The isolation and genetic characterisation of a novel alphabaculovirus for the microbial control of Cryptophlebia peltastica and closely related tortricid pests

Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) is an economically damaging pest of litchis and macadamias in South Africa. Cryptophlebia peltastica causes both pre- and post-harvest damage to litchis, reducing overall yields and thus classifying the pest as a phytosanitary risk. Various control methods have been implemented against C. peltastica in an integrated pest management programme. These control methods include chemical control, cultural control and biological control. However, these methods have not yet provided satisfactory control as of yet. As a result, an alternative control option needs to be identified and implemented into the IPM programme. An alternative method of control that has proved successful in other agricultural sectors and not yet implemented in the control of C. peltastica is that of microbial control, specifically the use of baculovirus biopesticides. This study aimed to isolate and characterise a novel baculovirus from a laboratory culture of C. peltastica that could be used as a commercially available baculovirus biopesticide. In order to isolate a baculovirus a laboratory culture of C. peltastica was successfully established at Rhodes University, Grahamstown, South Africa. This is the first time a laboratory culture of C. peltastica has been established. This allowed for various biological aspects of the pest to be determined, which included: length of the life cycle, fecundity and time to oviposition, egg and larval development and percentage hatch. The results obtained from these studies found that the biology of C. peltastica was similar to that of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). Once the laboratory culture had reached high densities, larvae showing symptoms of baculovirus infection were observed. Symptomatic larvae were collected and examined for the presence of a baculovirus. An alphabaculovirus (NPV) was successfully isolated and morphologically identified using purified OBs that were sectioned and observed by transmission electron microscopy. This was then confirmed by amplifying the polyhedrin gene region using degenerate primers. A BLAST analysis found a 93% similarity to a partial polyhedrin gene sequence to be that of Epinotia granitalis (Butler) (Lepidoptera: Tortricidae). The alphabaculovirus was then genetically characterised by generating restriction profiles and sequencing the whole genome. Due to the novelty of the virus, no comparison could be made. The biological activity of the alphabaculovirus was then tested against C. peltastica and two closely related Tortricidae pests: T. leucotreta and Cydiapomonella (Linnaeous) (Lepidoptera: Tortricidae). The alphabaculovirus was highly virulent against all three species. The lethal concentrations (LC50 and LC90) for the virus against C. peltastica was 8.19 x 103 and 3.33 x 105 OBs/ml. The LC50 and LC90 for T. leucotreta was 2.29 x 103 and 9.97 x 104 OBs/ml, respectively and C. pomonella had a LC50 of 1.43 x 103 OBs/ml and LC90 1.26 x 104 OBs/ml. The virus was particularly virulent against T. leucotreta and C. pomonella as compared to C. peltastica. The biological activity of the alphabaculovirus was also tested against CpGV resistant European C. pomonella. From the results it was observed that the virus had the ability to overcome the resistance in C. pomonella and could potentially be used in the resistance management of C. pomonella. With the successful biological activity results obtained from this study, preliminary investigation were made into the mass production of the alphabaculovirus using both the in vivo and in vitro production methods. For in vivo production both the homologous host (C. peltastica) and a heterologous host (T leucotreta) were investigated. Preliminary studies focused on determining the biological activity in fifth instars of both hosts. Fifth instar LC50 and LC90 values for C. peltastica were 3.43 x 103 and 1.11 x 107 OBs/ml and for T. leucotreta the LC50 and LC90 values were 2.53 x 103 and 8.82 x 106 OBs/ml, respectively. The average yield of virus produced in each species was also determined. Cryptophlebia peltastica had the highest viral yield of 5.37 x 1010 OBs/larva and 2.93 x 1010 OBs/larva for T. leucotreta. The results obtained, from the preliminary investigation concluded that the virus could be produced in vivo in both C. peltastica and T. leucotreta, however further research is required into the mass production in both hosts. The in vitro production of the virus was also considered and the susceptibility of the virus was tested against the C. pomonella cell line, Cp14R. After infection of the Cp14R cells with budded virus collected from fifth instar C. peltastica larvae, OBs could be observed after three days. Thus, the alphabaculovirus is susceptible to the Cp14R cell line, thus has the potential to be produced in vitro and further characterised. This study is the first to report of the identification and characterisation of a novel alphabaculovirus isolated from a laboratory reared culture of C. peltastica and the potential for it to be commercially developed into a bipoesticide and used against Tortricidae pests

DNA-based identification of Lepidoptera associated with citrus in South Africa

A number of insects, primarily Lepidoptera, cause damage to citrus in South Africa. A major limitation to the management and control of these pests is their correct identification. The aim of this study was to develop a database of gene sequences to aid in the identification of these Lepidoptera. Multiple specimens of 12 species were sequenced for the ~650 bp of the cytochrome oxidase I gene. These sequence data were supplemented and validated using sequences available in public databases. Results showed that each species could be unambiguously identified, and neighbour-joining analysis based on K2P distances formed highly supported, distinct clusters for each species, i.e. the maximum intraspecific genetic distance was less than that of the minimum interspecific genetic distances. Thus, this data set provides a molecular means to successfully identify the most important Lepidoptera associated with citrus in South Africa

Morphological and genetic characterization of a South African Plutella xylostella granulovirus (plxy GV) isolate

Plutella xylostella (L.) (Lepidoptera: Plutellidae), also known as diamondback moth, is a destructive insect pest of cruciferous crops (Talekar and Shelton 1993; Shelton 2004). The pest occurs wherever its host plants are cultivated and the global annual cost of damage and control is estimated to be US$4-5 billion (Zalucki et al. 2012). The extensive use of synthetic pesticides for control combined with the high fecundity of P. xylostella has resulted in the pest developing resistance to nearly all classes of insecticides (Grzywacz et al. 2009). Moreover, these chemicals have negative environmental implications and may affect non-target species, some of which are natural enemies of the pest

