Bradley C. Flett

Analysis of the Fusarium graminearum species complex from wheat, barley and maize in South Africa provides evidence of species-specific differences in host preference

Species identity and trichothecene toxin potential of 560 members of the Fusarium graminearum species complex (FGSC) collected from diseased wheat, barley and maize in South Africa was determined using a microsphere-based multilocus genotyping assay. Although three trichothecene types (3-ADON, 15-ADON and NIV) were represented among these isolates, strains with the 15-ADON type predominated on all three hosts. A significant difference, however, was identified in the composition of FGSC pathogens associated with Gibberella ear rot (GER) of maize as compared to Fusarium head blight (FHB) of wheat or barley (P<0.001). F. graminearum accounted for more than 85% of the FGSC isolates associated with FHB of wheat and barley (N=425), and was also the dominant species among isolates from maize roots (N=35). However, with the exception of a single isolate identified as an interspecific hybrid between Fusariumboothii and F. graminearum, GER of maize (N=100) was exclusively associated with F. boothii. The predominance of F. graminearum among FHB isolates, and the near exclusivity of F. boothii among GER isolates, was observed across all cultivars, collection dates, and provinces sampled. Because these results suggest a difference in host preference among species of the FGSC, we hypothesize that F. graminearum may be less well adapted to infect maize ears than other members of the FGSC.

Diplonine, a Neurotoxin Isolated from Cultures of the Fungus Stenocarpella maydis (Berk.) Sacc. that Induces Diplodiosis

Diplodiosis is a neuromycotoxicosis of cattle and sheep caused by ingestion of maize infected with the ear-rot fungus Stenocarpella (= Diplodia ) maydis . Apart from ataxia, paresis, and paralysis, the toxin is responsible for stillbirths and neonatal losses characterized by the presence of spongiform degeneration in the white matter of the brain in the offspring of dams exposed to infected maize cobs. In the present study a toxin, named diplonine, which induced neurological signs in guinea pigs resembling some of those occurring in cattle and sheep, was isolated from S. maydis cultures. Purification of diplonine was achieved by methanol extraction followed by chromatographic separation on silica gel and RP-18 stationary phases. The structure and relative configuration of diplonine were defined by analysis of NMR and MS data as (S)-2-amino-2-[(1R,2S)-1-hydroxy-2-methylcyclopropyl]acetic acid or the (S)-2-amino-2-[(1S,2R)-diastereomer.

Field evaluation of resistance to aflatoxin accumulation in maize inbred lines in Kenya and South Africa

ABSTRACT Aflatoxin, a carcinogenic toxin, is produced mainly by Aspergillus flavus and Aspergillus parasiticus. Contamination of maize (Zea mays L.) grain by these fungi occurs before harvest, and the easiest strategy to prevent this is to develop/use maize varieties resistant to Aspergillus spp. and aflatoxin accumulation. The objective of this investigation was to identify potential sources of resistance among 23 maize inbred lines (13 obtained from the MAIZE Competitive Grants Initiative, International Maize and Wheat Improvement Centre and 10 from Agricultural Research Council, South Africa). The inbred lines were planted in a randomized complete-block design at two locations each in Kenya and South Africa. Maize ears were inoculated at silking with three toxigenic strains of A. flavus. The inoculated ears in each plot were harvested at 12–18% moisture, dried, and visually assessed for Aspergillus ear rot (AER). Aflatoxin concentration in the kernels was determined using liquid chromatography–tandem mass spectrometry. Significant variation for both AER and aflatoxin concentration existed among the inbred lines at both locations in Kenya and one location in South Africa. Combined analysis revealed a significant (p < 0.001) lines × locations interaction for both AER and aflatoxin concentration. Higher incidences of AER (0–86.0%) and aflatoxin concentration (0.21–6.51 µg/kg) were recorded at Kiboko in Kenya than at the other three locations. A stronger genetic correlation (rG = 0.936, p < 0.0001) between the AER and aflatoxin concentration was recorded in Potchefstroom than at the other three locations. Repeatability of aflatoxin concentration was high at Kiboko (0.87) and Potchefstroom in South Africa (0.74). Three inbred lines, CML247, CML444, and CML495, emerged as potentially useful sources of resistance to AER and aflatoxin accumulation as they showed low levels of aflatoxin contamination in both localities in Kenya and in South Africa.

