Department of Plant Pathology

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Browsing Department of Plant Pathology by browse.metadata.advisor "Flett, B. C."

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    Evaluation of maize breeding populations for resistance to Fusarium verticillioides and fumonisin contamination
    (Stellenbosch : Stellenbosch University, 2017-03) Netshifhefhe, Nakisani Elelwani Innocentia; Rose, Lindy J. ; Viljoen, Altus; Flett, B. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH SUMMARY: Maize is an important crop cultivated all around the world. It is the main source of carbohydrates for over 200 million people in Sub-Saharan Africa. Daily consumption rates can reach up to 500 g per person in certain regions of Africa. Maize production is threatened by several abiotic and biotic factors which include fungi that cause maize ear rots. Fusarium verticillioides, which causes Fusarium ear rot (FER), and Aspergillus flavus, which causes Aspergillus ear rot (AER), are the most common fungal species associated with maize produced in southern and eastern Africa, respectively. Moreover, F. verticillioides produces fumonisins and A. flavus produces aflatoxins which are toxic secondary metabolites associated with harmful effects on humans and animals. Although several management strategies can be used to reduce mycotoxin accumulation in grain, host resistance has been documented to be the most efficient, cost-effective and environmentally sound strategy to minimize contamination. This study focused on evaluating F1 hybrids for improved resistance to FER and fumonisin contamination under South African and Kenyan conditions. A number of hybrids exhibited improved resistance to FER, fungal and fumonisin contamination. In South Africa, hybrids R119W x CKL05015, CML495 x CKL05015 and CKL05015 x R119W were the most resistant to FER severity, F. verticillioides colonisation and fumonisin contamination, respectively. Under Kenyan conditions, fungal colonisation was lowest in hybrids CKL05015 x CML495 and MIRTC5 x CML495, while fumonisin concentrations were lowest in hybrids CML444 x MIRTC5 and R119W x CKL05015. Parental inbred line performance was indicative of F1 hybrids performance. CIMMYT inbred lines CKL05015 and CML495, previously characterised as resistant to AER, exhibited significant resistance to F. verticillioides and its fumonisins across both countries. These lines were also found to be good general combiners for resistance to fumonisin contamination. Furthermore, F2 populations were also evaluated and the resistant F2 populations identified in this study can be used to produce recombinant inbred lines to utilise in genetic fingerprinting and mapping of resistant genes. Significant genotype x environment interactions influenced FER severity, fungal and fumonisin accumulation in maize grain. General combining ability (GCA) and specific combining ability (SCA) were significant for all three infection parameters evaluated while additive gene effects were predominant in the inheritance of resistance in this set of hybrids. This study provided fundamental information on maize lines that could be used by breeders to develop resistant cultivars. Based on the findings of this study, breeding for resistance to F. verticillioides and its fumonisins should be successful and expedited if the parental material involved is resistant.
