OCCURRENCE OF WHEAT HEAD BLIGHT-CAUSING FUSARIUM SPECIES AND ASSOCIATED TRICHOTHECENE MYCOTOXINS IN KENYAN WHEAT 1Muthomi, J. W., 1Ndung’u, J. K. and 2Gathumbi, J. K 1Department of Plant Science and Crop Protection; 2Department of Veterinary Pathology, Microbiology and Parasitology University of Nairobi, P. O. Box 30197, Nairobi, Kenya. Abstract Freshly harvested wheat grain samples were collected during the 2004-growing season in different agro- climatic zones to determine the presence of Fusarium head blight-causing Fusarium species. Fungal contamination was determined by isolation on agar media while mycotoxin analysis was by direct competitive ELISA. The wheat grain samples were highly contaminated with fungi, especially Epicoccum, Alternaria and Fusarium species. The mean Fusarium infection rate varied from 13 to 18%, with the major head blight – causing species being F. poae, F. graminearum, F. equiseti, and F. avenaceum. Most grain samples were contaminated with mycotoxins, with a mean incidence rate of up to 75% for deoxynivalenol and 86% for T-2 toxin. Other mycotoxins detected were zearalenone and Aflatoxin B1. Co-occurrence of deoxynivalenol, T-2 toxin and zearalenone was found in up to 35% of the samples. The results suggested the presence of Fusarium head blight in Kenya and associated mycotoxin contamination, though at low but significant levels. The presence of the different mycotoxins, though at low levels, could pose chronic adverse health effects to human and livestock fed on the contaminated wheat products. Introduction Fusarium head blight re-emerged recently as a devastating disease of wheat and other small-grain cereals through out the world (McMullen et al, 1997; Windels, 2000). The disease results in grain yield reduction and loss of quality due to shriveled grain and mycotoxin contamination. Mouldy grain can cause allergy and breathing problems (McMullen and Stack, 1999). However, the presence of Fusarium head blight-infected grain does not automatically result in mycotoxin contamination. Mycotoxins associated with grain affected by Fusarium head blight include trichothecenes (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin) and zearalenone (Park et al., 1996). The occurrence, amount and kind of mycotoxin depend on the environment, species of the fungus present, severity of infection and crop variety. Most Fusarium species have potential to produce mycotoxins in grains but F. graminearum is the most aggressive on wheat. The main trichothecene- producing species are Fusarium graminearum, F. culmorum, F. sporotrichioides, F. poae and F. crookwellense (Marasas et al., 1984). All the Fusarium species that infect cereals are capable of surviving saprophytically on crop debris (Parry et al., 1995). During harvest, the chaff (infected debris) and light-weight kernels are left in the field and are incorporated into the soil and serve as important sources of inoculum during the subsequent seasons (Sutton, 1982; Parry et al., 1995). Harvested wheat grain from Kenya has been previously shown to be contaminated with mycotoxin-producing Fusarium species (Muthomi and Mutitu, 2003). However, mycotoxin levels of the naturally infected wheat grain have not been determined. Therefore, this study was carried out to determine the level of Fusarium contamination and associated mycotoxins in freshly harvested wheat from Nakuru and Nyandarua districts of Kenya. Materials and Methods Survey and survey area
A survey was carried out during 2004 harvesting season in wheat growing districts of Nakuru and Nyandarua, Kenya. Five agroecological zones in Nakuru and 4 agroecological zones in Nyandarua were selected. In each agroecological zone, 10 farms were randomly selected, giving a total of 89 farms. Wheat grain samples (1-2kg per farmer) were collected for mycological and toxin analysis. The samples were stored at 4OC until analysed.
