INTRODUCTION

Fusarium section Liseola has a wide host range and is widespread throughout the world. The fungus not only causes considerable damage on many plants, but also is parasitic on plants without production of visible symptoms. Especially, Fusarium moniliforme J. Sheldon is widely distributed on maize and sorghum ; the sexual stage (teleomorph) is Gibberella fujikuroi (41) Ito in Ito K. Kimura. In addition to maize and sorghum, members of the G. fujikuroi species complex may infect numerous crops worldwide.

The fungus nomenclature used to this section is not settled. Macroconidial morphology, the most commonly used trait in distinguishing species of Fusarium, is not useful for distinguishing the species within the Liseola section (36); consequently, some authorities (47, 48) have recognized only a single species (usually F. moniliforme J. Sheldon) within this section. Nelson et al (38) distinguished four species within Liseola based on the presence of monophialides and polyphialides and the presence of microconidia in long chains, short chains, or false heads. Using similar morphological characters, Nirenberg (38) recognized six species within this section. Leslie has taken another approach, using the formation of the sexual stage to distinguish species.

The Gibberella stages of this fungus was found first by Wineland (55) when she put two compatible mating types together in culture. The occurrence of Gibberella moniliforme Wineland in nature has been reported in Japan (18), Taiwan (41), the United States (52), Australia (10), and several nations.

Leslie showed that Gibberella fujikuroi contain at least seven different, genetically distinct biological species (= mating populations). The mating pattern within each of these mating population is heterothallic and is governed by two allegers at a single mating-type locus. The biological species were given variety names by Kuhlman but have also been designated with letters (A-G) as different mating populations by others (17, 19, 29). Members of the same mating population are potentially sexually-fertile with one another but are not cross-fertile with members of other mating population. These different mating populations were associated with different anamorphic (asexual) species. These distinctions are important because different mating populations are found preferentially on different hosts.

Isolates of F. moniliforme have the potential to produce significant levels of mycotoxin such as fumonisins. Equine leukoencephalomalacia and hepatosis have a long record of association with maize contaminated with Fusarium spp. from the Liseola section (1, 2, 3, 4, 5, 6, 17, 22, 23, 33, 38, 42, 54). More recently, leukoencephalomalacia has been found in deer that were believed to have consumed maize contaminated with F. moniliforme (16), and this fungus has been correlated with a pulmonary edema syndrome in swine (14,39). Fumonisin B₁ induced pulmonary edema in swine (14). The presence of this compound also has been correlated with cancer-promoting activity in rats (13). Determinations of toxic levels of the compound and its distribution in commercial feeds have just begun (40, 50, 53). Geographically diverse strains of F. moniliforme collected from various substrates in Africa, Asia, and North America produced significant levels of fumonisin B₁, but other strains, primarily from Africa and Australia, produced little, if any, fumonisin B₁ (35). The nearly universal distribution of these fungi in maize and sorghum (28, 34, 51) and their ability to be internally seedborn in symptomless, apparently healthy grain (12, 34, 51), suggest a significant potential for widespread contamination of human foods and animal feeds.

In a recent survey (28), isolates belonging to Fusarium spp. in the Liseola section were recovered from all maize and sorghum fields sampled ; F. moniliforme and F. proliferatum were predominated. In parallel studies (9,28), members of the A, D, and F mating populations were recovered most frequently, and they collectively accounted for more than 50% of the Fusarium isolates recovered from maize and sorghum. Isolates belonging to the B and E populations also were recovered but at a lower frequency. Thus, the members of all these mating populations are sufficiently widely dispersed to warrant a study of the relative ability of different members of there mating population to produce fumonisin B₁. Leslie et al. reported that most strains belonging to the A mating population of F. moniliforme produce fumonisin B₁ at high levels, whereas members of F mating population generally produce low levels of this toxin (30).

Our objectives in this study were to determine the mating populations and type of G. fujikuroi isolates commonly associated with maize in Korea, to detect capability of producing fumonisin B and to confirm the relationship between mating populations and the capability of fumonisin production.

MATERIALS AND METHODS

1. Identification of Mating type and population

1) Strains

Isolates of Fusarium section Liseola were collected from many maize ears and locations in Korea and from imported corn from United States during 1994-1996. These isolates were obtained from moldy or heathy-looking corn ears, and were single spore cultured. All strains (TABLE 1.). Fungal cultures were maintained on fusarium complete medium. Genetic nomenclature of isolates was followed the proposal suggested by Yorder et al for plant-pathogenic fungi except that for mating type designations, which were followed those of Leslie (28, 29, 30).

