Polyclonal antibody-based ELISA in combination with specific PCR amplification of internal transcribed spacer regions for the detection and quantitation of Lasiodiplodia theobromae, causal agent of gummosis in cashew nut plants

Members of Botryosphaeriaceae family are associated with serious diseases in different plants across the world. In cashew nut plants (Anacardium occidentale), the fungus Lasiodiplodia theobromae causes a severe group of symptoms related to gummosis that results in decreased nut production. The aim of this work was to develop an indirect enzyme-linked immunosorbent assay (ELISA) with sufficient sensitivity and specificity to detect the fungus both in vitro and in planta (artificially and naturally infected) and to increase the detection specificity within the fungi group using primers specific for the internal transcribed spacer (ITS) sequences. A collection of L. theobromae isolates was obtained, and antisera against the fungus were raised in rabbits. Cross-reactivity against Neofusicoccum sp., Colletotrichum gloeosporioides, Phomopsis anacardii and Pestalotiopsis guepinii was examined. Naturally and artificially infected vegetal material were employed in the ELISAs. The fungi ITS sequences were determined, and single nucleotide polymorphisms were identified and used for primer design. For the naturally infected plants, there was an approximately fourfold variation in the absorbance values. Some positive readings for asymptomatic samples were detected. For the artificially infected samples, an ELISA-based weekly time-course analysis was conducted, and the values for samples from 0 and 7 days were lower than the threshold value. Beginning on day 14, the infection could be detected, with rates varying from 40% on day 14 to 80% on day 21 and 100% by the end of the experiment. The ITS sequencing revealed few polymorphisms among the L. theobromae isolates, but for C. gloeosporioides, P. anacardii, P. guepinii and Neofusicoccum sp., the sequences were sufficient to permit reliable discrimination. The feasibility of ELISA as an early detection technique to assist in gummosis management was demonstrated. PCR amplification based on ITS regions increases and complements serological specificity.