Masters Degrees (Plant Pathology)

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Antifungal activity of endophytes from arctotis arctotoides (l.f.) o. hoffm against pythium and rhizoctonia root-rot diseases of maize (zea mays L.)
(2021) Ncumisa, Yekelo.; Yobo, Kwasi Sackey.; Kubheka, Bongani Petros.; Otang- Mbeng, Wilfred.
Maize (Zea mays L.) is one of the predominant crops worldwide, together with wheat (Triticum aestivum L.) and rice (Oryza sativa L.). Approximately 60% of maize produced in South Africa is white maize and is a staple food for many South Africans. About 40% of maize grown is yellow maize and is used for animal feed. About 73% of maize produced in South Africa is produced in the Free State, Mpumalanga, and North West provinces. Maize is grown under various climatic conditions, which sometimes become favourable for disease development. Various plant pathogens such as-, Fusarium spp., Rhizoctonia solani, and Pythium spp. cause diseases in maize. These diseases are usually controlled by cultural practices and fungicides-. However, these control strategies are not always effective, especially against root rot pathogens. Infection of maize plants by Pythium spp. causes brown root lesions, whereas R. solani causes dark-brown root lesions. As an alternative to cultural and chemical control, biological control can be used to mitigate plant diseases. Biological control is based on the premise that the biocontrol agents (BCAs) produce antimicrobial compounds that inhibit pathogens’ growth. BCAs also compete with pathogens for resources in the host plant and rhizosphere. Some BCAs induce systemic acquired resistance in host plants. Endophytes are microorganisms that dwell within tissues in their host plants without any visible symptoms, and can be used as BCAs against plant diseases. Medicinal plants are host to a distinctive microbiome and are an excellent source of bioactive compounds which can be applied in agriculture, medical and pharmaceutical fields. Previous studies have shown that endophytes from medicinal plants are involved in producing secondary metabolites in their host plants. These endophytes impact the functioning of antioxidant enzymes, resulting in activated defence mechanisms against pathogens. Arctotis arctotoides (L.f) O. Hoffm is a medicinal plant used as pastes or decoctions against wounds, epilepsy, ringworms and other ailments. There are no reports where this medicinal plant has been tested against plant pathogens hence this study is necessary. In this study, endophytes isolated from A. arctotoides were tested against R. solani and Pythium spp. root rot pathogens of maize. This is based on the premise that endophytes isolated from this plant will inhibit the growth of plant pathogens. Disease-free A. arctotoides plants were collected from various locations in the Eastern Cape Province, Republic of South Africa, and transported to the laboratory to isolate bacterial endophytes. Twenty-six (26) bacterial endophytes were isolated from the roots, stem, and leaves within 24 hours of sampling. These endophytes were screened in vitro for their antifungal activity against R. solani and Pythium spp. root pathogens of maize. The endophytes were identified using Internal Transcribed Spacers (ITS) sequencing. Results of the in vitro screening showed that ten bacterial endophytes were antagonistic to R. solani, whereas-, 11 were antagonistic to Pythium spp. The percentage inhibition ranged from 17-50% and 8-64% for R. solani and Pythium spp. respectively. Only three bacterial endophytes (Bacillus cereus NYR11, Morganella morganii L143 NYR3, and M. morganii KC-Tt-01 NYL20) inhibited the growth of both pathogens significantly. The antagonistic effect of the best ten bacterial endophytes against each root rot pathogen was further evaluated under greenhouse conditions. The bacterial endophytes were applied as seed treatments and pathogens inoculated in the rhizosphere except the control treatments. The parameters measured were: -plant height once a week for six weeks, root length, number of root lesions, root and shoot weight at harvesting. Maize plants treated with the endophytes Bacillus cereus NYR11, Proteus mirabilis NYR9, and Morganella morganii strain DG56-16 NYS3 against R. solani and Myroides odoratus strain 6G NYL18, Alcaligenes faecalis NYS7, and Ralstonia spp. NYR8 against Pythium spp. showed low numbers of root lesions, increased root length, root and shoot weights. These bacterial endophytes showed potential to be used as BCAs against R. solani and Pythium spp. The antagonistic effect of the best three bacterial endophytes against each pathogen was further evaluated as mixtures in the greenhouse. These were B. cereus NYR11, P. mirabilis NYR9, and M. morganii DG56-16 NYS3 