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    Interactive effect of busseola fusca and fusarium verticillioides on ear rot and fumonisin production in maize
    (Stellenbosch : Stellenbosch University, 2017-03) Ncube, Edson; Viljoen, Altus; Flett, B. C.; Van den Berg, J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Maize is a crop of great economic importance in southern Africa, and is widely consumed as a staple food and animal feed. Production of maize, however, is hampered by pathogens and pests such as Fusarium verticillioides and the African stem borer Busseola fusca, respectively. Fusarium verticillioides infection results in Fusarium ear rot (FER) and contamination of maize kernels with fumonisin mycotoxins, while B. fusca, causes significant damage to maize tissues during larval feeding. Despite attempts to control F. verticillioides, fungal infection and fumonisin production remains a threat to maize production due to a lack of resistant maize cultivars and the inability to target the pathogen with fungicides and biocontrol products. Planting Bt maize hybrids have become an important mechanism for the management of stem borers of maize. However, the recent discovery of B. fusca resistance to Bt maize with a single crystal protein MON810 gene, indicates that care should be taken not to solely rely on this technology for the management of B. fusca. The interactive effect of B. fusca and F. verticillioides on FER and fumonisin production in maize was investigated in this study. Maize ears were inoculated with F. verticillioides alone, with both F. verticillioides and B. fusca, and with B. fusca alone. Fusarium verticillioides isolate MRC826 was inoculated by injecting a spore suspension of the fungus into the silk channel of each primary ear at the blister stage. For B. fusca infestation, aliquots of 10-15 neonate larvae were deposited into the whorl of each plant at the 12-13th leaf stage before tasselling using a mechanical applicator. Maize ears were also mechanically wounded at the blister stage with a cork borer (different sizes and number of wounds) to mimic hail damage, and half of the wounds infected with F. verticillioides. Results from this study indicated that the impact of B. fusca infestation on FER varied seasonally, possibly due to its sporadic damage to maize ears. Busseola fusca, however, did not result in a significant increase in fumonisin production. The severity of wounding of maize ears was an important contributor to FER development and fumonisin production. The effect of host plant genetic modification and pesticide application on FER and fumonisin production in maize was investigated by studying the response of a Bt hybrid and its non-Bt isohybrid to F. verticillioides infection and B. fusca infestation; and by treating plants with Beta-cyfluthrin (non-systemic) and Benfuracarb (systemic) insecticides. The field trials were conducted over three seasons using a randomised complete block design with six replicates per treatment. Uninoculated, uninfested and undamaged control treatments were included. All ears were harvested at physiological maturity and FER, total fumonisin concentration, stem borer cumulative tunnel length (B. fusca damage) and target DNA of fumonisin-producing Fusarium spp. were quantified. Busseola fusca infestation had no effect on fungal colonisation and fumonisin production in maize. Bt and non-Bt kernels were equally contaminated with fungal DNA, but FER and fumonisin production were reduced in the Bt hybrid under natural farming conditions. Despite the evidence found in this study and others that Bt maize indirectly reduces FER and fumonisin production, this was also inconsistent over seasons. Benfuracarb controlled stem borers, and therewith indirectly reduced FER and fumonisin production. FER development and fumonisin production by F. verticillioides varied over seasons, indicating the importance of environmental conditions on FER and fumonisin production. A survey was also conducted at two sites in the North West province and one site in the Free State province of South Africa to analyse mycoflora in B. fusca frass. The exposure of B. fusca larvae to F. verticillioides in stem borer frass was also evaluated in both greenhouse and field trials. Maize whorls were inoculated with a spore suspension of F. verticillioides MRC826 4 weeks after plant emergence and infested with aliquots of 5-10 neonate B. fusca larvae 2 days later. The control treatment consisted of B. fusca infestation only. Several fungal species were associated with stem borer frass, including Acremonium zeae, Aspergillus flavus, A. niger, F. chlamydosporum, F. incarnatum-equiseti species complex, F. oxysporum, F. subglutinans, F. verticillioides, Mucor circinelloides, Rhizopus oryzae and Talaromyces flavus. The occurrence of A. niger in the frass suggests that further studies need to be conducted to determine the effect of A. niger infection on fumonisin production in maize in South Africa. DNA quantity of fumonisin-producing Fusarium spp. was significantly more in frass collected from greenhouse plants inoculated with F. verticillioides and infested with B. fusca larvae than in frass collected from the uninoculated and infested control, whilst the field trial showed no significant differences in quantity of target DNA in frass from inoculated and uninoculated plants infested with B. fusca larvae. This indicates that plants in the field were naturally infected with F. verticillioides. This study showed that Bt maize had no effect on infection of maize ears by fumonisinproducing Fusarium spp. and the subsequent production of fumonisin in F. verticillioidesinoculated maize ears, indicating that the effect of Bt maize on fumonisin production in maize ears is indirectly associated with its control of severe stem borer damage. Busseola fusca frass was a reservoir of different fungal species; some pathogenic to maize, and others antagonistic to maize pathogens. Moreover, B. fusca infestation of maize stems was associated with higher levels of fumonisin-producing Fusarium spp. in larval frass when F. verticillioides was present on the plant. Multiple large wounds created by cork borers resulted in significantly more FER symptoms and fumonisin production, irrespective of artificial F. verticillioides inoculation of maize ears whereas B. fusca infestation resulted in a significant increase in FER in only one of the three seasons, moreover, it had no effect on fumonisin production in all three seasons. This indicates that severe wounds that opens up husk coverage and exposes maize kernels; caused by factors such as insects, hail and bird damage, and damage by implements; are important entry points for F. verticillioides that may lead to the transition from symptomless infection to necrotrophic pathogenicity resulting in FER and concomitant fumonisin production in maize kernels. However, climatic conditions are also important in FER and fumonisin production in maize. Moreover, Acremonium zeae endophytes occurring in frass can be used for the biological control of F. verticillioides resulting in the management of FER and subsequent fumonisin production.
