Herbs and spices are used globally by most population groups across the world. In sub-Saharan Africa, they have medicinal significance in addition to their culinary uses. Although herbs and spices are often used in combination, there are few studies that report on their interactive antimicrobial effect. This study screened 17 culinary herbs and spices (crude extracts and essential oils) for antimicrobial activity, individually, followed by 1:1 combinations of the active extracts, to determine the outcome of combining these for antibacterial activity. The minimum inhibitory concentrations (MIC) were determined against six common foodborne pathogens using the broth microdilution assay. The design of experiments (DOE) approach was subsequently employed in MODDE 9.1((R)) software, to optimise the experimental runs so as to identify the best interaction that would produce the best possible antimicrobial effect. Phytochemical profiling of the most active extracts was achieved using ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) analysis. The results demonstrated that combining Rosmarinus officinalis with either Syzygium aromaticum, or Salvia officinalis methanol extracts produced synergistic antimicrobial effects towards B. cereus with Sigma FIC = 0.25 mg/ml and 0.31 mg/ml, respectively. The DOE predicted that a combination of higher ratios of R. officinalis (59.5 %), higher ratios of S. officinalis (40 %), and lower ratios of S. aromaticum (0.5 %) would produce the best antimicrobial effect with MIC = 0.17 mg/ml. This was experimentally confirmed and there was a strong correlation (r-value 0.73) between the predicted and experimental MIC values, leading to the identification of an optimal antimicrobial combination. The combination of R. officinalis (56 %) and S. officinalis (44%) produced the antimicrobial effect (MIC = 0.19 mg/ml) which can be recommended for future studies. (c) 2023 The Authors. Published by Elsevier B.V. on behalf of SAAB. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Busseola fusca and Fusarium verticillioides are considered the most important pest and pathogen, respectively, of maize in South Africa. Busseola fusca tunnels into stems and ears of maize plants, whereas F. verticillioides causes Fusarium ear rot and deposits fumonisins in maize kernels. Frass (excreta) deposited by B. fusca larvae in maize stems and ears during feeding could potentially be contaminated with pathogens such as F. verticillioides, thereby indirectly and passively contributing to plant diseases. The mycoflora present in frass of B. fusca larvae was thus investigated in this study, and the dissemination of fumonisin-producing Fusarium spp. by B. fusca larvae in maize stems determined. Busseola fusca frass was collected from plants with visible insect damage in three maize-growing districts in South Africa. The mycoflora in the frass was isolated and identified morphologically to genus level, followed by the sequencing of target genes for species identification. Species of Acremonium, Aspergillus, Fusarium, Mucor, Rhizopus and Talaromyces were associated with the B. fusca frass. The role of B. fusca larvae in disseminating fumonisin-producing Fusarium spp. was then determined in greenhouse and field trials. Maize whorls were inoculated with F. verticillioides MRC826 spores 4 weeks after plant emergence, and infested with B. fusca larvae 2 days later. The stems were split open after 3 weeks and frass collected from feeding channels, thereafter, target DNA of fumonisin-producing Fusarium spp. was quantified using real-time PCR. Target DNA of fumonisin-producing Fusarium spp. was significantly higher in frass collected from greenhouse plants inoculated with F. verticillioides than in frass collected from the uninoculated control, indicating that the inoculation was successful in the absence of soil-borne inoculum. Nevertheless, the field trial showed no significant differences in target DNA in frass from inoculated and non-inoculated plants. This is possibly due to natural F. verticillioides infection of maize plants in the field. This study further indicated that B. fusca frass also contained maize pathogens such as Aspergillus spp. and Fusarium spp., which could cause ear rot diseases. The occurrence of Acremonium zeae in frass has potential implications for the biological control of F. verticillioides, as the fungus produces pyrrocidines A and B antibiotics which are known to be antagonistic to F. verticillioides. The occurrence of Aspergillus niger in frass requires further investigation in order to clarify its role in fumonisin contamination of maize in South Africa.