Each sample was thoroughly mixed and a 100g sub-sample taken randomly for mycological analysis. The seeds were surface sterilized for 3 minutes in 5% sodium hypochlorite containing four drops of Tween 20 for 3 minutes then rinsed three times with distilled water. Ten surface sterilised kernels were plated on each plate containing low strength Potato Dextrose Agar (PDA) media amended with mineral salts (0.005 g CuSO4.5H2O,
0.01 g ZnSO4.7H2O) and antimicrobial agents (0.05 g Chloramphenicol, 50 mg penicillin, 50 mg tetracycline,
50 mg streptomycin) to suppress growth of fast-growing fungi and bacteria (Muthomi, 2001). A total of 100 seeds were plated per sample. The plates were incubated at room temperature 20±5OC for 7-14 days after which kernels showing fungal infection were recorded, and the different fungal colony types determined. The fungal genera growing on the kernels were identified based on cultural and morphological characteristics. The Fusarium isolates were identified to species level based on synoptic keys by Nelson et al, (1983). Fusarium
colonies were sub cultured on both Synthetic Nutrient Agar (SNA) (Nirenberg, 1981) and Potato Dextrose Agar (PDA). The cultures were incubated under near UV light for 14–21 days to induce sporulation. Microscopic identification was based on presence or absence of macroconidia and microconidia, conidia morphology, the way the conidia were produced on conidiophores, type of conidiophores, presence and arrangement of chlamydospores.
Mycotoxin content in the wheat grain was determined by direct competitive Enzyme-Linked Immunosorbent Assay (ELISA), (AOAC 1995; Gareis et al., 1989, Hack et al., 1989). A total of 80 samples were analysed for deoxynivalenol, T-2 toxin, zearalenone and 41 samples for aflatoxin B1. Each sample was homogenized and 100g ground to fine powder. Five grams of the ground sample was extracted with 25 ml of methanol: water (50:50v/v) for aflatoxin, zearalenone and deoxynivalenol and 70:20 (v/v) for T-2 toxin. The extract was de-fatted with 10ml hexane and 4ml of the methanolic layer was diluted to 10% using Phosphate Buffer Solution (PBS). For T-2 toxin, the methanolic extract was diluted with an equal volume of distilled water. The 96 well microtitre polystyrene (Maxisorp®, Nunc, Denmark) plates were coated with 100µl of anti-aflatoxin antiserum K147 (Gathumbi et al., 2001) for aflatoxin B1, anti-deoxynivalenol antiserum DON143/16 (Usleber et al., 1992) for deoxynivalenol and anti serum ZEA A37 (Usleber et al., 1992) for zearalenone in bicarbonate buffer (Ph = 9.6) per well. For T-2 toxin, a commercial kit (Ridoscreen, r-Biopharm, Germany) was used and the ELISA procedure performed following manufacturer’s recommendations. Absorbance was determined using the spectrophotometer Elisa reader (Uniskan II, Finland) at 450 nm. A calibration curve for the standards for each toxin dilutions was plotted using log10 of standards concentration against the percentage inhibition of the
Results Fungi associated with naturally infected wheat
The major fungal genera isolated from wheat grain samples were Epicoccum, Alternaria, Fusarium, Aspergillus and Penicillium (Tables 1a and b). The mean kernel infection rate was 98.4% but the level of contamination varied in different agro-ecological zones. The highest total Fusarium infection was observed in Nyandarua district, with the agro-ecological zones LH4 having the highest infection rates in both districts. Agro-ecological zones with high Epicoccum and Alternaria infection rates were found to have lower levels of Fusarium. Fusarium species isolated were F. poae, F. chlamydosporum, F. oxysporum, F. graminearum, F. equiseti, F. moniliforme, F. avenaceum, F. semitectum, F. crookwellense, F. lateratium, F. sporotrichioides, F. scirpi, F. sambucinum and F. solani (Table 2a and b). Fusarium poae, F. chlamydosporum and F. oxysporum were the most prevalent in all the agro-ecological zones while F. graminearum was isolated in 6 out of the 9 agro-ecological zones. The least prevalent species were F. semitectum, F. scirpi, F. solani and F. sporotrichioides.
