Masters Degrees (Plant Pathology)

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Integrated control of postharvest Fusarium solani of potatoes using UV-C irradiation and Moringa oleifera leaf extracts.
(2024) Buthelezi, Gcinokuhle.; Mbili, Nokwazi Carol.; Mditshwa, Asanda.
Potatoes are a source of food, income and important nutrients which are beneficial to human health. However, production of potato tuber may be hindered by postharvest losses, which reduce the quality and quantity of potatoes reaching consumers. Major losses of potatoes are caused by poor harvesting, sorting, cleaning, handling, and packing. Fusarium solani, a causal agent of dry rot disease that mainly affects potatoes occurs during storage as the pathogen invades the potato tuber through tissue injuries inflicted during lifting or grading. Fusarium dry rot has been mainly controlled by using chemicals. However, the frequent use of chemicals has been recently reported to enhance fungicide resistance to potato pathogens. Chemical fungicides also negatively affect human health and the environment, as they introduce residues in treated potatoes and soil. There is a need to investigate and develop sustainable agricultural strategies such as UV-C irradiation and plant extracts as alternative strategies which are human and environmentally friendly. Therefore, this research aimed to evaluate the effect of UVC irradiation and Moringa oleifera leaf extract, independently and their combined effect in controlling F. solani on potatoes in vitro and in vivo. The antifungal effects of UV-C irradiation against F. solani were evaluated in vitro and in vivo. UV-C treatment for 10 and 15 minutes at a 10 cm distance successfully inhibited the mycelial growth of F. solani by ≥ 50% at 7-day post-inoculation. The in vivo results showed that ‘Sifra’ potatoes treated with UV-C for 10 minutes and 15 minutes had a disease incidence ≤ 33%. The increase in the duration of UV-C exposure to potatoes lowered the disease incidence on potatoes. The scanning electron icrographs showed the breakage and shrinkage of the mycelia in vitro, and the disruption of spores in UVC -treated potatoes. Moringa leaf extracts were prepared and adjusted into different concentrations, MLE 1%, MLE 1.5%, MLE 2%, MLE 2.5%, and MLE 3%. These concentrations were evaluated for their efficacy against F. solani in vitro and in vivo. The findings demonstrated that MLE 1.5%, MLE 2.5%, and MLE 3% inhibited the mycelial growth of F. solani by ≥ 50% in vitro. The in vivo findings revealed that both MLE 2.5% and MLE 3% reduced incidence of dry rot in potatoes. The antifungal activity of moringa was increased at higher concentrations. The scanning electron micrographs showed mycelia distortion in samples treated with moringa and the disruption of F. solani spores on the treated potatoes. It also indicated the formation of biofilms in moringatreated potatoes. Furthermore, this study evaluated the effect of integrated control of F. solani using UVC and moringa leaf extracts. The in vitro results demonstrated that samples treated with MLE 2.5% and exposed to UV-C for 15 minutes inhibited the mycelial growth of F. solani by 100%. ‘Sifra’ potatoes exposed to UV-C for 15 minutes and treated with MLE 3% had the lowest disease incidence (8.33%). The scanning electron micrographs showed abnormal, shrinkage, disruption, aggregation and reduced hyphae length of mycelia in samples treated with both UV-C and MLE. Moreover, it indicated the formation of biofilms in UV and moringa-treated surfaces of potato wounds. These integrated treatments enhanced efficacy compared to the individual application of either treatment. UV-C can be integrated with moringa and be used as alternatives to synthetic fungicides to control dry rot of potatoes.
Biocontrol of postharvest pathogens infecting avocado using endophytic trichoderma species.
(2023) Mkhize , Londeka.; Laing, Mark Delmege.; Bancole, Wonroo Bernice Armellel.
Pre-harvest fungal infection causes both pre- and postharvest avocado (Persea americana Mill.) diseases. Some foliar diseases also cause avocado fruit spots, including those caused by Pseudocercospora and Cladosporium species. Pre- and postharvest rots of avocado fruit are largely caused byColletotrichum, Lasiodiplodia and Neofusicoccum species, and these are also latent pathogens that usually infect the fruit in the orchards before harvest. These fungal pathogens cause crop losses in SouthAfrican production in untreated fruit by about 50-90%. As a result, several agrochemicals including prochloraz, copper oxychloride, thiophanate-methyl and thiabendazole are being used by farmers for avocado disease management. However, the intensive use of chemicals does not prevent all diseases, and toxic residues affect the environment and consumers of fruit containing residues. As a result, there are growing restrictions on the use of agrochemicals being exported to the European Union (EU), due to reductions in theMaximum Residue Levels (MRLs) of most of these fungicides. The use of biological control agents (BCAs) such as Trichoderma species has been regarded as a promising and environmentally friendly approach to controlling plant diseases. Trichoderma species are opportunistic, avirulent plant symbionts and some strains have been developed commercially as BCAs for use on many crops against a wide range of plant pathogens. Their modes of action are complex and include antibiosis, mycoparasitism, producing bioactive secondary metabolites, inducing plant defensive mechanisms and promoting plant growth. Therefore, the overall objective of this study was to isolate and screen endophytic strains of Trichoderma species to control fruit diseases of avocado such as stemend rot, anthracnose and leaf spot diseases. Isolation and Identification of Fungal Pathogens Avocado fruit from different supermarkets in Pietermaritzburg (KwaZulu-Natal, South Africa) that displayed symptoms of avocado fruit rot and fruit spot were collected and brought to the Plant Pathology Laboratory at the University of KwaZulu-Natal for fungal isolation. A total of forty-five isolates were isolated from symptomatic avocado fruit with typical characteristics of anthracnose, stem-end rot, Cladosporium spot, and Cercospora spot. The isolates were identified based on their cultural and morphological characteristics using light microscopy and scanning electron microscopy (SEM). Koch’s postulates screening was performed on fresh avocado fruit by spraying the fruit with suspensions of the key fungal pathogens of anthracnose, stem-end rot, and leaf spot. Frequently isolated colonies causing anthracnose on avocado fruit were divided into 2 morphological groups (Cs1 and Cs2). Colonies of Group 3 (Ls1) constituted 35% of the fungal pathogens isolated from fruit displaying stemend rot. Group 4 (Ls2) was a Lasiodiplodia species that was provided by Majola (2020) and freshly isolated colonies of this genus. In Group 5 were isolates of Pseudocercospora and Cladosporium species, which constituted about 20% of the cultures. All the fungal isolates were subjected to a pathogenicity assay, conducted twice, to confirm the pathogenicity of each isolate. Anthracnose pathogens in Group 1 (Cs1) were the most virulent strains, while Group 2 (Cs2) were the least virulent. Stem-end rot pathogens in Group 3 (Ls1) were more virulent compared to Group 4 strains (Ls2). Some inoculated fruit did not show any symptoms, even after 14 days post inoculation (dpi). Morphological characterization showed that Pseudocercospora species and Cladosporium spp. were the least virulent strains. Molecular identification of the fungi was undertaken using ITS sequence analysis. Isolate Cs1 was identified as Colletotrichum cobbittiense and isolate Cs2 was identified as Colletotrichum henanense. The most virulent isolate causing stem-end rot was Isolate Ls1, which was identified as Neofusicoccum parvum, a pathogen that was not previously recognized as being of importance in KwaZulu-Natal, South Africa. This suggested that this fungus is probably causing severe postharvest losses for avocado farmers in KwaZulu-Natal. Isolate Ls2 was identified in a previous study as Lasiodiplodia mahajangana using ITS1 and ITS2 gene sequence analysis. Isolation and Endophytic Screening of Trichoderma species Avocado leaves were sampled from the five avocado trees of the cultivar Fuerte, which are growing at Ukulinga farm, University of KwaZulu-Natal, Pietermaritzburg. From these trees, sixty leaves were sampled, and five fresh avocado fruits from a local supermarket in Scottsville, Pietermaritzburg were used as plant material for the isolation of Trichoderma species. Colony colour differences and radial growth measurements were two characteristics that were used to differentiate between the strains of Trichoderma species. A screening for endophytic activity of the isolated Trichoderma strains was conducted on fresh avocado fruit and seedlings. Fifty-two avocado seedlings of the cultivar Edranol were transplanted into pots in a greenhouse and sprayed with benomyl to kill any natural endophytic Trichoderma spp. found in them. The seventeen Trichoderma strains isolated previously were used to prepare suspensions with a concentration of 1x106 conidia/ml-1. The Trichoderma suspensions were sprayed on the seedlings and after two weeks the seedling leaves were sampled randomly and surfacesterilised using 2% sodium hypochlorite and sterile distilled water. Small segments were placed on Trichoderma selective medium (TSM) agar plates to check for the presence of the Trichoderma. During this study, only eleven strains of Trichoderma spp. were found to be endophytic. Another screening was then done on fresh avocado fruits. The fruits were sprayed with suspensions of the eleven endophytic Trichoderma strains. After a seven-day waiting period, the fruit was surface-sterilized, and the skin of the fruit was sampled in ten places per fruit and plated onto TSM. Pure cultures were purified on Potato Dextrose Agar (PDA) and monitored every second day to record the growth. A total of nine isolates of Trichoderma demonstrated high endophytic ability. Assessment of the Effect of Endophytic Trichoderma Strains on Anthracnose and Stem-end rot in vivo An in vivo investigation was conducted to understand the antagonistic activity of endophytic Trichoderma as a potential biocontrol agent against anthracnose and stem-end rot. Fresh avocado fruit of the cultivar Hass were collected from a local farm in Richmond (KwaZulu-Natal, South Africa). The fruit were sprayed with Trichoderma isolates conidial suspensions at concentration 1x106 conidia/ml-1 and a commercial Trichoderma-based product (Eco77). The fruit were then air-dried and stored in boxes inside black plastic bags to create a high relative humidity at room temperature for 7 days. After 7 days post inoculation (dpi), the black plastic bags were removed and the fruit were inoculated with the pathogens by spraying the isolates Cs1, Cs2, Ls1, and Ls2 with concentration 1x106 conidia/ml-1. This experiment was conducted twice to confirm the antagonistic activity of the endophytic Trichoderma isolates. A rating scale of 1-3 was used to describe the disease severity caused by isolates Cs1, Cs2, Ls1, and Ls2 after the fruit were treated with endophytic Trichoderma strains. All the fruit inoculated with Trichoderma and the pathogen developed less anthracnose or stem-end rot symptoms than fruit inoculated with the pathogen only. Isolates UK1E, UK4C and Eco77 were able to reduce symptoms on the fruit inoculated with the pathogenic species isolated, including C. cobbittiense, C. henanense, N. parvum and L. mahajangana. Based on ITS1 and ITS2 gene sequence analysis, UK1E was identified as Trichoderma asperellum and UK4C as Trichoderma koningiopsis. These two strains have the potential to be commercialized as biocontrol agents against the Botryosphaeriaceae family associated with anthracnose and stem-end rot of avocado. The next research phase would be to undertake field trials to see if preventative inoculation of the best Trichoderma strains can provide seasonal protection of fruit from pre- and postharvest pathogens.
