Novel Enzyme Immunoassays for Specific Detection of Fusarium oxysporum f . sp . cucumerinum and for General Detection of Various Fusarium Species

Kitagawa, T., Sakamoto, Y., Furumi, K., and Ogura, H. 1989. Novel enzyme immunoassays for specific detection of Fusarium oxysporum f. sp. cucumerinum and for general detection of various Fusarium species. Phytopathology 79:162-165. Competitive types of two novel enzyme-linked immunosorbent assays including nine other strains of Fusarium, showed little cross-reactivity. (ELISA) for Fusarium species were developed. Antiserum against a strain When cell fragments of F. oxysporum FS01 attached to the balls were used (F504) of F. oxysporum was elicited in rabbits, and a highly specific, as a solid-phase antigen in a heterologous competitive ELISA, the modified sensitive, and accurate ELISA for the homologous strain was developed by system was a general assay for 10 strains of four Fusarium species with a using the antiserum withf3-o-galactosidase-labeled anti-rabbit IgG as the high sensitivity. The specificity of the heterologous competitive ELISA was secondary antibody and cell fragments of the strain attached to Aminoshown to be limited to Fusarium species. The reason for developing the Dylark balls as the solid-phase antigen. All other microorganisms tested, improved ELISA is also presented. Additional keywords: competitive assay, crop plants, enzyme immunoassay, general assay of fusarial species, pathogenic fungi, specific assay. The genus Fusarium includes a tremendous variety of partial hydrolysate of Dylark (22), the co-polymer of styrene and pathogenic fungi that infect a wide range of crop plants and maleate, were purchased from Sekisui Chem. Ind., Mishima-gun, numerous nonpathogenic saprophytes (16). Some are airborne Osaka, Japan. Other chemicals used were of reagent grade. pathogens and others are soilborne. The presence of soilborne Media. Buffers were 0.02 M sodium phosphate-buffered saline Fusarium spp. is one of the main impediments to continuous (PBS), pH 7.0; buffer A (PBS, containing 0.1 M NaCl, 1 mM cropping of fields (15). Because no serological method has been MgCI2, 0.1% bovine serum albumin, and 0.1% NaN 3); buffer B successful, Fusarium is identified by morphological characteristics. (0.05 M sodium phosphate, pH 7.4, containing 0.01 M Distinguishing pathogenic and saprophytic isolates of the same ethylenediaminetetraacetic acid and 0.1% bovine serum albumin). species has not been easy. Some Fusarium species are divided into For potato-sucrose broth, potatoes were cut into 1-cm cubes, and formae specialis, or races, based on their differential ability to 200 g of potato cubes was boiled with 1 L of water for 20 mm, parasitize specific hosts (2). In addition, some Fusarium species mashed, and squeezed through a muslin bag. Sucrose (20 g) was produce phytotoxins or hormones such as mycotoxins (16), dissolved in the above extract, which was made up to 1 L with zearalenone (17,25), butenolide (28), fusaric acid (11,26), and water, adjusted to pH 5.6, and then autoclaved. gibberelins (27). These contaminants present problems because of Strains tested. The 10 strains of Fusarium and 15 strains of other their toxicity to animals fed infected crops (18,21). Consequently, a microorganisms used included: Fusarium oxysporum f. sp. quick diagnostic method for the simple detection of Fusarium cucumerinum F501, F. o. cucumerinum F504, F. o. melonis F401, contaminants in crops to determine the race of the fungus or to F. o. lycopersici F1001, F. o. batatas F1201, F. oxysporum F105 measure quantitatively any species of Fusarium would be of value (saprophyte), F. solani f. sp. pisi F200 1, F. moniliforme F2501, F. to ensure the quality of animal feed and to study the occurrence of roseum F103, F. roseum F3301, Aspergillus niger, A. flavus, Fusarium in farm fields. Chaetomium sp., Myrothecium sp., Microthecium sp., Penicillium Progress in the development of enzyme immunoassays (BIA) frequentans, P. charlesii, Trichoderma harzianum (the stock has been rapid (3,7,9,12). An BIA is a useful means for quick cultures of Kochi University), Escherichia co/i K12, Xanthomonas diagnoses of various microorganisms, such as viruses and bacteria, campestris pv. citri, Streptomyces scabies Obama, S. scabies that can be prepared in homogeneous suspensions (4-6,8,10). Few Higashihara, Pyricularia oryzae 037 Ken 60-19, P. oryzae 001 Kyu BIA have been developed for fungi because these do not form 8205A, and P. oryzae Ine 72. homogeneous suspensions. However, a novel and rapid diagnostic Preparation of cell fragments of Fusarium species. Each isolate method for Fusarium has been developed in our laboratory. In the of Fusarium was cultured in potato-sucrose broth for 1 wk with present article, we report two highly sensitive and accurate EIAs shaking and harvested by centrifugation. The pellet was washed for Fusarium species. One is aspecific assay for F. oxysporum f. sp. twice with water before lyophilization. A suspension of the cucumerinum F504, and the other assay detected 10 strains of lyophilized cells of F. oxysporum F504 in PBS was placed in a common Fusarium species that show specific pathogenicities to sonic disruptor (Branson Sonifier, model W 185, USA) at 60 W for different plants. 15 mmn in an ice-water bath, and cell wall fragments were collected by centrifugation at 800 g for 15 mmn. The suspension of the MATERIALS AND METHODS pelleted fragments was used for immunization of rabbits as well as for preparing solid-phase antigens. Reagents. /3-u-Galactosidase (GAL)-labeled goat anti-rabbit Immunization. Two rabbits were given subcutaneous and IgG was prepared as previously (23). Commercial Amino-Dylark intramuscular injections at 10 points with a l-ml suspension of the balls (6 mm diameter) (23,24), prepared from ethylenediamine and fragments of F. oxysporum F504 (1 mg/ ml of PBS) emulsified in an equal volume of incomplete Freund's adjuvant. Four booster _____________________________________________ injections of one half the original dose were given in the same way © 1989 The American Phytopathological Society at biweekly intervals. The rabbits were bled from the ear veins 2 wk