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    Investigating maize inbred line responses following infection by the Mycotoxigenic fungus Fusarium verticilliodes
    (Stellenbosch : Stellenbosch University, 2016-03) Rose, Lindy Joy; Viljoen, Altus; Van der Vyver, Christell; Flett, B. C.; Stellenbosch University. Faculty of AgriScience. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Fusarium verticillioides is an important fungal pathogen of maize and is associated with the crop wherever it is produced. It is the most common fungal contaminant of South African maize grain and causes Fusarium ear rot (FER). The presence of F. verticillioides can reduce grain yield and quality by the visible moulding and/or discolouration of the kernels which reduces its grading at silos. The fungus may also contaminate grain without visible symptoms and produce harmful secondary metabolites known as fumonisins. Fumonisins have been associated with a number of noxious effects on humans and animals. Their widespread occurrence in maize and maize-based products has led to numerous countries imposing maximum allowable limits of fumonisins in food and feed. Cultural practises, mainly focussed on optimal plant production as a disease management strategy, have been shown to reduce fumonisin contamination. However, host-plant resistance is considered the most feasible, economical and environmentally sound approach to manage F. verticillioides and its fumonisins. In this study the response of elite breeding lines to infection and fumonisin deposition by F. verticillioides was evaluated in a multi-environment trial over 2 years. These lines were evaluated with inbred lines previously characterised for their response to FER and fumonisin accumulation. The stability of the inbred line response across locations was also determined in order to identify lines with broad or specific adaptability for disease resistance. Inbred lines CML 390, RO 424W, US 2540W and VO 617y-2 consistently exhibited low FER severity (≤5%), fungal target DNA (≤0.1 ng μL-1) and fumonisin levels (≤5 mg kg-1) at most test locations. Line RO 424W was the most stable in its response to disease and fumonisin accumulation across environments. The elite inbred lines were highly susceptible to F. verticillioides and fumonisin accumulation with only CB-222 and CB-248 having intermediate resistance. These inbred lines may serve as sources of resistance in breeding programmes but can also be used in genomic and proteomic studies to better understand the genetic basis of resistance to F. verticillioides and its fumonisins. The response of Kenyan inbred lines, previously characterised for resistance to Aspergillus flavus and aflatoxins, to F. verticillioides and fumonisin contamination was evaluated in South Africa and Kenya. Common resistance mechanisms to these pathogens have been reported. The AER/aflatoxin-resistant lines were compared to South African lines previously characterised as resistant to F. verticillioides and fumonisin accumulation. Kenyan inbred lines CML 495, CML 264 and CKL05015 were most resistant to FER, F. verticillioides colonisation and fumonisin accumulation across localities. The inbred line CML 495 was also the most stable in its resistance response to F. verticillioides infection and fumonisin deposition, making it an attractive source of resistance for inclusion into a South African breeding programme. This study, therefore, provided further evidence that AER/aflatoxin-resistant lines appear to be a significant source of resistance to F. verticillioides and fumonisins. Genetic variability for resistance to F. verticillioides and fumonisin accumulation was generated by gamma irradiation of seven elite maize lines. Following mass irradiation, the lines were field planted and self-pollinated for four consecutive seasons (M0-M4 generations). The response of the M1 to M4 populations to F. verticillioides was evaluated visually for FER severity annually, and ears expressing less than 10% FER were advanced to the next breeding cycle. The M4 selections were also evaluated for F. verticillioides colonisation and fumonisin content. A number of selections derived from each inbred line were more resistant to FER, F. verticillioides colonisation and fumonisin accumulation when compared to their non-irradiated controls. However, no improved selections were obtained from inbred I-35. The transcriptional changes in maize induced upon infection by F. verticillioides were evaluated by next-generation RNA sequencing