NATURAL OCCURRENCE OF Fusarium SPP. AND MODELING FUMONISIN CONTAMINATION USING WEATHER VARIABLES AND INSECT DAMAGE IN MAIZE (Zea mays L.) IN EASTERN ETHIOPIA

Abstract

Maize production is an important component of food security in Ethiopia and ranks first in productivity and second in area coverage after tef. Fusarium is one of the major fungal genera associated with maize in the world. This genus comprises several toxigenic species including F. verticillioides and F. proliferatum, which are the main fumonisins producers. Fumonisin occurrence in maize grains is a main concern for human and animal health. Hence the study was conducted with the objectives to: 1) determine occurrence of Fusarium spp. and fumonisin contamination in fresh harvested and three-month stored maize samples in eastern Ethiopia; 2) determine the effects of farmers’ practices on levels of fumonisin contamination in fresh harvested and three-month stored maize samples; 3) determine the effects of season, location and maize hybrid and their interaction on kernel infection by F. verticillioides and fumonisin contamination; 4) analyze environmental factors associated with F. verticillioides kernel infection and fumonisin contamination; 5) develop preliminary model that would assist in the prediction of fumonisin concentration in maize grain at harvest using weather variables around silking date and insect damage at harvest; and 6) assess the antagonistic effects of indigenous Trichoderma spp. against F. verticillioides under in vitro conditions. Two-round surveys were conducted to collect 127 fresh harvested and 127 three-month stored maize samples from five districts (Girawa, Goromuti, Haramaya, Meta and Tullo) of Eastern Ethiopia during 2017/18 main cropping seasons. All samples were analyzed for occurrence of Fusarium species and fumonisin contamination in maize grain in the study areas. During the surveys, farmers’ agronomic and post-harvest practices were recorded through questionnaires with selected farmers. In addition, six maize varieties (Alemaya Composite, BH 140, BH 545, BH 661, Gibe-2 and Raare-1) were planted in a randomized complete block design (RCBD) in three replications at Girawa, Haramaya and Hirna fields during 2017 and 2018 cropping seasons. At harvest, 65 plants were randomly taken from each plot, evaluated for insect damage severity, hand-shelled and analyzed for F. verticillioides kernel infection and fumonisin contamination. Fusarium species were isolated and identified using direct plating technique by plating 15 kernel on Malachite green agar (MGA2.5) and Carnation leaf agar (CLA), while fumonisin concentration was analyzed by xvii ELISA protocols. Isolation of antagonistic Trichoderma species from rhizosphere soil was made using serial dilution technique at 10−2 , 10−3 and 10−4 . Antagonistic potential of Trichoderma isolates against F. verticillioides was evaluated in dual culture assay on PDA. In this result, higher numbers of Fusarium isolates were recorded in fresh harvested (2376 isolates) than in three month stored maize samples (1897 isolates). They were recorded in 97 and 90% of fresh harvested and three-month stored maize samples along with average kernel infection of 62 and 50%, respectively. A total of seven Fusarium species (F. andiyazi, F. graminearum, F. oxysporum, F. proliferatum, F. solani, F. subgulitnans and F. verticillioides) were identified based on morphological characteristics. Fusarium verticillioides was dominant in both fresh harvested and three-month stored maize samples, followed by F. proliferatum and F. subgulitnans in fresh harvested and three-month stored samples, respectively. The frequencies of F. proliferatum and F. verticillioides were higher in fresh harvested maize samples than in three month stored maize samples. Fumonisin was detected in all fresh harvested and 92% of three month stored maize samples (concentration ranging from 105 to 5,460 µg kg−1 ). The average (2,509 µg kg−1 ) fumonisin concentration detected in fresh harvested maize samples was higher than that of three-month stored maize samples (1,668 µg kg−1 ). Similarly, percentage of samples with fumonisin level above the regulatory limit was the highest in fresh harvested maize samples than in three-month stored maize samples. Thus, 84% of the fresh harvested and 94% of the three month stored maize samples met the standard set by US