Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: In order to test the effect of different plant growth-promoting strategies on Triticum aestivum L. (wheat), we investigated the ability of biochar and a grain-associated soil yeast, to improve the growth of this crop. Our first goal was to study the effect of biochar amendments to sandy soil on the growth and nutrition of wheat in the presence of mycorrhizal fungi. This was accomplished by amending soil with 0%, 1%, 2.5%, 5% and 10% (w/w) biochar and cultivating wheat plants in these soil-biochar mixtures. After harvesting, plant growth and mycorrhizal colonization of roots were measured. In addition, we studied the nutritional physiology of these plants with regards to nitrogen (N), phosphorous (P) and potassium (K) concentrations, as well as the growth efficiencies and uptake rates of these nutrients. We found that wheat growth was improved by biochar amendments to soil, probably as a result of elevated K levels in the plant tissues supplied by the biochar amendments. The second goal of this study was to obtain a soil yeast from the rhizosphere of another monocot in the family Poaceae, i.e. Themeda triandra Forssk. (red grass), and then evaluate this isolate for its ability to improve wheat performance. Three different Cryptococcus species were isolated from the rhizosphere of wild grass, i.e. Cryptococcus zeae, Cryptococcus luteolus and Cryptococcus rajasthanensis. Since C. zeae was previously isolated from maize, an isolate representing this species was selected to be used in further experimentation. With the ultimate goal of testing the ability of this yeast to improve wheat growth, its effect on wheat germination was investigated and compared to that of two other soil yeasts, i.e. Cryptococcus podzolicus CAB 978 and Rhodotorula mucilaginosa CAB 826. These three yeasts were subsequently tested for their ability to improve wheat growth in pot cultures in a greenhouse. After one and two months of growth, the culturable yeasts present in the rhizosphere and bulk soil were enumerated. The effects of these yeasts were elucidated by measuring wheat growth in terms of dry weight, as well as root and shoot relative growth rates (RGR). Changes in wheat nutrition were evaluated by determining the concentrations, growth efficiencies and uptake rates for P, K, zinc (Zn) and iron (Fe). During this study, it was found that only C. zeae CAB 1119 and C. podzolicus CAB 978 were able to enhance seed germination. Similarly, it was shown that C. zeae CAB 1119 was able to improve wheat growth during the first and second month of cultivation, whereas C. podzolicus CAB 978 only improved growth during the first month, and R. mucilaginosa CAB 826 had no effect on growth. This improved growth could be attributed to C. zeae CAB 1119 improving the P, K, Zn and Fe growth efficiency of wheat, which positively influenced the root and shoot RGR, and subsequently wheat growth. Our final goal was to test whether C. zeae CAB 1119 could affect wheat growth and nutrition when cultivated in sandy soil, which contained natural microbial consortia and 10% (w/w) biochar. Plants treated with viable or autoclaved cells of C. zeae CAB 1119, were subsequently cultivated in soil only or soil amended with biochar. After one month, plants were harvested and growth was measured with regards to dry weight, root RGR and shoot RGR. In addition, the concentrations of P, K, Zn and Fe were analyzed for these plants, where after the growth efficiencies and uptake rates were calculated for these four nutrients. Results indicated that plants growing in soil amended with biochar, and treated with viable C. zeae CAB 1119, showed the best growth. The increased root and shoot RGR witnessed in these plants was probably due to increased concentrations of P and K in the plants. This study opens new avenues of research with regards to the bio-fertilizers of wheat. / AFRIKAANSE OPSOMMING: Die uiteindelike doel van die studie was om die effek van verskillende plantgroei bevorderende metodes op die groei van Triticum aestivum L. (koring) te ondersoek. Dus het ons die vermoë van houtskool en ‘n graan-geassosieerde grondgis getoets om die groei