Immunoassay-based Techniques for Early and Specific Detection of Latent Postharvest Anthracnose in Mango

The postharvest anthracnose pathogen Colletotrichum gloeosporioides inciting latent or quiescent infection of mango was detected in early stages using immunoassay methods. Twenty-five pathotypes isolated from different agroclimatic zones of Tamil Nadu, Karnataka and Pondicherry, India, revealed the variation in protein profile analysis (SDS-PAGE). The polyclonal antibodies (PCA) were raised against the unfractioned mycelial protein (UMP) and a 40-kDa polypeptide present in all pathotypes. Standardization of antigen and antiserum dilutions revealed that an antigen dilution of 1:200 (protein concentration of 20g/ml) and antiserum dilution of 1:100 (protein concentration of 40g/ml raised against UMP) and 1:200 (protein concentration of 20g/ml raised against 40kDa polypeptide) was found to be optimum for the detection of anthracnose pathogen. Both antisera detected the C.gloeosporioides antigen in enzyme-linked immunosorbent assays (ELISAs), dot immunobinding assays (DIBAs) and Western blots. The specificity in reaction was compared by isolating other Colletotrichum spp. from various hosts viz., C.lindemuthianum (beans), C.falcatum (sugarcane), C.musae (banana), C.capsici (chillies) and Botryodiplodia theobromae (mango). The antisera generated against UMP revealed the cross-reaction with other host isolates and mango stem end rot pathogen (B.theobromae). The PCA raised against 40-kDa polypeptide exhibited the specific reaction with C.gloeosporioides isolates in all the immunoassay techniques. By utilizing both PCA, the presence of latent infection was observed in healthy-looking leaves, flowers and fruits in orchard conditions. The fruit tissues recorded high absorbance values followed by flowers and leaves in all the detection methods. The ELISA technique was also useful in assessing the pathogen inoculum at various biocontrol formulations sprayed mango trees under field conditions. The fluorescent pseudomonad strains mixture (KFP1+FP7) amended with chitin sprayed at 30-day intervals revealed the significant reduction in pathogen load than other formulations and unsprayed control.