Soybean (Glycine max [L.] Merr.) is economically the most important protein crop grown worldwide. However, Europe largely depends on soybean imported from the Americas (European Commission 2019; Haupt and Schmid 2020). In Germany, soybean production was not formally recorded before 2016, but since then a steady increase along with an expansion of the growing area from the south of Germany to northern states occurred. In 2019 an area of 29,000 hectares was under soybean cultivation (Federal Ministry of Food and Agriculture (Germany) 2019). In the state of North Rhine-Westphalia (NRW, western part of Germany) farmers have started in recent years to cultivate soybean, making it increasingly important to monitor pathogens associated with this new crop. At the beginning of October 2019, shortly before harvest, rows of black spots on pods and stems of soybean plants cv. Viola throughout a field site near Jülich (NRW) were observed. Close observation identified them as pycnidia with similarity to symptoms reported from soybean in Austria in 2015 (Hissek and Bedlan 2016). The collected samples were thoroughly surface sterilized (two washes with 70 % EtOH, a rinse in 0.5 % sodium hypochlorite solution and a final wash in sterile double distilled water) and placed on plates containing potato dextrose agar (PDA) at 22 °C in the dark. Fungal colonies were transferred to malt extract agar plates (MEA) and examined by microscopy. Thus, 34 of 41 isolates looked morphologically similar, producing colonies that appeared dark grey with white aerial mycelium and round to irregular margins. A single spore isolate was generated and designated IPP1903. Spores derived from IPP1903 were unicellular and mostly oblong to cylindrical with a mean width of 2.6±0.3 µm and a mean length of 5.9±0.8 µm (N=50, mean value ± standard deviation). Colonies on MEA were 5.4 to 5.8 cm in diameter after growth for seven days at 20 to 25°C with a photoperiod of 12 h and 3.3 to 3.7 cm in diameter after growth for seven days in the dark at 22°C. These morphological observations led to the conclusion that the isolate may belong to the genus Phoma. To test this hypothesis, we performed a drop test with 5 M NaOH which is used routinely to check for the presence of a genus-specific metabolite. We observed a change in color, indicating a positive test result. The color change was even more pronounced on the plates incubated in the light, further confirming the presence of "metabolite E" (Boerema et al. 2004; Kövics et al. 2014). Next, DNA was extracted and PCR was performed with primers specific for ITS regions (GenBank MT397284), LSU (MT397285), rbb2 (MT414713) or tub2 (MT414712). Sequencing results of PCR products were used to create a combined phylogenetic tree, including sequences published previously (Chen et al. 2015). Our sequencing results together with the morphological observations clearly identified the fungal isolate to be Boeremia exigua var. exigua. The isolate is publicly available in the CBS collection of the Westerdijk Fungal Biodiversity Institute with the accession no. CBS 146730. Koch's postulates were fulfilled by inoculating a spore suspension of the isolate IPP1903 (5x105 ml-1 in 0.05% Tween 20 solution in distilled water) onto healthy primary leaves of twenty 14 days old soybean plants of the cultivar Abelina. While the mock-inoculated plants (inoculated with 0.05% Tween 20 solution in distilled water) stayed healthy, the inoculated plants developed lesions on the leaves after seven days. Six weeks after inoculation the fungus could be reisolated from cuttings of the infected leaves after surface-sterilization. Fungal colonies were confirmed to be B. exigua var. exigua by morphological examination and via NaOH drop test. To our knowledge, this is the first report of B. exigua var. exigua causing disease on commercially grown soybean in Germany.
Keywords: Causal Agent; Crop Type; Field crops; Fungi; Pathogen detection; Pathogen diversity; Subject Areas; oilseeds and legumes.