First Report of Maize Ear Rot Caused by Fusarium boothii in Serbia

Fusarium graminearum species complex (FGSC) includes at least fifteen species which are some of the most significant fungi that infect maize in temperate areas (Sarver et al. 2011). Agroecological conditions in Serbia are suitable for the development of infection by members of FGSC and therefore during the period of 1993-2010, maize samples collected from northern Serbia (46°5'55" N, 19°39'47" E) showed typical symptoms of gibberella ear rot. Twenty isolates were selected for study for FGSC identification. Appearance of colonies and macroconidia on potato dextrose agar (PDA) of all isolates (average 37.75-45.35×4.35-5.35 µm, No 50) were consistent with descriptions of F. graminearum (O'Donnell et al. 2004). Monosporal isolates were grown on PDA and used for molecular analyses. DNA isolation was performed using DNeasy Plant Mini Kit. Identification of FGSC was performed on the basis of the TEF-1α gene amplified with primers ef1/ef2 (Geiser et al. 2004) and sequenced in both directions. In four selected isolates two additional genomic regions (histone H3 and β-tubulin) were further analyzed using primers H3-1a/H3-1b and T1/T22, respectively (O'Donnell et al. 2000, Glass and Donaldson 1995). Nucleotide sequences of TEF-1α, β-tubulin, histone H3 have been deposited in GenBank under accession numbers: isolate 914 (MF974400, MG063784, MF999140), 1495 (MF974405, MG063789, MF999145), 2812 (MF974408, MG063792, MF999148), 2822 (MF974409, MG063793, MF999149), respectively. Isolates were molecularly identified using BLAST tool from NCBI and with phylogenetic analyses (Maximum Parsimony method with 1000 bootstrap replications on concatenated sequences of all three genes-TEF-1α, β-tubulin and histone H3) using MEGA 11 software package (Tamura et al. 2011). In BLAST analyses isolate 2822 shared 100% nucleotide identity with reference isolate Fusarium boothii NRRL26916 (GQ915470) originating from Central America based on histone H3 gene. Isolates 914, 1495 and 2812 shared 99% to 100% nucleotide identity with F. graminearum isolates. Phylogenetic analyses showed that all equally parsimonious trees made of 32 selected sequences of species within FGSC available in GenBank database and our four isolates, rooted with external outgroup species F. pseudograminearum, grouped three isolates (914, 1495, 2812) with F. graminearum isolates (NRRL28336, 29169, 28439) and one isolate (2822) grouped with F. boothii isolates (NRRL 29020, 26916). Pathogenicity of isolates was confirmed using the method of Reid et al. (1996). Artificial inoculation of maize ears was performed on the third day after silking by injecting 2 ml of a conidial suspension with a concentration of 1x105 conidia/ml into the silk channel. The same procedure was applied to control plants, using sterile water instead of inoculum. After three weeks, symptoms of Gibberella ear rot appeared and the pathogen was again successfully isolated. The fungus was identical to original isolates, thus completing Koch's postulates. Control plants did not show any symptoms. Based on available literature, this is the first report of species F. boothii in Serbia. Future research should be focused on determining distribution, aggressiveness, synthesis of mycotoxins, as well as damage caused by F. boothii on grain yield and quality compared to F. graminearum. This will contribute to a better understanding of biodiversity of FGSC, which may lead to a successful strategy for controlling these harmful pathogens.