First Report of Fusarium pseudograminearum Causing Crown Rot of Wheat in Europe

In May 2016, Fusarium crown rot (FCR) of wheat (Triticum aestivum) was identified in a commercial field located in Córdoba, Spain. Affected plants showed necrosis and dry rot of the crown and basal stems. Three foci and at least 10 plants per focus were surveyed. Necrotic tissues were washed under running tap water. Subsequently, they were surface disinfested in a sodium hypochlorite solution (Cl at 5 g/liter) in sterile distilled water (SDW) for 1 min and washed twice with SDW. Small pieces from the basal stem were plated onto potato dextrose agar (PDA), acidified with 0.06% lactic acid, and incubated at 25°C in darkness for 7 days. According to the morphology of the colony, fungal species belonging to the Fusarium graminearum species complex were isolated, showing seven morphological types of colonies. All isolates were transferred onto PDA and carnation leaf agar (CLA) and incubated as described above. One of these isolates, ColPat-351, was first identified morphologically as F. pseudograminearum (Aoki and O’Donnell 1999). On PDA, this isolate produced abundant aerial mycelium, densely floccose, reddish-white to white with deep-red, brownish-violet to reddish-white reverse pigmentation. On CLA, it produced 3- to 5-septate slightly curved macroconidia, with an elongated arcuate apical cell and a distinct basal foot cell, 32.5 to 57.5 × 3.0 to 5.0 μm. Chlamydospores were observed after 3 weeks. No microconidia or sexual structures were formed. Sequence of the translation elongation factor 1α gene was amplified with primers EF1/EF2 (O’Donnell et al. 1998) and deposited in GenBank (accession no. MG570082). This sequence was identical (100%) to the sequence of F. pseudograminearum (JX118971). Following the morphological and molecular detection methods described above, the other six isolates were identified as F. avenaceum, F. culmorum, and F. graminearum, three well-known species associated with FCR complex of wheat in Spain (Marín 1986). Finally, polymerase chain reaction was conducted using the species-specific primer pair Fp1-1/Fpl-2 (Aoki and O’Donnell 1999) to confirm the identification. Only isolate ColPat-351 generated a 523-bp product, which is characteristic of F. pseudograminearum. Pathogenicity tests were conducted with inoculum produced on wheat seeds that were soaked for 12 h in flasks filled with SDW. Two 7-day-old mycelial plugs of the isolate ColPat-351 growing on PDA were placed in the flask, incubated at 25°C for 2 weeks, and shaken twice a week. Wheat seedlings were grown in individual plastic pots filled with 30 ml of sterilized peat moss for 1 month. After 1 month of growing, seedlings were inoculated by mixing 1 g of inoculated seeds with the substrate of each pot and placed in growth chamber at 23°C with a 12-h photoperiod. Controls were inoculated with sterile noninfested seeds using the same procedure. Ten replicated plants in individual pots were used, with an equal number of control seedlings. The experiment was repeated once. Necrosis and dry rot were consistently observed on the crown and basal stems in all inoculated seedlings 1 month after inoculation. F. pseudograminearum was reisolated from necrotic tissues, completing Koch’s postulates. No symptoms were observed on control plants. F. pseudograminearum was first described in Australia in 1999 but is currently a common species causing FCR of wheat and barley in many parts of the world (Scott and Chakraborty 2006). FCR is a common disease of wheat in central-southern Spain, causing significant yield losses. However, to our knowledge this is the first report of F. pseudograminearum associated with the FCR complex syndrome in Spain as well as in the rest of Europe. This information will be helpful to improve our knowledge on the etiology of FCR complex of wheat.