Browsing by Author "Laing, Mark Delmege."

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A study of bruchid resistance and its inheritance in Malawian dry bean germplasm.
(2007) Kananji, Geoffrey Acrey Duncan.; Melis, Robertus Johannes Maria.; Derera, John.; Laing, Mark Delmege.
Dry bean (Phaseolus vulgaris L.) is economically and nutritionally an important legume, not only in Malawi, but in many parts of Africa and Latin America. Unfortunately, two bruchid species (Acanthoscelides obtectus Say, and Zabrotes subfasciatus Boheman) are known to cause extensive damage in storage, reducing the economic importance, food value and planting value of the crop. The aim of this study was to: i) ascertain farmers’ perceptions of the importance of bruchids as storage pests, and to identify their preferred varietal traits in dry beans; ii) screen Malawian dry bean landraces for effective and adaptable sources of resistance to the two bruchid species; iii) determine the gene action and inheritance of bruchid resistance. Farmers’ perceptions on the importance of the two bruchid species to beans both in the field and in storage were established using a participatory rural appraisal (PRA) in three extension planning areas (EPAs) in Lilongwe agricultural development division (ADD). Results confirmed that the two bruchid species are important storage pests, causing serious storage losses among smallholder farmers. In the absence of any control measures, farmers indicated that more than 50% of their stored beans could be lost to bruchids. Indigenous bruchid control measures are not very effective, making it necessary to search for other control methods. It was also clear from the PRA results that breeders need to consider both agronomic and culinary traits in bean cultivar development. This would enhance uptake of newly developed varieties. To address the problem of bruchid damage experienced by smallholder farmers, a total of 135 dry bean genotypes, comprising 77 landraces and 58 improved varieties (obtained from collaborating partners) were tested under laboratory infestation (nochoice test methods) and field infestation (free-choice test methods). The objective of this study was to identify effective sources of resistance to the two bruchid species. Results of the study showed that there was a wide variation among the genotypes for resistance to the two bruchid species. Overall results showed that 88% of the genotypes ranged from susceptible to highly susceptible to Z. subfasciatus and only 12% of the genotypes were moderately resistant to resistant. Genotype screening for resistance to A. obtectus showed that only 12.5% were resistant, whereas 87.5% were moderately to highly susceptible. All of the improved genotypes were 100% susceptible to A. obtectus in storage. One landrace, KK35, consistently showed a high level of resistance to both bruchids under laboratory infestation, with results similar to the resistant checks (SMARC 2 and SMARC 4), while another landrace, KK90, displayed stable resistance under both laboratory and field infestation. However, performance of most genotypes was not consistent with field and laboratory screenings, suggesting that mechanisms of bruchid resistance in the field are different from that in the laboratory and field screening should always be used to validate laboratory screening. Resistance in the field was not influenced by morphological traits. The seed coat played a significant role in conferring resistance to both bruchid species in the laboratory, whereas arcelin did not play any significant role in conferring resistance in the landraces. The inheritance of resistance to A. obtectus was studied in a 6 x 6 complete diallel mating design, involving crosses of selected Malawian dry bean landraces. The F1 crosses, their reciprocals, and six parents were infested with seven F1 generation (1 to 3 d old) insects of A. obtectus in a laboratory, no-choice test. There were significant differences among genotypes for general combining ability (GCA) and specific combining ability (SCA). However, SCA accounted for 81% of the sum of squares for the crosses, indicating predominance of the non-additive gene action contributing to bruchid resistance. A chi-square test for a single gene model showed that 5 of the 13 F2 populations fitted the 1:2:1 segregation ratio of resistant, intermediate and susceptible classes, respectively indicating partial dominance. The eight F2 populations did not conform to the two gene model of 1:4:6:4:1 segregation ratio of resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible classes, respectively. Average degree of dominance was in the partial dominance range in five F3 populations, but in general resistance was controlled by over-dominance gene action in the F2 populations. The additive-dominance model was adequate to explain the variation among genotypes indicating that epistatic effects were not important in controlling the bruchid resistance. The frequency distribution of the 13 F3 populations for resistance to A. obtectus provided evidence for transgressive segregation, suggesting that resistance is conditioned by more than one gene. Reciprocal differences were not significant in the F2 generation seed; but were significant in four crosses in the F3 generation seed for adult bruchid emergence, suggesting that maternal effects or cytoplasmic gene effects also played a role in the inheritance of resistance to the common bean weevil. Through this study, important sources of bruchid resistance in dry bean have been identified in Malawian landraces (KK35, KK90 and KK73). These resistant sources will be used in a breeding programme to develop bruchid resistant bean cultivars, as well as improve resistance in susceptible commercial bean cultivars currently grown by farmers in Malawi.
Agronomic studies on edamame (vegetable soybean) in KwaZulu-Natal.
(2015) Arathoon, Arthur James.; Laing, Mark Delmege.
Abstract available in PDF.
Aspects of management of poplar rust in South Africa.
(2001) Hawke, Georgina Frances.; Laing, Mark Delmege.
