Doctoral Degrees (Plant Pathology)

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Application of transmissible agents to control citrus tree size.
(1997) Van Vuuren, Stephanus Petrus.; Da Graca, John Vincent.
Establishing citrus orchards at higher densities has become standard practice in South Africa. These plantings make more efficient use of inputs such as land, fertilizer, irrigation and spraying, and they are capable of high early production and economic returns. However, disadvantages develop over time, depending on tree density and climate. These include overcrowding between and in rows, which induces difficult access by vehicles, poor insect control in dense canopies, and a decrease in yield and quality due to dieback and overcrowding. It is thus important to find a method to control tree size to maintain the benefits of such plantings. All three main tree size controlling methods viz. the use of genetic dwarf plant material, management practises and the use of transmissible agents are currently under investigation in the country. The aim of this study was to identify transmissible agents that can be used successfully to control tree size without detrimental effects. Research was done to identify transmissible agents as well as their application as dwarfing agents for commercial use. An important safety measure that should be considered before a transmissible agent is introduced for tree size control of citrus, is that such an agent should be endemic in the industry. Citrus tristeza virus (CTV) is a well known endemic disease in Southern Africa and virus-free shoot-tip grafted (STG) material is pre-immunised with avirulent isolates to reduce its effect. STG is also used to remove citrus viroids (CVd's) and the use of such agents as dwarfing agents is in contradiction with the aims of the Southern African Citrus Improvement Programme. However, research over many years in Australia has shown that avirulent CVd's can be used succesfully to control tree size. It is thus important to identify suitable CVd isolates for commercial application but it is also important to know which CVd's occur in commercial orchards. The occurrence of CVd's in old commercial citrus orchards, planted prior to the introduction of shoottip grafted material, was established by biological indexing with Etrog citron (Citrus medica L. cv. Arizona-861-S-1). Twenty-one percent of the trees indexed tested positive for CVd's. Sequential polyacrylamide electrophoresis (sPAGE) was employed to establish which viroids are most commonly present. Only two groups of CVd were detected, viz. citrus exocortis viroid (CEVd) and CVd-III. The latter CVd was the most common (40%) while CEVd occurred in only 8% of the samples. More than a third of the samples were infected with both CVd's. Two CVd isolates, a mild and a severe isolate according to Etrog citron reactions, were bud-inoculated to Delta Valencia on rough lemon, Poncirus trifoliata and four trifoliate hybrid rootstocks. Growth, production and occurrence of disease symptoms of these trees were compared to trees on the same rootstocks without CVd. All the trees were pre-immunized with the standard tristeza virus isolate. Results of sPAGE analysis of nucleic extracts indicated that the severe isolate (CD 11) contained CEVd only, and the mild isolate (CD 12) contained CVd-III. Overall, both isolates caused a significant reduction in tree size. The cumulative production over five years of the CD 11 infected trees did not differ from the CVd-free trees although the trees were smaller. This was due to a significantly higher production efficiency (kg/m(3) canopy). The production efficiency of the CD 12 trees was similar to the CVd-free trees, but the smaller trees resulted in a significantly lower production. Disease symptoms occurred with both isolates, but symptoms differed. Poncirus trifoliata var. Rubidoux was more sensitive to CVd isolates than four trifoliate hybrid rootstocks. Marsh grapefruit trees on Troyer citrange rootstock were bud-inoculated with different CTV isolates prior to planting in the field. Selected CTV isolates GFMS 2, GFMS 10, GFMS 12, GFMS 19, GFMS 25, GFMS 27 and GFMS 35, free of citrus viroids, were bud-inoculated into the virus-free plants. A severe isolate (GFSS 1) and plants that were left virus-free served as controls. Tree size, production, fruit size and tree health were determined. Fifteen years after planting, canopy volumes of trees with three isolates, GFMS 2, GFMS 19 and GFMS 25, were significantly smaller than the control trees as well as trees with isolates GFMS 10, GFMS 12 and GFMS 35. Trees with isolate GFMS 19 had a larger diameter than those with isolates GFMS 2 and GFMS 25. Together with a slightly higher yield efficiency, GFMS 19 trees resulted in a cumulative yield equal to that of the control and the GFMS 12 trees. Considering fruit size and their value, the performance of trees with isolate GFMS 19 equalled that of the larger trees. Tree health was also similar which makes this isolate suitable for use in high density plantings. A projection was made which showed that the production and crop value of to trees with isolate GFMS 19 were similar to those of trees with isolates GFMS 10, GFMS 12 and GFMS 35. However, benefits such as easier and better spray application and easier harvesting can increase profits when trees with isolate GFMS 19 are planted. The dwarfing characteristics of four isolates, CD 4, CD 8, CD 9 and CD 10, derived from healthy looking dwarfed field trees were evaluated. They were bud-inoculated to Delta Valencia trees on Yuma citrange rootstock prior to planting in the field. Five years after planting, isolates CD 4 and CD 9 successfully reduced canopy volumes by 60%, and CD 10 by 30%, without any detrimental effects. No CVd's could be detected biologically or by sPAGE in these three isolates. Isolate CD 8 however, contained two viroids, CEVd and CVd-III, but had no deleterious effects on the rough lemon rootstock. CTV was the only other pathogen present in the isolates. Indexing for cachexia, psorosis, impietratura and tatter leaf was negative. The dwarfing abilities of the isolates are therefore attributed to isolates of citrus tristeza virus. Production was according to tree size and the yield efficiency of the inoculated trees was equal to that of the uninoculated control trees. External and internal fruit quality was not affected. The trees were naturally infected with huanglongbing (greening) five years after planting, but the disease remained low for several years in trees with isolate CD 4. Three transmissible isolates (CD 4, CD 9, CD 10), derived from dwarfed field trees, were compared with two CVd isolates (CD 8, CD 12) for their abilities to control tree size of sweet orange. The isolates were bud-inoculated to Valencia on rough lemon, two Poncirus trifoliata and three trifoliate hybrid rootstocks, and compared to uninoculated trees on the same rootstocks. Isolates CD 4, CD 9 and CD 10 gave no reaction on Etrog citron and sPAGE of nucleic acid extracts failed to detect known CVd's. The two CVd isolates gave severe and mild reactions on Etrog citron and sPAGE showed that CD8 contained CEVd and CVd-III while CD 12 contained only CVd-III. The effect of the isolates on tree size, production, production efficiency and disease occurrence were monitored. Overall, CD 4, CD 9 and CD 10 did not reduce canopy volumes while trees with CD 8 and CD 12 were significantly smaller. However, CD 4 significantly reduced canopy volumes where Yuma citrange was used as a rootstock. In contrast, CD 8, containing CEVd, did not reduce canopy volumes on this rootstock, while CD 12 reduced it significantly. None of the trees on the other rootstocks were affected by either CD 4, CD 9 or CD 10. Canopy volume reductions by CD 8 and CD 12 differed from each other with all the trifoliate rootstocks. The production efficiency of trees with the two CVd isolates was significantly higher than the control trees as well as those with CD 4, CD 9 and CD 10. The higher efficiency of these trees resulted in cumulative production equal to the uninoculated trees. Disease symptoms occurred where all the isolates were inoculated, however, symptoms as well as the susceptibility of the rootstocks, differed among each other. Delta Valencia trees on Yuma citrange rootstock were inoculated respectively with two mild (GFMS 12 and T55), an intermediate (GFMS 10) and a severe (GFSS 1) CTV isolate prior to planting in the field. The same CTV isolates were also inoculated in combination with a CVd- III isolate. A virus-free control was included in the trial. All the CTV isolates were without the Seedling Yellows component of CTV. Seven years after planting, canopy volumes of the trees with the two mild isolates and the control were smaller than those of trees with the intermediate and severe isolates. This was in contradiction to what was expected. Overall, the CVd isolate had an additional reducing effect on canopy size, but only those trees with mild isolate T55 and severe isolate GFSS 1 were significantly affected. Production on a per tree basis was according to the canopy sizes, thus, the trees carrying the intermediate CTV isolate were the highest producers. The production efficiency (kg/m(3) canopy), however, did not differ among trees with the CTV isolates and the control. The CVd isolate generally increased the production efficiency. The internal quality of the fruit was not affected by any treatment. The lack of a suitable genetic dwarfing rootstock for citrus makes it essential to evaluate alternative methods to reduce tree size for high density plantings. Four transmissible dwarfing factors, derived from dwarfed trees, were evaluated for commercial application in a hot (Malelane) and intermediate (Nelspruit) production area. Generally, trees in the hot production area were more vigorous with a lower production efficiency than trees in the intermediate area. The dwarfing effects of isolates CD 4, CD 9 and to a lesser extent, CD 10, were reduced in the hot area. Isolate CD 8 caused no dwarfing at either location. The reduction of dwarfing at the hot site may be attributed to the suppression of CTV by high temperatures. Currently some of the isolates which were tested in this investigation are applied on a larger scale in different climatic areas as commercial trials. Formal trials are continuing and they are aimed to elucidate the dwarfing characteristics as well as the inducement of disease of the four viroids in the CVd-III group.
Aspects of post-harvest seed physiology and cryopreservation of the germplasm of three medicinal plants indigenous to Kenya and South Africa.
(2002) Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman W.
The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5.
Banana bunchy top virus in South Africa: the distribution, molecular relationship and transmission studies.
(2021) Ximba, Sinethemba Patience Fanelwe.; Jooste, Anna Elizabeth Catharina.; Gubba, Augustine.
The first report of Banana bunchy top virus (BBTV) in KwaZulu-Natal (KZN), South Africa in 2016 has raised the need to study the virus in South Africa. The aim of this research project was to conduct surveys across banana-producing provinces, including KwaZulu-Natal, Mpumalanga and Limpopo in South Africa, to determine the spread of BBTV in banana production regions across the country. To date, the virus has been localized within the province of KZN in the South Coast region. Once positive samples were obtained, the genetic relationships between the South African isolates and those collected globally was investigated. This was done by studying five (DNA-C, -S; -N; -M; -U3) of the six components of the BBTV genome. No major differences between the isolates were observed. As the virus is only transmitted through infected planting material and through the vector, Pentalonia nigronervosa, BBTV transmission studies were conducted. Such studies have been conducted in different countries on this topic with conflicting results and BBTV transmission studies were included here as well. Plant species namely Colocasia esculenta, Alocasia macrorrhizos, Alpinia zerumbet and Strelitzia reginae, that are usually found growing around banana plantations, were investigated to determine if these plants act as reservoirs of the virus vector and also to determine if these potential alternative host plant can be hosts of the virus. Banana plants were included as controls in the experiment. A qPCR was optimised to test for BBTV in the plants and aphids at low concentrations. BBTV was detected in all of the plants except A. macrorrhizos. It was concluded that A. zerumbet was an alternative host of the banana aphid while C.esculenta and S. reginae are assumed to be intermediary hosts of the virus vector while A. macrorrhizos is neither a host of the vector nor of the virus.