The effect of permanent protective netting on insect pest prevalence in citrus orchards in South Africa

The use of protective netting is becoming an increasingly popular practice in the citrus industry in South Africa. However, data on its effects on biotic factors, particularly insect pests, are limited. This study focused on the effect nets have on key citrus pests in the Eastern Cape province. Orchards under nets and open orchards, of similar cultivars, ages and management practices, were monitored at several sites over two seasons for pest infestation and damage. Weekly monitoring was conducted for Thaumatotibia leucotreta infestation. Other pests were monitored either monthly or once a season. During the first season, T. leucotreta infestation was higher in orchards under nets, probably because, unlike the open orchards, the nets provided protection for the existing high levels of T. leucotreta. No T. leucotreta infestation was recorded in both orchard types in 2019. This was due to generally lower than usual pest abundance and dramatically improved area-wide management of T. leucotreta. Pheromone traps were used to monitor T. leucotreta males, including sterile moths used in a sterile insect technique programme. Although higher catches of wild T. leucotreta moths were recorded in orchards under nets, so too were there higher numbers of sterile moths and a higher ratio of sterile to wild moths, indicating the potential for better pest suppression for orchards under nets. Various other key pests were monitored during this time, with variable results for each species. Pests that were elevated under nets include Planococcus citri and Aonidiella aurantii. Pests that seemed to be lower under nets were Ceratitis capitata and Scirtothrips aurantii. Nets had no effect on Empoasca distinguenda, Penthimiola bella and Eriophyes sheldoni. These differences in pest levels in netted and open orchards trigger an important debate on whether nets are beneficial for or detrimental to the successful implementation of an integrated pest management programme

The influence of citrus orchard age on the ecology of entomopathogenic fungi and nematodes

A three-year survey of the ecology of entomopathogenic nematodes (EPN) and entomopathogenic fungi (EPF) was undertaken on soils from citrus orchards of different ages to determine the influence of orchard age on the ecology of entomopathogenic fungi and nematodes. The influence of mulch and irrigation method on the occurrence of EPN and EPF was also determined. Most of the isolates recovered (n = 810) were Beauveria sp. (87.88% of all isolates), followed by Metarhizium sp. (11.87% of all isolates). Only 0.24% of soil samples collected during this study tested positive for EPN. All EPN isolates recovered were Heterorhabditis bacteriophora. No significant differences in EPF occurrence were recorded between orchards under drip and micro-sprinkler irrigation. EPF occurrence was significantly lower (P = 0.016) in orchards covered by mulch (31.85% ± 2.07% occurrence) than in orchards with no covering (38.57% ± 1.57% occurrence). EPF occurrence of 40.33 ± 2.13% was highest in non-bearing orchards, followed by mature orchards (nine years or older) (36.76 ± 2.05% of samples) with the lowest EPF occurrence of 25.30 ± 2.02% reported in juvenile orchards (four to eight years old). Juvenile orchards sustain significantly less EPF than mature and non-bearing orchards because of the combined negative impact of less favourable environmental conditions (lower shade density) and fungicide applications

CpGV-M Replication in Type I Resistant Insects: Helper Virus and Order of Ingestion Are Important

International audienceThe genetic diversity of baculoviruses provides a sustainable agronomic solution when resistance to biopesticides seems to be on the rise. This genetic diversity promotes insect infection by several genotypes (i.e., multiple infections) that are more likely to kill the host. However, the mechanism and regulation of these virus interactions are still poorly understood. In this article, we focused on baculoviruses infecting the codling moth, Cydia pomonella: two Cydia pomonella granulovirus genotypes, CpGV-M and CpGV-R5, and Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV). The influence of the order of ingestion of the virus genotypes, the existence of an ingestion delay between the genotypes and the specificity of each genotype involved in the success of multiple infection were studied in the case of Cydia pomonella resistance. To obtain a multiple infection in resistant insects, the order of ingestion is a key factor, but the delay for ingestion of the second virus is not. CrpeNPV cannot substitute CpGV-R5 to allow replication of CpGV-M

The influence of citrus orchard age on the ecology of entomopathogenic fungi and nematodes

A three-year survey of the ecology of entomopathogenic nematodes (EPN) and entomopathogenic fungi (EPF) was undertaken on soils from citrus orchards of different ages to determine the influence of orchard age on the ecology of entomopathogenic fungi and nematodes. The influence of mulch and irrigation method on the occurrence of EPN and EPF was also determined. Most of the isolates recovered (n = 810) were Beauveria sp. (87.88% of all isolates), followed by Metarhizium sp. (11.87% of all isolates). Only 0.24% of soil samples collected during this study tested positive for EPN. All EPN isolates recovered were Heterorhabditis bacteriophora. No significant differences in EPF occurrence were recorded between orchards under drip and micro-sprinkler irrigation. EPF occurrence was significantly lower (P = 0.016) in orchards covered by mulch (31.85% ± 2.07% occurrence) than in orchards with no covering (38.57% ± 1.57% occurrence). EPF occurrence of 40.33 ± 2.13% was highest in non-bearing orchards, followed by mature orchards (nine years or older) (36.76 ± 2.05% of samples) with the lowest EPF occurrence of 25.30 ± 2.02% reported in juvenile orchards (four to eight years old). Juvenile orchards sustain significantly less EPF than mature and non-bearing orchards because of the combined negative impact of less favourable environmental conditions (lower shade density) and fungicide applications