Thesis (M.Sc.(Plant Protection)) -- University of Limpopo, 2022 / Runner beans are extremely sensitive to root-knot (Meloidogyne species) nematodes. Phytonematicides had been consistently used in managing population densities of Meloidogyne species in various crops, with the application technologies being restricted to the ground leaching technology (GLT) and botinemagation technology, each with its own advantages and disadvantages. The use of seeds as carriers of active ingredients of phytonematicides and then drying prior to sowing, is being considered as another potential application strategy in seeds with hypogeal germination. In such seeds, during seedling emergence the seed cover and the endosperm remain below the soil surface, just above the developing root system. As a result, in phytonematicide-primed seeds, the seed structures could serve as carriers for the active ingredients of phytonematicides. In cucurbitacin phytonematicides, Nemarioc-AL and Nemafric-BL phytonematicides contain cucurbitacin A and B, respectively as active ingredients. The objectives of the study were two-fold, namely, to determine whether runner bean (Phaseolus coccineus L.) seeds would (1) serve as carriers of active ingredients of cucurbitacin-containing phytonematicides without affecting seed germination under in vitro conditions, (2) serve as carriers of cucurbitacins intended for suppression of M. incognita population densities under greenhouse and microplot conditions. Two separate studies were conducted under laboratory conditions, with seven treatment solutions at 0, 2, 4, 8, 16, 32 and 64% Nemafric-BL or Nemarioc-AL phytonematicide. After exposure to separate solutions for 2 h, runner bean seeds were dried on laboratory benches for 72 h. Treatments were arranged in a completely randomised design (CRD), with 8 replications. Two layers of filter papers were placed inside each Petri dish seeded with 10 primed and dried seeds. Petri dishes were incubated inside LABCON growth chamber at 25ºC and 75% relative humidity. Successful seed germination, viewed as emergence of radicle from the testa, was recorded daily for a period of 10 days, with each count being removed from Petri dish to avoid re-counting. Under greenhouse and microplot conditions, primed-and dried seeds were sown in plastic pots containing 2 700 ml steam-pasteurised sandy loam soil, arranged in a randomised complete block design, replicated six times and eight times, respectively. Each seedling was inoculated by distributing 5 000 eggs and second-stage juveniles (J2) of M. incognita race 4 using a 50 ml plastic syringe. Originally, pots were irrigated using 500 ml chlorine-free tapwater, which was reduced to half after seedling emergence at every other day. Plant variables were collected at 56 days after inoculation and data were subjected to the Curve-fitting Allelochemical Response Dose algorithm model. In vitro, germination percentage (R 2 = 0.96), radicle length (R 2 = 0.89) and plumule diameter (R 2 = 0.96) versus Nemarioc-AL phytonematicide exhibited positive quadratic relations. Similarly, the variables versus Nemafric-BL phytonematicide, exhibited positive quadratic relations. In vitro, Mean Concentration Stimulation Point (MCSP) value of Nemarioc-AL phytonematicide on runner bean seeds was 1.05%, whereas for Nemafric-BL phytonematicide MCSP value was 0.58%. Under greenhouse conditions, plant height (R 2 = 0.97), chlorophyll content (R 2 = 0.92), dry shoot mass (R 2 = 0.98), dead nodule number (R 2 = 0.90), total pod number (R 2 = 0.97) and active nodule number (R 2 = 0.93) versus Nemarioc-AL phytonematicide exhibited positive quadratic relations., Similarly, chlorophyll content (R 2 = 0.95), gall rating (R 2 = 0.82), dry shoot weight (R 2 = 0.69), stem diameter (R 2 = 0.85) and total nodule number (R 2 = 0.86) versus Nemafric-BL phytonematicide exhibited positive quadratic relations. Under greenhouse conditions, MCSP values for Nemarioc-AL and Nemafric-BL phytonematicides were 4.18 and 3.69%, respectively. Under microplot conditions, total number of nodules (R 2 = 0.88), number of nodules dead (R 2 = 0.99), number of nodules active (R 2 = 0.95), fresh root mass (R 2 = 0.99), and fresh pod mass (R 2 = 0.99) versus Nemarioc-AL phytonematicide, exhibited positive quadratic relations, whereas plant height (R 2 = 0.85), number of nodules dead (R 2 = 0.87), dry shoot mass (R 2 = 0.97), fresh root mass (R 2 = 0.97) and total number of nodules (R 2 = 0.63) versus Nemafric-BL phytonematicide exhibited positive quadratic relations. Under microplot conditions, MCSP values for Nemarioc-AL and Nemafric-BL phytonematicides were 3.76 and 3.93%, respectively, each with ∑k = 0. All degrees of Nemarioc-AL and Nemafric-BL phytonematicides profoundly reduced nematode numbers under greenhouse and microplot trials. Based on the information obtained from this study, it was confirmed that runner bean (P. coccineus) is sensitive to Nemafric-BL and Nemarioc-AL phytonematicides supported by the Curve-fitting Allelochemical Response Dose (CARD) model results due to most plant variables that had sensitivity values of zero. In conclusion, the priming technology should be developed further since it has the potential of being successful in nematode management in seeds with hypogeal germination / National Research Foundation (NRF) and Potatoes South Africa