An assessment of infection on poplar clones grown at the Lion Match Company Redclyff Nursery in Seven Oaks, KwaZulu-Natal was made to ascertain the nature of infection of the three common species of Melampsora infecting poplars in South Africa. These three species are M. larici-populina, M. medusae and the hybrid, M. medusa-populina. Their contrasting wall echinulations were used to differentiate these species using a scanning electron microscope. A visual rating scale measuring percentage leaf area infected (LAI) was used to determine disease severity. Rust development was slow in new material obtained from New Zealand, indicating rust resistance. This new material was not infected with M. larici-populina. Plant material from Europe showed severe susceptibility to M. larici-populina. Melampsora medusae-populina was the most prevalent species found at Lion Match Company plantations, Seven Oaks, KwaZulu-Natal, South Africa in the survey conducted from January to April, 1998. A once-off survey of rust infected poplar leaves from ten different locations (over 1 500 km apart) was conducted to ascertain the effect of geographical and meteorological conditions on the presence and severity of rust on poplars. The most popularly grown clones in South Africa are Clone 65/29, Clone 65/31, Clone 1488, Clone 129 and the old clone Populus deltoides var. missouriensis. Clone 65/31 had the greatest severity of disease (10.4%) closely followed by Clone 1488 (9.5%). Clone 129 had the least amount of disease (1.8%). The most common rust species occurring in South Africa was M. larici-populina. The hybrid, M. medusae-populina, was the least prevalent, although race studies found this species to be the most virulent. Four trials were conducted to determine the potential of fungicides to control rust infections on poplars. Sixteen fungicides were tested. Naturally infected poplar trees of the clone 65/31, grown in pots, were used as test material. The first trial had 16 fungicide treatments and an untreated control. Four treatments were significantly more effective than others: Alto (cyproconazole) (at 0.3 ml/L) with and without the adjuvant Armoblen 600 (at 0.75 ml/L), Anvil (hexaconazole) (at 0.2 ml/L) and Early Impact (flutriafol + carbendazim) (at 0.6 ml/L), respectively. Oxycarboxin appeared to have enhanced disease progression. Two experimental strobilurins, Stroby WG and Quadris FL (kresoxim-methyl, BASF and azoxystrobin, Zeneca) (at 0.12 ml/L, and O.4 ml/L, respectively) and a new class of fungicide, Astor WG40 (experimental, Novartis) (at 2g/L) controlled rust poorly. Four treatments were used in the second trial: Quadris as a foliar spray (O.4 ml/L), Impact applied on superphosphate granules «1 ml + 5g)/tree), and two controls; superphosphate alone (5g/tree) and untreated. The key finding of the second trial was that Impact gave complete control as a granular application over a 56 day period. Superphosphate alone enhanced rust development slightly. A third trial was conducted which corroborated results obtained in the first two trials: Alto plus Armoblen 600 was the best treatment, Early Impact the next best, then Alto, Anvil, superphosphate coated with Impact, Impact alone, the untreated control, Duett and lastly, superphosphate alone. The superphosphate treatment again slightly increased the disease percentages. A fourth trial was conducted with different rates of Alto (0.1, 0.2, 0.3, 0.5, 0.7 ml/L, and an untreated control), applied with the standard rate of Armoblen 600. All rates of Alto gave control of the disease, the highest rate being the most effective. A 3 x 3 x 3 factorial design was used to determine the effect of nitrogen (N), phosphorus (P) and potassium (K) on the growth of poplar trees and development of rust infection. Nitrogen (limestone ammonium nitrate (LAN) at 28% N) was applied at 0, 15.5 and 31 kg/ha, K (KCI at 50% K) was applied at 0, 16.7 and 33.3 kg/ha and P (single superphosphate at 10% P) at 0, 5.3 and 10.6 kg/ha. Over one year the single best tree grew 4.1 m, having received a treatment of 31 kg N/ha, 10.6 kg P/ha and 16.7 kg K/ha. This same treatment gave the best mean growth of 3.1 m. The poorest treatment was 15.5 kg N/ha, 5.3 kg P/ha and 33.3 kg K/ha, with a mean of 1.7m in growth. The treatment of 15.5 kg N/ha, O kg P/ha and 33.3 kg K/ha resulted in the lowest disease level with a mean of 23.5% leaf area infected (LAI). Treatment with 31 kg N/ha, O kg P/ha and 33.3 kg K/ha resulted in the highest disease level with 39.2% LAI. The results suggested that higher N applications increased disease susceptibility, although this trend was not significant.
Assessment of maize germplasm lines for genetic diversity, cultivar superiority and combining ability.
(2012) Khoza, Suzan.; Derera, John.; Laing, Mark Delmege.
Maize (Zea mays L.) is an important crop in the world; however, its yield is compromised by new production challenges leading to poor yield in sub-Saharan Africa. This calls for a need to enhance maize adaptation to changing climate and challenging environments. The new maize varieties should be richly endowed with high frequency of genes that confer high yield under stress and non-stress conditions. Currently, such maize is not available, prompting research into development of new germplasm lines for use in developing new hybrids. The objective of the study was to determine i) the level of genetic diversity using SSR molecular markers and phenotypic data in a set of 60 maize inbreds from the breeding program, ii) genotype by environment interaction in maize hybrids, iii) cultivar superiority, iv) combining ability effects, v) the relationship between yield and secondary traits and vi) the relevant genetic parameters that underpin genetic gains in a breeding program. To study genetic diversity present in the germplasm, phenotypic data and 30 SSR markers were used to estimate the genetic distance between the inbreds. The results indicated that inbred lines which were put in the same cluster were related by pedigree and origin. To assess the level of genotype by environment interaction (GXE) and cultivar superiority of the new germplasm lines, hybrids were planted in five environments with two replications. Data were analysed using the REML and AMMI tools in GenStat 14th edition. The results revealed significant differences between hybrids and environments for grain yield. However, GXE interaction was also significant indicating possible challenges which can be encountered in selecting new hybrids. To determine combining ability estimates two different testers were used. The REML tool from GENSTAT was used to perform the line X tester analysis. Results indicated that both additive and non-additive gene action were important for grain yield. The direct selection strategy for yield was recommended because heritability of grain yield was high. Overall, results suggested that the information on genetic diversity will assist in defining heterotic groups; which will enable effective and efficient management of the germplasm lines to produce new maize hybrids.
Behavioural, reproductive and growth studies on Oreochromis mossambicus (Peters 1852)
(2010) Weber, Raimund Michael.; Laing, Mark Delmege.
A major obstacle facing the successful creation of an African aquaculture industry, based upon Oreochromids, is the irregular supply of good quality fish seed. There are several causative biological processes behind its irregular supply. The aim of this research was therefore to determine the basic requirements for the establishment and maintenance of a small breeding facility, for O. mossambicus. The goal was to make a unit that was simple and which could be easily replicated in rural, satellite aquaculture seed stations. The results obtained illustrate that a small reproduction unit can produce large quantities of healthy 90-day fry. Asynchronous hatching of the eggs and spawning asynchrony in female Oreochromis mossambicus are two elements which negatively affect uniformity in the fry produced. Typical fish seed production uses large ponds partitioned into breeding allotments or a series of breeding pools. While the earthen ponds provide a substrate in which a nest can be excavated, its presence is not required for mating success in the closely related O. niloticus (Linneaus 1758). Female mate choice, as well as apparent fecundity, according to nest size has been clearly recorded in related cichlids but no investigations have been made as to nest size and spawning synchrony in O. mossambicus. The main focus of this investigation was to ascertain whether O. mossambicus would accept artificial nest substitutes in preference to their own constructed ones and secondly, whether different alternatives would elicit different levels of acceptance. The observed results indicate a ready acceptance for artificial nest alternatives, with nest lip height being prioritised by the fish . The implications thereof are discussed in relation to the potential for optimization of breeding arenas for O. mossambicus by the provision of artificial nests whose dimensions satisfy both male and female preferences. In established communities, Oreochromis mossambicus display various complex and ritualised behaviours during stable and disruptive events. The aim of this research was primarily to produce a glossary of behaviours defining these interactions, particularly with reference to male-male behaviours. Three males and six females were allowed to acclimatise over one month, with various social groupings being established within the first few days. Results from this study illustrated not only a dynamic social structure, signaled via various chemosensory and visual methods, but also supported recent findings in apparent male-male courtship and the underlying ii causes. Furthermore, the observed male-male activity of the nestholder malesfirmly corroborate the current practice in aquaculture whereby only one male is allocated per breeding arena. The use of artificial incubation of Oreochromis spp. eggs has become widespread in high intensity fish seed production. Various types of incubator exist, and their selection is dependent on the specific attributes of the egg to be incubated. Currently available incubators are typically of a funnel (up-flow) or round bottomed (down-flow) design. Neither permits easy access to the eggs, which is particularly important when dealing with poor quality water as is typically found in rural areas. The aim of this study was to devise and test an easy-to-use incubator, applicable to rural seed production projects, which offers advantages over currently available incubator types. The final design, WETNURSE Type II, offered improved hatching rates over Type I, with a mean hatching success of 75%. While falling short of the desired 80% success rate (Rana 1986), the various other benefits provided by the design justify further optimization and testing. Three distinct populations of O. mossambicus, representing populations of inbred, randomly mated and genetically unknown (wild-caught) pedigree were analysed according to their food conversion efficiency (FCE). The intra- and inter-sample crosses were done with single males in order to produce half-sib progeny batches which allowed for the assessment of sire influences on the FCE of the progeny batches. The results show that the population of unknown pedigree is comparable to that of the randomly mating population, indicating the presence of sufficient genetic variation to permit further selection; the genetic contribution of the males to their respective progeny was insignificant in relation to that made by the female.