against R. solani and M. odoratus strain 6G NYL18, A. faecalis NYS7, and Ralstonia spp. NYR8 against Pythium spp. The mixtures were applied as seed treatments and pathogens inoculated in the rhizosphere except the control treatments. The parameters measured were-, plant height once a week for six weeks, root length, number of root lesions, root and shoot weight at harvesting. B. cereus NYR11 + M. morganii DG56-16 NYS3, and P. mirabilis NYR9 + M. morganii DG56-16 NYS3, significantly reduced the number of root lesions, increased root length and root weight in the presence of R. solani. In maize plants inoculated with Pythium spp. the single applications of Ralstonia spp. NYR8 and M. odoratus 6G NYL18 were better treatments than mixtures. These endophytes, especially M. odoratus 6G NYL18 increased root length, root and shoot weight, reduced the number of root lesions when applied individually. The M. odoratus 6G NYL18 + A. faecalis NYS7 + Ralstonia spp. NYR8 mixture was a better treatment than other mixtures, even though it was not better than the single application M. odoratus NYL18. The potential mode of action of the best three endophytes against each pathogen were evaluated. Modes of action assessed in this study were siderophore production, protein, chitin, and cellulose degradation. Out of six bacterial endophytes evaluated, only Ralstonia spp. NYR8 did not produce cellulase and siderophores. P. mirabilis NYR9 and M. odoratus 6G NYL18 did not produce protease. All the bacterial endophytes were unable to degrade chitin. Other modes of action used by the bacterial endophytes against the pathogens can be further evaluated.
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.
Bacteria and yeasts as potential biocontrol agents for the management of blue mould and green mould diseases of ‘Valencia’ oranges caused by Penicillium italicum and Penicillium digitatum.
(2024) Zondi, Onosizo Asimdumise.; Laing, Mark Delmege.; Bancole , Wonroo Bernice Armelle.
Citrus fruits can be infected by microorganisms that affect the quality of fruit. Penicillium moulds caused by Penicillium digitatum (green mould) and Penicillium italicum (blue moulds) are two of the most problematic plant pathogens that affect the Rutaceae. Penicillium digitatum is a mesophilic fungus that can produce a potential mycotoxin called citrinin that has the potential to be carcinogenic to animals and humans. Management strategies in place to control these pathogens such as synthetic fungicides provide control, however they have impacts on the environment which does not appeal to consumers. This study aimed to assess the efficacy of bacterial and yeast strains isolated from leaves and peels of lemon (Citrus lemon), oranges (Citrus sinensis) and limes (Citrus aurantiifolia) in controlling P. digitatum and P. italicum in laboratory and fruit storage trials. In the in vitro studies, 102 isolates were screened against P. digitatum and P. italicum using the dual culture assay on petri dishes. The best seven isolates were selected for secondary in vitro screening against both fungi. The best two isolates from the secondary screening against P. italicum were UK37 (96.89%) and SCO13 (94.23%) and for P. digitatum, UK37 (98.83%) and SCO13 (95.29%). Isolates UK37 and SCO13 were sent to Inqaba Biotech Industries (Pty) Ltd for molecular characterization and identification. Both isolates were identified as Bacillus amyloliquefaciens. Bacillus amyloliquefaciens strain SCO13 and B. amyloliquefaciens strain B UK37 were used in in vivo experiments to assess their efficacy in controlling P. digitatum and P. italicum on oranges ‘Valencia’ stored at 25 ℃ for ten days. Bacillus amyloliquefaciens strain SCO13 showed potential as a biological control agent against both P. digitatum and P. italicum, with a low disease incidence of 19.54% and 35.87% respectively. Two yeast strains of Meyerozyma guilliermondii that were previously isolated, were used in in vivo experiments to assess their efficacy in controlling P. digitatum and P. italicum on oranges ‘Valencia’ stored at 25 ℃ for ten days. Yeast treatments provided the least control of the Penicillium moulds on oranges. The SEM images of the mycelial growth of P. digitatum Meyerozyma spp. displayed antagonism by growing on P. digitatum hyphae and colonizing orange fruit surfaces. micrographs showed the bent hyphae and reduced number of conidia in vitro against B. amyloliquefaciens spp., with B. amyloliquefaciens B13 completely colonizing and growing over P. digitatum in vitro for seven days, incubated at 25℃. In conclusion, B. amyloliquefaciens demonstrated potential in controlling P. digitatum and P. italicum infection on oranges. These Bacillus spp. have the potential in the integrated pest management of Penicillium spp.
