Alternaria spp. were isolated from various types of asymptomatic and symptomatic tissue of pistachio trees in South African orchards. Isolates were subdivided into four morphotypes based on colony morphology. Growth studies at different temperatures however, failed to differentiate the isolates into the same morphotypes. Fifty isolates from asymptomatic nuts, flower buds, leaves and twigs, as well as diseased nuts were submitted to a molecular assay using AFLP. These isolates were compared with five type species of Alternaria from the Centraalbureau voor Schimmelcultures. All Alternaria isolates from pistachio fell within a single A. alternata/A. arborescens group, and did not cluster according to the morphotypes. Due to the endophytic/quiescent nature of Alternaria spp. that cause disease on pistachio, the time of fungicide application is as critical as the formulation and concentration of the chemical used. Fungicide trails were conducted to determine the effect of repeated spraying throughout the year, making use of a system where all fungicides that proved to be effective against A. alternata isolates in vitro were sequentially used in a spray programme. Primary infection seemed to take place in the young developing flower buds. The use of fungicides early in the season is therefore critical if disease of the nuts is to be prevented. Further sequential sprays continued right up to the time of nuts being harvested. The two best performing fungicided treatments included all the fungicides that was found to be effective against Alternaria spp. in vitro, used sequencially from early in the season right up to harvesting. Doubling the dosage in the most successful application did not significantly reduce incidence of the fungus in nuts. Use of bud break oils as done in pistachio orchards in California and Australia was replaced by canopy wetting at Green Valley Nuts in order to increased the number of chilling hours needed for optimal fruit set. Isolations conducted from asymptomattic flower buds, asymptomatic midseason nuts or asymptomatic mature nuts revealed that none of the treatments increased the isolation frequency of quiescent Alternaria infection from pistachio tissue.

Physical damage and disease are known to cause changes in the lipid composition of plants. The biochemical pathways that lead to changes in the lipid composition were investigated in a comparative study using Russian wheat aphid (RWA) (Diuraphis noxia) infested and uninfested resistant (âTugela DNâ, âGariepâ and âBettaâ DN) and near isogenic susceptible (âTugelaâ, âMolopoâ and âBetta â) wheat cultivars. Lipoxygenase (LOX) activity was determined spectrophotometrically while LOX proteins were analysed by means of Western blots. Lipid peroxidation was determined by measuring malondialdehyde levels spectrophotometrically. To investigate the involvement of prostanoids in the RWA resistance response, prostanoid biosynthesis was inhibited with indomethacin where after the activities of defence related enzymes peroxidase (POD) and LOX were determined spectrophotometrically. Lipid-like products were analysed by means of gas liquid chromatography (GC/MS) and mass spectrometry. Western blots were used to analyse the cyclooxygenase (COX) proteins. LOX, which catalyzes the first step of the lipoxygenase pathway, was selectively induced in the infested resistant wheat. This increase in LOX activity coincided with lipid peroxidation. Several lipid-like compounds were found to be newly induced after infestation and the synthesis of others were enhanced after infestation in the resistant wheat. Many of these lipid products were identified as hydroxyl and keto fatty acids. Some of these fatty acids could be detected as early as 1 h.p.i. (hours post infestation). The levels of some fatty acids increased as infestation proceeded while the levels of others reached a peak 48 h.p.i. and declined towards 96 h.p.i. Hydroxyl and keto fatty acids are important signalling compounds during defence responses. A second pathway, COX pathway, which was believed to exist only in mammals, was also found during the wheat-RWA interaction. The COX protein was selectively induced in resistant wheat after infestation . It has high homology to mammalian COX, which is responsible for prostanoid synthesis. The involvement of prostanoids in the RWA resistance response was confirmed by inhibition studies of prostanoid biosynthesis. Downstream defence reactions e.g. LOX and peroxidase (POD) activities were inhibited upon inhibition of the prostanoid biosynthetic pathway. These results emphasize the importance of two lipid biosynthesis pathways (LOX and COX) that may be essential for the establishment of a successful defence response in wheat to the RWA.

