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

Abstract

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 timecourse 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.

Analysis of phylogeny, distribution, and pathogenicity of Botryosphaeriaceae species associated with gummosis of Anacardium in Brazil, with a new species of Lasiodiplodia

“…To date only L. theobromae has been found associated with cashew gummosis (Freire et al 2002;Cardoso et al 2004;Muniz et al 2012;Moreira et al 2013). However, identification of causal agents was based on analysis of fungal morphology and cultural characteristics, which are today considered insufficient for species identification in the genus Lasiodiplodia (Phillips et al 2013).…”

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

“…For instance, sporulation efficiency may be negatively compromised by certain temperature, light, and substrate regimes. Previously, a method developed for pycnidiospore production involved the use of PDA medium supplemented with 10% mung bean extract (1000 g mung beans boiled in 2-L deionized water) and incubation of the cultures at 25°C for 5 days (Muniz et al, 2012). Since this method proved to be efficient, employment of these structures as the biological target for ATMT seems to be appropriate.…”

“…The strain was stored at 5°C on potato dextrose agar (PDA) (Muniz et al, 2012). Escherichia coli strain DH5α was used as a host for the propagation of plasmid DNA.…”

“…Before transformation, cultures obtained from germinated pycnidiospores (Muniz et al, 2012) were submitted to the minimum inhibitory concentration of hygromycin B. Cultures were inoculated on PDA, containing different concentrations of hygromycin B (0,50,100,150,200,250, and 300 µg/mL) and, then, incubated at 28°C for 14 days. For each treatment, 5 Petri dishes were used.…”

“…L. theobromae monosporic strain Lt19 was grown on PDA at 28°C for 14 days. Pycnidiospores were obtained according to the method of Muniz et al (2012), were collected by flooding the Petri dish with sterile water, and counted under a Zeiss microscope with a Neubauer chamber. The final pycnidiospore concentration was adjusted to 10 7 spores/mL in saline solution.…”