Thesis (M.Sc. Agriculture (Plant Protection)) -- University of Limpopo, 2021 / In use of phytonematicides as an alternative to synthetic chemical nematicides, the major challenge had been the development of appropriate application technologies, which are currently limited to the ground leaching technology (GLT) and botinemagation (BNT) systems. The former is labour-intensive, whereas the latter requires infrastructure that could be costly for smallholder farmers. The priming of seeds with hypogenous germination properties in phytonematicide solutions could serve as an alternative method of the application of phytonematicides, where the cotyledons would serve as carriers of the active ingredients that are leached into the rhizosphere for suppression of nematode numbers. However, since germination is a chemical process, it is not known whether the active ingredients in cucurbitacin containing phytonematicides would interfere with germination and the subsequent emergence of the seedlings through the incidence of phytotoxicity as observed in the use of the products in crop production. The objectives of the study, therefore, were (1) to investigate the sensitivity and overall sensitivity of pea (Pisum sativum L.) plants to Nemarioc-AL and Nemafric-BL phytonematicides, and (2) to determine the mean concentration point (MCSP) for pea-inoculated with Meloidogyne incognita under greenhouse and microplot conditions, where seeds were previously primed in phytonematicide solutions. Two separate trials were conducted with seven treatments, namely, 0, 2, 4, 8, 16, 32 and 64% Nemarioc-AL or Nemafric-BL phytonematicide, arranged in completely randomised design (CRD), with 8 replications each. Pea seeds were primed in Nemarioc-AL and Nemafric-BL phytonematicide solutions for two hours and shade dried prior to sowing. In vitro trial, 10 seeds were spread uniformly on a moistened filter paper in sterilised petri-dishes with lids and placed in an incubator at 25oC. In vivo trials were under greenhouse and micro-plot conditions, pea seeds were sown in 25-cm and 30-cm diameter plastic pots, respectively. Pots were filled with pasteurised loam soil. Seedlings were inoculated with 5 000 eggs + second-stage juveniles (J2) of M. incognita. Treatments in each case included priming seeds as explained earlier, arranged in a randomised complete block design (RCBD), with 6 replications under greenhouse conditions and 8 replications under micro-plot conditions. In all cases, plant growth variables were assessed using the Curve-fitting Allelochemical Response Dose (CARD) model to generate biological indices which were used to calculate MCSP and the overall sensitivity (Σk). Nematode variables in inoculated trials were assessed using the regression model. In vitro trials, germination variables had positive quadratic relation versus Nemafric-BL phytonematicide, with MCSP= 0.62 % and ∑k = 34 units. In contrast, tested germination variables exhibited negative quadratic relations versus Nemarioc-AL phytonematicide. In greenhouse trials, MCSP values for Nemarioc-AL and Nemafric-BL phytonematicides were 0.62 and 2.18 %, respectively, with ∑k = 0. Plant height (R2 = 0.86), stem diameter (R2 = 0.93) and chlorophyll content (R2 = 0.85), exhibited positive quadratic relationship against Nemarioc-AL phytonematicide, whereas, plant height (R2 = 0.95), stem diameter (R2 = 0.92), chlorophyll content (R2 = 0.89), number of flowers (R2 = 0.93) and dry shoot mass (R2 = 0.94), exhibited positive quadratic relationship against Nemafric-BL phytonematicide. In micro-plot trials, MCSP values for Nemarioc-AL and Nemafric-BL phytonematicides were 0.71 and 2.45 %, respectively, with ∑k = 0. Plant height (R2 = 0.95), stem diameter (R2 = 0.98), chlorophyll content (R2 = 0.98), and gall ratings (R2 = 0.98), exhibited positive quadratic relationships against Nemarioc-AL phytonematicide, while chlorophyll content (R2 = 0.97) and gall ratings (R2 = 0.96) exhibited positive quadratic relationships against Nemafric-BL phytonematicide. All degrees of Nemarioc-AL and Nemafric-BL phytonematicides profoundly reduced nematode numbers under greenhouse and micro-plot trials. In conclusion, both Nemarioc-AL and Nemafric-BL phytonematicides could be applied through the priming technology on pea seeds which have hypogenous germination properties in suppression of nematode population densities. / National Research Foundation (NRF)