Biocontrol of postharvest pathogens infecting avocado using endophytic trichoderma species.
(2023) Mkhize , Londeka.; Laing, Mark Delmege.; Bancole, Wonroo Bernice Armellel.
Pre-harvest fungal infection causes both pre- and postharvest avocado (Persea americana Mill.) diseases. Some foliar diseases also cause avocado fruit spots, including those caused by Pseudocercospora and Cladosporium species. Pre- and postharvest rots of avocado fruit are largely caused byColletotrichum, Lasiodiplodia and Neofusicoccum species, and these are also latent pathogens that usually infect the fruit in the orchards before harvest. These fungal pathogens cause crop losses in SouthAfrican production in untreated fruit by about 50-90%. As a result, several agrochemicals including prochloraz, copper oxychloride, thiophanate-methyl and thiabendazole are being used by farmers for avocado disease management. However, the intensive use of chemicals does not prevent all diseases, and toxic residues affect the environment and consumers of fruit containing residues. As a result, there are growing restrictions on the use of agrochemicals being exported to the European Union (EU), due to reductions in theMaximum Residue Levels (MRLs) of most of these fungicides. The use of biological control agents (BCAs) such as Trichoderma species has been regarded as a promising and environmentally friendly approach to controlling plant diseases. Trichoderma species are opportunistic, avirulent plant symbionts and some strains have been developed commercially as BCAs for use on many crops against a wide range of plant pathogens. Their modes of action are complex and include antibiosis, mycoparasitism, producing bioactive secondary metabolites, inducing plant defensive mechanisms and promoting plant growth. Therefore, the overall objective of this study was to isolate and screen endophytic strains of Trichoderma species to control fruit diseases of avocado such as stemend rot, anthracnose and leaf spot diseases. Isolation and Identification of Fungal Pathogens Avocado fruit from different supermarkets in Pietermaritzburg (KwaZulu-Natal, South Africa) that displayed symptoms of avocado fruit rot and fruit spot were collected and brought to the Plant Pathology Laboratory at the University of KwaZulu-Natal for fungal isolation. A total of forty-five isolates were isolated from symptomatic avocado fruit with typical characteristics of anthracnose, stem-end rot, Cladosporium spot, and Cercospora spot. The isolates were identified based on their cultural and morphological characteristics using light microscopy and scanning electron microscopy (SEM). Koch’s postulates screening was performed on fresh avocado fruit by spraying the fruit with suspensions of the key fungal pathogens of anthracnose, stem-end rot, and leaf spot. Frequently isolated colonies causing anthracnose on avocado fruit were divided into 2 morphological groups (Cs1 and Cs2). Colonies of Group 3 (Ls1) constituted 35% of the fungal pathogens isolated from fruit displaying stemend rot. Group 4 (Ls2) was a Lasiodiplodia species that was provided by Majola (2020) and freshly isolated colonies of this genus. In Group 5 were isolates of Pseudocercospora and Cladosporium species, which constituted about 20% of the cultures. All the fungal isolates were subjected to a pathogenicity assay, conducted twice, to confirm the pathogenicity of each isolate. Anthracnose pathogens in Group 1 (Cs1) were the most virulent strains, while Group 2 (Cs2) were the least virulent. Stem-end rot pathogens in Group 3 (Ls1) were more virulent compared to Group 4 strains (Ls2). Some inoculated fruit did not show any symptoms, even after 14 days post inoculation (dpi). Morphological characterization showed that Pseudocercospora species and Cladosporium spp. were the least virulent strains. Molecular identification of the fungi was undertaken using ITS sequence analysis. Isolate Cs1 was identified as Colletotrichum cobbittiense and isolate Cs2 was identified as Colletotrichum henanense. The most virulent isolate causing stem-end rot was Isolate Ls1, which was identified as Neofusicoccum parvum, a pathogen that was not previously recognized as being of importance in KwaZulu-Natal, South Africa. This suggested that this fungus is probably causing severe postharvest losses for avocado farmers in KwaZulu-Natal. Isolate Ls2 was identified in a previous study as Lasiodiplodia mahajangana using ITS1 and ITS2 gene sequence analysis. Isolation and Endophytic Screening of Trichoderma species Avocado leaves were sampled from the five avocado trees of the cultivar Fuerte, which are growing at Ukulinga farm, University of KwaZulu-Natal, Pietermaritzburg. From these trees, sixty leaves were sampled, and five fresh avocado fruits from a local supermarket in Scottsville, Pietermaritzburg were used as plant material for the isolation of Trichoderma species. Colony colour differences and radial growth measurements were two characteristics that were used to differentiate between the strains of Trichoderma species. A screening for endophytic activity of the isolated Trichoderma strains was conducted on fresh avocado fruit and seedlings. Fifty-two avocado seedlings of the cultivar Edranol were transplanted into pots in a greenhouse and sprayed with benomyl to kill any natural endophytic Trichoderma spp. found in them. The seventeen Trichoderma strains isolated previously were used to prepare suspensions with a concentration of 1x106 conidia/ml-1. The Trichoderma suspensions were sprayed on the seedlings and after two weeks the seedling leaves were sampled randomly and surfacesterilised using 2% sodium hypochlorite and sterile distilled water. Small segments were placed on Trichoderma selective medium (TSM) agar plates to check for the presence of the Trichoderma. During this study, only eleven strains of Trichoderma spp. were found to be endophytic. Another screening was then done on fresh avocado fruits. The fruits were sprayed with suspensions of the eleven endophytic Trichoderma strains. After a seven-day waiting period, the fruit was surface-sterilized, and the skin of the fruit was sampled in ten places per fruit and plated onto TSM. Pure cultures were purified on Potato Dextrose Agar (PDA) and monitored every second day to record the growth. A total of nine isolates of Trichoderma demonstrated high endophytic ability. Assessment of the Effect of Endophytic Trichoderma Strains on Anthracnose and Stem-end rot in vivo An in vivo investigation was conducted to understand the antagonistic activity of endophytic Trichoderma as a potential biocontrol agent against anthracnose and stem-end rot. Fresh avocado fruit of the cultivar Hass were collected from a local farm in Richmond (KwaZulu-Natal, South Africa). The fruit were sprayed with Trichoderma isolates conidial suspensions at concentration 1x106 conidia/ml-1 and a commercial Trichoderma-based product (Eco77). The fruit were then air-dried and stored in boxes inside black plastic bags to create a high relative humidity at room temperature for 7 days. After 7 days post inoculation (dpi), the black plastic bags were removed and the fruit were inoculated with the pathogens by spraying the isolates Cs1, Cs2, Ls1, and Ls2 with concentration 1x106 conidia/ml-1. This experiment was conducted twice to confirm the antagonistic activity of the endophytic Trichoderma isolates. A rating scale of 1-3 was used to describe the disease severity caused by isolates Cs1, Cs2, Ls1, and Ls2 after the fruit were treated with endophytic Trichoderma strains. All the fruit inoculated with Trichoderma and the pathogen developed less anthracnose or stem-end rot symptoms than fruit inoculated with the pathogen only. Isolates UK1E, UK4C and Eco77 were able to reduce symptoms on the fruit inoculated with the pathogenic species isolated, including C. cobbittiense, C. henanense, N. parvum and L. mahajangana. Based on ITS1 and ITS2 gene sequence analysis, UK1E was identified as Trichoderma asperellum and UK4C as Trichoderma koningiopsis. These two strains have the potential to be commercialized as biocontrol agents against the Botryosphaeriaceae family associated with anthracnose and stem-end rot of avocado. The next research phase would be to undertake field trials to see if preventative inoculation of the best Trichoderma strains can provide seasonal protection of fruit from pre- and postharvest pathogens.
