The history of the development in Natal of a forecasting service to warn of outbreaks of late blight disease caused by Phytophthora infestans is presented. The late blight pathogen and Alternaria solani, the causal organism of early blight disease, interact on potatoes and tomatoes to form a blight disease complex. Evidence is presented to show that it is expedient to manage this blight complex as a whole rather than to direct control at only one of the components in ignorance of the consequential enhancement of the potential of the other. In a search for an improved blight complex management strategy, factors concerning the possible existence of an annual migration of Phytophthora infestans inoculum, first postulated in the 1960's, along an east-west route across Natal, are collected and collated. Corroboration of the existence of the Phytophthora-pathway is given, inasmuch as it represents a serial outbreak of late blight along a temporal gradient. The possibility that the pathway is a manifestation of disease resulting from the erruption of pre-existing inoculum along an environmental gradient, can not specifically be excluded. However, the peculiar pattern of anabatic and katabatic winds along a river-valley network, superimposed on a continuous cropping pattern and its concomitant opportunity for blight to be endemic in the province, supports the postulated Phytophthora-inoculum pathway A fungicide spray trial was conducted in order to investigate the possibility of us i ng the pathway phenomenon as the framework for an improved blight control strategy and to explore the nature and level of the competitive interaction between Phytophthora infestans and Alternaria solani. This trial revealed that the interaction between the components of the blight complex was differentially altered by weather patterns and fungicide combinations. Treatments in which metalaxyl (Ridomil) alone was used for the control of late blight, gave a yield similar to those with propineb (Antracol), which inhibits A. solani primarily but also hus some negative effect on P. infestans. The yields from both these treatments were siguificant ly (p < 0,05) better than the yields recorded in the unsprayed control plots. A treatment in which Ridomil and Antracol were combined such that each was applied according to its recommended concentration, gave yield increases of 32,3% over the unsprayed control, although the yield from the Ridomil/Antracol treatment was not significantly greater (p < 0,05) than the yields recorded where either Ridomil or Antracol were used. A computer simulator, named GAUSE, was developed to simUlate the consequences of the competition between various combinations of P. infestans and A. solani. Results simulated by GAUSE corroborated those obtained from the field trial and support the conclusion that diseases of complex etiology require more than simplistic, univariate analysis of single cause-and-effect pathways. The competition quotient CQ is developed as a new parameter of competitive interactions. It is calculated as the ratio of the amount of disease in the absence of competition, to the amount of disease when the causal pathogen is competing with another pathogen in the same niche. The CQ may be calculated from various standard epidemiology statistics and it is used to demonstrate that the competitive displacement phenomenon places constraints on the interpretation and application of Vanderplank's basic epidemiology equations. A new pathosystems management concept namely the pathotope (pathos = suffering; topos = place0 concept, is introduced, having developed from the notion that epidemics have spatial as well as temporal attributes. Accordingly, an area in which individual farms are at the same level of probability at risk to disease, delimits the pathotope. The concept can be described at many integration lsvels and is presented as an important quantitative unit of comparative epidemiology. The pathotope concept accomodates such notions as are contained in the postulated Phytopnthora-pathway and is especially suited to integration with disease forecasting methods. An example of the application of the pathotope approach is presented and a strategy is proposed by which fungicide spraying is initiated and applied synchronously as determined by the degree of communal risk to attack and epidemic increase of disease. Within a pathotope, several common factors collectively determine the vulnerability of the group to disease. If a coherent, uniform strategy is to be developed and implemented by pathotope members, it is necessary that all members have access to the relevant information and that it be collected and disseminated conveniently and rapidly. A computer-based disease monitoring and mapping system which achieves these objectives is presented. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1980.