Sampling variation in the quantification of fumonisins in maize samples

Fumonisins produced by F. verticillioides and F. proliferatum cause mycotoxicoses in horses, swine and rats and have been associated with oesophageal cancer in humans. Accurate measurement of mycotoxins is essential for determining the safety of grain and their products for consumption. Four sources of variation were studied, namely sub-sample size, variation within a single maize sub-sample, number of replicates and toxin detection techniques used by independent laboratories. Variation in detected fumonisin levels within a single maize sample was high using the 25 g sub-samples proposed in the Neogen Veratox protocols. A 250 g sub-sample significantly reduced variation in fumonisin levels of samples. An incremental increase in sample size also improved the number of positive samples recorded. Increasing the number of replicates using the recommended sub-sample size (25 g) did notreduce variation except when the sample had high fumonisin levels. Improved accuracy was recorded when a 250 g sub-sample was used in conjunction with increased replicates. Data from laboratory analyses indicated that ELISA reactions (Agricultural Research Council – Grain Crops Institute) correlated significantly with HPLC results of the Medical Research Council (MRC), but neither of these correlated with results from an independent laboratory. Concentrations determined using ELISA were consistently higher than those from the HPLC (MRC) technique. Quantification technique, sample size, replicate number and laboratory where analyses are conducted, appear to be important sources of variation for quantification of fumonisins.

Variation in the identification of Fusarium spp. in maize samples due to enumerator and growth medium

Fusarium spp. have variable phenotypic and morphological features when cultured on different media. Inaccurate identifications could result in variation in the quantification of Fusarium spp. associated with ear rot of maize when two or more researchers identify the same Fusarium isolates based on morphological characteristics. Two hundred and fifty kernels from maize samples collected from each of five localities were surface-sterilized and plated on selective rose Bengal-glycerine-urea (RbGU) medium from which isolates were subsequently transferred to split plates containing Carnation Leaf Agar (CLA) and Potato Dextrose Agar (PDA) for further identification. Three different enumerators independently identified and enumerated Fusarium spp. on RbGU and CLA/ PDA media to determine the relative frequency of Fusarium spp. within maize samples, to compare the accuracy of species identification on different media and to detect and quantify bias among enumerators (inter-enumerator reliability). It was concluded that enumerators were consistent in the identification and quantification of F. verticillioides and F. subglutinans on both RbGU and CLA/PDA media. F. proliferatum enumerations yielded an interaction between locality and enumerator with no significant differences for media. The significant locality x enumerator interaction for F. proliferatum suggests that enumerators had difficulty to distinguish between F. proliferatum and F. verticillioides. The use of RbGU and/or CLA/PDA media were eliminated as a possible source of variation in the identification of Fusarium spp. The inter-enumerator reliability study indicated that Fusarium spp. enumerations by three different enumerators were reliable and accurate and were not a source of variation in the quantification of Fusarium spp. associated with maize ear rot.

Alternaria alternata, the causal agent of leaf blight of sunflower in South Africa

Sunflower (Helianthus annuus L.) is an important oilseed crop in South Africa, and is grown in rotation with maize in some parts of North West, Limpopo, Free State, Mpumalanga and Gauteng provinces. Alternaria leaf blight is currently one of the major potential disease threats of sunflower and is capable of causing yield losses in all production regions. Alternaria helianthi was reported as the main cause of Alternaria leaf blight of sunflower in South Africa; however small-spored Alternaria species have been consistently isolated from leaf blight symptoms during recent surveys. The aim of this study was to use morphological and molecular techniques to identify the causal agent(s) of Alternaria blight isolated from South African sunflower production areas. Alternaria helianthi was not recovered from any of the sunflower lesions or seeds, with only Alternaria alternata retrieved from the symptomatic tissue. Molecular identification based on a combined phylogenetic dataset using the partial internal transcribed spacer regions, RNA polymerase second largest subunit, glyceraldehyde-3-phosphate dehydrogenase, translation elongation factor and Alternaria allergen gene regions was done to support the morphological identification based on the three-dimensional sporulation patterns of Alternaria. Furthermore, this study aimed at evaluating the pathogenicity of the recovered Alternaria isolates and their potential as causal agents of Alternaria leaf blight of sunflower. Pathogenicity tests showed that all the Alternaria alternata isolates tested were capable of causing Alternaria leaf blight of sunflower as seen in the field. This is the first report of A. alternata causing leaf blight of sunflower in South Africa.

