Asian Fusarium langsethiae and the two subgroups of European F. langsethiae Tapani YliMattila 1, lga Gavrilova 2, Kerry Donnell 3, Todd Ward 3, Robert Proctor 3, and Tatiana Gagkaeva 2 1 University of Turku, Turku, Finland (http://users.utu.fi/tymat/projects.htm). 2 AllRussian Institute of Plant Protection (VIZR), St.PetersburgPushkin, Russia 3 USDAARS, Peoria, USA.
Women harvesting barley under the snow in East Karelia during World War II in 1942 (Rosen, 1998) Information regarding the effects of T2 mycotoxin on people has been collected from many accidents caused by moldy wheat or corn. ne of them took place in Ural region in the former Soviet Union during the World War II, when wheat could not be harvested before the winter. The mouldy wheat, which was harvested after the winter, caused the clinical syndrome alimentary toxic aleukia (ATA), with a mortality rate of 1060 % (Sarkisov, 1954; Joffe, 1974). Sarkisov produced the disease in cats by feeding millet infected with F. sporotrichioides that had been isolated from infected grain. Alcohol and ether extracts of F. sporotrichioides also caused necrotic lesions on rabbit skin. In 1968 the trichothecene T2 toxin was isolated from F. sporotrichioides and it was found to cause necrotic lesions on rabbit skin. So, T2 toxin is probably connected to ATA outbreaks in Soviet Union.
A and Btype trichothecenes T2 HT2 DAS Deoxynivalenol (DN) Nivalenol (NIV) Fusarenon X (FX) H H Ac Ac H H T2 toxin Longer group Carbonyl group DN riginally type A and B trichothecenes were detected based on their color on thinlayer chromatographic plates under UVA light.
In F. sporotrichioides, genes that encode trichothecene biosynthetic enzymes are located at 3 loci Tri16 connected to T2 synthesis Tri7 and Tri13 connected to NIV synthesis TRI8 TRI7 TRI3 Core TRI cluster TRI4 TRI6 TRI5 TRI10 TRI9 TRI11 TRI12 TRI13 H Ac Ac TRI101 locus TRI101 TRI1TRI16 locus TRI1 TRI16 Ac Ac Ac Proctor et al. (2008)
F. sporotrichioides Function of TRI1TRI16 locus Ac TRI1 TRI16 8 7 8 7 8 7 H Ac Ac Ac Ac Ac Ac T2 Toxin H Ac Tri16 is nonfunctional in in F. poae, which may explain its weak T2 producion (Beremand et al. 2003, Brown et al. 2003, McCormick et al. 2004, 2006, Proctor 2008, unpulished results)
Asian F. langsethiae and the two subgroups of European F. langsethiae Tapani YliMattila, Galina Kononenko, lga Gavrilova and Tatiana Gagkaeva Norway 2008 Turku 2006
SgI Sg II Wagner parsimony consensus tree for combined IGSRFLP data from complete IGS sequences (modified from Konstantinova & YliMattila, 2004). The bootstrap values are shown. Poy consensus tree of combined partial betatubulin and IGS and complete ITS sequences (modified from YliMattila et al., 2004) 18S ITS 1 5.8 S ITS 2 28 S IGS 18S
Identification of European and Asian Fusarium isolates Fusarium spp. Morphology Chemotaxonomy Pathogenicity Speciesspecific primers Sequences EF1 F. poae F. langs. F. sporot. F. sporot. S S/L S + S Asian F. sporot. S S/L S + + S F. poae P P P/L + P European F. langs. L S/L L/P ++ L Asian F. langs. L S/L L/P + + S P = F. poae, S =F. sporotrichioides, L = F. langsethiae
European F. langsethiae Morphology of colonies Isolate 53409 53410 53411 53412 53413 53414 53417 53418 53419 53420 53436 53437 53438 53439 rigin Finland, EteläKarjala Finland, Satakunta Finland, Satakunta Finland, Uusimaa Finland, Uusimaa Finland, Häme Finland, Uusimaa Finland, EteläPohjanmaa Finland, Satakunta Russia, Krasnodar Russia, rel Russia, Pskov Russia, Kaliningrad Russia, Vologda 53418 Subgroup II 53414 Subgroup II Black colour Subgoup I Red colour Subgroup II 53419 Subgroup I 53417 Subgroup I
F. langsethiae, subgroup I Turku F. langsethiae, subgroup II In Europe subgroup II has also been found in Austria, Czech Republic, Norway and Denmark, while subroup I has been found in Norway, Denmark, Austria, Czech Republic, Netherlands and UK (Konstantinova & YliMattila, 2004).
