Browsing by Author "Yobo, Kwasi Sackey."

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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.
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 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.
Characterisation of a scum in sport drink and determination of the effects of preservation factors on its development.
(2013) Mapompo, Odwa Mcebisi.; Siwela, Muthulisi.; Kolanisi, Unathi.; Yobo, Kwasi Sackey.
The development of a scum in a commercial sports drink is of concern because the product would be of poor quality, which may result in financial losses due to consumer rejection of the product and hence a decrease in the firm’s market share. The scum could be harmful to health and as such the firm could be litigated. Several factors, including microbial proliferation, may be the cause of the development of a scum in sports drink, but the actual cause seems not to have been established. The aim of this study was to characterise the scum in sports drink and determine the effects of preservation factors (pasteurisation, chemical preservatives and refrigeration) on its development. Samples of the sports drink were taken at different stages of processing to determine the effect of preservatives, pasteurisation and storage temperature on scum development. Some samples were kept at room temperature (approx. 25°C) and others were kept in the refrigerator (approx. 4ºC) during the study. A total of 150 samples were analysed over a period of four months. The structural characteristics of the scum that developed in the sports drink were determined by scanning electron microscopy (SEM) and elemental analysis. The sports drink samples were analysed for their microbial load and microbial types. Consumer acceptability of pasteurised and non-pasteurised drink was compared by conducting sensory evaluation using a consumer panel of 60 panellists. Customer complaints recorded by the sports drink manufacture that were due to scum development in the drink were also reviewed to establish the impact of scum development on consumer acceptability of the drink. The results of the study indicated that scum development was due to microbial contamination of the drink. The causative organism of the scum was identified as Acinetobacter baumanii. Acinetobacter baumanii is a gram negative non-spore forming coccobacilli and does not ferment sucrose. Acinetobacter baumanii forms the scum in sports drink as a means of protection from environmental stresses. The scum was found to be a compound of C, Si and O. The non-pasteurised samples were slightly more acceptable to consumers compared to the pasteurised samples. The consumer acceptability of pasteurised drink samples was negatively affected by the loss of aroma and flavour during pasteurisation. The preservation factors (chemical preservatives, pasteurisation and refrigeration) had no effect on scum development. To prevent post pasteurisation contamination, it is recommended that the pasteurisation process be done at the filling stage instead of at the holding stage. The frequency of changing rubbers and gaskets on the filling line should be at least every two months. The drink is pasteurised at 90ºC for 20 seconds, this needs to be reduced to a level where it will not have an influence on the loss of taste and aroma of the pasteurised drink, but without reducing the effectiveness of pasteurisation.
Characterisation of fluorescent Pseudomonas species causing foliar diseases of tomato in South Africa.
(2019) Langa, Nokubonga Angel.; Yobo, Kwasi Sackey.; Goszczynska, Teresa.
Dissertation summary available in pdf.
Development of a biocontrol agent, Lecanicillium attenuatum (Zare & Gams), of wheat leaf rust (Puccinia triticina Eriks.)
(2018) Nxumalo, Thembani.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Abstract available in PDF file.
Development of an integrated management approach to controlling bacterial speck of tomato.
(2015) Ncwane, Nonduduzo Charity.; Yobo, Kwasi Sackey.; Laing, Mark Delmege.
Bacterial speck of tomato caused by Pseudomonas syringae pv. tomato (Pst) is an economically important bacterial diseases in many tomato growing regions worldwide. The development of bacterial speck epidemics is favoured by cool temperatures, high humidity and prolonged leaf wetness. As a result of infection, dark-brown to black coloured lesions surrounded by halos that eventually lead to premature defoliation are observed. Yield reduction results from the reduced photosynthetic capacity of infected leaves, resulting in flower abortion. Infected tomato fruit become unattractive and unsuitable for sale on the fresh market or for processing. In this study 250 bacterial and 100 yeast isolates were obtained from diseased and healthy tomato leaf samples. These were screened in vitro for activity against Pst. Thirty bacterial and 20 yeast isolates demonstrated significant inhibition of Pst. During the secondary in vitro screening, 10 bacterial and 7 yeast isolates successfully inhibited the growth of Pst on tryptone soy agar (TSA) plates and were selected for further studies under greenhouse conditions. Bacterial Isolates LN17, LN24 and LN10 showed clear zones of inhibition against Pst ranging from 26–29 mm in