Thesis (M.Sc. (Zoology & Entomology)) - Rhodes University, 2008.

Thesis (M.Sc. (Zoology & Entomology)) - Rhodes University, 2008.

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.

Cryptophlebia leucotreta granulovirus (CrleGV-SA) is a baculovirus specifically pathogenic to the citrus pest false codling moth, Thaumatotibia leucotreta. CrleGV- SA is formulated as a commercial biopesticide, Cryptogran® (River Bioscience, South Africa). The virus has a stable, proteinaceous crystalline occlusion body (OB) that protects the nucleocapsid. The major limitation to the use of baculoviruses is their susceptibility to the ultraviolet (UV) component of sunlight, which rapidly and greatly reduces their efficacy as biopesticides. The UVA and UVB components are the most destructive to biological organisms. To date no publication has reported the effect of UV on the structure and virulence of CrleGV, or the effectiveness of the OB as a UV protectant. In this study the effect of UV irradiation on the structure and infectivity of pure CrleGV-SA and Cryptogran® was investigated using scanning electron microscopy (SEM), Raman spectroscopy, qPCR, and bioassays. The project included laboratory and field studies. In the laboratory, CrleGV-SA and Cryptogran® were exposed to either UVA or UVB for periods of 24 hours to 7 days before analysis. In the field, Cryptogran® was applied to trees in a citrus orchard with young fruit. The fruit were collected from 24 hours to 28 days after application and bioassays conducted to assess the effect of sunlight over time on virus structure and efficacy when applied to the northern or southern sides of the trees. No surface morphological changes to the virus were detected using SEM. However, small compositional changes were detected by Raman spectroscopy. qPCR and bioassays demonstrated that UV irradiation damaged the viral DNA, greatly reducing the infectivity of pure CrleGV-SA and Cryptogran®. Exposure to UVB reduced the virulence of the virus more than UVA. The field studies revealed that the activity of CrleGV-SA decreased more on the northern side of the trees than on the southern side.

False codling moth is a known pest of economic importance to many cultivated crops in South Africa and Africa south of the Sahara, and is particularly severe on citrus. If the fruit is infested just before harvest the chances of detecting signs of infestation are very low. As a result, the risk of packaging infested fruit and exporting them as healthy fruit is high. It is therefore a priority to develop a post-harvest technique for detection of False codling moth in citrus fruit at different levels of infestation in order to reduce phytosanitary risk. Compounds released and detected were indicative of infestation and were not insect produced but naturally produced fruit volatiles emitted at higher levels as a result of the insect within the fruit. Five major volatile compounds of interest were released by the infested oranges. These major volatile compounds include D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene. Limonene was one of the most abundant volatile compounds released by the infested citrus fruit. Naphthalene, which is possibly produced due to larval feeding and development within the fruit maintained higher concentrations than controls throughout the infestation within the fruit. Naphthalene would be a good indicator of False codling moth infestation, however, not primarily for early infestation detection. A significantly higher concentration of D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene and naphthalene was detected using the SEP over the SPME technique. The application of an SPME procedure and the utilization of this method for detection of volatiles present in the headspace of intact infested fruit are evaluated and the possible volatile compounds diagnostic of Thaumatotibia leucotreta infestation of orange fruit and differences in volatile compound response in different orange varieties is discussed.

False codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyr) (Lepidoptera: Tortricidae), is one of the most important pests on citrus. The Cryptophlebia leucotreta granulovirus (CrleGV) has been developed into a successful biological control agent, registered under the name Cryptogran, and is currently the preferred product for the control of FCM on citrus in South Africa. A prerequisite to the continued success of Cryptogran as a means of controlling false codling moth is to understand the factors affecting field persistence of the virus, and to find ways to improve it. The aim of this study was to gain a clearer understanding of the product and the abiotic and biotic factors affecting its persistence in the field, and to investigate methods to improve this persistence. The effect of UV-irradiation on the virus was determined, and various products were tested as UV protectants in laboratory bioassays. Lignin was the most effective additive, and was tested in several field trials, where it also enhanced the efficacy of Cryptogran. Laboratory trials indicated that Cryptogran is rainfast. Cryptogran applications early in the season had a longer period of residual activity than sprays applied closer to harvest. Daytime applications were less effective that evening sprays. Sprays applied coinciding with peaks in pheromone moth trap catches were more effective than those applied between peaks. Biotic factors influencing persistence were investigated. Residual efficacy was longer when treatments were applied to blocks than as single tree treatments. Attempts were made to quantify the effect of the navel end of a navel orange on the field persistence of Cryptogran. Cryptogran was shown to be compatible with many agricultural chemicals used on citrus. Economic thresholds and various cost-benefit analyses are discussed. A list of practical recommendations to growers was drawn up, and possibilities for future research are presented.