Biocontrol of three fusarial diseases.
(2004) Kidane, Eyob Gebrezgiabher.; Laing, Mark Delmege.
Over the past one hundred years, research has repeatedly demonstrated that phylogenetically diverse microorganisms can act as natural antagonists of various plant pathogens. Interest in biological control research continues reflecting the desire of multiple constituencies to develop sustainable methods for controlling plant diseases. The review of the literature comprises information on the epidemiology, economic importance and the different control options available against Fusarial diseases of cabbage, maize and pine, and the safety of microorganisms intended for use as biocontrol agents, their management and strategy of control. Trichoderma and Bacillus isolates used as biocontrol agents were obtained mainly from the rhizosphere of cabbage, maize and pine with a view that they would be adapted to those habitats where they would eventually to be used as innundative biocontrol agents. Preliminary selection was made based on in vitro antagonism of those isolates towards Fusarium oxysporum f. sp. conglutinans (Wollenweb.) W.C. Snyder & H.N. Hans. Ultrastructural studies of mycoparsitism of Trichoderma Isolates ET23, ET13 and Trichoderma harzianum Eco-T® which caused significant reduction in disease incidence and severity on later study under greenhouse conditions, were investigated on the vascular pathogen, F. oxysporum f. sp. conglutinans. Although the mode of action of the three isolates wall not fully elucidated, certain mechanisms such as mycoparasitism and antibiosis or production of antimicrobial substances, which cause cell wall degradation and lysis, have been identified. Twenty Trichoderma and 18 Bacillus isolates which showed antagonism towards F. oxysporum f. sp. conglutinans were tested against the same pathogen on cabbage under greenhouse conditions. Trichoderma isolates were delivered to the soil in two different ways, i.e., seed treatment and drenching, while Bacillus isolates were only drenched as spore suspensions. More than two-third of the biocontrol isolates caused significant reductions in disease incidence and severity of the vascular wilt disease. Application of Trichoderma isolates by drenching resulted in better control of the disease than when applied as a seed treatment. Of the 38 Bacillus and Trichoderma isolates tested against the cabbage yellows fungus, three Trichoderma and four Bacillus isolates were selected for further testing against Fusarium sp. and Rhizoctonia solani Kuhn on maize and Fusarium circinatum on Pinus patula seedlings. Since none of the Fusarium isolates obtained from diseased kernels and cobs of maize were pathogenic to the two maize cultivars, yellow maize and PAN 6479, provided by Pannar® seed company, biocontrol experiments on Fusarium diseases of maize could not be conducted. Only Trichoderma Isolate ET23 and T. harzianum Eco-T® were found to significantly control Rhizoctonia preemergency damping-off on maize while none of the Bacillus isolates caused significant increase in seedling emergence. In the test against F. circinatum on pine, in most cases, significant reduction in seedling mortality was observed in the first 4 to 8wk, however, after 12wk they were no longer effective. Improvement in the survival of pine seedlings were observed when T. harzianum Eco-T® was applied prior to the introduction of F. circinatum. There was almost a direct relationship between the inoculation time and percentage of survival of seedlings. Prior inoculation gives the biocontrol agent time to colonize the potential infection courts for the pathogen in the root area and to be established in the rhizosphere of the pine seedlings. It has been reported that the inconsistent and poor performance of biocontrol agents in the field can be improved with the use of mixtures of biocontrol agents to mimic the naturally suppressive soils which comprise numerous saprophytic microorganisms. However, these organisms have co-evolved for many years that they are adapted to live together in the same soil ecosystem. Therefore, when combinations of biocontrol organisms are used, the compatibility between these isolates is important. Compatibility tests between and among Bacillus and Trichoderma isolates were carried out in vitro. The tests revealed that the Bacillus and Trichoderma isolates are not all compatible. Trichoderma Isolate ET13 showed antagonism towards Isolates ET23 and T. harzianum Eco-T®; Bacillus Isolates B81 and BF011 were slightly antagonistic to Isolates EXR and JR01, and Isolate JR01 was slightly antagonistic to Isolate EXR. Comparisons of single versus mixtures of Bacillus or Trichoderma isolates showed that mixtures of Bacillus or Trichoderma isolates did not result in significantly greater reduction in disease incidence and severity of cabbage yellows.
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 characterization of South African bacteriophages infective against Streptococcus uberis, a causal agent of bovine mastitis.
(2019) Pillay, Caleb.; Laing, Mark Delmege.; Basdew, Iona Hershna.