Accumulation of toxigenic Fusarium species and Stenocarpella maydis in maize grain grown under different cropping systems

Mycotoxigenic fungi such as Fusarium graminearum, Fusarium verticillioides and Stenocarpella maydis infecting maize grain can be detrimental to both humans and animals due to the toxins they produce. Disease management strategies include tillage practices and crop rotations, however, these have not been sufficiently evaluated in South Africa. The effect of cropping systems on ear rot accumulation and mycotoxin contamination in maize grain was investigated in two localities over a four and six-year period. Cropping systems evaluated were: 1) monoculture maize conventional tillage, 2) monoculture maize no-till, 3) two, and 4) three-year rotation systems consisting of maize/cowpea and maize/cowpea/babala (all no-till), respectively. In Buffelsvallei, two additional crop rotations, maize/sunflower and maize/sunflower/babala (all no-till) were included. Naturally infected trials were visually evaluated for disease severity or incidence while fungal and mycotoxin contamination of maize grain was quantified. Disease incidence and mycotoxin contamination were inconsistent throughout the study period due to seasonal and geographical differences. In Buffelsvallei, cropping system had a significant effect (P < 0.05) on the accumulation of fumonisins and F. graminearum for 2010/11, deoxynivalenol (2011/12) and S. maydis incidence (2013/14). Fusarium graminearum and fumonisin accumulation was significantly higher in the three-year maize/cowpea/babala rotation and two-year sunflower rotation in the 2010/11 season, respectively. Deoxynivalenol levels in monoculture maize, using conventional tillage (2011/12) was significantly higher when compared to all other cropping systems and S. maydis incidence was significantly higher in maize conventionally tilled, no-till and two-year maize/cowpea and maize/sunflower cropping systems in the 2013/14 season. Cropping systems had no significant effects on fungal infection or mycotoxin accumulation in maize grain obtained from trials conducted at Erfdeel. The results of this study indicate that Conservation Agriculture systems under the environments evaluated, did not increase the risk of maize ear rots and mycotoxin production.

Evaluating three commonly used growth media for assessing fumonisin analogues FB1, FB2 and FB3 production by nine Fusarium verticillioides isolates

ABSTRACT Maize is most often infected by the fumonisin-producing Fusarium verticillioides. Total fumonisins of natural infected grain is made up of FB1, FB2 and FB3 with FB1 occurring naturally at higher levels. A maize plant can be infected with more than one F. verticillioides isolate, and finding a reliable method to elucidate the toxigenic potential of these isolates is important to extrapolate the possible fumonisin risk to consumers of grain. It is not clear whether F. verticillioides produces similar fumonisin levels, as well as fumonisin analogue ratios, across media. In this study, nine F. verticillioides isolates were subjected to three methods of fumonisin testing using liquid media, maize patties and a field trial (silk inoculation of grain) in Potchefstroom, South Africa. Spore concentrations of 1 × 106 conidia ml–1 of each isolate were used to inoculate the different media and levels fumonisin analogues were measured using HPLC. Fumonisin production per isolate was highly variable and was influenced by the two-way interaction of F. verticillioides isolate × growth media. Total fumonisins produced in the liquid medium ranged from 0 to 21.3 ppm, on maize patties fumonisins they ranged from 0 to 21.5 ppm, and in the silk inoculation technique they ranged from 0 to 15.5 ppm. The fumonisin analogue FB1 occurred at higher levels followed by FB3 in both in vitro studies. In the silk inoculation technique, fumonisin analogue FB2 was the second highest occurring analogue after FB1. Isolate GCI 282 produced higher FB2 and FB3 levels than FB1 in the patties and grain, respectively. In order not to miscalculate the fumonisin and analogue ratio levels per F. verticillioides isolate, the growth medium will have to be optimised for each isolate and more than one growth medium used.

Studies towards optimising the isolation of diplonine, a neurotoxin isolated from cultures of Stenocarpella maydis (Berk.) Sacc.

Diplonine, a mycotoxin that induces neurotoxic clinical signs in the guinea pig, resembling those occurring in cattle and sheep with diplodiosis, was isolated previously from a Stenocarpella maydisculture. Knowledge of the chemical properties of the toxin, which was characterised as a substituted ß-cyclopropylamino acid, enabled amendments in the present study to the initial steps of the isolation procedure. Extraction with water and fractionation by cation exchange chromatography improved the efficiency of isolation, potentially allowing the preparation of larger amounts of the toxin.Diplonine, a mycotoxin that induces neurotoxic clinical signs in the guinea pig, resembling those occurring in cattle and sheep with diplodiosis, was isolated previously from a Stenocarpella maydisculture. Knowledge of the chemical properties of the toxin, which was characterised as a substituted ß-cyclopropylamino acid, enabled amendments in the present study to the initial steps of the isolation procedure. Extraction with water and fractionation by cation exchange chromatography improved the efficiency of isolation, potentially allowing the preparation of larger amounts of the toxin.