3rd Int. FHB Symposium Szeged, Hungary, 2008 FUSARIUM LANGSETHIAE ON KERNELS OF WINTER WHEAT IN POLAND – OCCURRENCE AND MYCOTOXIGENIC ABILITIES Aleksander LUKANOWSKI – Czeslaw SADOWSKI Department of Phytopathology, University of Technology and Life Sciences, Ks. A. Kordeckiego 20, 85-225 Bydgoszcz, Poland, e-mail: [email protected] Abstract: The object of the study was mycological analysis of settlement of winter wheat grain samples collected in 2005/2006 and 2006/2007 cropping seasons with the special respect to a new species – Fusarium langsethiae. Its presence was noted in 38 samples in relatively low intensity (from 0.5 up to 3.5%). PCR assay confirmed identity of 38 isolates as F. langsethiae. Mycelium of tested strains of F. langsethiae was whitish with powdery appearance. Microconidia were formed napiform or globose, nonseptate, formed in heads. Macroconidia, sclerotia, and chlamydospores were not present after 3 weeks of incubation. Growth rates ranged from 5.4 to 10.3 mm/day. No isolate had potential ability to type B trichothecenes. Detailed chemical analyses of two strains showed them as producers of T-2 and HT-2 toxins at high concentrations. Key words: Fusarium langsethiae, PCR, wheat, mycotoxins Introduction Fungi from genus Fusarium, one of the most dangerous pathogens of cereals, cause serious loses of yield and significantly decrease its quality and wheat shows relatively high susceptibility to infection by these fungi. During research on mycotoxins and the occurrence of Fusarium spp. in cereal grain in Norway (Kosiak et al. 1997; Langseth and Rundberget 1999), it was found a Fusarium species of uncertain identity morphologically resembling F. poae but producing T-2 and HT-toxins typical for Fusarium sporotrichioides. Its mycelium had powdery appearance and new isolates were named ‘‘powdery F. poae” (Torp and Langseth 1999). As reported by Torp and Langseth (1999), the morphological features of the new species compared to any recorded mycotoxin profile have not been described for any other Fusarium species. Finally, results of detailed morphological surveys conducted by Torp and Nirenberg (2004) let to describe a „powdery F. poae” as a new species - Fusarium langsethiae. Materials and methods The objects of the study were 136 grain samples of winter wheat grown in different regions of Poland in 2005/2006 and 2006/2007 cropping seasons (Tab.1). Out of collected grain samples, 400 kernels from each sample were taken to determine their settlement by Fusarium spp. according to method described by Lukanowski and Sadowski (2002). Outgrowing colonies were transferred on agar test tubes and identified with mycological keys. During identification it was found that some individual isolates did not represent any known Fusarium species. They resembled “powdery” form of F. poae, described in 2004 by Torp and Nirenberg as a new species – F. langsethiae, thus all of them were initially identified as this species. To confirm correctness of species identification, PCR assay with SCAR primers (Wilson et al. 2004) was conducted. 453 DOI: 10.1556/CRC.36.2008.Suppl.B.40 Vol. 36, 2008, Suppl. B Cereal Research Communications Nine single-spore isolates of a new species were the objects of detailed morphological analysis. The strains were grown in darkness at 25°C in plastic Petri dishes on PDA to diagnose colony color, odor, and growth rate. They were also incubated on Saltwater Nutrient Agar (SNA) at 25°C for 21 days in near-UV light and darkness in a 12/12h cycles to promote formation of conidia, chlamydospores and sclerotia. The average mycelial growth rate per day was calculated on the basis of the difference between the colony diameters after 4 and 7 days of incubation measured in millimeters. There was also examined potential ability of F. langsethiae to produce group B trichothecenes with use of PCR assays testing the presence and functionality of Tri7 and Tri13 genes (Chandler et al. 2003). Additionally two F. langsethiae strains (Fl-6 and Fl 23-1) were analyzed for synthesis of T-2 and HT-2 toxins. Sterile rice grain (40% moisture) was inoculated in 250 ml Erlenmayer’s flasks with mentioned isolates. They were grown for four weeks at 23°C, and than rice was analyzed for presence of toxins with HPLC- MS/MS method. Results and discussion Out of 76 grain samples collected in four locations, analyzed in 2006, F. langsethiae was isolated from 27 samples. Generally, grain settlement by this species ranged from 0.5% to 1.8% with the exception for two samples where it was 3.5%. In 2007, out of 60 grain samples, 11 showed presence of F. langsethiae; the