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Browsing Theses and Dissertations by Author "Gwata, E. T."
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Item Open Access Combining Ability for Ear Prolificacy and Response of Prolific Maize (Zea May L.) Hybrids to Low Nitrogen Stress(2018-09-21) Makhumbila, Penny; Gwata, E. T.; Mashingaidze, K.Smallholder farmers in Sub-Saharan Africa still obtain low grain yields in maize largely due to low soil fertility. The soils are inherently low in nitrogen (N) that is required for the proper development of the maize plant. Currently there are no commercial cultivars for low N tolerance locally. The combining ability approach can be used as a tool for breeding desirable cultivars. In order to improve grain yield in maize, it is important to consider ear prolificacy which is a major yield component. Therefore this study was designed to estimate combining ability in maize. Exotic germplasm from the International Maize and Wheat Improvement Center and the Institute of Tropical Agriculture as well as the local germplasm from the Agricultural Research Council was used in the study to generate crosses. One hundred and two crosses were evaluated together with a standard commercial check under low N and optimum N conditions. The specific objectives of the study were to determine general and specific combining ability for prolificacy among local and exotic inbred lines and evaluate the response of prolific hybrids to low N conditions. The hybrids were planted in the 2014/2015 summer season under irrigation in Potchefstroom, Cedara and Taung in field plots consisting of 0.75m x 0.25m spacing in a 0.1 alpha lattice design replicated twice. Data for agronomic attributes were recorded and subjected to analysis of variance using SAS version 9.1.3. Genetic correlations were analyzed using the Principal Components Analysis and factor analysis based on the correlation analysis and major traits. The results showed variation in agronomic performance among the inbred lines and their F1 hybrids. Inbred lines including TZEI63, T1162W, L15 and L17 showed positive GCA estimates for ear prolificacy at the different locations. Specific combining ability for prolific hybrids was positive at all locations and environments. The GCA:SCA ratio was close to unity; indicating that the number of ears per plant showed highly significant (P<0.01) correlation with grain yield. The hybrids showed ear prolificacy under the low N conditions. This trait can be used effectively in stress tolerance maize breeding programmes.Item Open Access The effect of chemomutagenesis on root nodulation and seed protein in tepary bean (Phaseolus acutifolius)(2018-05-18) Mashifane, Dipoo Charity; Gwata, E. T.; Bessong, PascalTepary bean (Phaseolus acutifolius) is an important food legume originating from South America and the South-western parts of the United States. The crop is produced in many countries worldwide including South Africa. It is highly tolerant to drought and the seed contains a wide range of vitamins, minerals and protein of high nutritional quality. The genetic base of tepary bean is narrow but can be widened by chemical mutagenesis. However, there are no reports on the impact of chemical mutagenesis on the root nodulation and seed storage proteins in tepary bean. Therefore, this study was designed to examine root nodulation attributes and seed storage proteins of three tepary bean genotypes in the early mutagenic generations (M2 to M4) derived through treatment with varying doses (0.0, 0.5, 1.0, 1.5 and 2.0 v/v) of ethyl methanesulfonate (EMS). The experiment on root nodulation attributes was laid out as a 3 x 5 x 3 (genotypes x EMS doses x mutant generations) factorial design replicated three times. At harvest, shoot height (SHT), primary root length (PRL), dry weights (shoot, root and nodule), number of nodules per plant (NNP) and grain yield components such as the number of pods per plant (NPP) and number of seeds per pod (NSP) were measured. Highly significant (P≤0.01) dose effects were observed for SHT, PRL, shoot dry weight (SDW) and root dry weight (RDW). Highly significant (P≤0.01) interaction effects of mutant generation x genotype x dose were observed for NSP. A highly significant (P≤0.01) positive linear relationship was observed between the NNP and nodule dry weight (NDW). Increase in the PRL suggested that tepary bean mutants could be important in drought tolerance. EMS treatment led to an enhanced partitioning of dry matter (assimilates) to the shoots and roots. There was a three fold increase in most of the root nodulation traits at the 0.5% EMS dose.The Kjeldahl method was used for crude protein determination whereas the sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS PAGE) was utilized in determining the protein banding patterns of the bean. There were highly significant (P≤0.01) differences among the genotypes in crude protein accumulation. Highly significant (P≤0.01) mutant generation x genotype x dose were observed for seed protein accumulation. ‘Genotype 3’ attained the highest protein content (24.23%) at 1.5% EMS dose in the M4 generation. EMS doses ≥0.5% positively stimulated protein accumulation in all genotypes but high EMS doses (2.0%) depressed protein content. There were significant variations in seed storage protein profiles among the genotypes and mutant generations. ‘Genotype 6’ showed a distinct 15.0kDa protein fragment which was absent in the majority of the remaining genotypes. The presence of distinct protein subunits in the three genotypes could be used in varietalItem Open Access The effects of relative planting dates of legumes on productivity of cassava - legume intercrop(2017-08-18) Legodi, Khutso Debra; Ogola, J. B. O.; Gwata, E. T.See the attached abstract belowItem Open Access Evaluation of Diverse Cowpea (Vigna unguiculata [L.] Walp.) Germplasm for Field Performance and Drought Tolerance(2018-05-18) Nkoana, K. D.; Gwata, E. T.; Gerrano, A. S.Item Open Access Evaluation of Grafting Rootstocks on Plant Growth, Fruit Yield and Quality in Tomato (Lycopersicon esculentum Mill).