The results obtained indicate a good correspondence between the two methods (Table 2). These results suggest that the sensitivity reached for this procedure allow determining very low level of B. cinerea antigens in apparently healthy fruit that can deteriorate suddenly due to the development of latent or quiescent infection into visible disease. Also, the DNA quantified by the method developed

by González et al. [33] from uninfected and infected fruit extracts samples was amplified by PCR, with the purpose of verify if the same correspond to specific DNA of B. PLX4032 clinical trial cinerea [34]. The Figure 3A shows the DNA-B. cinerea from infected fruit extracts samples (apples, table grapes and pears respectively). The bands observed in the lane 1 correspond to a standard of molecular weight marker (MW); in the lanes 2, 3 and 4 correspond to a molecular marker (IGS) for each fruit extracts; in the lanes 5, 6 and 7 correspond to the Boty transposable element for each fruit extract and in the lanes 8, 9 and 10 correspond to the Flipper transposable element for each fruit extract. The Figure 3B shows control extracts made from uninfected fruits. There, only were observed bands in the lane 1 which correspond to a standard of molecular weight marker (MW) indicating clearly the absence of B. cinerea. Figure 3 Gels show one

sample of each kind of infected fruit extract with conidial suspensions (1 × 10 5 spores mL -1 ) and a control per each kind of uninfected fruit extract sample. (A) PCR product analysis of infected fruit extracts samples. Lane 1: standard molecular weight marker (MW). Lanes 2, 3 and 4: molecular marker IGS (ribosomal intergenic

C59 wnt spacer). Lanes 5, 6 and 7: Boty transposable element. Lanes 8, 9 and 10: Flipper transposable element. (B) PCR product analysis of uninfected fruit extracts samples. Lane 1: standard molecular weight marker (MW). Lanes 2, 3, 4, 5, 6, 7, 8, 9 and 10: not observed any bands, indicating clearly the absence of B. cinerea. The presence of both transposable elements (Boty and Flipper) indicates that B. cinerea can be molecularly out characterized as subpoblation transposa-type [35, 36]. Conclusions In the present study, a specific and sensitive indirect competitive ELISA for the quantification of B. cinerea in commercial apple, table grape and pear samples was developed and validated. This inexpensive and simplified method can be applied for 96 fruit samples, per each microtiter plate with a total time for the assay of 35 min. Preparations of immobilized antigen on surface microtiter plates were perfectly stable for at least 4 months assuring the reproducibility of the assay. This is one important advantage for the possible commercialization of the developed ELISA. The results obtained suggest that the sensitivity reached for this procedure allows determining very low levels of B. cinerea antigens in apparently healthy fruits.