Table 1a– Proportion (%) of fungi isolated from wheat kernels from different agro-ecological zones of Nakuru District, Kenya, during the 2004 cropping season.
(P ≤ 0.05), Values followed by different letters within columns are significantly different.
Table 1b–Proportion (%) of fungi isolated from wheat kernels from different agro-ecological zones in Nyandarua district, Kenya, during the 2004 cropping season.
LH3 15.29c 16.18d 42.15a 10.93a 14.57a LH4 24.32a 29.70c 41.25b 4.23d 0.29d UH3 14.05d 53.26a 22.92d 7.62c 1.96b UH4 18.41b 36.19b 34.35c 10.62b 0.44c Mean 18.02 33.83
(P ≤ 0.05), Values followed by different letters within columns are significantly different. Table 2a–Mean percentage isolation of different Fusarium species from wheat samples collected from different agroecological zones of Nakuru district during the 2004 cropping season.
LSD = 0.32, (P ≤ 0.05) Values followed by different letters are significantly different within columns
Table 2b–Mean percentage isolation of different Fusarium species from wheat samples collected from different agroecological zones of Nyandarua district, Kenya, during the 2004 cropping season. Fusarium species
LSD = 0.32, (P ≤ 0.05) Values followed by different letters are significantly different within columns
Mycotoxin contamination of wheat grain
Most of the wheat grain samples were contaminated with mycotoxins deoxynivalenol, T-2 toxin and zearalenone (Table 3a and b). The incidence and levels of the mycotoxins varied in samples from different agro-ecological zones. The most prevalent mycotoxin was T-2, followed by deoxynivalenol, zearalenone and aflatoxin B1. However, deoxynivalenol was detected in the highest concentrations of up to 302µg/kg while 2 toxin was in 35% of the samples analyzed. Samples from Nakuru District had higher mean levels of deoxynivalenol (132.65µg/kg) while samples from Nyandarua contained higher mean levels of T-2 toxin (29.8µg/kg) and zearalenone (7.1µg/kg). High variation in mycotoxin content was also observed among wheat grain samples from different agro-ecological zones in both districts. Samples with a high proportion of total Fusarium infection contained higher deoxynivalenol and T-2 toxin levels.
Table 3a–Percent incidence, range and mean of deoxynivalenol, zearalenone, T-2 and aflatoxin B1 content (µg/kg) in wheat grain samples from Nakuru district during the 2004 cropping season.
*Indicates the mean mycotoxin concentration in the all the samples analysed Table 3b–Percent incidence, range and mean of deoxynivalenol, zearalenone, T-2 and aflatoxin B1 content (µg/kg) in wheat grain samples from Nyandarua District.
*Indicates the mean mycotoxin concentration in the all the samples analysed
Discussion The wheat samples were highly contaminated with Epicoccum, Alternaria and Fusarium. Agroecological zones with high levels of Fusarium species isolated had low levels of Epicoccum and Alternaria,suggesting antagonistic effects between Epicoccum, Alternaria and Fusarium. The multiple contamination of wheat with different fungi indicates a potential risk of contamination of the grain with different mycotoxins like trichothecenes, T-2 toxin, zearalenone, fumonisins, moniliformin, alternariol monomethyl ether, altenuene, diacetoxyscirpenol, aflatoxinsand ochratoxins (Moss 1996; Placinta et al., 1999).