(2018-09-21) Mothapo, M. R.; Mchau, G. R. A.; Gwata, E. T.Grafting is the process of combining two parts of plants to form a single plant. Grafting is a common technique in trees and vine crops and is becoming popular in annual vegetable production in order to control biotic and abiotic stresses, improve fruit yield and quality. The objectives of the study were to determine the (a) compatibility of the tomato scion x rootstocks combinations (b) effect of rootstocks on vegetative growth parameters (c) effect of tomato rootstocks on fruit quality and yield of tomato. The experiments were conducted under a protected environment (in a high tunnel) at the University of Venda, Thohoyandou, South Africa. Four tomato rootstocks (‘Powapak’, ‘Everest’, ‘Matterhorn’ and ‘Golf’) and two scion cultivars (‘Money Maker’ and ‘Rodade’) were used to develop 10 scion x rootstock combinations. The tube grafting method was used. Seedlings were transplanted in the polyethylene bags inside the high tunnel. Growth parameters, including plant height, stem diameter, number of leaves per cluster as well as yield attributes were measured. In addition, the chemical fruit quality parameters including the total soluble solids and pH were determined. A randomized complete block design with four replications was used for the study. Quantitative data sets of the variables were subjected to analysis of variance. There was a high plant survival rate and the number of clusters per plant showed marked improvement in some grafting combinations particularly in ‘Rodade x Everest’ and ‘Money Maker x Everest’. A few individual stunted plants were observed in the tunnel. A significant seasonal effect was observed on some of the growth and productivity attributes. The pH showed a narrow range of values and there was significant interaction between sampling time and genotype. The results demonstrated the viability of producing grafted tomatoes under tunnel conditions.Item Open Access Genetic Study of Pod Shattering Resistance in Soybean (Glycine max (L.) Merrill) Plant Populations Derived from Exotic x Local Germplasm(2016-05) Nevhudzholi, Khuliso Marine; Gwata, E. T.; Mchau, G. R. A.See the attached abstract belowItem Open Access Genotypic variation in water use efficiency, gaseous exchange and yield of four cassava landraces grown under rainfed conditions in South Africa(2020-08-20) Malele, Kgetise Petros; Ogola, J. B. O.; Gwata, E. T.Agricultural production under rain-fed conditions is largely dependent on the availability of water stored in the soil during rainfall events. The production of cassava (Manihot esculenta Crantz) under rain-fed conditions in the north-eastern part of South Africa is constrained by low and erratic rainfall events. Improving cassava production in the area requires the use of cassava varieties which are efficient in the use of limited soil moisture. The current climate change and increasing population growth on the planet will place more pressure on agriculture to produce more food using less water. Therefore, previously under-researched and underutilised crop like cassava could be used to bridge the food gap in the future. Although the crop currently occupies low levels of utilisation in South Africa and it is cultivated by smallscale farmers in the Low-veld of Mpumalanga, Limpopo and Kwazulu-Natal provinces using landraces with no improved varieties available in the country. Information on the actual pattern of water extraction, water use and water use efficiency of cassava landraces grown in the dry environments of South Africa is limited. Therefore, the objective of the study was to determine the differences in water use efficiency, gaseous exchange and yield among four cassava landraces grown under rain-fed conditions. Two field experiments were conducted during the wetter (2016/2017) and drier (2017/2018) cropping season at the University of Venda's experimental farm. The trials were laid in a Randomized Complete Block Design (RCBD) consisting of four cassava landraces (ACC#1, ACC#2, ACC#3, and ACC#4) replicated three times. Mature cassava stem cuttings of 30 cm long, were planted manually at a spacing of 1 m x 1 m in both seasons. Each experimental unit consisted of six plant rows of 6 m length (36 m2) and 8 rows of 8 m length (64 m2) in the 2016/17 and 2017/2018 cropping season, respectively. The experiments were under rain-fed conditions without fertilizer additions and the plots were kept weed-free throughout the experimental period. Data collected in the field included soil moisture content, gaseous exchange parameters (net leaf ܥܱଶ uptake, stomatal conductance, and intracellular carbon dioxide concentration), chlorophyll content index (CCI), maximum photochemical quantum yield of PSII (Fv/Fm), effective quantum yield of PSII (ФPSII) and photosynthetic active radiation (PAR). Yield and yield components (root length (cm), root girth (cm), number of storage roots and mean root weight (g plant-1), root yield and aboveground biomass), as well as water use efficiency (WUE), were determined at harvest. Soil moisture content was measured at seven-day interval from sowing until harvest using a neutron probe. Soil moisture data were used to determine crop water use using the water balance approach. There was no variation in the root yield and yield components amongst the landraces in 2017/2018 cropping season but, genotypes affected aboveground biomass, root girth, number of roots per plant and root yield in 2016/2017 cropping season. There was a significant difference (P<0.01) in number of roots (per plant) 81% and 62% greater in ACC#3 and ACC#2 (6.7 & 6.0, respectively) compared with ACC#1 and ACC#4, which both recorded 4 roots per plant. Similarly, root girth was greater in ACC#3 (17.8 cm) and ACC#2 (18.2 cm) compared to ACC#1 (14.1 cm) and ACC#4 (12.9 cm), which were statistically the same. In contrast, total biomass (P<0.01) and root yield (P<0.05) were greater in ACC#3 (20.7 and 11.9 t ha-1, respectively) and ACC#1 (22.0 and 11.3 t ha-1, respectively) compared to ACC#2 and ACC#4 with root yields of 10.2 and 9.5 t ha-1, biomass of 17.1 and 16.3 t ha-1, respectively. Although the genotype x cropping season interaction did not affect root yield and yield components, root yield (by 33.8%; 2.7 t ha-1) and yield components were greater in the wetter compared to the drier season as expected. Water