Meloidogyne incognita (Kofoid and White) is a major pest of soybean in South Africa and due to its high level of pathogenicity to the crop it is quintessential that research in this regard should receive priority. Root-knot nematode control has in the past mostly included the use of nematicides, while crop rotation and inclusion of cultivars with genetic host plant resistance (henceforth referred to as resistance only) to these pests were also used. Since no synthetically-derived and/or biological agents are registered locally as nematicides on soybean, the use of resistant cultivars represents one of the most viable and environmentally-friendly strategies to protect local soybean crops against damage resulting from parasitism by M. incognita. Although numerous exotic soybean cultivars have been identified with resistance to M. incognita, only a few locally adapted ones have proved to exhibit resistance to the latter species. Moreover, at present Egret is the only cultivar still available for commercial use in South Africa. Little and fragmented information is, however, available on the use of plant enzymes, that are interrelated in biochemical pathways that are expressed in root-knot nematode resistant cultivars, for its use as an additional parameter to exploit such a trait. Therefore, the present study was undertaken to identify M. incognita resistance in selected, locally adapted soybean cultivars by quantifying and exploiting the latter trait by using enzyme activities as an additional parameter. In addition, resistance to M. incognita in selected resistant soybean cultivars was also verified by means of histopathological studies to identify cellular changes associated with the trait. In the first part of the present study, 31 locally adapted soybean cultivars of which 23 were commercially available in the 2012 growing season were evaluated for resistance to M. incognita. The latter was done by means of traditional screening protocols for which M. incognita-gall rating, egg and second-stage juvenile as well as the reproductive factor data per root system for each cultivar screened were recorded. Two greenhouse experiments were subsequently conducted concurrently, one of which the abovementioned nematode parameters were recorded 30 and the other 56 days after inoculation. Reproduction factor values were used as the main criterium to identify M. incognita resistance in local soybean cultivars since it is considered as a more reliable parameter for this specific type of evaluations. Reproduction factor values equal to and lower than one, indicating resistance to the M. incognita population used in this study, were recorded only for cultivar LS5995, as well as seven pre-released GCI cultivars. These eight cultivars also had very low egg, as well as egg and second-stage juvenile counts per root system, all of which differed significantly from the susceptible control, as well as a number of other cultivars. Root gall indices, on the other hand, did not show consistent results in terms of the identification of the host status of the 31 cultivar screened during this study. Using reproduction factor values, local farmers can thus be supplied with information on the resistance of commercially-available soybean cultivars. Eventually, such M. incognita-resistant cultivars can be used to reduce population levels of this nematode pest in fields of producers and also as valuable germplasm sources in breeding programs to introgress/stack this trait in newly-developed soybean cultivars. The second part of the study aimed to verify and exploit M. incognita-resistance in soybean either identified as resistant or susceptible during the screenings experiments, using enzymatic activity as biochemical markers. Cultivar LS5995 was included as the resistant and Dundee as the susceptible standard. The activity of three enzymes, namely guaiacol peroxidase, lipoxygenase and catalase were recorded at different time intervals in roots and leaf samples of the latter cultivars, of both nematode-inoculated and nematode-free plants of each cultivar. Significant (P ≤ 0.05) increases in guaiacol peroxidase activity in leaf and root samples of the M. incognita-resistant cultivars GCI7 and LS5995 (inoculated with J2) were recorded 24 hours (h) after onset of the experiment. Use of this enzyme thus emanated as a useful parameter to identify soybean cultivars that exhibit resistance against M. incognita, especially in leaves, which could substantially reduce the time needed to screen cultivars. In terms of lipoxygenase activity recorded, substantial variation existed between the cultivars tested. The M. incognita-susceptible cultivar Egret was the only cultivar for which a significant (P ≤ 0.05) increase in lipoxygenase activity in the roots was evident 24 h after inoculation. However, during the 48 h sampling time, significant (P ≤ 0.05) differences in lipoxygenase activity were also recorded for the two M. incognita resistant cultivars GCI7 and LS5995. Although the increase in lipoxygenase activity for the susceptible cultivar Egret was unexpected, it may indicate that some level of resistance is present in the latter cultivar, which has in previous studies been identified as resistant to M. incognita. Other factors such as a different M. incognita populations used and temperature differences in greenhouse conditions that applied in this study compared to that for an earlier study may, however, serve as explanations for the latter differences in host status identification of cultivar Egret. In terms of catalase activity recorded in leaf samples of the M. incognita-resistant cultivar LS5995, substantial reductions of as much as 35.6 % were recorded for J2-inoculated plants compared to those of the J2-free control plants. In leaf samples of the susceptible cultivars, Egret and Dundee, catalase was also reduced, but to a lesser extent and ranged from 6 to 26 %. Conversely, catalase activity in the leaves of J2-inoculated plants of the highly susceptible cultivar LS6248R was substantially increased by as much as 29.3 %. Enzyme data obtained as a result of the current study thus generally complemented those of traditional screening assays in which resistance in locally adapted cultivars were identified to a certain degree. It is, however, recommended that enzyme activity, to be used as bio-markers, still needs further refinement and more investigation to optimise their use in