Mastitis is an inflammatory disease of bovine mammary glands and is the most economically important disease affecting dairy herds in South Africa, and globally. The incidence and history of mastitis in South Africa has been extensively documented and found to be bacterial in origin. Streptococcus uberis (S. uberis) is the most common environmental causal agent of mastitis from clinical and subclinical samples in several countries, including Australia, the United Kingdom, New Zealand, and Belgium. Due to the causal agents ability to exist in cells, it remains protected from antibiotics. The quest for antibiotic free dairy management has funded the research into integrated strategies, predominantly proactive udder health management. Biological control methods are not widely used, specifically the use of bacteriophage (phage) therapy. Phages are ubiquitous (found in all biospheres) and are the most abundant organisms on earth. Understanding the interaction between phages and their hosts is vital to their manipulation for therapeutic conditions. This study aimed to isolate phages from unpasteurized milk of dairy cows, sequentially screened these against S. uberis isolates demonstrating antimicrobial resistance at the time. The phages have been screened for robust lytic characteristics for the of intent of a phage-based therapy. Bacterial strains of S. uberis were isolated from unpasteurized milk by Allerton laboratories submitted by dairy farms in KwaZulu-Natal, South Africa. The samples were screened for S. uberis using the following tests: haemolysis patterns on blood agar, catalase reactions using hydrogen peroxide (5%), and Gram reaction. The identity of the strains was then confirmed by Inqaba Biotechnical Industries (Pty) Ltd via sequencing of the 16s ribosomal RNA. The six S. uberis strains were screened against 8 commonly used antibiotics in the dairy industry: β-lactam (ampicillin, penicillin G, cefalexin, oxacillin and amoxicillin), macrolides (erythromycin), tetracyclines (tetracycline) and glycopeptide (vancomycin). Using the Kirby Bauer method and The European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breaking points zone diameters, version 10.0, 2020. Every strain of S. uberis was susceptible to vancomycin, which is not available for intra-mammary infections. Resistance to the 7 other antibiotics varied amongst the strains. This was not a survey study but an isolation of S. uberis strains to challenge the efficacy of phage therapy. Approximately 2,000 mastitic milk samples were screened for phages resulting in the isolation of 95 phages. This was further reduced by screening for constant lytic ability to five phages that were characterized for their phage titre, host range, single step growth phase and lethal dose activity. The titre of the five selected phages varied considerably. Phages CP1, CP2, CP76, CP79 and CP80 produced titres of 59x102, 56x105, 47x106, 50x104 and 35x104 pfu.ml-1, respectively. There was a differential interaction between the five phages isolates and the six strains of S. uberis. One strain of S. uberis was susceptible to all five phages, Strain 21A, whereas Strains 17D and 78B were not susceptible to any of the five phages. Phage CP2 was virulent to four of the six S. uberis strains. The single step growth assay illustrated the cyclic replication of the isolated phages takes between 50 - 60 minutes. In a lethal dose assay, Phages CP1 and CP2 were each able to reduce S. uberis counts by 86% and 83%, respectively. Of the 5 phages screened Phages CP1 and CP2 showed potential as stand-alone treatments, whereas Phages CP76, CP79 and CP80 would offer better control when combined in a phage cocktail, and this would broaden the host range. Phage samples were examined using transmission electron microscopy (JEOL 1400). Various negative stains were used to view the virus particles: 2% uranyl acetate (UA), 2% phosphotungstic acid (PTA) and 0.05-5% ammonium molybdate (AM). The AM staining provided the best images of infected bacterial cells and phage surface structures. All micrographs obtained, illustrated similar viral particle structures suggesting the phages screened belong to one family. A virus particle was measured to have a 50-65nm diameter icosahedral head and a short tail ranging from 25-35nm in length from the EM micrograph. The virus particles exhibited Podoviridae morphology. However, only a complete genomic sequencing will confirm the identity of these virus particles to a species level.
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 fusarium wilt of pine seedlings using endophytic microorganisms and silicon.
(2015) Gqola, Bomikazi Nobesuthu.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Globally, pitch canker fungus (Fusarium circinatum) poses a serious threat to the softwood forest industry. In South Africa, F. circinatum has characteristically been a nursery pathogen, causing a seedling wilt, and has attacked primarily Pinus patula and P. radiata seedlings and cuttings. With P. patula being the most commercially important species in southern Africa, this creates a substantial economic problem. There are no effective control methods to date. The overall study objective, therefore, was to isolate endophytic microorganisms from healthy pine seedling and seeds, and to screen these for activity against F. circinatum in pine seedlings. A second primary objective was to test soluble silicon (Si), applied as potassium silicate for its potential to control of Fusarium wilt of pine seedlings caused by F. circinatum. For the study to be carried out, standard methodologies and procedures had to be followed, which included the isolation of the pathogen and endophytes that were used in experiments reported in the subsequent chapters. A pathogenic strain of F. circinatum was isolated. One hundred and fifty isolates of bacterial and fungal endophytes were also isolated from the seeds of seven different species of pine and 110 seedlings and cuttings of various species of pine. For the successful screening of resistant cultivars, and control agents against F. circinatum, a reliable and representative artificial inoculation technique was needed. A study was undertaken to test various inoculation techniques, aiming to develop a reliable inoculation technique that would mimic the natural infection process of F. circinatum in the field, and to investigate the spore load required to initiate disease, without applying an excessive inoculum. Three inoculation techniques were tested using six P. patula hybrids/species. These included drenching with a conidial suspension, injection of the crown with conidia, and a wounding technique as developed by FABI, University of Pretoria, which involves cutting off a few centimetres of the apical shoot and inoculating conidia onto the wounded tissue. Using a haemocytometer, the spore load was adjusted to two inoculum levels, namely 102 and 106 conidia ml-1. The concentration of conidia had a significant effect (p<0.05) on the Area Under the Disease Progress Curve (AUDPC). A concentration of 106 conidia ml-1 caused more severe F. circinatum symptoms and more severe disease. Inoculation techniques also had a significant effect on AUDPC (p<0.001). The highest infection levels were achieved when plants were wounded by cutting of the top or by injection. However, drenching was a simple and reliable inoculation technique. The interactions between concentration of conidia and inoculation techniques was not significant (p>0.05). Choice of P. patula hybrid had a significant effect (p<0.05) on the AUDPC. There was a significant interaction (p<0.05) between the hybrids and the inoculation technique, where drenching was more effective at discriminating the different levels of resistance of the six tested hybrids. Wounded seedlings were all equally diseased, which reflects the failure of these two inoculation techniques to provide satisfactory inoculation. Endophytic microorganisms that were isolated from selected healthy pine seedlings, cuttings and seeds were screened for their potential as biological control agents against Fusarium wilt. Young P. patula seedlings were drenched weekly for four weeks with 5 ml of each endophyte (106 c.f.u ml-1). A pathogenic strain of F. circinatum was then inoculated onto the plant and the plants were subjected to drought stress for a week. The primary screening of the endophytes produced up to 60% reduction of the disease. The eighteen best endophyte isolates were selected for further screening. In the secondary screening, Isolates E56, E8 and E51 were the most effective biological control agents, while Isolate E85 was the least effective. Isolates E 141, E12, E13 and E27 provided a limited but significant level of control.
Biological control of gastrointestinal nematodes of small ruminants, using Bacillus thuringiensis (Berliner) and Clonostachys rosea (Schroers).
