Browsing by Author "Kubheka, Bongani Petros."

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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.
In vitro and in vivo screening of Bacillus spp. for biological control of Rhizoctonia solani.
(2003) Kubheka, Bongani Petros.; Hunter, Charles Haig.; Laing, Mark Delmege.
The increasing concerns about chemical pesticides that are environmentally hazardous and the continuous development of resistance by palhogens to chemical pesticides have led to this study. Many studies have shown that some Gram-negative bacteria, such as Pseudomonas flouresens, control plant diseases and promote plant growth. In this study Gram positive bacteria, Bacillus sp., were chosen because of their ability to produce endospores. Endospores can be used in stable, dry formulations. The advantage of using endospores is their ability to survive harsh conditions such as droughts and high temperatures, which give a long shelf life to the biological control agent. Bacillus isolates were recovered from the rhizosphere of 12 different crops, and were subsequently screened in vitro for their antimicrobial activity. Of 130 isolates, 87 exhibited antimicrobial activity against the test organisms: Rhizoctonia solani, Pythium sp., Phytophthora cinnamoni, Fusarium sp., and single representatives of Gram negative and Gram positive bacteria, namely, Erwinia carotovora and Staphylococcus aureus respectively. The Bacillus isolates B77, B81 and B69 inhibited all the test organisms investigated, which suggests that they produced broad spectrum antimicrobial compounds or more than one antimicrobial compound. Of the isolates that showed antimicrobial activity, 78 of them did not inhibit Trichoderma harzianum K D, which is a registered biological control agent; indicating their potential for combined application. Selected Bacillus isolates were tested for the biological control of R. solani under greenhouse conditions in wheat, cabbage, tomato, maize, and cucumber seedlings. Bacillus isolates were applied as seed treatments, and the inoculated seeds were planted in R. solani infested speedling trays. Shoot dry weight measurement of seedlings indicated that 12 out of 19 Bacillus isolates showed significantly different shoot dry weight in wheat whereas all the isolates tested in tomato and cucumber gave significantly different shoot dry weight. No significantly different shoot dry weight was obtained for maize or cabbage. Seed emergence findings indicated that none of the Bacillus isolates gave significantly different emergence percentage on wheat, cabbage, tomato, and maize but all of them showed significantly different emergence percentage on cucumber. The results indicate that both the pathogen and the biological control agents exhibited varying levels of specificity on each crop tested. The biological control potential of the best Bacillus isolates was tested on bean and maize crops in the field. Green bean and maize seeds were coated with the selected Bacillus isolates and then sown under field conditions. For each isolate, four replicate treatment plots were established, with and without a R. solani inoculum. Percentage emergence, plant survival levels to harvesting and yield of maize cobs and green beans pods were measured. For all parameters measured the positive and negative controls were not significantly different thereby rendering the results for the entire field study inconclusive. However, Bacillus isolates B77, BII, R5 and R7 improved green bean pod yield and Bacillus Isolate B8I increased maize yield, indicating their potentials as plant growth promoting rhizobacteria (PGPR).
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.