Thesis (MSc (Genetics))--University of Stellenbosch, 2011. / Includes bibliography / ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development. / AFRIKAANSE OPSOMMING: Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie-spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP-aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus-bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd.

Doctor of Philosophy / Department of Agronomy / Gina Brown-Guedira / Allan K. Fritz / Aegilops tauschii, the D-genome donor of modern wheat, has served as an important source of genetic variation in wheat breeding. The objective of this research was to characterize and utilize multiple Ae. tauschii-derived pest resistance genes contained in the germplasm KS96WGRC40. Two Ae. tauschii-derived genes, H23 and Cmc4, provide resistance to the Hessian fly (HF) and wheat curl mite (WCM), respectively. A linkage analysis of a testcross population estimated 32.67% recombination between H23 and Cmc4 on chromosome 6DS in wheat indicating that the two genes are not tightly linked as previous mapping reports show. Haplotype data of recombinant lines and physical mapping of linked microsatellite markers located Cmc4 distal to H23. Haplotype data indicated that both KS89WGRC04 and KS96WGRC40 have the distal portion of 6DS derived from Ae. tauschii. Microsatellite primer pairs BARC183 and GDM036 were more useful than the previously published linked markers in identifying lines carrying Cmc4 and H23, respectively. Through phenotypic selection and advancement within the testcross population, three TC1F2:4 lines were identified as homozygous resistant for H23 and Cmc4 and have the complete terminal segment of 6DS from Ae. tauschii. Two lines are more desirable than the original germplasm releases and can serve as a source of resistance to both HF and WCM in an elite background. A linkage analysis of a segregating recombinant inbred line population identified an Ae. tauschii-derived gene of major effect conferring resistance to Septoria leaf blotch (STB) and another Ae. tauschii-derived gene of major effect conferring resistance to soil-borne wheat mosaic virus (SBWMV) in the germplasm KS96WGRC40. The STB resistance gene in KS96WGRC40 is located in the distal 40% of the short arm of chromosome 7D flanked by microsatellite markers Xgwm044 and Xbarc126. Two previously reported STB genes, Stb4 and Stb5, have been mapped to 7DS in the same region as the STB resistance gene in KS96WGRC40. The uniqueness of the STB resistance genes on 7DS is questionable. The SBWMV resistance gene in KS96WGRC40 is located on chromosome 5DL linked to microsatellite marker Xcfd010. The SBWMV resistance gene within KS96WGRC40 was derived from TA2397 via KS95WGRC33.

T. aestivum ssp. spelta Iranian type has long been thought to potentially be the direct non-free threshing hexaploid progenitor. I evaluated a RIL population derived from a cross between CS and Iranian spelta accession P503 to identify loci suppressing free-threshabilty in P503. Identification of QTL associated with threshability in region known to harbor the Tg2A gene, and an inactive tg2D allele supported the hypothesis of Iranian spelta being derived from a more recent hybridization between free-threshing hexaploid and emmer wheat. Parastagonospora nodorum is an important fungal pathogen and secretes necrotrophic effectors that evoke cell death. In this research, a DH population segregating for Snn5 was used to saturate Snn5 region of chromosome 4B with molecular markers. The physical distance between Snn5 flanking markers was narrowed to 1.38 Mb with genetic distance of 2.8 cM. The markers developed in this study will provide a strong foundation for map-based cloning of Snn5.

Thesis (M.Sc. Agriculture (Agronomy) -- University of Limpopo, 2019 / Erratic rainfall and insect infestation are some of the factors that limit cowpea production in Limpopo (Asiwe, 2009). Improved cowpea varieties available for dryland production in Limpopo do not meet the farmer’s needs. Specific trait crosses are needed to be made with adapted varieties for the purpose of developing high yielding, pest tolerant and adapted varieties. Varieties with desired specific traits were planted in isolation plots for outcrossing by insects. F1 lines derived from parental lines that were half sib seeds were harvested and planted as F2, and seeds obtained from F2 generation were used to develop F3 segregating population at University of Limpopo Experiential Farm, Syferkuil, and planted along with parents. This F3 generation forms the current evaluation on which this report is based. Data collected included; aphid severity (measured on a 9-point scale), number of days to 90 % maturity, grain yield, 100 seed weight and number of genotypes selected for advancement. Results showed significant variation among progeny over parents therefore expressing transgressive segregation. Genotypes derived from pedigree TX08-30-5 were five while pedigree IT98K-205-8 and IT97K-499-35 had four genotypes each that expressed early maturity (80-90 days) which are desirable for evading drought. Twenty-three genotypes from pedigree GEC and three from pedigree IT97K-499-35 obtained grain yield (>101 g/plot and >161 g/plot) that was better than their parents. Only one genotype derived from pedigree TX08-30-1 expressed high resistance to aphids better than the parent with a mean aphid score range of 1-2. Four genotypes from pedigree TX08-30-5 obtained bigger 100 seed weight (>18.6 g) compared to parent. These progeny also proved to be high yielders as they expressed heterosis for all yield attributes than their parents. Eighty-two genotypes were selected for advancement. Significant variations were observed among progeny giving opportunity to make selections. In most cases, genotypes outperformed their parents which indicate positive heterosis. The 82 promising genotypes selected will be advanced for further selections from multi location testing for stability and adaptation.