(2011) Baloyi, Mahlatse Annabella.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Gastrointestinal nematode parasites cause great losses in the production of small ruminants through reduced productivity and the cost of preventive and curative treatments. Because of the threat of anthelmintic resistance, biological control of sheep nematodes has been identified as an alternative to anthelmintic drugs. Bacillus thuringiensis (Bt) (Berliner) and Clonostachys rosea (Schroers) have been widely studied as biocontrol agents. B. thuringiensis has been used for the biocontrol of insects and C. rosea has been successfully used as biocontrol agent of Botrytis cinera (De Bary) in plants. B. thuringiensis and C. rosea strains were isolated from soil collected from the Livestock Section at Ukulinga Research Farm, University of KwaZulu Natal, Pietermaritzburg. Twenty-five strains of Bt and 10 strains of C. rosea were successfully isolated. The Bt colonies were identified by their circular, white, flat and undulate character, and the gram-positive and rod-shaped endospores. C. rosea was identified by white colonies on Potato-dextose agar and the characteristic conidiophores, which were branched and showed phialides at the tips. In vitro screening of the isolates was undertaken to select the best isolates. The isolates that caused significantly greater mortality were Bt isolate B2, B10 and B12 and C. rosea isolates P1, P3 and P8. These isolates caused substantial nematode mortality in both faeces and water bioassay. Nematode counts were reduced by 28.5% to 62% and 44% to 69.9% in faecal bioassay for Bt and C. rosea, respectively. In the water bioassay, nematode counts were reduced by 62% to 85% for Bt and by 62.7% to 89.3% for C. rosea. The best inoculum level at which the best isolates were most effective, and the optimum frequency of application were determined. The trial was conducted using bioassays with faeces and water. Inoculum levels of 10(6), 10(8), 10(10), 10(12) spores ml-1 for Bt and 10(6), 10(8) and 10(10) conidia ml-1 for C. rosea was used in the faecal bioassay. The inoculum levels tested in water bioassay were 10(6), 10(8), 10(10) and 10(12) spores ml-1 for Bt and 10(9), 10(10), 10(11), 10(12) conidia ml-1 for C. rosea. In the faecal bioassay, B2 was the most effective Bt isolate at an inoculum level of 10(10) spores ml-1. Isolate P3 was the best C. rosea isolate at 10(8) conidia ml-1. In the water bioassay, Isolate P3 caused a mortality of 85% at inoculum levels of 10(9), 10(10) and 10(11) conidia ml-1. The performance of biological control agents in the field is sometimes inconsistent. Combining different biocontrol agents may be a method of improving their reliability and performance. However, the combination of most of the isolates was antagonistic, with efficacy less than that of either individual biocontrol agent. In particular, Isolate P3 was more effective when used alone than when combined with any other isolates. Therefore, the combination of biocontrol agents does not always result in synergistic interaction. There were some additive interactions between two bacterial isolates, and with one bacterial and fungal combination. The effect of feeding the best of the biocontrol agents, or diatomaceous earth (DE), was evaluated in sheep. Two doses of Bt (1g and 2g kg-1BW) and C. rosea (1g kg-1BW) reduced the numbers of L3 nematode larvae in sheep faeces. The DE product (at 15% of feed) also reduced L3 numbers but it was less effective than either the Bt or the C. rosea products. Nematode counts were reduced by 74.6%, 75.1%, 84.6%, 68.5% and 27.5% for Bt 1g kg-1BW, Bt 2g kg-1BW, C. rosea (1g kg-1 BW), DE and control, respectively.
Biological control of head rot and damping-off of sunflower using yeasts, Bacillus spp. and Trichoderma spp.
(2021) Moody, Alison Jade.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
The soil-borne fungi Sclerotinia sclerotiorum, Sclerotium rolfsii and Rhizoctonia solani are ubiquitous plant pathogens with a wide host range. They are among the most widespread pathogens, and cause destructive diseases of many crops, including sunflower (Helianthus annus L.), an economically important oilseed crop. Chemical fungicides are available for control of seedling damping-off caused by these pathogens, but there are environmental concerns and the possibility of developing tolerance in pathogen populations, which have led to a drastic reduction in their usage and increased the pressure to find alternative means of disease control. Additionally, there are no registered fungicides that effectively control Sclerotinia head rot of sunflower caused by S. sclerotiorum in South Africa. Successes in biological control (biocontrol) and plant growth promotion research have led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of three strains of Bacillus spp., one yeast and one commercial strain of Trichoderma spp., and their respective combinations, on Sclerotinia head rot on sunflower. An additional commercial strain of Trichoderma spp. was also evaluated for the control of damping-off. In vitro biological control and growth promotion studies were carried out under greenhouse conditions with the use of foliar spray treatment as the method of application for head rot, and seed and soil drench treatments for damping-off. In vitro screening was undertaken to select the best Bacillus and yeast isolates from 136 Bacillus spp. and 100 yeasts isolated from local wild sunflower heads. Dual-culture bioassays were undertaken and isolates were assessed for antagonism by examining the radial growth of S. sclerotiorum mycelium. A scale was used to group the isolates, based on their inhibition ability in order to select the best isolates to screen in vivo. Seventeen Bacillus isolates achieved a Class 3 rating (≥70% inhibition of pathogen mycelial growth), while only 4 yeast isolates achieved a Class 2 rating (41-69% inhibition). The isolates, along with T. atroviride strain 77 (T77), were further screened in vivo under greenhouse conditions for antagonistic activity against Sclerotinia head rot of sunflower cv. PAN7080 plants, when plants were at the R6 reproductive stage. Disease incidence was recorded 14 days after inoculation with BCAs and S. sclerotiorum, and grain was harvested, dried and weighed 85-115 days after planting. A total of 20 yeast and Bacillus isolates were screened against S. sclerotiorum and 4 Bacillus isolates and 1 yeast isolate reduced disease incidence by ≥50%, compared to the disease control. Bacillus B16 resulted in complete disease suppression, followed by B24, B26 and T77, which reduced disease incidence to 12.5%. Seven of the 20 yeast and Bacillus isolates, along with T77, significantly improved grain yield. B16 resulted in the highest grain yield, followed by T77. The effect of inoculum concentration was evaluated for the best performing yeast and Bacillus spp. isolates. A concentration of 1 x 108 cells mL-1 for yeast Y79, and 1 x 109 cfu mL-1 for B16, B24 and B26 caused the greatest disease suppression and improvement in grain yield. In comparison to the Bacillus isolates, Y79 was the poorest performing biocontrol agent (BCA), reducing the incidence of head rot the least. In addition, it was not as effective at improving grain yield and failed to perform consistently between the first, second and third greenhouse screening. Sunflower heads treated with single and combined inoculations of T77, Y79 and B16, B24 and B26 exhibited improved grain yield. Combined inoculations of B16 + B26 and B26 + B24 provided over 10.0% increase in grain yield (12.8% and 15.5%, respectively) over the disease-free control. Y79, when inoculated in combination with B16 and B24, scored reduced disease incidences of 62.5% and 37.5% as well as improved grain yields of 15.8 g and 36.0 g, respectively. In vitro dual-culture assays carried out with T. asperellum strain kd (Tkd) showed effective antibiosis activity and marked mycoparasitism of S. sclerotiorum, R. solani and S. rolfsii, despite the BCA performing poorly according to the Bell rating scale in dual culture plates. Greenhouse trials were carried out in Speedling 24® trays, and Tkd was applied as a seed treatment alone and/or a monthly-bimonthly soil drench. Various other greenhouse trials were set up to evaluate the potential of Tkd to suppress damping-off of sunflower caused by the three pathogens, and several growth parameters were measured. Seed treatment in combination with a monthly or bimonthly soil drench significantly increased seedling, shoot, root and head dry weight, along with root area, when tested against all three pathogens- effectively reducing disease incidence. Reduced disease incidence and enhanced seedling and plant growth were also achieved when Tkd was applied as a seed treatment alone, drench at planting alone, and drench at planting + bimonthly drench, but at lower levels. A number of methods were adapted from studies carried out in other parts of the world with the objective of finding a fast and reliable method of inducing sclerotia of S. sclerotiorum to germinate carpogenically and produce ascospores. However, none of the published techniques worked under the conditions tested. Only one method, adapted from a study conducted by a fellow South African researcher, resulted in stipe formation, but not in ascospore production. The failure of these published techniques to work under the local conditions may be attributed to the fungus having stringent requirements for environmental conditioning before it will sporulate carpogenically. It appears that these requirements vary with the geographic source of the sclerotia, and that effective conditioning parameters in one place may not work in other geographic locations. The results presented in this dissertation confirm the concept of biological control by Trichoderma spp. and Bacillus spp. as a viable disease control strategy to manage S. sclerotiorum of sunflower. Furthermore, this dissertation forms a basis for further Trichoderma-Bacillus-Yeast interaction studies to determine whether strains of these three organisms could be combined to enhance biocontrol and plant growth promotion.