Biocontrol agents in combination with moringa oleifera leaf extract for integrated control of botrytis cinerea of tomato.
(2022) Malevu, Nonkanyiso.; Mbili, Nokwazi Carol.; Magwaza, Lembe Samukelo.
Tomatoes and tomato-based foods provide essential nutrients beneficial to human health. Despite these benefits from tomatoes, postharvest losses result in unprofitable tomato production in some parts of the world. During ripening and harvesting, tomato becomes susceptible to diseases resulting in shorter shelf life. Susceptibility of tomato plants to Botrytis cinerea which causes grey mould infection can occur at any growth stage and the most susceptible growth stage is during ripening and senescing. Factors such as mechanical injuries, inadequate storage conditions, inappropriate handling, and transport affect tomato quality. The use of chemicals not only negatively affects farmers' yield by further enhancing pesticide resistance to crop pathogens but also influences other sectors of communities through contamination of drinking water sources which is an environmental hazard. There is a need to emphasise and encourage sustainable agricultural strategies such as biological control and plant extracts as alternative strategies which are eco-friendly and economically sustainable. Therefore, the main aim of this research was to examine the effect of biocontrol agents and Moringa oleifera leaf extract, individually and in combination, to control B. cinerea on tomatoes in vitro and in vivo. A total of 48 biocontrol agents were isolated from different parts of tomato leaves, citrus leaves, mushrooms and erect prickly pear. The isolates were screened against B. cinerea for the inhibitory effect and as potential control of the pathogen on potato dextrose agar (PDA) and tomato fruits. Serratia marcescens, Bacillus pumilus and Bacillus safensis inhibited B. cinerea by more than 50% in vitro. During in vivo screening, Serratia and Bacillus isolates inhibited grey mould incidence on ‘Jam’ tomatoes by more than 70%. The scanning electron microscopy images of the pathogen samples treated with biocontrol agents showed swollen and lysed mycelia. Moringa leaf extracts (MLE) were prepared into four concentrations MLE 1%, MLE 2%, MLE 3% and MLE 4%. The MLE concentrations were tested for their antifungal activity on the pathogen growth during in vitro studies. High concentrations were found to have some inhibitory effect on the mycelial growth of B. cinerea. There was no significant difference observed in the control, MLE 1% and MLE 2% since no mycelial inhibition was observed after 7 days at 25°C. For in vivo studies, all the concentrations had some inhibitory effect against grey mould on ‘Jam’ tomatoes. This was evidenced by lower disease incidence observed on the fruits treated with the moringa leaf extract compared to the control treatment. Scanning electron micrographs showed morphological changes in the hyphae on the samples treated with concentrations of MLE and there was also a breakage on the pathogen hyphae. Furthermore, this study evaluated the integrated control of B. cinerea using S. marcescens, B. safensis and B. pumilus integrated with MLE 2% and MLE 3% in vitro and in vivo. S. marcescens, B. safensis and B. pumilus integrated with MLE 2% and MLE 3% successfully suppressed mycelial growth of B. cinerea in vitro. Treating tomato fruits with S. marcescens, B. safensis and B. pumilus integrated with MLE 2% and MLE 3% reduced the disease incidence of grey mould compared to the control. The SEM images of the mycelial growth of B. cinerea showed shrinkage, and breakage of pathogen mycelia and the spores were damaged showing breakage and immature spores both in vitro and in vivo. Integrating moringa leaf extract, Serratia spp. and Bacillus spp. have the potential to be an alternative to synthetic fungicides to control postharvest pathogens.
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 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 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 gray leaf spot (Pyricularia grisea (cooke) sacc.) of ryegrass.
(2017) Dammie, Nompumelelo.; Yobo, Kwasi Sackey.