van dié plant te bevorder. Die eerste doel van die studie was om die effek van houtskool toedienings tot sanderige grond te evalueer. Dit is bewerkstellig deur 0%, 1%, 2.5%, 5% en 10% (w/w) van die houtskool by die sand toe te voeg en koring in die houtskool-sand mengsels te kweek. Na die verlangde groei tydperk is die koring geoes en die mikorrizale kolonisasie op en in die koring wortels bepaal. Gedurende hierdie studie is die effek van bogenoemde toedienings op die fisiologie van die plante ondersoek deur die konsentrasies, opname tempo’s, en groei ekonomie van die plante vir stikstof (N), fosfaat (P) en kalium (K) te bepaal. Ons het gevind dat die groei van koring deur die toediening van houtskool bevorder is en dit blyk dat dié effek weens die teenwoordigheid van hoë K vlakke in die plantweefsel is. Die tweede doel van ons studie was om ‘n gis vanuit die risosfeer van ‘n monokotiel wat aan die familie Poacea behoort, naamlik Themeda triandra Forssk. (rooigras) te isoleer. Die vermoë van die isolaat om die groei van koring te bevorder was daarna getoets. Drie verskillende Cryptococcus spesies was vanuit die risosfeer van rooigras geïsoleer, nl. Cryptococcus zeae, Cryptococcus luteolus en Cryptococcus rajasthanensis. Omdat C. zeae in ‘n vorige studie vanaf mielies geisoleer was, is ‘n isolaat van hierdie spesie gebruik in verdere eksperimente. Met die doel om te bepaal of dié gisspesie koringgroei kan bevorder, was die effek van C. zeae op die ontkieming van koring bestudeer en vergelyk met dié van twee ander grond giste, nl. Cryptococcus podzolicus CAB 978 en Rhodotorula mucilaginosa CAB 826. Hierdie drie giste is ook ondersoek om die groei van koring in ‘n glashuis te bevorder. Na een en twee maande se groei was die getalle van giste teenwoordig in die risosfeer en grond verder weg van die wortels bepaal. Die effek van dié giste op die groei van koring is bepaal in terme van droë gewig asook die relatiewe wortel en halm groei tempos. Veranderinge in die nutrient status van koring is ondersoek deur die konsentrasies, groei-ekonomie en tempo van opname vir P, K, sink (Zn) en yster (Fe) te bepaal. Ons het gedurende dié studie gevind dat C. zeae CAB 1119 en C. podzolicus CAB 978 die ontkieming van koring kon verbeter. Ons het ook gevind dat C. zeae CAB 1119 die groei van koring gedurende die eerste en tweede maand van groei kon bevorder, terwyl C. podzolicus CAB 978 dit net gedurende die eerste maand kon vermag en R. mucilaginosa CAB 826 geen effek gehad het nie. Die verbeterde groei kon aan C. zeae CAB 1119, wat die P, K, Zn en Fe groei effektiwiteit van die plante verbeter het, toegeskryf word. Die verbetering van groei effektiwiteit het ‘n positiewe invloed op die relatiewe groeisnelheid van die wortels en halms gehad, en dus op koringgroei. Die laaste doel van die studie was om te bepaal of C. zeae CAB 1119 die groei van koring kon bevorder wanneer die koring in sand wat natuurlike mikrobiese populasies bevat en met houtskool aangevul is, gekweek word. Plante is met lewensvatbare of nielewensvatbare selle van C. zeae CAB 1119 behandel en gekweek in sanderige grond, en/of grond waarby 10% (w/w) houtskool toegevoeg is. Die plante is na een maand geoes en die groei bepaal in terme van droë massa en die relatiewe wortel en halm groei tempos. Die konsentrasies van P, K, Zn en Fe in die plante, asook die fisiologie van die plante, nl. groei ekonomie en tempo van opname, met betrekking tot P, K, Zn en Fe is bepaal, Ons het gevind dat plante wat in die houtskool-grond mengsel gekweek is en met lewensvatbare selle van C. zeae CAB 1119 behandel is die beste groei getoon het. Die verbeterde relatiewe groei tempos van die wortels en halms was mees waarskynlik die gevolg van verhoogde P en K konsentrasies in die plante. Hierdie studie toon nuwe resultate in verband met die gebruik van biologiese alternatiewes tot kunsmis.