Mycotoxins have been implicated in a range of human and/or animal diseases and occur in a variety of grains. In addition, mycotoxins cause losses accruing from condemned foods/feeds, decreased animal productivity and negative impact on internationally traded commodities. The ingestion of mycotoxins can produce both acute (short-term) and chronic (medium/long-term) toxicities ranging from death to chronic interferences with the function of the central nervous, cardiovascular and pulmonary systems, and of the alimentary tract (Marasas, 1991). Pathogenic and Mycotoxin-producing Fusarium species such as F. graminearum and F. poae were isolated at high levels from wheat grain samples. The results correspond with findings of Muthomi and Mutitu (2003) and Muthomi (2002) who isolated Fusarium species at high frequencies from 5 wheat growing areas in Kenya, such as Timau, Njoro, Laikipia, Narok and Nyandarua. This indicates prevalence of head blight infections on the ears in the field. The severely-infected kernels are light enough to be expelled with chaff in the field during harvest (Tuite et al., 1990). Most of the Fusarium species isolated are pathogenic to wheat ears, causing head blight and mycotoxin contamination (Parry et al., 1995). According to Marasas et al., (1991) Fusarium equiseti, F. graminearum, F. poae, F. moniliforme and F. sporotrichioides are considered the most toxic Fusarium species. F. graminearum occurs worldwide and is the most important producer of deoxynivalenol (DON) and zearalenone. Fusarium sporotrichioides and F. poae are the most producers of T-2
toxin. Aflatoxin-producing moulds Aspergillus flavus and A. parasiticus occur widely, on inadequately dried food and feed grains.
The incidence of Fusarium mycotoxin in the wheat samples varied, ranging from 57% for zearalenone, 68% for deoxynivalenol and 76% for T-2 toxin. The level of incidence of the different mycotoxins corresponded to the prevalence of F. poae and F. graminearum (23.75% and 9.35%, respectively),the two fungi that produce these mycotoxins. This indicates that wheat grain from the two districts is contaminated with low but significant levels of multiple Fusarium mycotoxins. The co-occurrence of the Fusarium graminearum toxins deoxynivalenol and zearalenone with the F. moniliforme toxins fumonisin B1 and B2, for example, has been reported (Miller, 1991) in southern Africa. The co-occurrence of mycotoxins can affect both the level of mycotoxin production and the toxicology of the contaminated grain. The presence of trichothecenes may increase the production of aflatoxin in stored grain, for example, whereas some naturally occurring combinations of Fusarium toxins are synergistic in laboratory animals. The legislative limits for Fusarium mycotoxins range from 500µg/kg-2000µg/kg for deoxynivalenol, 30-1000 µg/kg for zearalenone and 100µg/kg for T-2 toxin (Van Egmond 1989). This implies that the Kenyan wheat products could be contaminated with low but significant levels of the four mycotoxins. Low levels of mycotoxins cause weight loss, immuno-suppression, cancer, reduced milk yields and other sublethal effects (Bennet and Keller, 1997). The co-occurrence of the different Fusarium mycotoxinsmay result in additive and synergistic effects (Muller and Schwardof, 1993). Synergistic toxicity due to multiple mycotoxins is greater than the sum total of the toxicities of each mycotoxin (Speijers, 2004). The number and type of mycotoxins in a sample depends on Fusarium strains present and their toxigenicity as well as environmental factors.
Different strategies are required for the management of mycotoxin contamination in grains. Considerable effort has been expended on the development of crop varieties that are resistant to mould growth and/or mycotoxin production. It has been suggested that wheat has three types of resistance to Fusarium graminearum; resistance to the initial infection, resistance to the spread of the infection and resistance to mycotoxin (deoxynivalenol) production (Schroeder and Christensen, 1963; Miller et al., 1985; Mesterhazy, 1995). Attempts to exploit the resistance to mycotoxin production (through either the inhibition of synthesis or chemical degradation) may hold the most potential because of the limited number of genes which control this process (Bai et al., 1999; Snijders, 1990). Once the crop is infected, the contaminated grain needs has to be identified and segregation and the mycotoxins destroyed (detoxification). The post-harvest handling of grains present many more opportunities for controlling mycotoxin production. Grain must be effectively dried and stored at safe moisture content.
Acknowledgement This research was funded by the International Foundation for Science (IFS) grant No.E/3654-1 References
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