use efficiency of root yield (WUErt) and water use efficiency of biomass production (WUEb) varied with landraces in season I from 37.0 kg ha-1 mm-1 (ACC#4) to 46.60 kg ha-1 mm-1 (ACC#3), and between 71.30 kg ha-1 mm-1 (ACC#2) and 86.0 kg ha-1 mm-1 (ACC#1), respectively. Landraces did not differ in their water use and soil moisture extraction in both seasons but differed in season. However, there was a significant positive correlation between water use efficiency of root yield (WUErt) (0.963***) and water use efficiency of biomass production (WUEb) (0.847***). WUE of biomass production was greater in the drier than the wetter season partly because of dry matter accumulation per evapotranspiration within the landraces. Photosynthesis did not vary with landraces, however, stomatal conductance varied with landraces from 0.08 mmol m-2 s-1 (ACC#4) to 0.2 mmol m-2 s-1 (ACC#2). In contrast, ACC#1 and ACC#3 recorded the same value of stomatal conductance, which is 0.1 mmol m-2 s-1. The effective quantum yield of PSII photochemistry (ΦPSII) did not vary with landraces but the maximum photochemical quantum yield of PSII (Fv/Fm) varied with landraces from 0.652 (ACC#4) to 0.792 (ACC#3) in season II. The proportion of intercepted radiation was affected by landraces in 2017/2018 cropping season. Highest proportion of intercepted radiation was observed in ACC#3 and the lowest in ACC#2. Proportion of intercepted radiation varied with landraces from 22.62% (ACC#2) to 86.45% (#ACC#3). There were significant genotypic variations in chlorophyll content recorded in both season. Chlorophyll content varied with landraces from 33.1 CCI (ACC4) to 55.4 CCI (#ACC3) in the 2016/2017, and in 2017/2018 cropping season chlorophyll content varied with landraces from 36.9 CCI (ACC4) to 78.7 CCI (#ACC3). The highest genotypic variation in chlorophyll content was observed in ACC#3, whilst the lowest chlorophyll content was recorded in ACC#4 in both seasons.Item Open Access Multi-Location Field Evaluation of Bambara Groundnut (Vigna Subterranean (L) Verdc) for Agronomic Performance and Seed Protein.(2018-05-18) Mogale, Tlou Elizabeth; Gwata, E. T.; Zhou, M.Bambara groundnut (Vigna subterranea) is one of the most important legumes cultivated primarily for food by smallholder farmers in Africa. It is an affordable source of protein and contributes to income generation as well as soil fertility. Despite its importance, it is cultivated largely for subsistence purposes in South Africa. Growers use landraces. The agronomic performance of the traditional varieties depends on environmental factors prevailing in a particular area. In Limpopo and Mpumalanga Provinces, there is no adequate information regarding the performance of bambara groundnut germplasm. The objectives of the study were to (i) determine the agronomic performance of Bambara groundnut across three contrasting locations in Limpopo and Mpumalanga provinces over two cropping seasons (ii) determine the genotypic variation in the seed protein level among 42 bambara groundnut genotypes. Forty-two bambara groundnut genotypes were evaluated under three different environmental conditions (Syferkuil, Thohoyandou and Nelspruit) over two (2013/2014, 2014/2015) seasons in a 7 × 6 rectangular lattice design replicated three times. Eight agronomic traits including dry shoot weight (DSW), number of pods per plant (NPP), pod length (PL), number of seed per pod (NSP), pod weight per plant (PWT), seed weight per plant (SWT), 100 seed weight (100-SWT) and seed yield (SYLD) were measured. The results showed that there were significant genotype x location interactions which demonstrated that the prevailing agro-ecological conditions at the test locations were distinct from each other. Five genotypes (‘BGN-19‘, ‘BGN-11‘, ‘BGN-12‘, ‘BGN-4‘and ‘BGN-34‘) attained >25.0% seed yield advantage over the local check ‘BGN-39‘. The results also showed that light brown coloured genotypes attained relatively higher seed yield compared to the other seed colours types. The cultivar superiority index (CSI) showed that three genotypes (‘BGN-12‘, ‘BGN-19’ and ‘BGN-34’) were the most stable across the test locations and attained >900.0 kg/ha on average. There were significantly high positive correlations between PWT and each of the three other attributes (SWT, 100 SWT and SYLD). In terms of seed protein, the results showed a poor relationship between seed yield and protein levels. ‘BGN-12’ which produced the highest seed yield, attained the lowest percent seed protein while genotype. On average, the genotypes contained 21.72% protein. The highest and lowest seed protein quantities were attained by the genotypes ‘BGN-42’ (25.17%) and ‘BGN-12’ (19.89%) respectively.Item Open Access The Response of Tepary Bean (Phaseolus actifolius) Germplasm to Induced Mutation(2016-05) Thangwana, Andries; Gwata, E. T.; Zhou, M.See the attached abstract belowItem Open Access Selection of Efficient Indigenous Rhizobia Inoculants for the Production of Selected Tropical Legumes in Limpopo Province (South Africa)(2023-10-05) Nemaembeni, Phethani Muofhe; Gwata, E. T.; Phalane, F. L.; Maphosa, T. M.Tropical legumes are important food crops for human and animal nutrition as well as the improvement of soil fertility. In Southern Africa, tropical legumes are cultivated mostly by smallholder farmers partly because of their ability to thrive in poor soils and adverse weather conditions. Tropical legumes are useful in these cropping systems because of their ability to fix nitrogen (N) thus minimizing the necessity for chemical nitrogenous fertilizers. Soil rhizobia (as bio-inoculants) can enhance the productivity of these legumes through the improvement of soil fertility. However, both the compatibility and competency of individual rhizobial strains is important for attaining optimum crop productivity. Therefore, it is imperative to identify the best efficient rhizobial strain x legume genotype combinations for successful nodulation and optimum legume yield for the benefit of resource-limited farmers in smallholder farming systems and end-users. The aim of this study was to improve the productivity of tropical legumes. The specific objectives of the study were to (i) determine efficient rhizobial