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.
Application of transmissible agents to control citrus tree size.
(1997) Van Vuuren, Stephanus Petrus.; Da Graca, John Vincent.
Establishing citrus orchards at higher densities has become standard practice in South Africa. These plantings make more efficient use of inputs such as land, fertilizer, irrigation and spraying, and they are capable of high early production and economic returns. However, disadvantages develop over time, depending on tree density and climate. These include overcrowding between and in rows, which induces difficult access by vehicles, poor insect control in dense canopies, and a decrease in yield and quality due to dieback and overcrowding. It is thus important to find a method to control tree size to maintain the benefits of such plantings. All three main tree size controlling methods viz. the use of genetic dwarf plant material, management practises and the use of transmissible agents are currently under investigation in the country. The aim of this study was to identify transmissible agents that can be used successfully to control tree size without detrimental effects. Research was done to identify transmissible agents as well as their application as dwarfing agents for commercial use. An important safety measure that should be considered before a transmissible agent is introduced for tree size control of citrus, is that such an agent should be endemic in the industry. Citrus tristeza virus (CTV) is a well known endemic disease in Southern Africa and virus-free shoot-tip grafted (STG) material is pre-immunised with avirulent isolates to reduce its effect. STG is also used to remove citrus viroids (CVd's) and the use of such agents as dwarfing agents is in contradiction with the aims of the Southern African Citrus Improvement Programme. However, research over many years in Australia has shown that avirulent CVd's can be used succesfully to control tree size. It is thus important to identify suitable CVd isolates for commercial application but it is also important to know which CVd's occur in commercial orchards. The occurrence of CVd's in old commercial citrus orchards, planted prior to the introduction of shoottip grafted material, was established by biological indexing with Etrog citron (Citrus medica L. cv. Arizona-861-S-1). Twenty-one percent of the trees indexed tested positive for CVd's. Sequential polyacrylamide electrophoresis (sPAGE) was employed to establish which viroids are most commonly present. Only two groups of CVd were detected, viz. citrus exocortis viroid (CEVd) and CVd-III. The latter CVd was the most common (40%) while CEVd occurred in only 8% of the samples. More than a third of the samples were infected with both CVd's. Two CVd isolates, a mild and a severe isolate according to Etrog citron reactions, were bud-inoculated to Delta Valencia on rough lemon, Poncirus trifoliata and four trifoliate hybrid rootstocks. Growth, production and occurrence of disease symptoms of these trees were compared to trees on the same rootstocks without CVd. All the trees were pre-immunized with the standard tristeza virus isolate. Results of sPAGE analysis of nucleic extracts indicated that the severe isolate (CD 11) contained CEVd only, and the mild isolate (CD 12) contained CVd-III. Overall, both isolates caused a significant reduction in tree size. The cumulative production over five years of the CD 11 infected trees did not differ from the CVd-free trees although the trees were smaller. This was due to a significantly higher production efficiency (kg/m(3) canopy). The production efficiency of the CD 12 trees was similar to the CVd-free trees, but the smaller trees resulted in a significantly lower production. Disease symptoms occurred with both isolates, but symptoms differed. Poncirus trifoliata var. Rubidoux was more sensitive to CVd isolates than four trifoliate hybrid rootstocks. Marsh grapefruit trees on Troyer citrange rootstock were bud-inoculated with different CTV isolates prior to planting in the field. Selected CTV isolates GFMS 2, GFMS 10, GFMS 12, GFMS 19, GFMS 25, GFMS 27 and GFMS 35, free of citrus viroids, were bud-inoculated into the virus-free plants. A severe isolate (GFSS 1) and plants that were left virus-free served as controls. Tree size, production, fruit size and tree health were determined. Fifteen years after planting, canopy volumes of trees with three isolates, GFMS 2, GFMS 19 and GFMS 25, were significantly smaller than the control trees as well as trees with isolates GFMS 10, GFMS 12 and GFMS 35. Trees with isolate GFMS 19 had a larger diameter than those with isolates GFMS 2 and GFMS 25. Together with a slightly higher yield efficiency, GFMS 19 trees resulted in a cumulative yield equal to that of the control and the GFMS 12 trees. Considering fruit size and their value, the performance of trees with isolate GFMS 19 equalled that of the larger trees. Tree health was also similar which makes this isolate suitable for use in high density plantings. A projection was made which showed that the production and crop value of to trees with isolate GFMS 19 were similar to those of trees with isolates GFMS 10, GFMS 12 and GFMS 35. However, benefits such as easier and better spray application and easier harvesting can increase profits when trees with isolate GFMS 19 are planted. The dwarfing characteristics of four isolates, CD 4, CD 8, CD 9 and CD 10, derived from healthy looking dwarfed field trees were evaluated. They were bud-inoculated