Investigating the utilization of indigenous forestry as a pathway for commercial forestry, community upliftment and land restoration.
(2024) Mathenjwa, Kuhlekonke Khulani.; Mbili, Nokwazi Carol.; Burgdorf, Richard.; Laing, Mark Delmege.; Sardeshpande, Mallika.
Forests play a vital role in sustaining ecological balance and providing essential resources for human well-being. In South Africa, forestry has predominantly relied on non-indigenous tree species, leading to limited natural forests and an increasing dependence on commercial forest plantations. These plantations are mainly made up of commercial exotic monocultures constituting pine, wattle, and eucalyptus species. Some of these exotic species tend to colonize the natural environments of local indigenous species and grasslands where they ultimately alter the soil and water conditions to outcompete local species. In addition, due to their greater growth forms than indigenous tree species, they alter the structure of natural vegetation which negatively impacts nutrient cycles and water availability. As a result, the need to explore alternative approaches in forestry that prioritize indigenous tree species has become evident. While indigenous commercial forestry is practiced globally, South Africa has untapped potential due to the limited number of indigenous tree plantations in the country. To explore the use of indigenous trees, the main aim of the project was to investigate the use of indigenous trees for commercial forestry, community development, as well as land restoration by assessing and evaluating the growth and survival of indigenous species through the application of propagation methods and the incorporation of plant growth-promoting fungus Trichoderma asperellum (Eco-T). This was followed by assessing ecological benefits of indigenous trees such as biodiversity conservation, soil nutrient analysis, and evaluating cost-effective methods and market potential for indigenous tree propagation, as well as the recovery of trees in anthropogenic soils. This aim was firstly achieved by assessing the feasibility and potential of indigenous forestry in South Africa through a literature search and then further reported on indigenous tree species' importance and utilization along with their associated resources. Subsequently, this research investigated the utilization of indigenous tree species in South Africa, particularly within three peri-urban communities of the eThekwini municipality, KwaZulu-Natal. The first phase of sampling followed snowballing sampling strategy through assistance from local authorities (N=29), and a questionnaire was administered at one of the focus group discussions (N=10). The second phase of sampling used purposive sampling strategy to identify interviewees (N=10). Ethnobotanical data was collected and a total of 21 indigenous tree species were identified and reported across the three communities, emphasizing the utilization patterns and preferences of local species. Amongst the reported species, 12 species were utilized in either all three or two communities. Across all three sites, four indigenous tree species emerged as the most commonly utilized species, notably, Sclerocarya birrea, Prunas africana, Trichilia emetica and Ficus sur, accounting for 90% of participants (n=20). Fruits and medicinal products derived from bark and leaves were the most commonly utilized resource products from indigenous trees, constituting 60% of overall usage by participants. These findings highlighted that indigenous tree species with multiple uses were commonly utilized more than those species with one use, which elucidates the commercial value and economic potential of those. Treepreneurs emerged as pivotal components in this study that showcased profound knowledge and commitment to indigenous tree propagation and cultivation, expressing a clear preference for indigenous species over alien invasive plants. Hence, understanding the perspectives and needs of the local community is essential for the successful implementation of indigenous commercial forestry. The growth of indigenous tree species under field and greenhouse conditions was assessed. A total of five indigenous tree species namely Trichilia emetica, Harpephyllum caffrum, Sclerocarya birrea, Ficus sur and Canthium inerme were selected for this study based on the preliminary findings of the ethnobotanical survey. Tree seedling growth and survival were the important parameters evaluated in this study to investigate the impact of Trichoderma asperellum (Eco-T) on the growth of indigenous tree species. Noteworthy results obtained from the field growth establishment trial include the highest survival rate H. caffrum and the fastest growth in F. sur, accounting for 100% and 33.13 cm, respectively. However, the application of Trichoderma treatment did not have any significant impact on the growth and survival of the indigenous tree species. These findings lay a great foundation for the cultivation, survival, and growth of indigenous tree species under field conditions. Greenhouse cultivation of indigenous tree species was conducted using anthropogenic soils from degraded lands. Here, the growth forms of indigenous tree species compared to the exotic species Eucalyptus grandis were investigated. The application of Eco-T as a treatment was also examined on the survival and growth of the seedlings. Indigenous tree seedlings exhibited a high survival percentage except for S. birrea in both Eco-T treated and untreated samples, accounting for 67 and 92%, respectively. Notably, the overall highest change in growth of height was observed in seedlings of F. sur with 48.8 cm but not as high as that of the control sample accounting for 106.16 cm. However, the treatment did not have any significant impact on the growth and survival of indigenous tree species. These results highlight the potential expressed by indigenous tree species especially F. sur under field and greenhouse conditions, in forestry and land restoration. They further emphasize the need for the development of specific indigenous tree species of commercial and ecological value. Furthermore, this study investigated the effect of different rooting hormones in the growth of different indigenous tree species from stem cuttings and then assessed the feasibility of propagating indigenous tree species using cuttings. In this study, four treatments namely Seradix, Dip n Grow, Eco-T and control were tested on three cutting types of the five indigenous tree species. All treatments showed no significant impact on the survival and growth of the species. At the end of the growing period, only one out of five species had survived, sprouted and rooted, accounting for 16% of F. sur. High mortality was observed at the end of the growing period (12 weeks) and can be accounted by various factors such as oversaturation on growing medium, temperature, and moisture. The findings revealed to yield great propagation success, compatibility of rooting hormones, type of species and cutting type must be taken into consideration. The findings further reveal the slight feasibility of propagating indigenous tree species from stem cutting, but also express great potential for F. sur. Hence, more research and development are needed to explore the potential of F. sur as well as other important indigenous tree species that can add value in forestry and be utilized for commercial purposes. Lastly, this study could provide valuable information for forestry managers, researchers, and policy makers to support the conservation and sustainable management of indigenous tree species. Overall, it can serve as a blueprint for similar initiatives and can be applied in various contexts advocating for a more inclusive, ecologically sound, and sustainable approach in the forestry sector.
Control of the sweet potato weevil (cylas puncticollis) with entomopathogenic nematodes.
(2023) Zulu, Sinethemba.; Ramakuwela, Tshimangadzo.; Laing, Mark Delmege.
Sweet potato (Ipomoea batatas L) is one of the important crops worldwide because of its high calorific value. Sweet potato weevils (SPW) are the major pest affecting sweet potato, leading to a massive yield loss annually. An initial goal was to evaluate the diversity, incidence and damage severity caused to sweet potato by SPW in fields and in storage in three provinces, and the knowledge of small scale farmers about SPW. A survey was conducted in the Gauteng, North West and Limpopo provinces. Most of the farmers (96%) planted sweet potato for income generation, 3% farm for home consumption and 1% farm for employment. About 64% farmers knew about SPW, 28% had no knowledge and 8% were not sure. The crop was worst affected by SPW (79%), with rats, porcupines and other pests posing less of a problem (12%, 8% and 1%, respectively). Farmers were affected by SPW throughout the year. Farmers agreed that SPW had a negative impact on their production, with 34% of them expressing this sentiment, 28 were not sure and 14% did not think that SPW affected their crop yields. About 67% lost a quarter of their yield annually, 27% of the farmers lost a third of their yield and 6% lost half of their yield. All the three provinces are affected by SPW. Despite the majority of the farmers being aware of SPW damage, they had little knowledge of how to control the pest. Entomopathogenic nematodes (EPNs) have potential as biocontrol agents of economically important pests. This study was conducted to explore the potential of EPNs for the biological control of the SPW, Cylas puncticollis (Boheman) 1883, in an artificially infested field using larvae-infested sweet potato pieces placed in perforated containers. A total of six treatments were applied in the field, namely A - control, B - chemical insecticide, C - Steinernema tophus Cimen isolate ROOI 352 Formulation 1 with 2% Barricade® gel, D - S. tophus Formulation 2 (cadavers) E - Heterorhabditis bacteriophora Poinar, isolate SGI 245 Formulation 1 with 2% Barricade® gel, F - H. bacteriophora Formulation 2 (cadavers). After two weeks, larvae, pupae and adult SPW numbers were counted to determine the SPW populations. Heterorhabditis bacteriophora (Treatment F, cadaver formulation) was the most effective treatment with 0% live larvae and 36% dead larvae. Larvae, pupue and adult weevils were evaluated, Treatment F obtained 0% for live LPW and 48% for dead LPW. This short-term field trial demonstrated a successful reduction of all life stages of SPW by H. bacteriophoraSGI 245. Further trials of this EPN for the control of SPW are needed throughout the crop production cycle. Entomopathogenic nematodes (EPNs) are of current research interest because of their ability to kill insect pests as biocontrol agents. However, EPNs are sensitive to ultraviolet (UV) light, high temperatures and desiccation. The aim of one study was to analyse the survival and efficacy of two strains of EPNs, produced either in vivo or in vitro, and formulated in either Barricade® gel or Potassium polyacrylate hydrogel (PPH) formulations. EPNs were produced in vivo using Galleria mellonella (Fabricius) 1798 (the greater wax moth), and in vitro using an artificial medium containing ground, desiccated larvae of Musca domestica (Linnaeus), the housefly. IJs of the three selected strains of EPNs were suspended in 2% gel formulations of Barricade® gel or PPH by mixing 98 ml of water containing infective juveniles (1000 IJs / ml) with 2 g of Barricade® gel or PPH. The formulations were stored in 2 ml Eppendorf tubes held at 15˚C. The survival of the IJs was then tested at two week intervals. The efficacy of formulated IJs was tested by infecting 10 mealworms with IJs (1000 IJs / ml), incubated for 72 hours at 25oC, and counting the number of dead mealworms at two weeks intervals. On Day One, the survival of the EPNs of both isolates, in both formulations, and the controls were 100%. After two weeks, the controls had zero IJs survival for all the isolates, produced either in vivo or in vitro. The three isolates in both the Barricade® gel and PPH formulations had a zero IJs survival after eight weeks. On Day One, the mortality of mealworms was 100% for all the formulations. Both formulations of the three isolates caused zero mortality of mealworms after eight weeks. The use of Barricade® gel and PPH in formulating three isolates of EPNs enhanced their survival for up to six weeks. The in vitro-produced EPNs had a higher level of survival than the in vivo produced EPNs. However, the in vivo production resulted in EPNs that were more effective at killing mealworms than the in vitro produced EPNs. However, these differences were marginal. Differences between the three strains of EPNs were also marginal. Entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae, coupled with their symbiotic bacteria, are utilised for the biological control of a wide range of agricultural insect pests. Persistence of EPNs in the field is affected by a number of abiotic factors such as UV light, fluctuation in temperature and desiccation. The aim of this study was to assess the persistence of EPNs under field conditions, applied in two different formulations (cadaver formulation and Barricade® gel formulation). Persistence was assessed after one and two months post treatment application by baiting soil with the greater wax moth larvae (Galleria mellonella) and observing the insect mortality. Heterorhabditis bacteriophora (SGI 245) cadaver formulation showed better survival in all the five sites, followed by H. bacteriophora Barricade® gel formulation. Persistence declined drastically two months post treatment from a maximum 100% to 0%. The findings suggest that EPNs post application survival in the field can be enhanced by both cadaver and Barricade® gel formulations. Sweet potato weevils is one of the most important insect pests of sweet potato. The control of SPW is difficult due to the cryptic nature of the larvae, and night activity of the adults. In Africa, control of SPW relies primarily on the use of synthetic insecticides. However, the insect has developed some level of resistance against a wide range of insecticides. Entomopathogenic nematodes (EPN), which are cosmopolitan soil-borne entomopathogens, have gained interest as potential biological control agents of various economically important insect pests. The main aim of this study was to evaluate the potential of EPN isolates from the families Heterorhabditidae and Steinernematidae as biological control agents of SPW and their effect on different sweet potato cultivars, under field conditions. Plant resistance has also been viewed as an efficient, cost-effective and environmentally-safe form of pest control methods of SPW under field conditions. Four different cultivars were planted and six biocontrol treatment were applied after a month. Overall, both the cadaver and Barricade® gel formulations of H. bacteriophora SGI 245 were more effective in reducing the plant damage caused by SPW than the Barricade® gel formulation of S. tophus ROOI 352. Overall, the cultivar Monate suffered less insect damage (0.103) than the cultivars Blesbok and Bophelo, and it was the best yielding cultivar. A combination of Treatment F of H.bacteriophora and the sweet potato cultivar Monate can be recommended for further field testing.