Mycotoxigenic fungi are common pathogens of maize and groundnuts; they produce mycotoxins which reduce the yield and quality of these grain crops. Numerous agricultural practices including crop rotation and storage methods have been shown to impact mycotoxin accumulation. Therefore, the farming and storage practices in maize and groundnut subsistence farming systems in Pongola, Vryheid, Jozini, Manguzi and Mbazwana Districts of northern KwaZulu-Natal (South Africa) were surveyed to determine their potential role in promoting or mitigating mycotoxin contamination. A questionnaire about agricultural farming practices and storage facilities was presented to 65 subsistence maize and/or groundnut farmers. At least 90% of the farmers surveyed were not aware of mycotoxins and their consequences to animal and human health. The majority of the farmers did not practise crop rotation. However, they practised intercropping and sorted damaged and mouldy grain (maize and groundnuts) before storage. The damaged or mouldy grain was largely used as animal feed, thereby exposing animals to an increased risk of mycotoxicoses. Metal tanks and inqolobane (a type of wooden structure) were identified as the most common storage structures. Harvested homegrown maize was mostly used for the farmers’ own consumption but also sometimes sold to the local community. The implementation of mycotoxin awareness campaigns is necessary, particularly in these districts. The storage facilities used by the subsistence farmers allowed increased moisture and insect invasion. The need for the surveillance of mycotoxins in subsistence-farmed food crops is vital. Significance: The main finding of this study is the extent of post-harvest losses and mycotoxin contamination of maize produced by smallholder farmers in South Africa. We further identify methods to manage the risk of mycotoxin exposure to smallholder farmers and their communities as well as reduce post-harvest losses.

Maize production in South Africa is negatively affected by Fusarium verticillioides, an endophytic maize pathogen, as well as by Busseola fusca larval damage. Fusarium verticillioides causes ear, stem and root rot, and also produces fumonisin mycotoxins which are toxic to humans and livestock. The African stem borer (Busseola fusca) is a pest of economic importance in maize plants in South Africa. In this study, the interaction between E verticillioides and B. fusca was investigated to elucidate its effects on Fusarium ear rot and fumonisin production in a Bt hybrid (MON810 event) and its B. fusca-susceptible non-Bt isohybrid. Field trials were conducted over three seasons using a randomised complete block design with six replicates per treatment. The effect of Beta-cyfluthrin (non-systemic, granular) and Benfuracarb (systemic, seed treatment) insecticide applications on the incidence of Fusarium ear rot and fumonisin production in maize was also determined in an unrelated conventional hybrid. For B. fusca infestations, larvae were dispensed into the whorl of each plant at the 12th leaf stage prior to tasselling, while a E verticillioides MRC826 spore suspension was inoculated through the silks at the Bilking stage. Maize ears were harvested at physiological maturity and Fusarium ear rot, total fumonisin levels, stem borer damage and target DNA of fumonisin-producing Fusarium spp. quantified. Significantly less Fusarium ear rot and fumonisin were produced in the Bt maize hybrid compared to the non-Bt isohybrid under natural farming conditions, but fungal colonisation and fumonisin production under artificial E verticillioides inoculation did not differ significantly between the Bt and non-Bt maize. Fumonisin production correlated moderately with the quantity of target DNA of fumonisin-producing Fusarium spp. extracted from maize plants. Benfuracarb application to control stem borer infestation resulted in a significant reduction in Fusarium ear rot and fumonisin production while Beta-cyfluthrin did not. Moreover, B. fusca damage to maize ears significantly increased when both insecticides were not applied to the B. fusca-infested plants. This study indicated that Bt maize and the application of Benfuracarb reduce B. fusca damage to maize ears thereby indirectly reducing Fusarium ear rot and fumonisin production. However, this was not consistent over seasons due to differences in climatic conditions. (C) 2017 Elsevier Ltd. All rights reserved.