Asian F. langsethiae Morphology of colonies 53421 Isolate 53421 53422 53423 53424 53425 53426 53427 53428 53429 53430 53431 53432 53433 rigin Siberia, Tuva Siberia, Buryatia Siberia, Chita Siberia, Krasnoyarsk Siberia, Krasnoyarsk Siberia, Irkutsk Siberia, Irkutsk Siberia, Irkutsk Far East, Primorsk Far East, Chabarovsk Far East, Amursk Siberia, Irkutsk Siberia, Irkutsk 53424 53433
T2 production: Asian F. langsethiae 400053000 ng/ml European F. langsethiae 100037000 ng/ml F. sporotrichioides 50010000 ng/ml Asian F. langsethiae European F. langsethiae ATA during world war II Tuva Buryatia Krasnoyarsk Chita Irkutsk Chabarovsk Primorskiy Amursk
European F. langsethiae Asian F. langsethiae M subgroup II subgroup I IBT 9959 IBT 8051 sgi 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Gel electrophoresis of amplification products with primers CNL12/PulvIGSr (specific for F. sporotrichioides/f. langsethiae) on DNA from fungal species of section Sporotrichiella. Lanes 1 and 44: Molecular weight marker VI (Roche); lanes 2 to 24: F. langsethiae strains; lanes 25 to 37: F. sporotrichioides strains, ; lane 38: F. sporotrichioides var. minus strain; lane 39 F. kyushuense strain; lanes 40 to 42: F. poae strains; lane 43: negative control (MilliQ) (modified from Konstantinova &YliMattila, 2004) IBT 40011 M F. langs. F. sporotrichioides F. kyush. F. poae Intermediate between subgroups I and II:
European F. langsethiae subgrop I F. sporotrichioides European F. langsethiae subgrop II and subgroup I European F. langsethiae subgroup II F. sporotrichioides 53432 Asian F. langsethiae 53432 Asian F. langsethiae F. poae F. kyushuense F. sporotrichioides F. poae F. kyushuense UPGMA tree (left) and NJ consensus tree for combined betatubulin, elongation factor 1 alpha and ribosomal IGS sequences. Bootstrap values higher than 50 % are shown.
Investigation of the pathogenicity of F. sporotrichiodes, F. poae and F. langsethiae isolates Method: Scale Detached leaf segment have been used to study pathogenicity. Leaves were taken from seedlings of oats cv. Borrus grown for 2 weeks in pots. Segments 45 cm long were cut and placed in plastic boxes on filter paper wet with water containing 100 mg/l benzimidazole. Each segment was inoculated centrally with 10 µl drop of suspension (5x10 6 spores per ml) from 14dayold cultures of Fusarium. A single necrotic lesion developed below each inoculation, and lesion lengths were measured alter 5 days at 23 ºC. Measurements were made as necrosis and chlorosis by 6 score scale. 1 score 2 score 3 score 4 score 5 score
Pathogenicity of Fusarium (6 isolates/row) species to detached leaves of oat cv. Borrus (5 days, 23 ºC) F. sporotrichioides F. poae Asian F. langsethiae European F. langsethiae Control
Conclusions Acccording to morphology the Asian F. langsethiae isolates are similar to European F. langsethiae. It is not possible to separate F. langsethiae isolates from F. sporotrichioides isolates based on chemical data (trichothecenes). Speciesspecific primers for European F. langsethiae isolates do not give a clear positive reaction with Asian F. langsethiae isolates, but F. sporotrichioidesspecific primers give a positive reaction with Asian F. langsethiae isolates. Asian F. langsethiae isolates have one bp difference in betatubulin sequences and two bp differences in EF 1 alpha sequences as compared to F. sporotrichioides. European F. langsethiae isolates can be divided into two subgroups (2 bp difference) based on EF 1 alpha sequences. All Asian F. langsethiae isolates have a TG repeat of at least 36 bp in IGS region, which makes it possible to separate them from F. sporotrichioides and European F. langsethiae isolates by using specific primers. The subgroups of European F. langsethiae isolates can be separated by a deletion of > 100 bp and the subgroups are the same as were found based on EF 1 alpha sequences. It is possible to separate the two subgroups of European F. langsethiae by specific primes (same primers as can be used for identification of Asian F. langsethiae isolates), which have been designed on both sides of the deletion (Konstantinoa & YliMattila 2004). All these groups are supported by bootstrap values >99.6 % in the NJ consensus tree of the combined three sequences. Asian F. langsethiae isolates cause necrosis on the detached leaves of oat like F. poae and european F. langsethiae in contrary to F. sporotrichioides, which usually cause chlorosis.