The effect of biocontrol agents and plant extracts on postharvest quality of ‘kent’ mangoes.
(2023) Radebe, Siphokazi Anele.; Mbili, Nokwazi Carol.; Mditshwa, Asanda.
Mango is a tropical fruit grown in all parts of the world, hence is economically important. This fruit is beneficial as it has medicinal properties helpful to human health. Mango fruit is climacteric with a high respiration rate, leading to rapid ripening, and limiting the shelf life. This also limits the import and export, whilst the fruit is under duress, there are also fungal pathogens that infect this fruit both pre and postharvest. Anthracnose is one of the diseases affecting mangoes which is caused by the fungal pathogen Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. The susceptibility of mango fruit to fungal diseases affects the total yield and profits of the producers. Synthetic fungicides have been used to control the infection caused by fungal pathogens. However, excessive use of fungicides has a negative impact on human health and is hazardous to the environment. Notably, fungal pathogens develop resistance to fungicides over time. The chemicals such as prochloraz have also been used as a treatment in cold storage to prolong the shelf life of mangoes, and the risk of consumers ingesting these chemicals is reported. Contrary to fungicides, this study evaluated the efficacy of biocontrol agents and plant extracts against C. gloeosporioides and in prolonging the shelf life of mangoes. During the in vitro studies, 150 yeast and 150 Bacillus strains were isolated from different plant leaf materials and screened against C. gloeosporioides. A dual assay was conducted, and potato dextrose agar (PDA) was inoculated with the fungal pathogen and biocontrol agents and then stored at 28˚C for 7 days. The best two Bacillus isolates (SL and Sl) and 1 yeast isolate (Ba) that inhibited the pathogen by more than 65% and were selected from a dual culture assay in secondary screening and identified using BLAST. These isolates were further used as potential biocontrol agents against the pathogen in vivo on ‘Kent’ mango fruits. The BLAST results identified isolate Ba as Meyerozyma guilliermondii, isolates SL and Ss as Burkholderia contaminans. These isolates were overall the best treatments with mycelial growth inhibition of 67.91%, 70.00%, and 74.04%, respectively. M. guilliermondii and two B. contaminans inhibited anthracnose disease incidence on ‘Kent’ mango fruit by 50% compared to the control treatment (0%) after 7 days at 25˚C. The scanning electron microscopy (SEM) images showed breakage and shrinkage of C. gloeosporioides mycelia in vitro, and there was little damage to mycelia and no spore germination on mango fruit. The different plant extracts, Aloe vera, Tetradenia riparia, Pelargonium sidoides, and Moringa oleifera were prepared and screened against C. gloeosporioides. PDA was amended with different concentrations of the plant extracts (1%, 1.5%, 2%, 2.5%, and 3%) then inoculated with the pathogen, and stored at 28˚C for 7 days. P. sidoides was selected as the best-performing plant extract at 2.5% and 3% concentrations with 100% mycelial growth inhibition in vitro. Anthracnose disease incidence was significantly reduced by P. sidoides at 3% and 2.5% concentrations (≤50% and ≤75%, respectively) compared to the control fruit with 100% infection. The SEM interaction of 2.5% and 3% of P. sidoides with C. gloeosporioides in vitro; and mango fruit treated with 2.5% and 3% of P. sidoides and the pathogen had a limited number of spores and thin mycelia in comparison to the control. Furthermore, this study evaluated the individual and combined effect of B. contaminans, P. sidoides, and carboxymethyl cellulose (CMC) edible coating on mango fruits at 10˚C for 21 days, followed by 7 days at 25°C. Untreated fruit had the highest mass loss (18.53%) compared to all treatments (CMC, BCA, BCA+PE and PE) which had 15.3%, 14.64%, 14.18% and 14.04%, respectively. Mango fruit coated with PE had DPPH scavenging activity at 51.11 μM TE/g DM, whereas CMC treated fruit had 29.66 μM TE/g DM, even lower than untreated fruit which had 36.52 μM TE/g DM. Phenolic content was recorded at 116.4 μg GEA/g DM on PE treated fruit compared to 95.1 μg GEA/g DM for the control fruit. The concentration of AA was 51.95 mg/g on PE treated mango compared to 24.61 mg/g on the control fruit. This study showed that B. contaminans, P. sidoides, and CMC edible coating can be exploited as postharvest treatments to extend the shelf life of mango fruit.
The evaluation of the effect of biological control agents and melatonin against fusarium oxysporum infecting potatoes.
(2023) Mbatha, Londeka Akhona.; Mbili, Nokwazi Carol.
Potatoes (Solanum tuberosum L.) are essential to the South African agricultural industry. Postharvest diseases, including Fusarium dry rot (FDR) caused by Fusarium oxysporum (S.), are detrimental to the potato industry, resulting in yield and economic losses. Currently, fungicides are the primary means of controlling the disease commercially. However, the use of synthetic chemicals has reduced their efficacy against some Fusarium strains. Therefore, there is a need for safer, more sustainable, and environmentally friendly control methods for the management of postharvest fungal diseases such as FDR. Biological control agents (BCAs) and generally regarded as safe (GRAS) products have inhibitory effects against various pathogensand can be used as alternatives to synthetic chemicals. The aim of the study was to evaluate the efficacy of biological control agents (yeast and bacteria) and melatonin, individually and in combination, against F. oxysporum in vitro and in vivo. To evaluate the efficacy of the potential BCAs, 30 isolates were obtained from the exocarps and leaves of various crops, namely, potatoes(Solanum tuberosum), sweet potatoes (Ipomoea batatas), erect prickly pear (Opuntia stricta), Ganoderma mushrooms (Ganoderma resinaceum, Ganoderma austroafricanum,) and tomatoes (Solanum lycopersicum). The isolates were screened against F. oxysporum using the streaking method. Isolates NG1, NG2, NG3, NG4, and KG1 inhibited the growth of F. oxysporum on potato dextrose agar by more than 50% and were selected for secondary screening. In the secondary screening, isolates NGI (60%) and NG2 (62%) significantly inhibited mycelial growth of F. oxysporum on PDA compared to the control. In vivo, isolates NG1 and NG2 significantly reduced the severity of FDR on inoculated ‘Sifra’ potato tubers and had disease severity percentages of 47,2% and 46,1%, respectively, compared to the untreated control. BLAST prediction identified isolate NG1 as the bacterial species Burkholderia cenocepacia (strain LMG 16656) and isolate NG2 as Bacillus amyloliquefaciens (strain MPA 1034). The efficacy of melatoninwas determined by screening six concentrations (0 M; 1 M; 10 M; 15 M; 50 M and 100M) of the phytohormone against F. oxysporum using the disc fusion method. The best- performing melatonin concentrations were selected for secondary screening and progressed to in vivo screening. Melatonin concentration of 100 M had the highest inhibition percentage (40,83%) of F. oxysporum in vitro compared to the untreated control. The second and third best concentrations were 15 M and 50 M with inhibition percentages of 35,83% and 34,17%, respectively. The efficacy of melatonin as an antifungal agent, was significantly lower in vitro compared to the in vivo results. In vivo, the ‘Sifra’ potato tubers treated with 100 M showed the lowest FDR severity of 58,57%, followed by 50 M and 15 M with disease severity percentages of 59,54% and 60,31%, respectively. Subsequently, the effects of the integration of best-performing BCAs and melatonin were evaluated. BCAs, (Burkholderia cenocepacia and Bacillus amyloliquefaciens) were integrated with six melatonin concentrations and screened against F. oxysporum in vitro and in vivo. In vitro, treatment Bamy + MEL100 had the highest mycelial growth inhibition percentage (59,92%), followed by Bamy + MEL15 and Bamy + MEL50 with mycelial growth inhibition percentages of 56,12% and 55,27%, respectively. ‘Sifra’ potato tubers treated with treatment Bamy + 100 M melatonin had the lowest disease severity of FDR of 50,61% compared to control treatment after 9 days at 25℃. Bcen + 100 M melatonin and Bamy + 50 M melatonin exhibited the disease severity percentages of 52,63% and 59,72%, respectively. Overall, tubers treated with a combination of melatonin and B. amyloliquefaciens showed less severe symptoms of FDR across all concentrations. Furthermore, the effects of the exogenous application of B. amyloliquefaciens and melatonin on the quality parameters of the potato tubers were evaluated. The treatments were administered to tubers using the dipping method and stored at ambient temperature for 14 days. The treated tubers were sampled and prepared for phenolic, protein, and ascorbic acid determination assays. The exogenous application of 100 M melatonin combined with B. amyloliquefaciens was the most effective treatment with the highest phenolic content (144,1 mg GAE/g DW), and protein content (68 mg/g DM). Tubers treated with melatonin had the highest ascorbic acid content (5,48 mg AAE/100g DM). Findings indicated that the exogenous application of melatonin and B. amyloliquefaciens do not have adverse effects on the quality parameters of potato tubers. This study demonstrated for the first time, the efficacy of melatonin as an antifungal agent against F. oxysporum causing FDR on potatoes and the effectiveness of the combination of melatonin and the antagonistic microorganism, B. amyloliquefaciens against F. oxysporum.