Two isolates of peanut stripe virus (PStV), stripe and blotch, were compared ultrastructurally in peanut (Arachis hypogaea L. 'Florigiant') at several stages of leaf expansion. Ultrathin sections of young leaves infected with either isolate of PStV revealed pinwheel inclusions attached to the cell wall near plasmodesmata. The cytoplasm of infected cells were highly vesiculated. Virus particles amassed in crystalline arrays were observed in blotch infected cells. Virus particles were observed along the arms of pinwheel inclusions. Scroll inclusions appeared in PStV infected cells at a later stage of leaf expansion. In more mature leaves, pinwheel and scroll inclusions occurred in the cytoplasm in association with mitochondria. Virus particles were observed free in the cytoplasm as well as concentrated in linear arrays along the inner surface of the tonoplast. Membrane and organelle degradation was evident in cells infected with either isolate of the virus. Numerous cytoplasmic inclusions and virus particles were observed in cells from light green areas of the leaf. Cells from dark green areas did not contain cytoplasmic inclusions and contained few if any virus particles. Particle measurements show stripe and blotch isolates to have a mean length of 753 nm and 747 nm for leaf dip preparations and 746 nm and 745 nm for partially purified preparations, respectively. Both isolates had a modal length of 750 nm, regardless of the extraction procedure. The relative virus titer of each isolate was determined in peanut leaves at five stages of leaf expansion and in dark green and light green areas of infected leaves. Virus titer increased significantly from the closed to the fully expanded stage, at which time the virus titer peaked and then decreased slightly. Virus titer was consistently higher in leaves infected with the blotch isolate at all expansion stages. Virus titer was also higher in cells from light green areas of the leaf than from dark green areas of the leaf, regardless of isolate. / M.S.

An investigation of the effect of Meloidogyne hapla, M. arenaria, M. arenaria thamesi, M. incognita acrita, and M. incognita on Ligustrum ovalifolium, L. japonicum, and Abelia floribunda was conducted. With the exception of M. hapla on L. ovalifolium and M. arenaria thamesi on A. floribunda, all 5 species of nematodes were pathogenic on the 3 host species. Striking differences in growth of the L. ovalifolium plants inoculated with the 4 root-knot species that caused significant reduction could easily be seen. Reduction in weight of the plants inoculated with the 4 root-knot species varied from 39.8% to 63.6% of the controls, and the reduction in total length of stems of these plants varied from 37.2% to 58.5% of the controls at the termination of the experiment. Reduction in weight of the L. japonicum plants inoculated with root-knot species varied from 29.2% to 51.7% of the controls. All plants inoculated with root-knot were significantly reduced in weight compared to controls. There were no visual differences in growth among the A. floribunda treatments; however, omitting the M. arenaria thamesi treatment which was not significantly different from control, the reduction in weight of the inoculated plants varied from 26.81 to 41.71 of the controls. / M.S.

The state of leaf health of intact Penneagle creeping bentgrass leaves into which Curvularia lunata was able to ingress was determined by reducing cuticle/wax formation with trichloroacetic acid (TCA) and stressing plants with high air temperatures. Plants were grown until the third leaf had fully expanded and the fourth leaf had not yet emerged from the whorl. Plants were then treated with TCA for 6 times on alternate days. Half of the plants were high air temperature stressed at 38°C for 18 hr before plants were inoculated. Leaf health was estimated throughout the growing period of the plants by extracting chlorophylls and then regressing the values with respect to time. In this manner, leaves at each nodal position could be classified as either juvenile, mature or senescent. Also, selected leaves were examined by scanning electron microscopy. The addition of TCA to plants decreased leaf and plant life, increased tillering and reduced the deposition of leaf surface waxes. High air temperature stressing the plants caused a rapid entry of the leaf into senescence and higher levels of TCA accelerated this process. Plants were inoculated with either C. lunata, C. lunata var. aerea or Drechslera sorokiniana. Histochemical techniques were used to determine if penetration of the fungus into plant tissue had occurred. Inoculation with D. sorokiniana resulted in lesion formation within 2 days. Symptoms commonly attributed to Curvularia blight were present on plants treated with 0.047 or 0.14 mM TCA and then high air temperature stressed and inoculated with C. lunata. Histochemical procedures failed to show the presence bf mycelium Qf C. lunata· within the cells of Curvularia blight symptom areas. In a separate study, plants were grown, clipped and maintained at 2.0 cm and grown until 30 or 120 days of age. Plants were high air temperature stressed or not and clipped 128, 64, 32, 16, 8, 2, 1 or 0 hr before inoculation with C. lunata. Results again showed that C. lunata had the ability to colonize heat stressed and/or old leaves but did not have the ability to infect and colonize juvenile or mature tissues. The amount of the turf foliage that is susceptible to thinning by C. lunata depends on the physiologic age of the leaf tissue. As stresses of high air temperatures are placed on the leaf tissue, a greater percentage of the leaf blades are forced into advanced senescence, thereby increasing their susceptibility to infection and colonization by C. lunata. / Ph. D.

