Development of real time PCR assay for detection and quantification of teliospores of Tilletia indica in soil

摘要

Tilletia indica, commonly called Karnal bunt, is an internationally quarantined wheat fungal pathogen which affects commercial seed trading as well as the quality of wheat grain for consumption. The teliospores of Tilletia indica surviving in soil serve as the primary source of inoculum and play a major role in disease development. Proper identification and detection of T. indica teliospores based on morphological features and germination of teliospores is time consuming and tedious. In this study, we validated PCR based species-specific primer which amplified 570 bp fragments using ITSKB primers. Further, the real time PCR assay was developed and standardized for detection and quantification of teliospores in soil. The (R-2) correlation coefficient (0.994) between C-T values and DNA concentrations showed the accuracy of qPCR based quantification. The sensitivity of qPCR marker was 100 fg. Thirteen field soil samples were assessed by qPCR for quantification of teliospore DNA. Low fungal DNA (15135.61 fg) was detected in field soil (K10) from Karnal, Haryana, India while high DNA concentration (3.31 ng) was detected in field soil from IARI, New Delhi (K4). The qPCR assay was done to correlate DNA concentration and number of teliospores per gram soil. The 125.89 fg DNA concentration of T. indica detected corresponding limit of 14 teliospores. Minimum detection limit in terms of teliospores count was 14. The teliospores recovered from Karnal. and IARI farm soils by centrifugation method were 450 and 1341, respectively while the qPCR assay based analysis detected higher number of teliospores ranging 1762 to 368332 teliospores. Thus, the developed qPCR diagnostic marker could be used for accurate, reliable and rapid detection of teliospores in soil which would further help in monitoring, quantifying teliosporic load and threshold level of inoculum in soil.