identification, verification and exploitation of M. incognita resistance in soybean cultivars. The third and final part of the study encompassed a comparison of cellular changes induced by M. incognita in resistant and susceptible soybean cultivars to verify the resistant reactions expressed in the enzyme data. According to light- and transmission electron microscope observations, distinct differences in the appearance and development of giant cells in roots of the M. incognita-resistant cultivars LS5995 and GCI7 existed when compared to those in roots of the susceptible cultivars Dundee and LS6248R. In the latter cultivars, giant cells that formed were characteristically large and contained a dense cytoplasm, with thick irregularly surfaced cell walls. Cell walls also displayed thick aggregations that appeared to be cell-wall ingrowths. These giant cells are optimal to facilitate M. incognita development and reproduction. In contrast, giant cells that were associated with the resistant cultivars LS5995 and GCI7 were small, irregularly shaped and contained increased amounts of deposited cell-wall material in the cytoplasm known as cell wall inclusions. Necrosis was also present in M. incognita-infected root cells of both cultivars. Such giant cells have been associated with retarded feeding, development and reproduction of the latter root-knot nematode species. However, it was evident that neither GCI7 nor LS5995 are immune to M. incognita since J2 survived and developed to third- and fourth and ultimately mature females that reproduced in their roots. Optimal giant cells that were formed in the roots of the M. incognitasusceptible cultivars Dundee and LS6248R thus supported the nutritional needs of the developing M. incognita individuals and led to significant increases in M. incognita populations 56 days after inoculation as was evident from the high reproduction factor values that were obtained for such cultivars during host status assessments that represented the first part of this study. The opposite was recorded the M. incognita-resistant cultivars LS5995 and GCI7 since sub-optimal giant cells in their roots could not sustain high offspring from such mature females. The presence of necrotic root tissue adjacent to giant cells, furthermore, indicated that hypersensitive reactions occurred in the latter resistant cultivars. Enzyme data obtained in the second part of this study supported the presence of hypersensitive reactions in root cells of the latter resistant cultivars. Guaiacol peroxidase and lipoxygenase inductions in particular in plant tissues have been reported to play integral roles in hypersensitive reactions that are exhibited by cultivars that are resistant to pests and diseases. Finally, results obtained from the different parts of this study complemented each other. It resulted in the resistance that was identified in the GCI7 pre-released cultivar being verified and exploited against that of the resistant standard LS5995. Research that was done during this study also represented the first investigations into the use of enzymes as biochemical markers of resistance against M. incognita in soybean in South Africa. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Meloidogyne incognita (Kofoid and White) is a major pest of soybean in South Africa and due to its high level of pathogenicity to the crop it is quintessential that research in this regard should receive priority. Root-knot nematode control has in the past mostly included the use of nematicides, while crop rotation and inclusion of cultivars with genetic host plant resistance (henceforth referred to as resistance only) to these pests were also used. Since no synthetically-derived and/or biological agents are registered locally as nematicides on soybean, the use of resistant cultivars represents one of the most viable and environmentally-friendly strategies to protect local soybean crops against damage resulting from parasitism by M. incognita. Although numerous exotic soybean cultivars have been identified with resistance to M. incognita, only a few locally adapted ones have proved to exhibit resistance to the latter species. Moreover, at present Egret is the only cultivar still available for commercial use in South Africa. Little and fragmented information is, however, available on the use of plant enzymes, that are interrelated in biochemical pathways that are expressed in root-knot nematode resistant cultivars, for its use as an additional parameter to exploit such a trait. Therefore, the present study was undertaken to identify M. incognita resistance in selected, locally adapted soybean cultivars by quantifying and exploiting the latter trait by using enzyme activities as an additional parameter. In addition, resistance to M. incognita in selected resistant soybean cultivars was also verified by means of histopathological studies to identify cellular changes associated with the trait. In the first part of the present study, 31 locally adapted soybean cultivars of which 23 were commercially available in the 2012 growing season were evaluated for resistance to M. incognita. The latter was done by means of traditional screening protocols for which M. incognita-gall rating, egg and second-stage juvenile as well as the reproductive factor data per root system for each cultivar screened were recorded. Two greenhouse experiments were subsequently conducted concurrently, one of which the abovementioned nematode parameters were recorded 30 and the other 56 days after inoculation. Reproduction factor values were used as the main criterium to identify M. incognita resistance in local soybean cultivars since it is considered as a more reliable parameter for this specific type of evaluations. Reproduction factor values equal to and lower than one, indicating resistance to the M. incognita population used in this study, were recorded only for cultivar LS5995, as well as seven pre-released GCI cultivars. These eight cultivars also had very low egg, as well as egg and second-stage juvenile counts per root system, all of which differed significantly from the susceptible control, as well as a number of other cultivars. Root gall indices, on the other hand, did not show consistent results in terms of the identification of the host status of the 31 cultivar screened during this study. Using reproduction factor values, local farmers can thus be supplied with information on the resistance of