Vegetative propagation of Corymbia henryi and its hybrids in South Africa through cuttings and mini-grafting techniques.
(2019) Mkhize, Millicent Smisele.; Laing, Mark Delmege.
Eucalyptus species are the most commonly planted hardwood species in South Africa, composing 42% of the total plantation area in the country but as the forestry planation areas in South Africa are not expanding, the only way to increase timber supply is to use the available land productively. This also increases the demand to efficiently propagate eucalyptus species, in an easy and economically viable way. This can be achieved through optimising vegetative propagation techniques which ensures mass multiplication of superior genotypes/phenotypes and the maintenance of individual characteristics in order to ensure that a plant is genetically identical to the original/donor plant. Spotted gum (Corymbia species) and their inter-specific hybrids have been identified as promising taxa for commercial forestry in South Africa due to their superior survival and growth across a broad range of edaphic and climatic conditions. The major benefits of these hybrids include superior growth characteristics, disease and insect resistance and frost tolerance, making them desirable for propagation. The hybrids of Corymbia torelliana and C. citriodora are some of the extensively used Corymbia hybrids since they combine frost tolerance (C. citriodora) and disease resistance (C. torelliana). The widely used vegetative propagation techniques include propagation through rooted cuttings and grafting. The aim of the study was to investigate the vegetative propagation of Corymbia henryi (C. henryi) hybrids through the rooting cuttings, and the grafting of C. henryi. The propagation of the hybrids through cuttings was carried out in three experiments, comparing cutting material collected from coppice stumps and ramets. The cuttings were collected from 14 C. henryi hybrid genotypes grown in Zululand. The cuttings were maintained in a rooting tunnel for four weeks before being placed in greenhouse conditions to acclimatize to the natural environment. The use of ascorbic acid (AA) (40 mg L-1) with Seradix® 2 (IBA 3g kg-1) yielded the highest rooting incidence, ranging from 37.5-55.5%, with the lowest rooting resulting from the application of propiconazole with Seradix®2 (IBA 3g kg-1) at >2%. The combination of AA with IBA has shown to be synergistic, since AA acts by protecting the rooting hormone from oxidation therefore allowing it to enhance tolerance of the plants to greenhouse conditions and alleviate stress. The cuttings selected from coppice had a higher rooting survival than those collected from hedge material. The genotype had an effect on the rooting success of cuttings, with the highest rooting percentage occurring with C. torelliana x C. citriodora subsp. variegata hybrids, with rooting ranging from 25-70%, while a C. torelliana x C. henryi hybrid had the least rooting success (%). The Corymbia species are considered difficult-to-root therefore the results have shown that these species can be propagated through rooting if the better rooting hybrid genotypes are selected. There is still a need to perform more trials to test the genotypes that have been found to root better in order to reach the commercial requirements of rooting rate of 70% and above. The propagation through grafting allows for the union of more than one genotype, whether belonging from the same species or different species and offers propagation of species which may be hard-to-root therefore cannot be produced through cuttings. The grafting experiment was carried out to optimize grafting techniques by comparing mini-grafting and conventional grafting techniques for Corymbia henryi. The rootstocks were grown and maintained at the ICFR nursery until time of grating. The scion material was collected from the Zululand region from C. henryi provenance mix. The grafting and mini-grafting was carried out in the grafting tunnel at the Institute for Commercial Forestry Research (ICFR) nursery, which had continuous mist for the duration of the experiments. The methods applied on the grafts were cleft and splice grafting. The evaluations made were based on the grafting method applied, comparing age of the grafts (mini- grafting and conventional grafting) and the effect of different treatments applied onto the grafts which included the control treatment, use of Parafilm® to tie the graft union, covering the grafts with polyethylene plastic (for one week) and use of an antitranspirant, Vapor Guard®. The control treatment, where grafts were tied with Parafilm® had the highest graft survival (33.3%), with the lowest graft survival on grafts covered with polyethylene plastic (%).Grafting onto four- month old seedlings(mini-grafting) had the highest survival at 55.6%, compared to grafting onto 10-month old seedlings (conventional grafting), with a survival of 22.2%. Mini-grafting has been tested over some species due to its ease of handling and is being evaluated for its potential in the propagation of Corymbia henryi. This type of grafting offers advantages of efficient management of plants that are grown in the nursery, allowing ease in irrigation, nutrition and pest disease control. The use of younger seedlings allows for grafting to be a commercially viable technique due the reduced time to grow the plant, flexible and pliable cambium layers of younger rootstocks and rapidness of grafting these younger seedlings. Mini-grafting was evaluated with the use of commercially available anti-transpirants and antioxidants, to assess effect of these treatments on graft success. The different ancillary treatments applied were the control, use of Parafilm® to tie the graft union, the use of anti-transpirants such as Nu-Film 17®, Vapor Gard® and Greenstim®, and the use of an antioxidant, ascorbic acid. Foliar application of ascorbic acid had the highest graft survival (60%) compared to the other treatments applied. The use of anti-transpirants in grafting of C. henryi was not successful therefore more research needs to be done on the commercially available anti-tranpirants in alleviating water stress in Eucalyptus species, as each crop may have different requirements for anti-transpirant application and doses may differ from one crop to another. Vegetative propagation of Corymbia henryi and its hybrids shows some potential for future use, but more research needs to be done to optimise these techniques to be able to have an impact on the commercial scale and in research outputs.
Management of aspergillus flavus link infections ingroundnut using bacterial endophytes as biological control agents.
(2023) Sibisi, Nokulunga Nompilo.
Microorganisms deteriorate groundnut seeds pre- and post-harvest. One of these microorganisms includes a fungus known as Aspergillus flavus Link. This pathogen reduces seed germination rate, see total oil content, carbohydrates, and proteins. Aspergillus flavus is a mycotoxigenic fungus that produces aflatoxins as secondary metabolites. As a result, seeds infected or colonized by A. flavus are considered not biologically unsafe for consumption and processing. Current management strategies have only provided temporary relief and increased susceptibility to the pathogen. Therefore, the use of environmentally friendly strategies is important. This study aimed to evaluate the efficacy of potential endophytic bacterial strains isolated from seeds, roots, leaves and stems of beans (Phaseolus vulgaris L.), peas (Pisum sativum L.), and groundnuts (Arachis hypogaea L.) in controlling A. flavus during storage and greenhouse trials. During the in vitro studies, 106 endophytic bacterial isolates were isolated and screened against A. flavus using the disc diffusion method. The best 10 isolates were selected for secondary screening against A. flavus in the dual culture assay. Only 13 (12%) of the isolates inhibited the growth of A. flavus in vitro after nine days of incubation. The best isolates from the screening trials include Isolate ALA (55.2%), followed by Isolates KI (40.9%) and KG (40.2%) inhibitions, respectively. Isolate ALA was obtained from Phaseolus vulgaris L. (dry bean) leaves, while Isolates KI and KG were isolated from groundnut stems and leaves. From the secondary screening, the best five isolates were sent to Inqaba Biotech Industries (Pty) Ltd for molecular characterization and identification at the species level. Three of the isolates KI, KG, and BB that were gram-positive were identified as either Bacillus siamensis, Bacillus velezensis or Bacillus amyloliquefaciens. Isolate BB also had similarities to B. subtilis. Isolate GNLA and ALA were identified as Pantoea dispersa and Pseudomonas fluorescens, respectively. Three of these strains (Bacillus siamensis/ Bacillus velezensis/Bacillus subtilis/Bacillus amyloliquefaciens strains BB, KG, and KI) were then used in in vivo experiments to assess their efficacy in controlling A. flavus on two groundnut cultivars (Akwa and Sellie-Plus) during a storage trial. In a short-term trial (7-day period), culture filtrate of B. amyloliquefaciens/B. siamensis/ B. velezensis strain KG showed potential as a biological control agent against A. flavus. An average percentage infection of 38.2% on Cultivar Akwa and 33.0% and Cultivar Sellie-Plus were observed. In contrast, the rest of the treatments (bacterial cells and crude lipopeptide extract) provided the least control. In the medium-storage trials (21-day period), the best endophytic bacterial treatment was the acidic cultured filtrate of B. amyloliquefaciens B. siamensis/ B. velezensis strain KG, which provided constant control for 21 days. The interaction of these isolates with A. flavus was studied using Scanning Electron Microscopy (SEM). In the greenhouse trials, three treatments were evaluated. These were (i) B. amyloliquefaciens/B. siamensis/ B. velezensis strain KG was used as a seed treatment, (ii) P. fluorescens strain ALA as a foliar spray treatment, and (iii) the combination of both seed treatment and foliar spray to control A. flavus during the greenhouse trial using the two groundnut cultivar Akwa and Sellie-Plus. Both cultivars performed somewhat differently for each treatment. The highest number of seeds and seed weight was recorded for the seed treatment for Sellie-Plus. In contrast, the combination of seed and foliar spray treatment recorded the highest number of seeds and seed weight for Cultivar Akwa. However, the combination of seed and foliar spray treatment showed potential as the best treatment against A. flavus with an average percentage disease incidence of 54.8% and 46.8% for Akwa and Sellie-Plus respectively. In conclusion, the endophytic bacterial strains demonstrated potential in controlling A. flavus infection in groundnut seeds. Adjusting the pH of cultured filtrate provided a constant and enhanced control against the pathogen. Furthermore, the combination of seed and foliar spray treatment using the two bacterial strains showed potential as a biological control agent against A. flavus.