Colonization of Pine Christmas trees by Verticicladiella procera Kendr. causes Procera root disease. Little is presently known regarding the pattern and effects of fungal development within colonized trees. The present studies were undertaken to elucidate the developmental pattern of the fungus in colonized trees, to gather information on possible mechanisms and physiological effects of disease development, and to explore the relationship between V. procera and other, well documented bluestain fungi. The presence of cellulose was demonstrated in the cell walls of X. procera, indicating the probable genetic relatedness of this fungus with Ophiostoma (Ceratocystis) bluestain fungi. Inoculation studies revealed that the fungus could penetrate wounded sapwood, and that colonized seedlings had lower water potentials than uncolonized seedlings. In addition, it was found that the fungus could persist in resinous stem lesions for 22 months without foliar symptoms, and resinous stem lesions with the fungus were significantly longer and deeper than wound lesions. An intensive isolation study revealed that the initial point of colonization in a tree is apparently at the root collar, progressing acropetally in both directions. Analysis of radial growth from increment cores showed that colonized trees had grown more slowly for the preceding three years than uncolonized trees. The sapwood moisture content of these cores was also significantly reduced in the colonized trees, indicating that the stem was drying out as symptoms developed. Histological examination of colonized sapwood showed that U fungal colonization of tissues progressed along rays and resin ducts, in a fashion similar to that of bluestain fungi. Permeability measurements demonstrated that symptomatic sapwood, either resin-soaked or black-stained, had significantly reduced water movement relative to asymptomatic sapwood. / Ph. D.

Erwinia carotovora subsp. Carotovora (Ecc) incites soft-rot on many plants. It is believed that soft-rot is due to the concerted activity of extracellular enzymes. Recombinant DNA techniques were used to study the molecular basis of pathogenesis incited by Ecc. Specifically, a clone library of Ecc strain EC14 DNA in plasmid pBR322 was constructed and transformed into Escherichia coli strain HB101. Some of the E. coli strains that contain these hybrid plasmids produce pectinases or cellulase(s). Plasmid pDR1 contains a 3.4 kilobase (kb) EC14 DNA fragment and mediates the production of endo-pectate lyases with isoelectric points (pI) of 9.5 and 7.5 in strain HB101. The pI 9.5 enzyme is believed to be the major extracellular pectolytic enzyme in soft-rot while the pI 7.5 enzyme has no documented counterpart in EC14. Subclone and transposon tn5 analyses of pDR1 indicate that 1.5 kb is necessary for the production of the pI 9.5 and pI 7.5 enzymes and that these enzymes are produced independently of other EC14 pectate lyase enzymes. Plasmid pDR30 contains a 2.1 kb EC14 DNA insert that mediates the production of an endo-polygalacturonase and an exo-pectate lyase in HB101. The exo-pectate lyase encoded by pDR30 produces an inducer of endo-pectate lyase synthesis as a reaction product. The endo-polygalacturonase encoded by pDR30 is thought to play a role in plant cell wall pectic polymer degradation. Restriction endonuclease and Southern hybrididizatian analyses indicate that the EC14 genes on plasmids pDR1 and pDR30 are not part of the same operon. Escherichia coli strain HB101 containing plasmid pDR1 or plasmid pDR30 is unable to macerate potato tuber slices. However, HB101 containing plasmids pDR1 and pDR30 can cause limited maceration of potato tuber slices. There appears to be a genetic interaction between plasmids pDR1 and pDR30 in maceration of potato tuber tissue. However, the EC14 gene(s) contained on plasmid pDR1 are transcribed independently of the EC14 genes contained on plasmid pDR30. It is possible that transcription of certain pectolytic enzymes independent of other pectolytic enzymes provides a flexible system for plant cell wall pectic polymer degradation. / Ph. D.