Avocado sunblotch disease is a graft-transmissible disorder known for over 60 years and has now been recorded in at least eight countries around the world. Affected trees develop yellow, depressed streaks on young stems and fruit, marked rectangular cracking of the mature bark and a decumbent style of growth. Often a tree with symptoms produces completely symptomless shoots, termed 'recovery' growth, which are latently infected. There is a reported 95 to 100% transmission of sunblotch through the seed of such branches, and "the resultant seedlings are themselves symptomless. Indexing for sunblotch to ensure that scion and, in view of seed transmission, especially rootstock material is free of the disease is very important . The standard method used for many years has been to graft tissue onto healthy indicator seedlings and observe for symptom development for 18 months to two years. One aim of the study presented in this thesis was to develop more rapid methods for detecting the sunblotch agent. By conducting the standard indexing method in a glasshouse at controlled high temperatures of 30/28º C (day/ night) and by cutting back the indicator plants every three months, the time was reduced from two years to eight months. While this represents a considerable saving in time, the ideal must be to develop a laboratory diagnostic test that requires no more than a few days, at most, to complete. A comparative study was therefore initiated on the phenol metabolism of healthy and sunblotch-infected avocados to determine whether infection causes any major change that may reliably serve as a marker for diagnostic purposes. Significantly increased peroxidase (PO) and phenylalanine ammonia-lyase (PAL) activities, decreased indoleacetic acid (IAA) oxidase activity and higher sinapic acid levels were detected in bark tissue showing sunblotch symptoms, but not in symptomless 'recovery' growth. In contrast, increased polyphenoloxidase (PPO) activity and isoenzymes, total soluble protein levels, water soluble phenols and reduced ferulic acid levels were found in the bark of all infected trees tested, both with symptoms and symptomless. However, these latter changes have been associated with other plant-virus systems and are therefore not necessarily specific for sunblotch. Neither is any sufficiently large to be definitive as a diagnostic test. Two unidentified phenols were detected in infected, mature bark, but not in infected young bark and leaves. introduced the possibility of rapid disease detection by polyacrylamide gel electrophoresis (PAGE) of extracted RNA's as used for known viroids. In this study the presence of previously reported small molecular weight sunblotchassociated RNA's was confirmed using PAGE methods requiring two to four days to complete. This thesis presents as a further development a more rapid method of PAGE detection of RNA's enabling indexing for sunblotch to be completed in under six hours. Whilst the biochemical studies did not reveal diagnostically meaningful differences between healthy and infected avocados, there were tendencies towards differences between healthy and symptomless carrier tissues, further investigation of which may lead to a future understanding of symptom development and the symptomless condition. These include apparent higher PO and lower PAL activities in symptomless carrier tissue, as well as higher PO isoenzyme a[1] and lower IAA oxidase isoenzyme a[1] activities. General studies on sunblotch-infected avocados showed that fruit from symptomless 'recovery' growth branches are significantly larger and have a higher oil content than those from healthy or diseased branches, the latter finding possibly indicating a more advanced state of maturity of 'recovery' growth fruit due to earlier flowering. The avocado sunblotch agent was shown to have an in vivo thermal inactivation point of 55º C, a temperature higher than the avocado tissue can withstand thereby eliminating the possibility of thermotherapy of infected twigs. In a host range study four lauraceous plant species, Persea Schiedeana, Cinnamomum zeylanicum, C. camphora and Ocotea bullata, were successfully infected with sunblotch by grafting from infected avocado. This is the first demonstration of any host other than avocado. A phanerogametic member of the same family, Cassytha filiformis, was shown to be able to transmit the disease from avocado to avocado. No hosts from other families were found. During an electron microscope study of sunblotch-infected avocado leaf tissue, gross alterations of the chloroplasts in the yellow areas were observed. These changes included organelle swelling, loss of grana and stroma lamellae, rearrangement of remaining membranes and presence of vesicles. Also in the yellow areas paramural bodies were encountered in higher numbers and displaying altered structure than in healthy and symptomless infected leaf tissue. This study on avocado sunblotch disease was successful in both of its aims. Firstly with regard to quicker indexing techniques, the standard method using indicator plants was shortened from two years to eight months, while a rapid, six-hour test based on PAGE analysis, was developed. Secondly, more light has been shed on the biochemical and ultrastructural effects of sunblotch on its host, the avocado, as well as providing information regarding the thermal sensitivity and the host range of the agent. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1980.