to Delta Valencia trees on Yuma citrange rootstock prior to planting in the field. Five years after planting, isolates CD 4 and CD 9 successfully reduced canopy volumes by 60%, and CD 10 by 30%, without any detrimental effects. No CVd's could be detected biologically or by sPAGE in these three isolates. Isolate CD 8 however, contained two viroids, CEVd and CVd-III, but had no deleterious effects on the rough lemon rootstock. CTV was the only other pathogen present in the isolates. Indexing for cachexia, psorosis, impietratura and tatter leaf was negative. The dwarfing abilities of the isolates are therefore attributed to isolates of citrus tristeza virus. Production was according to tree size and the yield efficiency of the inoculated trees was equal to that of the uninoculated control trees. External and internal fruit quality was not affected. The trees were naturally infected with huanglongbing (greening) five years after planting, but the disease remained low for several years in trees with isolate CD 4. Three transmissible isolates (CD 4, CD 9, CD 10), derived from dwarfed field trees, were compared with two CVd isolates (CD 8, CD 12) for their abilities to control tree size of sweet orange. The isolates were bud-inoculated to Valencia on rough lemon, two Poncirus trifoliata and three trifoliate hybrid rootstocks, and compared to uninoculated trees on the same rootstocks. Isolates CD 4, CD 9 and CD 10 gave no reaction on Etrog citron and sPAGE of nucleic acid extracts failed to detect known CVd's. The two CVd isolates gave severe and mild reactions on Etrog citron and sPAGE showed that CD8 contained CEVd and CVd-III while CD 12 contained only CVd-III. The effect of the isolates on tree size, production, production efficiency and disease occurrence were monitored. Overall, CD 4, CD 9 and CD 10 did not reduce canopy volumes while trees with CD 8 and CD 12 were significantly smaller. However, CD 4 significantly reduced canopy volumes where Yuma citrange was used as a rootstock. In contrast, CD 8, containing CEVd, did not reduce canopy volumes on this rootstock, while CD 12 reduced it significantly. None of the trees on the other rootstocks were affected by either CD 4, CD 9 or CD 10. Canopy volume reductions by CD 8 and CD 12 differed from each other with all the trifoliate rootstocks. The production efficiency of trees with the two CVd isolates was significantly higher than the control trees as well as those with CD 4, CD 9 and CD 10. The higher efficiency of these trees resulted in cumulative production equal to the uninoculated trees. Disease symptoms occurred where all the isolates were inoculated, however, symptoms as well as the susceptibility of the rootstocks, differed among each other. Delta Valencia trees on Yuma citrange rootstock were inoculated respectively with two mild (GFMS 12 and T55), an intermediate (GFMS 10) and a severe (GFSS 1) CTV isolate prior to planting in the field. The same CTV isolates were also inoculated in combination with a CVd- III isolate. A virus-free control was included in the trial. All the CTV isolates were without the Seedling Yellows component of CTV. Seven years after planting, canopy volumes of the trees with the two mild isolates and the control were smaller than those of trees with the intermediate and severe isolates. This was in contradiction to what was expected. Overall, the CVd isolate had an additional reducing effect on canopy size, but only those trees with mild isolate T55 and severe isolate GFSS 1 were significantly affected. Production on a per tree basis was according to the canopy sizes, thus, the trees carrying the intermediate CTV isolate were the highest producers. The production efficiency (kg/m(3) canopy), however, did not differ among trees with the CTV isolates and the control. The CVd isolate generally increased the production efficiency. The internal quality of the fruit was not affected by any treatment. The lack of a suitable genetic dwarfing rootstock for citrus makes it essential to evaluate alternative methods to reduce tree size for high density plantings. Four transmissible dwarfing factors, derived from dwarfed trees, were evaluated for commercial application in a hot (Malelane) and intermediate (Nelspruit) production area. Generally, trees in the hot production area were more vigorous with a lower production efficiency than trees in the intermediate area. The dwarfing effects of isolates CD 4, CD 9 and to a lesser extent, CD 10, were reduced in the hot area. Isolate CD 8 caused no dwarfing at either location. The reduction of dwarfing at the hot site may be attributed to the suppression of CTV by high temperatures. Currently some of the isolates which were tested in this investigation are applied on a larger scale in different climatic areas as commercial trials. Formal trials are continuing and they are aimed to elucidate the dwarfing characteristics as well as the inducement of disease of the four viroids in the CVd-III group.
Aspects of 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.
Aspects of post-harvest seed physiology and cryopreservation of the germplasm of three medicinal plants indigenous to Kenya and South Africa.
(2002) Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman W.
The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5.
Banana bunchy top virus in South Africa: the distribution, molecular relationship and transmission studies.
(2021) Ximba, Sinethemba Patience Fanelwe.; Jooste, Anna Elizabeth Catharina.; Gubba, Augustine.