Abstract available in PDF file.
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.
Characterisation of fluorescent Pseudomonas species causing foliar diseases of tomato in South Africa.
(2019) Langa, Nokubonga Angel.; Yobo, Kwasi Sackey.; Goszczynska, Teresa.
Dissertation summary available in pdf.
Characterization of potato virus Y (PVY) isolates infecting solanaceous vegetables in KwaZulu-Natal (KZN), Republic of South Africa (RSA)
(2009) Ibaba, Jacques Davy.; Gubba, Augustine.
Potato virus Y (PVY) is an economically important virus worldwide. In South Africa, PVY has been shown to be a major limiting factor in the production of important solanaceous crops, including potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana spp). The variability that PVY displays, wherever the virus occurs, merits the study of the isolates occurring in KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). This characterization will provide a clear understanding of strains/isolates from local vegetables and how they relate to the other PVY strains already identified, as well as information that can be used to manage the diseases they cause. Hence, the aim of this project was to study the biological and genetic properties of PVY isolates infecting potato, tomato and pepper in KZN. Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and reverse transcription polymerase chain reaction (RT-PCR) using primers specific to all PVY strains were used to detect the virus in plant material showing PVY-like symptoms collected from various locations in KZN. A total of 39 isolates (18 isolates infecting tomato, 12 infecting potato and 9 infecting pepper) were further differentiated into strains by means of ELISA using strain specific antibodies and RT-PCR using primers specific to the different strains of PVY identified around the world. All PVY isolates infecting tomato and pepper tested positive for the ordinary PVYO strain with both ELISA and RT-PCR. PVY isolates infecting potato were more diverse and comprised the PVYN, PVYNTN and PVYNWilga strains, with mixed infections noted in some cases. The biological properties were studied by mechanically inoculating Chenopodium quinoa, Nicotiana tabacum cv Xanthi, N. tabacum cv Samsun, N. glutinosa, and N. rustica with leaf extracts from plants infected with the different PVY strains detected in this study. All inoculated C. quinoa plants did not show symptoms. All tobacco plants showing symptoms were tested for the presence of PVY by means of ELISA using monoclonal antibodies targeting all strains and electron microscopy using the leaf dip technique. Not all the inoculated tobacco tested positive with ELISA. The symptoms observed were therefore divided into PVY-related and PVY non- related. PVY-related symptoms included vein clearing, mosaic chlorosis, stunting, and vein necrosis. PVY non-related symptoms included wrinkles and leaf distortions. Potyvirus-like particles of about 700 nm were observed under the transmission electron microscope (TEM) from plants showing PVY-related symptoms while rod shaped viral particles of sizes varying between 70 and 400 nm were observed from plants showing non-PVY related symptoms. A portion of the virus genome (1067 bp) covering part of the coat protein gene and the 3’ non-translated region (NTR) of three PVYO isolates infecting tomato, one PVYO isolate infecting pepper and one PVYNWilga isolate infecting potato were amplified, cloned and sequenced. The 5’ NTR, P1, HC-Pro and part of P3 regions (2559 bp) of a PVYN isolate infecting potato were also amplified, cloned and sequenced. Sequence data was compared with selected PVY sequences from different geographical locations around the world. These were available on the NCBI website and subsequently used for phylogenic analyses. The sequenced genomic regions of the PVYN isolate were found to be 99% similar to the New Zealand PVYN isolate (GenBank accession number: AM268435), the Swiss PVYN isolate CH605 (X97895) and the American PVYN isolate Mont (AY884983). Moreover, the deduced amino acid sequence comparison of the genomic regions of the PVYN isolate revealed the presence of five distinct amino acids residues. The three amino acid residues (D205, K400, and E419), which determine the vein necrosis phenotype in tobacco, were also identified. The coat protein and 3’ NTR sequences of all KZN PVYO isolates infecting pepper and tomato were closely similar to each other than to KZN PVYNWilga isolate infecting potato. The phylogenic analysis clustered the KZN PVYN isolate with the European sublineage N, PVYNWilga isolate infecting potato with the American PVYO isolate Oz (EF026074) in the O lineage and all PVYO isolates infecting tomato and pepper in a new sublineage within the O lineage. Taken together, these results point to the presence of PVY in solanaceous vegetables cultivated in KZN and they lay the foundation for the formulation of effective control measure against PVY diseases in KZN.