and monitored up to 7 days after inoculation (dai). Although an initial induction of defence-related transcripts associated with pathogen recognition, signalling molecules, pathogenesis-related genes, cell wall restructuring and secondary hormone-based signalling genes was observed 24 hours post inoculation (hpi), these were down-regulated 48 hpi. Plant responses did not prevent F. verticillioides from colonising maize kernels, as the target DNA of the pathogen continued to increase. At 72 hpi, genes involved in pathogenesis, G-coupled receptor signalling and response to oxidative stress were induced and may have resulted in the reduction of fungal contamination 7 dai. The transcriptional changes in maize suggest a delayed plant response to F. verticillioides infection and imply a pathogen-associated molecular pattern response characterised by a basal immunity. Several genes including PRRs, signalling molecules (protein kinases, calcium-dependant molecules, GTP-signalling and redox-associated molecules), PR protein-coding genes and those involved in secondary hormone signalling (auxins) that influence maize response to F. verticillioides warrant further investigation by genomic and proteomic approaches.
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    Monitoring fusarium, gibberella and diplodia ear rots and associated mycotoxins in maize grown under different cropping systems
    (Stellenbosch : Stellenbosch University, 2017-03) Mabuza, Londiwe; Rose, Lindy J. ; Janse van Rensburg, B.; Flett, B. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH SUMMARY: Maize ear rots represent a significant problem in most maize production areas resulting in reduced yield and quality due to visible fungal infection and mycotoxin contamination of maize grain. Mycotoxigenic fungi affecting cereal grains are particularly important for humans and animals as they pose food safety and security concerns. Increased maize productivity relies on integrated management strategies which include limiting soil erosion and water runoff. Therefore, agricultural practices that involve no-till and the retention of previous crop residues and/or cover crops are steadily increasing in maize production areas in South Africa. The relationship between no-till, the presence of crop residue in the field and maize ear rot disease severity and mycotoxin contamination is not well understood. The increase in the use of cropping systems that support the retention of crop residues in the field could have substantial impacts on maize production and food safety in South Africa. Adequate understanding of the role of agricultural practices in disease outbreaks can assist in enhancing management of maize ear rot pathogens. In this study, the influence of different cropping systems on F. verticillioides and F. graminearum accumulation, Diplodia ear rot (DER) incidence as well as mycotoxin contamination in maize grain was determined. Cropping systems did not significantly affect F. verticillioides accumulation, zearalenone and nivalenol contamination in all the years of evaluation. Fusarium graminearum accumulation, DER incidence and deoxynivalenol contamination were, however, significantly affected in certain years when disease development was favoured. A survey to establish the effect of no-till and conventional tillage practices on Fusarium ear rot, Gibberella ear rot and DER in maize grain and resultant mycotoxin contamination in maize grain was also conducted in commercial farms in South Africa. Additionally, the survival of F. graminearum and F. verticillioides as well fumonisin contamination in crop residue samples collected from conservation and conventional tillage commercial farms in South Africa was also investigated. Tillage practices did not have an effect of fungal accumulation, disease incidence and mycotoxin contamination in maize grain. The results from this study indicate that under local conditions, conservational agricultural practices can be used without the potential risk of enhanced disease accumulation and mycotoxin contamination. Fusarium graminearum and F. verticillioides accumulation and traces of fumonisins were quantified from all analysed crop residues and did not differ between tillage practices. The recovery of these ear rot-causing fungi from crop residues is an indication of its potential to act as inoculum reservoirs for these fungi. Although the levels of fungal target DNA quantified from the crop residues was low, the fungi may reproduce, survive and infect subsequent hosts.