Biological and molecular characterization of South African bacteriophages infective against Staphylococcus aureus subsp. aureus Rosenbach 1884, casual agent of bovine mastitis.
(2012) Basdew, Iona Hershna.; Laing, Mark Delmege.
Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification.
Biological control and plant growth promotion by selected trichoderma and Bacillus species.
(2005) Yobo, Kwasi Sackey.; Laing, Mark Delmege.; Hunter, Charles Haig.
Various Trichoderma and Bacillus spp. have been documented as being antagonistic to a wide range of soilborne plant pathogens, as well as being plant growth stimulants. Successes in biological control and plant growth promotion research has led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of six Trichoderma spp. and three Bacillus spp. and their respective combinations, for the biological control of Rhizoctonia solani damping-off of cucumber and plant growth promotion of dry bean (Phaseolus vulgaris L.). In vivo biological control and growth promotion studies were carried out under greenhouse and shadehouse conditions with the use of seed treatment as the method of application. In vitro and in vivo screening was undertaken to select the best Trichoderma isolates from 20 Trichoderma isolated from composted soil. For in vitro screening, dual culture bioassays were undertaken and assessed for antagonisms/antibiosis using the Bell test ratings and a proposed Invasive Ability rating based on a scale of 1-4 for possible mycoparasitic/hyperparasitic activity. The isolates were further screened in vivo under greenhouse conditions for antagonistic activity against R. solani damping-off of cucumber (Cucumis sativus L.) cv. Ashley seedlings. The data generated from the in vivo greenhouse screening with cucumber plants were analysed and grouped according to performance of isolates using Ward‟s Cluster Analysis based on a four cluster solution to select the best isolates in vivo. Isolates exhibiting marked mycoparasitism of R. solani (during ultrastructural studies) viz, T. atroviride SY3A and T. harzianum SYN, were found to be the best biological control agents in vivo with 62.50 and 60.06% control of R. solani damping-off of cucumber respectively. The in vitro mode of action of the commercial Trichoderma product, Eco-T®, and Bacillus B69 and B81 suggested the production of antimicrobial substances active against R. solani. In vitro interaction studies on V8 tomato juice medium showed that the Trichoderma and Bacillus isolates did not antagonise each other, indicating the possibility of using the two organisms together for biological control and plant growth promotion studies. Greenhouse studies indicated that combined inoculation of T. atroviride SYN6 and Bacillus B69 gave the greatest plant growth promotion (43.0% over the uninoculated control) of bean seedlings in terms of seedling dry biomass. This was confirmed during in vivo rhizotron studies. However, results obtained from two successive bean yield trials in the greenhouse did not correlate with the seedling trials. Moreover, no increase in protein or fat content of bean seed for selected treatments was observed. In the biological control trials with cucumber seedlings, none of the Trichoderma and Bacillus combinations was better than single inoculations of Eco-T®, T. atroviride SY3A and T. harzianum SYN. Under nutrient limiting conditions, dry bean plants treated with single and dual inoculations of Trichoderma and Bacillus isolates exhibited a greater photosynthetic efficiency that the unfertilized control plants. Bacillus B77, under nutrient limiting conditions, caused 126.0% increase in dry biomass of bean seedlings after a 35-day period. Nitrogen concentrations significantly increased in leaves of plants treated with Trichoderma-Bacillus isolates. However, no significant differences in potassium and calcium concentrations were found. Integrated control (i.e. combining chemical and biological treatments) of R. solani damping-off of cucumber seedlings proved successful. In vitro bioassays with three Rizolex® concentrations, viz., 0.01g.l-1, 0.1g.l-1 and 0.25g.l-1 indicated that the selected Trichoderma isolates were partly sensitive to these concentrations whereas the Bacillus isolates were not at all affected. In a greenhouse trial, up to 86% control was achieved by integrating 0.1g.l-1 Rizolex® with T. harzianum SYN, which was comparable to the full strength Rizolex® (1g.l-1) application. Irrespective of either a single or dual inoculations of Trichoderma and/or Bacillus isolates used, improved percentage seedling survival as achieved with the integrated system, indicating a synergistic effect. The results presented in this thesis further reinforce the concept of biological control by Trichoderma and Bacillus spp. as an alternative disease control strategy. Furthermore, this thesis forms a basis for Trichoderma-Bacillus interaction studies and proposes that the two organisms could be used together to enhance biological control and plant growth promotion.
Biological control of sorghhm and rice stem borers, chilo partellus and sessamia calamistis using endophytic strains of beauveria bassiana.
(2018) Bancole, Wonroo Bernice Armelle.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Sorghum and rice are two of the major cereals grown across the world. Both of these crops are subjected to a range of abiotic and biotic constraints. Insect pests are important biotic stress factors, which affect both of the crops at all of their growth stages. Stem borers from the family of Lepidoptera, e.g. Chilo partellus (Lepidoptera: Pyralidae) and Sesamia calamistis Hampson (Lepidoptera: Noctuidae) are important pests that attack these cereals. Control of C. partellus and S. calamistis has largely been with pesticides. However, chemical pesticides are too expensive for most small-scale farmers in Africa, leaving their crops unprotected. Biological control is one of the measures that have been advocated for the management of stem borers. Various strains of Beauveria bassiana (Vuillemin) have been documented as being endophytes infecting a wide range of plants, as well as being pathogenic on numerous insect pests. Successes in biological control research have led to the development of various B. bassiana products, which are available commercially, but these are largely epiphytic strains. Biological control studies were therefore conducted with several endophytic strains of B. bassiana against sorghum stem borer, C. partellus, and the rice stem borer, S. calamistis. The fungi were tested by endophytic behaviour and the ability to control the 3rd larval instars of both stem borers, in the laboratory and greenhouse. The interactions of B. bassiana strains and a commercially available Trichoderma product, Eco-T®, were tested in sorghum and rice plants. In vivo and in vitro screening were initially undertaken to evaluate the endophytic behaviour of 20 B. bassiana strains, using two inoculation methods. Subsequently, the best endophytic B. bassiana strains and the best inoculation method were tested at 30 and 60 days after inoculation. The strains were screened in vivo using seed treatments and foliar sprays, under greenhouse conditions, for endophytic behaviour in sorghum and rice plants. There were highly significant differences between the B. bassiana strains (P = 0.0001). Depending upon the inoculation method, the B. bassiana strains that successfully colonized the sorghum and rice plants could be selected after 30 and 60 days. Five strains of B. bassiana strains (Bb3, Bb4, Bb10, Bb21 and Bb35) were found to be endophytic in both crops, and to provide biological control against the two borers. The best five B. bassiana strains were tested for their pathogenicity on the 3rd instar larvae of C. partellus and S. calamistis. Out of the five endophytic strains of B. bassiana, two (Bb35 and Bb3) were the most pathogenic on C. partellus, with the greatest mortality of 80 % being achieved within 28 days after treatment. The B. bassiana strains Bb35 and Bb4 were the most effective strains against S. calamistis, killing 93.33 and 76.66% of the 3rd larval instar at 28 days, respectively. The cumulative mortality of the 3rd instar larvae of both stem borers increased over time at 21 days after inoculation for all five B. bassiana strains. A field trial was conducted to evaluate the biocontrol efficacy of the five best endophytic strains of B. bassiana against C. partellus, compared to pyrethroid pesticide, Karate. Three of the endophytic strains of B. bassiana strains were as effective as Karate sprays when they were applied as seed treatments, reducing damage by C. partellus as much as Karate did. In vitro and in vivo screening were carried out under laboratory and greenhouse conditions, using various inoculation methods, to assess the interaction between the five B. bassiana strains and a commercially available Trichoderma harzianum product, Eco-T®. In the in vitro dual culture bioassay, one of the five endophytic B. bassiana strains (Strain Bb35) was not inhibited by T. harzianum Strain Kd (TKD) at 15 days after inoculation at 30 days after in vitro inoculation. None of the five endophytic B. bassiana strains grew in the presence of TKD. Only the TKD grew all over the plates. In greenhouse trials, various interactions occurred between the two fungi, according to the inoculation methods. When a mixture of conidia of the two fungi was used at the same time as a seed treatment, there was a strong inhibitory effect by TKD toward the five B. bassiana strains. However, if sorghum plants were seed treated with the five B. bassiana strains, followed by drenching of the plant roots with a TKD suspension, then the B. bassiana strains appeared to be able to colonize the stems of the plants whilst the TKD colonized the roots. Sorghum roots were rapidly colonized by the TKD when it was used alone for the seed treatment. The endophytic behaviour of some strains of B. bassiana in sorghum and rice plants can be used as powerful tool to enhance their biological control activity against stem borers of these crops. However, the tested TKD and B. bassiana strains were not compatible in the same space, such as the rhizosphere, but could be used sequentially to secure the benefits of insect control by the B. bassiana strains, as well as the biological control and plant growth stimulation activities provided by T. harzianum strains.
Biological control of the common house fly (Musa domestica L.) using Bacillus thuringiensis (Ishiwata) berliner var. Israelensis and Beauveria bassiana (Bals.) vullemin in caged poultry facilities.
(2008) Mwamburi, Lizzy A.
