The challenges faced by the agricultural sector especially around improving production yields using environmentally friendly solutions have received market attention. Biological intervention can range from application of biological products to enhance the nutritional value of crops or to control plant pathogens. Biostart, a biological product that demonstrated growth enhancement when applied in lettuce crops is currently in the market. The product is comprised of a consortium of bacterial isolates (Bacillus licheniformis, Brevibacillus laterosporus and Bacillus laterosporus) but the contribution of the individual isolates to growth enhancement had not been elucidated. Green house experiments on lettuce seedlings with individual and mixed treatments were commissioned to determine such contribution. There was either no or marginal growth enhancement observed in the experiments. The results showed that the product was effective as a consortium and not as individual isolates. Further isolation and screening for potential Bacilli with antifungal properties was undertaken. An isolate identified as Bacillus subtilis that demonstrated inhibition against a wide spectrum of fungi, and especially the phytopathogenic Verticillium dahliae and Fusarium oxysporum, was successfully identified. The isolate was cryo-preserved and cultivated to significant levels at bench scale. A characterized comparison of different putative products with known systematic fungicide showed potential application even of heat treated products. The product showed control V. dahliae when tested in green houses with potatoes and tomatoes as test crops. This isolate has been targeted for further development as a biological control product.

Orientador: Pedro Luiz Martins Soares / Coorientador: Jaime Maia dos Santos / Banca: Bruno Flávio Figueiredo Barbosa / Banca: Rita de Cássia Panizzi / Resumo: A cultura de cana-de-açúcar é de grande importância para a economia brasileira. Os fitonematoides são um dos fatores limitantes na maioria dos canaviais causando grandes perdas à cultura. O manejo dos nematoides é feito através de nematicidas químicos que são prejudiciais ao homem, meio ambiente e produtos colhidos, devido sua alta toxicidade. O controle biológico tem sido uma alternativa viável e sustentável para o manejo dos nematoides. O objetivo do presente trabalho foi avaliar espécies de Bacillus no controle de Meloidogyne incognita e M. javanica in vitro e na cana-de-açúcar. Foram instalados experimentos em condições de laboratório com delineamento inteiramente casualizado com os tratamentos: Cadusafós 200 CS (14 L/ha); B. subtillis; B. firmus e B. amyloliquefaciens (1 e 10 L/ha), além de uma testemunha (água). Os ensaios em vasos, à céu aberto, tiveram delineamento inteiramente casualizado, com os tratamentos B. subtilis (10 L/ha), B. firmus (10 L/ha), B. amyloliquefaciens (10 L/ha), Carbofurano 350 SC (5 L/ha - tratamento padrão) e uma testemunha, com cinco repetições e duas avaliações (100 e 150 dias após inoculação e aplicação dos tratamentos). No ensaio in vitro, B. firmus (10 L/ha) é o mais eficiente para diminuir a eclosão de J2 de M. javanica, sendo estasticamente semelhante ao produto químico testado. Quanto a motilidade de J2 de M. javanica, B. subtilis (10 L/ha) e B. amyloliquefaciens (1 e 10 L/ha), causam a mortalidade do nematoide, porém inferior ao padrão químico testado. Para M. incognita, B. firmus (10 L/ha) proporciona maior eclosão de J2. Quanto a motilidade de J2 de M. incognita, Cadusafós 200 CS (14 L/ha) causou a maior mortalidade. Nos ensaios em vasos, todos os tratamentos aumentam o número de perfilhos, mas não controlam os nematoides / Abstract: The sugar-cane crop is very important to Brazilian economy. Phytonematodes are among the main limitating factors of such culture causing great losses. Chemical nematicides are harmful to humans, environment, harvested products, and are the main method used to manage the populations of these nematodes. Biological control have been a sustainable alternative to manage these nematodes. The aim of the present study was to evaluate Bacillus species for control Meloidogyne incognita and M. javanica in vitro and in sugar cane plants. We set assays in laboratory conditions in a completely random design with the treatments: Cadusafós 200 CS (14 L/ha), B. subitilis, B. firmus, B. amyloliquefaciens (1 and 10 L/ha) and a control (water). The pot assay, in a semi-field condition, had a completely random design with the treatments: B. subtilis (10 L/ha), B. firmus (10 L/ha), B. amyloliquefaciens (10 L/ha), Carbofurano 350 SC, with five replicates and two evaluation periods (100 and 150 days after inculation and application of treatments). At laboratory assay B. firmus (10 L/ha) was the most efficient in decreasing the M. javanica J2 eclosion and were statistically similar to the chemical nematicide tested. For M. javanica J2 mobility, B. subtilis (10 L/ha) and B. amyloliquefaciens (1 and 10 L/ha) cause nematode mortality, but lower than that presented by the chemical. To M. incognita, B. firmus (10 L/ha) provides higher J2 eclosion. For the M. incognita J2 mobility, Cadusafos 200CS (14 L/ha) caused higher mortality. In the pot assay, all treatments increase the number of tillers but do not control the nematodes / Mestre

