with IPM programmes has enabled their usage as safe microbial insecticides (biopesticides). Two baculovirus-based biopesticides, Cryptogran and Cryptex, which have been formulated with Cryptophlebia leucotreta granulovirus (CrleGV) have been registered for the control of false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa and have been successfully incorporated into IPM programmes. However, several studies have indicated that insects can develop resistance to baculovirus-based biopesticide as was shown with field populations of codling moth (CM), Cydia pomonella (L.), which developed resistance to the biopesticide Cydia pomonella granulovirus (CpGV-M) in Europe. Other studies have shown that, under laboratory conditions, FCM populations differ in their susceptibility to Cryptogran and Cryptex. In order to investigate difference in susceptibility as well as protect against any future resistance by FCM to Cryptogran and Cryptex, a search for novel CrleGV-SA isolates from diseased insects from different geographic regions in South Africa was performed. Six geographic populations (Addo, Citrusdal, Marble Hall, Nelspruit, Baths and Mixed colonies) of FCM were established and maintained in the laboratory. Studies on the comparative biological performance based on pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and duration of life cycle of the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies revealed a low biological performance for the Citrusdal colony. This was attributed to the fact that FCM populations found in the Citrusdal area are not indigenous and may have been introduced from a very limited gene pool from another region. When insects from five colonies, excluding the Baths colony, were subjected to stress by overcrowding , a latent baculovirus resident in the Addo, Nelspruit, Citrusdal, Marble Hall and Mixed colonies was brought into an overt lethal state. Transmission electron micrographs revealed the presence of GV occlusion bodies (OBs) in diseased insects. DNA profiles obtained by single restriction endonuclease analysis of viral genomic DNA using BamH 1, Sa/1, Xba1 , Pst1, Xh01 , Kpn1, Hindlll and EcoR1 revealed five CrleGV-SA isolates latent within the insect populations. The new isolates were named CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl, CrleGVSA Nels and CrleGV-SA Mix isolates. The novelty of the five CrleGV-SA isolates was confirmed by the presence of unique submolar bands, indicating that each isolate was genetically different. PCR amplification and sequencing of the granulin and egt genes from the five isolates revealed several single nucleotide polymorph isms (SNPs) which, in some cases, resulted in amino acid substitutions. DNA profiles from RFLPs, as well as phylogenetic analysis based on granulin and egt sequencing showed the presence of two CrleGV-SA genome types for the CrleGV-SA isolate. Cryptex and CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl and CrleGV-SA Mix were placed as members of Group one CrleGV-SA, and Cryptogran and CrleGV-SA Nels isolate were placed into Group two CrleGV-SA. In droplet feeding bioassays, the median survival time (STso) for neonate larvae inoculated with Group one and two CrleGV-SA were determined to range from 80 - 88 hours (3.33 - 3.67 days), for all five colonies. LDso values for Group one and two CrleGV-SA against neonates from the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies varied between some populations and ranged from 0.80 - 3.12 OBs per larva, indicating some level of variation in host susceptibility. This is the first study reporting the existence of genetically distinct CrleGV baculovirus isolates infecting FCM in different geographical areas of South Africa. The results of this study have broad-ranging implications for our understanding of baculovirus-host interactions and for the application of baculovirus basedbiopesticides.
- Full Text:
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