A produção brasileira de alimentos cresce em ritmo vertiginoso, havendo previsão de expansão nos próximos anos. Nota-se, porém, que o modelo atual empregado ao controle de doenças e pragas agrícolas, tem causado diversos problemas de ordem ambiental, social e econômica. Uma alternativa à redução de tais impactos, tem sido a implementação do controle biológico no modelo de manejo. Neste trabalho, avaliou-se o controle biológico do fungo fitopatogênico Botrytis cinerea por compostos produzidos pela linhagem ASBV-1T. Os screenings \"in vitro\" demonstraram que a linhagem produz quitinases e metabólitos secundários ativos, conhecidamente descritos como fatores importantes ao controle de fitopatógenos. A caracterização parcial do complexo enzimático, indicou que as quitinases produzidas pela linhagem ASBV-1T apresentam maior atividade em meio alcalino (pH 6.8-10.0), temperatura de 30°C e possuem estimativa de peso molecular superior a 100 kDa. Os ensaios \"in vivo\", realizados em morangos comerciais (cv. Oso Grande) demonstraram a efetividade dos subprodutos bioativos de caráter ionóforos (monactina, dinactina, trinactina, tetranactina e valinomicina) em controlar a infestação de B. cinerea, durante a fase de pós-colheita, em condições de armazenamento. Por meio da suplementação do meio de cultivo por sais inorgânicos, a via biossintética responsável pela expressão dos compostos de interesse pode ser ativada. A adição de MgS04 resultou em um aumento de 400% na produção de macrotetralídeos e 20% de valinomicina. Contudo, a expressão dos macrotetralídeos foi completamente inibida pela adição de ZnSO4. dobrando a produção de valinomicina. De acordo com os estudos de taxonomia polifasica, o isolado ASBV-1T, apresenta caracteristicas quimiotaxonômicos e moleculares pertinentes ao gênero Streptomyces, formando uma nova linha filética, no subclado das espécies S. albolongus, S. cavourensis subsp. cavourensis e S. celluloflavus. Diante do supra citado, propõem-se que o isolado seja reconhecido como a linhagem tipo de Streptomyces araujoniae sp. nov. Os resultados obtidos neste trabalho, permitem afirmar que a linhagem S. araujoniae sp. nov. apresenta potencial de ação contra o fitopatógeno B. cinerea, podendo auxiliar no controle desta doença durante a fase de pós-colheita da cultura de morangos. Ainda resta, para estudos futuros, o desenvolvimento de formulação específica para o emprego destes compostos no manejo da cultura, bem como ensaios toxicológicos, assegurando a viabilidade de aplicação de tais compostos. / The Brazilian food production has been growing fast in recent years and is expected to expand even more. However, the models used for the control of pests and diseases in agriculture has been widely questioned because they cause problems of environmental, social and economic. To reducing the use of chemical agents, has been the implementation of biological control to the management model to reduce this problematic. In this study, we evaluated the biological control of fungal pathogen Botrytis cinera of active compounds produced by strain ASBV-1T. In vitro assays indicated the presence of chitinase and antifungal metabolites in compounds produced by strain ASBV-1T and may been contributed to the antagonistic activity againts the fungal pathogenic. Partial characterization of the enzymatic complex showed the highest production of chitinase under alkalines conditions at pH (6.8-10.0), 30°C and has estimated molecular weight above 100 kDa. The test in vivo, preformed in commercial strawberries (cv. Oso Grande) demonstrated the effectiveness of bioactive products of character ionophores (monoactin, dinactin, trinactin, tetranactin and valinomycin) controlling the infestation of B. cinerea during the post-harvest, in store conditions. The biosynthetic pathway responsible for the expression of the compounds of interest can be activated by supplementations of the culture medium by inorganic salts mainly with MgSO4 resulted in increse of 400% in the production of macrotetralides and 20% valinomycin. However, the expression of the macrotetralides was inhibited when added ZnSO4, bending the production of valinomycin. According to polyphasic taxonomic studies, the isolated ASBV-1T has chemotaxonomic and molecular characteristics of the genus Streptomyces, which formed a new phyletic line, in subcalde of the S. albolongus, S. cavourensis subsp. cavourensis e S. celluloflavus species. Then propose that the isolate is recognized as the type strain of Streptomyces araujoniae sp. nov. The results obtained in this study revealed that S. araujoniae sp. nov. has action potential against the pathogen B. cinerea, can help control this disease during the post-harvest strawberries. Remains for future studies, the development of specific formulation of the employment of such compounds to crop management and toxicological tests, ensuring the feasibility of using such compounds.