Bacillus cereus is a group of ubiquitous facultative anaerobic spore forming Gram-positive rods commonly found in soil. It has been detected and implicated in several contaminated food products and raw milk in dairy farms and it causes foodborne gastroenteritis by producing several toxins. This study is aimed at characterizing virulence determinants of B. cereus from cow‟s raw milk. A total of 400 raw milk samples were collected in Fort Hare Dairy Trust and Middledrift Dairy Farm; and cultured on Polymyxin pyruvate Egg-Yolk Mannitol Bromothymol Agar (PEMBA) for 48 hours at 37°C. DNA was isolated from the isolates and 16S rDNA was amplified and sent to Central Analytical Laboratory for sequencing. The gyrB gene of B. cereus was also used to confirm the identity of the isolates. Antibiotic susceptibility profiles of the isolates together with virulence genes were investigated. Multilocus Sequence typing was used to investigate the genetic relatedness of some selected isolates. Furthermore, spores of the isolates were produced, harvested and their concentrations determined. All (100%) of the isolates were identified as having a 96-99% similarity to B. cereus, B. thuringiensis and B. anthracis using sequencing; while gyrB gene was observed in all (100%) of the isolates. Three virulence genes nheA, nheB, nheC encoding for non haemolysin enterotoxin were amplified in all (100%) the isolates. All (100%) of the isolates were susceptible to doxycycline, gentamycin, tetracycline, ciprofloxacin, chloramphenicol and streptomycin. Resistance to rifampicin and penicillin G was predominant with equal rate of 100%, while susceptibility to erythromycin, clindamycin and doxycycline ranged from 60% to 100%. The selected isolates were related and are descendants of the same ancestor. All (100%) the isolates produced spores. The B. cereus isolates contain virulence genes, has multiple antibiotic drug resistance and produce spores, which poses a health risk to the public and cannot be used as probiotics.

Rhizoctonia solani and Pythium spp. are aggressive soil-borne fungal pathogens responsible for seed rot and seedling damping-off of many crops. With increased environmental and public concern over the use of chemicals, biological control of these diseases has been attracting more attention. However, success with this strategy depends on the development of effective antagonists, which requires repeated in vitro and in vivo tests. Bacillus spp. were isolated from a soil sample obtained from a field where sorghum and tef had been grown for at least two years. Potential Bacillus isolates were screened for their ability to inhibit in vitro growth of R. solani and Pythium sp. Among 80 isolates tested, endospore forming Bacillus spp. H44 and H51 gave highest antifungal activity against the two test-pathogens in three consecutive tests. Results demonstrated that both H44 and H51 have potential as biocontrol agents against diseases caused by these two pathogenic fungi. The interaction between three isolates of Trichoderma (T. harzianum Eco-T, Trichoderma spp. SY3 and SY4) and Pythium sp. were investigated using in vitro bioassays together with environmental scanning electron microscopy (ESEM). Visual observation on the dual culture tests revealed that hyphal growth of Pythium was inhibited by these antagonists soon after contact between the two organisms within 3-4 days of incubation. The ESEM investigations showed that all three isolates of Trichoderma grew toward the pathogen, attached firmly, coiled around and penetrated the hyphae of the pathogen, leading to the collapse and disintegration of the host's cell wall. Degradation of the host cell wall was postulated as being due to the production of lytic enzymes. Based on these observations, antibiosis (only by Eco-T) and mycoparasitism (by all three isolates) were the mechanisms of action by which in vitro growth of Pythium sp. was suppressed by these Trichoderma isolates. The reduction of seedling diseases caused by R. solani and a pythium sp. were evaluated by applying the antagonists as seed coating and drenching antagonistic Bacillus spp. (B81, H44 and H51) and Trichoderma (T. harzianum Eco-T and Trichoderma spp. SY3 and SY4). On both crops, R. solani and Pythium sp. affected stand and growth of seedlings severely. With the exceptions of H51, applications all of isoltes to seeds reduced damping-off caused by R. solani in both crops. Application of Eco-T, H44 and SY3 to sorghum controlled R. solani and Pythium sp. effectively by yielding similar results to that of Previcur®. On tef, biological treatments with Eco-T and SY4 reduced seedling damping-off caused by R. solani and Pythium sp., respectively, by providing seedling results similar to the standard fungicides, Benlate® and Previcur®. Most other treatments gave substantial control of the two pathogens on tef. Overall, Bacillus sp. H44 and T harzianum Eco-T were the best biocontrol agents from their respective groups in reducing damping-off by the two pathogens. In all instances, effects of application method on performance of biocontrol agents and adhesive on emergence and growth of seedlings were not significant. A field trial was conducted at Ukulinga Research Farm at the University of Natal, Pietermaritzburg, South Africa, to determine efficacy of biological and chemical treatments on growth promotion and reduction of damping-off incited by R. solani and Pythium sp., and to evaluate the effects of a seed coating material, carboxymethyl cellulose (CMC), on seedling emergence and disease incidence. Seeds of sorghum and tef were treated with suspensions of antagonistic Bacillus H44 or T harzianum Eco-T, or sprayed with fungicides, Benlate® or Previcur®. Application of Benlate® and Previcur® during planting significantly increased the final stand and growth of sorghum seedlings. Seed treatments with both H44 and Eco-T substantially controlled damping-off caused by Pythium, resulting in greater dry weights of seedlings than the standard fungicide. However, they had negative effects when they were tested for their growth stimulation and control of R. solani. The CMC had no significant effect on germination and disease levels. These results showed that these antagonists can be used as biocontrol agents against Pythium sp. However, repeated trials and better understanding of the interactions among the antagonists, the pathogens, the crop and their environment are needed to enhance control efficiency and growth promotion of these antagonists. Some of these biocontrol agents used in this study have the potential to diseases caused by R. solani and Pythium sp. However, a thorough understanding of the host, pathogen, the antagonist and the environment and the interactions among each other is needed for successful disease control using these antagonists. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Fourteen spore-forming bacterial strains were isolated and screened for entomopathogenic activity. Five displayed toxicity towards the common mealworm, Tenebrio molitor L., (Coleoptera: Tenebrionidae). The majority of the isolates were obtained from insect larvae and insect rich environments. The three bacterial species identified were Bacillus thuringiensis Berliner, Brevibacillus laterosporus Laubach and Bacillus cereus Frankland and Frankland. Bioassays were conducted using T. molitor larvae. The one isolate of B. cereus required the highest concentration of bacterial cells to achieve its LC50, whereas one of the isolates of B. laterosporus required the lowest cell concentration to achieve its LC50. Dose response curves were generated for the five best isolates, which showed that the isolate of B. laterosporus (NDR2) was substantially more toxic than the other isolates. / Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2009.