Thesis (MScAgric)--Stellenbosch University, 1956. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

The Labiatae is a large family that occurs worldwide and have species that are adapted to almost all habitats and altitudes. Plectranthus is in this family. Plectranthus species are beautiful South African shrubs. The genus Plectranthus belongs to subfamily Nepetoideae of tribe Ocimeae. The test microorganisms were chosen carefully as each one belonged to a different taxonomic group of fungi and bacteria. Biologically active mono- and sesquiterpenoids are frequently found in many species of Plectranthus but there are little published data that directly link the presence of specific compounds in a species with the traditional uses of that species. Various Plectranthus spp. were collected and dried, followed by chemical extraction using various solvents. Dichloromethane extracts of P. fruticosus and P. ecklonii were screened for antibacterial and antifungal activities using the agar well and trench diffusion methods. It was found that both methods produced inconsistent results. The trench method required a bigger volume of plant extract to be filled into the well, hence, better biological activity was observed with that method. The well method required a smaller volume therefore poor activity was noted with this assay. The size of inhibition zones are dosage dependent. Overall, both plant extracts exhibited antibacterial but no antifungal properties. The pure compound (1), 11-Hydroxy-2-(4-hydroxybenzoyl)-5,7,9(11),13-abietatetraen-12-one, isolated from P. ecklonii was found to be the same as compound (10) which was isolated from P. lucidus. P. hadiensis was extracted using dichloromethane and hexane. The dichloromethane extract proved to contain much higher biological activity than the hexane extract. Three pure compounds, identified as diterpenes, were isolated from the crude dichloromethane extract of P. hadiensis. 6,7-Dihydroxyroyleanone-6,7,12-trihydroxy-8,12-abietadiene-ll,14-dione (2) and 7(alpha)-formoxy-6(beta)-hydroxyroyleanone (3) exhibited good antibacterial and antifungal activity but not against all the test organisms. The remaining pure compound, 7(alpha)-acetoxy-6(beta)hydroxyroyleanone (4), exerted good antifungal activity. This was measured by the inhibition zone which measured up to 14mm when compound 4 was tested against S. sclerotiorum. When testing the hexane extract against the Bacillus formulations, the pellets that were suspended once in Ringer's solution produced bigger inhibition zones than the pellets that were suspended twice. This could be due to bacterial cells washing out of the suspension. The dichloromethane extract of P. praetermissus proved to be very active against X campestris, producing an inhibition zone of 8 - 20mm. Two pure compounds were isolated from the crude extract and identified as diterpenes. Compound 5, 20(10--> 5)-abeo1( 10),6,8,11,13-abietapentaene-11,12,16-triol, and compound 6, 11,12,15-trihydroxy-20( 10-->5)-abeo-abieta-1-(10),6,8,11,13-pentaene are both known compounds which have previously been isolated from Salvia apiana. P. cilatus was extracted with chloroform and tested against various microorganisms for antifungal and antibacterial activities. It showed poor biological activity overall, except against S. sclerotiorum. The crude dichloromethane extract of P. zuluensis exhibited good antibacterial activity, which was limited to the Gram negative test organism. The extract produced an inhibition zone of 10-12mm when tested against X campestris. Pure compound 7, 2-hydroxy-4,6dimethoxyacetophenone, exerted excellent inhibition against B. subtilis and S. sclerotiorum. Neither compound 8, 1,2,4-trimethoxy-5-(2-propenyl)-benzene, nor compound 7, inhibited Candida spp., F. oxysporum and R. solani. Two diterpenes were isolated from the aerial plant parts of P. lucidus with dichloromethane and their structures elucidated by spectroscopic means. The pure compound 9, 11-hydroxy19-( 3-methyl-2-butenoyl)-5,7,9(11), 13-abietatetraen-12-one, showed moderate antifungal activity whereas compound 10, 11-hydroxy-2-(4-hroxybenzoyl)-5,7,9(11),13-abietatetraen12- one, showed high antifungal activity against R. solani, S. sclerotiorum and F. oxysporum. The crude and the pure compounds (formerly isolated from P. parviflorus) showed inhibition against X campestris. The dichloromethane extracts of P. purpuratus subsp. purpuratus and P. purpuratus subsp. tongaensis exhibit similar levels of biological activity when tested against the same test organisms. Poor antibacterial activity was noted with both extracts. However, excellent antifungal activity was depicted when both plant extracts were tested against F. oxysporum, R. solani and S. sclerotiorum. However, the highest biological activity was noted by R. solani which was totally inhibited by both dichloromethane extracts. The pure compound (11) isolated from P. purpuratus subsp. purpuratus was found to have the same chemical structure as compound (9) previously isolated from P. lucidus. The bioautography assay was used to detect and activity-guide the fractionation of antimicrobial compounds from all the Plectranthus spp. tested. The TLC fingerprint showed a zone of clearing around the lower bands of P. fruticosus and P. ecklonii when the plate was sprayed with a suspension of B. subtilis. This result is consistent with the agar well diffusion method. Clear zones were also noted on some bands of the extracts of P. zuluensis, P. ciliatus, P. hadiensis and P. praetermussis. Clear zones indicate inhibition of growth. Other plant extracts tested for biological activity were from the family Lamiaceae, however, not of the genus Plectranthus. Persicaria senegalensis, Pycnostachys reticulata and Ficus sur possessed moderate biological activity overall. It is interesting to note that P. senegalensis and F. sur exert high biological activity against Candida spp. This could be useful as herbal remedies for yeast infections. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The study was aimed at determining the metabolic fate of sucrose in intact sugarcane internodal tissue. Three aspects of the fate of sucrose in storage tissue of whole plants formed the main focus of the work. These were the rate of sucrose accumulation in the developing culm, the characterisation of partitioning of carbon into different cellular organic fractions in the developing culm and the occurrence of sucrose turnover in both immature and mature stem tissues. Specific attention was paid to confirming the occurrence of sucrose turnover in both immature and mature internodal tissue. This sucrose turnover has been described previously in both tissue slices and cell suspension cultures. However, certain results from previous work at the whole plant level have indicated that sucrose turnover does not occur in mature internodal tissue. Radiolabeled carbon dioxide (14CO2) was fed to leaf 6 of sugarcane culms of a high sucrose storing variety (Saccharum spp. hybrid cv. Nco376). All plants were of similar age (12 months) and were grown under similar conditions. The movement and metabolic fate of radiolabeled sucrose was determined at four time points, (6 hours, 24 hours, 7 days and 6 weeks) during a 6 week period. The metabolic fate of sucrose was determined in internodes number 3, number 6 and number 9. Internode 3 was found to have a relatively high hexose sugar content of 42 mg glc&fruc fw g-1 and a low sucrose content of 14 mg suc fw g-1. In contrast the sucrose content of internode 9 was much higher at 157 mg suc fw g-1 and the hexose sugar content much lower at 4.3 mg glc&fruc fw g-1. Based on previous work, the sugar content of internode 3 and internode 9 are characteristic of immature and mature tissues respectively. Internode 6 occupies an intermediary position between internode 3 and 6 with its sucrose content higher than its hexose sugar content, but with the hexose sugar content still being notable at 15 mg glc&fruc fw g-1. Although the metabolic fate of sucrose within sink tissue was the focal point of the study, the experimental design also allowed for certain aspects of sucrose production in the source to be investigated. The average photosynthetic rate for leaf 6 in full sunlight was estimated at 48 mg CO2 dm-2 s -1. During photosynthesis, only 30% of the fixed carbon was partitioned into the storage carbohydrate pool while the remaining 70% was partitioned into sucrose for immediate export from the leaf. This high rate of carbon fixation combined with a high rate of carbon export is characteristic of C4 plants such as sugarcane. On entering the culm, translocation of radiolabeled sucrose was predominantly basipetal with relatively little acropetal translocation. The majority of the radiolabeled carbon was found to be stored in mature internodes. No significant loss of radiolabeled carbon was observed in mature and elongating internodes over the study period. A 22% loss of total radiolabeled carbon was observed in immature internodes over the same period. This can probably be attributed to the higher rates of cellular respiration known to occur in immature tissues. There appear to be three phases of sucrose accumulation in the developing culm. Initially, the accumulation rate in rapidly growing tissue, as internode 3 develops into internode 6, is relatively low. This is followed by a rapid increase in the rate of sucrose accumulation during internode elongation, as internode 6 becomes internode 9. Finally, a decrease in the rate of sucrose accumulation is observed during late maturation, as internode 9 becomes internode 12. Determination of the sucrose content in internodes 3, 6 and 9 revealed that there is a notable increase in sucrose content during internode maturation. It is proposed that the higher sucrose content of mature tissue is not merely a consequence of the longer growth period of mature tissue, but is due to the increased rate of sucrose accumulation observed during internode elongation. Short-term (24 hours) analysis of carbon partitioning revealed that intemodal maturation was associated with a redirection of carbon from non-sucrose cellulal organic fractions to sucrose storage. In immature internodes only 20% of the total radiolabeled carbon was present in the sucrose pool 24 hours after feeding. In elongating internodes the figure increased to 54% while in mature internodes as much as 77% of the total radiolabeled carbon was retained in the sucrose pool. Concomitant with the increased carbon partitioning into stored sucrose down the developing culm is a decrease in carbon partitioning into the hexose sugar pool. In immature tissue, 42 % of the total radiolabel is present in the hexose sugar pool, while in mature tissue the percentage drops to 11%. This decrease is probably indicative of decreased levels of carbon cycling between the sucrose and hexose sugar pool as a result of internode maturation. Internode maturation was also found to be associated with a decrease in the amount of carbon in the water insoluble matter pool and the amino acid/ organic acid/ sugar phosphate pool. Thus, internode maturation is associated with a redirection of carbon from total respiration to sucrose storage. Long-term (6 weeks) analysis of carbon partitioning confirmed that sucrose storage in mature tissue is greater than that in immature tissue. From the 6 hour time point to the 6 week time point, an 87% reduction in the stored radiolabeled sucrose content was observed in immature internodes. During the same period only a 25% reduction in the stored radiolabeled sucrose was observed in mature internodes. Radiolabel loss from the radiolabeled sucrose pool in both mature and immature internodes was accounted for by relative radiolabel gains in other cellular organic fractions. At all time points during the study, and in all three tissues studied, radiolabel was found in the sucrose pool, the hexose sugars pool, the ionic pool and the water insoluble matter pool. The occurrence of radiolabel in the non-sucrose tissue constituents suggests that sucrose turnover is occurring in both immature, and mature internodal tissue. / Thesis (M.Sc.)-University of Natal, Durban, 2000.

