Abstract: The objective of this study was to determine the mycotoxigenic potential of 12 Fusarium isolates (10 species), including six isolates (4 species) from sorghum. The species were: F. thapsinum, F. semitectum, F. proliferatum and F. chlamydosporum isolated from molded sorghum seed; F. poae, F. graminearum and F. sporotrichioides from barley seed with Fusarium head blight; F. acuminatum from wheat seed; F. verticillioides from infected corn seed; and F. nygamai isolated from soil. Fumonisin and zearalenone concentrations were measured following incubation on autoclaved sorghum seed for 21 days at 25°C, while fusaric acid was measured in mycelia harvested from Czapek Dox broth cultures. F. thapsinum (SC8 and CS121) and F. semitectum (SC7) produced fusaric acid only (4.59-64.13 mg g-1). F. graminearum (KB172) and F. semitectum (CS152) produced zearalenone only (73.4 and 799.3 μg g-1, respectively). F. proliferatum (CS183), F. verticillioides (TX02) and F. nygamai produced both fumonisin (1.92-6.05 μg g-1) and fusaric acid (39.4-234.17 mg g-1). F. poae (KB652), F. acuminatum (Ark), F. chlamydosporum (CS102) and F. sporotrichioides (KB662) did not produce any of these three mycotoxins. Five of the six Fusarium isolates (three species) isolated from sorghum had mycotoxigenic potential. Fusarium spp. naturally occurring on sorghum in the field have the potential to contribute to mycotoxin contamination, either singly or in combination.
INTRODUCTION
The mycotoxins, fumonisin, zearalenone and fusaric acid, are secondary metabolites produced by a number of Fusarium species and are frequent contaminants of grains of several plants (Abbas et al., 1999; Benneth and Klich, 2003; Desjardins, 2006; Miller, 1995; Sweeney and Dobson, 1998). Fumonisin contamination of sorghum has been identified in many locations, including Brazil (da Silva et al., 2000), India (Bhat et al., 1997; Shetty and Bhat, 1997), Ethiopia (Ayalew et al., 2006) and Botswana (Siame et al., 1998). Zearalenone-contaminated sorghum poisoned pigs in Australia (Blaney et al., 1984). Zearalenone contamination of sorghum has been documented in Colombia (Diaz and Céspedes, 1997), as well as in the United States (Bowman and Hagler, 1991; Hagler et al., 1987; McMillian et al., 1983; Schroeder and Hein, 1975; Shotwell et al.,1980).
Fusaric acid, produced by several Fusarium species, was first noticed for its phytotoxicity to rice (Desjardins, 2006). This toxin is a pathogenicity factor, causing wilting in tomato plants infected with F. oxysporum f. sp. lycopersici (Fakhouri et al., 2003). To date, no outbreaks of human or animal disease have been linked to consumption of foods or feeds contaminated with fusaric acid (Desjardins, 2006). However, a synergistic toxic interaction of fusaric acid and fumonisin on developing chicks has been reported (Bacon et al. (1995). There is a lack of information of the ability of Fusarium species isolated from sorghum to produce fusaric acid, in comparison with fumonisin and zearalenone. Thus, this study was initiated to assess the ability of four Fusarium species isolated from sorghum to produce these mycotoxins, in comparison with six other species from other sources.
Single-spore cultures of F. thapsinum, F. semitectum, F. proliferatum and F. chlamydosporum isolated from sorghum seed with grain mold, F. poae, F. graminearum and F. sporotrichioides from barley kernels with symptoms of Fusarium head blight, F. acuminatum from wheat, F. verticillioides from corn kernels and F. nygamai isolated from soil and maintained on dried, colonized Whatman No. 2 filter papers stored in a freezer at -7°C were used in this study. Identities of isolates from sorghum were confirmed by Jane Juba at the Fusarium Research Center, The Pennsylvania State University.
Fumonisin and zearalenone production by isolates was evaluated by growing them on sorghum seed. Three hundred milliliters of water was added to 100 g of sorghum seed in 500 mL Erlenmeyer flasks and placed on laboratory benches for 36-48 h. The flasks were then drained and autoclaved at 121°C for 30 min and then autoclaved again on the following day. Flasks were inoculated with plugs from agar cultures and incubated for 21 days at 25°C. Every 3 days, the flasks were shaken thoroughly to facilitate complete colonization by the isolate. Three flasks were used per isolate. Non-inoculated, autoclaved sorghum was included as a control. The experiment was repeated once. Fumonisin and zearalenone were quantified using a competitive direct enzyme linked immunosorbent assay in a microwell format, following recommended protocol (Neogen Corp., Lansing MI).