The entomopathogenic fungus Beauveria bassiana and the bacterium Bacillus thuringiensis var. israelensis (Bti) have been widely studied for their role in biocontrol against many arthropods and extensively exploited for insect pest control. The purpose of this study was to evaluate the effect of four B. bassiana and two Bti formulations and their respective combinations, for the biological control of the common house fly, Musca domestica L., a major pest in poultry facilities. In vitro screening was undertaken to select the best B. bassiana isolates from 34 B. bassiana isolates and two Paecilomyces isolates. All the isolates of B. bassiana were found to be effective against adult house flies, but were marginally effective in controlling fly larvae. The Paecilomyces isolates were non-pathogenic towards both adult house flies and larvae. The best four isolates R444, 7320, 7569 and 7771 caused >90% mortality within 2d and were subjected to dose-mortality bioassays. Microscopic studies using light and scanning electron microscopy indicated the different durations of the lifecycle of B. bassiana development on the house fly. High temperature was found to delay conidial germination. Spore germination and mycelial growth were also inhibited by high adjuvant concentrations. Laboratory baseline bioassay data established, a dose-time response relationship using a waterdispersible granules (WDG) Bti formulation that demonstrated that the susceptibility of M. domestica larvae to a given concentration of Bti increased as the duration of exposure increased. In the laboratory studies, the LC50 and LC90 values of Bti for the larvae ranged between 65 - 77.4 and 185.1 - 225.9?g ml-1, respectively. LT50 and LT90 values were 5.5 and 10.3d respectively. In the field, a concentration of 10g Bti kg-1 (bran formulation) of feed resulted in 90% reduction of larvae for 4wk post-treatment. A higher concentration (2g L-1) of Bti in spray (WDG) applications was not significantly more effective than the lower concentration of 1g L-1. Thus, adding Bti to chicken feed has potential for the management and control of house flies in cagedpoultry facilities. The impact of oral feed applications of a bran formulation of Bti and a commercial chemical larvicide, Larvadex®, were compared with respect to their efficacy on the control of house fly 3 larval populations in poultry manure. The sublethal effects were manifested in terms of decreasing emergence of adult house flies. Although Larvadex® reduced larval density and caused significant reductions in emergence of adult house flies, it generally exhibited weaker lethal effects than Bti. The reduction levels achieved as a result of feeding 250mg Bti kg-1 at 5wk were similar to those achieved as a result of feeding twice the amount of Larvadex® at 4wk to the layers. From both an efficiency and economic perspective, comparisons to assess the impact of combining different concentrations of the two Bti formulations were carried out to evaluate their success in controlling house fly larvae and adults in poultry houses. The percentage mortality of larvae accomplished as a result of using a combination of 250mg kg-1 Bti in feed and 2g L-1 spray applications was equivalent to that obtained as a result of combining 500mg kg-1 Bti in feed and 1g L-1 spray application. The cost-benefit analysis (expressed in terms of mortality of larvae) indicated that the most effective combination for control of house fly larvae and fly emergence was the 500mg kg-1 in feed and 2g L-1 spray application combination that resulted in 67% larval mortality and 74% inhibition of adult house fly emergence. This study presents commercial users with possible combinations of applications of the two Bti formulations. Comparisons of larval mortalities and house fly emergence resulting from the Bti - B. bassiana treatments with those from Larvadex® - B. bassiana treatments, showed better control levels compared to any of the individual agents alone. The Bti treatments were more effective at controlling larval populations and inhibiting the emergence of house flies than Larvadex®, even when Larvadex® was applied together with B. bassiana. The effects of the Bti - B. bassiana and the Larvadex® - B. bassiana interactions were additive. These trials suggest that the efficacy of Bti in the control of house fly larvae may be improved with frequent applications of B. bassiana.
Biological control of the two-spotted spider mite, Tetranychus urticae Koch (Acari : tetranychidae).
(2009) Gatarayiha, Mutimura Celestin.; Laing, Mark Delmege.; Miller, Raymond Martin.
The two-spotted spider mite (TSM), Tetranychus urticae Koch, is an important pest of many greenhouse and field crops worldwide. The development of resistance in TSM populations to chemical acaricides, allied with public health concerns about pesticide residues, has motivated the search for alternative control measures to suppress the pest. Hyphomycetous fungi are promising agents for mite control and the fungus Beauveria bassiana (Bb) (Balsamo) Vuillemin was investigated in this study as a biocontrol agent. The principal objectives of this study comprised: a) screening Bb strains for their pathogenicity against T. urticae; b) testing the effect of adjuvants on the efficacy of Bb; c) studying the effect of plant type on persistence of Bb and the efficacy of control of Bb against T. urticae; d) evaluating the field efficacy of Bb applications against T. urticae; e) testing the compatibility of Bb with selected fungicides; and f) assessing the synergy between Bb and soluble silicon for T. urticae control. Screening bioassays of sixty-two strains of Bb identified the two most effective strains, PPRI 7315 (R289) and PPRI 7861 (R444), that caused mortality levels of more than 80% of adult mites at 9 d post-inoculation with 2 × 108 conidia ml-1. These strains performed significantly better than the Bb commercial strain PPRI 5339, in laboratory bioassays. The two strains also attacked mite eggs, causing 53.4% and 55.5% reduction in egg hatchability at 2 × 108 conidia ml-1 respectively. However, PPRI 7861 showed relatively higher production of conidia in culture and was, therefore, selected for further trials under greenhouse and field conditions. Greenhouse evaluations of the effects of two adjuvants (Break-thru® and a paraffin oil-based emulsion) on efficacy of Bb demonstrated a higher efficacy of the biocontrol agent (BCA) when it was applied with Break-thru® or the oil solution than with water alone. Moreover, Bb conidia applied in Break-thru® solution resulted in greater control of TSM than conidia applied in the mineral oil. There was also a dose-response effect and the control of TSM by Bb increased when the concentration of conidia was increased. The control of TSM by Bb in beans (Phaseolus vulgaris L), cucumber (Cucumis sativus L.), eggplant (Solanum melongena L.), maize (Zea mays L.) and tomato (Solanum lycopersicum L.) was tested in greenhouse trials. On these crops, the persistence of conidia declined over time. The rate of decline was significantly higher on maize. However, TSM mortality was positively correlated with the amount of conidia deposited on leaves immediately after spraying, rather than their persistence over time. Higher levels of mortality of TSM due to Bb application were observed on beans, cucumber and eggplants, suggesting that the type of crop must be taken into consideration when Bb is applied as a BCA. Field efficacy of Bb against mites was evaluated in two trials on eggplants. Based on assessment of population densities of mites and leaf damage assessments; both trials showed that the strain PPRI 7861 controlled TSM in the field. Two commonly used fungicides, azoxystrobin and flutriafol, were investigated in vitro tests on culture medium and laboratory bioassays on detached bean leaves (Phaseolus vulgaris L.) for their effects on Bb. Azoxystrobin (a strobilurin) was less harmful to Bb while flutriafol was found to be inhibitory. Another important finding of this study was the substantial enhancement of Bb efficacy by soluble silicon. When Bb was combined with soluble Si, the control of TSM was better than when either of the two products was applied alone. Moreover, application of soluble Si as a plant fertilizer in hydroponic water nutrient increased accumulation of peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase enzymes in leaves of plants infested with TSM. Increased activity of these defense enzymes in leaves deters feeding behaviour of mites. We suggested that feeding stress renders them susceptible to Bb infection, which would explain the synergy observed between the two agents.
Biological control of three grain storage pests: maize weevil, Sitophilus zeamais (Motschulsky), almond moth, Ephestia cautella (Walker) and cigarette beetle, Lasioderma serricorne (Fabricius), using novel strains of Beauveria bassiana (Balsamo) Vuillemin in powder formulation.
(2017) Saeed, Mohamed Baha Eldeen Eltayed Elhadi Mohamed.; Laing, Mark Delmege.; Miller, Raymond Martin.
Abstract available in PDF file.
Bioremediation of arsenic contaminated groundwater.
(2008) Teclu, Daniel Ghebreyohannes.; Wallis, Frederick Michael.; Tivchev, George V.; Laing, Mark Delmege.
Sulphate-reducing bacteria (SRB) mediate the reduction of metals/metalloids directly or indirectly. Bioremediation of arsenic contaminated water could be a cost-effective process provided a cheap carbon source is used. To this end, molasses was tested as a possible source of carbon for the growth of sulphate-reducing bacteria (SRB). Its chemical composition and the tolerance of SRB toward different arsenic species [As (III) and As (V)] were also investigated. Batch culture studies were carried out to assess 1, 2.5 and 5 g l-1 molasses as suitable concentrations for SRB growth. The results indicate that molasses does support SRB growth, the level of response being dependent on the concentration; however, growth on molasses was not as good as that obtained when lactate, the usual carbon source for SRB, was used. The molasses used in this study contained several metals including Al, As, Cu, Fe, Mn and Zn in concentrations ranging from 0.54-19.7 ìg g-1, but these levels were not toxic to the SRB. Arsenic tolerance, growth response and sulphate-reducing activity of the SRB were investigated using arsenite and arsenate solutions at final concentrations of 1, 5 and 20 mg l-1 for each species. The results revealed that very little SRB growth occurred at concentrations of 20 mg l-1 As (III) or As (V). At lower concentrations, the SRB grew better in As (V) than in As (III). Batch cultures of sulphate-reducing bacteria (SRB) in flasks containing pine bark, sand and polystyrene as support matrices and Postgate medium B were used to study formation of biofilms. The effects of the support matrices on the growth of the organisms were evaluated on the basis of pH and redox potential change and the levels of sulphide production and sulphate reduction. Characterisation of the matrix surfaces was done by means of environmental scanning electron microscopy (ESEM). A consortium of SRB growing on polystyrene caused a 49% of original sulphate reduction whereas on sand a 36% reduction occurred. Polystyrene was further examined for its durability as a long-term support material for the growing of SRB in the presence of As(III) and/or As(V) at concentrations of 1, 5 and 20 mg l-1. Both sulphate reduction and sulphide production were greater in this immobilised system than in the matrix-free control cultures. With pine bark as support matrix no significant sulphate reduction was observed. The kinetics of sulphate reduction by the immobilised cells were compared with those of planktonic SRB and found to be superior. The leaching of organic compounds, particularly phenolic substances, from the pine bark had a detrimental effect on the growth of the SRB. Different proportions of pine bark extract were used to prepare media to investigate this problem. Growth of SRB was totally inhibited when 100% pine bark extract was used. Analysis of these extracts showed the concentration of phenolics increased from 0.33 mg l-1 to 7.36 mg l-1 over the extraction interval of 15 min to 5 days. Digested samples of pine bark also showed the presence of heavy metals. The effects of nitrate, iron and sulphate and combinations thereof were investigated on the growth of a mixed culture of sulphate-reducing bacteria (SRB). The addition of 30 mg l-1 nitrate does not inhibit the production of sulphide by SRB when either 50 or 150 mg l-1 sulphate was present. The redox potential was decreased from 204 to -239 mV at the end of the 14 day batch experiment in the presence of 150 mg l-1 sulphate and 30 mg l-1 nitrate. The sulphate reduction activity of the SRB in the presence of 30 mg l-1 nitrate and 100 mg l-1 iron was about 42% of original sulphate, while if no iron was added, the reduction was only 34%. In the presence of 20 mg l-1 either As(III) or As(V), but particularly the former, growth of the SRB was inhibited when the cells were cultured in modified Postgate medium in the presence of 30 mg l-1 nitrate. The bioremoval of arsenic species [As(III) or As(V)] in the presence of mixed cultures of sulphate-reducing bacteria was investigated. During growth of a mixed SRB culture adapted to 0.1 mg l-1 arsenic species through repeated sub-culturing, 1 mg l-1 of either As(III) or As(V) was reduced to 0.3 and 0.13 mg l-1, respectively. Sorption experiments on the precipitate produced by batch cultured sulphate-reducing bacteria (SRB-PP) indicated a removal of about 77% and 55% of As(V) and As(III) respectively under the following conditions: pH 6.9; biomass (2 g l-1); 24 h contact time; initial arsenic concentration,1 mg l-1 of either species. These results were compared with synthetic iron sulphide as adsorbent. The adsorption data were fitted to Langmuir and Freundlich isotherms. Energy dispersive x-ray (EDX) analysis showed the SRB-PP contained elements such as sulphur, iron, calcium and phosphorus. Biosorption studies indicated that SRB cell pellets removed about 6.6% of the As(III) and 10.5% of the As(V) from water containing an initial concentration of 1 mg l-1 of either arsenic species after 24 h contact. Arsenic species were precipitated out of synthetic arsenic-contaminated groundwater by reacting it with the gaseous biogenic hydrogen sulphide generated during the growth of SRB. The percentage removal of arsenic species was dependent on the initial arsenic concentration present. Lastly, laboratory scale bioreactors were used to investigate the treatment of arsenic species contaminated synthetic groundwater. A mixed culture of SRB with molasses as a carbon source was immobilised on a polystyrene support matrix. The synthetic groundwater contained either As(III) or As(V) at concentrations of 20, 10, 5, 1 or 0.1 mg l-1 as well as 0.1 mg l-1 of a mixture with As(III) accounting for 20, 30, 40, 60 and 80% of the total. More that 90% and 60% of the As(V) and As(III) respectively were removed by the end of the 14-day experiment. At an initial concentration of 0.1 mg l-1 total arsenic had been reduced to below the WHO acceptable level of 10 ìg l-1 when the proportion of As(III) was 20 and 30%, while at 40% As(III) this level was reached only when the treatment time was increased to 21 days. The efficiency of As(III) removal was increased by first oxidising it to As(V) using MnO2.
