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 (R2=0.8303) was found between pg fungal DNA and fumonisin content. A similar correlation (R2=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 B1+B2 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.

Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic for Clavibacter michiganensis subsp, michiganensis, the causal agent of bacterial canker on tomato, C. m. subsp. sepedonicus, the causal agent of ring rot on potato, and Xanthomonas campestris pv. vesicatotqa, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.

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The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed. Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358 100-fold.

We developed a multiplex PCR for the identification of the most frequently occurring species of Fusarium on wheat in Western Europe. Surveys suggest that Fusarium graminearum has replaced Fusarium culmorum as the dominant species on winter wheat in the Netherlands during the 1990s. Quantitative PCR was used to monitor Fusarium populations during the growing season. Populations on lower leaves consisted primarily of Microdochium nivale, but F. graminearum dominated in heads and on harvested grain, suggesting that the inoculum for the infection of the heads did not come from the leaves. A quantitative PCR recently developed for Fusarium verticillioides from maize may help to reduce the threat posed by fumonisin in large communities in Africa that rely on maize as a staple food.