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Good quality water for agricultural use is rapidly becoming a luxury due to competition for this water among the municipal, industrial and agricultural sectors. This has often forced growers to use poor quality water for irrigation. Salinity is one of the main sources of poor water quality and high electrical conductivities (EC’s) due to salinity may become a problem. The aim of this study was to compare the response of South African spring wheat and South African spring barley at germination, seedling growth, vegetative growth, reproductive growth and maturity stage to salinity stress caused by irrigation with saline water. This study was conducted in the laboratory and under controlled glasshouse conditions at the University of Stellenbosch in the Western Cape Province of South Africa. Treatments in trial 1 (incubation trial) were made up of three wheat cultivars (SST 027, SST 056 and SST 087) and three barley cultivars (Nemesia, Erica and Hessekwa) exposed to five EC levels of NaCl solutions (4, 8, 12, 16 and 20 dS m-1) and a control (0 dS m-1) of distilled water, during the germination phase. In trial 2 (pot trial), wheat cultivar SST 027 and barley cultivar SVG 13 were also subjected to the above solutions, but plants were grown till the tillering stage. In trial 3 (pot trial) cultivars used in trial 2 were subjected to five nutrient solutions with EC levels of 1.6, 3, 6, 9 and 12 dS m-1 and allowed to grow till maturity (harvesting stage). Fully balanced nutrient solution with EC = 1.6 dS m-1 was used as a control and NaCl was added to the solutions to obtain the needed EC. In trial 1, final germination percentage (FGP), salt tolerance (ST) and germination rate (GR) were measured at 7 days after incubation. The study showed that when the EC level was increased, FGP, ST and GR of all wheat and barley cultivars tested were decreased. However, significant reduction was only observed at high EC levels with regard to FGP and ST. Wheat cultivars recorded faster GR compared to barley cultivars and tended to be less sensitive to salinity in the germination stage. Cultivars from the same species did not show significant differences. In trial 2, shoot length (SL), root length (RL), shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW) and root dry weight (RDW) were measured at 35 days after planting (DAP). In general, the study showed that salinity had a significant (P0.05) effect on seedling growth of all measured parameters of both wheat and barley. Mean values for most growth parameters were higher for barley cultivar SVG 13 as compared to wheat cultivar SST 027. However, little evidence was found to show that barley is more salt tolerant than wheat at the seedling stage. In trial 3, selected growth parameters were measured at tillering (28 DAP), booting (54 DAP), flowering (71 DAP) and maturity stage (150 DAP). The study showed that salinity had a significant (P0.05) effect on the vegetative growth, reproductive growth and grain yield of both wheat and barley. Although barley generally produced higher dry weights especially at the early growth stages no clear evidence was found that South African spring barley is more salt tolerant than South African spring wheat. / AFRIKAANSE OPSOMMING: Besproeiingswater met ‘n goeie kwaliteit vir landboukundige gebruik word vinning baie skaars weens kompetisie, a.g.v menslike en industriële gebruik. Produsente word dus dikwels gedwing om water met ‘n swak kwaliteit te gebruik vir besproeiing. ‘n Hoë sout inhoud (brakwater) soos gemeet deur ‘n hoë elektriese geleidingsvermoë (EC), mag dus ‘n problem wees. Die doel van hierdie studie was om te bepaal hoe Suid Afrikaanse lente koring en gars gedurende ontkieming asook saailing-, vegetatiewe-, reproduktiewe- en rypwordingstadiums reageer teenoor soutstremming wat veroorsaak is deur besproeiing met brakwater. Die studie is uitgevoer in laboratoriums en onder gekontrolleerde glashuistoestande by die Universiteit van Stellenbosch in die Weskaap Provinsie van Suid Afrika. Behandelings in die eerste proef (inkubasie studie) het bestaan uit drie koring kultivars (SST 027, SST 056 en SST 087) en drie gars kultivars (Nemesia, Erica en Hessekwa) wat tydens ontkieming benat is met vyf NaCl-oplossings met EC waardes van 4, 8, 12, 16 en 20 dS m-1 onderskeidelik, asook ‘n kontrole met gedistilleerde water (0 dS m-1). In die tweede proef is die koring kultivar, SST 027 en die gars kultivar SVG 13 in ‘n potproef ook aan bogenoemde