2) Crossing procedure

Carrot agar with slight modifications, was used for sexual crosses. Fresh carrots (400 g) were washed, diced, and autoclaved 10 min in 400 ml distilled water. The carrots and liquid was blended into a pureed, mixed with an additional 500 ml distilled water and 20 g agar, autoclaved an additional 30 min, and dispensed into 60 ¡¿ 15 mm plastic Petri dishes at 15-17 ml per dish. Initially, all crosses were made with the standard testers

as the female parents. Culture plates were transfer agar block of the maternal parent and incubated at 25¡É with a 12 dark and light cycle. After 10-14 days, microconidial and mycelial suspension of paternal parent was poured on the lawn of maternal plates and spread with a sterile glass rod. After 10 to 20 days mature dark blue perithecia were observed with oozing ascospores. Once mating type and mating population were identified, strains were tested for female fertility by reciprocol crosses in which the field isolate functioned as the female parent and the standard tester as the male parent. All crosses were repeated at least twice.

2. Analysis of Fumonisin production.

1) Cultural Conditions

Fungal isolates were cultured on complete medium for 1 to 2 weeks on an alternation light-dark schedule at 25¡É. The plates were washed with sterile distilled water to produce conidial suspensions. Erlenmeyer flasks (300 ml) with cotton plug were filled with 50 g of wheat grains and 30 ml distilled water and then autoclaved at 121¡É for 1 hr on each of two consecutive days. Each flask was inoculated with 10⁷ conidia and shaken once or twice daily for 3 days to distribute the inoculum evenly. The flask cultures were incubated in the dark at 25¡É ¡¾ 2¡É for 30 days. After that, the cultures were dried at room temperature for 5 days, ground to a fine meal in a blender, and stored at -20¡É until analyzed.

2) Extraction procedures

Fumonisins were extracted and analyzed from Fusarium cultures according to the modified procedure of Xie et al. (56). A 10 g of the culture was extracted with 50 ml of acetonitrile-water (1:1, vol/vol) on a shaker for 1 hr. A 2 ml aliquot of the filtered extract was diluted with 6 ml of distilled water and acidified to ca. pH 4.0 with 1 N HCl. The solution was applied to a Waters C₁₈ Sep-Pak Plus solid phase extraction (SPE) catridge (Waters, Milford Co.) previously washed with 5 ml of acetonitrile followed by 5 ml of distilled water. The catridge column was washed with 10 ml of acetonitrile-water (3:17, vol/vol) and eluted with 10 ml of acetonitrile-water (7:3, vol/vol). The eluted was concentrated to dryness and dissolved in 2 ml of methanol. A 0.5-ml aliquots, which was evaporated to dryness under a stream of nitrogen at ca. 40¡É, was dissolved in 50 §¡ of methanol and derivatized with 150 §¡ of OPA (Complete solation, Sigma Chemical Co.). The derivative was applied to the instrument of HPLC within 1 min after derivertizing. For the analysis of HPLC, the following equipment and conditions were used: instrument, TSP Spectra (Thermo Separation Products Inc., San Jose, CA., U.S.A.); Bondclone10 C18 column (4.9 mm by 300 mm; particle size, 10 §­; Phenomenex Co., Torrance, CA., U.S.A.); mobile phase, an acetonitrile gradient containing 1% acetic acid beginning at 20:80 of acetonitrile-water with a hold of 1 min and changing linearly to 80:20 of acetonitrile-water at 30 min with a final hold of 10 min; flow rate, 1 ml/min; FL3000 fluorescence detector, excitation at 336 nm and emission at 440 nm wavelength. The quantitation of fumonisins was performed by PC1000 program controlling HPLC system.

Mating population, type and female fertility of isolates used in this study

^a Names of provinces in Korea where collected. Unites States: a total of 30 isolates was collected from imported maize from United Stetes.

^b ND = not determind

^c Total number of isolates

DISCUSSION

Three mating populations, termed A, B, and C, were described by Hsieh et al. (17) and a fourth, D, was added by Kathariou (19) described all four mating population as varieties with over lapping characters. Mating population E and F was added by Leslie (30,31). which has a Fusarium nagamai anamorph, has reported. A summary of the known mating population and the anamorphs with which they have been associated is given in TABLE. A number of population of G. fujikuroi (7, 8, 9, 20, 21, 26, 27, 28, 29, 30, 31, 39, 45, 46, 49) have been analyzed for one or more genetically important traits et al. (30).

