M.Sc. / Please refer to full text to view abstract

MSc., Faculty of Science, University of the Witwatersrand, 2011 / The behaviour and physiology of every insect, during all developmental stages, is largely determined by temperature. Metabolic rate, flight activity, nutrition, growth rate, oviposition and longevity can all be correlated to temperature. Consequently, insect development occurs within a definite temperature range which can be experimentally determined. This serves as a basis from which models that estimate insect growth, development and reproduction can be formulated. Such studies on temperature-dependent development are therefore important for understanding predator-prey relationships and insect population dynamics relevant in epidemiology, pest management and biological control of weeds and insect pests. The biological control of water hyacinth, Eichhornia crassipes (Pontederiaceae), in South Africa currently relies on six established agents. However, the results of this programme do not compare well with the achievements made elsewhere. This has been attributed to a number of constraining factors, chief among which is a wide variety of climatic regions, low minimum temperatures and a high incidence of frosting which slows the build-up of natural enemy populations. This research verified and augmented the thermal tolerance data available for three of South Africa’s more efficacious agents used against water hyacinth, namely Neochetina eichhorniae, N. bruchi (Curculionidae) and Eccritotarsus catarinensis (Miridae). Using these data, plant productivity and insect activity was modelled against fine-scale temperature data incorporating three distinct microclimates from 14 field sites distributed throughout South Africa’s climatic regions. Water hyacinth and its natural enemies were found to be negatively affected by low average temperatures. However, the relative consequences for each species at a population level were quite different. Similar thresholds for development, close to 10°C, meant that periods available for growth in areas where temperature is limiting were roughly the same for both plant and insects. Nevertheless, although plant growth largely ceased each winter and aerial parts were often extensively damaged from frost, low temperatures rarely led to significant plant mortality. By contrast, reduced insect recruitment coupled with a high susceptibility to cold- and frost-induced mortality of all life-history stages, pushed insect populations into winter bottlenecks and even caused local extinctions. The ability to overwinter effectively appears to the primary cause for limited control in colder regions. Surviving post-winter insect populations were therefore small, inflicted minimal damage due to reduced feeding rates, and were generally asynchronous with the recovery of water hyacinth. This asynchronous development translated into a lag period of roughly 42 days between the onset of water hyacinth growth and the time at which the plant was subjected to meaningful herbivory. Free from early season herbivory, coupled with the fact that vegetative reproduction continued through winter, water hyacinth populations were able to quickly recover and outpaced the detrimental affects caused by insect feeding well into the growth season. The implications for supplementary management strategies are also discussed in light of these outcomes.

Six pathogenic fungal species were isolated from naturally-infected Echinochloa species and evaluated as biological control agents for E. crus-galli, E. colona, and E. glabrescens in rice. Bipolaris sacchari, Curvularia geniculata, and Exserohilum monoceras were non-pathogenic to rice and caused high mortality of Echinochloa species. E. monoceras was selected for further study. Under regulated greenhouse conditions, an inoculum dose of 2.5 $ times$ 10$ sp7$ conidia/m$ sp2$ killed E. crus-galli and E. glabrescens seedlings while 5.0 $ times$ 10$ sp7$ conidia/m$ sp2$ caused 100% mortality of E. colona seedlings. The 1.5-leaf stage was the most susceptible growth stage for all three Echinochloa species. E. glabrescens was most susceptible to E. monoceras infection, E. crus-galli had an intermediate susceptibility, and E. colona was least susceptible. The optimum temperature for 100% mortality was between 20 and 30 C for all Echinochloa species, whereas the minimum dew period for 100% mortality was 16 h for E. colona, 12 h for E. crus-galli, and 8 h for E. glabrescens. Under screenhouse conditions and in the absence of an artificial dew period, over 90% of Echinochloa seedlings were killed when inoculum was sprayed in an oil emulsion or when applied as a dry powder to the water surface of a simulated paddy field. Maximum conidia production occurred on V-8 juice agar or centrifuged V-8 juice agar, at 28 C in the dark. No conidia were produced in liquid media. Of various agricultural products tested as solid substrates, the highest sporulation (1.81 $ times$ 10$ sp6$ conidia/g dry weight) occurred on corn leaves. Host range tests on 54 plant species in 43 genera and 19 families showed that Rottboellia cochinchinensis, was also highly susceptible to this fungus. Of the crops tested, only corn seedlings were lightly infected under optimum greenhouse conditions but no disease occurred on corn under field conditions. Bipolaris sacchari, Exserohilum monoceras, and E. oryzae

