Real-time PCR mediated monitoring of Fusarium foetens in symptomatic and non-symptomatic hosts

Abstract

Fusarium foetens is a recently described aggressive vascular pathogen of Begonia x hiemalis. Since 2004, it has caused severe losses for Begonia growers in Northern Europe and North America. F. foetens is likely to be of exotic origin. Little is known about the accumulation of the fungus in Begonia plants before and during symptom expression and about its host range. We have optimised a molecular detection method for F. foetens by only using the plant part containing the largest amount of the pathogen and by optimising the tissue maceration and DNA extraction techniques. This allowed a reliable detection limit of 2310 spore equivalents per plant and a theoretical detection limit of as low as 84 to 167 spore equivalents per plant. Using this method, we demonstrated exponential accumulation of F. foetens DNA in Begonia roots, resulting in symptoms at a threshold of approximately 10 7 spore equivalents and levelling off at 10 9 spore equivalents per plant. The observed rate of accumulation and the amount of pathogen DNA in non-symptomatic plants can be combined to determine whether the cuttings were infected after delivery at the Begonia nursery and to calculate the estimated timing of symptom development. To test the host range, we applied the optimised molecular detection technique. During these tests, only Begonia x hiemalis plants became symptomatic, but many other plant species supported growth of the pathogen. This information can be used to aid pathogen control and has implications for pest risk assessment.

Real‐time and qualitativePCRfor detectingPseudomonas syringaepv.actinidiaeisolates causing recent outbreaks of kiwifruit bacterial canker

“…Real-time PCR experiments based on 10-fold serial dilutions of leaf spiked samples and bacterial gDNA were run in parallel to determine the relationship between C t values of the Psa gDNA dilution series and the CFU dilution series, as previously described by Huvenne et al (2011). The amount of DNA could then be linked to CFU and the pathogen quantified in terms of CFU.…”

“…The standard quantification procedure was performed following Huvenne et al (2011). The equation of the real-time PCR dose-response curve, obtained by using the Psa CRA-PAV 1530 gDNA, was C t = À3Á378 9 log (amount of DNA in fg) + 41Á058, with an R 2 of 0Á999.…”

PM 7/111 (1) Fusarium foetens

“…DNA can be extracted from the pellets of macerated root tissue using commercial DNA extraction kits, following the manufacturer's recommendations. In Huvenne et al (2011), the Invisorb Spin Plant Mini Kit (Stratec, Germany) performed significantly better for recovery of F. foetens DNA from infected plant tissue than the other methods tested, but other commercial kits might result in adequate DNA recovery. The DNA extract is eluted in 100 lL and used in real-time PCR as described below (2.2) • Positive isolation control (PIC) to ensure nucleic acid of sufficient quantity and quality is isolated: nucleic acid extraction and subsequent amplification of the target organism or a matrix sample that contains the target organism (e.g.…”

“…(1) Confirmation with molecular tests in case of doubts (2) Isolation optional note that risks of false negative tests results are high with asymptomatic plant material pathogenicity tests, such as 25-28°C, 16 h light and 65% relative humidity (Huvenne et al, 2011). This will increase the pathogen concentration in the plants and avoid false negatives (Huvenne et al, 2011). For guidance on sampling, refer to ISPM 31 Methodologies for sampling of consignments (FAO, 2008).…”

“…A pest risk assessment for F. foetens was published in 2002 (Baayen et al, 2002) and updated recently (van der Gaag & van Raak, 2010). In a study by Huvenne et al (2011), 16 potential host species were inoculated, including Cyclamen persicum, Rosa mini, Exacum affine, Saintpaulia ionantha, Euphorbia pulcherrima, Calathea spp., Spathiphyllum 'Alfa', Delphinium, Campanula isophylla, Rudbeckia and Valeriana officinalis. They found that although F. foetens can sustain itself on all plant species tested, and even accumulate to high levels in the root system of some of these species, it produced symptoms only on Begonia.…”