SPAT: ERA-NET EUPHRESCO II: Bewertung und Testung von Krankheitserregern an Erdbeere

Strawberry (Fragaria × ananassa) is an important food crop that is susceptible to a range of pests, including fungi, bacteria, viruses and nematodes. The main fugal pathogens in Europe are Phytophthora cactorum, Colletotrichun acutatum, Verticillium dahliae, Botrytis cinerea, Mycosphaerella fragariae (purple stain), and Sphaerotheca macularis, while the main bacterial disease is Xanthomonas fragariae (angular leaf spot). The objectives of the project were to make an overview of literature on strawberry diseases in partner countries and on all of the testing methods that are applicable in testing for X. fragariae and P. fragariae. Another objective was to develop and update diagnostic protocols and evaluate their strength and weaknesses by a ring test. The amount of literature of strawberry diseases at national level is relatively poor (except in Russia). The questionnaires and the testing of field samples conducted within the project gave some information of the occurrence of strawberry diseases in the partner countries. The status of virus diseases has only been surveyed in Russia, Austria and Ireland. Their importance has not been surveyed in this project. Leaf diseases occur in Estonia only on fields of small producers. Over a period of 5 years (2008-2013) they were important in Ireland, Spain and Lithuania (mainly P. aphanis). For 60 % of respondents in Lithuania grey mold were the most important disease, it is also a main disease in Austria and Ireland. 25% of producers in Spain would name grey mold as second most important disease. Macrophomona phaseolina was only mentioned by one of five Spanish producers. Fusarium spp. were only observed in Spain during the relevant period. In 2013 Verticillium-diseases occurred mainly in Austria and Lithuania, during 2008-2013 they were also observed in Spain and Lithuania. Crown rot (P. cactorum) emerged (frequently) in Spain, Lithuania, Austria and Ireland. The available publications suggest that P. fragariae is of major commercial concern in Ireland. X. fragariae only occurred in Austria in field but has also been reported as present in Ireland and Spain. More field inspections would be necessary to gain a representative picture of the occurrence and relevance of strawberry diseases. Available diagnostic methods were reviewed and analysed as a systematic review of PCR-based methods used for detection or quantification of the most important strawberry pathogens, including Fusarium spp., P. fragaria, C. acutatum, V. dahliae, B. cinerea, M. phaseolina and X. fragariae. Using appropriate subject headings, all scientific databases were searched from their inception up to April 2014. A total of 259 titles and abstracts were reviewed of which 23 full texts met all the inclusion criteria. The accuracy and sensitivity of PCR diagnostic methods was the focus of most studies included in this review. From a systematic review of the currently available published literature, real-time PCR (rtPCR) was shown to be a particularly promising technique for diagnosing and quantifying pathogen populations in strawberry. This technique allows an accurate, reliable and high throughput detection of target DNA in symptomless strawberry leaves and various environmental samples in real time. For example, amount of microsclerotia of V. dahlia in soil samples can be quantified by real-time PCR in order to replace the time consuming wet sieving technique followed by a classical plating and counting of grown microsclerotia. The outcome is that the wet sieving technique is to date the only suitable method for quantification of V. dahliae microsclerotia from soil. Real-time PCR is much faster, but sensitivity of a practicable procedure is too low for quantification from soil in order to give recommendations on the choice of strawberry varieties concerning susceptibility to V. dahliae. Common diagnostic methods (PCR and rtPCR) to quarantine and emerging strawberry pathogens were developed in several partner countries. Optimized PCR and rtPCR protocols were evaluated for effective detection of pathogens: Ph. fragariae, Ph. cactorum, V. dahliae, V. albo-atrum, M. phaseolina, F. oxysporum f. sp. fragariae, F. solani and B. cinerea. There was a pronounced variation in the percentage of correctly detected samples (56-96%) among the participating labs and between the assays tested. False negative results could be attributed to a reduced sensitivity due to processes of lyophilization or vacuum concentration of primers and/or extracted DNA from samples, which were decided on to simplify transportation of the material tested. Contamination during the rehydration of samples and/or primers or handling with the PCR mix may have led to false positive results. However, these assumptions would have to be examined in more detail. Under certain conditions, using freshly extracted DNA and primers, all tested assays should be suitable to detect the selected diseases directly from diseased strawberry plants. This could be shown in WP 4 when methods were optimized in the different labs, respectively. However, the ring test pointed out that for implementation of these molecular methods in different labs some optimization processes might be necessary under the different conditions to obtain an assay, which gives satisfactory results in the hands of each lab personal and equipment. The data generated in this ring test can be perfectly used for validation processes. More research is still needed to get the adequate overview of the pathogens occurrence. Also, although RT-PCR is the best suitable detection method, the preanalytical critearia still need analysing. The new diagnostic protocols need some additional optimization to give same results in different locations. But as often several diseases and conditions form a complex there is a need for a universal approach. In addition to the studied pathogens, the role of nematodes in strawberry diseases and quantification of soil-borne pathogens need to be assessed.