Field trials were conducted in 1988 to evaluate the effectiveness of Steinernema (=Neoaplectana) carpocapsae (Weiser) in controlling the fire ant. Infective juveniles (IJ) of the nematode were applied as drench on 235 and 422 mounds, respectively for 2-month summer and 6-week fall evaluation periods. In comparative trials, amidinohydrazone (Amdro) was applied to 249 (summer) and 65 (fall) active mounds, with 245 (summer) and 78 (fall) untreated active as controls. Nematode treatments resulted in an average of 47% control (Abbott's formula) in summer trials and 19-88% control in the fall trials, compared with 39% and 47% control, respectively with amidinohydrazone. Active mounds treated with nematodes or amidinohydrazone had significantly fewer individuals than control mounds in summer trials.

Several ant species may be associated with the same honeydew producing insect in citrus orchards. Recently antagonism and competition between two such ants were reported from Letaba. This report stimulated the present study, the purpose of which is to ascertain which ant species are most numerous in citrus orchards ln the Eastern Cape Province; the reasons for these ants being able to occupy orchards; and, finally, their relationships. A survey of the Sundays River Valley and the Fish River Valley revealed that three species - Anoplolepis custodiens Smith, Anoplolepis steingroeveri Forel, and Pheidole megacephala Fabricius - were commonly found in citrus orchards in association with Soft Brown Scale insects. The territorial relationships of these ants were determined by plotting their distribution at intervals for over a year. It was found that this distribution could be correlated with seasonal climatic conditions, the effects of farming practices, brood rearing requirements and behavioural differences between the three species. The ant, A.steingroeveri, had not been previously studied, and since foraging is an integral part of territorial behaviour, some aspects of its biology, and more especially its foraging activity, were investigated to compare it with the known biology of the other two species. Observations on the foraging activity of A. steingroeveri were made at monthly lntervals for almost a year, and correlated with climatic conditions prevailing at the time. Some seasonal and dally variations in foraging behaviour could not be readily explained in terms of the effects of temperature and humidity on the ants. Other factors, includ1ng trophallactic stimulation and overcast skies, were found to influence ant activity. Owing to the apparent lack of information in the literature and possibility of periodic honeydew production by Soft Brown Scale insects controlling ant activity was investigated in the laboratory. The results of this study showed that it was not likely that honeydew excretion restricted the activity of the ants. Several investigators have also found that many interrelated climatic factors correlated with ant foraging behaviour, but have expressed dissatisfaction at not being able to reach the crux of the matter. To clarify the issue a constant temperature chamber was constructed in which a colony of Crematogaster ants was housed and the ants were subjected to a series of temperatures at different humidities, all other factors were eliminated or kept constant. The effects of these two factors on the thermokinetics and foraging activity of the ants were registered. This thesis contains several sections and at the end of each the results are discussed in the light of the available literature and comparisons are drawn between the species under investigation. The main results are given in the summary. After the summary two appendices are included. One is an attempt to compare the effect on ant size of the honeydew diet of orchard colonies with the proteinaceoue diet of veld ants. The other appendix is an experiment to control citrus ants with an insecticide.