The first report of Banana bunchy top virus (BBTV) in KwaZulu-Natal (KZN), South Africa in 2016 has raised the need to study the virus in South Africa. The aim of this research project was to conduct surveys across banana-producing provinces, including KwaZulu-Natal, Mpumalanga and Limpopo in South Africa, to determine the spread of BBTV in banana production regions across the country. To date, the virus has been localized within the province of KZN in the South Coast region. Once positive samples were obtained, the genetic relationships between the South African isolates and those collected globally was investigated. This was done by studying five (DNA-C, -S; -N; -M; -U3) of the six components of the BBTV genome. No major differences between the isolates were observed. As the virus is only transmitted through infected planting material and through the vector, Pentalonia nigronervosa, BBTV transmission studies were conducted. Such studies have been conducted in different countries on this topic with conflicting results and BBTV transmission studies were included here as well. Plant species namely Colocasia esculenta, Alocasia macrorrhizos, Alpinia zerumbet and Strelitzia reginae, that are usually found growing around banana plantations, were investigated to determine if these plants act as reservoirs of the virus vector and also to determine if these potential alternative host plant can be hosts of the virus. Banana plants were included as controls in the experiment. A qPCR was optimised to test for BBTV in the plants and aphids at low concentrations. BBTV was detected in all of the plants except A. macrorrhizos. It was concluded that A. zerumbet was an alternative host of the banana aphid while C.esculenta and S. reginae are assumed to be intermediary hosts of the virus vector while A. macrorrhizos is neither a host of the vector nor of the virus.
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 and molecular characterization of South African bacteriophages infective against Staphylococcus aureus subsp. aureus Rosenbach 1884, casual agent of bovine mastitis.
(2012) Basdew, Iona Hershna.; Laing, Mark Delmege.
Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification.
Biological characterization of South African bacteriophages infective against Streptococcus uberis, a causal agent of bovine mastitis.
(2019) Pillay, Caleb.; Laing, Mark Delmege.; Basdew, Iona Hershna.
Mastitis is an inflammatory disease of bovine mammary glands and is the most economically important disease affecting dairy herds in South Africa, and globally. The incidence and history of mastitis in South Africa has been extensively documented and found to be bacterial in origin. Streptococcus uberis (S. uberis) is the most common environmental causal agent of mastitis from clinical and subclinical samples in several countries, including Australia, the United Kingdom, New Zealand, and Belgium. Due to the causal agents ability to exist in cells, it remains protected from antibiotics. The quest for antibiotic free dairy management has funded the research into integrated strategies, predominantly proactive udder health management. Biological control methods are not widely used, specifically the use of bacteriophage (phage) therapy. Phages are ubiquitous (found in all biospheres) and are the most abundant organisms on earth. Understanding the interaction between phages and their hosts is vital to their manipulation for therapeutic conditions. This study aimed to isolate phages from unpasteurized milk of dairy cows, sequentially screened these against S. uberis isolates demonstrating antimicrobial resistance at the time. The phages have been screened for robust lytic characteristics for the of intent of a phage-based therapy. Bacterial strains of S. uberis were isolated from unpasteurized milk by Allerton laboratories submitted by dairy farms in KwaZulu-Natal, South Africa. The samples were screened for S. uberis using the following tests: haemolysis patterns on blood agar, catalase reactions using hydrogen peroxide (5%), and Gram reaction. The identity of the strains was then confirmed by Inqaba Biotechnical Industries (Pty) Ltd via sequencing of the 16s ribosomal RNA. The six S. uberis strains were screened against 8 commonly used antibiotics in the dairy industry: β-lactam (ampicillin, penicillin G, cefalexin, oxacillin and amoxicillin), macrolides (erythromycin), tetracyclines (tetracycline) and glycopeptide (vancomycin). Using the Kirby Bauer method and The European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breaking points zone diameters, version 10.0, 2020. Every strain of S. uberis was susceptible to vancomycin, which is not available for intra-mammary infections. Resistance to the 7 other antibiotics varied amongst the strains. This was not a survey study but an isolation of S. uberis strains to challenge the efficacy of phage therapy. Approximately 2,000 mastitic milk samples were screened for phages resulting in the isolation of 95 phages. This was further reduced by screening for constant lytic ability to five phages that were characterized for their phage titre, host range, single step growth phase and lethal dose activity. The titre of the five selected phages varied considerably. Phages CP1, CP2, CP76, CP79 and CP80 produced titres of 59x102, 56x105, 47x106, 50x104 and 35x104 pfu.ml-1, respectively. There was a differential interaction between the five phages isolates and the six strains of S. uberis. One strain of S. uberis was susceptible to all five phages, Strain 21A, whereas Strains 17D and 78B were not susceptible to any of the five phages. Phage CP2 was virulent to four of the six S. uberis strains. The single step growth assay illustrated the cyclic replication of the isolated phages takes between 50 - 60 minutes. In a lethal dose assay, Phages CP1 and CP2 were each able to reduce S. uberis counts by 86% and 83%, respectively. Of the 5 phages screened Phages CP1 and CP2 showed potential as stand-alone treatments, whereas Phages CP76, CP79 and CP80 would offer better control when combined in a phage cocktail, and this would broaden the host range. Phage samples were examined using transmission electron microscopy (JEOL 1400). Various negative stains were used to view the virus particles: 2% uranyl acetate (UA), 2% phosphotungstic acid (PTA) and 0.05-5% ammonium molybdate (AM). The AM staining provided the best images of infected bacterial cells and phage surface structures. All micrographs obtained, illustrated similar viral particle structures suggesting the phages screened belong to one family. A virus particle was measured to have a 50-65nm diameter icosahedral head and a short tail ranging from 25-35nm in length from the EM micrograph. The virus particles exhibited Podoviridae morphology. However, only a complete genomic sequencing will confirm the identity of these virus particles to a species level.