strains that combine with tropical legume species to produce optimum crop productivity and (ii) determine efficient tropical legume species x rhizobial genus combinations that produce optimum crop productivity. The study consisted of two experiments both of which were carried out at the Agricultural Research Council (ARC), Plant Health and Protection (Pretoria) greenhouse and laboratory facilities (25° 61’ 547” S, 28° 36’ 435” E). The conditions in the greenhouse were set at a 14 h day temperature of 28° C and 10 h night temperature of 15 °C. In the first experiment, four tropical legumes (pigeonpea; soybean; tepary bean; bambara groundnut) were used in the study. At planting, each legume species was inoculated separately with each of 15 rhizobial strains and allowed to grow for six weeks after which a range of N fixation variables were evaluated. The experiment was laid out in a split plot design with legume species as the main factor and rhizobial strain as the sub-factor. The quantitative data sets on leaf color score (LCS), nodule dry weight (NDW), shoot dry weight (SDW) and root dry weight (RDW) were subjected to standard analysis of variance (ANOVA) procedures and GGE biplot analysis. In the second experiment, three tropical legumes and four rhizobial strains (each from a distinct genus) which were selected from experiment 1 were used. A split-split plot design with the rhizobial strain as the main factor, legume species as the sub-factor and legume variety as sub-sub factor with two replications was used and the data sets of the N fixation variables were analyzed following the same procedure as described above. The results showed that there was variation in legume species x rhizobial strain compatibility and the pattern of root nodulation varied within the legume species depending on the specific strain used. Tepary bean nodulated poorly with most of the rhizobial strains. There were significant differences among the legume species for all the traits that were measured. However, there were no significant (P < 0.05) differences among the rhizobial strains for NDW, RDW and SDW. The highest NDW (0.05 g) and SDW (0.42 g) were attained by bambara groundnut. The mean LCS for the trial was (19.26) while the highest (23.65) and lowest (14.23) LCS were associated with the rhizobial strains Bradyrhizobium elkanii (R4) and Phyllobacterium leguminum (R11), respectively. Among the Paraburkholderia species, the rhizobial strain Paraburkholderia phenolyruptix (R1) was associated with the highest (22.08) LCS. The results also revealed that the rhizobial strains Rhizobium leucaenae (R8) and Rhizobium sp (R6) induced the highest (20.89) and lowest (15.24) LCS, respectively. However, the rhizobial strain Bradyrhizobium elkanii (R14) which attained a relatively high LCS, was associated with the heaviest NDW among all the strains. In contrast, there were detectable nodules associated with four rhizobial strains. Two distinct rhizobial species, namely Paraburkholderia sp (R2) (designated N362) and Bradyhizobium lupini (R5) were associated with the heaviest shoots across the legume species. In contrast, the control strain achieved the lowest (0.25 g) SDW. In addition, all the three strains from the genus Paraburkholderia and the single strain from the genus Phyllobacterium as well as all the five from the genus Bradyrhizobium (R4; R5; R12; R13 and R14) were associated with significantly (P< 0.05) heavier SDW than the control. The results also revealed highly significant (P < 0.01) positive correlations between the LCS and NDW. However, the LCS was negatively but significantly (P < 0.05) correlated with the RDW. In addition, there were highly significant positive correlations between the SDW and each of NDW and RDW. The variety x legume species interaction was highly significant (P < 0.001) for all the attributes that were measured except for NDW. Inoculation with Bradyrhizobium sp (33a-PP4) showed varietal differences in the pattern of N fixation indicators in bambara groundnut and pigeonpea. Nonetheless, some of the soybean varieties formed no nodules after inoculation with the Paraburkholderia sp and Phyllobacterium leguminum strains. Similarly, soybean responded poorly to inoculation with Rhizobium sp. (34a-PP5) and Bradyrhizobium sp (33a-PP4). In pigeonpea, all the four varieties that were used in the study showed similar LCS values. The SDW was used for determining the ideal rhizobial genus and legume species by applying the GGE biplot method in which the legume species and rhizobial strains were coded as environment scores and genotypes scores, respectively. The GGE biplot analysis indicated that 60.0% of the rhizobial strains were distributed in the top left quadrant but none in the bottom right quadrant. The rhizobial strains ‘R2’ (Rhizobium sp; 34a2-PP5) and ‘R1’ (Paraburkholderia sp; N362), were positioned far away from the origin suggesting that they uniquely influenced the legume species. The biplot analysis also revealed that the legume species (coded as environment scores), particularly ‘E1’ (pigeonpea) and ‘E2’ (soybean), were separated by acute angles between them and grouped in the same top right quadrant. In contrast, the remainder of the pairs of legume species were separated by obtuse angles with each suggesting that they were negatively related to each other in terms of the SDW trait. The legume species ‘E3’ (tepary bean) showed the shortest absolute projection suggesting that it was the most stable in performance across the rhizobial strains. In determining the ‘which- won where’, the biplot analysis explained 95.23% total variation of which PC1 and PC2 accounted for 84.41% and 10.82%, respectively. The results also revealed that among the rhizobial strains (depicted as genotypes) on the vertices of the polygon ‘R2’ (Rhizobium sp; 34a2-PP5) and ‘R4’ (Bradyrhizobium elkanii; 33a-PP4), performed best with pigeonpea (E1) and soybean (E2), respectively (Fig. 10). Bambara groundnut (E4’) showed the longest vector, suggesting that it had a high discriminating ability. The rhizobial strain ‘R2’ (Rhizobium sp; 34a2-PP5) was positioned in the innermost concentric circle, thus representing the ideal and most stable strain for SDW among the strains. The study findings