Biological control of Phytophthora root rot of citrus seedlings and cuttings.
(2005) Abraham, Abraha Okbasillasie.; Laing, Mark Delmege.; Bower, John Patrick.
With an increasing realization that many agrochemicals are hazardous to animals and humans, came the desire to replace these chemical agents with biological approaches that are more friendly to the environment and human health. Microorganisms play an important role in plant disease control, as naturally occurring antagonists. Microorganisms may also have beneficial effects on plant development when applied to plant roots. Research efforts worldwide have recorded successes in biological control and growth stimulation on many crops, particularly when using members of the genera Bacillus and Trichoderma. Their use on citrus rootstock could be advantageous to nurserymen and growers in reducing the incidence of seedling mortality and increasing production. To achieve these objectives, laboratory and tunnel experiments were conducted to develop effective biocontrol agents for citrus seedlings and cuttings. Nineteen 0 ut 0 f 23 Trichoderma isolates tested in vitro against Phytophthora p arasitica sp showed antagonistic activity by hyperparasitism and four out of eight Bacillus isolates resulted in antagonism by forming inhibition zones. The positive in vitro activity of Trichoderma and Bacillus isolates on Phytophthora provided motivation step for further trials in the greenhouse to evaluate their biological control activity on citrus seedlings and cuttings. A greenhouse trial was carried out to evaluate the biological control potential of 23 Trichoderma isolates (drenched at 5 x 105 spores / rnI) and two Bacillus isolates (drenched at 1 X 106 or 1 X 108 colony forming units (CFU) / rnI) to suppress Phytophthora parasitica sp. of rough lemon (Citrus jambhirini Lush.) seedlings. Five isolates ofTrichoderma (AA12, AA5, Trichoderma harzianum (AA16), SY3F and Eco-T~ were highly effective in suppressing Phytophthora root rot, with AA12 providing the best control. The Bacillus isolates also suppressed the pathogen but were not as effective as the Trichoderma isolates. This trial was used to test for growth stimulation activity by some of the biocontrol agents. To verify these results, a further trial was carried out to evaluate growth stimulation capabilities in the absence of any pathogen. Trichoderma Isolates AA13 and AA17 caused no 111 change in seedling growth, while other Trichoderma and Bacillus isolates had an inhibitory effect on the seedling growth. This trial indicated that the biocontrol activity was affected by inoculum densities, and as a result in vitro sporulation capacity was evaluated. TrichodermaIsolate AA16 was the largest spore producer, followed by Eco-T®. Spore production was lowest from Trichoderma isolates AA4 and AA12. Growth stimulation responses of Trichoderma Isolates AA4, AA16, Eco-TID and SYN6 were further studied at four different doses (1 X 103, 1 X 104, 5 X 105 or 1 X 106 spores / ml) on rough lemon and trifoliate orange seedlings. Trifoliate oranges responded positively to 1 X 104 and 5 X 105 spores / ml of Eco-TID, but rough lemon responded negatively to all dosages of the Trichoderma isolates applied. This indicates that the inoculum density responses may be host specific. Higher population density of 1 X 106 spores / ml of all tested Trichoderma isolates had a stunting effect on seedling growth of both species. Based on t he positive results 0 f individual applications of some Trichoderma and Bacillus isolates, of the biological control agents on rough lemon seedlings against Phytophthora parasitica in an earlier greenhouse trial, their combined effect in the control of the pathogen was performed. Before carrying out a greenhouse trial, activities of the isolates to be combined were evaluated in vitro. This trial showed that Trichoderma Isolates AA16 and Eco-T®were compatible. Trichoderma isolates AA16 and Eco-T®were also found to be compatible with Bacillus Isolates B77, B81 and PHP. As a result, further in vivo trials were conducted. The tunnel trials were carried out as two separate experiments: In the first experiment, a combination of two Trichoderma Isolates A A 16 and Eco-T®was conducted assayed at 5 X 105 or 1 X 106 spores / ml, on rough lemon seedling, and cuttings and trifoliate orange and sour orange seedlings. A combination of Trichoderma isolate AA16 and Eco-T®at 5 X 105 spore / ml increased significantly the new flush biomass of rough lemon cuttings compared to AA16 alone, but was not different from Eco-TID alone. The combination of AA16 and Eco-T® achieved no change of biomass of rough lemon and trifoliate orange seedlings. The combination of AA16 and Eco-TID did not increase the root biomass of sour orange compared to AA16 or Eco-r® alone. The combination of AA16 and Eco-r® at higher doses (1 x 106 spores / ml) showed significantly better suppression of Phytophthora root rot of rough lemon cuttings but did not show disease suppression in all seedling species verities tested. In a second experiment, individual and combined effects of Trichoderma isolates (drenched at 5 X 105 spores / ml) with Bacillus isolate (drenched at 1 X 106 colony forming units (CFU) / ml) for suppression of Phytophthora root rot on rough lemon and trifoliate orange seedlings was performed. The combination of Trichoderma Isolate AA16 and Bacillus Isolate B81 increased root biomass on rough lemon seedlings compared to the combination of Trichoderma AAI6 or Bacillus PHP but was not significantly different to Trichoderma AA16 alone. Bacillus PHP combined with Trichoderma AA16 or singly had no effect on rough lemon seedlings. Combining Trichoderma Eco--r® and with Bacillus B8I or PHP did not increase biomass of rough lemon seedlings compared to Trichoderma Isolate Eco--r® alone. There was no statistically significant differences in the effects of the combinations of the Trichoderma and Bacillus isolates compared to their individual applications on the biomass of trifoliate oranges. This study established the antagonistic potential of several South African isolates of Trichoderma and Bacillus as a viable alternative to agrochemicals for controlling Phytophthora parasitica. The growth stimulation capabilities of Trichoderma isolates in terms of seedling development was also demonstrated.