Biocontrol agents in combination with moringa oleifera leaf extract for integrated control of botrytis cinerea of tomato.
(2022) Malevu, Nonkanyiso.; Mbili, Nokwazi Carol.; Magwaza, Lembe Samukelo.
Tomatoes and tomato-based foods provide essential nutrients beneficial to human health. Despite these benefits from tomatoes, postharvest losses result in unprofitable tomato production in some parts of the world. During ripening and harvesting, tomato becomes susceptible to diseases resulting in shorter shelf life. Susceptibility of tomato plants to Botrytis cinerea which causes grey mould infection can occur at any growth stage and the most susceptible growth stage is during ripening and senescing. Factors such as mechanical injuries, inadequate storage conditions, inappropriate handling, and transport affect tomato quality. The use of chemicals not only negatively affects farmers' yield by further enhancing pesticide resistance to crop pathogens but also influences other sectors of communities through contamination of drinking water sources which is an environmental hazard. There is a need to emphasise and encourage sustainable agricultural strategies such as biological control and plant extracts as alternative strategies which are eco-friendly and economically sustainable. Therefore, the main aim of this research was to examine the effect of biocontrol agents and Moringa oleifera leaf extract, individually and in combination, to control B. cinerea on tomatoes in vitro and in vivo. A total of 48 biocontrol agents were isolated from different parts of tomato leaves, citrus leaves, mushrooms and erect prickly pear. The isolates were screened against B. cinerea for the inhibitory effect and as potential control of the pathogen on potato dextrose agar (PDA) and tomato fruits. Serratia marcescens, Bacillus pumilus and Bacillus safensis inhibited B. cinerea by more than 50% in vitro. During in vivo screening, Serratia and Bacillus isolates inhibited grey mould incidence on ‘Jam’ tomatoes by more than 70%. The scanning electron microscopy images of the pathogen samples treated with biocontrol agents showed swollen and lysed mycelia. Moringa leaf extracts (MLE) were prepared into four concentrations MLE 1%, MLE 2%, MLE 3% and MLE 4%. The MLE concentrations were tested for their antifungal activity on the pathogen growth during in vitro studies. High concentrations were found to have some inhibitory effect on the mycelial growth of B. cinerea. There was no significant difference observed in the control, MLE 1% and MLE 2% since no mycelial inhibition was observed after 7 days at 25°C. For in vivo studies, all the concentrations had some inhibitory effect against grey mould on ‘Jam’ tomatoes. This was evidenced by lower disease incidence observed on the fruits treated with the moringa leaf extract compared to the control treatment. Scanning electron micrographs showed morphological changes in the hyphae on the samples treated with concentrations of MLE and there was also a breakage on the pathogen hyphae. Furthermore, this study evaluated the integrated control of B. cinerea using S. marcescens, B. safensis and B. pumilus integrated with MLE 2% and MLE 3% in vitro and in vivo. S. marcescens, B. safensis and B. pumilus integrated with MLE 2% and MLE 3% successfully suppressed mycelial growth of B. cinerea in vitro. Treating tomato fruits with S. marcescens, B. safensis and B. pumilus integrated with MLE 2% and MLE 3% reduced the disease incidence of grey mould compared to the control. The SEM images of the mycelial growth of B. cinerea showed shrinkage, and breakage of pathogen mycelia and the spores were damaged showing breakage and immature spores both in vitro and in vivo. Integrating moringa leaf extract, Serratia spp. and Bacillus spp. have the potential to be an alternative to synthetic fungicides to control postharvest pathogens.
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.
Identification and characterization of viruses infecting soybean (glycine max. L) in KwaZulu-Natal, South Africa.
(2021) Ndlovu, Nobuhle Sithembile.; Gubba, Augustine.
Soybean (Glycine max L.) is the world's most important seed legume, primarily used as an oil crop and protein source. Plant viruses are a major limiting factor to soybean production worldwide. Many destructive plant viruses have been discovered to infect soybean on a global scale. In South Africa, few viruses have been detected infecting soybean crops. The studies of viruses on soybean were undertaken several years ago and it is possible that the virus population structure may have evolved over time. The aim of the study was to identify and characterise viruses infecting some soybean cultivars grown in KwaZulu-Natal. Additionally, to determine the incidence of seed transmitted viruses. The first part of the study was undertaken to detect and identify viruses presently infecting soybean grown in the province of KwaZulu-Natal, South Africa and determine the incidence of any seed transmitted viruses. Fifty-four soybean leaf samples exhibiting virus-like symptoms were collected from breeding lines growing in a Plant Pathology disease garden and greenhouses at the University of KwaZulu-Natal during the 2018 - 2019 and 2019 - 2020 growing seasons. Mechanical inoculation using inoculum prepared from the soybean field samples was done on Nicotiana tabacum L. to propagate the viruses in the collected samples. Symptom development was monitored on inoculated N. tabacum plants for 2-3 weeks after inoculation. The field samples were also subjected to Reverse Transcription Polymerase Chain Reaction (RT-PCR) and PCR to detect viruses known to infect soybean worldwide. Generic and specific primers were used to target specific coding regions of the viruses tested. Antibodies specific to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) were used to test for virus presence in the field samples using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The study also focused on determining the incidence of seed transmitted viruses by planting different soybean varieties/lines and testing for virus presence after the plants had germinated. Virus presence was based on symptoms exhibited by the germinated plants and by DAS-ELISA. Results of this study showed that the inoculated N. tabacum plants developed virus-like symptoms. Soybean mosaic virus (SMV), TMV, CMV, and hibiscus chlorotic ringspot virus (HCRSV) were identified in the field samples based on PCR results. Seed transmission assays did not demonstrate the presence of viruses based on symptomatology and DAS-ELISA tests. The second part of the study was undertaken by using Next Generation Sequencing (NGS) to analyse the complete genome sequence of HCRSV infecting soybean in the province of KwaZulu-Natal, South frica Total RNA extracted from soybean samples exhibiting virus-like symptoms was combined into one sample and used as template for NGS analysis. The sequence data generated was analysed using Genome Detective Virus Tool version 1.133. The HCRSV complete genome sequence obtained was compared with other HCRSV sequences from GenBank database using BLASTN. Pairwise and Multiple sequence alignments of the sequences were done using ClustalW tool available in MEGA X. Phylogenetic analysis was done using nine closely related HCRSV sequences including turnip crinkle virus (TCV) which was used as an outgroup. The open reading frames (ORFs) for the HCRSV genome were determined using ORF finder and protein sizes were measured using Protein Molecular Weight software. Recombination events were analysed using RDP4 software. NGS data analysis revealed that HCRSV, CMV and TMV were present in the infected soybean samples. Results from the phylogenetic analysis showed that the NdlovuNS_HCRSV-SA isolate from this study (Accession number: OK636421) was closely related to isolate XM from China with a bootstrap value of 99%. Genome organisation analysis of the NdlovuNS_HCRSV_SA isolate compared with other HCRSV isolates suggested high levels of similarity. The BLAST analysis correlated with the results from the genome organisation data, with the HCRSV isolates sharing 87.87% - 97.10% nucleotide identity. Recombination analyses showed a single event confirming that the NdlovuNS_HCRSV-SA isolate is a recombinant strain. Accurate detection and identification of viruses plays an important role in virus disease management. Undetected viruses many occur and cause severe losses in soybean production. In this study, molecular detection techniques were used to accurately detectand identify the viruses infecting soybean field samples. It is important to emphasize that accurate and early detection of viruses is crucial for application of proper and effective control measures. The findings of this study will contribute to the body of knowledge on viruses infecting soybean in South Africa and will help in developing effective control measures.
Biological control of root knot nematodes (meloidogyne spp.) using bacterial and fungal antagonists.
(2014) Pambuka, Gilmore Taenzaniswa.; Laing, Mark Delmege.
Root-knot nematodes are an important pest of many crops worldwide. Chemical nematicides are the main control methods used to reduce damage caused by nematode pests on crops. However, there are aims of reducing the use of chemical nematicides, resulting in a shift towards the use of biological control, which is an environmentally friendly and safer method of control. Potential antagonists of Meloidogyne javanica were isolated from grazing pastures of livestock, and the rhizosphere of tomato plants grown under glasshouse conditions. A total of 94 bacterial and 22 fungal isolates were screened in vitro by means of microwell bioassays. Twenty bacterial and eight fungal isolates showed nematicidal activity, causing root-knot nematode second-stage juvenile (J2) mortalities of between 47.0% and 65.4%, and 33.0% and 66.3%, respectively. Five bacterial and three fungal isolates caused J2 mortalities of more than 60%. In vitro studies were conducted to evaluate the efficacy of these isolates (Bacillus spp., Hypocrea lixii-the teleomorph of Trichoderma harzianum) and Trichoderma spirale, together with two previously isolated biocontrol agents, Hypocrea lixii Strain Eco-T® and Clonostachys rosea, on the root-knot nematode M. javanica. All the bacterial isolates and fungal treatments caused significant levels of J2 mortality of M. javanica of between 59.0% to 94.0% after 12, 24 and 48 h. Bacillus thuringiensis (Isolate BG25) and H. lixii (Isolate Cr5) caused the highest mortality of J2. B. thuringiensis (Isolate BG25) and H. lixii (Isolate Cr5), when applied as a seed dressing or as a soil drench significantly (P<0.001) reduced penetration of M. javanica J2into the roots of tomato plants. The two isolates also reduced disease severity and significantly (P<0.001) reduced formation of galls, production of egg masses and the number of eggs per root. Growth parameters in terms of shoot length, shoot weight and dry shoot weight were significantly (P<0.001) increased by seed dressing and soil drench treatments of all bacterial and fungal isolates. B. thuringiensis (BG25) and H. lixii (Cr5) caused the greatest effect on growth parameters measured on tomato plants under greenhouse conditions.
Identification and characterization of viruses infecting tobacco (Nicotiana tabacum L.) in South Africa.
(2021) Ndaba, Bongeka Sylvia.; Gubba, Augustine.; Mafongoya, Paramu L.