diameter. During in vitro screening, seven yeast isolates were selected, based on their ability to reduce the development of bacterial speck lesions on tomato leaves over a period of 7 d using a detach-leaf technique. Yeast Isolates IB7, Y54 and Y21 moderately suppressed bacterial speck lesions and were rated Class 2 on a five class rating system. Isolate Y25 was the best isolate and was rated Class 1. Scanning electron microscopy revealed that yeast cells colonised the leaf surface. Ten bacterial and seven yeast isolates selected from in vitro screening were further screened under in vivo under greenhouse conditions for their ability to control bacterial speck of tomato. Two of the bacterial isolates were identified as Bacillus cereus; one as B. thuringiensis; 5 as Bacillus spp.; and 2 as unidentified Bacillus spp. Two of the yeast isolates were identified as Rhodotorula glutinis; two as Rhodotorula mucilaginosa; and the remaining three isolates as Cryptococcus magnus, C. diffluens and Rhodosporidium babjevae, respectively. Bacterial isolate Bacillus thuringiensis LN1 and Bacillus sp. LN10 reduced AUDPC units by 25 and 52% and by 51 and 48% in Experiments 1 and 2, respectively compared to the pathogen inoculated control. Yeast isolates Rhodotorula glutinis Y25 and Bacillus cereus Y14 caused reductions in AUDPC units by 95 and 86% and 42% and 58% in Experiments 1 and 2, respectively, compared to the pathogen inoculated control. Based on the results from the greenhouse studies, two isolates (one bacterial and one yeast) were selected for further studies under nursery conditions. These two isolates were Bacillus sp. Isolate LN10 and R. glutinis Isolate Y25. Reduced concentrations of a plant activator, acibenzolar-S-methyl (ASM) and a plant sanitiser didecyl-dimethyl-ammonium chloride (DDAC), were evaluated for their effect on bacterial speck under greenhouse conditions in an effort to use them together with the two selected biological control agents as an integrated strategy to manage bacterial speck. Treatment with 25% of the recommended concentration of acibenzolar-S-methyl caused significant disease suppression (81.2% control) in both Experiments 1 and 2. Treatment with 25% of the recommended concentration of didecyl-dimethyl-ammonium chloride reduced disease severity by 6.3% and 9.3% in Experiments 1 and 2. The best control strategies were selected for integrated disease management studies under greenhouse and nursery conditions. Two biological control agents, Bacillus sp. LN10 and R. glutinis Y25, the plant sanitizer DDAC (at 25% of recommended strength) and the plant defence inducer ASM (at 25% of recommended strength) were used. It was found that any combination with 25% ASM caused significant disease reduction. 25 % ASM + Bacillus sp. LN10 + R. glutinis Y25 had a synergistic effect and gave disease reduction of 99.1% and 92.62% under greenhouse and nursery conditions, respectively, and was more effective than the copper bactericide control. All combinations with 25% DDAC provided no significant disease control. The combination treatment of 25% DDAC + Bacillus sp. LN10 + R. glutinis Y25 was ineffective under greenhouse and nursery conditions. The combination treatment of the two biological control Bacillus sp. LN10 + R. glutinis Y25 was ineffective under greenhouse and nursery conditions.
Efficacy of mon 89034 bt trait in conferring fall armyworm resistance in high yielding three-way and single-cross maize hybrids.
(2020) Chingombe, Pretty Nyaradzo.; Derera, John.; Yobo, Kwasi Sackey.
Maize production, especially in tropical sub-Saharan Africa, is hampered by the fall armyworm (FAW) posing a serious threat to food security and livelihoods. Many methods of control including pesticide use have been tried against FAW but without sustainable success. The main objective of this study was to investigate whether or not the Bt trait (MON89034) could be successfully integrated in high yielding tropical hybrids and confer effective resistance to FAW when deployed in three-way and single cross hybrids. The study was conducted under natural FAW hotspot conditions and under field conditions representative of farmer’s situation. Conventional non-genetically modified (non-GM) tropical single cross hybrids and inbred lines were crossed to four WEMA Bt lines. The resultant three-way and single cross hybrids were evaluated at three sites, in South Africa. The results indicated adequate discrimination of hybrids according to FAW resistance and grain yield, under both FAW infestation and at two other sites with limited FAW pressure. The experimental Bt hybrids displayed high yields exceeding 5 t/ha and higher FAW resistance, which was comparable to standard genetically modified (GM) control hybrids. In sharp contrast, the conventional non-GM control hybrids recorded yield as low as 0 t/ha, under FAW infestation. They were highly susceptible to FAW which was indicated by high damage scores. Therefore, the event MON89034 was effective in conferring FAW resistance in both three-way and single cross hybrids. Although the environment main effects were highly significant (P<0.001) for grain yield, the three-way cross hybrids were relatively stable and showed non-significant (P>0.05) genotype x environment interaction (GxE) effects. In sharp contrast, GxE effects were highly significant (P<0.001) for grain yield of single cross hybrids, indicating that they were less stable than their three-way counterparts. New Bt hybrids with high cultivar superiority index and combining high yield potential and FAW resistance were identified. These included (H3WX3167Bt) (HSX5054Bt), (HSX5368Bt) and (H3WX3194Bt). The three-way experimental hybrid (H3WX3167Bt) had yield advantage of 64% above WEMA GM checks, 33% above local GM hybrid checks and 22% above conventional non-GM checks. The single cross experimental hybrid (HSX5368Bt) exhibited yield advantage of 127% above mean of conventional non-GM checks, 100% above mean of WEMA checks and 99% above mean of local GM checks, under FAW infestation. In addition, secondary traits, such as ear prolificacy and number of ears harvested per plot, which had significant direct and indirect effects for grain yield under FAW infestation were identified for use in construction of a viable selection index. Overall, the study was successful and showed efficacy of the Bt trait (MON89034) in conferring FAW resistance when deployed in tropical high yielding three-way and single cross hybrids. The best performing experimental Bt hybrids with high yield and high FAW resistance, and out-yielded both GM and non-GM standard commercial hybrids, would be advanced in the breeding program that targets the GM market segment in tropical Africa. A survey of the literature has not revealed prior studies on evaluation of FAW resistance in three-way cross hybrids. The trait is deployed predominantly in single cross hybrids, in the GM maize production lead countries, such as Argentina, Brazil, China, South Africa and USA. Therefore, this study formed a significant baseline for revealing useful information on the efficacy of the Bt trait in conferring FAW resistance in three-way cross hybrids which are predominantly deployed to smallholder farmers in tropical Africa.
Evaluating the effect of phenolic compounds on the growth of phyllosticta citricarpa, the casual organism of citrus black spot.
(2016) Hlatshwayo, Zinhle Valitha.; Bertling, Isa.; Yobo, Kwasi Sackey.
Abstract available in PDF file.
Evaluation of integrated control of postharvest grey mould and blue mould of pome fruit using yeast, potassium silicate and hot water treatments.
(2012) Mbili, Nokwazi Carol.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
The public concern over synthetic pesticides in foods and the environment has created an interest to find effective and safe non-fungicide means of controlling postharvest pathogens. The overall objective of this thesis was to evaluate the effect of potassium silicate, yeast antagonists and hot water dip treatment to control postharvest grey mould and blue mould of pome fruits, caused by Botrytis cinerea and Penicillium expansum, respectively. Botrytis cinerea and Penicillium expansum were isolated from infected strawberry and pear fruits, respectively. These isolates were found to be non-resistant to YieldPlus® (Anchor yeast, Cape Town, South Africa), a biofungicide containing a yeast Cryptococcus albidus. A total of 100 epiphytic yeast isolates were obtained from the fruit surface of “Golden Delicious” apples and “Packham’s Triumph” pears, and screened against B. cinerea and P. expansum. Fifteen yeast isolates reduced grey mould incidence by > 50%, when applied four hours before inoculation with B. cinerea. Similarly, seven yeast isolates reduced blue mould incidence by > 50%, when applied four hours before inoculation with P. expansum. YieldPlus® and yeast Isolate YP25 provided the best control of B. cinerea, while Isolate YP60 and YieldPlus® provided the best control of P. expansum on “Golden Delicious” apples. A mixture of YP25 and YP60 provided complete control of both B. cinerea and P. expansum, when applied to “Golden Delicious” apples before inoculation with either B. cinerea or P. expansum. Electron microscopy studies showed that yeast Isolates YP25 and YP60 inhibited the mycelial growth of B. cinerea and P. expansum, respectively. Preventative and curative application of potassium silicate resulted in reduced incidence of B. cinerea or P. expansum of “Golden Delicious” apples. Electron microscopy studies indicated that potassium silicate inhibited the growth of B. cinerea and P. expansum. Furthermore, treatment of “Golden Delicious” apples with either potassium chloride or potassium hydroxide resulted in reduced incidence of both B. cinerea and P. expansum. In vivo tests showed that the disease incidence of P. expansum and B. cinerea on “Golden Delicious” apples was reduced by hot water dip treatments at 58-60°C for 60 to 120 seconds, compared with the control fruit treated with sterile distilled water, without causing skin damage. The use of potassium silicate, yeasts (Isolates YP25 and YP60), YieldPlus® and the antagonists mixture (YP25+YP60) in combination, resulted in the control of B. cinerea and P. expansum of “Golden Delicious” apples compared with Imazalil® treated fruit.
Evaluation of selected free-living diazotrophic bacteria for plant growth promotion and biological control of damping-off fungi.
(2013) Otanga, R.R.N.; Yobo, Kwasi Sackey.; Laing, Mark Delmege.