No description available.

The use of biological control agents (BCAs) in the past has shown limited success as its application has often been done incorrectly, and in addition, management practices are rarely altered to incorporate BCAs. Criteria for the correct application of BCAs have been devised as part of the research, and companies selling these products may use the said criteria. Such application will ensure the correct BCAs are used and, more specifically, used under the correct conditions. The powdery mildew (PM) fungus is often seen to develop resistance to synthetic fungicides and, therefore, alternative control measures are required. BCAs as an alternative pose less risk to the environment, workers and the consumer. A pre-trial has been conducted with a range of BCAs to see if they can control powdery mildew (PM) in a greenhouse environment on hydroponically grown cucumber (Cucumis sativus L.) plants using the variety Baccara that has only a moderate tolerance to PM. The BCAs have been compared to the control (synthetic fungicide: Bravo). Comparative work includes Coyier's model, which has been modified and adapted for these trials to determine the percentage of leaf area covered by the PM infection. Furthermore, the number of fruit harvested per treatment, kilogram yield, total mass of yield and average fruit mass is also used to determine the efficacy of the BCAs as these factors have economic significance to commercial growers. The pre-trial showed promise until the fertigation computer failed, resulting in a nutrient shortage and imbalance, confirming that BCAs alone cannot control PM. Synthetic fungicides were applied until control of PM and plant nutrition was regained. BCAs were re-introduced and used until the end of crop production. The confirmation from the pre-trial has led to the inclusion of silicon in conjunction with the BCAs in the two subsequent trials (Trials 1 & 2). Silicon was applied with the BCAs as a foliar spray on a weekly basis. In trials 1 and 2, the cucumber variety, Palladium, with a high genetic tolerance to PM is used, as this variety is suited to form part of the holistic approach used for trials 1 and 2. Trial 1 showed that treatment A, containing Streptomyces griseovirdis and Streptomyces aureofaciens, had the highest yield. Both of these are bacterial BCAs and demonstrated their adaptability to varied climatic conditions, notably when low humidity was experienced. In treatment B, Trichoderma harzianum strains, Rifai and Uppington, show the slowest rate of PM development. In trials 1 and 2, the best actual PM control was obtained by two fungal based BCAs (Trial 1, treatment C was Ampelomyces quisqualis) and (Trial 2, treatment B was Trichoderma harzianum strains, Rifai and Uppington), showing that fungal BCAs have a place for this application, but the growth-enhancing properties of bacterial based BCAs make economic sense and would make them attractive to growers. Treatment A (Streptomyces spp.) had the most number of fruit for the entire growing period and the best overall yield (kg yield) again. Two of the BCA / silicon treatments have marginally better PM control compared to that of the control (E) treatment, although not statistically significant. Treatment E (control) has the highest average fruit mass in this instance but does not have the highest yield (kg yield) when compared to treatments A and B, possibly due to the growth-enhancing properties of most of these BCAs. Therefore, most of these BCA treatments give fairly inconsistent results that vary possibly according to season, humidity and temperature, making it difficult to predict their efficacy. Using combinations or weekly alternations of these BCAs with extremes of climatic adaptation will probably be the most reliable method of obtaining consistent results. Bacterial BCAs are shown to have lower humidity requirements and produce the most consistent results in terms of fruit number, yield and fruit mass and a combination of bacterial and fungal based BCAs would possibly be the best as this would control PM and yet still have the growth enhancing properties from the bacterial based BCAs. From the research, it can be said that some BCAs in trials 1 and 2 produce results similar to that of the control in terms of percentage leaf area covered by PM and some are shown to have improved yields. Results produced from certain BCA treatments are thus equal to the control; yet provide an environmentally friendly alternative to synthetic fungicides. Silicon is listed as a beneficial element rather than an essential element; however, literature claims it to be highly effective in treating PM in cucurbits. Results from trials 1 and 2 show that control of PM is possible in most cases, when a holistic approach is used. This approach includes a cucumber variety with a high PM tolerance, optimum nutrition, cultural practices and silicon in combination with the BCAs. A complete change of management practices is necessary to implement such a BCA program. / Agriculture, Animal Health & Human Ecology / M. Tech. (Nature Conservation)