South Africa hosts one of the largest cactus pear germplasm collections in the world. However, not all the varieties have been fully characterised, and evaluated for fruit quality, nutritional quality for use as fodder, and disease resistance. In this study, 38 South African cactus pear (Opuntia ssp.) varieties were characterised using AFLP markers to circumvent G X E effect on phenotypic characterisation. With the use of nine primer combinations, the varieties were grouped into four main clusters based on 346 fragments (per sample) of which 48% were polymorphic between samples. The dendrograms generated indicated that commercially cultivated varieties were dispersed amongst the different clusters indicating that they represent the genetic diversity within the germplasm. Genotype specific fragments were generated using six primer combinations, allowing the unique identification of nine varieties, three of which are commercially cultivated (Meyers, Roedtan, and Santa Rosa). Varieties that are recommended for commercial cultivation in the Mokopane district of the Limpopo Province, based on fruit quality and yield are Gymno Carpo, Malta, Algerian, Morado, Meyers, and Roedtan. These varieties meet the minimum requirements for cactus pear fruit production in South Africa. Nutritional quality evaluation of pruned cladodes from a commercial orchard in the Free State Province indicated that the varieties, Malta, Gymno Carpo, and American Giant ranked the highest in terms of CP content. Varieties that yielded the highest DM content were Messina, Nepgen, and Cross X. Varieties that ranked the highest for OM content were Cross X, Nepgen, and Sicilian Indian Fig. Gymno Carpo and Malta are amongst the varieties recommended for cultivation for fruit, as such they can be used as dual purpose crops for the production of both fodder and fruit. Evaluation for disease resistance indicated a quantitative mode of resistance across all varieties for all three fungal pathogens tested. The most resistant varieties surveyed in this study across all three fungal pathogens were Amersfoort, Meyers, and Algerian. Roly Poly, Direkteur, and Zastron were the more susceptible varieties. Of the three fungal pathogens tested, P. virens was the least affected by the antagonistic activity of the yeast isolates. Isolate 25 (Rhodotorula mucilaginosa) performed well against all three pathogens, whilst the remainder of the isolates displayed inhibition at varying degrees.

An attempt was made during this study to assign a putative role for At-RLK3 in Arabidopsis thaliana. Receptor-like protein kinases (RLKs) are a group of proteins that was described in various plants. These proteins have a very characteristic structure. They are integral plasma membrane proteins where a hydrophobic transmembrane domain links an extracellular ligand binding domain with an intracellular protein kinase domain. RLKs are involved in various plant processes including development, disease resistance and hormonal signaling. At-RLK3 is a single copy gene in A. thaliana that is expressed during various stress conditions. Oxidative and osmotic stress, infection with pathogens and exposure to salicylic acid (SA) all led to the induced expression of the gene. A similar increase in At-RLK3 protein levels was found after the respective treatments. The kinase domain shares high homology with several other protein kinases and has all 11 conserved sub-domains characteristic of plant protein kinases. It was proven that At-RLK3 is an active protein kinase that specifically phosphorylates serine and threonine amino acids within its active domain. It preferentially uses Mg 2+ as cofactor. The enzyme is located at the plasma membrane and is rapidly activated upon treatment with hydrogen peroxide (H2O2), menadione and osmotic stress conditions, but not after SA treatment. The extracellular domain has two conserved cysteine rich regions that form part of two âdomain of unknown functionâ (DUF) regions. These cysteines are thought to be involved in the formation of disulphide bridges and to be responsive to changes in the redox conditions surrounding the cell. Plants expressing an antisense copy of the gene showed an altered response to treatments with H2O2 and SA compared to the wild type plants. Whereas the wild type plants activated the plant defense shortly after treatment with both H2O2 and SA, only SA managed a similar activation in the transgenic plants. The transgenic plants treated with H2O2 did not show this initial activation. In addition, after both treatments the wild type plants exhibited an amplified and sustained activation of the defense reaction 48 h after treatment which was completely absent in the transgenic plants. This led to the hypothesis that At-RLK3 is responsible to detect changes in extracellular H2O2 levels in A. thaliana most probably due to a change in redox conditions. This then leads to the activation of the enzyme and the subsequent activation of the plant defense response.

Puccinia triticina (leaf rust) is an obligate biotrophic fungus. It is a major pathogen of Triticum aestivum (wheat) all over the world. In a previous study, two differentially expressed genes were cloned from resistant wheat infected with P. triticina. According to the sequence analysis, the first encoded a cell wall invertase (TaCwi01) and the other a monosaccharide transporter (TaMst01). These two genes and their encoded proteins appear to play a role during sugar signaling. Sugar signaling has already been described in several plant-pathogen interactions. In this study, the presence of sugar signaling as well as the roles of TaCwi01, TaMst01 and a hexokinase gene in the leaf rust â wheat interaction were investigated. Gene expression studies of all three genes showed both early as well as late changes in expression. TaCwi01 gene expression showed an early induction in IS plants at 3 hpi followed by an immediate and complete inhibition until 36 hpi. Expression in IR plants was repressed for the duration of the study with a transient increase at 21 hpi. Enzyme activity analysis revealed a similar pattern of the expression with a significant activation in both IS and IR plants at 27 and 24 hpi respectively. Results of TaMst01 expression revealed both an early induction at 3 to 6 hpi and a late induction at 33 hpi in IS and IR plants. The later induction in expression of TaMst01 was reflected in the significant increase in MST activity at 30 hpi. Hexokinase expression analysis showed a slight increase in expression from 0 to 6 hpi in both IS and IR plants. This was again associated with increased enzyme activity shortly after infection. Furthermore it was shown that due to a SCPU domain in the polypeptide sequence of TaCwi01, it is possible that this gene could have originated from the pathogen. It was concluded that a putative sugar signaling took place during the infection of wheat with leaf rust and that all three genes played a definite role.