Chemical control of soybean rust (Phakopsora pachyrhizi) on soybeans.
(2005) Du Preez, Eve Diane.; Caldwell, Patricia May.; Laing, Mark Delmege.
Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. is an aggressive wind dispersed fungal disease which has spread around the world at an alarming rate in the last decade. The disease was first reported in South Africa (SA) in 2001. It has become well established in the province of KwaZulu-Natal. Reports are occasionally made from eastern Mpumalanga, late in the growing season, in years with good rainfall. Yield losses of 10 - 80% have been reported due to SBR infection. Literature was reviewed to better understand the pathogen in an attempt to find suitable disease management strategies. Many strategies involve delaying, rather than preventing, SBR infection. Of the two strategies to prevent infection, the use of fungicides was the only option for disease control in SA, as no resistant cultivars are available. Field trials were conducted to determine which fungicides are effective in controlling SBR. Further research was conducted to determine the timing, frequency and rate of fungicide applications for optimal control of SBR. Trials were evaluated for disease severity, seed yield and the effect of fungicides on seed quality. Fungicides from the triazole class of the sterol biosynthesis inhibiting group of fungicides were found to be the most effective in controlling SBR. A fungicide from the strobilurin group was found to be less effective than the triazoles at the suggested rate, but was found to be as effective when evaluated at higher dosage rates. Triazoles premixed with fungicides from the benzimidazole and strobilurin groups were also effective in controlling SBR. Timing of application was found to be critical for strobilurin fungicides, but not for triazole fungicides, which have a curative ability, unlike strobilurins. Strobilurin fungicides applied preventatively, before the appearance of disease symptoms were as effective as triazole fungicides applied after disease symptoms, but before infection levels had reached 10%. Across both wet and dry seasons two fungicide applications applied at 21d intervals at the R2 growth stage resulted in effective disease control. In wet seasons, a third fungicide application resulted in yields that were higher, albeit not statistically significant, than two fungicide applications. Assessments of individual fungicides for optimal dosage rate found that registered rates were already optimal for some fungicides, but for others it appeared as if alterations were necessary to the rate suggested for registration. This study was one of the first to extensively evaluate the efficacy of the new triazole and strobilurin fungicides on SBR control. The results have been shared globally, but particularly with newly affected countries in South and North America. Although this research has been groundbreaking, there are still many aspects of fungicide control which need to be studied in order to further optimise chemical control of SBR.
Control of insect-transmitted viruses in cucurbit crops in KwaZulu- Natal.
(1998) Cradock, Kenwyn Roan.; Da Graca, John Vincent.; Laing, Mark Delmege.
The production of cucurbits (Cucurbitaceae) in KwaZulu-Natal faces the constant threat of viral diseases. These can be so severe as to severely limit or prevent production in the latter part of the growing season (December-April). The important viruses in this regard are zucchini yellow mosaic potyvirus (ZYMV), watermelon mosaic 2 potyvirus (WMV2), watermelon mosaic potyvirus - Morocco strain (WMV-M), papaya ringspot potyvirus - type W (PRSV-W), cucumber mosaic cucumovirus (CMV), and squash mosaic comovirus (SqMV). The potyviruses and CMV are vectored by aphids (Homoptera: Aphididae) and SqMV is vectored by cucumber beetles (Coleoptera: Coccinelidae). PRSV and SqMV were found to be absent from the region, while CMV was found not to be a serious threat to cucurbit production. ZYMV, WMV2 (now confirmed to occur in South Africa) and WMV-M are the major viral pathogens of cucurbits in KwaZulu-Natal. The distribution of these viruses and methods for their control were investigated. Investigations of aphid morphology using the scanning electron microscope were undertaken to determine if taxonomic studies could be conducted using this form of microscopy. The best form of specimen preparation was the cryo-fixation technique, which resulted in less collapse of the body wall and general damage to the specimen when compared to the critical point drying technique. Due to the lack of mobility of the specimen while viewing, this form of microscopy is rejected as a means of identifying aphids to the species level. ZYMV was found to occur in a number of weed species (Galinsoga parviflora, Malva