oplossings blootgestel maar toegelaat om tot die stoelstadium te ontwikkel. In die derde proef is genoemde twee kultivars besproei met vyf voedingsoplossings met EC-waardes van 1.6, 3, 6, 9 en 12 dS m-1 en toegelaat om tot oesstadium te ontwikkel. ‘n Volledig gebalanseerde voedingsoplossing met EC = 1.6 dS m-1 is as kontrole gebruik en NaCl is by ander oplossings gevoeg om die verlangde EC te verkry. In die eerste proef waar die finale ontkiemingspersentasie (FOP), sout toleransie (ST) en ontkiemingstempo (OT) na 7 dae gemeet is, is gevind dat FOP, ST en OT van al die koring en gars kultivars wat getoets is, met toenemende EC gedaal het. Statisties betekenisvolle afnames in FOP en ST is egter slegs by hoë EC waardes waargeneem. Koring kultivars het vinniger ontkiem as gars kultivars en was geneig om meer tolerant teenooor sout stremming te wees vergeleke met gars. Verskille tussen kultivars van dieselfde spesie was egter weglaatbaar klein. In die tweede proef waar plante toegelaat is om te groei tot die stoelstadium (35 dae na plant) is al die gemete planteienskappe (stingel- en wortellengte, asook vars en droë massas van stingels en wortels) van beide gars kultivar, SVG 13 en koring kultivar, SST 027, betekenisvol verlaag deur ‘n toename in EC van die besproeiingswater. Hoewel gars ten opsigte van die meeste gemete eienskappe groter gemiddeldes as koring getoon het, is weinig bewys gevind wat daarop dui dat die getoetsde gars kultivar SVG 13 meer souttolerant is as die koring kultivar SST 027. In die derde proef waar dieselfde koring en gars kultivars vanaf plant tot oestyd besproei is met genoemde voedingsoplossings en metings tydens stoelstadium (28 dae na plant), stamverlenging (54 dae na plant), blomstadium (71 dae na plant) en oesrypstadium (150 dae na plant) gedoen is, is alle gemete vegetatiewe-, reproduktiewe- en opbrengskomponente van beide spesies verlaag deur die soutstremming. Hoewel gars ook in hierdie proef veral gedurende vroeë groeistadiums groter droë massas as koring geproduseer het, is geen konkrete bewyse gevind wat daarop dui dat die getoetsde Suid Afrikaanse lente gars kultivar SVG 13 meer sout tolerant is as die koring kultivar SST 027.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Global food security is hampered by a variety of insects/pest and plant diseases. In wheat, the Russian wheat aphid (RWA) is a significant pest problem in many areas of the world. Wheat has developed defensive mechanisms against the RWA over time which are activated upon feeding. One such mechanism is the hypersensitive response (HR) which is effective against phloem-feeding insects i.e. D. noxia (Diuraphis noxia, Kurdjumov, RWA). In this study, two genes associated with the hypersensitive response i.e. ascorbate peroxidase (APX) and glutathione S transferase (GSTF6b) were investigated to elucidate their function in the defensive mechanism of wheat using a reverse genetic approach i.e. particle bombardment. This study has succeeded in the established of a tissue culture and transformation system which generated three genetically modified wheat plants with decreased resistance to RWA feeding due to gene silencing. The establishment of this system enabled to test the association of defensive related genes in wheat to RWA resistance. Expression analysis performed on obtained transgenics before and after RWA infestation reavealed that the silenced plants were more susceptible to RWA feeding. Chlorosis was observed in the Gamtoos-S-APX transgenic plant which is an indicator of oxidative damage to the photosynthetic machinery of the plant. Decreased GSTF6b transcripts was found in the transgenic Gamtoos-S-GSTF6b and transgenic Gamtoos-R-GSTF6b transgenic plants but no visible symptoms of infestation was observed in these two plants. Resistance breeding could be strengthened by developing broad spectrum resistance plants by incorporating wheat defensive related genes with known function into the breeding programs. The use of this transformation system will allow rapid identification and introduction of agronomically important genes by upregulating these genes to enhance bread wheat against aphid infestation.