Tobacco (Nicotiana tabacum L.) is one of the most economically important crops which contributes more than R17 billion to the national GDP of South Africa each year. Production, yield and quality of tobacco have been seriously affected by a number of factors including the emerging and recurrent plant viruses. A number of viruses are known to infect tobacco resulting in substantial yield losses. Orthotospoviruses belong to the group of emerging viruses which have shown to spread rapidly over the last decade. These viruses are among the top ten pathogens known to infect a number of crops worldwide. In 2019, large thrips populations accompanied with severe necrotic, ringspot and chlorotic symptoms typical of Orthotospovirus infections were observed in tobacco fields across South Africa (SA). Plant material showing these virus-like symptoms were collected from the three provinces of Limpopo, Northwest and Western Cape for laboratory analysis. A total of 22 leaf samples of different cultivars exhibiting orthotospovirus-like symptoms were first tested using Enzyme-Linked Immunosorbent Assay with Orthotospovirus specific antibodies, of which 19 of the 22 tested positive. The same samples were further tested with a reverse-transcriptase polymerase reaction (RT-PCR) using 2 sets of primers, firstly with orthotospovirus genericprimers, and then with tomato spotted wilt virus (TSWV) specific primers. RT-PCR results showed that 19 of the 22 samples tested positive for Orthotospoviruses and 11 of the 22 samples tested positive for TSWV. Nineteen positive sample PCR products PCR products of the positive samples were sent for Sanger sequencing. The sequences obtained were subjected to Basic Local Alignment Search Tool (BLAST) and two orthotospoviruses were detected; TSWV and groundnut ringspot virus (GRSV). Eleven of the 19 sequences matched with TSWV and eight matched with GRSV. According to our knowledge, this is the first report of GRSV infecting tobacco in SA. To determine if the symptoms observed on the tobacco plants were due to mixed virus infections, samples were subjected to Next Generation Sequencing (NGS). For this technique the total RNA was extracted from the frozen symptomatic leaf samples using a Quick-RNA™ Plant Miniprep kit (ZYMO Research, USA). To save costs, all the extracted RNA from the 22 samples was mixed together into one sample and sent to Agricultural Research Council (Biotechnology Platform) for NGS library preparation and sequencing. The NGS data was analysed using the online pipeline software, Genome Detective Virus Tool Version 1.133. The results showed that 11 other plant viruses from different genera, namely; tobacco mosaic virus (TMV), west African asystasia virus 1 (WAAV1), potato virus Y(PVY), tobacco vein clearing virus (TVCV), tomato leaf curl Uganda virus (ToLCUV), petunia vein clearing virus (PVCV), cucumber mosaic virus (CMV), beet mosaic virus (BMV), beet western yellows virus (BWYV), beet cryptic virus 2 (BCV2), and beet cryptic virus 3(BCV3). These viruses that co-infected tobacco with orthotospoviruses resulting in the severe symptoms observed in the field. NGS analysis also detected the presence of a third orthotospovirus; tomato chlorotic spot (TCSV) in the samples. NGS also was able show the presence of partial and complete sequences for the viruses mentioned above with a coverage between 70-100%. The phylogenetic analysis was done to determine the relationship of each these viruses with other nucleotide sequences of the same species or genus from GENEBANK. Viruses were grouped into clusters according to the sequences of the closest relatives in the genus, and they had a nucleotide identity ranging from 56 to 99 percent to their closest species, which indicates the occurrence of some new isolates The information presented in this study shows that viruses constitute a significant threat to the economic production of tobacco in SA. Several emerging viruses from different genera that infect tobacco in South Africa were detected using NGS. Most of the viruses detected by NGS are being reported on tobacco in SA for the first time. This study demonstrates the importance and effectiveness of using NGS for plant virus identification without any prior knowledge or based on the symptoms of the virus. Unlike other methods used to identify viruses infecting plants, NGS can detect mixed virus infections. The accurate identification of all viruses infecting tobacco at any given time is crucial for developing effective and sustainable control strategies.
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.
Management of aspergillus flavus link infections in groundnut seeds using hot water treatment and biological control agents.
(2021) Mazibuko, Phiwokuhle Zasemangweni Phelele.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Groundnut (Arachis hypogaea L.) is the 13th most important crop and classified as a cash flow crop. Groundnuts’ economic importance includes food and fodder purposes in the agriculture and food industries. The major constraint in the cultivation of groundnuts is Aspergillus flavus Link contaminating the kernels, subsequently resulting in aflatoxin contamination. Aflatoxins are highly toxic secondary metabolites produced by fungi of the genus Aspergillus. Under favourable conditions, A. flavus grows and develop in groundnuts at pre and postharvest. A. flavus causes yellow mould disease and can cause severe damage to the kernel. Cultural management practices are used to control yellow mould; however, they are often ineffective. Fungicides are considered the best available method for managing yellow mould, but they are inadequate in achieving A. flavus inhibition; therefore, alternative control strategies and integrated strategies are needed to properly manage the disease. This study was conducted to evaluate the efficacy of using potential yeast and Bacillus spp. with hot water treatment (HWT) in controlling A. flavus in groundnut seeds at optimum temperature x time combination without affecting seed germination rate and seed vigour. The efficacy of the treatments in minimizing aflatoxin concentration in groundnuts were also evaluated. In vitro screening trials were conducted to select the best yeast and Bacillus spp. antagonists from 169 yeast isolates and 60 Bacillus spp. isolated from leaves of different plant species. In vitro screening was carried out using the dual culture technique, and data was presented as average percentage inhibition. Both Bacillus and yeast isolates were grouped according to their in vitro performance, and the percentage inhibition data was subjected to Analysis of Variance (ANOVA) using Statistical Analysis System (SAS Version 9.4.). The best 10 yeast and best 10 Bacillus isolates were selected for secondary screening. The best two Bacillus spp. and best two yeast isolates were used as potential biological control agents in the in vivo experiments. The in vivo trial was repeated once. From the screening trials, the best performing isolates were; Isolate CC1y (yeast) with 72.6% inhibition, Isolate PF3y (yeast) with 70.8% inhibition, Isolates LM1b (Bacillus spp.) with 70.3% inhibition and Isolate PTP1b (Bacillus spp.) with 68.6%. inhibition. Only 1.7% of the 169 yeast isolates provided A. flavus mycelial inhibition greater than 70%. The best performing yeast antagonists were isolates from the spider plant, Chlorophytum comusum (Thunb.), Jacques and protea flower, Protea cynaroides (L.) L. Moreover, only 1.6% of the 60 Bacillus isolates provided a more significant mycelial inhibition with average inhibition of 70%. The best performing Bacillus species were isolated from citrus, Citrus x limon (L.), and pink purslane, Portulaca pilosa (L.). For in vitro hot water treatment (HWT) experiments, the best temperature x time combination provided the least percentage infection, with a significant reduction in disease intensity over time (AUPDC) and a non-significant reduction in seed germination rate. The best temperature x time combination was at 40oC for 60 seconds, followed by 40oC for 20 seconds. The 40oC for 60 seconds showed the least mycelial growth of A. flavus (in vivo), with the least disease progress over time and stimulating the best germination rate of treated seeds. The 40oC for 60 seconds was the best combination of all the 17 treatments with the least/worst treatment at 75oC for 60 seconds. The number of seed infections was recorded over two weeks. From the in vitro screening trials, the best two performing yeast spp. and best two Bacillus spp. were sent to Inqaba Biotechnical Industries (Pty) Ltd for molecular characterization and identification to species level. Isolates were identified as follows: Isolate CC1y as Suhomyces kilbournensis KU751783, Isolate PF3y as Rhodotorula mucilaginosa MK267619.1, KY076610.1, Isolate LM1b as Bacillus cereus JX218990.1, and Isolate PTP1b as Alcaligenes faecalis MG746621.1. The best performing combination treatment was HWT + Suhomyces kilbournensis in the inhibition of A. flavus in groundnut seeds. HWT + S. kilbournensis achieved 52% control after 12 weeks storage. Bacillus cereus alone provided the lowest aflatoxin concentration of 0.00840 mgkg-1 in treated seeds. Under storage conditions, the best treatment for disease reduction did not necessarily produced the least concentration of AFB1 and the lowest percentage of infection. In conclusion, the tested yeast, Bacillus spp., and hot water treatment were effective in reducing A. flavus infections in groundnut seeds. Moreover, the combination of these treatments provided enhanced disease control. For the first time, this study reports the application of hot water treatment combined with biological control agents on groundnuts to manage aflatoxin B1 and A. flavus infections in groundnut seeds.
Investigating the effect and efficiency of steel slag as a source of silicon for plant uptake.
(2021) Dadabhay, Adeel.; Laing, Mark Delmege.; Basdew, Iona Hershna.
