Thesis (MScAgric (Conservation Ecology and Entomology)--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The obscure mealybug, Pseudococcus viburni (Signoret) (Pseudococcidae), is one of the common and serious pests of apples and pears in South Africa. The management of this pest in South Africa is dominated by the use of insecticides, while research into using natural enemies for biological control of mealybugs is still ongoing. Increasing concern over the environmental impact, pesticide residues in fruits, resistance, and expense associated with frequent use of insecticides make it necessary to investigate alternative biological control methods, such as the use of entomopathogenic nematodes, for the control of mealybugs. Entomopathogenic nematodes have proven comparable or even superior to chemicals in controlling certain insect pests, without residue problems or a harmful effect on the environment. An important aspect of using endemic nematodes includes the identification of species of nematodes and their symbiotic bacterial cells. A study was carried out to describe a new species of Steinernema, which was recovered during a previous survey in citrus orchards in three provinces of South Africa. Morphometrics, morphology, crossbreeding, drawings, light microscopy and Scanning Electron Microscopy (SEM) photographs were used to describe the new species. A cryopreservation method has been simplified and optimised for the long-term storage of Steinernema khoisanae (SF87) and Heterorhabditis zealandica (J34). Different cryoprotectants used included 15% glycerol, 8% ethylene glycol and 8% dimethyl sulfoxide (DMSO), in which S. khoisanae was incubated at room temperature for periods of two, three, four and five days, followed by a methanol wash. An optimum survival rate of 69% was obtained for S. khoisanae after a four-day incubation period in 15% glycerol. This technique has been used for the cryopreservation of H. zealandica, with a 78% survival rate. The thawed nematodes of both species were able to infect Galleria mellonella larvae after 42 days of cryopreservation (-196ºC) and were able to complete their life cycles. / AFRIKAANSE OPSOMMING: Die ligrooswitluis, Pseudococcus viburni (Signoret) (Pseudococcidae), is een van die algemene en ernstige peste van appels en pere in Suid-Afrika. Die bestuur van hierdie pes word tans in Suid-Afrika deur die gebruik van insekdoders gedomineer terwyl navorsing oor die gebruik van natuurlike vyande vir die beheer van P. viburni nog aan die gang is. Die verhoogde kommer oor die omgewing, residue in vrugte, weerstand, en die koste verbonde aan die gereelde gebruik van chemiese middels maak dit nodig om alternatiewe biologiese metodes van beheer, soos die gebruik van entomopatogeniese nematodes vir die beheer van witluis, te ondersoek. In ander lande is reeds aangetoon dat entomopatogeniese nematodes onder sekere omstandighede en vir sekere insekte gelykwaardige of selfs beter beheer kan gee as chemiese middels. ʼn Belangrike aspek van die gebruik van endemiese nematodes vir die beheer van insekte sluit die korrekte identifikasie van die spesies met hul geassosieerde bakteriese simbionte in. ʼn Nuwe spesie van Steinernema is uit ʼn vorige opname van entomopatogeniese nematodes in sitrusboorde in drie provinsies van Suid-Afrika geïsoleer. Morfometrie, morfologie, kruisteling, ligmikroskoop en SEM fotografie is gebruik om ʼn nuwe spesies te beskryf. ʼn Kriopreserveringsmetode is ontwikkel en ge-optimaliseer vir die langtermyn bewaring van Steinernema khoisanae (SF87) en Heterorhabditis zealandica (J34). Verskillende kriobeskermingsmiddels insluitend 15% gliserol, 8% dimetiel sulfokied (DMSO) en 8% etileen glikol, waarin S. khoisanae vir periodes van twee, drie, vier, en vyf dae geïnkubeer is, is teen kamertemperatuur, getoets, gevolg deur ʼn metanolbad. Optimum oorlewing van 69% is verkry vir S. khoisanae nadat die infektiewe larwes (IJ) vir vier dae in 15% gliserol gehou is. Hierdie tegniek is ook toegepas op H. zealandica, met 78% oorlewing van die IJ. Die ontvriesde nematodes van beide spesies was in staat om Galleria mellonella larwes suksesvol te infekteer en hulle lewensiklus te voltooi nadat hulle vir 45 dae onder kriopreservering gehou is teen -196ºC.

Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.