The use of biological control agents (BCAs) in the past has shown limited success as its application has often been done incorrectly, and in addition, management practices are rarely altered to incorporate BCAs. Criteria for the correct application of BCAs have been devised as part of the research, and companies selling these products may use the said criteria. Such application will ensure the correct BCAs are used and, more specifically, used under the correct conditions. The powdery mildew (PM) fungus is often seen to develop resistance to synthetic fungicides and, therefore, alternative control measures are required. BCAs as an alternative pose less risk to the environment, workers and the consumer. A pre-trial has been conducted with a range of BCAs to see if they can control powdery mildew (PM) in a greenhouse environment on hydroponically grown cucumber (Cucumis sativus L.) plants using the variety Baccara that has only a moderate tolerance to PM. The BCAs have been compared to the control (synthetic fungicide: Bravo). Comparative work includes Coyier's model, which has been modified and adapted for these trials to determine the percentage of leaf area covered by the PM infection. Furthermore, the number of fruit harvested per treatment, kilogram yield, total mass of yield and average fruit mass is also used to determine the efficacy of the BCAs as these factors have economic significance to commercial growers. The pre-trial showed promise until the fertigation computer failed, resulting in a nutrient shortage and imbalance, confirming that BCAs alone cannot control PM. Synthetic fungicides were applied until control of PM and plant nutrition was regained. BCAs were re-introduced and used until the end of crop production. The confirmation from the pre-trial has led to the inclusion of silicon in conjunction with the BCAs in the two subsequent trials (Trials 1 & 2). Silicon was applied with the BCAs as a foliar spray on a weekly basis. In trials 1 and 2, the cucumber variety, Palladium, with a high genetic tolerance to PM is used, as this variety is suited to form part of the holistic approach used for trials 1 and 2. Trial 1 showed that treatment A, containing Streptomyces griseovirdis and Streptomyces aureofaciens, had the highest yield. Both of these are bacterial BCAs and demonstrated their adaptability to varied climatic conditions, notably when low humidity was experienced. In treatment B, Trichoderma harzianum strains, Rifai and Uppington, show the slowest rate of PM development. In trials 1 and 2, the best actual PM control was obtained by two fungal based BCAs (Trial 1, treatment C was Ampelomyces quisqualis) and (Trial 2, treatment B was Trichoderma harzianum strains, Rifai and Uppington), showing that fungal BCAs have a place for this application, but the growth-enhancing properties of bacterial based BCAs make economic sense and would make them attractive to growers. Treatment A (Streptomyces spp.) had the most number of fruit for the entire growing period and the best overall yield (kg yield) again. Two of the BCA / silicon treatments have marginally better PM control compared to that of the control (E) treatment, although not statistically significant. Treatment E (control) has the highest average fruit mass in this instance but does not have the highest yield (kg yield) when compared to treatments A and B, possibly due to the growth-enhancing properties of most of these BCAs. Therefore, most of these BCA treatments give fairly inconsistent results that vary possibly according to season, humidity and temperature, making it difficult to predict their efficacy. Using combinations or weekly alternations of these BCAs with extremes of climatic adaptation will probably be the most reliable method of obtaining consistent results. Bacterial BCAs are shown to have lower humidity requirements and produce the most consistent results in terms of fruit number, yield and fruit mass and a combination of bacterial and fungal based BCAs would possibly be the best as this would control PM and yet still have the growth enhancing properties from the bacterial based BCAs. From the research, it can be said that some BCAs in trials 1 and 2 produce results similar to that of the control in terms of percentage leaf area covered by PM and some are shown to have improved yields. Results produced from certain BCA treatments are thus equal to the control; yet provide an environmentally friendly alternative to synthetic fungicides. Silicon is listed as a beneficial element rather than an essential element; however, literature claims it to be highly effective in treating PM in cucurbits. Results from trials 1 and 2 show that control of PM is possible in most cases, when a holistic approach is used. This approach includes a cucumber variety with a high PM tolerance, optimum nutrition, cultural practices and silicon in combination with the BCAs. A complete change of management practices is necessary to implement such a BCA program. / Agriculture, Animal Health and Human Ecology / M. Tech. (Nature Conservation)