Public tendency, of late, is to reduce liberal use of harmful synthesized chemicals for promoting plant health. Today, biological control is becoming a commonly cited disease control option. Biological control agents (BCAs) not only control disease , but also promote plant growth. Application of biological control is based largely on knowledge of control mechanisms employed by antagonists, as well as the means of application that will ensure that an antagonistic population is established. Knowing the advantages is not the only factor that should be considered before application commences as, the disadvantages must be clearly outlined and explored further before a constructive decision as on implementation of biological control. A literature review was undertaken to provide the necessary technical information about biological control, its potential uses, methods of application, mechanisms of action employed, advantages and disadvantages associated with biological control application, public perceptions and the potential future of biological control. Diseases encountered within the KwaZulu-Natal Midlands on pasture and turf grasses were determined by a once-off survey conducted over 1999/2000. The aim of the survey was to determine broadly the management practices of farmers and groundsmen in KwaZulu-Natal and the potential impact of these on the occurrence of weeds, insects and diseases. The survey also addressed the level of existing knowledge about biological control and willingness to apply such measures. In the pasture survey, farmers were questioned about: soil type, grass species common used, irrigation , fertilization and liming, grazing programs and weed, insect and disease occurrences and control measures implemented. The same aspects were addressed in a survey to a representative sample of groundsmen (turfgrass production) , including also: topdressing, greens base used, drainage systems, mowing practices and decompaction principles. The survey showed correlation between pest incidence and management practices implemented. In terms of pest control, both farmers and groundsmen indicated a stronger preference to the use of herbicides , insecticides and fungicides. Use of fungicides for disease control by farmers is considered an often unfeasible expense, rather more emphasis was placed on implementing cultural control methods. At present farmers do not apply biological control strategies, but they did indicate much interest in the topic. Alternatives to current, or lack of current, disease management strategies are important considerations, with two new diseases identified in the KwaZulu-Natal Midlands just within the period of this thesis. Biological control strategies are implemented by 8% of the groundsmen surveyed, with emphasis being placed on augmenting the already present natural predators rather than the introduction of microbial antagonists. Although often mis-diagnosed by farmers Helminthosporium leaf spot is a common disease in the KwaZulu-Natal Midlands on Pennisetum clandestinum (kikuyu), This disease reduces pasture quality and detracts from the aesthetic appearance and wearability of turfgrasses. Helminthosporium leaf spot is incited by a complex of causal agents , Bipolaris was confirmed as the casual agent of Helminthosporium leaf spot on kikuyu at Cedara. Disease control by two BCAs, Bacillus (B. subtilis Ehrenberg & Cohn.) and Trichoderma (T. harzianum Rifai), as commercial formulations was tested against the fungicide, PUNCH EXTRA®. In vitro, Trichoderma was shown to be aggressive in controlling Bipolaris sp. In vivo, disease control achieved with Trichoderma kd was comparative with PUNCH XTRA® but not statistically different (P>=0.05). Trichoderma and Bacillus provided better disease control in comparison to an untreated control. Improved growth of Lolium sp. was determined in vitro, with Trichoderma kd and Bacillus B69 treatments. The microbe-based treatments accounted for growth stimulation, with significant (P<=O.05) growth differences noted. A microbial activator, MICROBOOST®was added to the treatments to improve microbial efficiency. Improved plant growth with MICROBOOST® applications was shown. Improved growth associated with microbial treatments, Trichoderma harzianum kd; Bacillus subtilis B69 and Gliocladium virens Miller, Gibens, Foster and con Arx. ,was also determined in vivo at Cedara, on L.perenne L., Festuca rubra L. and Agrostis stolonifera L. Establishment of a suppressive soil with antagonistic microbes resulted in significant (P<=O.05) effects on final grass coverage (except G. virens), as well increased root and shoot lengths (P<=O.05). Increased germination rates, as expressed in vitro, were not shown in vivo. Microbial activity with the application of MICROBOOST® showed little effect on germination but increased root and shoot lengths significantly (P<=O.05). Increased weed growth