The characterisation of turkey rhinotracheitis virus from chickens and the development of a suitable vaccine.
(1994) Maharaj, Sanjay Balkishore.; Da Graca, John Vincent.
Three turkey rhinotracheitis virus-like (TRTV-like) isolates were isolated from chickens with swollen heads. All grew well via the yolk sac (y/s) , chorioallantoic membrane (CAM), tracheal organ culture (TOC) , chicken embryo fibroblast (CEF) , and Vero cell routes. Affected embryos were stunted and severely congested. No pathological alterations were detected in allantoic sac (a/s) inoculated embryos. The CEF and Vero cells required trypsinisation for five consecutive passages before any visible cytopathic effects (CPE) were observed. Intra-cytoplasmic eosinophilic inclusions were observed in Vero and CEF monolayers . Only isolate 652/93 caused 100% ciliostasis in TOC. The other two isolates were able to cause a maximum of only 70-80% ciliostasis. The isolates were inactivated by chloroform treatment and exposure to 56°C for 1 h. Long term storage could be achieved at -70°C or in liquid nitrogen but not at 4°C or at -20°C. The isolates did not agglutinate chicken red blood cells and were found to contain genomes of RNA. They were able to elicit TRTV antibodies in specific pathogen free (SPF) birds as measured with the Pathasure enzyme linked immunosorbent assay (ELISA) kit. They could also be neutralised by TRTV-specific antisera. Electron microscopy of infective allantoic fluid (A/F) revealed particles of 100-300 nm in diameter with a helical nucleocapsid component approximately 15 nm in diameter and a fringe of approximately 12 nm long spikes. The processes of VLP development and maturation in TOC's and Vero cells were similar with accumulations of virus-like nucleoprotein close to the plasma membrane, forming the nucleocapsid. Virus-specified spikes were then inserted into the plasma membrane after which the VLP budded from the plasma membrane, incorporating this membrane with spikes as its own. Nine viral polypeptides with molecular weights of 200kDa, 83kDa, 53kDa, 40kDa, 37kDa, 28kDa, 19kDa and 15kDa were detected by SDS-PAGE in samples of the three isolates. The 83kDa and 53kDa polypeptides were also detected by western blotting using TRTV specific antisera. Both, a TRTV and a 652/93 isolate non-radioactive DNA probe, appeared specific for TRTV and TRTV-like isolates. The 652/93 probe detected 652/93 virus in SPF chickens for 19 days post-inoculation. A vaccine produced in SPF eggs using the attenuated 652/93 isolate, was able to protect vaccinates against virulent virus in laboratory challenge studies. In field trials, birds vaccinated at day-old or at day-old and again at 14 days, showed slightly improved performance compared to non-vaccinated birds. However, this benefit was not statistically significant. It is suggested that other environmental and disease factors mask the benefit provided by the vaccine in field trials. The three TRTV-like isolates appear to be chicken strains of TRTV and vaccination with an autogenous vaccine appears to afford some benefit to vaccinates.
Development of a new entomopathogenic nematode species, steinernema innovation : biological characterization and mass production.
(2014) Ramakuwela, Tshimangadzo.; Laing, Mark Delmege.; Hatting, Justin Louis.; Hazir, Selçuk.
Entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis are employed as environmental friendly biopesticides to control key pests in high value agricultural crops. Their successful application in developed countries has proved their effectiveness. The production of EPN for large scale commercial application has been restricted to developed countries because high capital cost of setting up liquid fermentation, and the high running costs of such units. If EPNs are to be commercialized in developing countries, then this will probably be achieved using in vivo culturing in insect larvae, or by using in vitro solid culture on a small to medium scale. The Republic of South Africa bans the importation of exotic EPN products by Amendment 18 of Act 36 of 1983. Therefore EPN products in South Africa will need to be developed from indigenous species. This study focused on optimizing the solid culture production and biological characterization of an indigenous nematode, Steinernema innovationi, as a biocontrol agent. A good production system must achieve high yield, maintain high virulence and the product must have a reasonable shelf-life. Optimum production temperature was determined using in vivo culturing of S. innovationi inside Galleria mellonella larvae. Performance was measured by quantifying the first day of emergence of infective juveniles (IJs) from cadavers, yield of IJs and IJ length, at five temperatures ranging from 18°C to 25°C. Optimum temperature was selected as 22°C based on high yield and slow emergence. There was a yield of 92,756 ± 28,089 IJs/larva, and 84,056 ± 27,832 IJs/larva, at 22°C and 25°C, respectively, which were not significant different. However, IJ emergence was significantly slower at 22°C, which provided more time for nutrient uptake and therefore these IJs had a greater nutrient reserve. There was no correlation observed between IJ length vs. temperature and IJ length vs. yield. A medium containing a puree of the larvae of the common house fly (Musca domestica) and 3% canola oil produced the highest yield of IJs (781,678 ± 221 IJs/5g), the highest level of live IJs (>84%) and the lowest level of adults (<10%) remaining in the medium at the time of harvest, compared to five other media formulations. All media were subjected to in vitro mass production at 22°C. A liquid inoculum of the EPN gave higher yields than a solid culture inoculum, irrespective of concentration. The length of IJ did not affect virulence against last instar larvae of G. mellonella, which was >90% in all experiments. A study characterizing optimum storage temperature of S. innovationi was carried out at five temperatures ranging from 5°C to 25°C in aqueous suspension over a period of 84 days. Survival was highest and most stable at 15°C, ranging from 84% to 88% after 84 days storage. Storage of the EPN in a sponge at a concentration of 2.5 million IJs in 15ml 0.1% formalin solution was successful, with an improvement of 6% compared to aqueous storage at 15°C. Furthermore, storage of the EPN in a sponge at 25°C, after a period of low temperature (15°C) storage for 84 days, did not have a detrimental impact on IJ survival and infectivity (87% and 95%, respectively). The new EPN (S. innovationi) was characterized by studying traits related to its infectivity (infectivity under a range of temperatures [10°C to 35°C], foraging behaviour & persistence under field conditions). Time till death was shortest at 25 and 30°C (average 1 day). The highest number of established IJs was recovered at 25°C (mean = 27°C). The nematode infected Galleria mellonella larvae at all depths and was capable of covering a distance of up to 15cm in 24 hours. There was no statistical difference between in vitro (40%) and in vivo (27%) cultured IJ persistence 4 weeks post application. Furthermore, there was no statistical difference in nematode recovery after 1 and 4 weeks post application of in vitro (33% & 40%, respectively) and in vivo (60% & 27%, respectively) produced IJs. Thermal activity was optimal at 25°C, the new species was classified as a cruiser and proved to survive under field conditions for at least 4 weeks. Susceptibility of larvae and/or pupae of Eldana saccharina (Walker), Sesamia calamistis (Hampson), Chilo partellus (Swinhoe), Tenebrio molitor (Linnaeus), Galleria mellonella (Linnaeus), Cydia pomonella (Linnaeus), Plutella xylostella (Linnaeus), and Gryllidae acheta (Linnaeus) representing three orders (Coleoptera, Lepidoptera & Orthoptera) was tested at a low and high concentration of 50 and 500 IJs, respectively. The hosts G. acheta, C. partellus and P. xylostella showed least susceptibility with maximum mortalities at the 500 IJs concentration of 28%, 45% and 92%, respectively. All other hosts suffered 100% mortality. Pupal mortality ranged from 47% to 68%. An LC50 and LC70 of 3 IJs/larva and 31 IJs/larva, respectively, was calculated for the black cutworm, Agrotis ipsilon (Hufnagel). These results provided a broad guideline on the relative pathogenicity of this new species against different hosts. A cost analysis was calculated for in vitro solid culture of the EPN, including the cost of rearing an insect-based nutrient component. An estimated retail price was then compared to the costs of market products around the globe. An estimated retail price (R90.61) for S. innovationi was considerably lower than the market price for other Steinernema species, which ranged from R271.50 to R458.55, in South African rands. The production system developed in this study for S. innovationi offers a highly competitive small to medium scale production method to produce EPN products without having to invest in large scale liquid fermentation equipment, by using relatively cheap production media ingredients, and simple solid culture growing conditions.
The development of transgenic sweet potato (Ipomoea batatas L.) with broad virus resistance in South Africa.
(2013) Sivparsad, Benice.; Gubba, Augustine.