Biological control and plant growth promotion by selected trichoderma and Bacillus species.
(2005) Yobo, Kwasi Sackey.; Laing, Mark Delmege.; Hunter, Charles Haig.
Various Trichoderma and Bacillus spp. have been documented as being antagonistic to a wide range of soilborne plant pathogens, as well as being plant growth stimulants. Successes in biological control and plant growth promotion research has led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of six Trichoderma spp. and three Bacillus spp. and their respective combinations, for the biological control of Rhizoctonia solani damping-off of cucumber and plant growth promotion of dry bean (Phaseolus vulgaris L.). In vivo biological control and growth promotion studies were carried out under greenhouse and shadehouse conditions with the use of seed treatment as the method of application. In vitro and in vivo screening was undertaken to select the best Trichoderma isolates from 20 Trichoderma isolated from composted soil. For in vitro screening, dual culture bioassays were undertaken and assessed for antagonisms/antibiosis using the Bell test ratings and a proposed Invasive Ability rating based on a scale of 1-4 for possible mycoparasitic/hyperparasitic activity. The isolates were further screened in vivo under greenhouse conditions for antagonistic activity against R. solani damping-off of cucumber (Cucumis sativus L.) cv. Ashley seedlings. The data generated from the in vivo greenhouse screening with cucumber plants were analysed and grouped according to performance of isolates using Ward‟s Cluster Analysis based on a four cluster solution to select the best isolates in vivo. Isolates exhibiting marked mycoparasitism of R. solani (during ultrastructural studies) viz, T. atroviride SY3A and T. harzianum SYN, were found to be the best biological control agents in vivo with 62.50 and 60.06% control of R. solani damping-off of cucumber respectively. The in vitro mode of action of the commercial Trichoderma product, Eco-T®, and Bacillus B69 and B81 suggested the production of antimicrobial substances active against R. solani. In vitro interaction studies on V8 tomato juice medium showed that the Trichoderma and Bacillus isolates did not antagonise each other, indicating the possibility of using the two organisms together for biological control and plant growth promotion studies. Greenhouse studies indicated that combined inoculation of T. atroviride SYN6 and Bacillus B69 gave the greatest plant growth promotion (43.0% over the uninoculated control) of bean seedlings in terms of seedling dry biomass. This was confirmed during in vivo rhizotron studies. However, results obtained from two successive bean yield trials in the greenhouse did not correlate with the seedling trials. Moreover, no increase in protein or fat content of bean seed for selected treatments was observed. In the biological control trials with cucumber seedlings, none of the Trichoderma and Bacillus combinations was better than single inoculations of Eco-T®, T. atroviride SY3A and T. harzianum SYN. Under nutrient limiting conditions, dry bean plants treated with single and dual inoculations of Trichoderma and Bacillus isolates exhibited a greater photosynthetic efficiency that the unfertilized control plants. Bacillus B77, under nutrient limiting conditions, caused 126.0% increase in dry biomass of bean seedlings after a 35-day period. Nitrogen concentrations significantly increased in leaves of plants treated with Trichoderma-Bacillus isolates. However, no significant differences in potassium and calcium concentrations were found. Integrated control (i.e. combining chemical and biological treatments) of R. solani damping-off of cucumber seedlings proved successful. In vitro bioassays with three Rizolex® concentrations, viz., 0.01g.l-1, 0.1g.l-1 and 0.25g.l-1 indicated that the selected Trichoderma isolates were partly sensitive to these concentrations whereas the Bacillus isolates were not at all affected. In a greenhouse trial, up to 86% control was achieved by integrating 0.1g.l-1 Rizolex® with T. harzianum SYN, which was comparable to the full strength Rizolex® (1g.l-1) application. Irrespective of either a single or dual inoculations of Trichoderma and/or Bacillus isolates used, improved percentage seedling survival as achieved with the integrated system, indicating a synergistic effect. The results presented in this thesis further reinforce the concept of biological control by Trichoderma and Bacillus spp. as an alternative disease control strategy. Furthermore, this thesis forms a basis for Trichoderma-Bacillus interaction studies and proposes that the two organisms could be used together to enhance biological control and plant growth promotion.