Chromolaena odorata (L.) King and Robinson is an alien invasive weed to most of the Old World tropical regions of the earth, including South Africa where it is morphologically distinct from most other C. odorata plants examined from both its native and invasive range. It is thought that these morphological differences are related to difficulties encountered in successful establishment of biological control agents on the South African population of C. odorata. It has been postulated that the source population of the South African population will harbour potential biocontrol agents that will be suited to successful establishment on the South African plants. Several morphological, cytological and isozyme studies have been attempted to identify the source population of the South African population, but these have failed to identify the origin of the South African population. In this dissertation two PCR-based methods were attempted, in an investigation into whether the morphological differences and difficulties in establishment of biocontrol agents have a genetic basis. The two techniques attempted were: Inter Simple Sequence Repeat (ISSR) amplification, and DNA sequencing. Results could not be obtained using the ISSR method, and the reason for this was not discovered despite extensive trials. The internal transcribed spacer region and the external transcribed spacer region sequences were obtained from five samples, and compared. It was found that the ETS region gave more phylogenetic signal at the intraspecific level than the ITS region. However, due to difficulties in amplification of the external transcribed spacer region, work here focussed on obtaining Internal Transcribed Spacer sequences for 61 samples. Each of the samples sequenced had a unique ITS sequence, displaying a high level of intraspecific genetic diversity. The degree of this diversity is discussed with reference to the possible influences of polyploidy and concerted evolution on genetic structure. The ITS data indicated that some of the physical traits used to define ‘morphotypes’ of C. odorata were not correlated to genotype. From discussion and comparison of morphological character distributions and the ITS-based phylogeography it is suggested that the geographical origin of the South African population is Greater Antilelan, rather than from the continents of North and South America, which is where the Australasian, West African and Mauritian infestations are suggested to have originated.

The biology of key citrus pests Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Ceratitis rosa Karsch (Diptera: Tephritidae) includes their dropping from host plants to pupate in the soil below citrus trees. Since most EP fungi are soil-borne microorganisms, the development and formulation of alternative control strategies using these fungi as subterranean control agents, targeted at larvae and pupae in the soil, can potentially benefit existing IPM management of citrus in South Africa. Thus, a survey of occurrence of entomopathogenic fungi was undertaken on soils from citrus orchards and natural vegetation (refugia) on conventionally and organically managed farms in the Eastern Cape Province in South Africa. A method for baiting soil samples with citrus pest T. leucotreta and C. capitata larvae, as well as with the standard bait insect, Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), was implemented. Sixty-two potentially useful entomopathogenic fungal isolates belonging to four genera were collected from 288 soil samples, an occurrence frequency of 21.53%. The most frequently isolated entomopathogenic fungal species was Beauveria bassiana (Balsamo) Vuillemin (15.63%), followed by Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin (3.82%). Galleria mellonella was the most effective insect used to isolate fungal species (χ2=40.13, df=2, P≤ 0.005), with a total of 45 isolates obtained, followed by C. capitata with 11 isolates, and T. leucotreta with six isolates recovered. There was a significantly (χ2=11.65, df=1, P≤ 0.005) higher occurrence of entomopathogenic fungi in soil samples taken from refugia compared to cultivated orchards of both organically and conventionally managed farms. No significant differences were observed in the recovery of fungal isolates when soil samples from both farming systems were compared. The physiological effects and host range of 21 indigenous fungal isolates obtained in the Eastern Cape were investigated in the laboratory to establish whether these isolates could be effectively used as biological control agents against the subterranean life stages of C. rosa, C. capitata and T. leucotreta. When