Biological control of root knot nematodes (meloidogyne spp.) using bacterial and fungal antagonists.
(2014) Pambuka, Gilmore Taenzaniswa.; Laing, Mark Delmege.
Root-knot nematodes are an important pest of many crops worldwide. Chemical nematicides are the main control methods used to reduce damage caused by nematode pests on crops. However, there are aims of reducing the use of chemical nematicides, resulting in a shift towards the use of biological control, which is an environmentally friendly and safer method of control. Potential antagonists of Meloidogyne javanica were isolated from grazing pastures of livestock, and the rhizosphere of tomato plants grown under glasshouse conditions. A total of 94 bacterial and 22 fungal isolates were screened in vitro by means of microwell bioassays. Twenty bacterial and eight fungal isolates showed nematicidal activity, causing root-knot nematode second-stage juvenile (J2) mortalities of between 47.0% and 65.4%, and 33.0% and 66.3%, respectively. Five bacterial and three fungal isolates caused J2 mortalities of more than 60%. In vitro studies were conducted to evaluate the efficacy of these isolates (Bacillus spp., Hypocrea lixii-the teleomorph of Trichoderma harzianum) and Trichoderma spirale, together with two previously isolated biocontrol agents, Hypocrea lixii Strain Eco-T® and Clonostachys rosea, on the root-knot nematode M. javanica. All the bacterial isolates and fungal treatments caused significant levels of J2 mortality of M. javanica of between 59.0% to 94.0% after 12, 24 and 48 h. Bacillus thuringiensis (Isolate BG25) and H. lixii (Isolate Cr5) caused the highest mortality of J2. B. thuringiensis (Isolate BG25) and H. lixii (Isolate Cr5), when applied as a seed dressing or as a soil drench significantly (P<0.001) reduced penetration of M. javanica J2into the roots of tomato plants. The two isolates also reduced disease severity and significantly (P<0.001) reduced formation of galls, production of egg masses and the number of eggs per root. Growth parameters in terms of shoot length, shoot weight and dry shoot weight were significantly (P<0.001) increased by seed dressing and soil drench treatments of all bacterial and fungal isolates. B. thuringiensis (BG25) and H. lixii (Cr5) caused the greatest effect on growth parameters measured on tomato plants under greenhouse conditions.
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 Musca domestica L. in horse stables, using Bacillus thuringiensis serovar israelensis and Beauveria bassiana.
(2013) Martins, Cheralyn.; Chimonyo, Michael.; Laing, Mark Delmege.
House flies (Musca domestica L.) are common pests affecting horses and their owners. Control of house flies in stable yards is currently based on the use of pesticides. However, the development of resistance by these flies to most pesticide groups has motivated horse owners to seek alternative methods of fly control. An entomopathogenic fungus, Beauveria bassiana (Bb) and an entomopathogenic bacterium, Bacillus thuringiensis var. israelensis (Bti) are two biological agents known to have activity against house flies. The broad objective of this study was to evaluate the effect of these two biological control agents on house flies in an equine environment. Using a structured questionnaire, presented in Chapter 2, thirty horse owners in KwaZulu-Natal were asked about the nuisance value of house flies, their current control measures, the potential market for biocontrol agents against house flies, and each owner’s perception of biocontrol methods. The horse owners were using three methods of house fly control namely, physical, chemical and biological. Most horse owners (97%) wanted access to effective biocontrol agents for control of house flies. Most horse owners (80%) stabled their horses at night, some or all of the time. The resultant manure piles in the stable yard were considered to be the primary cause of house fly problems. About 64% of the horse owners were dissatisfied with the currently available methods of controlling house flies in this situation. Chapter 3 covers two observational trials in which varying doses of Bacillus thuringiensis var. israelensis (Bti) were fed to horses, in order to identify a baseline dosage to give to horses in order to adequately control house fly populations growing in horse manure. The bacterium Bti, grown on wheat bran, was fed to six miniature horses at doses of 0, 0.125, 0.25, 0.5, 0.75, and 1.0 g per meal in Trial 3a, and at 0, 0.5, 1, 2, 4 and 8 g per meal in Trial 3b. Faeces were collected three times a week for 11 weeks and placed in incubation trays to allow the number of emerging adult house flies and closed pupae to be counted. In Trial 3a, there was a significant reduction in the number of closed pupae with an increase in Bti in the feed. The regression equation suggests that there will be 3.1 times as many closed pupae in the faeces when horses are fed 1 g of Bti in their feed, than when horses are fed no Bti. This dosage is the minimum baseline dosage for future trials. v Using manure from horses dosed in Trials 3a and 3b, the survival of the bacterium through the gut of horses was evaluated using a standard isolation technique. The growth of Bt colonies on the manure after the Bt isolation technique showed that some of the bacterial cells survived transition through the digestive tract of the horse. This study was qualitative in nature and did not attempt to quantify the level of Bti spore survival. These two observations suggest that Bacillus thuringiensis var. israelensis has the potential to be used as a biocontrol agent, applied via horse feed, for the control of house flies in stable yards. Future clinical trials, with appropriate replication, should be conducted using 1 g Bti/meal as the lowest test dosage. The objective of Chapter 4 was to determine whether spraying Bti or Bb on to horse manure is effective in the control of house flies. Over a six week period, two spraying trials were conducted in which increasing doses of Bb and Bti were sprayed on to 500 g samples of horse manure. Counts of house fly pupae and adults were taken. The doses of Bb and Bti tested were 0, 1, 2, 4 g in Trial 4a, and 0, 4, 8 and 12 g in Trial 4b. The research reported in Chapter 4 was characterized by the unexpectedly high levels of biological variation in egg, larvae and pupae numbers that were found in samples of horse manure, taken from the same skip two days apart. The statistical design of the two trials conducted was inadequate to cope with the high level of variation about treatment means for fly and larval counts. However, despite the lack of significant differences between treatment means, there is observational evidence that suggests that both Bb and Bti do have an effect on house fly survival. A simplified statistical model, which compared the number of hatched house flies on untreated manure, with the number on manure treated with any level of Bb (1 to 4 g /250 ml water), found a significant reduction in the number of hatched flies on treated manure. There was no significant corresponding reduction in the number of closed pupae, which suggests that Bb acts primarily before the larva pupates. The optimal dose of Bb and Bti to be sprayed on to manure could not be determined because of the high variation about treatment means. It is suggested that, in future trials similar dosages for Bb could be tested, but that higher dosages of Bti (starting at 2 g/250 ml water) should be used. Trial periods should be extended and replication increased dramatically to reduce variation about treatment means. Transformation of data before analysis may also be necessary to equalize variation about treatment means.
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.