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance. / AFRIKAANSE OPSOMMING: Die Russiese-koringluis (RWA) is ‘n pes wat ‘n belangrike ekonomiese invloed op koring opbrengste het en infestasie kan tot grootskaalse skade en oes verlies lei. Verskeie studies, onder andere komplimentêre DNA amplifiseerde fragment polimorfismes (cDNA-AFLPs), onderdrukkende onderskeidende hibridisaie (SSH) en mikro-reekse wat voorheen op transkriptomiese vlak gedoen is, het moontlike gene wat by RWA weerstand betrokke is, geïdentifiseer. Alhoewel hierdie gene reeds geidentifiseer was, hulle rol is nogtans onbekend. Dié gene moet nog getoets word, duur funksionele genetiese benaderingste maak. In hierdie studie is ‘n gars streep mosaïek virus vektor (BSMV) gebruik om kandidaat-gene van belang in ‘n Dn1-weerstandige geen-bevattende kultivar (TugelaDN) te onderdruk. Ondrukking van gene het deur middel van virus geïnduseerde geen onderdrukking (VIGS) plaasgevind. In hierdie studie is die hipotese gestel dat die gene betrokke by die hipersensitiewe reaksie (HR) ‘n invloed op plantweerstand kan hê en is dus geteiken vir geen-onderdrukking-studies. Hierdie gene het die volgende ingesluit: glutatioon-S-transferase (GST), superoksied dismutase Cu/Zn (SOD) en askorbien peroksidase (APX). Egter, omdat luisinfestasie ook tot verwonding aanleiding gee, is die onderdrukte gene ook onder alleenlik verwondingstoestande getoets. Luis vrugbaarheid is gebruik as indikator van betrokkenheid omdat meer vatbare plante ‘n hoër luis vrugbaarheid tot gevolg het. In die studie is gevind dat onder alleenlik verwondingkondisies, plante wat Dn1 bevat, ‘n groter hipersensitiewe respons vertoon, as vatbare kontroles. Daar is verder gevind dat askorbien peroksidase ‘n belangrike rol tydens verwondings-geïnduseerde weerstand in Dn1-plante speel. Daar is verder bevind dat die onderdrukking van superoksied dismutase Cu/Zn (SOD) en ‘n tilakoïed-geassosïeerde askorbien peroksidase (tAPX). Onder luis-infestasie kondisies, geen effek op luisvrugbaarheid gehad het nie en dus nie direk by die weerstandsrespons betrokke is nie. Die onderdrukking van ‘n phi-klas glutatioon-S-transferase F6 (TaGSTF6) het egter ‘n groot invloed op luis-vrugbaarheid gehad en kan dus ‘n rol in Dn1-weerstand speel. Die moontlike weerstands gene, geïdentifiseer as nukleotied bindings proteïen (NBP) en weestandsgeen anoloog 2 (T10rga2-1D), is getoets onder luis-infestasie kondisies. Die analise van NBP het getoon dat dit ‘n integrale deel van die yster homeostase meganisme in die sitosol, wat vir Fe-kluster samestelling verantwoordelik is, vorm. Onderdrukking van beide die NBP en T10rga2-1D het tot die uitdrukking van ‘n vatbare fenotipe aanleiding gegee. T10rga2-1A is ‘n NBS-LRR proteïen wat bekend is om noodsaaklik te wees tydens roes weerstandigheid in teenwoordigheid van die weerstandsgeen Lr10. T10rga2-1D-onderdrukte behandelings het tot vatbaarheid aangeiding gegee en daartoe gelei dat plante na luis-infestasies doodgaan. Hierdie resultate dui dus ‘n rol vir T10rga2-1D in Dn1-weerstandigheid aan, en suggereer verder dat hierdie geen ‘n goeie stroom-op kandidaat in Dn1-weerstandigheid is.

Gaeumannomyces tritici is a soil-borne, highly destructive, wheat root pathogen, causing take-all disease. Some modern, elite, winter wheat cultivars possess a genetic trait promoting low take-all inoculum build-up (LowTAB). This leads to reduced disease if wheat is grown in the same field the next year. This PhD aimed to test if genetics (LowTAB) and chemistry will individually or synergistically influence take-all fungal inoculum build-up in first wheats as methods to control second wheat take-all disease. The underlying mechanism, epidemiology, agronomy and genetics of the TAB (take-all build-up) trait in eight first wheat field trials was investigated. This identified two minor QTLs conferring the LowTAB trait, in a doubled haploid mapping population. This PhD also confirms the highly complex cultivar-year-field interactions that underpin this trait. Root phenotyping experiments in the field and laboratory highlight that the TAB trait is not likely to be the result of root system architecture variation. Future field trials are planned to confirm