Selection for and Analysis of UV-Resistant Cryptophlebia Leucotreta Granulovirus-SA as a Biopesticide for Thaumatotibia leucotreta

Cryptophlebia leucotreta granulovirus-SA (CrleGV-SA) is used as a commercial biopesticide for the false codling moth, Thaumatotibia leucotreta, in citrus and other crops. The virus is sensitive to UV irradiation from sunlight, which reduces its efficacy as a biopesticide in the field. We selected a UV-resistant CrleGV-SA isolate, with more than a thousand-fold improved virulence compared to the wild-type isolate, measured by comparing LC50 values. CrleGV-SA purified from infected T. leucotreta larvae was exposed to UV irradiation under controlled laboratory conditions in a climate chamber mimicking field conditions. Five cycles of UV exposure, followed by propagating the virus that retained infectivity in vivo with re-exposure to UV, were conducted to isolate and select for UV-resistant virus. Serial dilution bioassays were conducted against neonates after each UV exposure cycle. The concentration-responses of the infectious UV-exposed virus populations were compared by probit analysis with those from previous cycles and from the original CrleGV-SA virus population. NGS sequences of CrleGV-SA samples from UV exposure cycle 1 and cycle 5 were compared with the GenBank CrleGV-SA sequence. Changes in the genomes of infective virus from cycles 1 and 5 generated SNPs thought to be responsible for establishing UV tolerance. Additional SNPs, detected only in the cycle 5 sequence, may enhance UV tolerance and improve the virulence of the UV-tolerant population.

An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus

A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field.

Population dynamics and distribution of exotic and native frugivorous insects of citrus in Nsukka, Nigeria

Invasion of the exotic Bactrocera dorsalis (Hendel, 1912) (Tephritidae) has drastically reduced the abundance and distribution of native Ceratitis anonae Graham, 1908 (Tephritidae) and false codling moth Thaumatotibia leucotreta (Meyrick, 1913) (Tortricidae) in Nigeria. There is an overlap in resource use among the three frugivorous insects which could result in interspecific competition. Knowledge of the population dynamics and distribution of the three frugivorous insects in the study area is lacking and such data are indispensable for effective management of the tephritid and tortricid pests. The population dynamics and distribution of the three frugivorous insects were investigated to predict the route of invasion and the outcome of interactions among the three frugivorous insects. Bactrocera dorsalis occurred in all the seven Local Government Areas (LGAs) sampled while C. anonae and T. leucotreta occurred together in two LGAs and separately in two other LGAs. The population of B. dorsalis is significantly higher than either C. anonae or T. leucotreta in the study area. The three frugivorous insects are negatively associated with one another but only the association between B. dorsalis and C. anonae was significantly different. Bactrocera dorsalis invaded the study area from southern Nigeria and C. anonae is being driven towards northern Nigeria.

Biological Control of a Phytosanitary Pest (Thaumatotibia leucotreta): A Case Study

Thaumatotibia leucotreta, known as the false codling moth, is a pest of citrus and other crops in sub-Saharan Africa. As it is endemic to this region and as South Africa exports most of its citrus around the world, T. leucotreta has phytosanitary status for most markets. This means that there is zero tolerance for any infestation with live larvae in the market. Consequently, control measures prior to exporting must be exemplary. Certain markets require a standalone postharvest disinfestation treatment for T. leucotreta. However, the European Union accepts a systems approach, consisting of three measures and numerous components within these measures. Although effective preharvest control measures are important under all circumstances, they are most critical where a standalone postharvest disinfestation treatment is not applied, such as within a systems approach. Conventional wisdom may lead a belief that effective chemical control tools are imperative to achieve this end. However, we demonstrate that it is possible to effectively control T. leucotreta to a level acceptable for a phytosanitary market, using only biological control tools. This includes parasitoids, predators, microbial control, semiochemicals, and sterile insects. Simultaneously, on-farm and environmental safety is improved and compliance with the increasing stringency of chemical residue requirements imposed by markets is achieved.