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The potential of hot water dipping, vapour heating or ultraviolet irradiation to eliminate the use of chemicals for control of sprouting and post-harvest diseases of the potato (Solanum tuberosum L.) was examined. The microorganisms on which these treatments were tested were the fungal dry rot (Fusarium solani) and the bacterial soft rot (Erwinia carotovora pv. carotovora), two major post-harvest pathogens of potatoes. The study focused on short-term storage (three months) at 8$ sp circ$C or 18$ sp circ$C, which are representative of storage temperatures used by producers in northern temperate and semi-arid tropical regions, respectively. / Response surface methodology was used in experimentation to facilitate analysis of data and identification of optimal operating conditions for the treatments. The following parameters were used to assess tuber quality after the treatments and 3-month storage: firmness, color and structure. / It was found possible to obtain 100% control of sprouting and diseases for the three-month storage without resorting to the use of chemicals. This was achieved without significantly altering the quality attributes of the tubers under certain conditions of hot water, ultraviolet radiation or combinations of these two with storage at 8$ sp circ$C for three months. Although 100% control was not possible for the storage at 18$ sp circ$C, treated tubers performed much better than the controls. Vapour heat (50-70$ sp circ$C) was much less effective at controlling sprouting and was therefore not tested on the pathogens. / A numerical model of the heat transfer phenomenon in the tuber was also developed. It was used to predict the transient temperature distribution in the tuber. The model was solved using the line-by-line technique and model simulations were validated against experimental data.

Five cultivars, all belonging to the family Cucurbitaceae, have been tested for the ability to regenerate shoots or somatic embryos from cotyledonary explants. The influence of several combinations of growth regulators on regeneration from cotyledonary and other explants was tested. No regeneration was obtained from the two cultivars Cucurbita maxima Duch. cv A-Line and Cucurbitapepo L. cv Rolet. Somatic embryos developed on Cucurbita maxima Duch. cv Chicago Waited, a Hubbard squash. A shoot regeneration response was observed for the cultivar Cucumis sativus L. cv Ashley, but the frequency was low and results could not be repeated in subsequent experiments. A reliable shoot regeneration protocol was developed for Cucumis melo L. cv Hales Best 36. The influence of the antibiotics kanamycin sulphate and cefotaxime on shoot regeneration from cotyledonary explants of Cucumis melo L. cv Hales Best 36 was tested. The plasmid pBI121 was transferred from Escherichia coli strain HB101 into Agrobacterium tumefaciens strain LBA4404 via a triparental mating. The plasmid pBI121, contains the screenable marker gene β-glucuronidase (GUS) and the selectable neomycin phosphotransferase-II gene (NPT-II) that confers kanamycin resistance. Cotyledonary tissue was transformed using this Agrobacterium tumefaciens transformation system. The influence of co-cultivation time, inoculation time and the wound factor acetosyringone on transformation was established. Rooted plantlets were regenerated from transformed cotyledonary tissue placed on kanamycin supplemented regeneration media. Plantlets tested positive for the presence of the GUS gene, using fluorometric and histochemical assays. The developed protocol was used to transform Cucumis melo cv Hales best 36 with the pat gene that provides resistance to the herbicide Ignite®. A selection medium was developed containing phosphinothricin, the active ingredient of the herbicide Transformants were selected on this medium and five lines were recovered. These plants were acclimatized and the herbicide resistance was confirmed in greenhouse spray tests. The ploidy level of these plants was deduced from indirect evidence of micro- and macroscopic characteristics that have been shown to have a correlation with the chromosome number of melon plants. The five lines were subjected to molecular analysis. The polymerase chain reaction was used to give an indication of the transformed nature of the selected plants. Agarose gel electrophoresis confirmed that the correct size band could be obtained from the putative transformants and the presence of pat in the product was verified using a non-radioactive system for nucleic acid analysis. Stable gene insertion into the genome of the plant was verified with a Southern blot of the total genomic DNA. This was achieved by hybridising a radioactively labelled ³²P probe specific for the pat gene to a blot of restriction digested plant DNA. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