Sweet potato (Ipomoea batatas Lam.) is ranked as the seventh most important food crop in the world and its large biomass and nutrient production give it a unique role in famine relief. However, multiple virus infection is the main disease limiting factor in sweet potato production worldwide. The main objective of this research project was to develop a transgenic sweet potato cultivar with broad virus resistance in South Africa (SA). A review of current literature assembled background information pertaining to the origin, distribution and importance of the sweet potato crop; viruses and complexes infecting sweet potato; and the strategies used in sweet potato virus detection and control. A survey to determine the occurrence and distribution of viruses infecting sweet potato (Ipomoea batatas Lam.) was conducted in major sweet potato-growing areas in KwaZulu-Natal (KZN). A total of 84 symptomatic vine samples were collected and graft inoculated onto universal indicator plants, Ipomoea setosa Ker. and Ipomoea nil Lam. Six weeks post inoculation, typical sweet potato virus-like symptoms of chlorotic flecking, severe leaf deformation, stunting, chlorotic mosaic, and distinct interveinal chlorotic patterns were observed on indicator plants. Under the transmission electron microscope (TEM), negatively stained preparations of crude leaf sap and ultra-thin sections from symptomatic grafted I.setosa plants revealed the presence of elongated flexuous particles and pinwheel type inclusions bodies‟ that are characteristic to the cytopathology of Potyviruses. Symptomatic leaf samples from graft-inoculated I. setosa and I. nil were assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato virus G (SPVG), Sweet potato mild speckling virus (SPMSV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato latent virus (SPLV), Cucumber mosaic virus (CMV), and Sweet potato C-6 virus (C-6) using the nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). The majority of leaf samples (52%) tested positive for virus disease and showed the occurrence of SPFMV, SPMMV, SPCSV, SPCFV, SPVG, SPMSV, and SPCaLV. Of these 7 viruses, the most frequently detected were SPFMV (39%), SPVG (30%), followed by SPCSV (13%) and SPMMV (12%). SPCaLV and SPCFV at 10% and SPMSV at 7% were found exclusively in samples collected from one area. SPFMV, SPVG, SPCSV, and SPMMV were identified as the most prevalent viruses infecting sweet potato in KZN. The genetic variability of the three major viruses infecting sweet potato (Ipomoea batatas Lam.) in KZN was determined in this study. A total of 16 virus isolates originating from three different locations (Umbumbulu, Umfume and Umphambanyomi River) in KZN were analyzed. These comprised of 10 isolates of Sweet potato feathery mottle virus (SPFMV), five isolates of Sweet potato virus G (SPVG) and one isolate of Sweet potato chlorotic stunt virus (SPCSV). The phylogenetic relationships of the SPFMV, SPVG and SPCSV isolates from KZN relative to isolates occurring in SA and different parts of the world were assessed. The division of SPFMV into four genetic groups (strains) according to the phylogenetic analysis of coat protein encoding sequences revealed mixed infections of the O (ordinary) and C (common) strains in sweet potato crops from KZN. All SPFMV isolates showed close lineage with isolates from South America, East Asia and Africa. The SPVG isolates showed high relatedness to each other and close lineage with other isolates, especially those from China and Egypt. Analysis of the partial sequence of the Heat shock protein 70 homologue (Hsp70h) gene indicated that the SPCSV isolate from KZN belongs to the West African (WA) strain group of SPCSV and showed close relatedness to an isolate from Argentina. The knowledge of specific viral diversity is essential in developing effective control measures against sweet potato viruses in KZN. Multiple virus infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KZN. In order to address the problem of the multiplicity and synergism of sweet potato viruses in KZN, this study aimed to develop transgenic sweet potato cv. Blesbok with broad virus resistance. An efficient and reproducible plant regeneration protocol for sweet potato (Ipomoea batatas Lam.) cultivar Blesbok was also developed in this study. The effect of different hormone combinations and type of explants on shoot regeneration was evaluated in order to optimize the regeneration protocol. Coat protein (CP) gene segments of SPFMV, SPCSV, SPVG and SPMMV were fused to a silencer DNA, the middle half of the nucleocapsid (N) gene of Tomato spotted wilt virus (TSWV) and used as a chimeric transgene in a sense orientation to induce gene silencing in the transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring a modified binary vector pGA482G carrying the plant expressible neomycin phosphotransferase ll gene (nptll), the bacterial gentamycin-(3)-N-acetyl-transferase gene and the expression cassette. A total of 24 putative transgenic plants were produced from the transformed apical tips via de novo organogenesis and regeneration into plants under 50mg/L kanamycin and 200 mg/L carbenicillin selection. Polymerase chain reaction (PCR) and Southern blot analyses showed that six of the 24 putative transgenic plants were transgenic with two insertion loci and that all plants were derived from the same transgenic event. The six transgenic sweet potato plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV- infected Ipomoea setosa Ker. Although virus presence was detected using NCM-ELISA, all transgenic plants displayed delayed and milder symptoms, of chlorosis and mottle of lower leaves when compared to the untransformed control plants. These results warrant further investigation under field conditions.
The effects of Trichoderma (Eco-T) on biotic and abiotic interactions in hydroponic systems.
(2003) Neumann, Brendon John.; Laing, Mark Delmege.; Caldwell, Patricia May.
The following body of research provides a detailed overview of the interactive effects of biocontrol agents and environmental factors and how these influence both the host plant and pathogen populations within hydroponic systems. Pythium and other zoosporic fungi are pathogens well suited to the aquatic environment of hydroponics. Motile zoospores facilitate rapid dispersal through fertigation water, resulting in Pythium becoming a yield reducing factor in most hydroponic systems and on most crops. With increasing trends away from pesticide use, biocontrol is becoming an ever more popular option. Unfortunately, much of our knowledge of biocontrol agents and their formulation can not be directly transferred to the widely differing environments of hydroponic systems. Paulitz (1997) was of the opinion that if biocontrol was to be successful anywhere, it would be in hydroponics. This is primarily due to the increased ability, in hydroponics, to control the growing environment and to differentiate between the requirements of the pathogen versus those of the host plant and biocontrol agent. Key environmental factors were identified as soil moisture, root zone temperature, form of nitrogen and pH. A review of the literature collated background information on the effects of biocontrol agents and environmental manipulation on plant growth and disease severity in hydroponic systems. A commercial formulation of Trichoderma (Eco-T(R1)) was used as the biocontrol agent in all trials. Dose responses in Pythium control and plant growth stimulation in lettuce were first determined using a horizontal trough system (closed system). In such systems optimum application rates were found to be lower than in field application (1.25x10[to the power of 5] spores/ml). This is probably because Trichoderma conidia are not lost from the system, but re-circulate until being transported into the root zone of a host plant. No significant growth stimulation was observed, although at high doses (5x10[to the power of 5] and 2.5x10[to the power of 5] spores/ml) a significant reduction in yield was recorded. Possible reasons for this growth inhibition are suggested and a new theory is proposed and investigated later in the thesis. In an open system of cucumber production (drip irrigated bag culture) no statistically significant results were initially obtained, however, general trends still showed the occurrence of positive biocontrol activity. The initial lack of significant results was mostly due to a poor knowledge of the horticulture of the crop and a lack of understanding of the epidemiology behind Trichoderma biocontrol activity. These pitfalls are highlighted and, in a repeat trial, were overcome. As a result it could be concluded that application rates in such systems are similar to those used in field applications. Management of soil moisture within artificial growing media can aid in the control of Pythium induced reductions in yield. A vertical hydroponic system was used to determine the interactive effects of soil moisture and Trichoderma. This system was used because it allowed for separate irrigation regimes at all 36 stations, controlled by a programmable logic controller (PLC). With lettuce plants receiving optimum irrigation levels, no significant reduction in yield was observed when inoculated with Pythium. However, after Pythium inoculation, stresses related to over- or under-watering caused significant yield losses. In both cases, Trichoderma overcame these negative effects and achieved significant levels of disease control, especially under higher soil moisture levels. Growth stimulation responses were also seen to increase with increasing soil moisture. Similar results were obtained from strawberry trials. These results show that Pythium control is best achieved through the integration of Trichoderma at optimum soil moisture. However, where soil moisture is above or below optimum, Trichoderma serves to minimize the negative effects of Pythium, providing a buffering capacity against the effects of poor soil moisture management. Pythium, root zone temperature and form of nitrogen interact significantly. In greenhouse trials using horizontal mini troughs with facilities for heating or cooling recirculating water, nitrate fertilizer treatments resulted in statistically significant results. Lettuce growth was highest at 12°C, although no significant differences in yield were observed between 12-24°C. Pythium was effective in causing disease over the same temperature range. Pythium inoculation did not result in yield reduction at 6 and 30°C. Trichoderma showed a slight competitive advantage under cooler temperatures (i.e., 12 degrees C), although significant biocontrol occurred over the 12-24 degrees C range. Ammonium fertilizer trials did not generate statistically significant data. This is possibly due to complex interactions between root temperature, ammonium uptake, and competitive exclusion of nitrification bacteria by Trichoderma. These interactions are difficult to replicate over time and are probably influenced by air temperature and available light which are difficult to keep constant over time in the system used. However, the data did lead to the first clues regarding the effects of Trichoderma on nitrogen cycling as plants grown with a high level of ammonium at high temperatures were seen to suffer more from ammonium toxicity when high levels of Trichoderma were added. In further trials, conducted in the recirculating horizontal mini trough system, it was determined that Trichoderma applications resulted in an increase in the percentage ammonium nitrogen in both the re-circulating solution and the growing medium. This was a dose-related response, with the percentage ammonium nitrogen increasing with increasing levels of Trichoderma application. At the same time an increase in ammonium in the root tissue was observed, corresponding with a decrease in leaf nitrate levels and an increase in levels of Cu, Na, Fe and P in leaf tissue. In independent pot trials, populations of nitrifying bacteria in the rhizosphere were also seen to decrease with increasing Trichoderma application rates. This led to the conclusion that the increase in ammonium concentration was as a result of decreased nitrification activity due to the competitive exclusion of nitrifying bacteria by Trichoderma. The possibility that Trichoderma functions as a mycorrhizal fungus and so increases the availability of ammonium for plant uptake is not discarded and it is thought that both mechanisms probably contribute. Water pH provides the most powerful tool for enhancing biocontrol of Pythium by Trichoderma. Trichoderma shows a preference for more acidic pHs while Pythium prefers pHs between 6.0 and 7.0. In vitro tests showed that Trichoderma achieved greater control of Pythium at pH 5.0, while achieving no control at pH 8.0. In greenhouse trials with the recirculating horizontal mini trough system, yield losses resulting from Pythium inoculation were greatest at pH 6.0 and 7.0, with no significant reduction in yield at pH 4.0. Biocontrol activity showed an inverse response with greatest biocontrol at pH 5.0.