Biological control of fusarium wilt of pine seedlings using endophytic microorganisms and silicon.
(2015) Gqola, Bomikazi Nobesuthu.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Globally, pitch canker fungus (Fusarium circinatum) poses a serious threat to the softwood forest industry. In South Africa, F. circinatum has characteristically been a nursery pathogen, causing a seedling wilt, and has attacked primarily Pinus patula and P. radiata seedlings and cuttings. With P. patula being the most commercially important species in southern Africa, this creates a substantial economic problem. There are no effective control methods to date. The overall study objective, therefore, was to isolate endophytic microorganisms from healthy pine seedling and seeds, and to screen these for activity against F. circinatum in pine seedlings. A second primary objective was to test soluble silicon (Si), applied as potassium silicate for its potential to control of Fusarium wilt of pine seedlings caused by F. circinatum. For the study to be carried out, standard methodologies and procedures had to be followed, which included the isolation of the pathogen and endophytes that were used in experiments reported in the subsequent chapters. A pathogenic strain of F. circinatum was isolated. One hundred and fifty isolates of bacterial and fungal endophytes were also isolated from the seeds of seven different species of pine and 110 seedlings and cuttings of various species of pine. For the successful screening of resistant cultivars, and control agents against F. circinatum, a reliable and representative artificial inoculation technique was needed. A study was undertaken to test various inoculation techniques, aiming to develop a reliable inoculation technique that would mimic the natural infection process of F. circinatum in the field, and to investigate the spore load required to initiate disease, without applying an excessive inoculum. Three inoculation techniques were tested using six P. patula hybrids/species. These included drenching with a conidial suspension, injection of the crown with conidia, and a wounding technique as developed by FABI, University of Pretoria, which involves cutting off a few centimetres of the apical shoot and inoculating conidia onto the wounded tissue. Using a haemocytometer, the spore load was adjusted to two inoculum levels, namely 102 and 106 conidia ml-1. The concentration of conidia had a significant effect (p<0.05) on the Area Under the Disease Progress Curve (AUDPC). A concentration of 106 conidia ml-1 caused more severe F. circinatum symptoms and more severe disease. Inoculation techniques also had a significant effect on AUDPC (p<0.001). The highest infection levels were achieved when plants were wounded by cutting of the top or by injection. However, drenching was a simple and reliable inoculation technique. The interactions between concentration of conidia and inoculation techniques was not significant (p>0.05). Choice of P. patula hybrid had a significant effect (p<0.05) on the AUDPC. There was a significant interaction (p<0.05) between the hybrids and the inoculation technique, where drenching was more effective at discriminating the different levels of resistance of the six tested hybrids. Wounded seedlings were all equally diseased, which reflects the failure of these two inoculation techniques to provide satisfactory inoculation. Endophytic microorganisms that were isolated from selected healthy pine seedlings, cuttings and seeds were screened for their potential as biological control agents against Fusarium wilt. Young P. patula seedlings were drenched weekly for four weeks with 5 ml of each endophyte (106 c.f.u ml-1). A pathogenic strain of F. circinatum was then inoculated onto the plant and the plants were subjected to drought stress for a week. The primary screening of the endophytes produced up to 60% reduction of the disease. The eighteen best endophyte isolates were selected for further screening. In the secondary screening, Isolates E56, E8 and E51 were the most effective biological control agents, while Isolate E85 was the least effective. Isolates E 141, E12, E13 and E27 provided a limited but significant level of control.
Biological control of gastrointestinal nematodes of small ruminants, using Bacillus thuringiensis (Berliner) and Clonostachys rosea (Schroers).