parviflora, Amaranthus sp., Solarium spp.), which could serve as reservoirs of virus. WMV-M and CMV were also found in some weed species. All tests for the potyviruses and SqMV were done using the double-antibody sandwich (DAS) ELISA technique. CMV was tested for using indirect ELISA tests. A third of the plants tested were found to be infected with more than one virus which could have implications for disease severity. Disease severity was found to increase at about midway through the growing season (December- January). This was concurrent with a massive increase in the general aphid population in the experimental area. As no aphids were seen on the cucurbits in the fields, these vectors are believed to be transient inhabitants of the crop at first testing and then rejecting the plants as a food source. All control measures applied in the trial were aimed at reducing the numbers of aphids in the plots. Aphids were trapped using yellow sticky traps. Cucurbita pepo (zucchini) was used in the trials due to its bush growth habit and good virus symptom expression. The success of the treatments was determined by monitoring the numbers of aphids present in the plots, and the use of a rating scale which assessed the severity of virus disease in the plots. The two best treatments were the white reflective mulch and the straw mulch. In the cultivar trial which assessed ten different cultivars for their virus resistance/tolerance. The best three cultivars were 'SQ 229', 'Puma', and 'SQ 228'. 'SQ 229' and 'SQ 228' were withdrawn from the market by the seed company for unknown reasons. From the results obtained from these investigations, a disease management programme can be suggested. All cucurbit crops should be grown over a white reflective mulch, drip irrigation should be used to reduce agitation of the plants which could unnecessarily disturb feeding aphids, and a resistant or tolerant cultivar should be used in the latter half of the growing season. The effectiveness of any treatment can be assessed by comparing the number of aphids caught with the number caught in the control plots.
Control of the sweet potato weevil (cylas puncticollis) with entomopathogenic nematodes.
(2023) Zulu, Sinethemba.; Ramakuwela, Tshimangadzo.; Laing, Mark Delmege.
Sweet potato (Ipomoea batatas L) is one of the important crops worldwide because of its high calorific value. Sweet potato weevils (SPW) are the major pest affecting sweet potato, leading to a massive yield loss annually. An initial goal was to evaluate the diversity, incidence and damage severity caused to sweet potato by SPW in fields and in storage in three provinces, and the knowledge of small scale farmers about SPW. A survey was conducted in the Gauteng, North West and Limpopo provinces. Most of the farmers (96%) planted sweet potato for income generation, 3% farm for home consumption and 1% farm for employment. About 64% farmers knew about SPW, 28% had no knowledge and 8% were not sure. The crop was worst affected by SPW (79%), with rats, porcupines and other pests posing less of a problem (12%, 8% and 1%, respectively). Farmers were affected by SPW throughout the year. Farmers agreed that SPW had a negative impact on their production, with 34% of them expressing this sentiment, 28 were not sure and 14% did not think that SPW affected their crop yields. About 67% lost a quarter of their yield annually, 27% of the farmers lost a third of their yield and 6% lost half of their yield. All the three provinces are affected by SPW. Despite the majority of the farmers being aware of SPW damage, they had little knowledge of how to control the pest. Entomopathogenic nematodes (EPNs) have potential as biocontrol agents of economically important pests. This study was conducted to explore the potential of EPNs for the biological control of the SPW, Cylas puncticollis (Boheman) 1883, in an artificially infested field using larvae-infested sweet potato pieces placed in perforated containers. A total of six treatments were applied in the field, namely A - control, B - chemical insecticide, C - Steinernema tophus Cimen isolate ROOI 352 Formulation 1 with 2% Barricade® gel, D - S. tophus Formulation 2 (cadavers) E - Heterorhabditis bacteriophora Poinar, isolate SGI 245 Formulation 1 with 2% Barricade® gel, F - H. bacteriophora Formulation 2 (cadavers). After two weeks, larvae, pupae and adult SPW numbers were counted to determine the SPW populations. Heterorhabditis bacteriophora (Treatment F, cadaver formulation) was the most effective treatment with 0% live larvae and 36% dead larvae. Larvae, pupue and adult weevils were evaluated, Treatment F obtained 0% for live LPW and 48% for dead LPW. This short-term field trial demonstrated a successful reduction of all life stages of SPW by H. bacteriophoraSGI 245. Further