Inoculation with free-living diazotrophic bacteria is well documented to enhance vegetative growth and yield increases of various crops coupled with suppression of sublethal pathogens. The use of microbial inoculants has been identified as an alternative or supplement to use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. The search for effective free-living diazotrophic bacterial strains for formulation as biofertilizers has been on going since the 1970’s and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 250 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within KwaZulu-Natal, province, Republic of South Africa. These were evaluated for plant growth-promotion and biological control of damping-off fungi initially by in vitro screening. The growth promotion traits tested included, phosphate-solubilization, production of indole-3-acetic acid, production of ammonia and acetylene reduction. Biocontrol traits evaluated included siderophore-production, antibiosis, and production of hydrogen cyanide (HCN). Biochemical and molecular bioassay tests were conducted to identify the twenty most promising isolates selected in the in vitro study. The twenty isolates were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of maize (Zea mays L.) and wheat (Triticum aestivum L.) under greenhouse conditions. The five most promising isolates identified for growth promotion under greenhouse conditions for each crop were assessed for their effects on the germination of maize in vitro and growth and yields of two maize and two wheat cultivars, when combined with a low dose of nitrogenous fertilizer in field trials. The five Bacillus subtilis (Ehrenberg) Cohn isolates that suppressed the growth of a wide range of pathogenic fungi in vitro were tested for their efficacy against damping-off of wheat caused by Rhizoctonia solani Kühn in the greenhouse. These isolates were further studied for their modes of action against R. solani in vitro. The modes of action tested included antibiosis, production of siderophores, extracellular enzymes, production of hydrogen cyanide (HCN) and antibiotic resistance. The twenty most promising bacterial isolates identified from the in vitro screening reduced acetylene to ethylene, produced indole-3-acetic acid and siderophores, one isolate solubilized phosphate, and 9 inhibited the growth of R. solani. These twenty isolates enhanced growth of maize and wheat above the Uninoculated Control under greenhouse conditions. The growth enhancements varied with bacterial isolate x crop species interactions, which identified five different isolates for each of the two crops. Relative to the Uninoculated Control, the best five isolates significantly (P = 0.001) enhanced the growth of maize and wheat at all fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry biomass of maize and wheat, and enhanced root development of maize in the greenhouse. Inoculation of maize and wheat with the two most promising isolates identified from the field trial for each crop, in combination with 65% and 50% of the recommended amount of nitrogenous fertilizer for maize and wheat, respectively, caused the same increases in shoot biomass as the Fully Fertilized Control. Application of a combination of the best bacterial isolates and 35% nitrogenous fertilizer resulted in the same or greater shoot dry biomass and yields of both maize and wheat under field conditions. Shoot dry biomass of wheat increased by 75% above the Uninoculated Control and 30% above the Fully Fertilized Control. The wheat yield increased by 95% above the Uninoculated Control and 43% above the Fully Fertilized Control. Seed inoculation with the best isolates combined with 35% N increased yields of maize by 41% above the Uninoculated Control and 15% above the Fully Fertilized Control. The best isolates significantly (P < 0.001) increased plant height, chlorophyll levels and shoot biomass of maize relative to the Uninoculated Control. There was a positive correlation between chlorophyll level and yield, chlorophyll level and shoot dry biomass, height and shoot dry biomass and height and yield of maize at P = 0.01 with r values of 0.87, 0.77, 0.92 and 0.81, respectively. The isolates that exhibited multiple plant-growth promoting traits in vitro, increased shoot biomass of both maize and wheat in the greenhouse and field, and caused yield increases in the two crops under field conditions. Five B. subtilis isolates inhibited the growth of some of the pathogenic fungi tested in vitro up to 95%. Seed inoculation with the same isolates significantly (P = 0.001) suppressed R. solani damping-off of wheat under greenhouse conditions and exhibited multiple mechanisms of disease control in vitro. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising the yields. The B. subtilis isolates obtained in this study can effectively control R. solani damping-off of wheat, fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture. Key words: Free-living bacteria; plant growth-promotion; diazotrophs; biological nitrogen fixation; phosphate-solubilization; siderophores; indole-3-acetic acid; biocontrol; damping-off
Integrated control of Fusarium head blight of wheat.
(2015) Mngadi, Zandile Nothile Consolate.; Yobo, Kwasi Sackey.