An epidemiological analysis of the Phytophthora and Alternaria blight pathosystem in the Natal Midlands.
(1980) Putter, Christoffel Antonie Johannes.; Martin, Michael Menne.; Rijkenberg, Fredericus Hermanus Johannes.
The history of the development in Natal of a forecasting service to warn of outbreaks of late blight disease caused by Phytophthora infestans is presented. The late blight pathogen and Alternaria solani, the causal organism of early blight disease, interact on potatoes and tomatoes to form a blight disease complex. Evidence is presented to show that it is expedient to manage this blight complex as a whole rather than to direct control at only one of the components in ignorance of the consequential enhancement of the potential of the other. In a search for an improved blight complex management strategy, factors concerning the possible existence of an annual migration of Phytophthora infestans inoculum, first postulated in the 1960's, along an east-west route across Natal, are collected and collated. Corroboration of the existence of the Phytophthora-pathway is given, inasmuch as it represents a serial outbreak of late blight along a temporal gradient. The possibility that the pathway is a manifestation of disease resulting from the erruption of pre-existing inoculum along an environmental gradient, can not specifically be excluded. However, the peculiar pattern of anabatic and katabatic winds along a river-valley network, superimposed on a continuous cropping pattern and its concomitant opportunity for blight to be endemic in the province, supports the postulated Phytophthora-inoculum pathway A fungicide spray trial was conducted in order to investigate the possibility of us i ng the pathway phenomenon as the framework for an improved blight control strategy and to explore the nature and level of the competitive interaction between Phytophthora infestans and Alternaria solani. This trial revealed that the interaction between the components of the blight complex was differentially altered by weather patterns and fungicide combinations. Treatments in which metalaxyl (Ridomil) alone was used for the control of late blight, gave a yield similar to those with propineb (Antracol), which inhibits A. solani primarily but also hus some negative effect on P. infestans. The yields from both these treatments were siguificant ly (p < 0,05) better than the yields recorded in the unsprayed control plots. A treatment in which Ridomil and Antracol were combined such that each was applied according to its recommended concentration, gave yield increases of 32,3% over the unsprayed control, although the yield from the Ridomil/Antracol treatment was not significantly greater (p < 0,05) than the yields recorded where either Ridomil or Antracol were used. A computer simulator, named GAUSE, was developed to simUlate the consequences of the competition between various combinations of P. infestans and A. solani. Results simulated by GAUSE corroborated those obtained from the field trial and support the conclusion that diseases of complex etiology require more than simplistic, univariate analysis of single cause-and-effect pathways. The competition quotient CQ is developed as a new parameter of competitive interactions. It is calculated as the ratio of the amount of disease in the absence of competition, to the amount of disease when the causal pathogen is competing with another pathogen in the same niche. The CQ may be calculated from various standard epidemiology statistics and it is used to demonstrate that the competitive displacement phenomenon places constraints on the interpretation and application of Vanderplank's basic epidemiology equations. A new pathosystems management concept namely the pathotope (pathos = suffering; topos = place0 concept, is introduced, having developed from the notion that epidemics have spatial as well as temporal attributes. Accordingly, an area in which individual farms are at the same level of probability at risk to disease, delimits the pathotope. The concept can be described at many integration lsvels and is presented as an important quantitative unit of comparative epidemiology. The pathotope concept accomodates such notions as are contained in the postulated Phytopnthora-pathway and is especially suited to integration with disease forecasting methods. An example of the application of the pathotope approach is presented and a strategy is proposed by which fungicide spraying is initiated and applied synchronously as determined by the degree of communal risk to attack and epidemic increase of disease. Within a pathotope, several common factors collectively determine the vulnerability of the group to disease. If a coherent, uniform strategy is to be developed and implemented by pathotope members, it is necessary that all members have access to the relevant information and that it be collected and disseminated conveniently and rapidly. A computer-based disease monitoring and mapping system which achieves these objectives is presented.
The epidemiology and control of Leptosphaeria maculans cause of Crucifer Blackleg, in KwaZulu-Natal.
(1996) Laing, Mark Delmege.; Putter, Christoffel Antonie Johannes.; Rijkenberg, Fredericus Hermanus Johannes.
The perfect stage of Leptosphaeria maculans is reported for the first time in South Africa. Viable pseudothecia and pycnidia were found on dead, weathered tissue, sometimes in close association, whereas only pycnidia were found on live tissue. Some seedlots of imported cabbage seed were found to be internally infected with L. maculans at low levels and Alternaria brassicicola at higher levels. Fungicides iprodione (dicarboximide), triforine and propiconazole (sterol-biosynthesis inhibitor) eliminated both pathogens from infected seed. In a field trial of eight cabbage and two cauliflower cultivars, incidence of stem infection by L. maculans ranged from 16-80%. Two seedlots of the cabbage cultivar Gloria Osena differed in blackleg stem susceptibility. No correlation was found between stem lesion incidence and foliar infection counts of each cultivar, or stem lesion incidence and each cultivar's average days-to-harvest. In a second trial, incidence of stem infection ranged from 50% (Rotan) to 95% (Dynasty) in cabbage, and 64.2 to 96.6% in cauliflower cultivars. All Brussels sprouts and broccoli cultivars tested were highly susceptible. The cultivars of turnip and tyfon tested were observed to be immune to blackleg, whereas the swedes, Japanese radish, chou moullier and red cabbage cultivars tested were highly susceptible. No correlation was found between stem length and incidence of stem infection. Different seedlots within several cabbage and cauliflower cultivars differed in their blackleg susceptibility. A third cultivar trial with 10 replicates of four seedlots of one cabbage cultivar confirmed that different seedlots of a single cultivar may vary significantly in their susceptibility to blackleg. Benomyl was applied to cabbage at the seedling stage only, or at the seedling stage followed by field applications every 14 d. Relative to an untreated control, multiple applications of benomyl resulted in a 33% reduction in stem infection, a ten-fold reduction in plants killed and a 50% reduction in the proportion of non-harvestable heads, relative to an untreated control. Seedling treatment resulted in a lower infection level, a lower mortality rate and a greater mean head mass than those of the untreated control. However, none of these differences were statistically significant. In a debris degradation trial, more than 90% of buried debris (cabbage stems infected by L. maculans) had decomposed after 2.5 yr, whereas 80% of surface debris had decomposed over the same period. The susceptibilities of seedbed transplants (SBT) and container-grown seedlings (CGS) were compared using different forms of L. maculans inoculum. "Dunk" inoculation of SBT into a pycnidiosporial suspension resulted in a stem infection level of 50% greater than an uninocu1ated control. Contamination of seedbeds resulted in an infection level of 46%. "Dunk" inoculation of CGS resulted in infection level of 22%. When CGS were grown in contaminated trays an infection level of 33.4% resulted. Interplot interference ill the form of inoculum dispersal over a 1 m border was low (1.8 and 2.7% for SBT and CGS, respectively) . In a further trial examining the relationship of inoculum level and blackleg, a strong interaction was found between inoculation technique and inoculum level. Inoculation of field plots with infected debris was a more efficient technique than dipping seedlings into a pycnidiospore suspension prior to transplanting. Twenty nine blackleg epidemics were surveyed over 11 yr. Seedbed transplants (SBT) had been used in 83% of cases. Two cases (7%) had involved direct drilled seedlings (DDS). However, excess seedlings had been transplanted, making DDS epidemiologically equivalent to SBT. Three cases (10%) had involved container-grown seedlings (CGS) grown on mono cropped cabbage lands. Disease occurred in two patterns: in crops grown from SBT and DDS, blackleg occurred down the lines. In all CGS cases, disease occurrence was randomly patterned. In all cases, diseased debris was found in seedbeds and production fields. Disease spread in the field was limited to the two plants on either side of the initially infected plant, 1.3 m or less, suggesting that infection had resulted from splash dispersed pycnidiospores. The disease cycle was mono- or oligo cyclic but not polycyclic. Over a period of 6 yr, cabbage fields of 26 farms were each examined once for cruciferous weeds infected with L. maculans. No viable blackleg lesions were discovered on cruciferous weeds, suggesting that weeds play no role in the local crucifer blackleg pathosystem. A theory is proposed that windows of disease susceptibility open and shut during the different phenological stages of a crucifer's life, and that the susceptibility of different plant organs vary with the phenological state of the plant. It is also postulated that blackleg is a "low sugar disease". Disease incidence was lower in well fertilized cabbage plants than minimally fertilized plants. Organoleptic tests of cabbage cultivars correlated superior flavour and texture in cabbage with a high susceptibility to blackleg. An integrated management strategy is proposed, based on seed treatment with fungicides, the use of container-grown seedlings rather than seedbed transplants, a 3 yr rotation of crucifer lands with non-cruciferous crops, implementation of either deep-ploughing or accelerated biodegradation to eliminate debris, the development of higher levels of horizontal resistance to L. maculans in cruciferous vegetables, application of field fungicides in high risk areas (benzimidazoles or triazoles, or combinations), and the minimization of stress and optimization of host nutrition.
Epidemiology and management of grey leaf spot : a new disease of maize in South Africa.
(1996) Ward, John Michael Julian.; Cairns, Andrew Lawrence Patrick.; Rijkenberg, Fredericus Hermanus Johannes.; Laing, Mark Delmege.