provided new information in the patterns of N fixation among a set of tropical legumes that were inoculated with distinct rhizobial genera. The information will be useful in future for formulating bio-inoculants that may improve legume productivity in Limpopo Province (South Africa) where all the legume species that were used in this study are cultivated. The validation of the symbiotic efficiency of Bradyrhizobium elkanii (33a-PP4) and Phyllobacterium leguminum; 31b-PP4) with more diverse bambara groundnut germplasm will be merited.Item Open Access Soil texture and mineralogy influence on the productivity of selected tropical legumes(2022-07-15) Mashaba, Lacaster Themba; Wakindiki, I. T. C.; Gwata, E. T.Cowpea (Vigna unguiculata) and tepary bean (Phaseolus acutifolius) are essential legumes which provide food for many people in South Africa. However, the productivity of these major tropical legumes in Limpopo Province is low partly because of poor soil fertility and soil degradation. This study aimed to determine the productivity of tropical legumes (cowpea and tepary bean) in different soils in Limpopo Province. Two pot experiments were conducted at the University of Venda experimental farm. The experimental design was a Completely Randomized Design (CRD) arranged in a 2 × 4 factorial treatment structure replicated three times. The aim of the first experiment was to determine the effect of soil types on nodule dry weight (NDW), root dry weight (RDW) and above-ground biomass (ADB) of the legumes. Modified plastic pots (2.0 L) were used for planting the legumes. The second experiment was aimed at determining the effect of soil types on productivity variables including the number of branches per plant (NB), plant height (PHT), plant vigour (PV), number of pods per plant (NPP), number of seeds per pod (NSP), pod length (PL), pod weight per plant (PWT) and seed weight per plant (SWTP) of the two legume species. Similarly, 5.0 L plastic pots were filled with soil (4.5 kg) and used for sowing the seed of legumes. Soil type had highly significant (P≤0.001) effects on NDW, ADB and RDW of legume species. The highest NDW (0.2133 g), ADB (3.6767 g) and RDW (2.1067 g) of cowpea was observed on the Leptsols. There were no nodules in tepary bean. For tepary bean, the highest ADB (1.6933 g) was observed in Leptsols whereas, the highest RDW (0.7433 g) was observed in Luvisols. In the second experiment, the results showed highly significant (P≤0.001) effects of Luvisols, Leptsols, Ferralsols and Fluvisols on PHT, NB, NPP, PWT, SWTP and PV. However, soil type had no significant (P >0.05) effects on PL and NSP. Leptsols was the most productive soil type for cowpea in all the measured parameters when compared to other soil types. The results in both experiments could be attributed due to variations in soil properties. It was concluded that Leptsols is the most productive soil for cowpea. However, field experiments are recommended to validate the results.Item Open Access A Study of the Genetics of Root Nodulation in Pigeonpea (Cajanus cajan) Using Indigenous Rhizobia(2023-10-05) Phalane, Francina Lebogang; Gwata, E. T.; Mchau, G. R. A.; Hassen, A. I.Pigeonpea (Cajanus cajan) is an important grain legume, which is grown in many African countries largely for human and animal consumption. Leguminous crops such as pigeonpea fix atmospheric nitrogen (N) symbiotically in the root nodules thus elimination of the need for expensive chemical nitrogenous fertilizers. The determination of host plant x microsymbiont combinations that produce optimum crop productivity is important in the pigeonpea value chain. In addition, the characterization of both symbionts is necessary for exploiting the N fixation in tropical legumes including pigeonpea. There is a dearth of information regarding the agronomic performance of pigeonpea and other common legume species such as Bambara groundnut (Vigna subterranea), soybean (Glycine max) and tepary (Phaseolus acutifolius) that are cultivated in South Africa. Therefore, the aim of this study was to evaluate the symbiotic effectiveness of indigenous rhizobia on pigeonpea.. The specific objectives of the study were to: (i) collect rhizobial strains that are associated with root nodulation in pigeonpea from diverse locations across South Africa (ii) perform molecular characterization of the rhizobia that are associated with root nodulation in pigeonpea from diverse locations in South Africa (iii) to sequence the whole genome of a selected rhizobial strain derived from pigeonpea and determine its molecular characteristics and (iv) determine the effectiveness of the rhizobial strains with pigeonpea and other common tropical legume species. In the first objective of the study, forty soil samples were collected from diverse locations across the country and used for inoculating separately the seed of each of five randomly selected pigeonpea genotypes. The pigeonpea plants were raised in a N-depleted growth medium in the greenhouse. A split-plot experimental design with two replications was used in the study. After six weeks of growth, the plants were harvested to isolate rhizobia from the root nodules. Several morphological characteristics of the rhizobial colonies including shape and growth habit (type) were determined. In addition, a range of N fixation variables of the host pigeonpea plants was measured including the nodule dry weight (NDW) and shoot dry weight (SDW) per plant. A variety of the colony morphologies ranging from tiny to medium as well as cream white and large, watery oval colonies was observed. Two hundred and eighty putative pigeonpea rhizobial strains were obtained from the root nodules of the plants. Based on their morphological characteristics on YMA-CR, nutrient agar and peptone glucose agar, the isolates were deposited into the South African Rhizobium Culture Collection gene bank. There was ˃40.0 % difference in the number of nodules between ‘Genotype-5’ and ‘Genotype-4’ but the difference in NDW between the two genotypes was ˃80.0 %. In contrast, the heaviest dry shoots (0.4513 g) that were attained by ‘Genotype-3’, weighed 52.0% more than the lightest dry shoots that were observed for ‘Genotype-4’. The results indicated that the soil