Fusarium verticillioides is most frequently associated with maize in South Africa. It colonises maize roots, stems and ears endophytically and causes diseases such as Fusarium ear rot (FER) and stalk rot. Fusarium verticillioides can produce fumonisins, which are toxic secondary metabolites harmful to humans and animals. It is, however, unknown whether endophytic and pathogenic isolates from distinct maize tissues differ in their ability to cause disease and produce fumonisins. In this study, Fusarium spp. were collected from maize roots, stems and kernels for phylogenetic analysis and the F. verticillioides isolates were subjected to pathogenic and toxigenic comparison. The translation elongation 1-α (TEF1) gene of the isolates was sequenced, and a phylogenetic tree constructed with maximum likelihood (ML) and Bayesian interference (BI) inferred. Fumonisin production of F. verticillioides isolates was determined in vitro and in planta by using high performance liquid chromatography, and virulence of the isolates was determined by silk channel inoculation of maize ears under field conditions. F. verticillioides was the species with the highest number of isolates followed by F. temperatum and then F. subglutinans. Phylogenetic analyses clustered the different Fusarium spp. according to species. Fumonisin production by F. verticillioides isolates varied from 0 to 21.3 mg/kg in vitro, and 0–16.2 mg/kg in planta. All the F. verticillioides isolates produced FER symptoms, including isolates from roots and stems. Fusarium verticillioides isolates in South Africa thus presented a species highly diverse in toxigenicity, but not in virulence. This finding has implications for managing mycotoxins in maize, as visible symptoms might be misleading to the actual toxin level present, for example low level of disease severity might represent high fumonisin levels and vice versa. The high numbers of F. temperatum, also a mycotoxin producer highlights the concern that kernels could be contaminated with more than one mycotoxin. Integrated disease management of not only F. verticillioides but all Fusarium spp. should thus focus strongly on reducing fungal contamination of maize and the detoxification of grain with focus on using regionally adapted maize varieties.

Fusarium verticillioides and Busseola fusca are among the most significant biotic constraints to maize production in South Africa. In this study, the effect of B. fusca damage and mechanical wounding to maize ears on Fusarium ear rot development and fumonisin production was investigated. The effect of the interaction on Fusarium ear rot and B. fusca damage was studied by inoculating maize ears with F verticillioides isolate MRC826 and infesting plant whorls with aliquots of 10-15 neonate larvae at the 12th leaf stage prior to tasselling. To simulate hail damage, maize ears were mechanically wounded at the blister stage with cork borers of different sizes and number of wounds, with and without F verticillioides inoculation. Uninoculated, uninfested and undamaged control treatments were included. Field trials were conducted over three seasons using a randomised complete block design with six replicates per treatment. In all seasons, most Fusarium ear rot developed on ears inoculated with F verticillioides. This study indicated that B. fusca infestation was not associated with higher Fusarium ear rot incidence in two of the three seasons. Moreover, B. fusca infestation was not associated with high fumonisin production in any season. This indicates that the presence of B. fusca was not highly associated with Fusarium ear rot or fumonisin production, possibly due to its characteristic feeding nature that causes sporadic wounds on maize ears, but also the strong impact of weather variability. Fusarium ear rot development and fumonisin production by F verticillioides inoculum varied seasonally indicating the importance of environmental conditions on Fusarium ear rot and fumonisin production. Fusarium ear rot and fumonisin production significantly increased with the severity of cork borer wounding in both naturally infected and artificially inoculated maize ears. Therefore, the prevention of severe injuries to kernels, whether by mechanical damage or insects, should be considered as important in reducing Fusarium ear rot and fumonisin production in maize. (C) 2017 Elsevier Ltd. All rights reserved.

Using micro high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) a simple and fast method for the quantitative determination of 26 mycotoxins was developed. Sample preparation consists of a single extraction step and a dilute-and-shoot approach without further cleanup. With a total run time of 9 min and solvent consumption below 0.3 mL per chromatographic run, the presented method is cost-effective. All toxins regulated by the European Commission with maximum or guidance levels in grain products (fumonisins B-1 and B-2 (FB1 and FB2)); deoxynivalenol (DON); aflatoxins B-1, G(1), B-2, and G(2) (AFB(1), AFG(1), AFB(2), and AFG(2)); ochratoxin A (OTA); T-2 and HT-2 toxins; and zearalenone (ZEN) can be quantified with this method. Furthermore, the enniatins B, B1, A, and A1 (EnB, EnB1, EnA, and EnA1); beauvericin (BEA); 3-acetyl- deoxynivalenol (3-AcDON); fusarin C (FusC); sterigmatocystin (STC); gliotoxin (GT); and the Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), and altertoxin I (ATX I) can also be quantified. For all regulated compounds, recoveries ranged between 76 and 120 %. For all other toxins, the recovery was at least 51 %. The method was applied for the analysis of 42 maize samples from field trials in South Africa.