Grey leaf spot is a relatively new fungal disease of maize in South Africa. It has become well established in the province of KwaZulu-Natal, and is capable of reducing grain yields by 20 to 60%. The disease is spreading to neighbouring provinces and countries. This study was conducted to establish solutions to the problem that could be easily implemented by maize farmers. Available literature was reviewed to establish the most appropriate epidemiologically based control measures that might be applicable in South Africa. Field trials were conducted to determine the effects of stubble and conventional tillage practices, cultivar susceptibility, fungicides, the correct time and frequency of fungicide treatment. and the financial benefits of fungicide treatment on grey leaf spot severity. The trials were evaluated for disease severity and grain yields. No commercial hybrids were identified to be resistant to grey leaf spot in the maize hybrid response to grey leaf spot trial. However, subsets of high-yielding hybrids less-susceptible to disease were identified - including PAN 6480, CRN 3584, SNK 2154 and PAN 6578. The most susceptible hybrids were identified to include RS 5206, PAN 6552, A 1849, PAN 6528 and PAN 6140. Fungicides containing carbendazim/flusilazole, were found to be most effective in controlling disease and increased maize yields. Hybrids such as RS 5206 and RS 5232 highly susceptible to disease and showed the highest grain yield response to fungicide treatment, whilst least-susceptible hybrids, such as PAN 6480, had the lowest response. The tillage trial aimed at management practices to reduce grey leaf spot indicated fungicides to be more effective in managing disease than tillage practices aimed at a reduction of initial inoculum. Trials on chemical control of grey leaf spot identified fungicides of the triazole and benzimidazole chemical groups to be effective in controlling disease, but only combination products of these chemical groups, were registered, in support of the pathogen resistance strategy. Products registered were carbendazim/flusilazole, carbendazim/flutriafol and carbendazim/difenoconazole. The frequency and timing of fungicide applications for the control of grey leaf spot in maize studies identified spray treatments initiated when disease had progressed to the basal five leaves and, before the exponential phase of the epidemic, provided the most effective disease control and concomitant high grain yields. Further spray treatments were necessary with early disease infections, in order to provide disease control until crop physiological maturity. The final study on the economic benefits of fungicide treatment of grey leaf spot in maize in KwaZulu-Natal indicated that the highest added yield response was not necessarily the best parameter to justify fungicide treatment. Rather, the expected added profit was a better parameter. In this study the highest added profits were R1 400 ha(-1) from the triple-spray programme in 1993/94 and R439 ha(-1) from a single-spray in 1992/93. The optimum treatment choice depended on the individual's risk-return preferences, which reflect his level of risk-aversion. An integrated approach using tillage practices, crop rotations, hybrids less- susceptible to the pathogen and the judicious use of fungicides is likely to be the most successful in controlling the disease. In the long term, the cornerstone of the integrated approach will be the development and use of hybrids resistant to the disease.
Ethnopharmacological study on plants used for skincare and beauty by some Xhosa communities.
(2018) Thibane, Vuyisile Samuel.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
The paraphrase “beauty lies in the eyes of the beholder” by Plato, a Greek philosopher, has echoed through the fabrics of time and still echoes in our generation. The statement simply ought to refer to that the observer gets to decide what is “beautiful” in their eyes. The decision on what and who is beautiful is heavily influenced by one’s surroundings. In a digital-market environment the concept of beauty enhancement is paraded by bigger forces to drive their own agenda. The desire to enhance one’s facial appearance has significantly contributed to the observed growth of the beauty industry. The notable growth of the industry has in some cases resulted in unpleasant consequences due to the side effects of some of the products in the market. In this global-blooming industry products are traded and exchanged at a rapid rate. Introduction of safer natural beauty enhancement products are required for the South African market if we were to alleviate those with undesirable side effects and could combat some socio-economic challenges through job creation. The use of plants as the source of natural compounds has proven to be a reliable strategy in ethnopharmacological applications. The Eastern Cape Province has a rich plant biodiversity and the communities have immense indigenous knowledge (IK) on the use of these plants. There is a need to explore the pharmacological application of these plants by introducing beauty enhancement product formulations made from local resources. The project was aimed at documenting and conductingethnopharmacological evaluation of plants used for skincare and beauty for their potential in formulation of beauty enhancement products. An ethnobotanical survey was conducted to document plants that are used by communities in the Raymond Mhlaba municipality for skincare and beauty. Knowledge holders were identified by purpose sampling method and the interviews were conducted in isiXhosa using a structured questionnaire. Information on demographics, names of the plant, type of plant, plant part used, method of preparation and administration and frequency of use was collected and captured in the questionnaires. The Asphodelaceae and Asteraceae were the most represented families of the plants used for skincare and beauty. The communities used sustainable harvesting practices as the leaves were the most utilized plant parts. The most reported beauty enhancement uses were for achieving desired skin complexion and for skin smoothness, with both accounting up to 50% of the reported plant usages. Sixteen plants with the highest frequency index (FI) were selected from the ethnobotanical survey for ethnopharmacological studies related to beauty enhancement. This included Acokanthera oblongifolia (Hochst.) Codd, Aloe ferox Mill, Arctotis arctotoides (L.f) O.Hoffm, Bulbine frutescens (L.) Willd, Cassipourea flanaganii (Schinz) Alston, Chenopodium album L, Clausena anisata (Willd.) Hook.f ex Benth, Haemanthus albiflos Jacq, Marrubium vulgare L, Ilex mitis (L.) Radlk, Plantago lanceolata L, Rorippa nasturtium-aquaticum (L.) Hayek, Sonchus asper L, Symphytum officinale L, Ruta graveolens L and Urtica urens L. The antimicrobial activity of plant extracts was assessed using the microdilution bioassay to determine the minimum inhibitory concentrations (MIC). The antimicrobial activity of petroleum ether (PE), dichloromethane (DCM), 70% aqueous ethanol (v/v) and water extracts of the selected plants were assessed against infectious skin microorganisms including Bacillus subtilis ATCC 6051, Brevibacillus agric ATCC 51663, Staphylococcus aureus ATCC 12600, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 11775, Klebsiella pneumoniae ATCC 13883, Candida albicans ATCC 10231 and the dermatophytes Microsporum canis ATCC 36299, Trichophyton mentagrophytes ATCC 9533 and Trichophyton tonsurans ATCC 28942. The majority of the tested plant extracts were effective and inhibited the skin commensal bacteria E. coli with MIC values less than 100 μg/mL. Prolonged infections by commensal bacteria can condition the skin environment and provide favourable conditions for more opportunistic bacteria such as the Staphylococci genus. Ethanol extracts of C. flanaganii and U. urens expressed high antibacterial activity against S. aureus with MIC values less than 100 μg/mL. Ethanol extracts of R. graveolens and dichloromethane extracts of A. arctotoides were effective at inhibiting S. epidermidis and S. aureus, respectively. Inhibition of two opportunistic bacteria has a positive effect on skin tone, due to the scarring and darkening associated with infection by the Staphylococci genus. There was notable activity recorded against C. albicans and dermatophytes M. canis, T. mentagrophytes and T. tonsurans by extracts of A. oblongifolia, A. arctotoides, C. flanaganii, I. mitis and R. graveolens at different polarities with MIC’s less than 1000 μg/mL. The phenolic content and antioxidant activity of the plants were determined by assessing 50% aqueous methanol extracts (v/v) for their total phenolic and flavonoid content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) and the coupled oxidation of linoleic acid and bleaching of β-carotene. The antioxidant mechanism of phenolic compounds associated with beauty enhancement has been proposed to be due to their free radical chain breaking capabilities, metal chelation, oxidant quenching and inhibition of enzymatic activity. The total phenolic content of A. ferox, I. mitis and C. flanaganii were significantly high with recorded values ranging from 37.87 to 50.34 mgGAE/g. The flavonoid content of C. flanaganii, A. oblongifolia and P. lanceolata were significantly high. Methanol extracts of R. graveolens and C. flanaganii expressed the highest antioxidant activity, with IC50 values comparable to the standard antioxidant when assessed for their DPPH radical scavenging activity. The presence of antioxidants in the skin structural layers has a positive effect on the health and function of the skin. Extracts of U. urens, A. ferox, C. flanaganii, B. frutescens, P. lanceolata, H. albiflos, M. vulgare, C. anisata, S. officinale and R. nasturtium-aquaticum expressed good metal chelating potential. The highest oxidative protection in the β-carotene linoleic acid model with comparative oxidation rate ratio (ORR) to the positive control was observed for C. flanaganii, S. officinale and U. urens. The results indicate the ability of the plant extracts to provide protection against increased levels of lipid peroxidation in the skin, an important factor in beauty enhancement, due to delaying the age process. The photo-protective effect of the plant extracts was measured by calculating the sun protection factor (SPF). The SPF is the ratio of ultraviolet (UV) radiation required to produce minimal erythema dose (MED) in protected skin to unprotected skin with higher values indicative of increased protection from photo damage. Ethanol extracts of P. lanceolata, C. flanaganii, A. oblongifolia, I. mitis and A. arctotoides exhibited SPF values of more than 15, which translated to photo protection of the skin against UVB radiation by more than 93.3%. The plant extracts demonstrated the highest absorbance of UVB radiation at a wavelength region between 300 – 305 nm. These will protect the skin against UV-induced oxidative damage and enhance the skin’s health and function. The inhibition of enzymes with beauty enhancement potential by the plant extracts was assessed against tyrosinase, secretory phospholipase A2 (sPLA2), lipoxygenase (15-LOX) and cyclooxygenase (COX-1 and COX-2). Ethanol extracts of R. nasturtium-aquaticum, C. anisata, S. officinale and C. flanaganii expressed good anti-tyrosinase activity. The coupled protection against UV-induced damage and modulation of the tyrosinase enzyme activity can be exploited to achieve the desired skin complexion. The anti-inflammatory studies revealed the potential of extracts of C. flanaganii, P. lanceolata and R. nasturtium-aquaticum to serve as dual inhibitors of 15-LOX and COX-2 enzymes. The inhibition of 15-LOX and COX-2 is effective at resolving psoriasis, a skin-inflammatory associated disease which has a negative effect on the health and beauty of the skin. The effectiveness of ethanol extracts of C. flanaganii, C. album, C. anisata and R. nasturtium-aquaticum in maintaining the cells health and function was examined on human epidermal melanocytes (HEM) cell lines. Ethanol extracts of C. flanaganii, C. album, C. anisata and R. nasturtium-aquaticum were able to inhibit cellular tyrosinase activity and therefore reduce melanin production. The effective concentrations of the extracts were further reported as non-toxic to melanocytes. The observed anti-tyrosinase activity of the extracts against HEM cell lines contribute positively in achieving the desired skin complexion while providing photo protection against UV-induced damage. Therefore, plant extracts that are efficient and safe to use can be incorporated into formulations intended for beauty enhancement and further analysed under clinical trials. The study further suggests that the model undertaken be promoted to individuals and corporations interested in formulation of cosmeceuticals to ensure the safety and efficiency of their products.
Evaluation of diazotrophic bacteria as biofertilizers.