(2011) Baloyi, Mahlatse Annabella.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Gastrointestinal nematode parasites cause great losses in the production of small ruminants through reduced productivity and the cost of preventive and curative treatments. Because of the threat of anthelmintic resistance, biological control of sheep nematodes has been identified as an alternative to anthelmintic drugs. Bacillus thuringiensis (Bt) (Berliner) and Clonostachys rosea (Schroers) have been widely studied as biocontrol agents. B. thuringiensis has been used for the biocontrol of insects and C. rosea has been successfully used as biocontrol agent of Botrytis cinera (De Bary) in plants. B. thuringiensis and C. rosea strains were isolated from soil collected from the Livestock Section at Ukulinga Research Farm, University of KwaZulu Natal, Pietermaritzburg. Twenty-five strains of Bt and 10 strains of C. rosea were successfully isolated. The Bt colonies were identified by their circular, white, flat and undulate character, and the gram-positive and rod-shaped endospores. C. rosea was identified by white colonies on Potato-dextose agar and the characteristic conidiophores, which were branched and showed phialides at the tips. In vitro screening of the isolates was undertaken to select the best isolates. The isolates that caused significantly greater mortality were Bt isolate B2, B10 and B12 and C. rosea isolates P1, P3 and P8. These isolates caused substantial nematode mortality in both faeces and water bioassay. Nematode counts were reduced by 28.5% to 62% and 44% to 69.9% in faecal bioassay for Bt and C. rosea, respectively. In the water bioassay, nematode counts were reduced by 62% to 85% for Bt and by 62.7% to 89.3% for C. rosea. The best inoculum level at which the best isolates were most effective, and the optimum frequency of application were determined. The trial was conducted using bioassays with faeces and water. Inoculum levels of 10(6), 10(8), 10(10), 10(12) spores ml-1 for Bt and 10(6), 10(8) and 10(10) conidia ml-1 for C. rosea was used in the faecal bioassay. The inoculum levels tested in water bioassay were 10(6), 10(8), 10(10) and 10(12) spores ml-1 for Bt and 10(9), 10(10), 10(11), 10(12) conidia ml-1 for C. rosea. In the faecal bioassay, B2 was the most effective Bt isolate at an inoculum level of 10(10) spores ml-1. Isolate P3 was the best C. rosea isolate at 10(8) conidia ml-1. In the water bioassay, Isolate P3 caused a mortality of 85% at inoculum levels of 10(9), 10(10) and 10(11) conidia ml-1. The performance of biological control agents in the field is sometimes inconsistent. Combining different biocontrol agents may be a method of improving their reliability and performance. However, the combination of most of the isolates was antagonistic, with efficacy less than that of either individual biocontrol agent. In particular, Isolate P3 was more effective when used alone than when combined with any other isolates. Therefore, the combination of biocontrol agents does not always result in synergistic interaction. There were some additive interactions between two bacterial isolates, and with one bacterial and fungal combination. The effect of feeding the best of the biocontrol agents, or diatomaceous earth (DE), was evaluated in sheep. Two doses of Bt (1g and 2g kg-1BW) and C. rosea (1g kg-1BW) reduced the numbers of L3 nematode larvae in sheep faeces. The DE product (at 15% of feed) also reduced L3 numbers but it was less effective than either the Bt or the C. rosea products. Nematode counts were reduced by 74.6%, 75.1%, 84.6%, 68.5% and 27.5% for Bt 1g kg-1BW, Bt 2g kg-1BW, C. rosea (1g kg-1 BW), DE and control, respectively.
Biological control of 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.
Biological control of sorghhm and rice stem borers, chilo partellus and sessamia calamistis using endophytic strains of beauveria bassiana.
(2018) Bancole, Wonroo Bernice Armelle.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Sorghum and rice are two of the major cereals grown across the world. Both of these crops are subjected to a range of abiotic and biotic constraints. Insect pests are important biotic stress factors, which affect both of the crops at all of their growth stages. Stem borers from the family of Lepidoptera, e.g. Chilo partellus (Lepidoptera: Pyralidae) and Sesamia calamistis Hampson (Lepidoptera: Noctuidae) are important pests that attack these cereals. Control of C. partellus and S. calamistis has largely been with pesticides. However, chemical pesticides are too expensive for most small-scale farmers in Africa, leaving their crops unprotected. Biological control is one of the measures that have been advocated for the management of stem borers. Various strains of Beauveria bassiana (Vuillemin) have been documented as being endophytes infecting a wide range of plants, as well as being pathogenic on numerous insect pests. Successes in biological control research have led to the development of various B. bassiana products, which are available commercially, but these are largely epiphytic strains. Biological control studies were therefore conducted with several endophytic strains of B. bassiana against sorghum stem borer, C. partellus, and the rice stem borer, S. calamistis. The fungi were tested by endophytic behaviour and the ability to control the 3rd larval instars of both stem borers, in the laboratory and greenhouse. The interactions of B. bassiana strains and a commercially available Trichoderma product, Eco-T®, were tested in sorghum and rice plants. In vivo and in vitro screening were initially undertaken to evaluate the endophytic behaviour of 20 B. bassiana strains, using two inoculation methods. Subsequently, the best endophytic B. bassiana strains and the best inoculation method were tested at 30 and 60 days after inoculation. The strains were screened in vivo using seed treatments and foliar sprays, under greenhouse conditions, for endophytic behaviour in sorghum and rice plants. There were highly significant differences between the B. bassiana strains (P = 0.0001). Depending upon the inoculation method, the B. bassiana strains that successfully colonized the sorghum and rice plants could be selected after 30 and 60 days. Five strains of B. bassiana strains (Bb3, Bb4, Bb10, Bb21 and Bb35) were found to be endophytic in both crops, and to provide biological control against the two borers. The best five B. bassiana strains were tested for their pathogenicity on the 3rd instar larvae of C. partellus and S. calamistis. Out of the five endophytic strains of B. bassiana, two (Bb35 and Bb3) were the most pathogenic on C. partellus, with the greatest mortality of 80 % being achieved within 28 days after treatment. The B. bassiana strains Bb35 and Bb4 were the most effective strains against S. calamistis, killing 93.33 and 76.66% of the 3rd larval instar at 28 