samples contained diverse rhizobial isolates with distinct morphological characteristics and significant differential N fixation ability of the pigeonpea genotypes suggesting that there was a potential to select for optimum host genotype x rhizobial strain combinations for N fixation in this legume species. In the second objective of the study aimed at the molecular characterization of the rhizobial strains derived from pigeonpea, two housekeeping bacterial genes (namely 16S rRNA and recA) were used to identify each rhizobial strain to the species level. In addition, the phylogenetic relationships among these rhizobial strains were determined. The results showed that 56 strains were confirmed as rhizobia and deposited into the national rhizobia collection bank. Two primers successfully amplified both the Rhizobium strain (30bp3) as well as several Bradyrhizobium strains (16a2p3, 15bp3, 11a2p3, 13bp3, 33ap4 and 19a1p3). Two novel genera of rhizobia (Phyllobacterium and Paraburkholderia), were associated with root nodulation in pigeonpea. There was a considerable variation in the size of the sequences of both the 16S rRNA and recA genes among the rhizobial isolates. The sequences of the 16S rRNA genes across the four genera averaged 1015.73 bp. The 16S rRNA Rhizobium phylogenetic tree showed that the rhizobial isolates obtained from pigeonpea were dispersed in six different clusters and grouped with several type species of the genus Rhizobium including R. tropici with a high (77.0%) similarity grouping. The 16S rRNA based phylogenetic tree showed that the novel Paraburkholderia rhizobial isolate ‘30a2p3’, grouped with several type strains including P. rhizosphaerae (with a similarity grouping >50%) but in the recA based phylogenetic tree, the same isolate was grouped in Cluster IV with 93.0% similarity grouping. The study concluded that the sequences of the two genes (16S rRNA and recA) of the isolates from pigeonpea could provide sufficient phylogenetic information about the isolates up to the species level and confirmed that this legume is promiscuous in diverse soils from South Africa. The objective focusing on the whole genome sequencing of a selected rhizobial strain derived from pigeonpea to determine its molecular characteristics selected the rhizobial strain 10ap3 (SARCC-755) (that was originally derived from pigeonpea in the trapping experiments). Upon DNA from the strain, the DNA libraries were prepared using the Nextera protocol (Illumina, USA) and paired-end (300bp x 2) sequenced on a MiSeq (Illumina) sequencer at the Biotechnology Platform, Agricultural Research Council-Onderstepoort Campus (Pretoria, South Africa). The genome was a large circular chromosome (6,297,373 bp) and containing the overall G + C content of 60.0%. The total number of genes in the genome of the strain was 6,013 of which 99.13% were coding sequences. However, only 5,833 of the genes were associated with proteins that could be assigned to specific functions. Several important genes that were found on the genome, included the genes for N metabolism, stress response, phosphorus metabolism and iron acquisition as well as adenosine monophosphate nucleoside for purine conversion. The nodulation gene (nolR), which functions as a DNA binding transcription factor was located on contig 12. Precursor genes for purine synthesis, for instance, inosine-5-monophosphate and adenylosuccinate, which are also responsible for nodule formation, were also present on the genome. The results showed that the genome of this strain (Rhizobium tropici SARCC-755) does not contain common nod and nif genes suggesting that an alternative pathway involving a purine derivative was involved in its symbiotic association with pigeonpea. The genome also possessed some genes that are associated with abiotic stresses and mineral nutrient acquisition thus making it a candidate for future formulation of commercial inoculants especially when considering its high symbiotic efficiency with pigeonpea. In the fourth study objective, which focused (i) on determining the relative performance of individual tropical legume species when inoculated separately with each of the specific rhizobial strains that were previously derived from pigeonpea, (ii) quantify the magnitude of the effects of interactions between the host tropical legume species x rhizobial strain on a range of N fixation variables and (iii) identify the winning (superior) rhizobial strains with the specific test legume species. Thirty-six rhizobial strains which were previously isolated from pigeonpea root nodules were used in the study. There was at least one strain representing each of four distinct rhizobial genera namely Bradyrhizobium, Paraburkholderia, Phyllobacterium and Rhizobium. The experiment was laid out as a split plot design with legume species as the main factor and rhizobial strain as the sub-factor. Each treatment was replicated twice. The data sets of several N fixation variables including NDW and SDW were measured and subjected to the analysis of variance and Pearson’s correlation analysis using SAS statistical software (version 9.3) followed by mean separation using LSD test at the 5.0% probability level. Further analysis using the GGE biplot model was carried out to understand better the relationship between the host plants and the microsymbionts. For objective (i) of the fourth study, three healthy seeds of each legume species were planted in a plastic pot filled with 1.65 kg sterile river sand saturated with Hoagland solution. At seven weeks after germination, each plant was harvested and gently washed with tap water before detaching the nodules carefully from the roots. Similarly, the shoot was separated from the roots for each plant prior to oven drying all the harvested plant parts at 70o C for 48 h followed by weighing to determine the dry weights. The results showed marked variability in the responses of the legume species to inoculations with individual rhizobial strains. Tepary bean showed poor nodulation as indicated by the chlorotic plants which contrasted sharply with those of Bambara groundnut. Pigeonpea responded differentially to each individual rhizobial strain resulting in marked differences in the nodule load per plant. Some