the QTLs identified and to test for links between TAB and root-soil-microbial interactions. The effect of foliar applied chemistry (fungicide: Amistar, active ingredient: azoxystrobin and plant growth regulator: Moddus, a.i. trinexapac-ethyl) combined with genetics (TAB) on first wheat take-all inoculum build-up and second wheat disease was investigated. To complement this, laboratory screens were performed checking for common target site mutations to the azoxystrobin fungicide, in new and historic G. tritici isolates. For the first time, legacy effects of first wheat foliar chemistry on second wheat disease were identified, however no synergy with genetics were found. Early first wheat Amistar sprays reduced second wheat take-all disease, whilst later sprays and plant growth regulator, Moddus; had no effect. However, first wheat inoculum reduction by Amistar, could not be directly linked to the second wheat disease outbreaks observed. No evidence of fungicide resistance was found in 40 UK isolates, thus the varied efficacy of Amistar is linked to soil dose rate at the different application times. The collective PhD findings of the effect of first wheat chemistry and genetics make a significant contribution to the control of take-all disease in commercial second wheat crops.

Le blé tendre, Triticum aestivum, est une des céréales les plus cultivées au monde et est d’une importance considérable pour l’alimentation humaine, fournissant environ un cinquième des calories consommées par l’Homme. Le rendement en grain chez les céréales dépend majoritairement du nombre et de la taille des grains. Chez le riz (Oryza sativa), le gène GW2 a été isolé dans un locus à effet quantitatif majeur pour la taille et le poids du grain. Ce gène code pour une enzyme E3 ligase de type RING, qui régule négativement la taille et le poids du grain de riz. L’homologue de GW2 chez le blé tendre, le gène TaGW2, est exprimé par trois copies TaGW2-A,TaGW2-B et TaGW2-D, portées par chacun des génomes homéologues A, B et D. Les trois copies présentent des profils d’expression distincts au cours du développement du grain. TaGW2-A a été cartographié dans une région de QTLs pour le rendement, sur le chromosome 6AS ; et du polymorphisme dans sa séquence promotrice et intronique a été retrouvé associé au poids de 1000-grains dans une core collection mondiale de blé tendre. Afin de rechercher la fonction de TaGW2, l’extinction stable des trois copies TaGW2 a été entreprise par ARN interférence. De manière surprenante, les plantes transgéniques montrent des réductions significatives des dimensions et du poids du grain de blé (- 22,5 et - 30% du volume et de la masse du grain, respectivement), ainsi que du nombre de cellules de l’albumen (- 25%), comparé aux plantes témoins dans nos conditions ; suggérant que TaGW2 est un régulateur positif de la taille finale du grain chez le blé tendre. La protéine TaGW2-A a été caractérisée aux niveaux moléculaire et biochimique : elle est une E3 ubiquitine ligase fonctionnelle in vitro, et s’accumule dans la cellule au niveau du nucléole, du nucléoplasme et du cytoplasme. Sa fonction E3 ligase semble notamment influencer sa localisation subcellulaire. Afin de déterminer la ou les voie(s) de signalisation dans la(es)quelle(s) intervient TaGW2, une banque ADNc de grains de blé a été construite et criblée par double-hybride avec 320 acides aminés de la protéine TaGW2-A. Les premiers interacteurs potentiels identifiés suggèrent d’une part un rôle de TaGW2 dans la régulation de la division cellulaire, et d’autre part une fonction E3 Nedd8 ligase, en plus de son activité E3 ligase. / Wheat, Triticum aestivum, is one of the world’s major cereal crops and is of considerable importance to human nutrition, supplying one-fifth of the calories consumed by humans. For important food crops such as wheat, rice and maize, grain yield mainly depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for grain size and weight, and encodes an E3 RING ligase that negatively regulates these yield components. Wheat has TaGW2 homologs in A, B and D genomes; and copies show distinct expression pattern during whole grain development in wheat. TaGW2-A was mapped