commercially-available soybean cultivars. Eventually, such M. incognita-resistant cultivars can be used to reduce population levels of this nematode pest in fields of producers and also as valuable germplasm sources in breeding programs to introgress/stack this trait in newly-developed soybean cultivars. The second part of the study aimed to verify and exploit M. incognita-resistance in soybean either identified as resistant or susceptible during the screenings experiments, using enzymatic activity as biochemical markers. Cultivar LS5995 was included as the resistant and Dundee as the susceptible standard. The activity of three enzymes, namely guaiacol peroxidase, lipoxygenase and catalase were recorded at different time intervals in roots and leaf samples of the latter cultivars, of both nematode-inoculated and nematode-free plants of each cultivar. Significant (P ≤ 0.05) increases in guaiacol peroxidase activity in leaf and root samples of the M. incognita-resistant cultivars GCI7 and LS5995 (inoculated with J2) were recorded 24 hours (h) after onset of the experiment. Use of this enzyme thus emanated as a useful parameter to identify soybean cultivars that exhibit resistance against M. incognita, especially in leaves, which could substantially reduce the time needed to screen cultivars. In terms of lipoxygenase activity recorded, substantial variation existed between the cultivars tested. The M. incognita-susceptible cultivar Egret was the only cultivar for which a significant (P ≤ 0.05) increase in lipoxygenase activity in the roots was evident 24 h after inoculation. However, during the 48 h sampling time, significant (P ≤ 0.05) differences in lipoxygenase activity were also recorded for the two M. incognita resistant cultivars GCI7 and LS5995. Although the increase in lipoxygenase activity for the susceptible cultivar Egret was unexpected, it may indicate that some level of resistance is present in the latter cultivar, which has in previous studies been identified as resistant to M. incognita. Other factors such as a different M. incognita populations used and temperature differences in greenhouse conditions that applied in this study compared to that for an earlier study may, however, serve as explanations for the latter differences in host status identification of cultivar Egret. In terms of catalase activity recorded in leaf samples of the M. incognita-resistant cultivar LS5995, substantial reductions of as much as 35.6 % were recorded for J2-inoculated plants compared to those of the J2-free control plants. In leaf samples of the susceptible cultivars, Egret and Dundee, catalase was also reduced, but to a lesser extent and ranged from 6 to 26 %. Conversely, catalase activity in the leaves of J2-inoculated plants of the highly susceptible cultivar LS6248R was substantially increased by as much as 29.3 %. Enzyme data obtained as a result of the current study thus generally complemented those of traditional screening assays in which resistance in locally adapted cultivars were identified to a certain degree. It is, however, recommended that enzyme activity, to be used as bio-markers, still needs further refinement and more investigation to optimise their use in identification, verification and exploitation of M. incognita resistance in soybean cultivars. The third and final part of the study encompassed a comparison of cellular changes induced by M. incognita in resistant and susceptible soybean cultivars to verify the resistant reactions expressed in the enzyme data. According to light- and transmission electron microscope observations, distinct differences in the appearance and development of giant cells in roots of the M. incognita-resistant cultivars LS5995 and GCI7 existed when compared to those in roots of the susceptible cultivars Dundee and LS6248R. In the latter cultivars, giant cells that formed were characteristically large and contained a dense cytoplasm, with thick irregularly surfaced cell walls. Cell walls also displayed thick aggregations that appeared to be cell-wall ingrowths. These giant cells are optimal to facilitate M. incognita development and reproduction. In contrast, giant cells that were associated with the resistant cultivars LS5995 and GCI7 were small, irregularly shaped and contained increased amounts of deposited cell-wall material in the cytoplasm known as cell wall inclusions. Necrosis was also present in M. incognita-infected root cells of both cultivars. Such giant cells have been associated with retarded feeding, development and reproduction of the latter root-knot nematode species. However, it was evident that neither GCI7 nor LS5995 are immune to M. incognita since J2 survived and developed to third- and fourth and ultimately mature females that reproduced in their roots. Optimal giant cells that were formed in the roots of the M. incognitasusceptible cultivars Dundee and LS6248R thus supported the nutritional needs of the developing M. incognita individuals and led to significant increases in M. incognita populations 56 days after inoculation as was evident from the high reproduction factor values that were obtained for such cultivars during host status assessments that represented the first part of this study. The opposite was recorded the M. incognita-resistant cultivars LS5995 and GCI7 since sub-optimal giant cells in their roots could not sustain high offspring from such mature females. The presence of necrotic root tissue adjacent to giant cells, furthermore, indicated that hypersensitive reactions occurred in the latter resistant cultivars. Enzyme data obtained in the second part of this study supported the presence of hypersensitive reactions in root cells of the latter resistant cultivars. Guaiacol peroxidase and lipoxygenase inductions in particular in plant tissues have been reported to play integral roles in hypersensitive reactions that are exhibited by cultivars that are resistant to pests and diseases. Finally, results obtained from the different parts of this study complemented each other. It resulted in the resistance that was identified in the GCI7 pre-released cultivar being verified and exploited against that of the resistant standard LS5995. Research that was done during this study also represented the first investigations into the use of enzymes as biochemical markers of resistance against M. incognita in soybean in South Africa. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014