A culture system was established for mass rearing of the Russian knapweed nematode, Subanguina picridis (Kirjanova) Brzeski. This system consisted of two parts; a shoot culture system used as the host plant source for the nematode culture, and a monoxenic nematode culture system. The nematode developed and reproduced in this system. Galls were induced on the leaves, stems, and shoot tips of cultured Russian knapweed (Acroptilon repens (L.) DC.) shoots. After 3 months in culture, the nematode number per petri dish increased from the initial 50 inoculated to 7,000-10,000, a 140 to 200 fold increase. This study represents the first time that an above-ground gall forming nematode has been propagated in vitro. It also represents a unique monoxenic nematode culture system to mass rear above-ground endoparasitic plant nematodes. / Various factors, including tissue type, tissue age, medium, and temperature, which affect the formation and development of galls, were examined. The nematode failed to reproduce in callus tissues, and it could not develop beyond the 4th stage in excised root cultures. The optimum incubation conditions determined were: 60-80 $ mu$molm$ sp{-2}$s$ sp{-1}$ light intensity, 20 C temperature, and 4-8 mm for shoot length. / The virulence of cultured nematodes was tested in the greenhouse on Russian knapweed seedlings and vegetative shoots from root segments. The results demonstrated the feasibility and application of this novel mass production system. Nematodes produced in this system were virulent and the growth rate of infested Russian knapweed were reduced.

There has been little documentation of the success of introduced agents for classical weed biological control. Field evaluation of an insect's establishment, spread and early host impact within its new environment must be performed before agent success can either be doucmented or predicted. Population attributes of the ednophagous biological control agent, Mecinus janthinus Germar (Coleoptera: Curculionidae), and interactions with its target weed, Dalmation toadflax, (Linaria dalmatica (L.) Mill.) (Scrophulariaceae), were explored across variable levels of resource availability and insect abundance. Patterns of population growth and impact of this biocontrol agent were very consistent throughout this study. Within four years of release, populations of M. janthinus achieved outbreak population levels and virtually eliminated the seed producing shoots from toadflax stands. There is a tight but flexible relationship between oviposition site selection and offspring performance in this endophagous herbivore, maximizing offspring survival even under moderate to high M. janthinus densities. These attributes allow M.janthinus to be an effective biocontrol agent under changing levels of resource availability. / ix, 134 leaves : ill. ; 28 cm.

Cyperus rotundus L. CYPRO (purple nutsedge) and Cyperus esculentus L. CYPES (yellow nutsedge) are problematic weeds on every continent. At present there is no comprehensive means of controling these weeds.. The primary means of control is herbicides, although the weeds are becoming more resistant. Bioherbicide control of purple and yellow nutsedge is an important avenue of research, with much of the focus being to increase the virulence of current fungal pathogens of C. rotundus and C. esculentus. The primary aim of this study was to increase the virulence of a fungal pathogen of C. rotundus and C. esculentus, with the objective of creating a viable bioherbicide. A possible means of increasing the virulence of a pathogen would be to increase the amount of amino acid produced by the fungus. This was proposed as a means of increasing the virulence of Dactylaria higginsii (Luttrell) M. B. Ellis. Overproduction of amino acids such as valine and leucine result in the feedback-inhibition of acetolactate synthase (ALS), an enzyme which is a target for many herbicides currently on the market. By applying various amino acids to tubers of purple nutsedge and comparing the results with a reputable herbicide, glyphosate, it was possible to determine the success of the amino acid applications. Only glutamine treatment at 600 mg.r1 resulted in significantly less (P<O.OOI) germination compared with the water control, while the glyphosate application resulted in no germination. Four treatments were significantly different (P<O.OOI) from the water control in terms of shoot length, but no pattern or conclusion could be drawn from the results. Injecting amino acids and glyphosate into the leaves of the plants gave similar results to those obtained with the tubers, with no visible damage on those plants injected with the amino acids and complete plant death of those injected with glyphosate. Amino acids had little effect on the growth of the C. rotundus plant or tuber. It was later determined by a colleague (Mchunu1 , unpublished) working on the same project, that D. higginsii does not infect the local ecotypes of C. rotundus in Pietermaritzburg, South Africa. A second fungus, Cercospora caricis Oud., was isolated from C. rotundus growing in the region, and confirmed as a Cercospora species by conidial identification. Like many Cercospora species, C. caricis produces a phytotoxin, cercosporin. An increase in production of cercosporin would theoretically lead to an increase in virulence of C. caricis. Mutation of hyphae by i J Makhosi Mchunu: Address: National department ofAgriculture; Private Bag 3917; Port Elizabeth; 6056 Email: Makhosimc@NDA.agric.za ultraviolet-C light was perfected on C. penzigii Sacc., where 5 min exposure to DV-C light resulted in approximately 99% cell death. Surviving colonies were analysed by spectrophoresis, and the surviving mutant gave an absorbance value of approximately 5% more than the median. Samples were analysed by high-performance liquid chromatography (HPLC) to determine the presence of cercosporin. No definitive result was obtained. Exposure of C. caricis to DV-C for 5 min. resulted in approximately 65% hyphal cell death, with 20 min. resulting in approximately 95% death. A spontaneous mutant was observed in a colony that had been exposed to DV-C. This mutant showed sectored growth with red and grey growth patterns. The red section of the mutant was subcultured and analysed by spectrophoresis and HPLC. The red C. caricis gave an absorbance reading of approximately 140 on HPLC compared with about 22 from the grey colony. HPLC analysis of the wild-type C. caricis did not produce a peak corresponding to that of the cercosporin standard, although no conclusion could be obtained on the presence or absence ofthe toxin. The virulence of the mutant C. caricis could not be determined as inoculation experiments were unsuccessful, and had to be discontinued due to time constraints. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