Quality protein maize (QPM) has high nutritional value, but production is threatened by downy mildew (DM) and maize streak virus disease (MSVD) among other constraints. There are few studies of DM and MSVD resistance in QPM cultivars. The objective of this study was to improve resistance to DM and MSVD in three QPM populations. This was realized through ascertaining farmers’ key production constraints and special preferences for cultivars; determining the utility of recurrent selection method for improvement of three QPM populations (SussumaS2, ZM521Q and Pop62SRQ); and determining grain yield potential. The study was conducted in Mozambique for DM and in Zimbabwe for MSV, during 2003 to 2006. Surveys were conducted in Manica and Angonia districts in Mozambique to ascertain farmers’ perceptions and preferences for maize varieties, especially QPM. Participatory rural appraisal tools that included semi-structured questionnaires and focus group discussions were used to collect data. Results showed that farmers predominantly grew open pollinated varieties and fewer normal maize hybrids (non-QPM), and grain yield was estimated to be very low (0.2 to 0.6 t ha-1). Results showed that drought and insect pests were the dominant constraints to maize productivity in Mozambique, while diseases were ranked third. Downy mildew disease and MSVD were considered to be the most important diseases reducing maize productivity. Farmers also showed high preference for high yielding and early maturity cultivars in all areas. Predominantly, farmers were still using their local landraces because of sweet taste, particularly for home consumption and flint grain for storage. Farmers’ access to improved cultivars was limited due to high seed prices on the local market. Research priorities as perceived by the farmers included breeding for resistance to drought, grain weevils and diseases and sweetness. Generally, farmers showed little knowledge of QPM varieties and the importance of this trait, but they observed that the few QPM varieties they knew had some weaknesses such as poor storability and susceptibility to DM and MSVD which required improvement. These results should be considered in breeding new cultivars, both normal and QPM. To improve DM and MSV disease resistance in QPM varieties, S1 recurrent selection was conducted in three QPM populations, Sussuma, ZM521Q and Pop62SRQ at Umbeluzi Research Station in Mozambique and at CIMMYT-Harare Research Quality protein maize (QPM) has high nutritional value, but production is threatened by downy mildew (DM) and maize streak virus disease (MSVD) among other constraints. There are few studies of DM and MSVD resistance in QPM cultivars. The objective of this study was to improve resistance to DM and MSVD in three QPM populations. This was realized through ascertaining farmers’ key production constraints and special preferences for cultivars; determining the utility of recurrent selection method for improvement of three QPM populations (SussumaS2, ZM521Q and Pop62SRQ); and determining grain yield potential. The study was conducted in Mozambique for DM and in Zimbabwe for MSV, during 2003 to 2006. Surveys were conducted in Manica and Angonia districts in Mozambique to ascertain farmers’ perceptions and preferences for maize varieties, especially QPM. Participatory rural appraisal tools that included semi-structured questionnaires and focus group discussions were used to collect data. Results showed that farmers predominantly grew open pollinated varieties and fewer normal maize hybrids (non-QPM), and grain yield was estimated to be very low (0.2 to 0.6 t ha-1). Results showed that drought and insect pests were the dominant constraints to maize productivity in Mozambique, while diseases were ranked third. Downy mildew disease and MSVD were considered to be the most important diseases reducing maize productivity. Farmers also showed high preference for high yielding and early maturity cultivars in all areas. Predominantly, farmers were still using their local landraces because of sweet taste, particularly for home consumption and flint grain for storage. Farmers’ access to improved cultivars was limited due to high seed prices on the local market. Research priorities as perceived by the farmers included breeding for resistance to drought, grain weevils and diseases and sweetness. Generally, farmers showed little knowledge of QPM varieties and the importance of this trait, but they observed that the few QPM varieties they knew had some weaknesses such as poor storability and susceptibility to DM and MSVD which required improvement. These results should be considered in breeding new cultivars, both normal and QPM. To improve DM and MSV disease resistance in QPM varieties, S1 recurrent selection was conducted in three QPM populations, Sussuma, ZM521Q and Pop62SRQ at Umbeluzi Research Station in Mozambique and at CIMMYT-Harare Research. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Maize ear rots are among the most important impediments to increased maize production in Africa. Besides yield loss, they produce mycotoxins in their host whose contamination has been linked to several human and animal mycoses. The main objectives of the studies reported on in this thesis were (i) to investigate farmer perceptions of maize ear rot disease and prospects for breeding for host plant resistance in Zambia; and (ii) to establish the levels of incidence and extent of maize ear rot infection as well as the level of mycotoxins in the maize crops of smallholder farms in central and southern Zambia; (iii) to appraise the field inoculation techniques and assess them for their suitability for the Zambian environmental conditions, (iv) to determine the combining ability of Zambian maize populations for resistance to ear rot and investigate the genetic basis of this resistance; and (v) to investigate both direct and indirect responses to full-sib selection for ear rot resistance in Zambian maize populations. A participatory rural appraisal (PRA) was conducted in four communities, involving a total of 90 farmers. Participatory methods were used, such as focused group discussions, group interviews, participant scoring and ranking. Farmers ranked and scored the various constraints affecting their maize production in general and the maize ear rots in particular. Ear rots were ranked as the third most important biotic stress and it was evident that although farmers were aware of the disease, they were not aware of mycotoxins. This was reflected in the way they disposed of rotten maize: either by feeding livestock or eating it in periods of hunger. The survey of ear rots and mycotoxins was carried out in the Southern and Central Provinces of Zambia. A total of 114 farms were covered in the survey: maize samples were collected and both ear rot fungi and mycotoxins were isolated. Fusarium and Stenocarpella were the most frequently isolated fungi from smallholder farms. The levels of fumonisins on these farms ranged from 0.05 to 192 ppm, while those of aflatoxins were between 1.5 and 10.6 ppb. In 50% of the farmsteads surveyed, the mycotoxins, i.e. fumonisins and aflatoxins, exceeded the recommended FAO/WHO 1limits of 2 ppm and 2 ppb, respectively. Five field inoculation techniques namely, colonised toothpick, leaf whorl placement, ear top placement, spore suspension spray, and silk channel injection, were evaluated over three seasons in a series of experiments. It was found that the leaf whorl placement of inoculums, followed by colonized toothpick method, gave a constant ranking of genotypes across locations and years compared to the other three methods. In addition, the use of a mixture of ear rots as inoculum was as effective as its principal single species constituents. In the population diallel analysis, five broad-based maize populations were crossed in a diallel and evaluated under artificial ear rot inoculation using an inoculum mixture of three ear rot fungi, Aspergillus flavus, Fusarium verticilloides and Stenocarpella maydis at four locations in Zambia. The purpose was to estimate general (GCA) and specific combining ability (SCA) and investigate genotype x environment interaction. GCA effects were found not to be significant for disease severity but were significant for grain yield across environments. Populations with a strong GCA effect for disease severity across sites included PRA783244c3, Pop25, MMV600, and ZUCASRc2. Across sites, the F1 combinations, MMV600 x Pop25, ZUCASRc2 X Pop25, and Pop25 x PRA783244c2 had strong SCA effects for root lodging, ear drooping, husk cover and ear insect damage. In a related diallel analysis of 10 full-sib families derived from these populations, it was observed that resistant x susceptible families and their reciprocal crosses performed better than their resistant parents, suggesting an over dominant expression of resistance. Both maternal and non maternal effects were observed to be influencing resistance to ear rots. There was a preponderance influence of non-additive gene action. A response to full-sib recurrent selection was conducted in four locations in Central Zambia. Out of the 343 families created in 2005/6 season, 10% were selected from each population and recombined to create five new populations. These, with the original populations, were evaluated in four sites during the 2007/8 season. There was a net reduction in ear rot incidence and rot severity in the new synthetic population. Pop10 had the largest reduction in disease severity. The predicted gain per cycle was -4.1% and realized gain was -2.5% for disease incidence, and 0.19% and 19.4% for grain yield. Genetic variability was maintained though with low heritability estimates. Negative but at times strong association between grain yield and ear rot disease severity was detected suggesting that in general selecting for ear rot resistance would enhance grain yield in the five populations. Overall the importance of the ear rots and mycotoxins in compromising yield and health of the communities in Zambia, respectively, were confirmed and support the call to improve maize varieties for resistance to ear rots. The results indicate that the five populations could be enhanced for ear rot resistance through population improvement procedures such reciprocal recurrent selection that exploit both additive and non-additive variation. Selection might be compromised by the large genotype x environment interaction effects, and large reciprocal effects and their interaction with the environments. To enhance repeatability genotypes should be artificially inoculated, by placing the inoculum in the leaf whorl followed by colonized toothpick inoculation, and screened in many environments to identify genotypes with stable resistance to ear rots. / Thesis (Ph.D) - University of KwaZulu-Natal, Pietermaritzburg, 2009.