these pests were treated with a fungal concentration of 1 x 10⁷ conidia ml⁻¹, the percentage of T. leucotreta adults which emerged in fungal treated sand ranged from 5 to 60% (F=33.295; df=21; P=0.0001) depending on fungal isolate and the percentage of pupae with visible signs of mycosis ranged from 21 to 93% (F= 96.436; df=21; P=0.0001). Based on fungal isolates, the percentage adult survival in C. rosa and C. capitata ranged from 30 to 90% and 55 to 86% respectively. The percentage of C. rosa and C. capitata puparia with visible signs of mycosis ranged from 1 to 14% and 1 to 11% respectively. Deferred mortality due to mycosis in C. rosa and C. capitata adult flies ranged from 1 to 58% and 1 to 33% respectively, depending on fungal isolate. Entomopathogenic fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult survival or adult and pupal mycosis were considered. The relative potency of the four most virulent Beauveria isolates as well as the commercially available Beauveria bassiana product, Bb Plus® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against C. rosa, C. capitata and T. leucotreta which all exhibited a dose-dependent response. Against fruit flies the estimated LC50 values of all five Beauveria isolates ranged from 5.5 x 10¹¹ to 2.8 x 10¹² conidia/ml⁻¹. There were no significant differences between the relative potencies of these five fungal isolates. When T. leucotreta was considered, isolates: G Moss R10 and G 14 2 B5 and Bb Plus® were significantly more pathogenic than G B Ar 23 B3 and FCM 10 13 L1. The estimated LC₅₀ values of the three most pathogenic isolates ranged from 6.8 x 10⁵ to 2.1 x 10⁶ conidia/ml⁻¹, while those of the least pathogenic ranged from 1.6 x 10⁷ to 3.7 x 10⁷ conidia/ml⁻¹. Thaumatotibia leucotreta final instar larvae were exposed to two conidial concentrations, at four different exposure times (12, 48, 72 and 96 hrs) and showed an exposure time-dependant relationship (F=5.43; df=3; P=0.001). At 1 x 10⁷conidia/ml⁻¹ two Beauveria isolates: G Moss R10 and G 14 2 B5 were able to elicit a response in 50% of test insects at 72 hrs (3 days) exposure. Although a limited amount of mycosis was observed in the puparia of both fruit fly species, deferred adult mortality due to mycosis was high. The increased incidence of adult mortality suggests that post emergence mycosis in adult fruit flies may play a more significant role in field suppression than the control of fruit flies at the pupal stage. The increased incidence of pupal mortality, as well as the relatively low concentrations of conidia required to elicit meaningful responses in T. leucotreta pupae may suggest that pre-emergent control of false codling moth will play a more significant role in field suppression than the control of adult life stages using indigenous isolates of entomopathogenic fungi. Various entomopathogenic fungal application techniques targeted at key insect pests within integrated pest management (IPM) systems of citrus are discussed.

[Introduction] In the Western Cape Province the mealybug, Planococcus citri (Risso), was first reported on vines in 1930 by Joubert (1943a). By 1935 this mealybug had spread to the Hex River Valley, and subsequently to all the main table grape producing areas of the Western Cape Province. At present Pl. citri is the most important insect pest of the table grape industry and can, if not effectively controlled, result in a loss of at least five per cent of the export table grape crop (Kriegler, 1954). Some preliminary work on the natural enemies of Pl. citri on vines was carried out by Stubbings in 1948, but no further work of this nature has been undertaken in this area since then. The fact that the natural enemies can be an effective check to this mealybug on vines in the Western Cape Province has been known for a number of years (Potgieter, 1937; Hattingh, 1943; Joubert, 1943a; Myburgh, 1951). The present work is an attempt to obtain some basic knowlege of the population fluctuations of the insects concerned in this biological control. Surveys undertaken have shown that there is a complex of primary, secondary and possibly tertiary Hynenopterous parasites associated with Pl. citri. The presence of hyperparasites reduces the efficiency of the primary parasitic Hymenoptera. The usefulness of these primary parasites is further reduced as they only occur in effective numbers for a short period of the year. On the other hand, although attacked by some parasites, the numerous coccinellids found preying on Pl. citri are of more importance in reducing the mealybug populations, as they are present on the vines in effective numbers for the greater part of the year.