To measure fusaric acid production by isolates, conidia (2x105) were added to 250 mL flasks containing 50 mL Czapek Dox liquid medium and incubated at ambient room temperature with shaking (250 rpm). Three flasks were used per isolate. Fusaric acid was quantified using a modification of the method of Notz et al. (2002). The concentration of fusaric acid in each flask was expressed in mg g-1 dry weight of mycelia after 15 days of incubation.
Data for the levels of fumonisin, zearalenone and fusaric acid were analyzed using the command PROC ANOVA (SAS version 9.1, SAS Institute, Cary, NC) to determine the differences in mycotoxin production by the Fusarium species. Mean comparisons were based on Tukey’s Studentized Range test at the 5% probability level.
Fumonisin was produced in sorghum seed cultures by F. nygamai, F. proliferatum and F. verticillioides, but not by the other Fusarium species (Table 1). Fusarium nygamai produced a significantly higher level of fumonisin than F. verticillioides. Zearalenone was produced in sorghum seed cultures only by F. semitectum (CS152) and F. graminearum (Table 1). Six of the 12 Fusarium species in this study produced fusaric acid in Czapek Dox broth (Table 1). Fusarium proliferatum produced the highest level of fusaric acid, 234.17 mg g-1 dry weight of mycelia; whereas F. thapsinum (CS121) produced the lowest level, 4.59 mg g-1 dry weight of mycelia. The other species producing fusaric acid included F. nygamai, F. semitectum (SC7), F. verticillioides and F. thapsinum (SC8).
This study was initiated to assess the mycotoxin-producing potential of ten Fusarium species when cultured on sorghum. Four species were obtained from sorghum kernels with grain mold. Six species were capable of producing at least one type of mycotoxin. Our findings confirm the mycotoxin-producing capability of Fusarium spp. reported by other workers (Abbas et al., 1999; Bennett and Klich, 2003; Desjardins, 2006; Sweeney and Dobson, 1998). Additionally, we report for the first time the production of fusaric acid by an isolate of F. semitectum (SC7).
We found quantitative differences in toxin-producing potential among isolates of F. thapsinum. This confirms earlier work with other species. All 8 isolates of Fusarium (Gibbosum and Semitectum) isolated from the sorghum seed produced varying amounts of zearalenone on autoclaved sorghum, with one isolate producing 3030 μg g-1 (McMillan et al., 1983). Three of five isolates of F. equiseti produced 0.363-0.667 μg g-1 zearalenone in sterile corn culture (Shotwell et al., 1980). Fumonisin concentrations on sorghum seed inoculated with F. moniliforme ranged from 8.25-125.31 μg g-1 (Bhat et al., 2000), while 47% of F. moniliforme isolates from Burundi sorghum samples produced 3-374 μg g-1 fumonisins in rice culture (Munimbazi and Bullerman, 1996). Leslie et al. (2005) evaluated five Fusarium species from sorghum for toxigenicity. These species, until recently, were grouped as F. moniliforme.
| Table 1: | Production of mycotoxins by Fusarium species |
| 1ND = non-detectable, 2Means within a column followed by the same letter (s) are not significantly different based on Tukey’s Studentized Range test at the 5% probability level | |
They found that F. nygamai and F. verticillioides produced fumonisin, while F. andiyazi, F. pseudonygami and F. thapsinum did not. F. graminearum isolated from sorghum in Australia produced zearalenone in culture on either maize or sorghum (Blaney and Dodman, 2002). The isolate of F. graminearum used in this study originated from barley.
Factors affecting Fusarium mycotoxin production in sorghum in the field are not well known. Hagler et al. (1987) found zearalenone contamination associated with wet weather during flowering and grain fill, but there was no head blight and grain mold was not severe. Grain mold was significantly correlated in two combined years with zearalenone contamination in several locations in North Carolina, with levels of contamination up to 3099 ng g-1, but contamination also occurred in the absence of grain mold (Bowman and Hagler, 1991).
Mixtures of mycotoxins in grains can occur and there is the possibility of interactions in some cases (Miller, 1995). In addition to contamination in the field, there is also the possibility of post-harvest accumulation of mycotoxins. Areas with poor food handling procedures, poor grain storage, the presence of toxigenic fungi and rain at the time of harvest are factors contributing to mycotoxin contamination (Abbas et al., 1999; Benneth and Klich, 2003; Bhat et al., 1997).
This study evaluated the mycotoxigenic potential of ten Fusarium species in pure culture. Toxigenicity under field conditions could be different and could be affected by factors such as weather, timing of infection and interaction with other microorganisms.
ACKNOWLEDGMENTS
We thank Andrea Howard, Adam Blackwelder and Sarah Simmons for their assistance with the fumonisin and zearalenone analysis.