range was from 0.8% to 2.0% (Tab. 1). Fusarium langsethiae was isolated more often from samples collected in northern Poland. Strains of a new species was collected so far in Austria, the Czech Republic, Denmark, England, Norway, the Netherlands and Italy. Fungus was isolated from infected oats, wheat and barley (Torp and Adler 2004). Classification and identification of Fusarium spp. makes many difficulties because of these methods base on morphology of the anamorphic mycelium, and most of perfect forms of Fusarium spp. is not found so far (Chelkowski and Witkowska 1999). Thus, in this study PCR assay was used to confirm the correctness of morphological identification of F. langsethiae. Specific 310 bp DNA fragment was obtained for all tested strains (Phot. 1) and its sequence was deposited in GenBank (Lukanowski et al. 2008). 454 3rd Int. FHB Symposium Szeged, Hungary, 2008 Table 1. Settlement of winter wheat kernels by Fusarium spp. including F. langsethiae Location No. of samples No. of samples % of kernels with with F. langsethiae Fusarium F. langsethiae spp. 2006 8, including: Bogatka 1.5 18.0 Milwus 1.0 11.1 Nadobna 0.5 17.7 Radostowo (18°44’E, 27 cultivars Pegassos 1.0 12.0 53°59’N) Ritmo 1.5 21.8 Sakwa 1.8 17.8 Turnia 0.5 10.5 Zyta 1.8 23.6 20 combinations 15 0.5 – 3.5 21.0 -38.8 Radostowo (cv. of chemical protection Ritmo) with fungicides 2, including: 27 in cropping systems Zyta (integrated 1.5 6.3 Osiny (21°34’E, (organic, integrated, system) 1.0 1.0 51°21’N) conventional, Kobra monoculture) (monoculture) Minikowo (17°44’E, 1, including: Trend 53°10’N) Trend 3.5 9.5 1, including: Sobiejuchy (17°43’E, Smuga 52°54’N) Smuga 1.0 1.5 2007 3, including: Stanowo (19°18’E, 53°54’N) 8 Anthus 1.0 17.8 wheat after wheat. Meteor 0.8 39.0 simplified cultivation Milvus 1.0 22.8 4, including: Hybred 2.0 26.8 Stanowo, wheat after 8 Milvus 1.3 25.3 wheat, full cultivation Turnia 1.0 45.0 Drifter 1.0 36.8 Stanowo, wheat after 8 0 0.0 17.5 – 24.7 rape, full cultivation 3, including: 13 combinations of 3 1.0 20.0 Stanowo (cv. Drifter) chemical protection 6 0.8 20.3 with fungicides 13 1.0 20.3 23 in cropping systems Osiny (21°58’E, (organic, integrated, 1, including: 51°25’N) conventional, Zyta (monoculture) 0.8 29.8 monoculture) 455 Vol. 36, 2008, Suppl. B Cereal Research Communications NC – negative control Phot. 1. Confirmation of analyzed strains as F. langsethiae Mycelium of tested strains of F. langsethiae grown on PDA medium was whitish, showing a powdery surface, 1 to 3 mm high producing no odor. Microconidia were napiform or globose, nonseptate, with an average length of 6.4 ȝm and width of 5.6 ȝm. Microconidia in heads, borne on unbranched or branched 8.5 to 16.3 ȝm long monophialides. There was no formation of macroconidia, sclerotia, and chlamydospores after 3 weeks of incubation. Growth rates ranged from 5.4 to 10.3 mm/day. These results were in accordance with report of Torp and Nirenberg (2004). Weather conditions in 2006 did not favor occurrence of Fusarium spp. on kernels. Grain settlement ranged from 1.0% up to 38.8%. Dominating species were F. poae (up to 12.8%) and F. tricinctum (11.0%) followed by F. sporotrichioides (up to 6.5%), F. avenaceum (up to 4.8%) and F. culmorum (up to 3.0%). More favorable conditions for growth and development of Fusarium Head Blight were noted in the next cropping season, when settlement of grain was from 5.0 to 60.3%. Among isolated species F. poae dominated (up to 30.0%), next F. tricinctum (up to 26.0%), F. avenaceum (up to10.0%), F. culmorum (up to 8.0%) and F. sporotrichioides (up to 3.0%). PCR assay results showed no potential ability of tested isolates of F. langsethiae to produce type B trichothecenes. It was also confirmed by chemical analysis of two isolates (Fl-6 and Fl 23-1) which synthesized T-2 and HT-2 toxins at concentrations: 1651 ppb, 5020 ppb and 108000 ppb and 8640 ppb respectively. These results are in accordance with reports stating that each species is able to produce a more or less characteristic toxin pattern, but intraspecific variation has also been found (Marasas 1991; Sugiura et al. 1993; Langseth et al. 1999). 456 3rd Int. FHB Symposium Szeged, Hungary, 2008 References Chandler, E.A., Duncan, R.S., Thomsett, M.A., Nicholson, P. 2003. Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis. Physiological and Molecular Plant Pathology 62:355-367. Chelkowski, J., Witkowska, I. 1999. Fungal pathogens of cerealis, their identification and studies on genetic diversity using polymerase chain reaction (PCR) (in Polish). PostĊpy Nauk Rolniczych 4:49-60. Kosiak, B., Torp, M., Thrane, U.