associated with the treatments (except G. virens) was considered a drawback of the microbial-treatments. Microbial treatments were also applied to pasture grasses. An in vitro grazing trial was established at Cedara, using L. multiflorum L. to evaluate the microbe-based treatments Trichoderma kd, Bacillus B69 and G. virens for improved pasture establishment and for increased grazing preference by Dohne Merino sheep. Trichoderma kd was associated with increased dry and wet biomass , but lower dry matter yields in comparison to the control. Only G. virens accounted for a higher dry matter percentage than the control. However, differences between the control and the microbial treatments was very small and not significant (P>=0.05). Of the three grazing observations made, sheep showed no grazing preference to plots with or without microbial treatments In general, the body of this research has shown that microbial treatments have the potential for increased disease control and growth stimulation of grasses. However, lack of significant differences between microbial treatments and controls has raised the question as to effect of external factors on microbial activity and survival, especially in vivo. This raises the question as to the validity of the use of microbial treatments where growth conditions cannot be controlled , remembering that the cost of establishment must be covered by the economic returns from utilization. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Thesis (PhD(Agric))--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The apple industry in South Africa currently relies entirely on chemical fungicides to control apple scab, caused by Venturia inaequalis. In this dissertation, alterative management strategies against V. inaequalis were tested for the first time in South Africa. New information on the behaviour of the sexual winter phase of V. inaequalis in different climatic conditions was found and sources of asexual inoculum overwintering in apple orchards were identified. The effect of leaf shredding on fruit and leaf scab incidence and severity was tested against a non-shredded, non-sprayed negative control, a positive control that followed a commercial fungicide programme and a combined treatment of a commercial fungicide programme with leaf shredding, from 2010 to 2013. Reductions in fruit and leaf scab incidence and severity in the leaf shredding treatment were significantly lower compared to the negative control. Quantitative real-time polymerase chain reaction (qPCR) of airborne ascospores trapped using volumetric spore traps was used to measure the reduction in airborne ascospores in the shredded plots, and confirmed the efficacy of shredding found by comparing scab incidence and severity on fruit and leaves. Shredding twice during leaf-drop increased the efficacy of the treatment. Results indicate that leaf shredding should be integrated into scab management strategies in future. However, practical considerations unique to South African orchards, e.g. timing of leaf shredding relative to leaf-drop and orchard layouts, need to be addressed. Pseudothecial densities (PD, number of pseudothecia per fertile lesion) and ascal densities (AD, number of asci per pseudothecium) were compared between in Koue Bokkeveld (KB), a cold winter region, and Elgin (EL), a warm winter region experiencing climate warming, in 2012 and 2013. Scabbed leaves were detached during leaf-drop and overwintered in their region of origin and in the other region. The PD in leaves collected in KB and overwintered in KB was significantly higher than for leaves collected in EL and overwintered in EL, and leaves collected in KB and overwintered in EL. These results agreed with what was expected, as temperature during pseudothecial formation (i.e. the first four weeks after leaf-drop) was significantly lower in KB than in EL. However, the PD for leaves collected in EL and overwintered in EL did not differ significantly from EL leaves overwintered in KB. AD values in all treatments did not differ significantly from one another. Results suggest that factors other than temperature may be involved in controlling PD, e.g. the EL population may include strains not present in the KB population, with higher optimal temperatures for pseudothecial formation. Apple buds and pygmy apples were collected and tested for presence, number and viability of conidia in 2010, 2011 and 2012. Pygmy apples are small, late season fruit that remain attached to the tree throughout winter, especially in regions with warmer winters where trees do not experience sufficient chilling to complete dormancy. High conidial numbers were found on outer bud tissue and low numbers on inner bud tissue, but viable conidia were only found on inner bud tissue, using microscopy, and generally in orchards with high scab levels in the previous season. Molecular methods using PCR-RFLP and qPCR confirmed the presence of high amounts of V. inaequalis DNA in outer bud tissues, although calculated conidial amounts were higher than data obtained when using microscopy, which could indicate presence of mycelia not detected during microscopic examination. Higher numbers of conidia with higher percentage viability were found on pygmy apples, which are a more likely source of asexual inoculum in South African apple orchards than the low number of viable conidia on inner bud tissue. