Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in fruit post-harvest tissues

MARTÍN A. FERNÁNDEZ-BALDO; JORGE G. FERNÁNDEZ; SIRLEY V. PEREIRA; GERMÁN A. MESSINA; ELOY SALINAS; JULIO RABA; MARÍA I. SANZ FERRAMOLA.

BMC MICROBIOLOGY

BIOMED CENTRAL LTD

Año: 2011 vol. 11 p. 2 - 8

1471-2180

 Background: Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, whichcauses substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparentlyhealthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitivemethods are necessary for its detection and quantification. This article describes the development of an indirectcompetitive enzyme-linked immunosorbent assay (ELISA) for quantification of B. cinerea in apple (Red Delicious),table grape (pink Moscatel), and pear (William’s) tissues.Results: The method was based in the competition for the binding site of monoclonal antibodies between B.cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent ontothe surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity,precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. Theimmobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The funguswas detected and quantified in any of the fruits tested when the rot was not visible yet. Results were comparedwith a DNA quantification method and these studies showed good correlation.Conclusions: The developed method allowed detects the presence of B. cinerea in asymptomatic fruits andprovides the advantages of low cost, easy operation, and short analysis time determination for its possibleapplication in the phytosanitary programs of the fruit industry worldwide.