Mating populations of Gibberella fujikuroi and their corresponding

Fusarium anamorphs

* Cited from J. F. Leslie (1995, Can. J. Botany)

Recovery of mating populations from different hosts based on Kansas State University Fusarium strain collection

* Cited from J. F. Leslie (1995, Can. J. Botany)

Recovery of mating populations from different geographic regions based on Kansas State University Fusarium collection

Isolates obtained from corn grains exhibited the similar frequencies of A+ and A- mating types. However, isolates obtained from a single ear have the same mating type. In other words, both A+ and A- mating type isolates exist in the same region or field, but only one mating type exists in a single ear (Fig.10). It has been strongly suggested that sexual recombination might occur in the field, because both mating type isolates exist in the same field or seed lots (31). However, our data imply, less significance of sexual recombination of F. moniliforme in the field condition. In previous study (Chapter¥°), isolates obtained from a single maize ear composed more than 1 VCG. Therefore, It is warranted to study the possibility of sexual crosses of F. moniliforme in the field in rear future.

Assignment of a strain to a mating may indicate the potential of that strain to produce fumonisins. It has been suggested that the strains in the A mating population produced significant levels of fumonisin while most E and F strains did not. However, quantitative variation in fumonisin levels may be quite large within a mating population, and the distributions observed for the different mating populations overlap substantially. In some cases, large differences in fumonisins production can be observed among strains recovered from the same field or even from the same plants. Most isolates belonging to A mating population produced significant levels of fumonisin in this experiment. However, no significant difference was observed between A+ and A- mating type. It appears that mating types do not implicate fumonisin production.

HIigh frequency of fumonisin-non producer was found in our collection of F. moniliforme mating population A. Although fumonisin-non producers have been reported previously, high frequency in our study is something different from many other studies (29, 30). It should be further characterized whether inability to produce fumonisin is inherited genetic characters or environmental cues affect the fumonisin production. We used rice grains as substrates for growing fungal cultures, but wheat has been frequently used in many other studies (30).

Recently, several analogues of fumonisin have been chemically characterized, but fumonisin B₁ was usually analyzed in population studies (30). We analyzed fumonisin B₁, B₂, and B₃ from all isolates tested and found that fumonisin B₁ accounted for 75-90% of total fumonisins. Generally, fumonisin B₁ is predominant and several times higher than B₂ or B₃ in concentrations. However, six isolates (Gf32, 33, 35, 36, 37, 38, 40) produced more fumonisin B₃ than fumonisin B₁ or B₂. This finding is significant on production pattern of fumonisin by F. moniliforme. Furthermore, these 6 isolates composed a distinct unique VCG and clustered into the same group in random amplified polymorphic DNA analyses (Chapter ¥° and Chapter ¥²). These all combined data suggest that there 6 isolates are geneticaly distinct from other isolates of F. moniliforme.

Biosynthesis pathway for fumonisin production is not clear at present time and little information is available on the derivatization of fumonisin in F. moniliforme. Combined efforts of crossing between fumonisin B₁ and B₃ producers, and molecular approaches would lead our insights on understanding of fumonisin production.

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Appendix 1. Isolation and Identification Media

1. Peptone/PCNB media

¡Ü Isolation medium for Fusarium

¡Ü Adjust pH to 6.0 ; autoclave, then add 20ml streptomycin stock (5g/100ml distilled water-filter sterilize), add 12ml neomycin stock (1g/100 ml distilled water-filter sterilized)

2. KCl media

¡Ü Idetification medium for Fusarium

To 1 liter of glass distilled water add:

Appendix 2. Complete Medium

To 1 liter of glass distilled water add:

¡ÜVitamin Stock Solution (for use in complete medium)

To 1 liter of glass 50% ethanol add:

* To reduce contaminats add 1ml chloroform and store at 2-5¡É.

Appendix 3. Nit mutant inducing Medium

Appendix 4. Minimal Medium (Puhalla,1985)

To 1 liter of glass distilled water add:

¡ÜTrace Elements Solution

To 95ml of distilled water add:

¡ÜFor other nitrogen source media substitute with one of five different nitrogen sources:

¡¡