trials of this EPN for the control of SPW are needed throughout the crop production cycle. Entomopathogenic nematodes (EPNs) are of current research interest because of their ability to kill insect pests as biocontrol agents. However, EPNs are sensitive to ultraviolet (UV) light, high temperatures and desiccation. The aim of one study was to analyse the survival and efficacy of two strains of EPNs, produced either in vivo or in vitro, and formulated in either Barricade® gel or Potassium polyacrylate hydrogel (PPH) formulations. EPNs were produced in vivo using Galleria mellonella (Fabricius) 1798 (the greater wax moth), and in vitro using an artificial medium containing ground, desiccated larvae of Musca domestica (Linnaeus), the housefly. IJs of the three selected strains of EPNs were suspended in 2% gel formulations of Barricade® gel or PPH by mixing 98 ml of water containing infective juveniles (1000 IJs / ml) with 2 g of Barricade® gel or PPH. The formulations were stored in 2 ml Eppendorf tubes held at 15˚C. The survival of the IJs was then tested at two week intervals. The efficacy of formulated IJs was tested by infecting 10 mealworms with IJs (1000 IJs / ml), incubated for 72 hours at 25oC, and counting the number of dead mealworms at two weeks intervals. On Day One, the survival of the EPNs of both isolates, in both formulations, and the controls were 100%. After two weeks, the controls had zero IJs survival for all the isolates, produced either in vivo or in vitro. The three isolates in both the Barricade® gel and PPH formulations had a zero IJs survival after eight weeks. On Day One, the mortality of mealworms was 100% for all the formulations. Both formulations of the three isolates caused zero mortality of mealworms after eight weeks. The use of Barricade® gel and PPH in formulating three isolates of EPNs enhanced their survival for up to six weeks. The in vitro-produced EPNs had a higher level of survival than the in vivo produced EPNs. However, the in vivo production resulted in EPNs that were more effective at killing mealworms than the in vitro produced EPNs. However, these differences were marginal. Differences between the three strains of EPNs were also marginal. Entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae, coupled with their symbiotic bacteria, are utilised for the biological control of a wide range of agricultural insect pests. Persistence of EPNs in the field is affected by a number of abiotic factors such as UV light, fluctuation in temperature and desiccation. The aim of this study was to assess the persistence of EPNs under field conditions, applied in two different formulations (cadaver formulation and Barricade® gel formulation). Persistence was assessed after one and two months post treatment application by baiting soil with the greater wax moth larvae (Galleria mellonella) and observing the insect mortality. Heterorhabditis bacteriophora (SGI 245) cadaver formulation showed better survival in all the five sites, followed by H. bacteriophora Barricade® gel formulation. Persistence declined drastically two months post treatment from a maximum 100% to 0%. The findings suggest that EPNs post application survival in the field can be enhanced by both cadaver and Barricade® gel formulations. Sweet potato weevils is one of the most important insect pests of sweet potato. The control of SPW is difficult due to the cryptic nature of the larvae, and night activity of the adults. In Africa, control of SPW relies primarily on the use of synthetic insecticides. However, the insect has developed some level of resistance against a wide range of insecticides. Entomopathogenic nematodes (EPN), which are cosmopolitan soil-borne entomopathogens, have gained interest as potential biological control agents of various economically important insect pests. The main aim of this study was to evaluate the potential of EPN isolates from the families Heterorhabditidae and Steinernematidae as biological control agents of SPW and their effect on different sweet potato cultivars, under field conditions. Plant resistance has also been viewed as an efficient, cost-effective and environmentally-safe form of pest control methods of SPW under field conditions. Four different cultivars were planted and six biocontrol treatment were applied after a month. Overall, both the cadaver and Barricade® gel formulations of H. bacteriophora SGI 245 were more effective in reducing the plant damage caused by SPW than the Barricade® gel formulation of S. tophus ROOI 352. Overall, the cultivar Monate suffered less insect damage (0.103) than the cultivars Blesbok and Bophelo, and it was the best yielding cultivar. A combination of Treatment F of H.bacteriophora and the sweet potato cultivar Monate can be recommended for further field testing.