in a genomic region on 6AS, encompassing previous reported QTLs for yield; and polymorphisms in TaGW2-A (promoter and intron 7) were associated with thousand-grain weight, in a worldwide wheat core collection. To investigate TaGW2 function, RNA interference was used to down-regulate TaGW2 transcripts levels. Surprisingly, transgenic wheat lines significantly showed decreased grain weight and size-related dimensions, and endosperm cell number compared to controls. The present study thus suggests that TaGW2 is a positive regulator of the final grain size in wheat, conversely to GW2 in rice. Biochemical and molecular analyses of the protein TaGW2-A revealed that 1) TaGW2-A is a functional E3 ubiquitine ligase in vitro, 2) TaGW2-A accumulates in the nucleolus, the nucleoplasm, and the cytosol, 3) E3 ubiquitine ligase activity seems to impact TaGW2-A subcellular localization. To investigate the TaGW2 signalling pathway(s), cDNA library from whole wheat grains was built and screened with the bait protein TaGW2(1-320). Preliminary results from the interactomic study suggest that TaGW2 may regulate cell division. Moreover, TaGW2 may also function as an E3 Nedd8 ligase, besides its E3 ubiquitin ligase function.

La productivité et la qualité des céréales biologiques sont soumises à deux principaux facteurs limitants dans les systèmes sans élevage : des déficits chroniques en azote (N) du sol et des infestations par les adventices. Des légumineuses telles que les trèfles ou les luzernes peuvent servir à la fois de plantes de couverture et d'engrais verts grâce à leur fixation symbiotique d'N atmosphérique. Cependant, leur substitution aux céréales présente un moindre intérêt économique dans les systèmes de grandes cultures en l'absence d'animaux pour les valoriser. L'association relais de couverts de légumineuses dans un blé d'hiver nous a semblé être une option intéressante pour à la fois enrichir le système sol-plante en N, couvrir le sol dès la récolte du blé associé et limiter le risque de compétition avec le blé en décalant au printemps la date de semis des légumineuses sous couvert de blé. Pour évaluer l'efficacité de ces associations, quatre espèces de légumineuses (Medicago lupulina L., M. sativa L., Trifolium pratense L. et T. repens L.) ont été semées au tallage du blé d'hiver sur huit parcelles réparties dans la région Rhône-Alpes. Leurs effets sur la maîtrise des adventices, l'enrichissement, la préservation et la restitution d'N au système sol-plante et les performances des cultures ont été observés, durant une succession blé d'hiver-culture de printemps. Les résidus des couverts ont été enfouis à la fin de l'hiver, 9 à 12 semaines avant le semis d'une culture de printemps. Nos travaux ont montré l'absence d'effet des couverts associés sur le rendement en grains du blé d'hiver. Mais des diminutions du taux protéique des grains sont apparues dans un tiers des situations d'association notamment avec M. lupulina et T. pratense, les espèces les plus développées à la récolte du blé. Notre suivi de la disponibilité des ressources trophiques principales (eau, N, lumière) nous a permis d'identifier une compétition pour l'eau et l'N du sol. Nous avons également noté l'efficacité des couverts de légumineuses dans le contrôle de la densité des adventices dès le stade de floraison du blé et de leur biomasse durant l'interculture. Le meilleur contrôle des adventices a été permis par M. lupulina et T. pratense, à la récolte du blé, et T. pratense et T. repens, à la fin de l'automne, associé aux biomasses aériennes observées les plus importantes. Enfin, nous avons observé une forte proportion d'N issu de la fixation symbiotique dans la biomasse aérienne des légumineuses à la fin de l'automne (80 à 94%), représentant un apport d'N exogène au système sol-plante évalué entre 37 et 77 kg N ha-1. Cet enrichissement en N n'a pas entrainé d'aggravation de la lixiviation d'N durant l'hiver. Les couverts de légumineuses n'ont pas non plus diminué la lixiviation comparativement à l'absence de couvert. Après leur destruction, les résidus des couverts ont restitué une partie de l'N accumulé (+28 à +42 kg ha-1 d'N minéral sur les 90 premiers centimètres de