Fusarium head blight (FHB) is a destructive fungal disease of wheat (Triticum aestivum L) world-wide. There are no resistant cultivars currently available, nor are there any effective fungicides against FHB of wheat. The use of biological control agents (BCAs) has been reported to be effective, if combined with other control measures. Therefore, this study was aimed at isolating and screening for antagonistic BCAs against F. graminearum Strain F.32 (F.32) in in vitro and in vivo bioassays. Subsequently, single and combined applications of potassium silicate (KSi) and BCAs treatments were tested for their ability to reduce FHB of wheat under greenhouse and field conditions. In vitro bioassays showed that eight Bacillus isolates and two Trichoderma strains inhibited F.32 on potato dextrose agar (PDA) plates. Bacillus Isolates B13, B14 and B15 inhibited mycelial growth of F.32 by between 41 - 49%, whereas B1, B7, B9 and B16 inhibited mycelial growth of F.32 by between 20 - 28%. Two commercial Trichoderma harzianum strains, Strains T.kd and T.77 inhibited mycelial growth of F. graminearum F.32 by 30% and 24%, respectively. None of the 16 yeast isolates tested inhibited the germination of F.32 macroconidia within 7 h when screened on yeast dextrose chloramphenicol agar, ¼ strength PDA and water agar plates. Eight Bacillus isolates, two commercial T. harzianum strains and four yeast isolates were tested against FHB under greenhouse conditions. Bacillus Isolate B7 significantly (p = 0.05) reduced FHB severity by 16% compared to the pathogen-inoculated Control treatment. None of the four tested yeast isolates significantly reduced FHB compared to the pathogen-inoculated Control treatment (p = 0.90). Single applications of T. harzianum Strains T.kd and T.77 did not significantly reduce FHB compared to the pathogen-inoculated Control treatment (p = 0.22). Repeated applications of T. harzianum Strains T.kd and T.77 did not significantly improve the reduction of FHB compared to the pathogen-inoculated Control treatment (p = 0.76). Granulated KSi fertilization at 3.0 and 4.5 g reduced FHB of wheat under greenhouse conditions by 49.0 and 54.0%, respectively, on Day 12 post-pathogen inoculation (ppi) compared to the pathogen-inoculated fertilization at 600 mg L-1 was not effective in reducing FHB of wheat under greenhouse conditions in the first 12 days after pathogen inoculation. There was no significant difference in the severity of FHB of wheat heads after treatments with KSi alone, and KSi combined with the T. harzianum Strains T.kd or T.77. None of the single and combined treatments with Bacillus Isolate B7, T. harzianum Strain T.kd and KSi treatments significantly reduced FHB severity and incidence compared to the pathogen-inoculated Control treatment on Day 20, Day 30 and Day 40 ppi under field conditions. None of the treatments tested under field conditions reduced deoxynivalenol levels in the wheat grains below 2 mg kg-1, which is the legally recommended limit. Biological control agents that demonstrated antagonistic potential against F.32 on agar plates were ineffective in reducing the severity of FHB by F.32 on wheat under field conditions. Potassium silicate demonstrated a limited capacity to reduce the levels of FHB under greenhouse conditions. The search for effective BCAs against FHB of wheat grown under South African conditions will need to continue.
Integrated management of early blight of tomato caused by alternaria solani.
(2016) Pute, Ncediwe.; Yobo, Kwasi Sackey.; Laing, Mark Delmege.
Abstract available in PDF file.
Integrating microdosing of fertilizers with biological control agents for maize production in the Eastern Cape, South Africa.
(2015) Kubheka, Bongani Petros.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Maize is an important staple crop in South Africa, and is also used for animal feed. In the Eastern Cape Province in South Africa a larger percentage of the farmers are small scale farmers and lack financial resources to apply the recommended levels of fertilizer inputs for optimal production. The currently promoted system of maize production in the Eastern Cape was designed specifically for commercial production, e.g., it is based on the use of agrochemicals to control plant diseases and pests, combined with the use of synthetic fertilizers to provide nutrients, and the application of large quantities of lime to solve soil acidity issues. The currently available mechanical equipment used to fertilize maize are only for row fertilization, whereas in between rows there may be losses of fertilizer due to the distance to the roots. Small scale farmers of this region do not apply lime. Consequently, maize yields are very low for small scale farmers in the Eastern Cape Province, relative to commercial farmers. Both biotic and abiotic factors combine to reduce maize yields. These include root diseases caused by Rhizoctonia solani Kühn and other root pathogens and poor soils (highly acidic soils with low nutrient content, especially of P and Mo). Given that the farmers do not treat the seed, lime the soil, and apply little or no fertilization, yields are consistently low. One goal of this study was to control root rot on maize caused by R. solani using a biocontrol agent, and a potassium silicate fertilizer as a priming agent of plant disease resistance. A commercial biocontrol agent, Eco-T® (a.i. Trichoderma harzianum Strain kd), is known to control most pathogenic root fungi, including R. solani. This treatment was evaluated alone and in combination with potassium silicate (KSil) in field trials over two seasons. Two KSil formulations were tested, namely a liquid and a slow release formulation. All treatments significantly reduced damage by R. solani, with T. harzianum plus the liquid formulation of KSil resulting in the highest level of control in Season 1, and T. harzianum alone providing the highest level of disease control in Season 2 (p = 0.018). There was no significant difference in the levels of control provided by T. harzianum and KSil applications when they were applied individually. All treatments significantly increased the maize yield relative to inoculated control. The treatment that gave the highest percentage difference relative to the inoculated control was