The study examined the larval parasitoids of Cryptophlebia leucotreta (Meyrick) on citrus in South Africa and aimed to improve the existing rearing techniques of C. leucotreta with a view to mass rearing of biological control agents. The biological characteristics of Agathis bishopi Nixon (Hymenoptera: Braconidae) were studied, with an emphasis on parasitism rates in the field, host stage preference, developmental rate, life span and offspring sex ratios. Two larval parasitoids, A. bishopi and Apophua leucotretae (Wilkinson) (Hymenoptera: Ichneumonidae), and an egg parasitoid, Trichogrammatoidea cryptophlebiae Nagaraja (Hymenoptera: Trichogrammatidae), were recorded on C. leucotreta on citrus. A. bishopi was the more abundant of the larval parasitoids and exhibited density dependent parasitism. The highest parasitism rates were observed in December with up to 38% in Sundays River Valley and 34% in Gamtoos River Valley, at a time when the highest false codling moth infestations were observed. Agathis bishopi was recorded only in the Eastern Cape Province. The sex ratio of A. bishopi was biased towards females throughout the study (77% in Gamtoos River Valley and 72% in Sundays River Valley). Agathis bishopi is a solitary, koinobiont, larval-pupal endoparasitoid. The species showed a preference for 1st and 2"d instar hosts. Females regulate the sex of their progeny according to the size and larval stage of the host, ovipositing unfertilised eggs in younger, smaller larvae (1st instars) and fertilised eggs in older, larger larvae (2nd instars). The developmental rate of A. bishopi is in synchrony with that of the moth and adults emerge when adult moths that have escaped parasitism emerge. Agathis bishopi and T. cryptophlebiae compliment each other because they have different niches and strategies of attack. Integrating A. bishopi and T. cryptophlebiae into the management of citrus orchards has potential to suppress false codling moth. Larger rearing containers seemed ideal for large-scale rearing of false codling moth. A higher percentage of adults was obtained from larvae reared in larger containers than in smaller ones. The width of the sponges used as stoppers prevented escape of the larvae. Media prepared in larger containers are easier and simpler to prepare than in smaller ones, thus eliminating many precautions otherwise necessary to prevent contamination. Moth production was greatly reduced by the high concentration of Sporekill used for egg decontamination.

The false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is one of the major pests of citrus in South Africa, the others being mealybug, Mediterranean fruit fly, bollworm and some mites. Due to problems such as the expense of pesticides, insects evolving pesticide resistance (Hogsette 1999), chemical residue on the skin of export fruit and the negative impact of pesticides on the environment, it became necessary to find alternative methods for pest control (Viggiani 2000). Agathis bishopi (Nixon) (Hymenoptera: Braconidae), a larval parasitoid of false codling moth known only from the Sundays River Valley area (Sishuba 2003), offers a means of control for the pest. A total of 11 389 navel oranges were collected from various orchards in the Addo/Kirkwood area, and false codling moth larvae infested 36.09% of the fruit. A single parasitoid species, A. bishopi, was reared from these larvae. In 2006 the highest parasitism rate, 11.43%, was recorded in May and in 2007, the highest parasitism rate, 13.27%, was in April. Agathis bishopi parasitizes larvae in instars 2 and 3, possibly due to the accessibility of these younger instars to the female parasitoid and possibly due to the length of the life cycle of this koinobiont. Second instar hosts yielded the highest number of parasitoids, and there was no emergence of parasitoids from fifth instar larvae. Females of A. bishopi live for 18.5 days (n = 20; S.E. = 3.1) and males for 8.25 days (n = 20; S.E. = 1.23). Females produce an average of 23 offspring in a lifetime, while female false codling moths produce about 800 eggs each. A high number of parasitoids will be required per hectare to reduce the population of false codling moth. Captive rearing of A. bishopi proved difficult due to viral and fungal contamination. Agathis bishopi has potential for use in an integrated pest management programme once the hurdle of mass-rearing has been overcome.

A granulovirus isolated from Cryptophlebia leucotreta larvae was shown through restriction endonuclease analysis to be a novel strain (CrleGV-SA). No more than one isolate could be identified from a laboratory culture of C. leucotreta. However, a preliminary examination of restricted DNA profiles of isolates from different geographical regions indicated some minor differences. In surface dose bioassays on artificial diet, LC50 and LC90 values with neonate larvae were estimated to be 4.095 x 103 OBs/ml and 1.185 x 105 OBs/ml respectively. LT50 and LT90 values with neonate larvae were estimated to be 4 days 22 h and 7 days 8 h, respectively. Detached fruit (navel orange) bioassays with neonate larvae indicated that virus concentrations that are likely to be effective in the field range from 1.08 x 107 to 3.819 x 1010 OBs/ml. In surface dose bioassays with fifth instar larvae LC50 and LC90 values were estimated to be 2.678 x 107 OBs/ml and 9.118 x 109 OBs/ml respectively. LT50 and LT90 values were estimated to be 7 days 17 h and 9 days 8 h, respectively. A new artificial diet for mass rearing the host was developed. Microbial contamination of diet was significantly reduced by adding nipagin and sorbic acid to the diet and by surface sterilising C. leucotreta eggs with Sporekill. Almost 20 % more eggs were produced from moths reared on the new diet compared to moths reared on the old diet. A further 9 % improvement in egg production and a reduction in the labour required to produce eggs, was made with the development of a new oviposition cage attached to the moth eclosion box. Virus was mass produced in fifth instar C. leucotreta larvae by surface inoculating diet with the LC90. When 300 individuals were placed onto inoculated diet, 56 % of them were recovered six to 11 days later as infected larvae. Mean larval equivalents was 1.158 x 1011 OBs/larva. When larvae and diet were harvested together, highest yields of virus were achieved at eight days after inoculation. Microbial contamination in semi-purified preparations of CrleGV ranged from 176211 to 433594 (OB:CFU ratio). Half-life of CrleGV in the field was estimated to be less than 1 day on the northern aspect of trees and between 3 - 6 days on the southern aspect. Original activity remaining (OAR) of the virus dropped below 50 % after 5 days on the northern aspect of trees and was still at 69 % on the southern aspect of trees after 3 weeks. In field trials, CrleGV reduced C. leucotreta infestation of navel oranges by up to 60 % for a period of 39 days. CrleGV in combination with augmentation of the C. leucotreta egg parasitoid, Trichogrammatoidea cryptophlebiae, reduced infestation by 70 %. The integration of CrleGV into an integrated pest management (IPM) system for the management of C. leucotreta on citrus is proposed.

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.