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood for infection by other pathogens. Knowledge about the epidemiology and especially inoculum sources of this disease is imperative for subsequent development of management strategies. Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through infected propagation material in South Africa. However, the infection pathways and inoculum sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen in various media is by means of isolation onto artificial growth media. This has proven to be problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it can be identified. The aim of this study was (i) to develop a protocol for the molecular detection of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora. A protocol was developed and validated for the molecular detection of Pa. chlamydospora in grapevine wood. Firstly, several previously published protocols were used to develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic DNA from grapevine wood. The protocol was validated using various grapevine material from 3 different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3 different nurseries, including grapevines that were subjected to hot water treatment. The basal end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial medium and molecular detection. The identity of PCR products obtained from a subset of samples, that only tested positive for Pa. chlamydospora based on molecular detection, was confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1% of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora was not isolated from hot water treated samples. The results confirm the importance of hot water treatment for proactive management of Petri disease in grapevine nurseries. However, Pa. chlamydospora DNA was molecularly detected in hot water treated samples in frequencies similar to that detected in non-hot water treated samples. As expected, the DNA in hot water treated plants was not destroyed and could be detected by the developed molecular detection protocol. This is an important consideration when using molecular detection for disease diagnosis or pathogen detection and shows that these methods should be used in conjunction with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10 to 15 times cheaper than commercial DNA extraction kits. Preliminary studies showed that the aforementioned molecular detection technique was not specific and sensitive enough for detection of Pa. chlamydospora in soil and water (unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. Rootstock cane sections and soil samples were taking from the mother blocks from several nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage and grafting. Scion and rootstock cuttings were also collected during grafting and soil were collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were Pa. chlamydospora specific, except for five bands obtained from callusing media and one band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12- hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the callusing medium samples. These media should therefore be considered as potential inoculum sources or infection points of the pathogen during the nursery stages. The results furthermore confirmed previous findings that Pa. chlamydospora is mainly distributed through infected rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or chemical amendments in the hydration water and callusing medium and wound protection from soil borne infections. / AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora is die hoof veroorsakende organisme van Petri se siekte wat lei tot die agteruitgang en terugsterwing van jong wingerdplante (1-7 jaar oud) en veroorsaak verhoogde vatbaarheid van hout vir infeksie deur ander patogene. Kennis oor die epidemiologie en veral die inokulumbronne van die siekte is noodsaaklik vir die daaropvolgende ontwikkeling van beheerstrategieë. Isolasies het getoon dat Pa. chlamydospora meestal versprei deur middel van geïnfekteerde voortplantingsmateriaal in Suid-Afrika. Die infeksieweë en inokulumbronne in wingerdkwekerye is egter steeds onbekend. Die enigste bestaande metode vir die opsporing van die patogeen, in verskeie mediums, is deur middel van isolasie op kunsmatige groeimediums. Dit is egter gevind om problematies te wees aangesien die swam uiters stadig groei (dit vat tot 4 weke vanaf isolasie tot identifikasie) en die kulture is telkens oorgroei deur ander organismes voordat identifikasie kan plaasvind. Die doel van die studie was (i) om ‘n protokol te ontwikkel vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout, en (ii) om die protokol te gebruik, saam met ander, om verskillende monsters (water, grond, onderstok- en bostok-ente en kallusmedium) te toets, wat versamel is van kwekerye in Suid- Afrika, tydens verskillende kwekerystadiums, vir die teenwoordigheid