This study focuses on the isolation and characterization of bacteria from lignocellulosic biomass obtained from the sediments of the Tyume River in Alice, Eastern Cape and to determine those bacterial isolates with good potential for modification and decomposition of lignocellulosic biomass for industrial application. Several bacterial isolates were recovered and screened for ability to degrade various lignocellulosic materials. Nine of the isolates were positive for lignocellulolytic activity. Four isolates were cellulase positive and six were xylanase positive. Moreover, one isolate (SB1) was positive for both xylanase and cellulase activities and showed the best hydrolysis zone on solid media. This isolate was then chosen as the best and identified molecularly. The 16S rDNA sequence analysis indicated that SB1 was a Bacillus cereus species. Factors affecting the cellulose and xylanase enzyme production by the organisms were studied. The organisms produced the enzymes maximally at earlier hours of incubation (12-30 hr) and optimally at acidic pH (3-5) and at moderate temperatures (35-45ºC). SB1 appears to hold promise in the decomposition of lignocellulosic wastes.

Maize is the staple food for most South Africans. This implies that any damage to the maize crop will affect food security of many South Africans. Although Eastern Cape Province is not a traditionally maize producing area, smallholder farmers in the province produce it mostly for subsistence purposes and some sell the surplus on the local market or use it to secure other good through barter trading. In South Africa, insect-resistant Bt maize/yieldgard has been used commercially for approximately 10 years now. Available impact studies on Bt maize reveal that, this technology is beneficial not only to farmers but consumers of maize products as well. Welfare gains as well as positive effects for human health are realised by both groups. Due to the costs and effectiveness associated with traditional and conventional maize stem borer control methods, Bt technology has the potential to be part of the solution. This thesis has attempted to investigate the economic viability of planting Bt maize seeds under smallholder farming conditions and identify factors as well as perceptions relating to attributes of Bt maize and to analyze the relationships between those perceptions and choices regarding use of Bt technology. Data was collected from 90 households who were selected using purposive sampling through the use of the snowball method. To collect data, a questionnaire was administered through face-to-face interviews. Gross margin analysis revealed that Bt maize is a more profitable option as compared to conventional maize seeds. Furthermore, econometric analyses, through use of the binomial regression model revealed that perceptions could be used to distinguish between users and non-users of Bt maize seed in the Eastern Cape Province. Results of inferential analysis indicate that the statistically significant variables at 5% level are gene erosion, quality and nutrition of products and food labels for Bt maize products perceptions. On the other hand, low expenses, seed market availability and farmers’ knowledge perceptions were significant at 10%. These findings suggest that an adjustment in each one of the significant variables can significantly influence the probability of Bt maize adoption. In view of the research findings, several policy proposals are suggested to support policy formulation. Key words: Bacillus thuringiensis (Bt) maize, yieldgard, smallholder farmers, perceptions, Flagstaff, gross margin analysis, binomial logistic regression model, Eastern Cape Province.