rhizobial strains, (for instance, Rhizobium strain ‘26a-PP3’) induced profuse nodulation in Bambara groundnut but not in the other legume species. The principal component (PC) analysis showed that the first two principal components accounted for 78.74% of the total variation. Four N fixation variables, including the NDW and SDW, were moderately associated with PC1. The GGE biplot of the rhizobial strain x legume species interaction for NDW explained 82.44% of the total variation. For NDW, the environments represented by E3 (soybean) and E4 (pigeonpea) were positively correlated since their vectors were separated by an acute angle. However, E1 (Bambara groundnut) and E3 (soybean) were negatively correlated since they were characterized by an obtuse angle between them for the SDW. The ‘which-won where’ biplot for NDW explained 82.44% of the total variation of which PC1 and PC2 accounted for 50.40% and 32.04% of the total variation, respectively. Two rhizobial strains on the vertices of the polygon (Rhizobium sp. 36a-PP5) and (Rhizobium sp. 26a2-PP5) performed best with Bambara groundnut (E1) and soybean (E3), respectively. E3 (soybean) consisted of the longest vector line suggesting that it possessed a high discriminating ability. Two rhizobial strains, namely, (Rhizobium sp.; 33a-PP2) and (Rhizobium multihospitium; 37a-PP4) were identified as ideal for RDW and SDW, respectively. The biplot analysis also revealed that for SDW, E1 (Bambara groundnut) and E4 (pigeonpea), in that sequence, were plotted closet to the epicentre. The GGE biplot analysis also revealed that both pigeonpea and Bambara groundnut provided the most ideal symbiotic activity for NDW but tepary bean lacked the discriminatory ability for NDW. Further testing and validation of the symbiotic activities of the rhizobial strains identified in this study in field trials on diverse legume species and in multiple agro-ecological locations is recommended. It will also be desirable to identify new bio-inoculants for improving tepary bean productivity.Item Open Access Variation in Drought Tolerance Attributes Among Tepary Bean (Phaseolus acutifolius) Germplasm(2023-05-19) Nong, Refilwe Aljareau; Gwata, E. T.; Gerrano, A. S.Tepary bean (Phaseolus acutifolius A. Gray) is an important food legume which originated from South America. In South Africa, it is cultivated by smallholder growers mainly in the drought prone Sekhukhune District of Limpopo Province. Currently, there are no significant breeding efforts aimed at cultivar development of this crop and it remains under-utilized despite the potential of the crop. Therefore, this study evaluated drought tolerance and growth attributes of the tepary bean emphasising on the leaf proline content that are associated with drought tolerance directly or indirectly. The study also determined the drought tolerance and growth relationships as well as identified potentially superior genotypes of tepary bean. The germplasm was evaluated before and after the soil moisture stress treatment which was imposed on the trial by withholding water for 21 days. A 6 x 7 rectangular lattice design replicated three times was used for evaluating 42 genotypes. The results showed that prior to soil moisture stress, there were significant (P<0.05) differences among the 42 genotypes for all the six phenotypic parameters that were measured. The highest (1.05 μmol/g dry weight) and lowest (0.32 μmol/g dry weight) leaf proline content (LPC) were observed for genotypes ‘Ac-35’ and ‘Ac-9’, respectively. The trial mean for proline was 0.69 μmol/g dry weight. The genotype ‘Ac-42’ attained the highest (27.85) leaf chlorophyll content (LCC) which was 48.94% higher than the check genotype (‘Ac-34’). The genotype ‘Ac-33’ achieved almost two-fold higher relative water content (RWC) (84.72%) than genotype ‘Ac-11’ which recorded the lowest (43.12%) RWC. The highest (68.70 mmol m-2s-1) stomatal conductance (SC) was three-fold more than for the check genotype (19.90 mmol m-2 s-1). At least four genotypes (‘Ac-6’, ‘Ac-7’, ‘Ac-22’ and ‘Ac-28’) attained significantly (P < 0.05) greater stem height (SH) than the trial mean (28.63 cm). After the soil moisture stress treatment, the results revealed that the LPC ranged from 1.26 to 0.36 μmol/g dry weight that were observed for genotype ‘Ac-35’ and ‘Ac-9’, respectively. The LPC showed a positive but not significant (P > 0.05) correlation with each of the other remaining attributes both before and after the moisture stress treatment. Similarly, after the soil moisture stress, the LCC maintained a highly significant (P < 0.01) positive correlation with the RWC but a negative correlation with the SH. In both soil moisture conditions, there was no discernible correlation between the SD and the SH. In general, the soil moisture stress lead to a variable increment in the LPC among the genotypes. An independent samples t-test which was used to determine the significance of the change in LPC showed that there was a highly significant (P < 0.00019) difference between the measurements of this amino acid before and after soil moisture stress. The results also showed a reduction in LCC during the soil moisture stress period but there was no clear pattern of the influence of the soil moisture stress on both the SC and RWC. The principal component analysis showed that before the soil moisture stress, the first two principal components accounted for 45.49% of the total variation and three traits (SC, LPC and SH) were highly associated with PC1. In addition, SC contributed the most variation for this component. However, PC2 was highly associated with LPC and RWC. In contrast, PC3 was dominated by SH. The results also showed that after the soil moisture stress, the first two principal components accounted for >50.0% of the total variation. The LPC and SH were highly associated with PC2 but PC3 was dominated by both LCC and SD. In the biplot analysis four genotypes (‘Ac-2’, ‘Ac-19’, ‘Ac-30’ and ‘Ac-41’) were clustered around the origin prior to the moisture stress treatment while five genotypes (‘Ac-3’, ‘Ac-9’, ‘Ac-11’, ‘Ac-28’ and ‘Ac-35’) were distinct and positioned far away from the origin. The genotypes in the right top quadrant (including ‘Ac-4’, ‘Ac-6’, ‘Ac-7’ and ‘Ac-28’) were associated and characterized by high leaf proline, high degree of stomatal opening and tall shoots. The tallest shoots were associated with the genotypes that were grouped in the left top quadrant while the remainder of the genotypes were characterized by thick stems and grouped in the left bottom quadrant. The tepary bean genotypes were grouped into three main clusters with the majority of the genotypes (64.28%) grouped in cluster III. Cluster I consisted of only seven genotypes including ‘Ac-40’ (which was associated with high LCC) as well as ‘Ac-2’, ‘Ac-35’, and ‘Ac-37’ (which were characterized by both LPC and RWC). The check (genotype ‘Ac-34’) was grouped in cluster III in a sub-cluster with genotype ‘Ac-20’. This study discusses the implications of the observed variability among the tepary bean genotypes for these phenotypic attributes and growth parameters. There will be merit in validating these results on a field basis together with grain yield evaluation and genotyping over multiple locations and seasons to determine elite germplasm that breeders and growers can utilize.Item Open Access Variation in Root Nodulation Traits among Parental Genotypes and Segregating f 2 Pigeonpea Plant Populations(2023-05-19) Mthombeni, Tinyiko; Gwata, E. T.; Gerrano, A. S.Pigeonpea (Cajanus cajan L. Millsp.) is an important grain legume that originated in the Indian sub-continent. In South Africa, it is grown either as single plants or as a hedge, mainly in Kwazulu-Natal, Limpopo, and Mpumalanga Provinces. The crop provides highly nutritious food for human consumption and fixes considerable amounts of atmospheric nitrogen (N) thus contributing to the improvement of soil fertility. Root nodulation in pigeonpea is an integral part of the symbiotic process that results in N fixation thus contributing to the productivity of the crop. Currently, there are no reports that determined the genetics of root nodulation in pigeonpea. Therefore, this study was designed to determine the mode of inheritance for selected root nodulation traits. The experiment was conducted in a greenhouse at the Agricultural Research CouncilPlant Health and Protection (ARC-PHP). The average day and night temperatures in the greenhouse were 28°C and 15°C- respectively, with 14 hours of daylight. A randomized complete block design with two replications was used to set up the experiment. Six pigeonpea genotypes were used in the study together with thirty-six rhizobia strains originating from soil that was collected from diverse locations across South Africa. The nodulation variables which were measured included leaf chlorophyll content (LCC), shoot dry weight (SDW), root dry weight (RDW) and nodule dry weight (NDW). The data sets for each of these quantitative variables were subjected to the analysis of variance followed by mean separation using the least significant difference at the 5% probability level using statistical software (Statistix 10.0), and subsequently to analysis of goodness of fit test using standard Chi-square procedures for various Mendelian ratios. The results revealed that the method which was employed to phenotype both the parental genotypes and the F2 progenies was effective and enabled a distinction between the phenotypic classes among the treatments hence a rapid, simple technique to identify contrasting parental genotypes for specific nodulation traits for use in the subsequent genetic study. The GGE biplot analysis revealed that the rhizobial strains 'R24', 'R28', 'R31' and 'R34' were clustered around the origin. In contrast, the rhizobial strains 'R7', 'RB', 'R10', 'R27' and 'R29'were positioned far away from the origin. The biplot also indicated that the pigeonpea parental genotypes (coded as environment scores), 'Gen-1' (E1 ), 'Gen-2' (E2), 'Gen-3' (E3) and 'Gen-5' (ES) were separated by acute angles between them and grouped in the same quadrant. The 'which-won where' biplot explained 56.05% total variation of which PC1 and PC2 accounted for 29.40% and 26.65% of the total variation, respectively. The results also revealed that the rhizobial strains (depicted as genotypes) on the vertices of the polygon 'R 1 0', 'R 11 ', 'R27' and 'R3S' performed best with the pigeonpea parental genotypes (depicted as environments) 'Gen-2' (E2), 'Gen-S' (ES), 'Gen-4' (E4) and 'Gen-6' (E6), respectively. The genotype 'Gen-S' (ES) showed the longest vector line, suggesting a high discriminating ability. The frequency distribution curve for the F2 plant population that was derived from the cross Pa x P1 showed approximately a normal distribution curve but with a slight skew to the right suggesting the presence of epistatic gene action for the LCC trait. The segregation ratio of 9 high:7 low chlorophyll content in the cross P4 x P2 (P-04-SST x P-02-DC) suggested duplicate recessive epistasis in which there is complete dominance at both gene pairs; but, when either gene is homozygous recessive, it masks the effect of the other gene. For SDW, the results also confirmed that the 9:7 ratio was the best fit. The segregation pattern, based on the LCC, of the F2 population in the cross P-04-SST x P-02-DC, best fitted the 9:7 ratio. The results showed that the 9:7 ratio was generally predominant for the traits that were studied thus indicating a high probability that more than one gene, with epistasis are involved in their genetic control. The LCC showed a weak negative correlation with each of NDW and SDW in the F2 progenies that were derived from P-04-SST x P-02-DC. However, there was a positive but weak correlation between NDW and SDW in this set of progenies. In contrast, there was a highly significant (P < 0.01) positive correlation between NDW and SDW in 'cross 2'. The LCC was positively correlated to both NDW and SDW in the F2 progenies that were derived from the cross involving Pax P1. It is recommended that future studies should include the determination of heritability values that can be used in breeding programs aimed at the genetic improvement of N fixation in pigeonpea. It may also be necessary to combine classical Mendelian genetics with modern genomics tools to gain a better understanding of the complex nature of N fixation in pigeonpea as well as its genetic manipulation.