Inhibition of Fusarium oxysporum f. sp. cubense race 4 by Burkholderia cepacia was evident when grown on various media (TSA, PDA, PSA, YM, KMB, PPM, NYGA, LA) with different carbon sources and under various pH and temperature conditions. In addition, B. cepacia was able to inhibit several fungal pathogens in vitro. Antagonism of B. cepacia against F. oxysporum f. sp. cubense occured at high levels of Fe³+, which may suggest that antagonism by B. cepacia did not involve siderophore production. Thin layer chromatogram (TLC) examination showed that B. cepacia produced several substances, one of which had similar R[f] value to that described for pyrrolnitrin. Cell-free supernatant of a 4-day culture of 6. cepacia was applied to an Amberlite XAD-2 column and inhibitory activity co-eluted with the 95% methanol (pH 9.5) fraction. The concentrated activated fractions showed inhibitory activity against F. oxysporum f. sp. cubense. A GC-MS chromatogram indicated numerous components in the antifungal extracts. The only compound identified in the Wiley 138 library, was 1,2- Benzenedicarboxylic acid, bis (2-Ethylhexyl) ester. Observations by light microscopy indicated that B. cepacia inhibited spore germination in F. oxysporum f. sp. cubense race 4 and retarded the mycelial growth. The interaction between the endophytic bacterium, B. cepacia and F. oxysporum f. sp. cubense race 4 was investigated with aid of scanning and transmission electron microscopy. This demonstrated that the bacterium was able to colonize the surface of hypha and macrospore of F. oxysporum f. sp. cubense. Mycelial deformation, terminal and/or intercalary swelling were evident. At later stages, hyphae of F. oxysporum f.sp. cubense, colonized by B. cepacia, were found to have collapsed. Further studies in vivo confirmed that B. cepacia colonized the hypha of F. oxysporum f. sp. cubense which had invaded banana roots. TEM observation showed that in the banana plant B. cepacia was closely associated with the healthy banana roots and a matrix was frequently found to be present between the bacterium and the plant surface. In addition, B. cepacia exists mainly in the intercellular space of the banana roots. UV irradiation treatment of B. cepacia resulted in a mutant that had lost inhibitory activity against F. oxysporum f. sp. cubense on TSA agar. Transposon mutagenesis of B. cepacia was performed by Tn5 insertion. Six mutants which had lost or had reduced inhibitory activity against F. oxysporum f. sp. cubense were generated. These mutants showed no inhibitory zones on TSA medium in the presence of the fungus. It was observed that one mutants. cepacia :: Tn5-188 appeared to lose the ability to colonize the fungal hypha, whilst a different mutant B. cepacia ::Tn5 - 217 was still able to colonize the fungal hyphae. TLC analyses showed that there was a decrease in antibiotic production in mutants B. cepacia :: Tn5 - 217 and B. cepacia - UV - 34, compared with the wild type. GC- MS analyses showed that there was no evidence of the peaks at 14.62 minutes, 20.0 minutes and 20.46 minutes in both chromatograms of mutants B. cepacia :: Tn5 -217 and 8. cepacia -UV - 34, compared with the wild type B. cepacia. No PCR products were detected using primers that were developed from sequences within the biosynthetic loci for Phi of P.fluorescens Q2-87(GenBank accession no. U41818) and PCA of P. fluorescens 2-79 (GeneBank no. L48616). Colony hybridization suggested that genomic DNA from B. cepacia could contain both Phi- and PCA probes. It was found that hybridization of genomic DNA digested with Cla-I of B. cepaca with Phl2a probe only occurred at low stringency. A hybridization signal was detected from a Cla-l fragment of approximately 2800bp. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

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.