Diallel crosses were evaluated in three agro-ecologies in Colombia and parents and families selected for conventional and molecular breeding trials conducted during the study. At the same time an inter-specific cross was evaluated for the effective introduction of higher DMC from wild cassava relatives. High positive and negative GCA and SCA values were estimated for the agronomic yield traits. Harvest index and DMC were under the influence of additive gene action, while ComRt, FRY and DRY were more influenced by non-additive effects. Moderate to high heritability estimates were obtained for the different agronomic traits. High heritability values were estimated for DRY, FRY, DMC, HI, RtPlt and moderate for RtWt. AMMI analysis ranked FRY, DRY, HI and RtPlt as stable, DMC relatively stable and RtWt as unstable across environments. In the seedling stage DRY, FRY, RtWt, and ComRt ranked parents in the same direction. Dry matter content and HI ranked parents similar but in the opposite direction for other yield characteristics. Overall rating ranked MPER 183 and CM 4574-7 as best parents followed by SM 1565-15 and SM 1665-2. Seedling nursery and the diallel analysis identified SM 1741-1 and MPER 183 as good parents for a number of traits. Replication and blocking during clonal evaluation greatly reduced the non-genetic factors resulting in high correlation among traits evaluated. Fresh root yield, DMC, HI, RtWt and RtPlt were found to be important in determining DRY. Root weight was found to be the most important indicator of DRY. Crossing of the elite cultivar MTAI-8 to the wild relative M. tristis resulted in high DMC values ranging from 34.39% to 42.73%. Bulk segregant analysis showed that parent SM 1741-1 was associated with favourable QTLs for DMC. Markers SSRY 150 (R2=18.1%) and SSRY 160 (R2=28.9%) in the diallel cross, SSRY 99 (R2=22.68), SSRY 141 (R235.89) and NS 169 (R2=20.01) in the wild cross and SSRY 11 (R2=34.57), SSRY 62 (R2=50.21) and NS 644 (R2=29.09) in the mapping population showed association with DMC with values high enough for practical use in a MAS breeding programme.

The aim of this study was to investigate the early events following the infection of wheat with leaf rust. An attempt was made to identify and characterize genes putatively involved in these early events. More specifically, the aim was to obtain genes whose role in infected plants could be linked to the resistance locus within the resistant plant. DDRTâPCR was used to isolate differentially expressed genes from the resistant Thatcher+Lr34 plants during the first 15 h after infection. Four cDNA fragments were cloned and sequenced. The first clone coded for a monosaccharide transporter, while the second clone encoded a cell wall invertase. Both these clones formed part of a different study to postulate a role for these proteins during infection. The third cDNA clone, coded for a putative heat shock protein. Heat shock proteins are molecular chaperones and are normally involved in ensuring cellular homeostasis by preventing the aggregation of denatured proteins and assisting in the folding and transport of new and denatured proteins. The fourth clone encoded an indoleâ3âglycerol phosphate lyase. The expression of the putative heat shock protein increased 86 fold within 9 h.p.i in infected resistant wheat and was chosen for further analysis. The gene shared very high sequence similarity to an O. sativa HSP70 gene and was called TaHlp01 (Triticum aestivum Heat shock Like Protein 01). TaHlp01 was inducibly expressed upon infection of resistant wheat with leaf rust as well as yellow rust but its expression remained constant in the infected susceptible cultivars. This indicated that the regulation of expression is dependent on the presence of the resistance locus within the resistant cultivars. A transient repression of TaHlp01 expression was found during the later stages of infection in both the IR and IS plants that were similar to a transient repression of TaHlp01 expression after Thatcher+Lr34 plants were treated with salicylic acid. TaHlp01 expression was also found to be induced by heat stress, indicating a possible role during heat stress. A possible interplant communication event was also examined. It was found that infected plants were able to induce the defence response of uninfected plants. This communication between infected and uninfected plants was more effective between plants of the same cultivars than between different cultivars. It appears as if resistant plants were able to induce a more controlled defence response than susceptible plants. When uninfected resistant and susceptible plants exposed to infected plants, was infected themselves, they exhibited a more resistant phenotype compared to plants that was not exposed. The communication event during this interaction most likely involves jasmonic acid.

