Polyclonal Antibody-based ELISA in combination with specific PCR amplification of ITS 1 regions for the detection and quantitation of Lasiodiplodia theobromae, causal agent of 2 gummosis in cashew nut plants

Members of Botryosphaeriaceae family are associated with serious diseases in different plants 18 across the world. In cashew nut plants (Anacardium occidentale L.), the fungus Lasiodiplodia 19 theobromae causes a severe group of symptoms related to gummosis that results in decreased nut 20 production. The aim of this work was to develop an indirect enzyme-linked immunosorbent 21 assay (ELISA) with sufficient sensitivity and specificity to detect the fungus both in vitro and in 22 planta (artificially and naturally infected) and to increase the detection specificity within the 23 fungi group using primers specific for the internal transcribed spacer (ITS) sequences. A 24 collection of L. theobromae isolates was obtained, and antisera against the fungus were raised in 25 rabbits. Cross-reactivity against Neofusicoccum sp., Colletotrichum gloeosporioides, Phomopsis 26 anacardii and Pestalotiopsis guepinii was examined. Naturally and artificially infected vegetal 27 material was employed in the ELISAs. The fungi ITS sequences were determined, and single 28 nucleotide polymorphisms were identified and used for primer design. For the naturally infected 29 2 plants, there was an approximately 4-fold variation in the absorbance values. Some positive 1 readings for asymptomatic samples were detected. For the artificially infected samples, an 2 ELISA-based weekly time-course analysis was conducted, and the values for samples from 0 and 3 7 days were lower than the threshold value. Beginning on day 14, the infection could be 4 detected, with rates varying from 40% on day 14 to 80% on day 21 and 100% by the end of the 5 experiment. The ITS sequencing revealed few polymorphisms among the L. theobromae isolates, 6 but for Colletotrichum gloeosporioides, Phomopsis anacardii, Pestalotiopsis guepinii and 7 Neofusicoccum sp., the sequences were sufficient to permit reliable discrimination. The 8 feasibility of ELISA as an early detection technique to assist in gummosis management was 9 demonstrated. PCR amplification based on ITS regions increases and complements serological 10 specificity