sol par rapport au témoin à l'émergence de la culture de printemps, 12 semaines après leur destruction). Cette restitution a permis un enrichissement en N des pailles et grains de la culture de printemps et une augmentation de 30% du rendement lorsqu'il s'agissait de maïs. Finalement nous concluons sur l'intérêt des couverts de légumineuses associés en relais dans un blé d'hiver pour apporter une réponse positive aux problèmes principaux des rotations de grandes cultures biologiques (adventices, déficits d'N et diminutions des performances des cultures). Nous terminons en proposant des voies d'évolution des associations testées, notamment pour limiter les risques de compétition durant l'association. Nous évoquons également les implications scientifiques et pratiques de ce travail pour de futures études sur ce sujet. / Cereal productivity and quality are subject to two main problems in organic stockless systems: chronic soil nitrogen (N) deficiencies and weed infestation. Legume species as clovers or alfalfas can be used as cover crops and green manures due to their natural ability to fix atmospheric N. Nevertheless, their substitution to cereals in crop rotations is less economically viable without animals to use it. Relay intercropping of legume cover crops (RIL) in winter wheat was expected to simultaneously enrich the soil-plant system in N, cover the soil from the wheat harvest onwards and limit the risk of competition with wheat by delaying the undersowing of legumes in spring. To evaluate the efficiency of RIL, four legume species (Medicago lupulina L., M. sativa L., Trifolium pratense L. and T. repens L.) were undersown at wheat tillering stage, in eight fields organically managed in the Rhône-Alpes region, France. Their effects on weed control, on N enrichment, preservation and restitution in the soil-plant system and on crop performance were observed during the succession of the winter wheat and a spring crop. RIL residues were incorporated in soil in late winter, 9 to 12 weeks before the sowing of spring crops. Our work illustrated the absence of detrimental effect of RIL on wheat grain yield despite the decrease of the grain protein content in one third of the situations. This decrease was mainly observed with M. lupulina and T. pratense as they were the most developed legume species at wheat harvest. Our monitoring of trophic resources (water, N and light) enlightened the competition for soil water and N during the intercropping period. We also showed the efficiency of RIL in the control of weed density from wheat flowering stage onwards and of weed aerial biomass in late autumn. The best weed control was observed with M. lupulina and T. pratense, at wheat harvest, and with T. pratense and T. repens in late autumn, in relation to the highest aerial biomasses observed with these species. Finally, we noted an important proportion of N derived from atmosphere (Ndfa) in legumes' shoots in late autumn (80 to 94%), representing an input of exogenous N in the soil-plant system ranging from 37 to 77 kg Ndfa ha-1. The N enrichment of the system did not increase mineral N lixiviation during winter. However, legume cover crops did not significantly prevent any lixiviation of mineral N relative to the control without cover crop. After their ploughing in, legumes residues returned a part of the accumulated N (+28 to +42 kg ha-1 of mineral N in the first 90 cm of soil compared to the control at the emergence of the spring crop, 12 weeks after ploughing). This restitution of mineral N allowed a significant N enrichment of straw and grains of the spring crop with all previous legumes cover crop species. With maize as spring crop, the grain yield increased by 30%, on average, compared to the control treatment. We finally conclude on the interest of RIL in winter wheat to bring a positive response to the problems of weed control, N deficiency and crop performances in organic crop rotations. We then suggest possible improvements of the RIL system, especially against the competition for trophic resources during the relay intercropping period. We also mention scientific and practical implications of this work for future studies on this subject.