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    Mycotoxin contamination of maize and groundnut produced by subsistence farmers in northern KwaZulu-Natal
    (Stellenbosch : Stellenbosch University, 2018-03) Phokane, Sylvia; Rose, Lindy J. ; Flett, B. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Subsistence farmers in South Africa face many production challenges including infection of their grain crops with mycotoxigenic fungi and concomitant mycotoxin contamination. Fusarium spp. and Aspergillus spp. are the most common fungal species infecting maize and groundnuts while plant-parasitic nematodes are also associated with groundnuts in South Africa. Maize and groundnut questionnaires regarding production practices were presented to subsistence farmers in Pongola, Vryheid, Jozini, Manguzi and Mbazwana districts of northern KwaZulu-Natal (KZN), South Africa. Maize and groundnut grain samples were also collected at harvest and after three months of storage during the 2012/13 and 2013/14 seasons. Groundnuts, roots and soil samples were collected before harvest during the 2013/14 season, only. Fusarium graminearum, F. verticillioides and A. flavus target DNA levels were quantified in maize using quantitative polymerase chain reaction and the presence of multi-mycotoxins were determined using the liquid chromatography tandem mass spectrometry. Nematodes were extracted using sieving method and identified microscopically. Questionnaires revealed that over 90% of farmers were not aware of mycotoxins or their implications on human and livestock health. Visually diseased grain was often fed to livestock sensitive to mycotoxicosis such as chickens. Production practices amongst some farmers including crop rotation and the well-ventilated storage of grain may contribute to reduced mycotoxin contamination. In maize grain the Fusarium graminearum levels were significantly higher than F. verticillioides and A. flavus levels in both seasons. Contrary to expectations, zearalenone, produced by F. graminearum, was very low (<0.02 μg/g) at harvest and storage during both seasons while deoxynivalenol and nivalenol was not detected. There were significant differences between districts (localities) and collection periods (harvest and storage) and localities per seasons (P < 0.05) for all mycotoxigenic fungi and mycotoxins evaluated. Maize sampled in Jozini district was the most contaminated with mycotoxigenic fungi and mycotoxins while Mbazwana and Manguzi districts were the least contaminated. Four plant-parasitic nematodes, namely D. africanus, Pratylenchus spp., Meloidogyne spp. and Helicotylenchus spp., were identified from groundnut samples obtained in Jozini, Manguzi and Mbazwana during the 2012/13 and 2013/14 seasons. Furthermore, Tylenchus spp. was identified for the first time in groundnuts, pegs and soil collected before harvest during the 2013/14 season. Results from this study showed that there is a need for mycotoxin awareness campaigns and additional surveillance to continuously monitor mycotoxin contamination and potential exposure. More in-depth analyses of all the potential factors contributing to mycotoxin contamination and exposure, particularly in the subsistence production are of northern KZN, is warranted.