KSil liquid formulation combination with T. harzianum, the combinations gave a significant 45% increased yield over the inoculated control. This means that this combination is an option for the farmers. Small scale farmers in the Eastern Cape produce maize in poor soils that have low pH levels, very high levels of acid saturation and low nutrient levels, especially of P. The second part of this study was to investigate achievable approaches to liming and fertilization for small scale farmers in the Eastern Cape. These included fertilization and liming by micro-dosing of 2:3:2 (34) fertilizer, superphosphate fertilizer and dolomitic lime using a cap of a soda bottle to measure out approximately 5g to each maize plant, applied directly into the planting hole. In order to fix atmospheric nitrogen and to solubilize phosphates in these acidic soils, a nitrogen-fixing isolate of B. megaterium was drenched into the planting holes. Micro-dosing of 2:3:2 (34) fertilizer increased maize yields by 64.6% and 13.6%, over the two seasons of the study. Micro-dosing with superphosphate fertilizer also significantly increased the maize yield (P = 0.001) by 50.5% and 37.4%. The combination of B. megaterium and 2:3:2 (34) fertilizer significantly increased the maize yield (P = 0.001) by 54.7% and 48.1% in season 1 and 2, respectively. The combination of B. megaterium, 2:3:2 (34) fertilizer and lime significantly (P = 0.018) increased maize yield, maize plant height, and stem diameter in both seasons. The increases in both seasons were consistent as a result of this combination compared to the 2:3:2 (34) fertilizer and lime combination. Whenever B. megaterium was included in the treatment combination, yields were increased, although not significantly. It was therefore concluded that micro-dosing of fertilizers can have a significant role in improving the yields for small scale farmers that cannot afford to apply the recommended levels of fertilizer or lime. It was also concluded that the use of B. megaterium is beneficial when combined with NPK and P fertilizers. Field experiments over three seasons were designed to evaluate the integration of the treatments applied in the field experiments mentioned above. The study was conducted in a field with a pH of 4.0 and an acid saturation of 54%. The methods included micro-dosing and spot application of fertilizers and lime. A strain of B. megaterium was used as a nitrogen fixer and phosphate solubilizer. For maize root disease control, the methods employed included the use of a biological control agent, T. harzianum and potassium silicate as a plant defense activator. The aim of the study was to reduce input costs whilst still providing adequate fertilization and root disease management. R. solani significantly reduced maize yields, by up to 34%, but treatment of maize seed with T. harzianum, or B. megaterium reduced losses over the three seasons from 34% to 16% and to 10%, respectively. In Season 1, the integration of all treatments (T. harzianum, B. megaterium and potassium silicate) increased maize yields by 130% relative to the R. solani inoculated control. The plots with the highest yields in the presence of R. solani were treated with T. harzianum (216%), followed by T. harzianum plus potassium silicate (214%), and lastly plots treated with T. harzianum plus B. megaterium (178%). A similar trend was observed over the three seasons. A cost benefit analysis of the integrated management of maize grown under acidic conditions and also in the presence of R. solani was undertaken after the three seasons of field experiments. The first experiment evaluated the control of R. solani using the T. harzianum, priming of plant resistance using potassium silicate, as well as the combination of T. harzianum and potassium silicate. The second experiment evaluated micro-dosing of 2:3:2 (34) fertilizer and lime, and the use of B. megaterium as a nitrogen fixer and phosphate solubilizer. The third experiment evaluated the integration of micro-dosing of 2:3:2 (34) fertilizer, superphosphate, lime and B. megaterium. The current retail prices were used. It was observed that the combination of T. harzianum and the potassium silicate liquid formulation consistently gave the highest returns on investment in controlling R. solani over the three seasons. Full fertilization consistently provided a negative return, with a mean loss of R3, 363 over three seasons, relative to the Untreated Control, which was not fertilized. Micro-dosing with lime plus 2:3:2 fertilizer gave the highest net return on investment. This was significantly different (p = 0.001) to both the Untreated Control and the Full Fertilization in Season 1. However in Season 2, the combination of B. megaterium plus 2:3:2 (34) fertilizer micro-dosed resulted in the highest net return that differed significantly from both the Untreated Control and the Full Fertilization. In the integration experiment all treatments in Season 1 gave a significantly higher yields and increased net returns on investment, relative to the R. solani inoculated control, with the lowest giving a 39% net return and the highest giving a 65% net return. In Season 2 none of the treatments resulted in significantly higher yields. In Season 3 a repeat of Season 1 results was seen where all treatments resulted in a significantly higher yields and net returns relative to the R. solani inoculated control, with the exception of B. megaterium in the presence of the pathogen. Two treatments, namely T. harzianum only and T. harzianum plus B. megaterium were consistently among the top three treatments that significantly controlled R. solani. The combination that gave consistently higher return on investment in the control of R. solani and also in the provision of nutrients was the T. harzianum plus B. megaterium plus micro-dosed 2:3:2 (34) and lime. It was therefore concluded that a cost effective method of fertilization and liming that will suit the Eastern Cape small scale farmers is micro-dosing rather than conventional method. Moreover incorporating B. megaterium improved yield consistently at little cost. For the concurrent control of root pathogens such as R. solani, it is recommended that the small scale farmers use T. harzianum, and possibly potassium silicate.