The effect of spore titer, virulency, and carrier systems on biological control of giant foxtail (Setaria faberi hermm.) with a species of Phoma isolated from this weed wereinvestigated. The lowest concentration of Phoma conidia which significantly affected (lowered) growth of giant foxtail was 1x106 conidia/ml. In conducting Koch's postulates, all but one (FF2) of the Phoma, isolates tested infected giant foxtail. Isolates FF1 and FF9 were the most virulent against giant foxtail. No correlation was apparent between virulency and fungus germination rate. No visible difference occurred with in vitro germination rates of spores incubated upon pea vsr water agar media; nutrient rich vs. nutrient poor media, respectively. These results suggest that Phoma spores already contain sufficient nutrients required for infection of foxtail. A definitive answer as to the effect of carrier system (e.g. surfactant, sticker, spreader, etc.) on efficacy of Phoma to infect foxtail could not be determined from results obtained, based upon statistical analysis of variance (ANOVA) for the single experiment performed. Several of the surfactant treatments (e.g. Silwet 77 and 408) did produce significant biomass losses against foxtail due to the phytotoxicity of the carrier system alone, and not reflective of fungal (Phoma) infection. Inoculation of foxtail plants with a conidial suspension amended with carriers of either Tween 20 (0.05%-0.1%) or methylcellulose (0.1%) should optimize chances for sufficient infection resulting in biological control of this weed. / Department of Biology

Inoculation studies were conducted in controlled environments with isolates of a Phoma sue. collected from leaf spot lesions on the weed giant foxtail. Limited host specificity studies resulted in this potential bioherbicide fungus killing all three foxtail species tested including Setaria faberi (giant), a. viridis (green) and S. lutescens (yellow). Several agronomically important plant species tested exhibited a hypersensitive-type response to infection, but these plants soon grew out of this symptom and appeared healthy. This bioherbicide preferred cool temperatures, exhibiting optimal biomass loss (100%) or death against foxtail seedlings following 120 hours leaf wetness with plants incubated at 20°C. At more conducive growth temperatures for the weed (25'C-300C), optimum biomass loss achieved was only 70% following 50 hours leaf wetness. Susceptibility to this Phoma sue. greatly decreased as foxtail seedlings attained 4 or more leaves per plant. To kill this weed, the author recommends inoculation of foxtail seedlings in early evening to take advantage of cooler temperatures and to inoculate plants between cotyledon to 3-leaf stages of growth. / Department of Biology

Water lettuce, Pistia stratiotes Lamarck (Araceae) is a South American plant that has the potential to be a very damaging and important aquatic weed in many tropical countries, including South Africa. It has the potential to rapidly multiply vegetatively and completely cover watercourses in a very short space of time outside of its natural range under ideal conditions and without its natural enemies. In such instances, the weed may cause hindrances to water transport and fishing, increasing chances of malaria, as well as affecting the natural ecology of the system. Water lettuce can also set seed, which may lay dormant for long periods, germinating when conditions are favourable. It is therefore very necessary to adopt control methods against the weed where it is a problem. However, water lettuce has also been effectively and completely controlled in many countries by the leaf-feeding weevil, Neohydronomus affinis Hustache. High nutrient levels in the form of nitrates and phosphates have been shown to have largely negative effects on biological control in several studies, with control being incomplete or taking longer than in similar areas with lower nutrient levels. The effectiveness of N. affinis on the biological control of water lettuce was investigated in a laboratory study, growing P. stratiotes plants with and without insects at different nutrient concentrations. In these studies biological control of water lettuce with N. affinis was found to be complete under eutrophic nutrient conditions, although control took longer when higher nutrient levels were tested. A field site study was conducted at a sewage settlement pond in Cape Recife Nature Reserve near Port Elizabeth, South Africa. This highly eutrophic system was used as a field example for the effectiveness of biocontrol of P. stratiotes by N. affinis under eutrophic conditions. The weevils at Cape Recife caused a massive and rapid crash in the percentage coverage of the weed, from 100% in May 2003, to approximately 0.5 % in September 2003. Plant growth parameters were also found to decrease considerably in size correspondingly with this crash from May 2003 until spring 2003. Plant size only again started to increase gradually but steadily through spring 2003 and into summer. In the laboratory studies, the fecundity of weevils was shown to be much higher on plants grown under higher nutrient concentrations than on plants grown in lower nutrient concentrations. The results from the wing-muscle analysis under different nutrient concentrations were not easy to interpret, and there were few differences in wing muscle state between most of the concentrations. From these findings it is suggested that nutrient concentration, particularly high levels of nitrates and phosphates is not a limiting factor in terms of effective biological control of P. stratiotes with N. affinis, but that under high nutrient conditions biological control might take longer.

From the introduction: The work on Mimorista is divided into two sections: a laboratory orientated study which describes the biology and rearing techniques and a field orientated study where the establishment of the insect and its impact on jointed cactus populations are examined. In the final chapters the integration of this insect in the current control program are discussed and recommendations regarding its future in South Africa are given.