Comparative studies on virulence, genetic variability and mycotoxin production among isolates of fusarium species infecting wheat

Abstract

Isolates of Fusarium species isolated mainly from wheat in Germany and Kenya were investigated for variation in mycotoxin production, virulence and PCR-based DNA characteristics. The isolates were tested for mycotoxin production on wheat ears and in culture while differences in virulence was tested on wheat ears and seedlings in pot and field experiments. Genetic variation was determined by random amplified polymorphic DNA (RAPD-PCR). Wheat varieties from Kenya were also tested for their susceptibility to Fusarium head blight after inoculation with Fusarium graminearum. Fusarium species isolated from the wheat grown in Kenya according to decreasing frequency were F poae (43%), F graminearum (39%), and F. avenaceum (8%). Other Fusarium species identified were F. equiseti, F. oxysporum, F. camptoceras and F. chlamydosporum. Different Fusarium species could be differentiated by RAPD-PCR. Epicoccum spp. 'and Alternaria spp. were isolated at high frequencies of 40% and 25% respectively. A high pressure liquid chromatography protocol was developed which could simultaneously detect nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3- acDON) and 15-acetyldeoxynivalenol (15-acDON) and zearalenone (ZEA). The detection limit was I ug/g. Solid cultures yielded higher levels of mycotoxins than liquid fermentation cultures. Fusarium cull1101'll111produced NIV, DON, 3-acDON and ZEA while F graminearum produced NIV, DON, 15-acDON and ZEA. 3-acDON and 15-acDON were produced during the active growth phases for both Fusarium species while DON was mainly produced during the stationary growth phase. This suggested that the acetylated derivatives are biosynthetic precursors of DON. Isolates of F. culmorum and F. graminearum showed high variation in amount and type of mycotoxins they produced in culture. Sixteen out of the 27 isolates of F. culmorum produced NIV (26.2 to 220.6 ug/g), 10 produced DON (27.2 to 180.8 ug/g) and 3-acDON (64.7 to 548.7 ug/g.). Only one isolate produced all the three mycotoxins (NIV, DON and 3- acDON). Chemotaxonomy of F. culmorum into NIY- and DON-chemotypes is suggested. All but two of the 27 isolates produced ZEA (0.23 to 68.4 11gig). Thirty five out of the 42 F. graminearum isolates produced DON (3.8 to 575 11gig), 27 produced 15-acDON (3.1 to 81.6 ~g/g) and 3 isolates produced NIV (147 to 338 ug/g). 15-acDON was only detected in cultures of DON-producing isolates and no isolate was found to produce both NIV and DON. AlI the F. graminearum isolates produced ZEA (5 to 637 ug/g), The production of DON was correlated to that of the acetyl DON (r = 0.5 to 0.99) for both F. culmorum and F. graminearum, but production of trichothecenes was not correlated to that of ZEA. The isolates of F. culmorum and F. graminearum differed in virulence to wheat ears. Colonization of the kernels by the fungal mycelium, determined as ergosterol content, also varied among the isolates of F graminearum (49 to 228 ug/g). The pattern of mycotoxin production in the kernels closely matched that observed in culture. The more virulent isolates produced mainly DON while the less virulent isolates produced mainly NIV, therefore suggesting involvement of DON in pathogenicity. Exposure of wheat seedlings to varying concentrations of pure DON and (3-acDON and 15-acDON) resulted in severe reduction in dry weight (17 to 77%). NIV and ZEA showed no significant effect therefore suggesting a phytotoxic effect of DON which might have contributed to virulence observed for some of the isolates. Random amplified polymorphic DNA (RAPD) PCR confirmed variation among isolates of F culntorum and F gram inearum , indicating that variation in mycotoxin production and virulence are genetically controlled. The NIV and DON producing chemotypes of F. graminearum could be clearly differentiated on RAPD-PCR profiles, while F culmorum could not. All the 15 wheat varieties tested were susceptible to Fusarium head blight but they differed in the level of susceptibility and the resulting mycotoxin contamination. Susceptibility measured as head blight severity, fungal colonization (ergosterol content) and yield reduction ranged from 29% to 68%, 67 ug/g to 187 ug/g and 23 to 57% respectively. Mycotoxin contamination measured as DON content varied from 5 ug/g to 31 ug/g.