Thesis (MScConsEcol (Conservation Ecology and Entomology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Pseudococcus viburni (Signoret) (Hemiptera: Pseudococcidae) (obscure mealybug), is a common and serious pest of apples and pears in South Africa. Consumer and regulatory pressure to produce commodities under sustainable and ecologically compatible conditions has rendered chemical control options increasingly limited. Information on the seasonal occurrence of pests is but one of the vital components of an effective and sustainable integrated pest management system needed for planning the initiation of monitoring and determining when damage can be expected. It is also important to identify which orchards are at risk of developing mealybug infestations while development of effective and early monitoring tools for mealybug populations will help growers in making decisions with regards to pest management and crop suitability for various markets. It is also essential to determine the presence and efficacy of naturally occurring biological control agents in orchards so as to ascertain the potential of biological control as a viable alternative in orchards. However, under the current integrated pest management protocol, it has been difficult to determine this, due to the sporadic and relatively low incidence of mealybug infestations in some orchards, or by simply relying on naturally occurring field populations of biocontrol agents. Knowledge of the environmental conditions under which P. viburni population levels may become destructive is also essential for timing the release of insectary reared natural enemies as well as understanding the population ecology of this pest and its natural enemies. Information was gathered regarding the seasonal phenology of P. viburni and its natural enemies in pome fruit orchards in the Western Cape Province during the 2007/08 and 2008/09 growing seasons. Seasonal population studies showed that P. viburni has multiple overlapping generations with all life stages present throughout the year. The highest orchard infestations occurred during the summer period until early winter (January to early June). This was followed by a decrease in population from late June to November, before another increase in December. Presence-absence sampling of mealybugs on the host plant revealed that woody parts of the tree, such as the trunk and old stems were the most preferred sites for mealybug habitation, due to the availability of protected refuge sites. Migration of mealybug populations to newer growth and the upper sections of the tree crown, such as the new stems, leaves and eventually the fruit, was observed from December throughout the summer period until the early winter in June. Fruit colonization in both apples and pears commenced in January, when the fruit had developed a size sufficient for P. viburni to penetrate and occupy spaces such as the fruit core, calyx and stem end. There was no evidence of P. viburni occurring beneath the soil surface or on the roots of host trees. Two natural enemies of mealybugs, namely Pseudaphycus maculipennis (Mercet) and Coccidoxenoides perminutus (Girault), were found to be active in apple and pear orchards in the Western Cape. However, the status of C. perminutus as a parasite of P. viburni still needs to be verified despite evidence of emergence from P. viburni mummies, which was not sufficient enough to suggest that it is a useful biological control agent. Seasonal abundance trends of the two natural enemies revealed that their lifecycle is synchronized with that of the host. However, there was no evidence of P. maculipennis activity in Ceres. No predators were found during the course of this study. The rate of P. viburni parasitism at harvest was 46.52%, with P. maculipennis and C. perminutus constituting 98.966% and 1.034% of the parasitoids recovered from mealybug mummies, respectively. Studies on the use of pheromone traps as early monitoring tools for P. viburni showed that there was a positive and significant relationship between the fruit infestation and number of P. viburni adult males caught in pheromone-baited traps (r2 = 0.454). The action threshold level was estimated to be 2.5 male P. viburni caught per trap per fortnight at an economic threshold of 2% fruit infestation. / AFRIKAANSE OPSOMMING: Pseudococcus viburni (Signoret) (Hemiptera: Pseudococcidae) (ligrooswitluis), is ‘n algemene en ernstige plaag van appels en pere in Suid-Afrika. Druk deur verbruikers en regulasies om kommoditeite onder volhoubare en ekologies verenigbare toestande te produseer het chemiese beheeropsies toenemend beperk. Inligting oor die seisoenale voorkoms van plae is een van die essensiële komponente van ‘n effektiewe en volhoubare geïntegreerde plaagbestuurprogram. Dit is in die aanvanklike beplanning van monitering en om te bepaal wanneer skade verwag kan word. Dit is ook belangrik om boorde vroegtydig te identifiseer wat die risiko het om witluisbesmettings te ontwikkel. Die ontwikkeling van effektiewe en vroeë moniteringstegnieke vir witluisbevolkings sal produsente help met besluitneming rakende plaagbestuur en die geskiktheid van gewasse vir verskeie markte. Dit is ook noodsaaklik om die teenwoordigheid en effektiwiteit van biologiese beheer agente wat natuurlik in boorde voorkom te bepaal ten einde die potensiaal van biologiese beheer as ‘n lewensvatbare alternatief vas te stel. Onder die huidige geïntegreerde plaagbestuurprotokol was dit egter moeilik om laasgenoemde te bepaal weens die sporadiese en relatiewe lae voorkoms van witluisbesmettings in sommige boorde of deur bloot staat te maak op die veldpopulasies van biologiese beheer agente wat natuurlik voorkom. Kennis van die omgewingstoestande waaronder P. viburni bevolkingsvlakke skadelik raak is ook noodsaaklik vir die beplanning van vrylating van biologiese beheer agente, asook om die bevolkingsekologie van hierdie plaag en sy natuurlike vyande te verstaan. Inligting oor die seisoenale fenologie van P. viburni en sy natuurlike vyande in sagtevrugte boorde in die Westelike Kaapprovinsie is gedurende die 2007/08 en 2008/09 groeiseisoene versamel. Seisoenale bevolkingstudies het getoon dat P. viburni verskeie oorvleuelende generasies het met alle stadia teenwoordig regdeur die jaar.