days, respectively. The cumulative mortality of the 3rd instar larvae of both stem borers increased over time at 21 days after inoculation for all five B. bassiana strains. A field trial was conducted to evaluate the biocontrol efficacy of the five best endophytic strains of B. bassiana against C. partellus, compared to pyrethroid pesticide, Karate. Three of the endophytic strains of B. bassiana strains were as effective as Karate sprays when they were applied as seed treatments, reducing damage by C. partellus as much as Karate did. In vitro and in vivo screening were carried out under laboratory and greenhouse conditions, using various inoculation methods, to assess the interaction between the five B. bassiana strains and a commercially available Trichoderma harzianum product, Eco-T®. In the in vitro dual culture bioassay, one of the five endophytic B. bassiana strains (Strain Bb35) was not inhibited by T. harzianum Strain Kd (TKD) at 15 days after inoculation at 30 days after in vitro inoculation. None of the five endophytic B. bassiana strains grew in the presence of TKD. Only the TKD grew all over the plates. In greenhouse trials, various interactions occurred between the two fungi, according to the inoculation methods. When a mixture of conidia of the two fungi was used at the same time as a seed treatment, there was a strong inhibitory effect by TKD toward the five B. bassiana strains. However, if sorghum plants were seed treated with the five B. bassiana strains, followed by drenching of the plant roots with a TKD suspension, then the B. bassiana strains appeared to be able to colonize the stems of the plants whilst the TKD colonized the roots. Sorghum roots were rapidly colonized by the TKD when it was used alone for the seed treatment. The endophytic behaviour of some strains of B. bassiana in sorghum and rice plants can be used as powerful tool to enhance their biological control activity against stem borers of these crops. However, the tested TKD and B. bassiana strains were not compatible in the same space, such as the rhizosphere, but could be used sequentially to secure the benefits of insect control by the B. bassiana strains, as well as the biological control and plant growth stimulation activities provided by T. harzianum strains.
Biological control of the common house fly (Musa domestica L.) using Bacillus thuringiensis (Ishiwata) berliner var. Israelensis and Beauveria bassiana (Bals.) vullemin in caged poultry facilities.
(2008) Mwamburi, Lizzy A.
The entomopathogenic fungus Beauveria bassiana and the bacterium Bacillus thuringiensis var. israelensis (Bti) have been widely studied for their role in biocontrol against many arthropods and extensively exploited for insect pest control. The purpose of this study was to evaluate the effect of four B. bassiana and two Bti formulations and their respective combinations, for the biological control of the common house fly, Musca domestica L., a major pest in poultry facilities. In vitro screening was undertaken to select the best B. bassiana isolates from 34 B. bassiana isolates and two Paecilomyces isolates. All the isolates of B. bassiana were found to be effective against adult house flies, but were marginally effective in controlling fly larvae. The Paecilomyces isolates were non-pathogenic towards both adult house flies and larvae. The best four isolates R444, 7320, 7569 and 7771 caused >90% mortality within 2d and were subjected to dose-mortality bioassays. Microscopic studies using light and scanning electron microscopy indicated the different durations of the lifecycle of B. bassiana development on the house fly. High temperature was found to delay conidial germination. Spore germination and mycelial growth were also inhibited by high adjuvant concentrations. Laboratory baseline bioassay data established, a dose-time response relationship using a waterdispersible granules (WDG) Bti formulation that demonstrated that the susceptibility of M. domestica larvae to a given concentration of Bti increased as the duration of exposure increased. In the laboratory studies, the LC50 and LC90 values of Bti for the larvae ranged between 65 - 77.4 and 185.1 - 225.9?g ml-1, respectively. LT50 and LT90 values were 5.5 and 10.3d respectively. In the field, a concentration of 10g Bti kg-1 (bran formulation) of feed resulted in 90% reduction of larvae for 4wk post-treatment. A higher concentration (2g L-1) of Bti in spray (WDG) applications was not significantly more effective than the lower concentration of 1g L-1. Thus, adding Bti to chicken feed has potential for the management and control of house flies in cagedpoultry facilities. The impact of oral feed applications of a bran formulation of Bti and a commercial chemical larvicide, Larvadex®, were compared with respect to their efficacy on the control of house fly 3 larval populations in poultry manure. The sublethal effects were manifested in terms of decreasing emergence of adult house flies. Although Larvadex® reduced larval density and caused significant reductions in emergence of adult house flies, it generally exhibited weaker lethal effects than Bti. The reduction levels achieved as a result of feeding 250mg Bti kg-1 at 5wk were similar to those achieved as a result of feeding twice the amount of Larvadex® at 4wk to the layers. From both an efficiency and economic perspective, comparisons to assess the impact of combining different concentrations of the two Bti formulations were carried out to evaluate their success in controlling house fly larvae and adults in poultry houses. The percentage mortality of larvae accomplished as a result of using a combination of 250mg kg-1 Bti in feed and 2g L-1 spray applications was equivalent to that obtained as a result of combining 500mg kg-1 Bti in feed and 1g L-1 spray application. The cost-benefit analysis (expressed in terms of mortality of larvae) indicated that the most effective combination for control of house fly larvae and fly emergence was the 500mg kg-1 in feed and 2g L-1 spray application combination that resulted in 67% larval mortality and 74% inhibition of adult house fly emergence. This study presents commercial users with possible combinations of applications of the two Bti formulations. Comparisons of larval mortalities and house fly emergence resulting from the Bti - B. bassiana treatments with those from Larvadex® - B. bassiana treatments, showed better control levels compared to any of the individual agents alone. The Bti treatments were more effective at controlling larval populations and inhibiting the emergence of house flies than Larvadex®, even when Larvadex® was applied together with B. bassiana. The effects of the Bti - B. bassiana and the Larvadex® - B. bassiana interactions were additive. These trials suggest that the efficacy of Bti in the control of house fly larvae may be improved with frequent applications of B. bassiana.