Fusarium spp. produce fumonisins - mycotoxins that are of importance to maize production in South Africa. Fumonisins have been associated with human oesophageal cancer and cause various diseases in animals that are of concern to the animal feed industry. Maize samples, collected from subsistence farm fields in the Eastern Cape, KwaZulu-Natal, Limpopo and Mpumalanga provinces of South Africa during the 2006 and 2007 growing seasons, were analysed for Fusarium spp. and contamination with fumonisins. Fusarium verticillioides was the most common Fusarium species in maize followed by F. subglutinans and F. proliferatum. Levels of contamination with fumonisins ranged from 0 μg/g to 21.8 μg/g, depending on the region where samples were collected. Levels of fumonisins were highest in northern KwaZulu-Natal (Zululand) where 52% and 17% of samples collected in 2006 and 2007, respectively, exceeded 2 μg/g. Regression analyses showed a positive correlation between fumonisin-producing Fusarium spp. determined by real-time polymerase chain reaction and concentration of fumonisins (r = 0.93). Many samples from Zululand, and some from Mokopane (Limpopo) and Lusikisiki (Eastern Cape), contained fumonisins at levels well above the maximum levels of 2 μg/g set by the Food and Drug administration (USA) and therefore also the limit of 1 μg/g set by the European Union for food intended for direct human consumption. Regulations governing contamination of grain with fumonisins are not yet implemented in South Africa. The high incidence of fumonisins in subsistence farming systems indicates the need for awareness programmes and further research.

Author: Ncube, E. Flett, B.C. Waalwijk, C. Viljoen, A. Vol 27 Issue 2 Publication: 2010 Page: 195-198 : Aflatoxins are carcinogenic mycotoxins produced by Aspergillus spp. in groundnut kernels. Forty-six groundnut samples were collected from subsistence farmers in three provinces of South Africa, namely KwaZulu-Natal (KZN), Mpumalanga (MP) and Limpopo (LP), in 2006 and 2007. Aflatoxin levels of groundnut kernels were quantified using an ELISA technique. The occurrence of A. parasiticus colonies was predominant and it was isolated at twice the frequency of A. flavus. Aflatoxins were present in groundnut produced in the northern parts of KZN, where aflatoxin levels of up to 131 parts per billion (ppb) were found. In Mpumalanga and Limpopo, the highest aflatoxin levels were 160 ppb and 2 ppb, respectively. In the Makhanisi and Mbazwane localities situated in KwaZulu-Natal, and in Boshoffontein in Mpumalanga, aflatoxin levels were higher than the maximum permitted level set by the Food and Drug Administration in the USA (20 ppb), the European Union (6 ppb) and the Department of Health in South Africa (10 ppb) for groundnut that is intended for direct human consumption. This study indicates the need for mycotoxin awareness campaigns and control programs to be implemented in rural areas of South Africa.

A quantitative detection tool was developed to enable the monitoring of fumonisin-producing fungi in food and feed commodities. To this end, a quantitative PCR (TaqMan) was developed that targets a conserved region in the polyketide synthase gene fum1, which is involved in the biosynthesis of fumonisin. Hence, this method specifically detected isolates from the fumonisin-producing species Fusarium verticillioides, F. proliferatum, F. nygamai and F. globosum whereas isolates of the fumonisin non-producing species F. equiseti, F. graminearum, F. oxysporum, F. semitectum and F. subglutinans that commonly occur on maize were not detected. Moreover, a few fumonisin non-producing F. verticillioides isolates did not generate any fluorescent signals and were therefore not detected. The correlation between quantitative PCR and mycotoxin content was determined using field samples collected at homestead farms in South Africa. Among 40 samples from the Eastern Cape collected in 2005 a good correlation (R 2 =0.8303) was found between pg fungal DNA and fumonisin content. A similar correlation (R 2 =0.8658) was found among 126 samples collected from four provinces in South Africa in 2007. These observations indicate that samples containing ≥ 40 pg fungal DNA/mg sample are suspected of also exceeding the 1 mg/kg total fumonisin level and therefore do not comply with the European Commission limit for fumonisins B 1 +B 2 for maize intended for direct human consumption that applies from 1 October 2007. Combined with the very high maize intake, our results indicate that fumonisin levels in maize from South African homesteads regularly exceed the tolerable daily intake for fumonisins.