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse appelbedryf is tans afhanklik van chemiese swamdoders vir die beheer van die appelskurf patogeen, Venturia inaequalis. In hierdie proefskrif is alternatiewe bestuurstrategiëe vir die eerste keer in Suid-Afrika ondersoek. Nuwe inligting te opsigte van die gedrag van die geslagtelike winterfase van V. inaequalis, is onder verskillende klimaatstoestande ingewin en bronne van die oorwinterende ongeslagtelike inokulum in appelboorde, is identifiseer. Die invloed van blaarversnippering op die voorkoms en erns van appelskurf op vrugte en blare, is vanaf 2010 tot 2013 ondersoek en met ʼn negatiewe kontrole (onversnipperde blare sonder spuitprogram), ʼn positiewe kontrole (ʼn kommersiële swamdoderspuitprogram is gevolg) en gekombineerde behandelings (kommersiële swamdoderspuitprogram en blaarversnippering) vergelyk. Daar was ʼn betekenisvolle verskil in die voorkoms en erns van skurf op vrugte en blare met blaarversnippering teenoor die negatiewe kontrole. Kwantitatiewe intydse polimerase kettingvermeerderingsreaksie (kPKR) van luggedraagde askospore, vasgevang in volumetriese lokvalle, is gebruik om die afname van luggedraagde askospore in versnipperde behandelings te meet. Die doeltreffendheid van versnippering as behandeling, is bevestig deur die voorkoms van appelskurf te vergelyk met die ernstigheidsgraad daarvan op vrugte en blare. Die uitvoer van blaarversnippering twee keer gedurende die blaarvalperiode het die effektiwiteit van hierdie behandeling verhoog. Hiervan kan dus afgelei word dat blaarversnippering voordelig sal wees vir die bestuur van appelskurf en in toekomstige bestuurspraktyke ingesluit moet word. Praktiese oorwegings, uniek aan Suid-Afrikaanse boorde, soos boorduitleg en die tydsberekening van blaarversnippering teenoor blaarval, moet egter in ag geneem word. Pseudothesiale digtheid (PD; die aantal pseudothesia per vrugbare letsel) en askale digtheid (AD; die aantal aski per pseudothesium) is gedurende 2012 en 2013 vir die Koue Bokkeveld (KB), 'n koue winterstreek, en warm winterstreek Elgin (EL), 'n winterstreek wat klimaatsverwarming ervaar, vergelyk. Blare, met skurf, is gedurende blaarval gepluk en oorwinter in hul gebied van oorsprong, asook in die ander klimaatstreek. Blare wat in KB versamel is en in KB oorwinter het, se PD was aansienlik hoër as dié wat in EL versamel is en in EL oorwinter het, sowel as dié wat in KB versamel is en in EL oorwinter het. Hierdie resultate stem ooreen met wat verwag is, om rede die temperatuur gedurende pseudothesiale vorming, d.w.s. die eerste vier weke na blaarval, aansienlik laer in KB as in EL was. Die PD van blare wat in EL versamel en daar oorwinter het, het egter nie betekenisvol verskil van blare wat in KB oorwinter het nie. Die AD-waardes tussen behandelings verskil nie noemenswaardig nie en word as onbeduidend beskou. Die verkrygde resultate dui aan dat daar ander faktore as temperatuur betrokke is by die beheer van PD, bv. die EL-skurfpopulasie, waar die warmer klimaat meer optimaal is vir pseudothesiale vorming, rasse wat nie in die KB-bevolking teenwoordig is nie, mag insluit. Appelknoppe en dwerg-appels is gedurende 2010, 2011 en 2012 versamel en vir die teenwoordigheid, aantal en lewensvatbaarheid van konidiospore getoets. Dwergappels is klein laatseisoen appeltjies wat reg deur die winter aan die boom bly hang; veral in die streke met warmer winters waar die bome nie die nodige koue ervaar om dormansie te voltooi nie. Met behulp van mikroskopie is ʼn hoë aantal spore op die buitenste knopweefsel en lae getalle in die binneweefsel bespeur; maar lewensvatbare spore is net in die binneweefsel van knoppe waargeneem, wat hoofsaaklik afkomstig is van boorde wat hoë vlakke van appelskurf in die vorige seisoen ervaar het. Molekulêre tegnieke, PKR-RFLP en kPKR, is gebruik vir bepaling van V. inequalis DNA hoeveelhede op die buitenste knopweefsel. Hoër getalle konidiospore is met die molekulêre analise gevind, as dié verkry met mikroskopiese ondersoek en dui op die moontlike teenwoordigheid van miselium wat nie met visuele waarneming sigbaar was nie. Meer konidiospore met 'n hoër vlak van lewensvatbaarheid is op dwerg-apples gevind en dit is moontlik 'n meer waarskynlike bron van ongeslagtelike inokulum in Suid-Afrikaanse appelboorde, as die lae getalle van lewensvatbare konidiospore op die binneweefsel van die appelknoppe.

Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Sulphate-reducing bacteria (SRB) mediate the reduction of metals/metalloids directly or indirectly. Bioremediation of arsenic contaminated water could be a cost-effective process provided a cheap carbon source is used. To this end, molasses was tested as a possible source of carbon for the growth of sulphate-reducing bacteria (SRB). Its chemical composition and the tolerance of SRB toward different arsenic species [As (III) and As (V)] were also investigated. Batch culture studies were carried out to assess 1, 2.5 and 5 g l-1 molasses as suitable concentrations for SRB growth. The results indicate that molasses does support SRB growth, the level of response being dependent on the concentration; however, growth on molasses was not as good as that obtained when lactate, the usual carbon source for SRB, was used. The molasses used in this study contained several metals including Al, As, Cu, Fe, Mn and Zn in concentrations ranging from 0.54-19.7 ìg g-1, but these levels were not toxic to the SRB. Arsenic tolerance, growth response and sulphate-reducing activity of the SRB were investigated using arsenite and arsenate solutions at final concentrations of 1, 5 and 20 mg l-1 for each species. The results revealed that very little SRB growth occurred at concentrations of 20 mg l-1 As (III) or As (V). At lower concentrations, the SRB grew better in As (V) than in As (III). Batch cultures of sulphate-reducing bacteria (SRB) in flasks containing pine bark, sand and polystyrene as support matrices and Postgate medium B were used to study formation of biofilms. The effects of the support matrices on the growth of the organisms were evaluated on the basis of pH and redox potential change and the levels of sulphide production and sulphate reduction. Characterisation of the matrix surfaces was done by means of environmental scanning electron microscopy (ESEM). A consortium of SRB growing on polystyrene caused a 49% of original sulphate reduction whereas on sand a 36% reduction occurred. Polystyrene was further examined for its durability as a long-term support material for the growing of SRB in the presence of As(III) and/or As(V) at concentrations of 1, 5 and 20 mg l-1. Both sulphate reduction and sulphide production were greater in this immobilised system than in the matrix-free control cultures. With pine bark as support matrix no significant sulphate reduction was observed. The kinetics of sulphate reduction by the immobilised cells were compared with those of planktonic SRB and found to be superior. The leaching of organic compounds, particularly phenolic substances, from the pine bark had a detrimental effect on the growth of the SRB. Different proportions of pine bark extract were used to prepare media to investigate this problem. Growth of SRB was totally inhibited when 100% pine bark extract was used. Analysis of these extracts showed the concentration of phenolics increased from 0.33 mg l-1 to 7.36 mg l-1 over the extraction interval of 15 min to 5 days. Digested samples of pine bark also showed the presence of heavy metals. The effects of nitrate, iron and sulphate and combinations thereof were investigated on the growth of a mixed culture of sulphate-reducing bacteria (SRB). The addition of 30 mg l-1 nitrate does not inhibit the production of sulphide by SRB when either 50 or 150 mg l-1 sulphate was present. The redox potential was decreased from 204 to -239 mV at the end of the 14 day batch experiment in the presence of 150 mg l-1 sulphate and 30 mg l-1 nitrate. The sulphate reduction activity of the SRB in the presence of 30 mg l-1 nitrate and 100 mg l-1 iron was about 42% of original sulphate, while if no iron was added, the reduction was only 34%. In the presence of 20 mg l-1 either As(III) or As(V), but particularly the former, growth of the SRB was inhibited when the cells were cultured in modified Postgate medium in the presence of 30 mg l-1 nitrate. The bioremoval of arsenic species [As(III) or As(V)] in the presence of mixed cultures of sulphate-reducing bacteria was investigated. During growth of a mixed SRB culture adapted to 0.1 mg l-1 arsenic species through repeated sub-culturing, 1 mg l-1 of either As(III) or As(V) was reduced to 0.3 and 0.13 mg l-1, respectively. Sorption experiments on the precipitate produced by batch cultured sulphate-reducing bacteria (SRB-PP) indicated a removal of about 77% and 55% of As(V) and As(III) respectively under the following conditions: pH 6.9; biomass (2 g l-1); 24 h contact time; initial arsenic concentration,1 mg l-1 of either species. These results were compared with synthetic iron sulphide as adsorbent. The adsorption data were fitted to Langmuir and Freundlich isotherms. Energy dispersive x-ray (EDX) analysis showed the SRB-PP contained elements such as sulphur, iron, calcium and phosphorus. Biosorption studies indicated that SRB cell pellets removed about 6.6% of the As(III) and 10.5% of the As(V) from water containing an initial concentration of 1 mg l-1 of either arsenic species after 24 h contact. Arsenic species were precipitated out of synthetic arsenic-contaminated groundwater by reacting it with the gaseous biogenic hydrogen sulphide generated during the growth of SRB. The percentage removal of arsenic species was dependent on the initial arsenic concentration present. Lastly, laboratory scale bioreactors were used to investigate the treatment of arsenic species contaminated synthetic groundwater. A mixed culture of SRB with molasses as a carbon source was immobilised on a polystyrene support matrix. The synthetic groundwater contained either As(III) or As(V) at concentrations of 20, 10, 5, 1 or 0.1 mg l-1 as well as 0.1 mg l-1 of a mixture with As(III) accounting for 20, 30, 40, 60 and 80% of the total. More that 90% and 60% of the As(V) and As(III) respectively were removed by the end of the 14-day experiment. At an initial concentration of 0.1 mg l-1 total arsenic had been reduced to below the WHO acceptable level of 10 ìg l-1 when the proportion of As(III) was 20 and 30%, while at 40% As(III) this level was reached only when the treatment time was increased to 21 days. The efficiency of As(III) removal was increased by first oxidising it to As(V) using MnO2. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5. / Thesis (Ph.D.)-University of Natal, Durban, 2002.