van Pa. chlamydospora. ‘n Protokol is ontwikkel en geverifieer vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout. Eerstens is verskeie protokols wat voorheen gepubliseer is, is as grondslag gebruik vir die ontwikkeling van ‘n ekonomiese en tydbesparende DNA ekstraksie protokol. Hierna is PKR (polimerase ketting reaksie) amplifikasie met spesie-spesifieke inleiers (Pch1 en Pch2) gevind om sensitief genoeg te wees om so min as 1 pg van Pa. chlamydospora genomiese DNA van wingerdhout op te spoor. Die protokol is geverifieer deur verskeie wingerdhoutmateriaal van 3 verskillende onderstokkultivars (101-14 Mgt, Ramsey en Richter 99) te gebruik, wat elk versamel is van 3 verskillende kwekerye. ‘n Aantal van die wingerstokke is ook onderwerp aan warmwaterbehandeling. Die basale kant van die onderstok is parallel geanaliseer vir Pa. chlamydospora deur gebruik te maak van isolasies op kunsmatige groeimedium asook molekulêre opsporing. Die identiteit van ‘n submonster van PKR produkte van verskeie monsters, wat slegs positief getoets het vir Pa. chlamydospora met die molekulêre opsporing, is bevestig om Pa. chlamydospora spesifiek te wees. Dit is gedoen deur middel van restriksie ensiem analise met AatII. Molekulêre opsporing is gevind om aansienlik meer sensitief te wees as isolasies, deurdat Pa. chlamydospora opgespoor is van positiewe sowel as negatiewe isolasies. Die molekulêre tegniek het Pa. chlamydospora in ‘n gemiddeld van 80.9% van die monsters opgespoor, terwyl slegs ‘n gemiddeld van 24.1% van die monsters postief getoets het vir Pa. chlamydospora, deur middel van isolasies. Pa. chlamydospora is nie geïsoleer van die monsters wat warmwaterbehandeling ondergaan het nie. Die resultate bevestig hoe belangrik warmwaterbehandeling is vir die proaktiewe beheer van Petri se siekte in wingerdkwekerye. Pa. chlamydospora DNA is met die molekulêre tegniek opgespoor, in warmwaterbehandelde monsters, in getalle wat ooreenstemmend is met die van niewarmwaterbehandelde monsters. Soos verwag, is DNA in warmwaterbehandelde plante nie vernietig nie en kon dit telke male opgespoor word deur die ontwikkelde molekulêre opsporing protokol. Dit is ‘n belangrike feit wat in ag geneem moet word wanneer molekulêre opsporing gebruik word in siekte diagnose en opsporing van patogene en dit is ‘n aanduiding dat die metodes gebruik moet word in samewerking met ander diagnostiese tegnieke. Die DNA ekstraksie protokol het getoon om tot en met 10 tot 15 kere goedkoper te wees as kommersiële DNA ekstraksie pakkette. Voorlopige studies het getoon dat die bogenoemde molekulêre opsporings tegniek nie spesifiek en sensitief genoeg is vir die opsporing van Pa. chlamydospora uit grond en water nie (ongepubliseerde data). Daarom is ‘n enkel-buis geneste-PKR tegniek geoptimiseer vir die opsporing van Pa. chlamydospora DNA wat geëkstraheer is vanaf grond, water, kallusmedium en wingerdhout. Dele van onderstokke en grond monsters is geneem vanaf moederblokke van verskeie kwekerye. Gedurende die voor-opberging en enting periodes is watermonsters versamel vanaf hidrasie en fungisied tenke. Bostok- en onderstokente is ook versamel gedurende enting en grond is versamel vanaf kwekerybeddens net voor uitplanting. Die enkelbuis geneste-PKR was sensitief genoeg om so min as 1 fg van Pa. chlamydospora genomiese DNA vanaf water en 10 fg vanaf hout, kallusmedium en grond op te spoor. PKR analise van die verskillende kwekerymonsters het getoon dat daar ‘n teenwoordigheid is van verskeie putatiewe Pa. chlamydospora spesifieke bande (360 bp). Daaropvolgende analise deur middel van DNA volgordebepaling en restriksie ensiem analise het bevestig dat al die 360 bp PKR bande wel Pa. chlamydospora spesifiek is, behalwe vir vyf bande wat verkry is vanaf kallusmedium en een band verkry vanaf water. As slegs Pa. chlamydospora spesifieke bande in ag geneem word, is daar met molekulêre opsporing die teenwoordigheid van Pa. chlamydospora gevind in 25% van die onderstokke, 17 % van die grond versamel vanaf moederblokke, 42% van die onderstokente versamel tydens enting, 16% van die bostokente, 40% van die watermonsters versamel voor die 12-uur hidrasie periode, 67% van die watermonsters versamel gedurende enting en 8% van die kallusmediummonsters. Hierdie mediums moet dus beskou word as potensiële inokulumbronne of infeksiepunte van die patogeen gedurende die kwekerystadiums. Die resultate bevestig ook verdere bevindinge wat aandui dat Pa. chlamydospora meestal versprei word deur geïnfekteerde onderstokke en ente. Geïnfekteerde bostokente is ook aangedui om potensiële draers van die patogeen te wees. Beheerstrategieë moet dus wondbeskerming van onderstok moederplante insluit, asook uitwissing van die patogeen vanaf onderstokente (bv. warmwaterbehandeling), toediening van biologiese of chemiese stowwe in die hidrasie water en kallusmedium en wondbeskerming teen grondgedraagde infeksies.