Currently, high numbers of genotypes are unnecessarily being carried from generation to generation in the early generations of wheat breeding programs, only to be discarded in the advanced generations because of not reaching the strict quality standards. If a simple and reliable test could be used as an indication for baking quality in the early generations of the program, a more effective way to do culling of poor quality lines would be possible. Advanced (elite) and early generation material (F4) were evaluated to determine the influence of genotype and environment on the variation detected in SDS sedimentation. Firstly, nine intermediate hard red wheat elite lines and two intermediate hard red cultivars were evaluated for three consecutive years over eight locations in the Eastern Free State. Secondly, six F4 populations and two hard red cultivars were evaluated for one season at three locations in the Eastern Free State. Single plants in the F2 generation were selected by taking SDS sedimentation into consideration (selected group) and without taking SDS sedimentation into consideration (unselected group) and the influence of selection on the subsequent F4 generations was determined. The following conclusions were reached in the study: · There is a significant correlation between baking volume and SDS sedimentation. SDS sedimentation is therefore a reliable predictor of baking volume. · SDS sedimentation successfully discriminated between the genotypes in both the F4 and elite material. The contribution by genotype was high enough to make effective selection for SDS sedimentation possible. The large genotype x year effect on performance in contrast to the genotype x location effect in the elites, indicated that testing of genotypes across years may be more important than testing of genotypes across locations. · There were significant differences between the groups where selection was done on SDS sedimentation (selected) and groups where selection was done without taking SDS sedimentation into consideration (unselected groups) in most of the populations. The difference in SDS sedimentation between selected and unselected groups for most populations remained constant from the F2 to the F4 generation. Early generation SDS sedimentation selection would therefore effectively enhance baking quality in later generations. This would decrease the amount of lines that have to be evaluated in later generations and would have practical and financial advantages for a breeding program. · In both the elite and F4 material, there was a strong relationship between SDS sedimentation and dough strength, presumably because gluten responsible for the dough strength includes the glutenin, which takes part in the flocculation action of the SDS sedimentation process. SDS sedimentation selection in the early generations of a breeding program can therefore result in excessive dough strength in subsequent generations of certain crosses if a trait evaluating dough strength does not accompany SDS sedimentation selection. · The relationship between protein quantity (content) and SDS sedimentation varied, possibly due to difference in the glutenin quantity and quality between the genotypes. The negative correlation between SDS sedimentation and grain yield was highly significant. This could possibly be attributed to the influence of protein content, because it is well known that a negative association exists between protein content and grain yield, which was also evident in the study. · Mixing development time and hardness index (F4 generation) and grain yield and mixing development time (elite material) are the most definable independent variance predictors for SDS sedimentation in the F4 material. Therefore, mixing development time has a large influence on SDS sedimentation or is related to the same aspect, presumably gluten. · To perform effective SDS sedimentation selection in early generations, testing at one to two locations is adequate. A further increase of locations will result in a very small increase in precision, but would have large practical and financial implications and is therefore not recommended. The gains in precision obtained by increasing the number of years were substantially more than by increasing the number of replications. It is recommended that four to five years is essential for reliable SDS sedimentation selection. This can be done by selecting in the F2 to F5 generations in a breeding program, which will result in a decrease in variation and an improvement in SDS sedimentation stability. This will result in a drastic decrease in the effect of genotype x environmental interaction, which will make further selection later in the breeding program for SDS sedimentation selection unnecessary.