Food and Drug Administration (FDA) in raw maize for human consumption (≤4000 µg kg−1 ), while 63% of fresh harvested and 83% of three-month stored maize samples met the standard (<2000 µg kg−1 ) set by East African Community (EAC). Farmers’ agronomic practices, such as intercropping, late planting, late harvesting and insect pest attack in the field, tended to increase the likelihood of fumonisin contamination at levels above 2000 µg kg−1 in both fresh harvested and three-month stored maize, while application of fertilizer decreased the likelihood of fumonisin contamination at levels above 2000 µg kg−1 in fresh harvested maize samples. Post-harvest practices, such as drying maize in the field and sorting, showed decreasing trend on likelihood of fumonisin contamination to the level above 2000 µg kg−1 in three-month stored maize. Since some field environmental conditions may not be controlled, farmers should adopt pre- and post-harvest practices that reduce fumonisins contamination to the level below the permissible regulatory limits. High Fusarium kernel infection and fumonisin contamination recorded in the study area could indicate risk of exposures to fumonisin as almost all maize produced in the study areas were used for human consumption. The observed prevalence of the other Fusarium species, indicates the possibility of contamination of maize kernels by several other mycotoxins. Thus, variety of harmful mycotoxins such as xviii deoxynivalenol, nivalenol and zearalenone should also be considered in the future assessments. Higher Fusarium species kernel infection and fumonisin contamination recorded in fresh harvested maize indicates Fusarium infection and fumonisin production was accorded in the field prior to harvest. Therefore, any future study regarding Fusarium species and fumonisin contamination should focus on pre-harvest environmental condition. In the two years of field experiments, the average F. verticillioides kernel infection (36.6%) and fumonisin concentration (2.24 µg g−1) detected in 2018 season was higher than kernel infection (34.6%) and fumonisin concentration (1.9 µg g−1) detected in 2017 season. Among the fields, maize harvested from Hirna field was highly (47.87%) infected by F. verticillioides and fumonisin concentration (3.45 µg g −1 ). On average, Alemaya Composite variety had the lowest (28.2%) kernel infection by F. verticillioides and the lowest fumonisin concentration (1.31 µg g−1 ). Using 36 data points (year x location x varieties), stepwise multiple linear regression detected several weather variables in four 7-day critical periods around silking date and insect damage as predictor of fumonisin contamination. Fumonisin was well predicted when data from both insect damage and weather variables were included in the model and the model explained 92% of the variation in fumonisin concentration. The current model did not take into account all maize growth stage and weather variables. Thus, other variables need to be identified and included in future cycles of model improvement and verification. A total of ten Trichoderma isolates (Tr1, Tr2, Tr3, Tr4, Tr5, Tr6, TrNS2, TrN3, TrNS4 and TrNS5) were identified and grouped into two Trichoderma spp. (T. harizanum and T. viride). All Trichoderma isolates were analyzed for antagonistic potential against F. verticillioides in in vitro condition in a dual culture assay after 6 day of incubation. Six Trichoderma isolates (Tr1, Tr3, Tr5, TrNS2, TrN3 and TrNS4) exhibited inhibition percentage ˃80% after six day of dual culture incubation. The tested Trichoderma isolates (T. harizanum and T. viridea) showed varying degrees of antagonism against F. verticillioides. However, further studies on the effectiveness of Trichoderma spp. to control F. verticillioides should be conducted under in vivo conditions. The overall results of this present study are important in creating new management strategies to prevent maize kernel infection by Fusarium spp. and grain contamination by fumonisins. Management decisions to use maize genotypes resistant for F. verticillioides infection and fumonisin accumulation are more likely to produce maize grain that meet tolerable fumonisin concentrations. Since there no regulatory limit for any mycotoxin contamination in food in Ethiopia at the moment, efforts are needed to intervene the situation in order to have safe food for consumer.