Submitted in fulfilment of the requirements for the degree of Master of Applied Science in Food Science and Technology, Durban University of Technology, 2015. / Listeria monocytogenes have been implicated as a public health concern worldwide. The study explored the survival of non-adapted, heat adapted and chlorine adapted L. monocytogenes on tomatoes; as well as the survival of non-adapted, heat adapted and chlorine adapted biofilms after exposure to sodium dodecyl sulphate (SDS), levulinc acid, sodium hypochlorite solution. Contact time of 1, 3 and 5 minutes was used. The survival of L. monocytogenes was monitored at 0, 24, 48 and 72 hours. The sanitizers were used individually or combined as follows; 1% sodium dodecyl sulphate individually; 0.5% levulinic acid individually; 200 ppm sodium hypochlorite solution individually and 0.5% levulinic acid/0.05% sodium dodecyl sulphate in combination (mixture). The samples were kept at 4 °C throughout the period of assessment. The effect of these sanitizers on pH, total soluble solids (TSS) and titratable acidity (TA) was also determined. Furthermore, the attachment of L. monocytogenes on tomatoes was investigated using a scanning electron microscope. Highest log reduction of non-adapted L. monocytogenes were observed on tomatoes treated with 1% SDS and least log reduction was achieved when tomatoes were treated with sodium hypochlorite solution. Though the log reduction achieved by 0.5% levulinic acid was higher that sodium hypochlorite solution, it was lower than log reduction achieved when 0.05% SDS / 0.5% levulinic acid mixture was used for all contact times. Using non-adapted L. monocytogenes, SDS was able to destroy all L. monocytogenes at 1, 3 and 5 minutes contact time. The trend was the same when heat adapted and chlorine adapted L. monocytogenes were used. There was no significant log reduction observed with biofilms. More favourable results were observed as contact time was increased from 1 to 5 minutes. Though there was a decrease in surviving bacteria from 1 to 3 minutes contact time, this decrease was not significant. The study investigated if exposure to sanitizer has an effect on pH, titratable acidity (TA) and total soluble solids (TSS) of the tomatoes. It was revealed that levulinic acid and mixture can have detrimental effect on pH, TA and TSS of tomatoes. The TA and TSS of samples treated with levulinic acid and mixture varied significantly (P ≤ 0.05) compared to the control sample. Although the TA and TSS of samples treated with SDS and sodium hypochlorite solution were different from the control, the differences were not significant. As much as sanitizers have the potential to reduce the bacterial population in fresh produce they may not completely destroy pathogens. Chlorine based sanitizers such as sodium hypochlorite though frequently used in the fresh produce industry, are not the best sanitizer to be used against food borne pathogens. Other sanitizers such as SDS used alone or in combination with another sanitizer can achieve better results than the widely used sodium hypochlorite solution as observed in this study. Stress adapted pathogens become less responsive to sanitizers during subsequent treatments. Through this research, it was established that biofilms are resistant to sanitizers. Though application of sanitizers in fresh produce is cheaper and simpler to apply, there is need to monitor varying concentrations of sanitizers, contact time and minimise contact with sub-surfaces as this could lead to sensory quality losses.

The objective of this research was to investigate the occurrence of mycoflora in rice plants and rice seeds in South Africa and their negative impact. A total of six species of Fusarium were isolated from diseased rice plants and rice seeds and identified as F. anthophilum, F. chlamydosporum, F. compactum, F. equiseti, F. fujikuroi and F. semitectum. In the translation elongation factor data set, Fusarium equiseti isolates grouped together within the F. incarnatum - equiseti Species Complex (FIESC). The isolates from rice clustered together in a single clade with the F. equiseti and F. incarnatum isolates forming two separate sub-clades.The isolates of F. equiseti present a new phylogenetically distinct species in FIESC. In the pathogenicity tests, isolates of both F. anthophilum and F. fujikuroi caused bakanae disease to rice plants. Fifty four rice cultivars and lines were tested by the standardized test tube inoculation method for resistance and susceptibility against bakanae isolate of F. anthophilum and the bakanae isolate of F. fujikuroi. None of the rice cultivars and lines was found to be resistant to bakanae isolates of Fusarium spp. The fungicide, benomyl was found to be most effective as a seed treatment for controlling bakanae disease of rice due to isolates of both F. anthophilum and F. fujikuroi. Thiram was found to be the least effective fungicide for controlling bakanae disease of rice caused by isolates of both the Fusarium spp. Apart from Fusarium species, other fungi that were also isolated from diseased rice plants and rice seeds were identified as Alternaria alternata, Alternaria longipes, Cochliobolus miyabeanus, Nigrospora sphaerica, Phoma eupyrena, Phoma jolyana, Phoma sorghina and Pithomyces sp. In mycotoxin tests, the isolates of both F. anthophilum and F. fujikuroi produced moniliformin. None of the isolates of F. anthophilum and F. fujikuroi produced fumonisins. This research is important as it identifies many fungal species in rice plants and seeds in South Africa for the first time. Currently, there is very little literature that makes reference to such findings under South African conditions. In addition, this investigation unravels previously unknown information on the resistance of rice to bakanese disease. Finally, information is provided on the effectiveness of commonly used fungicides (benomyl and thiram) to control rice diseases. This knowledge is crucial information that is useful to plant pathologists, the farming community and the scientists that are involved in strategies of fighting or reducing rice diseases so as to help contribute to food security. / Environmental Sciences / D. Phil. (Environmental Science)

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses. / AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.