Silicon (Si) is the second most abundant element on the planet, after oxygen, making about 25% of the Earth's crust. Since it exists in the Earth’s crust, many plants can accumulate it in large concentrations, in amounts similar to macronutrients. Si enhances growth and yield of some annual and vegetable crops, promotes upright growth (stronger and thicker stems, shorter internodes), prevents lodging, promotes favourable exposure of leaves to light, provides resistance to bacterial and fungal diseases and decreases the effects from abiotic stresses such as: high or low temperatures, salinity, heavy metal and aluminium toxicity and water deficiency. Si treatment effects on plant growth under disease and drought stress were analysed to determine the effectiveness of steel slag as a source of Si for plant uptake. Four Si treatments: slag products (SP1.7 and SP5.0); Agri-sil granular (AGS); and potassium silicate (Pots) were tested on rye grass, maize, zucchini, green pepper, broccoli and beans under disease and drought stress. Energy Dispersive X-ray (EDX) microanalysis was performed to determine the Si content within the leaves resulting from the different treatments over time. All treatments provided a positive Si uptake into the plant leaves. Maize had the highest rate of Si levels taken up into the leaves over a period of 180 days for all Si treatments, when compared to the other crops under disease stress. Si-treated plants accumulated Si into their leaves at a higher rate under disease stress than drought stress. Si treatments improved the growth of all test crops. Steel slag was an effective treatment for providing Si for the uptake in plants and to improve plant growth. The effect of pre-harvest Si application to inhibit Colletotrichum capsici on post-harvest pepper fruit (Capsicum annuum L.) was analysed. Pepper fruit were harvested from pepper plants (Capsicum annum L. cv. Revelation) that were Si treated to provide Si for plant uptake. Si treatments used were: Pots, which was used as a positive control; SP1.7; SP5.0; and AGS. The area covered by infection (%) on the fruit was recorded every seven days for a period of 21 days, to determine the disease progress. All Si treatments significantly reduced the rate of infection by the pathogen. By day seven, the disease progress was inhibited, with a recorded area of infection being below 3.5% compared to the control, which was at 8.4%. By day 14, it was inhibited from 33.6% (control) to below 16% and by day 21, it was inhibited from 57.4% (control) to below 31% for all Si treatments. Area under the disease-progress curve (AUDPC) value (%days) was the lowest for the SP1.7 treatment, which means it enhanced post-harvest disease resistance by the greatest amount. SP5.0 had the highest AUDPC value from all Si treatments. Pre-harvest application of Si reduced post-harvest anthracnose disease in green pepper fruit. The efficiency of steel slag as a source of Si for citrus and avocado uptake was analysed. Three different species: Citrus sinensis (Orange cultivars: Valencia and Navel); Citrus limon L. (lemon); and Persea Americana L. (avocado) were used for this study. Five Si treatments were tested: Pots; AGS; Agri-sil liquid (ASL); SP5.0; and SP1.7. EDX was performed to determine the Si content within the leaves resulting from the different treatments over time. All Si treatments provided a positive uptake of Si into the citrus leaves. Valencia trees treated with the SP1.7 had the highest rate of Si taken up into the leaves, with an area under curve (AUC) value of 210.24%days, followed by SP5.0 with an AUC value of 195.48. SP1.7 and AGS provided the highest rates of Si uptake into navel orange leaves with AUC values of 187.02 and 187.92, respectively. Lemon trees treated with SP1.7 and AGS had the highest rates of Si taken up into the leaves. Citrus trees treated with SP1.7 had higher rates of Si taken up into the tree leaves, with the exception of the AGS treatment having the highest rate of Si taken up in lemon. Avocado trees treated with SP1.7 had the highest rate of Si taken up into the leaves, with an AUC value of 28.29. Steel slag was an efficient and effective source of Si for the uptake in citrus and avocado leaves.
Integration of rapid hot water treatments and biocontrol agents to control postharvest pathogens of tomato.
(2019) Ziena, Luwam Weldegabir.; Laing, Mark Delmege.; Workneh, Tilahun Seyoum.
Anthracnose and sour rot caused by Colletotrichum and Galactomyces (syn. Geotrichum) species, respectively, are major fungal postharvest pathogens causing significant losses of tomato fruit. The growing public concern over human health and environmental risks posed by pesticides, the accumulation of chemical residues in fruit, and the production of secondary effects on fruit, as well as the development of resistant strains has reduced the available options of synthetic fungicides to control these pathogens. Finding alternatives or integrated approaches to provide disease control comparable to the use of synthetic fungicides is therefore needed, especially for the control of postharvest diseases, while maintaining a high quality of fruit during storage and marketing. The overall objective of this study was to develop an integrated treatment that combined rapid hot water treatments with biological control agents to control two postharvest pathogens of tomato, Colletotrichum and Galactomyces spp., and to track their impact on the postharvest quality of tomato fruit. The mechanisms of rHWTs and antagonist yeasts involved in decay control were also investigated, in passing. Isolation and Identity of the Pathogens Isolation and identification of fungal pathogens associated with tomato fruits were carried out to determine the most common fungi associated with tomato spoilage in South Africa. A total of 55 isolates were recovered from symptomatic tomato fruits with typical symptoms of anthracnose and sour rot. The cultural and morphological characteristics of all isolates were observed and compared with standard descriptions to establish their identity. Pathogenicity tests were performed. The effects of wound and non-wound inoculation methods on the infection process and disease development were studied by scanning electron microscopy (SEM). The identities of one of the most pathogenic isolates of each pathogen were then determined using the consensus sequences and the nucleotide Basic Local Alignment Search Tool (BLASTn) on The National Center for Biotechnology Information (NCBI) website. Out of the 55 isolates, 33 were Colletotrichum spp., and the other 22 isolates were Galactomyces speciesp. Colletotrichum isolates were further classified into Colletotrichum gloeosporioides and Colletotrichum acutatum, based on cultural and morphological analyses. All the Galactomyces isolates were similar and were identified as strains of Galactomyces candidum. Among the isolated strains, C24 and C37A from the Colletotrichum isolates, and G18, G23 and G29 from the Galactomyces isolates, were extremely pathogenic. SEM results showed that all wound and non-wound Colletotrichum inoculated fruits developed anthracnose, whereas non-wound Galactomyces inoculated fruits failed to develop sour rot, indicating that Galactomyces requires a wound for infection to occur. Molecular analyses confirmed the identities of the pathogens as Colletotrichum gloeosporioides (Penz.) and Galactomyces candidum Butler & Petersen (anamorph: Geotrichum candidum Link). The detection of these predominant fungal pathogens in this study indicated that both fungal pathogens are widely distributed on tomato fruit in KwaZulu-Natal. There is therefore a need to roll out effective and sustainable control strategies. Isolation, screening and identification of yeast strains A total of 148 yeast isolates were recovered from the surface of tomato fruits and were screened for antifungal activity in vitro using a dual culture assay. Only 25 isolates had strong antifungal activity against C. gloeosporioides and G. candidum. These isolates were then screened for phytotoxicity on healthy tomato fruits. Subsequently ten yeast isolates, which were non-phytotoxic to tomato fruits and which inhibited both pathogens, were selected for in vivo testing of their antifungal activity and their effects on tomato quality. The effects of delays between pathogen inoculation after yeast treatment, as well as the mechanism of decay control, were studied using SEM. Out of these 25 isolates, 4 were excluded for showing phytotoxic effect on the fruits. Isolates Y108, Y121 and Y124 showed strong antagonistic effects against both pathogens with no detrimental effect on the fruit. However, the application of the best 10 antagonist yeasts had no effect on the general quality parameters of the tomato fruits. The identity of the best three antagonist yeast isolates was then determined using molecular analysis of their sequences of the internal transcribed spacer (ITS) regions, which identified the best three isolates as strains of Meyerozyma guilliermondii (Wick) Kurtzman. The biocontrol efficacy of the yeast isolates was affected by the timing of their application. The yeast cells needed time to multiply, and thereby provide preventative protection. The sooner the application of the yeast treatments, the better was the biocontrol efficacy of the antagonist yeasts. Competition for nutrients, attachment to fungal hyphae and production of an extracellular matrix were among the probable modes of action of the antagonist yeasts in this study. The best isolates of M. guilliermondii, especially isolate Y108, were effective as biocontrol agents against C. gloeosporioides and G. candidum and could provide a sustainable alternative to the use of chemical pesticides. Hot water treatments with temperatures of 20, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71 and 80℃ were applied to tomato fruit for periods of 10, 20 and 30s on non-inoculated and inoculated fruit, in order to determine the optimal temperature x time combinations on pathogen control and postharvest quality traits of fruits. The effect of shorter times at the best working temperatures were also tested. The mechanism of heat treatments on decay control was then studied using the SEM. The temperature regimes at which no heat damage occurred on the skin of tomato fruits were 20℃, and from 44℃ to 59℃, at all exposure times, and at 62℃ for 20s. With increased temperature x time combinations above these levels, all treatments caused heat damage, which appeared as peeling, scalding, cracking and ageing either at the same time of treatment, or after 10 days of storage at 25℃. The best combinations of the rHWTs significantly reduced disease incidence, while maintaining fruit quality. These were: 56℃ x 20s, 59℃ x 10s and 62℃ x 10s. Moreover, the combinations of 56℃ x 15s, and 62℃ x 8s were even more effective. Heat treatments caused the melting of the wax platelets of the fruit, sealing cracks in the wax cover of fruit, which remained highly visible on control fruits. Induction of host defence, and inhibition of sporulation and mycelial growth were among the possible modes of action of HWTs in this study. The results have demonstrated the high potential of rHWTs to control C. gloeosporioides and G. candidum, while maintaining postharvest quality during storage, thus prolonging the shelf-life of tomato fruit. Therefore, rHWTs should be considered as a viable technology for the control of postharvest diseases of tomato fruits on a commercial level. rHWT, equivalent to pasteurization, is a rapid process, and avoids introducing a delay in the processing time of large volumes of fruit going through a commercial packhouse. The application of rHWTs and antagonist yeasts each provided significant control of both C. gloeosporioides and G. candidum. The combination of these two treatments enhanced the efficacy of both individual treatments. The integration of rHWTs at 62 x 8s with the yeast M. guilliermondii isolate Y108 resulted in the best disease control against both C. gloeosporioides and G. candidum, and delivered enhanced tomato fruit quality postharvest. This enhanced effect of rHWTs in combination with antagonistic yeasts could be the result of various interactions between the heat treatments, antagonist yeasts and the fruit. The results presented in this thesis highlight the potential to use biological and physical disease control management strategies, as stand-alone treatments or in combination, as alternative control measures against postharvest tomato anthracnose and sour rot. Although both rHWTs and antagonist yeasts reduced both C. gloeosporioides and G. candidum incidence, the combined treatment provided the best disease control with the best fruit quality. Heat treatments partially disinfect fruit, allowing for the successful colonization of the fruit surfaces and wound sites with antagonist yeasts, which then provide a residual disease control effect for the fruits. Integration of these treatments enhanced persistence and stability of each single treatments, which would be valuable in the tomato industry as part of an effective disease management strategy, which would be economically viable, readily implemented and environmentally sound. Further research is required to implement the technology at an industrial scale.
Biological characterization of South African bacteriophages infective against Streptococcus uberis, a causal agent of bovine mastitis.
(2019) Pillay, Caleb.; Laing, Mark Delmege.; Basdew, Iona Hershna.