Introgression of aflatoxin and fumonisin contamination resistance genes in maize hybrids.
(2014) Chiuraise, Nyashadzashe.; Derera, John.; Yobo, Kwasi Sackey.
Maize is the principal crop in Africa, particularly in southern Africa. However, food security in the region is constantly threatened by the contamination of maize grain through mycotoxins, such as aflatoxins and fumonisins caused by Aspergillus flavus and Fusarium verticillioides, respectively. Food security is defined as the capacity of a nation to ensure that all people, at all times, have physical, social and economic access to sufficient, safe and nutritious food that meets their dietary needs and food preferences for an active and healthy life. Aflatoxins and fumonisins are carcinogenic, teratogenic, mutagenic and immunosuppressive to both humans and livestock. Presently, breeding for reduced mycotoxin contamination is one of the best strategies to reduce aflatoxin and fumonisin contamination in maize grain. Although mycotoxin resistant maize inbred lines have been identified, currently, there are no aflatoxin and fumonisin resistant commercial maize hybrids available to farmers in southern Africa. Decades of research have resulted in the identification of maize inbred lines that are resistant to either aflatoxin or fumonisin accumulation but not to both. Therefore the current study aimed at stacking resistance genes to the two toxins in one germplasm line or hybrid. The first objective of this study was to determine the current picture of mycotoxin contamination in southern African maize germplasm. Thus, a survey on South African and regional experimental hybrids was carried out during 2012/13 and 2013/14 seasons to determine the natural incidences of different types of ear rots and to identify the associated fungi. The second objective was to stack the resistance genes in a single product through introgression of aflatoxin and fumonisin resistance genes from tropical inbred lines into adapted inbred lines used in the subtropical and temperate conditions of southern Africa. Consequently, the resultant 72 single cross hybrids were evaluated for fumonisin contamination and 44 three-way cross hybrids and their progenies (146 S2:3 families) were evaluated for both aflatoxin and fumonisin contamination under artificial inoculation, in South Africa. Survey results showed that F. verticillioides was the most prevalent ear rot causing fungi followed by Stenocarpella maydis, Fusarium graminearum and A. flavus. These pathogens have potential to cause fumonisins, dipliotoxins, vomitoxins and aflatoxins. Assessment of experimental hybrids indicated a significant variation (P <0.001) among hybrids for ear rot incidence, and contamination by mycotoxins. Five single cross hybrids accumulated consistently low fumonisin levels (<4 ppm) both in the greenhouse and field trials. Three 3-way cross hybrids displayed a combined low contamination level for both aflatoxins (<5ppb) and fumonisins demonstrating potential for stacking resistance genes in the end product. Four S2:3 families also accumulated low levels of both aflatoxins and fumonisins below the legal limits of 5 ppb and 4 ppm, respectively, further demonstrating that new maize inbred lines can be developed by stacking mycotoxin genes. Therefore the study indicated a significant progress towards breeding mycotoxin resistant hybrids. Recommendations for upscaling this achievement are discussed.
Investigating the antifungal efficacy of moringa leaf extracts against Fusarium oxysporum, a causal agent of fusarium dry rot.
(2021) Mncube, Carren Nonhlahla.; Bertling, Isa.; Yobo, Kwasi Sackey.