Phthorimaea operculella, commonly known as the Potato Tuber Moth, is an economically important agricultural pest worldwide. The baculovirus, Phthorimaea operculella granulovirus (PhoGV) has been considered as a means of control alternative to chemical control because of its host specificity and harmless impact on other organisms and ecosystems. An isolate of PhoGV obtained from a South African PTM population would be beneficial in the production of a biopesticide, which is not yet available. An efficient and cost-effective rearing method would be advantageous for potential commercial production. Commercial table and seed potato plantations and storage facilities located in Patensie, Bathurst, Howick and Ivanhoe were surveyed for PTM infestations. Patensie was the only site where milky discoloured larvae were found, a potential symptom of PhoGV infection. TEM analysis revealed no virus in these samples. Since no virus was found in the field-collected samples, PTM insects were collected to initiate rearing in the laboratory. PTM was raised by three different methods in the laboratory. A cost/benefit analysis, survival rate, fertility and sex ratio were recorded for each rearing method. Rearing method one was deemed unsuccessful for efficient commercial rearing, as survival percentage and fertility were low. Rearing methods two and three had high survival rates and high fertility, and were efficient and less labour intensive than rearing method one. Rearing method three was the most productive technique, but for commercial production rearing method two was considered the most manageable and efficient. The sex ratio was 1:1 for all three cultures. The cost analysis revealed that rearing methods two and three were less expensive than rearing method one because less labour was required to monitor insects. The success of rearing PTM for 19 months will enable these cultures to be up-scaled to a large production facility for mass rearing. Virus was not found in the field surveys or in laboratory cultures, therefore chemical, temperature, humidity and carbon dioxide stressors were used in an attempt to initiate a baculoviral infection. Symptoms were exhibited in larvae subjected to chemical, temperature and humidity treatments, but these were confirmed by TEM analysis not to be a result of PhoGV infection. The success of rearing PTM in the laboratory suggests that the method could be used in the commercial rearing of the insects in a large mass-rearing facility. The data obtained from induction protocols have allowed for better understanding for future induction for PhoGV and other baculoviruses in other insect species. The failure to isolate a South African PhoGV strain for developing a biopesticide against PTM has motivated further studies in obtaining a baculovirus in order for South Africa to develop a commercial product against this pest.

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a major pest of Brassica crops in many parts of the world. Because of its ability to develop resistance to virtually all major groups of insecticides, including Bacillus thuringiensis Berliner (Bt), much attention has therefore been given to biological control using parasitoids. South Africa has an abundance of parasitoids attacking this pest. Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae) is the most abundant larval parasitoid of P. xylostella in South Africa. In East Africa, its role in biological control of P. xylostella is insignificant, and the most abundant parasitoid there is Diadegma mollipla (Holmgren) (Hymenoptera: Ichneumonidae), a larvalpupal parasitoid. In South Africa, however, D. mollipla is out-competed by C. plutellae. Total parasitism of P. xylostella in East Africa rarely exceeds 15%, therefore there is a need to introduce more effective and heat-tolerant parasitoids of P. xylostella to that region. This study was therefore initiated to examine the potential of C. plutellae and D. mollipla as biological control agents by studying certain aspects of their biology in the laboratory, as well as the suitability of C. plutellae for introduction into East Africa. Biological aspects studied were: (i) host instar preference, fecundity, and searching efficiency of C. plutellae and D. mollipla at different host and parasitoid densities; (ii) effects of temperature on parasitism of P. xylostella by C. plutellae and D. mollipla, and on their rates of development and emergence. In addition, the role of parasitoids in controlling P. xylostella on unsprayed cabbage plots was investigated. Both C. plutellae and D. mollipla preferred to attack second and third instar hosts than fourth instars in choice and no-choice tests. However, D. mollipla attacked more fourth instar hosts than C. plutellae. Cotesia plutellae laid mainly female eggs in second and third instar hosts than in fourth instars, whereas D. mollipla laid more female eggs in fourth instar hosts than in second and third instar hosts. Diadegma mollipla was more fecund [82.57 ± 32.87, (mean ± s.d.) than C. plutellae (42.13 ± 12.2), and was long lived (7.13 ± 3.69 days) compared to the latter (5.23 ± 2.7 days). An increase in host density resulted in the reduction in the area of discovery (a) and the killing power (K) for both parasitoids. No significant differences were detected between the searching efficiency (t = -1.42NS, d.f. = 48, P < 