The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
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The characterisation of a South African isolate of Cryptophlebia leucotreta Granulovirus (CIGV)
- Authors: Singh, Shalene
- Date: 2002
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4067 , http://hdl.handle.net/10962/d1004929 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Description: The false codling moth (FCM), Cryptophlehia Leucatreta, causes widespread damage to economically important fruit crops throughout sub-Saharan Africa. Fruit are rendered unfit for consumption once they have been stung by FCM larvae. Larval infestation of fruit can lead to significant pre-harvest losses or post-harvest waste, posing a major problem to the citrus industry. Current control of the pest includes the use of chemical pesticides. The larval form of FCM is known to be infected by a granulovirus called Cryptophlebia leucotreta granulovirus (CIGV). Granuloviruses are highly specific against their hosts and are harmless to vertebrates, plants and the environment. The development of CIGV into a biological control agent would offer an attractive and safer alternative for the control of this pest. A full characterisation of CIGV is required prior to the virus being disseminated into the environment. In this project, the characteristics of CIGV will be examined. Viral DNA was extracted from infected larvae and the DNA analysed by restriction fragment length polymorphism (RFLP). Fragmentation profiles of the South African and Cape Verde (CV3) isolates of the virus were compared, revealing distinct differences between them. The size of the CIGV-SA genome was calculated to be 112 kbp, identical to the size of the CV3 isolate. Physical maps for five restriction enzymes were constructed for the CIGV-SA genome. The alignment of these maps with maps the CV3 isolate (for the same enzymes) further highlighted the differences between the isolates. The genetic engineering of granuloviruses could significantly improve the speed of kill of these viruses. Therefore essential genes like egt and granulin were isolated (by PCR) and their position located in the genome. Both genes were sequenced and their phylogeny with other granulin and egt genes investigated. Finally, tbe incidence of CIGV in natural populations of FCM larvae was investigated, by screening field-collected larvae for the presence of the virus. CIGV was successfully detected from dot blots of larval DNA using both radiolabelled and non-radiolabelled probes and by PCR. Trends regarding the incidence of CIGV in natural populations of larvae were also determined.
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