Biological control of the two-spotted spider mite, Tetranychus urticae Koch (Acari : tetranychidae).
(2009) Gatarayiha, Mutimura Celestin.; Laing, Mark Delmege.; Miller, Raymond Martin.
The two-spotted spider mite (TSM), Tetranychus urticae Koch, is an important pest of many greenhouse and field crops worldwide. The development of resistance in TSM populations to chemical acaricides, allied with public health concerns about pesticide residues, has motivated the search for alternative control measures to suppress the pest. Hyphomycetous fungi are promising agents for mite control and the fungus Beauveria bassiana (Bb) (Balsamo) Vuillemin was investigated in this study as a biocontrol agent. The principal objectives of this study comprised: a) screening Bb strains for their pathogenicity against T. urticae; b) testing the effect of adjuvants on the efficacy of Bb; c) studying the effect of plant type on persistence of Bb and the efficacy of control of Bb against T. urticae; d) evaluating the field efficacy of Bb applications against T. urticae; e) testing the compatibility of Bb with selected fungicides; and f) assessing the synergy between Bb and soluble silicon for T. urticae control. Screening bioassays of sixty-two strains of Bb identified the two most effective strains, PPRI 7315 (R289) and PPRI 7861 (R444), that caused mortality levels of more than 80% of adult mites at 9 d post-inoculation with 2 × 108 conidia ml-1. These strains performed significantly better than the Bb commercial strain PPRI 5339, in laboratory bioassays. The two strains also attacked mite eggs, causing 53.4% and 55.5% reduction in egg hatchability at 2 × 108 conidia ml-1 respectively. However, PPRI 7861 showed relatively higher production of conidia in culture and was, therefore, selected for further trials under greenhouse and field conditions. Greenhouse evaluations of the effects of two adjuvants (Break-thru® and a paraffin oil-based emulsion) on efficacy of Bb demonstrated a higher efficacy of the biocontrol agent (BCA) when it was applied with Break-thru® or the oil solution than with water alone. Moreover, Bb conidia applied in Break-thru® solution resulted in greater control of TSM than conidia applied in the mineral oil. There was also a dose-response effect and the control of TSM by Bb increased when the concentration of conidia was increased. The control of TSM by Bb in beans (Phaseolus vulgaris L), cucumber (Cucumis sativus L.), eggplant (Solanum melongena L.), maize (Zea mays L.) and tomato (Solanum lycopersicum L.) was tested in greenhouse trials. On these crops, the persistence of conidia declined over time. The rate of decline was significantly higher on maize. However, TSM mortality was positively correlated with the amount of conidia deposited on leaves immediately after spraying, rather than their persistence over time. Higher levels of mortality of TSM due to Bb application were observed on beans, cucumber and eggplants, suggesting that the type of crop must be taken into consideration when Bb is applied as a BCA. Field efficacy of Bb against mites was evaluated in two trials on eggplants. Based on assessment of population densities of mites and leaf damage assessments; both trials showed that the strain PPRI 7861 controlled TSM in the field. Two commonly used fungicides, azoxystrobin and flutriafol, were investigated in vitro tests on culture medium and laboratory bioassays on detached bean leaves (Phaseolus vulgaris L.) for their effects on Bb. Azoxystrobin (a strobilurin) was less harmful to Bb while flutriafol was found to be inhibitory. Another important finding of this study was the substantial enhancement of Bb efficacy by soluble silicon. When Bb was combined with soluble Si, the control of TSM was better than when either of the two products was applied alone. Moreover, application of soluble Si as a plant fertilizer in hydroponic water nutrient increased accumulation of peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase enzymes in leaves of plants infested with TSM. Increased activity of these defense enzymes in leaves deters feeding behaviour of mites. We suggested that feeding stress renders them susceptible to Bb infection, which would explain the synergy observed between the two agents.

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