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

Development of numerical techniques for evaluation of point-focus solar cavity receiver performance

Solar receiver cavities, which are designed to absorb large amounts of concentrated solar irradiation, form the central component of a solar collection plant. Since this receiver’s efficiency is directly proportional to the plant’s overall performance, the optimum design of these receivers is an important research field, as it is key to the maximisation of electricity output, while maintaining reasonable costs as an alternative to the high costs of fossil fuel energy generation technologies. Due to the high temperatures that are reached inside a solar receiver, the prediction of heat flux distribution and the subsequent effects on conjugate heat transfer have been key areas of research in the solar field. Initially dominated by experimental studies, research has trended towards numerical prediction using finite volume methods (FVM), due to the low turnaround time and cost-effective nature of this type of analysis. Owing to the need to accurately predict these heat flux distributions, a methodology to numerically simulate concentrated heat flux on complex surfaces of a solar receiver is developed. A combination of Monte Carlo ray tracing (MCRT) methods and computational fluid dynamics (CFD) is implemented to estimate system performance, while minimising computational time and expense, with limited sacrifice of accuracy. After successful validation of this method with experimental data, iterative performance simulations on a candidate geometry, implemented in a realistic solar-concentrating field, are performed to showcase the ability of the methodology to accurately predict system performance. The sample geometry is based on a number of implementations from various case studies and receivers that are used nowadays, with each iteration allowing for parameter adjustment to maximise optical and thermal efficiency. Key result outputs include the prediction of heat flux distributions and subsequent thermal stress raisers, such as hot spots, convective and re-radiation heat losses, and operating temperatures. Determining which of these thermal stress raisers from the implementation of this model can further improve and streamline designs.Dissertation (MEng)--University of Pretoria, 2018.Mechanical and Aeronautical EngineeringMEngUnrestricte

Central solar receiver CFD modelling utilising generated heliostat field heat flux maps

Paper presented to the 3rd Southern African Solar Energy Conference, South Africa, 11-13 May, 2015.A numerical method to couple Monte Carlo ray tracing data to a Finite Volume (FV) semi-transparent surface to allow for the determination of thermal efficiency due to an input heat flux profile and corresponding ray directions within a central cavity receiver is presented. A sample Biomass cavity receiver[1, 2] is used as a 2-D validation case to demonstrate that a CFD FV approach can be used as an accurate solution to the Radiative Transfer Equation (RTE). A 3-D representation of this cavity allows for the approximation of cavity thermal efficiency to be compared between various input heat flux profiles due to the addition of conjugate heat transfer. Results allow for deductions to be made on the benefits of more accurate representations of heat flux maps due to the point concentration of solar energy from a heliostat field. These representations of heat profiles can be used in future applications such as cavity and heliostat field optimization by creating the critical link between ray tracing and conjugate heat transfer solution methods to evaluate central tower cavity receiver designs.cf201

Endophyte isolations from Syzygium cordatum and a Eucalyptus clone (Myrtaceae) reveal new host and geographical reports for the Mycosphaerellaceae and Teratosphaeriaceae

Speci e s of Mycosphaerellaceae and Teratosphaeriaceae (Ascomycetes) cause important leaf, shoot and canker diseases globally on a broad range of hosts, including Eucalyptus and other Myrtaceae. Recently, species of the Mycosphaerellaceae and Teratosphaeriaceae have been isolated as asymptomatic endophytes. In this study, endophytic species of Mycosphaerellaceae and Teratosphaeriaceae were isolated from samples taken from healthy native Syzygium cordatum (Myrtaceae) and related non-native Eucalyptus grandis x E. camaldulensis (hybrid clone) growing in Mtubatuba, KwaZulu Natal, South Africa. Multi-locus sequence analysis (MLSA) using the Internal Transcribed Spacer (ITS) region, the partial Large Subunit (LSU; 28S nrDNA) of the nuclear ribosomal DNA operon and Translation Elongation Factor-1α (TEF-1α) genes were used to correctly identify the 22 resulting isolates. The isolates grouped in five clades representing Readeriella considenianae that was isolated only from the Eucalyptus hybrid clone, Mycosphaerella marksii and M. vietnamensis from S. cordatum and Pseudocercospora crystallina from both S. cordatum and the Eucalyptus hybrid clone. Interestingly, the serious canker pathogen T. zuluensis was isolated from Eucalyptus leaves, although it is known only from stem and branch cankers. Of the species found, R. considenianae and M. vietnamensis were found in South Africa for the first time, while M. marksii, M. vietnamensis and P. crystallina were shown to naturally infect native S. cordatum for the first time. Despite the limited number of trees sampled, the new host and distribution reports show that more intensive sampling, especially following an endophyte approach, will reveal more complete patterns of host preference and geographical distribution for these fungi.Thutuka Funding programme of the National Research Foundation (NRF), members of the Tree Protection Co-operative Programme (TPCP) and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB), South Africa.http://link.springer.com/journal/13313hb201

Finite‐volume ray tracing using Computational Fluid Dynamics in linear focus CSP applications