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    Mycotoxin levels in subsistence farming systems in South Africa
    (Stellenbosch : Stellenbosch University, 2008-03) Ncube, Edson; Waalwijk, C.; Flett, B. C.; Viljoen, Altus; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Fusarium spp. and Aspergillus spp. are toxin-producing fungi associated with maize and groundnut. Fusarium verticillioides produces fumonisins in maize, and Aspergillus flavus produces aflatoxins in maize and groundnut kernels. Both toxins are responsible for carcinogenesis in humans and animals. Contamination of maize and groundnut with mycotoxins is often most severe in rural areas where subsistence farmers are unaware of their existence and follow agricultural practices that might contribute to their production. A questionnaire was, therefore, compiled to investigate agricultural decisions in rural areas that may influence mycotoxin contamination of crops. During 2006 and 2007, maize and groundnut samples were collected in the Eastern Cape, KwaZulu-Natal (KZN), Limpopo, and Mpumalanga provinces. Mycotoxin levels were quantified using the ELISA technique, and the incidence of Fusarium spp. in maize grain was determined by plating maize kernels out on Fusarium selective medium. Fumonisin-producing Fusarium spp. were also quantified using real-time PCR (TaqMan). The incidence of A. flavus and A. parasiticus in groundnut was determined by plating out kernels on potato dextrose agar. Fumonisin contamination levels in maize samples ranged from 0-21.8 parts per million (ppm) and aflatoxin levels ranged from 0- 49 parts per billion (ppb), depending on the region where samples were collected. Aflatoxin levels in groundnut ranged from 0-160.1 ppb. Fusarium verticillioides was the most common Fusarium sp. in maize followed by F. subglutinans and F. proliferatum, respectively. Regression analyses showed a positive correlation between fumonisin-producing Fusarium species when determined by real-time PCR and fumonisin concentration (r2=0.866). Regression analyses further showed a highly significant positive correlation between A. flavus and aflatoxin contamination (r2=0.10235). Samples from northern KZN contained levels of mycotoxins that were far in excess of the maximum levels set by the Food and Drug Administration in the USA. In South Africa there are currently no regulations with regard to the maximum allowable levels of fumonisin in human food. The high incidence of mycotoxin contamination of human food in subsistence farming systems indicates the need for awareness programmes and further research.
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    Resistance in South African maize inbred lines to the major ear rot diseases and associated mycotoxin contamination
    (Stellenbosch : Stellenbosch University, 2014-12) Mouton, Marili; Viljoen, Altus; Rose, Lindy J. ; Flett, B. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Maize (Zea mays L.) is one of the most important grain crops produced globally and serves as the primary source of carbohydrates and vitamins to millions of people in Africa. Whenever environmental conditions are favourable, fungal species such as Fusarium verticillioides, Fusarium graminearum sensu lato, Aspergillus flavus and Stenocarpella maydis frequently infect the ears of maize, reducing yield and grain quality. Of greater economic concern is the contamination of maize kernels with mycotoxins produced by ear rot pathogens due to its association with mycotoxicoses and immune suppression in humans and animals. Outbreaks of ear rot diseases commence in the field, but their associated toxins can be produced along the value chain. Planting resistant cultivars, as part of an integrated management strategy, could provide effective means of controlling preharvest ear rot diseases and mycotoxin accumulation in maize. Maize cultivars resistant to the major ear rot fungi and their mycotoxins are not yet available in South Africa and therefore should be developed in plant improvement programmes where durable resistance is combined with useful agronomic traits. The first step in introducing resistance into maize cultivars would be to find sources of genetic resistance. Infertility or unwanted traits may be present in wild relatives or other species of maize, and therefore locally adapted breeding material would be the most desirable source. This research aimed to identify publically available maize genotypes with durable resistance to the major ear rot pathogens and their associated mycotoxins in South Africa. In this study, a collection of inbred lines with diverse genetic backgrounds and valuable agronomic characteristics were evaluated under a range of field conditions. Some inbred lines were resistant to Fusarium ear rot (FER) and fumonisin contamination during artificially inoculated trials over two years. Furthermore, these FER-resistant inbred lines have been tested for resistance to other important maize ear rot diseases including Gibberella ear rot, Diplodia ear rot and Aspergillus ear rot in a multi-location field trial. Inbred lines with low and high levels of resistance to multiple infections were identified, but significant inbred x location interactions were observed. This suggests that potentially resistant lines will require further testing in an extra season to confirm their resistant status. If confirmed, these sources could be used to investigate the underlying mechanisms conferring resistance, or to develop molecular markers to facilitate the transfer of resistance into commercially valuable cultivars.