Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml(-1) buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml(-1) buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.