Karakterizacija vrsta iz Fusarium fujikuroi kompleksa patogena zrna strnih žita u Srbiji

Abstract

Iz zrna obične pšenice, durum pšenice, ječma i tritikalea sakupljenih iz skladišta sa 55 lokaliteta u Srbiji tokom četiri godine (2014.-2017.) dobijeni su izolati vrsta Fusarium fujikuroi kompleksa. Odabrani izolati su identifikovani i okarakterisani na osnovu patogenih, molekularnih, morfoloških i toksikoloških karakteristika. Na strnim žitima identifikovane su tri vrste F. fujikuroi kompleksa - Fusarium verticillioides, Fusarium proliferatum i Fusarium subglutinans. Ova ispitivanja su potvrdila prisustvo ove tri vrste na zrnu pšenice i ječma u Srbiji. F. verticillioides, F. proliferatum i F. subglutinans su po prvi put opisane na zrnu durum pšenice u Srbiji, kao i na zrnu tritikalea u svetu. Molekularna identifikacija vrsta F. fujikuroi kompleksa urađena je na osnovu tri genska lokusa (ITS, TEF 1-α i RPB2). Sekvenciranjem tri genska lokusa dobijene su sekvence na osnovu kojih su rekonstruisana filogenetska stabla za svaki region posebno. Na osnovu TEF 1-α regiona uspešno su razdvojene sve tri vrste F. fujikuroi kompleksa, dok na osnovu druga dva regiona to nije bilo moguće. Identifikacija vrsta urađena je i primenom specifičnih prajmera (VER1/VER2, PRO1/PRO2, SUB1/SUB2, FV-F2/FV-FR, FS-F1/FS-R i FS-F2/FS-R). Na osnovu makroskopskih karakteristika kao što su izgled i boja micelije, pigmentacija podloge uočena je varijabilnost kod sve tri vrste F. fujikuroi kompleksa, ali na osnovu ovih karakteristika nije bilo moguće precizno razlikovati ove tri vrste. Makrokonidije i mikrokonidije obrazovale su sve tri vrste, dok je prisustvo mezokonidija zabeleženo samo kod izolata vrste F. subglutinans. Vrste F. fujikuroi kompleksa obrazovale su makrokonidije ravnih ivica sa zakrivljenom apikalnom ćelijom i bazalnom ćelijom u obliku stopala. Najduže makrokonidije obrazovala je vrsta F. subglutinans. Mikrokonidije su bile bez septi, ovalnog oblika. Najkraće mikrokonidije obrazovala je vrsta F. proliferatum. Vrsta F. verticillioides obrazovala je mikrokonidije u dugim lancima na monofijalidama, dok je vrsta F. proliferatum obrazovala kraće lance mikrokonidija na monofijalidama i polifijalidama. Kod vrste F. subglutinans mikrokonidije su se formirale u lažnim glavicama na monofijalidama i polifijalidama. Načini obrazovanja mikrokonidija mogu poslužiti u preliminarnoj identifikaciji ove tri vrste. Virulentnost vrsta ispitana je na listovima ječma, klasu durum pšenice i klasu pšenice, ječma i tritikalea. Na listovima ječma vrste F. proliferatum i F. subglutinans bile su jednako virulentne, dok je vrsta F. verticillioides imala najmanju prosečnu ocenu virulentnosti. Ispitivanja na klasu durum pšenice sprovedena su tokom dve godine. Rezultati su pokazali da je virulentnost bila veća u drugoj godini ispitivanja. Tokom obe godine najvirulentnija vrsta je bila F. verticillioides dok su vrste F. proliferatum i F. subglutinans bile su jednako virulentne. Tokom jedne proizvodne godine ispitana je virulentnost ovih vrsta na klasu pšenice, ječma i tritikalea, nakon čega je zaključeno da su sve tri vrste F. fujikuroi kompleksa bile jednako virulentne. Vrste ovog kompleksa su značajne zbog svoje sposobnosti da sintetišu mikotoksine od kojih su najznačajniji fumonizini. Koncentracije sintetisanih fumonizina ispitane su ELISA metodom. Dobijeni rezultati ukazali su na visoke koncentracije fumonizina kod izolata vrsta F. verticillioides i F. proliferatum, dok su izolati vrste F. subglutinans sintetisali fumonizine u niskim koncentracijama ili ispod praga detekcije.

Fusarium fujikuroi species complex isolates were obtained from grains of common wheat, durum wheat, barley and triticale collected from storages from 55 locations in Serbia during four years (2014-2017). Selected isolates were identified and characterized based on pathogenic, molecular, morphological and toxicological characteristics. Three species of F. fujikuroi complex were identified on small grains - Fusarium verticillioides, Fusarium proliferatum and Fusarium subglutinans. Results of these researches confirmed the presence of these three species on wheat and barley grains in Serbia. This is the first report of F. verticillioides, F. proliferatum and F. subglutinans on durum wheat grain in Serbia, as well as on triticale grain in the world. Molecular identification of the F. fujikuroi species complex was done on the basis of three gene loci (ITS, TEF 1-α and RPB2). By sequencing three gene loci, sequences were obtained on the basis of which phylogenetic trees were reconstructed for each region. All three species of the F. fujikuroi species complex were successfully separated based on the TEF 1-α region, while on the other two regions it was not possible. Species identification was also done using specific primers (VER1/VER2, PRO1/PRO2, SUB1/SUB2, FV-F2/FV-FR, FS-F1/FS-R and FS-F2/FS-R). Based on macroscopic characteristics such as appearance and color of the mycelium, pigmentation of the substrate, variability was observed in all three species of F. fujikuroi species complex, but based on these characteristics, it was not possible to accurately distinguish these three species. All three species produced macroconidia and microconidia, while the presence of mesoconidia was observed only in F. subglutinans isolates. Species of the F. fujikuroi complex produced straight-edged macroconidia with a curved apical cell and a foot-shaped basal cell. The species longest macroconidia were produced by F. subglutinans. Microconidia were without septa, oval in shape. The shortest microconidia were formed by F. proliferatum isolates. The species F. verticillioides formed microconidia in long chains on monophialids, while F. proliferatum formed shorter chains of microconidia on monophialids and polyphialids. Species F. subglutinans formed microconidia in false heads on monophialids and polyphialids. The methods of formation of microconidia can be useful in the preliminary identification of these three species. The virulence of the species was tested on barley leaves and spikes on durum wheat, wheat, barley and triticale. On barley leaves, F. proliferatum and F. subglutinans were equally virulent, while F. verticillioides had the lowest average virulence rating. Research on the durum wheat spikes was conducted in two year period. The results showed that virulence was higher in the second year of the trial. During both years, the most virulent species was F. verticillioides, while the species F. proliferatum and F. subglutinans were equally virulent. During one production year the virulence of these species was tested on wheat, barley and triticale spikes, after which it was concluded that all three species from F. fujikuroi complex were equally virulent. The species of this complex are significant for their ability to synthesize mycotoxins, of which the most important are fumonisins. The concentrations of synthesized fumonisins were tested using the ELISA method. The obtained results indicated high concentrations of fumonisins in isolates of F. verticillioides and F. proliferatum, while isolates of F. subglutinans synthesized fumonisins in low concentrations or below the detection rate.