Fungal diseases are amongst the most-destructive potato pathogens worldwide. Potato (Solanum tuberosum L.) is one of the most-common and -widely consumed crops in South Africa (SA). Population growth in SA is continuously putting pressure on the potato production, which has subsequently accelerated mono-cropping and chemical use, particularly in an attempt to control fungal diseases. There are currently no fusarium dry rot- resistant cultivars and no commercial biological control agents available to reduce occurrence of this disease. In an attempt to reduce agricultural pollution, and effectively control fusarium dry rot, plant extracts have been investigated for their antifungal properties. Moringa oleifera Lam. (moringa) is a tree well-known for its wide phytochemical composition and its antifungal abilities have been documented, but not on potato dry rot. This study was, therefore, carried out to investigate the antifungal potential of moringa leaf extracts against Fusarium oxysporum, the causal agent of fusarium dry rot of potato. Various potato cultivars were used, based on seasonal availability. Since the moringa plant is containing high amounts of secondary metabolites, in this study, first, the phytochemical composition of Moringa leaf excracts (MLEs) and the effect of MLE treatment on the phenolic concentration of tubers were analysed. The Moringa leaf powder was extracted with either (30%, 50% or 80%) of acetone, ethyl acetate, methanol and the other moringa powder eas extracted with distilled water. Various MLEs were tested for their capability to improve tuber quality post- harvest by assessing potato quality parameters, such as percentage mass loss and firmness. An in vitro assay was carried out to evaluate the moringa leaf extracts’ (MLEs’) inhibition activity on F. oxysporum using a disc diffusion method. The ability of MLEs to prevent fusarium dry rot development and to delay disease progression was also investigated in in vivo assays. Phytochemical analysis of MLEs revealed the presence of tannins, phenolics, flavonoids and glycosides. Tannins were, however, absent in ethyl acetate MLEs. Treatment with MLEs enhanced the concentration of free and bound phenolics in ‘BP1’ and ‘Mondial’ potatoes. Tubers coated with methanol-MLE had the highest concentration of both, free and bound phenolics. Treatment with MLEs slowed down average percentage mass loss (seemingly water loss) in both cultivars. Treatment with 70% ethyl acetate MLE, however, accelerated this water loss in both cultivars, but particularly in ‘Mondial’. This average reduction in mass was slightly less than the ii average reduction in mass of control tubers. Moringa treatment also preserved the healthy appearance of tuber skin and tuber firmness more so than the control. Macroscopic characteristics of F. oxysporum were pinkish colonies and a dark pigmentation, as observed on the PDA plates. The conodogenous cell was long and branched and the macroconidia had three to five septations. The fungus was found to be pathogenic on both cultivars used in this experiment, as tubers had dark depressions, typical for fusarium dry rot, and a white mycelium on the tuber surface. The efficacy of MLEs as antifungal agents was tested using a disc diffusion method. Three solvents were used to produce MLE stock solutions, namely acetone, ethyl acetate, ethanol, at three concentrations (30%, 50% and 70%). These solvent solutions were each further diluted to yield 2% and 4% concentrations, yielding eight MLE treatments. The dilutions of the MLEs produced with lower solvent concentrations (30% and 50%) had 100% fungal inhibition activity, while dilutions of MLEs extracted with the higher solvent concentration (70%) inhibited fungal development to a lesser degree. The 2% and 4% dilutions of 70% acetone, 70% ethyl acetate, and 70% methanol; resulted in 95.0%, 86.3% and 90.3% inhibition, respectively. Water-MLE, on the other hand, inhibited the fungus only by 87.5%. These results indicate that the extraction solvent and its concentration influence the antifungal efficiency of MLEs. In vivo assays demonstrated that MLEs were able to prevent disease development to a certain extent. Solvent type and concentration were influential in preventing fusarium dry rot development, as tubers treated with either 30% and 50% of acetone-MLE and methanol-MLE concentrations had the smallest average lesion diameter (3.7 mm and 3.4 mm for ‘Valor’ and ‘Mondial’, respectively). The 70% Ethyl acetate-MLE, on the other hand, was ineffective in controlling fusarium dry rot in both cultivars, resulting in an average lesion diameter of 14 mm; this was not significantly different from the control, which had an average lesion diameter of 15 mm (P>0.05) in ‘Valor’ tubers. In ‘Mondial’, 70% ethyl acetate-MLE treatment resulted in an average lesion diameter of 11 mm. Further, tubers of both cultivars treated with 50% and 70% ethyl acetate-MLE were prone to secondary infections by bacterial soft rot. The ability of MLE to delay and slow fusarium dry rot development is an indication of its antifungal potential. Response to MLE treatment was found to be cultivar-dependent, as following MLE treatment, ‘Mondial’ was more tolerant to fusarium dry rot than ‘Valor’. Solvent type and concentration were also found to influence MLE antifungal activity. Tubers treated with MLEs extracted with higher solvent concentrations (70%) as well as those treated with ethyl acetate MLEs were less tolerant to fusarium dry rot. This research, therefore, demonstrates that lower organic solvent (30% and 50%) concentrations should be used, when preparing antifungal extracts of moringa leaf powder.