In an effort to characterize the relationship between fungal pathogenicity and host genetic resistance, sorghum panicles of 11 genotypes were inoculated with five fungi frequently isolated from sorghum grain. Panicles were inoculated at anthesis with Fusarium graminearum, Fusarium thapsinum, Curvularia lunata, Phoma sorghina and Alternaria alternata spores. There were highly significant differences in the levels of fungal pathogenicity on different sorghum genotypes. These differences accounted for 58.4% of observed variation in ergosterol concentration. Genotype by pathogen (G x P) interactions accounted for 33.5% of the observed ergosterol concentration variation. The implication is that different genotypes reacted differently to different fungi. The genotypic reactions of the hosts accounted for 8.1% of the observed ergosterol concentration variation. Overall, fungal pathogenicity is the most important factor to consider in the evaluation of germplasm for grain mould resistance. Possible sources of resistance could be identified by use of biplot analysis of G x P interactions. Visual scoring for grain mould has limited value without identifying causal fungi. Fusarium thapsinum and Phoma sorghina were the most abundant fungi across all genotypes. Gene action and heritability for grain mould resistance in sorghum were investigated using a selection of 9 random pollen parents with varying levels of grain mould resistance to a different set of three random seed parents. Differences in ergosterol concentration were used as a measure of level of grain mould resistance among all genotypes. Use of ergosterol concentration as a measure of grain mould severity did not correlate with visual field scoring. Other traits measured include plant yield, plant height, kernel hardness, field grade score, days to flowering, glume color and seed color. The combined analysis of variance showed no genotypic variance for grain mould resistance. The expression of grain mould resistance was also not stable with significant genotype x location interaction. The analysis from Potchefstroom showed significant differences among genotypes whereas Cedara-1 and 2 showed no differences. Additive genetic variance was greater than dominance variance for all traits except grain mould resistance. A significant heterosis of -20.15% was observed for grain mould resistance indicating the importance of use of hybrid seed. LM124 (white seeded female) and LM130 (brown seeded female) produced resistant hybrids. Due to very high environmental variance, grain mould heritability could not be detected. Yield indicated the highest heritability of 0.41 at Cedara-1, plant height 0.39 at Cedara-2 and kernel hardness 0.38 at Potchefstroom. The significance of genotype by environment interaction was assessed over three environments. Differences in ergosterol concentration were used as a measure of level of grain mould resistance among all genotypes. Significant G x E interaction was detected after analysis of variance across all three locations. Single site analysis was then done to better explain the nature of the G x E interaction. Potchefstroom is the only location that showed significant genotypic responses to grain mould infection. Low grain mould pressure at Cedara-1 and Cedara-2 caused very low genotypic responses. A biplot was then used to indicate all genotypic performances across the three locations in a graphical design. Fungal species infecting sorghum kernels were isolated, counted and identified. Low genotypic responses at the Cedara locations seems to have been caused by prevalence of a less aggressive fungal species, Mucor spp. Weather variables did not correlate significantly with ergosterol concentration. Mycotoxin levels of aflatoxin, deoxyvalenol (DON), and zearalenone together with ergosterol concentration levels across 39 sorghum genotypes were measured. Varying levels of ergosterol and mycotoxins were observed across the three locations. There was no correlation between ergosterol concentration and any of the mycotoxins which indicates that mycotoxins concentration is not related to total fungal biomass. Mycotoxin concentration must therefore be related only to the biomass of the fungal species that is producing the mycotoxin among all fungal species infesting the grain. Farmers in Potchefstroom should be wary of aflatoxins and zearalenone while farmers in Cedara should choose varieties that tend to be low in DON and aflatoxins while not ignoring zearalenone.

Since conventional sources of resistance to stem borers are not adapted to South African conditions, resistance has to be introgressed into locally adapted breeding material. Combining ability for resistance possibly differs between heterotic groups, and susceptible elite material do not necessarily respond similarly in crosses with sources of resistance from different genetic backgrounds. From earlier work conducted under greenhouse conditions it was observed that larval development rate possibly differs with difference in time of the year. The objective of this study was to determine which combinations of resistant sources with local elite susceptible material could provide the best expression of resistance. Susceptible and resistant lines were crossed in various combinations to obtain semi-resistant single and double crosses with various resistance levels. These were compared to susceptible and resistant standards in field trials. Plants were artificially infested with neonate larvae. Evaluations of leaf feeding damage, stem damage, ear damage, plant height reduction and grain yield were subjected to factorial analysis, using planting date and infestation as factors. Two resistant lines and one semi-resistant single cross were identified that seemed to provide particularly high levels of resistance. However, the study did not successfully identify which resistance source should be used in combination with which heterotic group, neither did resistance derived from different sources seem to compliment each other. Using principal component analysis of data on double crosses, no pattern could be observed that indicated crosses comprising more than one resistant parent to be more resistant than those in which only one resistant parent featured, whereas variance in resistance did not seem to relate to heterotic patterns. A further aspect of the study involved investigation into the effect of variance in planting date on the expression of resistance. Various lines and hybrid combinations with different resistance levels were evaluated in a field trial comprising five planting date treatments and artificial infestation of plants. Data were subjected to factorial analysis as above. The expression of resistance was significantly affected by planting date. The incidence of leaf feeding damage, internal plant damage, larval survival and plant height reduction decreased with an increase in planting date, whereas larval mass and the incidence of ear damage increased with later planting. It appears that increased ear damage with later planting may result at least in part from an increase in larval development rate as affected by a decreasing photoperiod.