0.001) of the two parasitoids. An increase in parasitoid density also resulted in a decline in searching efficiency, but not the killing power, of both parasitoids. Cotesia plutellae completed development at all temperatures tested (21-33°C), whereas D. mollipla completed development at temperatures from 18-30°C, and C. plutellae had a lower threshold for development (8.14°C) compared to D. mollipla (10.23°C). At all tested temperatures, the generation time for C. plutellae was shorter compared to D. mollipla. The possible reasons for the dominance of C. plutellae over D. mollipla in the field are: shorter generation time, high production of female progeny in younger hosts, low interference among searching females, and relatively wide thermal tolerance. The role of parasitoids in regulating diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), populations was studied for two years (February 2000-January 2002) on unsprayed cabbage fields at Brits, North West Province, South Africa. Cabbage seedlings were transplanted in three consecutive times each year. Cabbage infestations by P. xylostella larvae and pupae, and their parasitoids, were monitored at weekly intervals. The flight activity of P. xylostella male moths was monitored using sex-pheromone traps. Trap catches indicated that the moths were active throughout the year. The flight activity of the moths corresponded with infestations on the crop. Trap catches and infestation levels were generally low from December to August and high from September to November. Eight hymenopteran parasitoids were reared: the larval parasitoids Cotesia plutellae (Kurdjumov) (Braconidae) and Apanteles halfordi (Ullyett) (Braconidae); the larval-pupal parasitoids Oomyzus sokolowskii (Kurdjumov) (Eulophidae) and Diadegma mollipla (Holmgren)(Ichneumonidae); the pupal parasitoid Diadromus collaris (Gravenhorst) (Ichneumonidae); and the hyperparasitoids Eurytoma sp. (Eurytomidae), Mesochorus sp. (Ichneumonidae), and Pteromalus sp. (Pteromalidae). Cotesia plutellae was the most abundant parasitoid of P. xylostella followed by O. sokolowskii, D. collaris, A. halfordi and D. mollipla. Parasitism of P. xylostella larvae was high reaching 100% on several occasions during late spring to end of autumn (November-May) each year. Parasitism was lower (<50%) in winter and early spring (June-September). Apanteles halfordi was absent in winter but re-appeared in spring. Parasitism of P. xylostella pupae by D. collaris was high only during spring (September-October). Hyperparasitism was generally low except when primary parasitoids were abundant in spring (September-November) and summer (December-February) when up to 25% of P. xylostella larvae and C. plutellae cocoons yielded hyperparasitoids. The role of other biotic and abiotic mortality factors on the population dynamics of P. xylostella is discussed.

Please read the Summary/Samevatting on pp 138-141 in chapter 6 of this document / Thesis (PhD (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

The woodwasp Sirex noctilio is one of the most serious invasive pests of Pinus plantations in the southern hemisphere. Extensive control programs have been developed to manage this pest, of which biological control has been a major component. This thesis examined the factors that could influence the control of S. noctilio in South Africa. A critical comparison of S. noctilio infestations and control efforts throughout the southern hemisphere revealed that control has not been uniformly effective, and local adaptation of control strategies is likely required as S. noctilio moves to new areas. The parasitic nematode Deladenus siricidicola is considered the primary biological control agent of S. noctilio. This nematode also feeds on the fungal symbiont of S. noctilio, Amylostereum areolatum. Possible factors influencing the success of this nematode in the summer rainfall areas of South Africa were examined. Data from a field trial revealed that moisture content of the wood influences inoculation success and this is influenced by the time of inoculation and the section of the tree inoculated. Laboratory-based assays revealed that incompatibility between the strain of A. areolatum and D. siricidicola was unlikely to be the cause of low inoculation success with the nematode, but that artificial inoculations could be affected by competition of A. areolatum with sapstain fungi. The parasitic wasp Ibalia leucospoides is another biological agent for S. noctilio. Mitochondrial and nuclear DNA sequence data reflected the extensive introduction of I. leucospoides into the southern hemisphere, followed by genetic bottlenecks that fixed only a few haplotypes in the introduced populations. Promoting awareness of S. noctilio in the forestry community has also been an important component of the control strategy. Data from a survey questionnaire showed that the awareness campaign had been generally successful, but the lack of basic knowledge to identify S. noctilio and its symptoms and the poor reach of the awareness media to some sectors of the forestry community, was of concern. This thesis has contributed towards understanding the factors that influence the control of S. noctilio in South Africa, with relevance to other regions where S. noctilio has been introduced. / Thesis (PhD)--University of Pretoria, 2010. / Zoology and Entomology / Unrestricted