(2013) Kifle, Medhin Hadish.; Laing, Mark Delmege.;
Inoculation with diazotrophic bacteria is well documented as a means to enhance growth and increase yields of various crops, especially when used as an alternative or a supplement to the use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. Nitrogen is the most limiting nutrient for increasing crop productivity, and the use of chemical sources of N fertilizers is expensive, and may contribute to environmental pollution. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. The search for effective diazotrophic bacterial strains for formulation as biofertilizers has been going on for over 40 years and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 195 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within the KwaZulu-Natal Province, Republic of South Africa. Ninety five of the isolates were selected for further screening because they were able to grow on N-free media using different carbon sources. Isolates that were very slow to grow on N-free media were discarded. Of these, 95 isolates were screened in vitro for growth promotion traits tests including tests for ammonia production and acetylene reduction. The best 20 isolates that were also able to reduce acetylene into ethylene were selected for growth-promotion trials on maize under greenhouse conditions. Of the 20 isolates, ten isolates enhanced (P = 0.001) growth of maize above the Un-inoculated Control. Molecular tests were conducted to identify the ten most promising isolates selected in the in vitro study. In the greenhouse study, these diazotrophic isolates were screened for their ability to enhance various growth parameters of maize (Zea mays L.), following various inoculation techniques (drenching, seed treatment, foliar spray and combination of these). Inoculations with the five best diazotrophic isolates by various methods of application increased dry weight and leaf chlorophyll content (P < 0.001, P = 0.001), respectively, compared to the Untreated Control. Although, all methods of application of diazotrophic inoculants used in this study resulted in increased dry weight and leaf chlorophyll content, combined methods of application (seed treatment + drenching) and sole application (seed treatment) were significantly more (P < 0.05) efficient. The best five most promising isolates were identified for growth promotion of maize under greenhouse conditions. They were also assessed for their effects on germination of wheat in vitro and were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of wheat (Triticum aestivum L.). These five isolates were also investigated for their potential to enhance growth and yields of maize and wheat crops in field trials, when combined with a low dose of nitrogenous fertilizer. These isolates were further studied for their contribution for enhancing plant growth through nitrogen fixation by predicting N content in leaves using a chlorophyll content meter (CCM-200) and correlated to extractable chlorophyll level at R2 = 0.96. In this study, relative to the Un-inoculated Control, the best five isolates enhanced growth of maize and wheat when combined with a 33% N-fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry weight of maize and wheat; and enhanced root and shoot development of these crops in both greenhouse and field conditions. The best contributions of diazotrophic bacteria was achieved by Isolate LB5 + 0% NPK (41%), V9 + 65% NPK (28.9%), Isolate L1 + 50% NPK (25%), Isolate L1 + 25%NPK (22%) and LB5 + 75% NPK (15%) undergreenhouse conditions. At 30 or 60 DAP, isolates with 33%N-fertilizer caused relatively higher dry weight than the 100%NPK. Inoculation of Isolate StB5 without 33N% fertilizer cuased significant (P<0.005) increases in stover dry weight. In field studies, inoculation of diazotrophic bacteria alone or with 33%N-fertilizer resulted in relatively greater increases of dry weight, stover dry weight, number of spikes and yield at different growth stages higher than the Un-inoculated or Unfertilized Control. However, the increases were not statistically significant. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising yields. The isolates obtained in this study can effectively fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture.
Evaluation of the molluscicidal activity of bacillus spp. isolates to control aquatic intermediate host snails of liver fluke (fasciola spp.).
(2024) Van Wyngaard, Matthew George Dennis.; Laing, Mark Delmege.; Hunter , Charles.
Aquatic snails are involved in harmful disease cycles of Fasciola (liver fluke) that affect both humans and livestock in agriculture. The focus of this study was to isolate and identify candidate bacterial isolates antagonistic to aquatic snails, with the ultimate goal of controlling the host snails responsible for the transmission of liver flukes in South Africa. A bacterial antagonist would offer a novel means of snail population control and reduce the dependence on chemicals, and the growing risk of resistance. Due to their molluscicidal capabilities reported in the literature, and the benefits of being endospore-formers, strains within the family Bacillaceae were targeted as candidate biocontrol agents. A population of the freshwater snail Physella acuta (Draparnaud, 1805) was established and used for screening bacterial candidates as an easily-reared, proxy species for the Fasciola spp. intermediate host snails. Aerobic endospore-forming bacteria were isolated from aquatic soil collected primarily in the KwaZulu-Natal province, South Africa, utilising several approaches, including a general endospore heat-shock isolation method and two Bacillus thuringiensis (Bt) (Berliner, 1915) specific isolation methods. Initial screening for molluscicidal activity of 1180 isolates did not yield any strong performers; however, a subset of 124 isolates demonstrated some evidence of potential activity in preliminary screening, that in the absence of any strong molluscicidal isolates, warranted a more in-depth investigation. Subsequently, when the bioassays were repeated none of the 124 isolates showed strong molluscicidal activity; however, the eight isolates causing the highest mortality rates (16.7 – 50%) were selected for further analysis to determine whether these isolates exhibited weak molluscicidal activity. When spent culture supernatant was evaluated in molluscicidal bioassays 12 of the 124 isolates demonstrated strong molluscicidal activity. Selected isolates underwent identification and characterisation using DNA-based methods for the purposes of species identification and de-duplication of isolates on the basis of sequencing data and end point PCR. Isolates were identified using the benchmark technique of 16S rDNA gene fragment sequencing. For discrimination of closely related isolates, sequence analysis of two additional housekeeping genes was performed using rpoB and dnaJ. In addition, a number of end point PCRs were used to support sequence data by the presence or absence of PCR products for the lipopeptide marker genes for surfactin, fengycin, bacillomycin and iturin, as well as a Bacillus velezensis (Ruiz-García et al., 2005) specific primer. All the isolates were confirmed to be members of the Bacillaceae. Isolates selected for their production of molluscicidal supernatants were found to be closely related and identified as strains of B. velezensis, Bacillus amyloliquefaciens (Priest et al., 1987) and Bacillus subtilis (Ehrenberg, 1835). Isolates with potential molluscicidal activity were found to be more diverse, with representatives of B. velezensis, Bacillus cereus (Frankland and Frankland, 1887), Bacillus mycoides (Flügge, 1886), Paenibacillus sp., Priestia sp., and Gottfriedia sp. being identified. Isolates were deduplicated by removal of replicates with identical results for the various gene sequence data and end point PCRs. From the original 12 isolates producing molluscicidal supernatant, 6 were retained and all 8 isolates with potential molluscicidal activity were carried forward for further investigation. Molluscicidal supernatant producers underwent lipopeptide extraction procedures and bioassays to determine whether this class of compounds may be responsible for their observed molluscicidal activity. The 8 potential molluscicidal isolates, along with the 6 molluscicidal supernatant producers, were assayed for molluscicidal activity against P. acuta snails in a longterm (i.e. 2 weeks feeding endospore-impregnated food pellets followed by 4 weeks observation) endospore exposure assay. Molluscicidal active fractions were successfully extracted and concentrated from broth culture supernatants via a lipopeptide acid precipitation extraction protocol. This suggested that lipopeptides, or compounds extractable by acid precipitation, were responsible for the observed molluscicidal activity. Lipopeptide extracts showed molluscicidal activity at between 25 and 200 μg.mL-1 concentrations. In contrast, extended endospore exposure assays yielded no significant molluscicidal results. For the 8 isolates brought forward based on their potential molluscicidal activity, this result confirmed the lack of direct molluscicidal activity identified in the initial screening. Additionally, for isolates that produced molluscicidal supernatant, exposure of snails to these isolates’ endospores did not result in any observable molluscicidal activity, suggesting that only the excreted metabolites were responsible for their molluscicidal activity and that this did not occur at sufficient concentrations in the snail-tank system to demonstrate an effect. Subsequent investigations focused on the extracted molluscicidal compounds, their characterisation and identification, as well as the measurement of their lethal concentrations. Crude lipopeptide extracts from Landy and TSB growth media with molluscicidal activity against P. acuta underwent UPLC-ESI-MS to identify the lipopeptide components. In addition, the dose response curves were measured for 24 h and 72 h contact times. The LC50 for crude lipopeptide extracts with a 24 h exposure ranged between 16.37 μg.mL-1 and 36.16 μg.mL-1, and the LC95 was between 20.05 and 48.68 μg.mL-1. The LC50 for the crude extracts with a 72 h exposure was between 11.89 μg.mL-1 and 27.21 μg.mL-1, and the LC95 was between 15.09 and 30.93 μg.mL-1. Analysis of the UPLC-ESI-MS spectra for each molluscicidal crude extract indicated the presence of various lipopeptide isoforms, including surfactin, iturin, fengycin and bacillomycin-D and -L. Surfactin was common to all molluscicidal crude lipopeptide extracts examined, which suggests that surfactin was a major contributor to the observed molluscicidal activity. The molluscicidal properties of pure surfactin was evaluated against two aquatic snail species, P. acuta, which was used in initial screening, and secondly against Pseudosuccinea columella (Say, 1817), a host snail of Fasciola liver flukes. The molluscicidal efficacy of the crude lipopeptide extracts and pure surfactin were assessed against a test fish species, Danio rerio (F. Hamilton, 1822), as a first step in evaluating ecotoxicity. Commercially-available pure surfactin was found to be molluscicidal against P. acuta and P. columella, with LC50 values of 10.04 μg.mL-1 and 16.58 μg.mL-1, respectively, and LC90 of 12.29 μg.mL-1 and 19.15 μg.mL- 1, respectively, with a 24 h contact time. Crude lipopeptide extracts demonstrated lethal effects on 24 hpf D. rerio embryos with an LC50 of between 10.19 and 44.93 μg.mL-1 with a 24 h contact time. Danio rerio exposure to pure surfactin was found to be lethal, with LC50 and LC90 values of 7.96 and 11.45 μg.mL-1, respectively, with a 24 h exposure period. In comparison to 24 hpf embryos, 24 hpf eggs are still encased in the chorion. The 96 hpf embryos showed lower sensitivity to surfactin with LC50 values of 13.96 and 12.26 μg.mL-1, and LC90 values of 17.61 and 16.87 μg.mL-1, respectively, with 24 h contact times. This is the first report of surfactin having molluscicidal characteristics but it does raise some ecotoxicological concerns for its potential use as a molluscicide. This research is the first broad-scale screening of aerobic endospore-forming bacteria for use as molluscicidal biocontrol agents. An initial screening of 1180 isolates did not reveal any strong molluscicidal isolates with direct activity on aquatic snails. However, an investigation into secreted metabolites determined that lipopeptide extracts exhibited molluscicidal effects, of which surfactin was common to all active fractions. Pure surfactin assays proved that this lipopeptide has molluscicidal activity at low concentrations, with LC50 values of 10.04 μg.mL- 1 and 16.58 μg.mL-1 for P. acuta and P. columella, respectively, with a 24 h exposure time. This is a novel finding, increasing the range of activity of this biotechnologically useful compound. While no isolates from this research have demonstrated utility as biocontrol agents, the finding of molluscicidal lipopeptides opens a new avenue for the biocontrol of aquatic snail pest species, which may impact the neglected tropical diseases fascioliasis and schistosomiasis, with further potential for the control of other aquatic snail pest species.

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