The Wheeler Orchid Collection is home to some of the most endangered species of orchids in the world. This fantastic reservoir of endangered species has been enhanced and broadened by its function as a plant rescue station for the U.S. customs service. Unfortunately, this responsibility increases the risk of bringing orchids, which harbor contageous diseases, into the greenhouse where sap transmitted diseases such as the Tobacco Mosaic Virus (TMV), can run rampant. Although manipulation of orchid characteristics is typically done by classical plant breeding techniques, genetic engineering is emerging as a useful technique for the introduction of desirable traits into the orchid genome. Through the use of genetic engineering techniques it may be possible to mitigate the symptoms associated with this destructive virus. Virus resistance may be achieved through the expression of either the sense or antisense viral coat protein gene in orchid tissues if an efficient means of orchid transformation is developed. In this research two transformation protocols were examined for their ability to efficiently transform orchid tissue. The first transformation protocol explored utilized the native ability of Aq bacterium tumefaciens to incorporate DNA into host plants to achieve transformation. The second mechanism explored was particle bombardment transformation.Many strains of A. tumefaciens were employed using direct exposure of Cattleya_ orchid protocorm and callus tissue. Particle bombardment using DNA coated 0.5 um diameter tungsten particles and high pressure helium tank acceleration was employed. The particle bombardment procedure employed the pG35barB plasmid which confers herbicide resistance to the herbicide basta when integrated and expressed in plant tissues.GUS fluorescence assays and PCR analysis indicate that T-DNA is present in orchid tissues, while Southern blot analysis was unable to display that integration had occurred. Particle bombardment yielded herbicide resistant orchid tissues which have yet to be analyzed by Southern blot analysis to confirm integration due to limited tissue quantities. / Department of Biology

The effect of temperature and duration of leaf wetness (DLW) on sporulation of Bremia lactucae was determined for lettuce cv. Ithaca. A single spore isolate of B. lactucae was produced and was identified using lettuce differential lines each of which had known gene for resistance. Potted lettuce plants were inoculated with the isolate and incubated at 15 C, at 100% relative humidity for 24 h and then at a relative humidity lower than 70%. Seven days later, the plants were submitted to five different temperatures (5, 10, 15, 20 and 25 C) and six durations of leaf wetness (4, 6, 8, 10, 12 and 14 h). The number of spores produced was determined at the end of each wet period. After 4 h of incubation no spores were observed at any of the temperatures. Highest number of spores was found at 10 and 15 C for more than 10 h of DLW. The rate of sporulation rapidly increased between 8 and 10 h for all the temperatures, including 25 C, where the amount of spores produced was very low. The observed number of spores was transformed into proportion of maximum sporulation (PMS) by providing each data by the maximum number of spores observed for each experiment. The Richards model was used to describe sporulation as a function of leaf wetness duration and the rate and maximum value expressed as a function of temperature. This approach resulted in a three-dimensional equation that explained 87% of the variation in the PMS. Spore viability was also estimated for each temperature and DLW. It was zero after 6 h of incubation and reached almost the maximum after 10 h for all the temperatures. The sporulation model was validated under field conditions and it predicted high, medium, low, or no sporulation in 8 out of 11 times.

This project investigated the impact of bird predation on insect pest populations within two cornfields of Southwestern Quebec. Birds were excluded from portions of these fields through the use of bird-proof netting. Rhopalosiphum maidis, Ostrinia nubilalis, Agrotis spp. and Sphenophorus spp. were all found at greater densities in plots where birds were excluded as compared with the non covered plots. Only the adult Diabrotica longicornis population was not reduced by birds. Results significantly suggest that birds can reduce insect pest populations. Bird visits in cornfields with respect to distance from field edge, type of edge habitat and corn phenology were also studied. Red-winged Blackbird, Song and Chipping Sparrows and American Robin were the most frequently observed bird species inside cornfields. Early in the summer, birds tended to stay closer to the field edge, especially when composed of mature coniferous trees. Different bird species visited the cornfields at specific periods during corn development.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 120-122). / Also available in microform / xvii, 128 leaves, bound ill. 29 cm

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