Early detection and quantification of Ramularia beticola in sugar beets using spore traps and real-time PCR

Abstract

Ramularia leaf spot is one of the most important leaf diseases in sugar beets (Beta vulgaris L.) in northern European countries. It is caused by the ascomycete Ramularia beticola Faut. & Lamb. and thrives in cool, temperate climate at relatively high humidity and temperatures between 17 and 20°C. If not controlled, yield losses can amount to 15 – 20 %. Over the last decades an increase of severe attacks of Ramularia leaf spot has been reported in Denmark, Sweden, and Finland. The disease is typically controlled preventatively using fungicides like epoxiconazole or epoxiconaozle + pyraclostrobin. According to IPM guidelines, fungicides in Denmark are presently recommended to be applied if a severity threshold of 5 % is exceeded, the first application normally around August the 1st. The second application is performed when new infections are observed with threshold 5-10 %. Typically 25-50% of the commonly used fungicide dose is recommended for each treatment. Preliminary field trials have indicated a more efficient control of fungal diseases and higher sugar yield if sugar beets are treated with fungicides before visual symptoms occur. For a better determination of the optimized timing of disease control, more advanced methods are needed. In collaboration between the Universities of Copenhagen and Aarhus and the R&D enterprise Nordic Beet Research (NBR) a study was conducted with the goals a) to develop an early detection system based on spore trapping and b) to develop a real-time PCR based method to detect and to quantify R. beticola DNA in air samples obtained from Burkard® spore traps. Primers were designed based on the ITS region sequences and tested on R. beticola isolates from 2010 and 2011. The specificity of the primers was tested on related fungi, including Cercospora beticola and other Ramularia fungi. Real-time PCR was used to measure the quantity of the DNA of R. beticola collected on tapes from the Burkard Spore Traps®. Three spore traps were set up in sugar beet fields at three different sites on the Danish island of Lolland. Based on real-time PCR, R. beticola DNA was detected in the air samples 14 and 16 days prior to first visible symptoms on untreated plants, which is in accordance with the reported latency of 2 weeks of the fungus. It was observed that the amount of DNA varied depending on weather conditions. It is further investigated how different amounts of DNA can be linked to weather conditions in order to be able to predict which conditions have been most conductive for spore production and spreading. The results from spore trapping are linked to fungicide treatments in field trials using different timings, in order to investigate if spore trapping can be linked to recommendations for spraying. It is expected that the results can lead to an IPM based recommendation where spraying only takes place when a real risk has been verified based on spore trapping or weather data

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