Cultivation of exotic and local mushroom species for commercial production.
(1995) Adey, Samantha.; Laing, Mark Delmege.
The project was undertaken primarily to determine the potential of growing Stropharia rugoso-annulata and Volvariella volvacea at a commercial level under South African conditions. Termitomyces umkowaani, an indigenous mushroom, was also investigated. Mushroom culturing, spawn production and optimizing fruiting substrates were determined. The feasibility of commercial and subsistence production in KwaZulu-Natal was investigated. Of the seven strains of S. rugoso-annulata purchased from CBS (Baarn, the Netherlands) that were tested, Strain 289.85 was the most vigorous. The best agar medium for culturing of S. rugoso-annulata was potato dextrose agar (PDA) followed by malt extract agar (MEA) and vegetable juice agar (V8). Maximum growth of the mushroom mycelium occurred from Day 7 to Day 14, as expected. The best spawn substrate tested was sorghum, and the best fruiting substrates tested were banana leaves and maize stover. As S. rugoso-annulata is a white-rot fungus capable of degrading lignin, this result was expected. S. rugoso-annulata grows best on substrates with a low C:N ratio. Strain 1665 of V. volvacea was the best of the three strains obtained from CBS. The best culture medium for V. volvariella was PDA amended with 1 % straw. V8 agar alone supported poor growth. In trials for suitable spawn and fruiting substrates for V. volvacea, banana leaves supported the best mycelial growth. Mycelium grown on V8 amended with 1% maize stover showed the greatest expansion on the substrates tested. A strain of T. umkowaani was isolated from a fresh basidiocarp collected in Pietermaritzburg, KwaZulu-Natal. Growth of T. umkowaani was most rapid on a Basal Medium but mycelial vigour was inferior to that of mycelium grown on Basal Medium amended with rabbit faeces, especially at the level of 2.5%. High levels of contamination occurred when more rabbit faeces was added to the Basal Medium. Microbial contamination was a major limiting factor to the productivity of this project. The primary sources of culture contamination were due to mites, inadequate sterile technique and a high level of Trichoderma spores in the Department due to an ongoing Trichoderma biocontrol project. Placing the bags of cultures on trays with the edges smeared with petroleum jelly effectively kept the mites away from the cultures. The contamination of a stock culture with Penicillium was solved with the use of Benomyl-amended agar media. Contamination problems in spawn production were the result of the inoculation process (especially when conducted by one person), the initial leaking of cotton wool caps, polypropylene bag seams and micropores and cracks in the walls of polypropylene bags. Treating the cotton wool in the caps with Busan 30A prevented the entry of contaminant bacteria and fungi through the caps. The problem of leaking seams of the bags was solved by using a longer heating period on the bag-sealer machine. SEM studies confirmed the presence of micropores and stress fractures in the walls of the bags used in this project. This problem can be solved by using high quality polypropylene with a reduced content of plasticiser. A systems analysis of exotic mushroom production, the process of mushroom production, steps in the process, sales and constraints in the South African context, and possible solutions are discussed. Spawn production by small growers is not economically feasible due to the capital required. Access to capital is a constraint of production in controlled environments but should not limit outdoor production. However, outdoor production is constrained by climatic requirements of the fungi. Based on optimum temperatures, GIS maps of KwaZulu-Natal were generated to display the potential areas for outdoor production. Overlap of suitable regions for production of S. rugoso-annulata and V. volvacea is limited. Outdoor production of V. volvacea will be limited to Northern coastal regions for only four months of the year. A computerized decision support system was developed to answer questions of feasibility of production to the mushroom growers, based on the requirements of the mushrooms. A current lack of marketing of speciality mushrooms is considered to be a major constraint to sales and therefore potential production, particularly for subsistence farmers.
Detection, differentiation and genome analysis of potato virus Y isolates infecting potato (Solanum tuberosum L.) in the Msinga area in KwaZulu-Natal, Republic of South Africa.
(2016) Ximba, Sinethemba Patience Fanelwe.; Gubba, Augustine.
Abstract available in PDF file.

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