The modelling of solar irradiation in concentrated solar power (CSP) applications is traditionally done with ray-tracing methods, e.g. the Monte Carlo method. For the evaluation of CSP receivers, the results from ray-tracing codes are typically used to provide boundary conditions to Computational Fluid Dynamics (CFD) codes for the solution of conjugate heat transfer in the receivers. There are both advantages and disadvantages to using separate software for the irradiation and heat transfer modelling. For traditional ray-tracing methods, advantages are the cost-effectiveness of the Monte Carlo method in modelling reflections from specular surfaces; the ability to statistically assign a sun shape to the rays; the statistical treatment of reflectivity and optical errors (e.g. surface slope errors), to name a few. When considering a complex mirror field and a complex receiver with secondary reflective surfaces, especially with selective coatings to enhance absorption and limit re-radiation losses, standard ray tracers may be limited in specifying emissivity and absorptivity, which are both specular and temperature dependent, and are hence not suitable as radiation analysis tool. This type of scenario can be modelled accurately using CFD, through the finite volume (FV) treatment of the radiative transfer equation (RTE) and a banded spectrum approach at an increased computational cost. This paper evaluates the use of CFD in the form of the commercial CFD code ANSYS Fluent v15 and v16 to model the reflection, transmission and absorption of solar irradiation from diffuse and specular surfaces found in linear CSP applications. 2-D CFD solutions were considered, i.e. line concentration. To illustrate and validate the method, two sources were used. The first source was test cases from literature with published solutions and the second a combined modelling approach where solutions were obtained using both FV and ray tracing (with SolTrace). For all the test cases, good agreement was found when suitable modelling settings were used to limit both ray-effect and false scattering errors.The University of Pretoria (South Africa) and the South African National Research Foundation (DST-NRF Solar Spoke).http://www.elsevier.com/locate/apenergy2017-12-31hb2017Mechanical and Aeronautical Engineerin

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

Development of a Postharvest Cold Treatment for Cryptophlebia peltastica (Lepidoptera: Tortricidae) for Export of Litchis From South Africa

The litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae), is endemic to sub-Saharan Africa and certain Indian Ocean islands. It is an important pest of litchis and to a lesser extent macadamias. Litchis are exported to certain markets that consider C. peltastica as a phytosanitary pest. Consequently, an effective postharvest phytosanitary treatment is required. This study sought to develop a cold disinfestation treatment for this purpose. First, it was established that the fifth instar was the most cold-tolerant larval stage, as it was the only instar for which there was still some survival after 12 d at 1°C. It was then determined that cold treatment trials could be conducted in artificial diet, as there was no survival of fifth instar C. peltastica in litchis after only 9 d at 1°C, whereas it took 15 d at this temperature before no survival of fifth instar C. peltastica was recorded in artificial diet. Consequently, cold susceptibility of fifth instar C. peltastica and the most cold-tolerant larval stages (fourth and fifth instar) of false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), were compared in artificial diet. There was no survival of C. peltastica after 13 d at 1°C, whereas this was only so for T. leucotreta after 16 d. Consequently, it can be concluded that any cold treatment that has been proven effective against T. leucotreta would be as effective against C. peltastica. Finally, it was confirmed that the cold susceptibility of T. leucotreta in artificial diet did not overestimate the effect of cold on T. leucotreta larvae in litchis

Morphological, genetic and biological characterisation of a novel alphabaculovirus isolated from Cryptophlebia peltastica (Lepidoptera: Tortricidae)

Cryptophlebia peltastica is an agricultural pest of litchis and macadamias in South Africa with phytosanitary status for certain markets. Current control methods rely on chemical, cultural and classical biological control. However, a microbial control option has not been developed. An Alphabaculovirus from C. peltastica was recovered from a laboratory reared colony and morphologically characterised by transmission electron microscopy (TEM). Analysis of occlusion bodies indicated a single NPV (SNPV) varying in size from 421 to 1263 nm. PCR amplification and sequencing of the polh gene region using universal primers followed by BLAST analysis revealed a 93% similarity to a partial polh gene sequence from Epinotia granitalis NPV. Further genetic characterisation involving single restriction endonuclease (REN) digestion of genomic DNA was carried out to generate profiles for comparison against other baculovirus species and potential new isolates of the same virus. The complete genome of the virus was sequenced, assembled and analysed for a more comprehensive genetic analysis. The genome was 115 728 base pairs (bp) in length with a GC content of 37.2%. A total of 126 open reading frames (ORFs) were identified with minimal overlap and no preference in orientation. Bioassays were used to determine the virulence of the NPV against C. peltastica. The NPV was virulent against C. peltastica with an LC50 value of 6.46 × 103 OBs/ml and an LC90 value of 2.46 × 105 OBs/ml, and time mortality ranging between 76.32 h and 93.49 h. This is the first study to describe the isolation and genetic characterisation of a novel SNPV from C. peltastica, which has potential for development into a biopesticide for the control of this pest in South Africa

Botryosphaeria dothidea : a latent pathogen of global importance to woody plant health

Botryosphaeria dothidea is the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales). Fungi residing in this order are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea one of the most common species on a large number of hosts. Its taxonomic circumscription has undergone substantial change in the past decade, making it difficult to interpret the large volume of literature linked to the name B. dothidea. This pathogen profile synthesises the current understanding of B. dothidea pertaining to its distribution, host associations and its role as a pathogen in managed and natural woody environments. The prolonged latent infection or endophytic phase is of particular importance as it implies that the fungus can easily pass undetected by quarantine systems in traded living plants, fruits and other plant parts. Infections typically become obvious only under conditions of host stress, when disease symptoms develop. This study also considers the knowledge emerging from the recently sequenced B. dothidea genome elucidating previously unknown aspects of the species, including mating and hostinfection strategies. Despite more than 150 years of research on B. dothidea, there is clearly much to be learned regarding this global tree pathogen. This is increasingly important given the stresses imposed on various woody hosts due to climate change.The National Research Foundation (NRF) of South Africa and members of the Tree Protection Co-operative Programme (TPCP).http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1364-37032018-05-31hb2017GeneticsMicrobiology and Plant Patholog