Mastitis is an inflammatory disease of bovine mammary glands and is the most economically important disease affecting dairy herds in South Africa, and globally. The incidence and history of mastitis in South Africa has been extensively documented and found to be bacterial in origin. Streptococcus uberis (S. uberis) is the most common environmental causal agent of mastitis from clinical and subclinical samples in several countries, including Australia, the United Kingdom, New Zealand, and Belgium. Due to the causal agents ability to exist in cells, it remains protected from antibiotics. The quest for antibiotic free dairy management has funded the research into integrated strategies, predominantly proactive udder health management. Biological control methods are not widely used, specifically the use of bacteriophage (phage) therapy. Phages are ubiquitous (found in all biospheres) and are the most abundant organisms on earth. Understanding the interaction between phages and their hosts is vital to their manipulation for therapeutic conditions. This study aimed to isolate phages from unpasteurized milk of dairy cows, sequentially screened these against S. uberis isolates demonstrating antimicrobial resistance at the time. The phages have been screened for robust lytic characteristics for the of intent of a phage-based therapy. Bacterial strains of S. uberis were isolated from unpasteurized milk by Allerton laboratories submitted by dairy farms in KwaZulu-Natal, South Africa. The samples were screened for S. uberis using the following tests: haemolysis patterns on blood agar, catalase reactions using hydrogen peroxide (5%), and Gram reaction. The identity of the strains was then confirmed by Inqaba Biotechnical Industries (Pty) Ltd via sequencing of the 16s ribosomal RNA. The six S. uberis strains were screened against 8 commonly used antibiotics in the dairy industry: β-lactam (ampicillin, penicillin G, cefalexin, oxacillin and amoxicillin), macrolides (erythromycin), tetracyclines (tetracycline) and glycopeptide (vancomycin). Using the Kirby Bauer method and The European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breaking points zone diameters, version 10.0, 2020. Every strain of S. uberis was susceptible to vancomycin, which is not available for intra-mammary infections. Resistance to the 7 other antibiotics varied amongst the strains. This was not a survey study but an isolation of S. uberis strains to challenge the efficacy of phage therapy. Approximately 2,000 mastitic milk samples were screened for phages resulting in the isolation of 95 phages. This was further reduced by screening for constant lytic ability to five phages that were characterized for their phage titre, host range, single step growth phase and lethal dose activity. The titre of the five selected phages varied considerably. Phages CP1, CP2, CP76, CP79 and CP80 produced titres of 59x102, 56x105, 47x106, 50x104 and 35x104 pfu.ml-1, respectively. There was a differential interaction between the five phages isolates and the six strains of S. uberis. One strain of S. uberis was susceptible to all five phages, Strain 21A, whereas Strains 17D and 78B were not susceptible to any of the five phages. Phage CP2 was virulent to four of the six S. uberis strains. The single step growth assay illustrated the cyclic replication of the isolated phages takes between 50 - 60 minutes. In a lethal dose assay, Phages CP1 and CP2 were each able to reduce S. uberis counts by 86% and 83%, respectively. Of the 5 phages screened Phages CP1 and CP2 showed potential as stand-alone treatments, whereas Phages CP76, CP79 and CP80 would offer better control when combined in a phage cocktail, and this would broaden the host range. Phage samples were examined using transmission electron microscopy (JEOL 1400). Various negative stains were used to view the virus particles: 2% uranyl acetate (UA), 2% phosphotungstic acid (PTA) and 0.05-5% ammonium molybdate (AM). The AM staining provided the best images of infected bacterial cells and phage surface structures. All micrographs obtained, illustrated similar viral particle structures suggesting the phages screened belong to one family. A virus particle was measured to have a 50-65nm diameter icosahedral head and a short tail ranging from 25-35nm in length from the EM micrograph. The virus particles exhibited Podoviridae morphology. However, only a complete genomic sequencing will confirm the identity of these virus particles to a species level.
The potential of combined rapid hot water treatment and yeast biocontrol for suppressing postharvest avocado anthracnose and stem-end rot diseases.
(2020) Majola, Thembeka Faith.; Laing, Mark Delmege.; Burgdorf, Richard Jörn.
Avocado (Persea americana Mill.) is a highly nutritious fruit, rich in vitamins, minerals, and antioxidants. In South Africa, the avocado industry experiences combined losses of about 50% due to anthracnose and stem-end rot. The fungi most commonly associated with these diseases are Colletotrichum gloeosporioides (Penz.) Penz. & Sacc and Lasiodiplodia theobromae (Pat.) Griffon & Maubl respectively. Acceptable control levels of these diseases have been achieved by postharvest treatments with prochloraz. However, a significant reduction of the maximum residue levels by the European Union has precluded the use of this fungicide from 2020. Therefore, this study aimed to develop an alternative treatment regime to control the primary postharvest diseases of avocado. The aim of the study was to optimize a rapid hot water treatment (RHWT) and to discover an effective yeast biological control followed by the integration of these two treatments. Temperatures tested for the RHWT ranged from 20 to 80°C, combined with exposure periods ranging from 10, to 180 seconds. These were applied to “Hass”, “Fuerte”, and “Pinkerton” fruit. Levels of disease occurrence were reduced when temperatures between 52°C and 58°C were combined with exposure times of 10 to 30 seconds, which also caused no heat damage of fruit. Overall, the best treatment was a temperature and time combination of 56°C for 10 seconds. More than 100 yeast isolates were isolated and screened against the two primary pathogens. Three yeasts performed exceptionally well, including a commercial yeast variety known as B13. The combination of RHWT and all four yeasts provided a level of control comparable with that provided by the fungicide prochloraz. Excellent and consistent control was achieved from the integration of yeast strain B13 and rapid hot water treatment of 56°C for 10 seconds.
A relook at the epidemiology of Cercospora spot on avocado in South Africa.
(2020) Kallideen, Reshika.; Laing, Mark Delmege.; Schoeman, Maritha.
Avocado (Persea americana Mill.) belongs to the family Lauraceae and is one of the most economically important subtropical fruit crops in the world. The South African avocado industry contributed approximately “R1.2 billion to the total gross value of subtropical fruits (R3.4 billion) during the 2017/18 season”, according to the latest available records. One of the most serious pre-harvest diseases affecting avocado in South Africa is Cercospora spot. Losses of up to 70% have been reported on unsprayed trees. This disease is commonly found in avocado producing regions where warm, humid and rainy conditions persist. It affects all commercial cultivars, with ‘Fuerte’ being recognized as the most susceptible cultivar. The plant pathogen responsible for this disease is Pseudocercospora purpurea (Cooke) Deighton. As with other Cercospora species, this fungus grows slowly and sporulate sparsely on artificial media. Typical disease symptoms are found on the leaves, stems and fruit. Lesions first appear on the underside surface of leaves. These lesions are minute and are brown in colour. As the disease progresses, lesions are observed on both sides of leaves and have distinctive chlorotic halos. On the fruit, small lesions form, later becoming sunken, irregular and brown to black in colour. The most commonly chemical control is copper oxychloride although there are other registered fungicides for use against Cercospora spot. The South African Avocado industry currently uses a predictive model, developed by Dr J.M. Darvas, in the early 1980s, to predict the number of conidia and the timing for the first spray. The model is based on the temperature and rainfall that occurred in the week preceding the calculation of the prediction. adaptions in the fungal populations over the years, it was vital to re-evaluate this model. The primary aims of this study were to determine whether the current Darvas 2 model is still valid for forecasting the first spray for effective control; secondly, to evaluate whether the inclusion of humidity and/or leaf wetness values into a model would enhance its predictive accuracy; and thirdly, to evaluate the size of fruit that was susceptible to infection by P. purpurea. In this study, spore trapping and critical infection trials (bagging trial) were conducted for two seasons. Spore traps were placed in two unsprayed ‘Fuerte’ orchards (HL Hall and Sons and the ARC-TSC) in the first season (2017/18). However, in the second season (2018/19), only one orchard (ARC-TSC) was used for both trials because no conidia were trapped as a result of a low disease incidence at the Halls orchard that was used in Season One. At harvest, fruit were assessed for Cercospora spot using a disease rating scale. The disease index data for both seasons (2017/18 and 2018/19) were correlated with weather data using multiple stepwise linear regression analysis. In both seasons, the critical infection period was in the beginning of the season. It was also established that fruit exposed to natural infection early in the season from October to November developed significantly more Cercospora disease symptoms than fruit exposed later in the season. The daily spore trapping results (2017/18 season) indicated that conidia were mostly trapped on days when rainfall occurred. The most significant correlation (r=0.893) was found between the weekly number of trapped conidia and weekly rainfall (September to December 2017). Based on the weekly spore trapping results of the 2018/19 season, for the period October to December, there was a strong correlation (r=-0.696) between conidia and mean maximum temperature. For the entire season (October to April 2018) the correlation between conidia and mean maximum temperature was slightly lower (r=-0.520). In the 2017/18 season, more rainfall fell and more conidia were trapped than in the 2018/19 season. Due to low rainfall during the 2018/19 season, a stronger correlation was found between conidia and temperature than conidia and rainfall. This negative correlation can be explained by the cooling effect of rain, as mentioned by Darvas (1982). For both seasons, the weekly weather parameters and the weekly spore trapping data were correlated with one another. Using multiple stepwise linear regression analysis of the weekly conidia trapped and weekly weather data, three models were developed for each season. It was found that all new models (for each season) followed a similar pattern to the Darvas 2 model, with some minor differences. The spore trapping results confirmed that rainfall and temperature were the dominant environmental parameters. However, leaf wetness and relative humidity were not factors in the release of conidia but played a role in disease development, probably in the step of host infection. The study found that the Darvas 2 model was still an effective forecasting tool. However, the selected model/s (current Darvas 2 model or the new models) must be used in combination with fruit size monitoring to determine accurate and cost-effective timing of the first spray. This study determined that the first spray should be applied around mid-October (depending on the geographic region, rainfall and Z values). In addition, it was also concluded that spraying should begin when the Z-value is 15, and fruit size is approximately 25mm in diameter, and not 40mm as previously recommended by Darvas (1982). This study showed that spraying when the fruit is 40mm in diameter would be too late to slow down disease development. In support of the primary aims, experiments were conducted to determine the growth requirement/s of P. purpurea. The growth of P. purpurea was evaluated on several artificial media (potato dextrose agar (self-made), potato dextrose agar (commercial), malt extract agar, potato sucrose agar, oatmeal agar (self-made), oatmeal agar (commercial), and V8 juice agar. The fungus was grown on these media at temperatures ranging from 5oC to 35oC. The radial growth was recorded by measuring the colony diameter for a period of 28 days at seven-day intervals. The results of the growth study indicated that oatmeal agar was the best agar medium, and that 25oC was the optimal temperature for the growth of P. purpurea on artificial media. In conclusion, this study showed that the Darvas 2 model is still an effective forecasting tool, irrespective of climate change and that when the model i.e., either the current Darvas 2 model or one of the newer models is used in combination with fruit size monitoring, we can achieve a more accurate and cost-effective time to apply the first fungicide spray.

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