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THE USE OF A SIMPLE ELISA FOR IDENTIFICATION OF MONILINIA FRUCTICOLA.AND MONILiNIA SPP..
THE USE OF A SIMPLE ELISA FOR IDENTIFICATION OF MONILINIA FRUCTICOLA. AND MONILiNIA SPP.. KJD HUGHESl, JN BANKSl, RH RIZVIl, J MCNAUGHTON 2, CR LANEl, L STEVENSON2 and RTA COOK l . l Central Science Laboratory MAFF, Sand Hutton, York Y04 1 LZ, UK. 2 Adgen Diagnostic Systems, Auchincruive, Ayr, KA6 5HW, UK. Background and objectives Brown rot of stone and pome fruit is caused by the fungi Monilinia fructicola, M. fructigena and M. laxa. Of these M. fructicoia is an EC listed quarantine fungus which is difficult to distinguish from the other two. Work done within an EU funded project has improved identification of these species by cultural and molecular methods [1]. However, these methods are either slow or require trained laboratory staff and are therefore unsuitable for routine monitoring purposes. Monocional antibodies (MAbs) have been raised with promising specificity towards the genus Monilinia and the species M. fructicoia[2]. The enzyme conjugate horse radish peroxidase (HRP), used for selecting MAbs [2] suffers from the disadvantage of requiring a substrate which may cause false positives with plant material containing high levels of polyphenol oxidase/ peroxidase [3]. This paper describes a preliminary experiment in development of MAbs for routine diagnostic work using simple antigen extraction procedures and a substrate suitable for testing plant material. Materials and methods Isolates of Monilinia spp., whose identity was confirmed by molecular and cultural studies [1], were grown for three weeks on potato dextrose agar, incubated at 220C and 12h near UV 1 12h dark. Fungal material for screening was obtained by scraping the surface of test cultures with a single edged razor blade. Scrapings were transferred to 1.5 mi phosphate buffered saline (PBS, pH 7.2), vortexed, allowed to stand overnight at 4"C then centrifuged at 17,000 g. In addition to MAbs raised previously [2] further MAbs (10,12 and 13) were produced by workers in [2]. The production and characterisation of these MAbs will be described elsewhere but tissue culture supernatants at 1/10 dilution were tested in an indirect ELISA against expressed liquid from plum fruit (plum juice), soluble extracts of the three Monilinia species and six commonly occurring fungal saprophytes of fruit. Methods were as previously described [2] except anti-mouse alkaline phosphatase (AP) conjugate was used (1g/ml) with p-nitrophenyl phosphate substrate. Absorbance was measured at 405nm after 1 h incubation at 22oC in the dark and values greater than twice PBS controls were recorded as positive. Results and conclusions In a preliminary experiment MAb 1 0 showed M. fructicoia specificity, reacting positively to 11 out of 16 M. fructicoia isolates. Four of these gave high absorbance values (0.95-- 1.89), two medium (0.43 and 0.77) and five weak (0.24---0.27). Negative results were observed for six isolates of M. fructigena, six M. laxa, the six fungal saprophytes and plum juice. We are investigating why some of the M. fructicoia isolates gave weak reactions and others were not detected. MAbs 12 and 13 showed genus specificity, reacting only with Monilinia isolates and not with saprophytic fungi or plum juice. The AP conjugate gave acceptable signals and was a suitable replacement for HRP. The specificity of the MAbs coupled with ease of extraction of fungal antigens and the use of AP in the ELISA will be of direct use in routine diagnostic work. The potential of genus and species-specific MAbs for use in a diagnostic kit is being evaluated. Funding provided by MAFF UK Plant Health Division and EU project (FAIR 1-CT95-0725). References 1 . Corazza L, Cook RTA, Lane CR, Fulton CE, van Leeuwen GCM, Pereira AM, Nazare-Pereira A, Melgarejo P, De Cal A, 1998. Abstract in ICPP98. 2. Banks JN, Rizvi RH, Lane C, Hughes KJD and Cook RTA, 1997. In: Diagnosis and Identification of Plant Pathogens. Eds. Dehne HW, et ai Kluwer Academic pp.391-393. 3. Converse RH and Martin RR, 1990. In: Serological methods for detection and identification of viral and bacterial plant pathogens. APS, p. 182.