Diversity, phylogeny, and pathogenicity of Lasiodiplodia spp. infecting avocado in India and development of sensitive point-of-care LAMP assay for detection of Lasiodiplodia pseudotheobromae

Abstract

Avocado (Persea americana) is a vital fruit crop cultivated in the high-humid tropical regions of the Western Ghats, India, which is significantly impacted by diseases caused by Lasiodiplodia species. These opportunistic fungal pathogens, belonging to the Botryosphaeriaceae family, primarily affect woody plants, including avocados. The study aimed to identify the causal agent of graft failure, root rot, stem canker, and dieback in avocado plants. A roving survey was conducted across the Western Ghats in Karnataka, Tamil Nadu, and Kerala to collect symptomatic nursery seedlings and adult plants. Initial identification was performed based on conidial morphology, followed by DNA sequence analysis of the ITS rDNA, translation elongation factor 1-α (tef1-α), and beta-tubulin (β-tub) gene regions. Concatenated analysis of the ITS, tef1-α, and β-tub genes identified ten isolates of Lasiodiplodia pseudotheobromae, two isolates of Lasiodiplodia brasiliensis, and two isolates of Lasiodiplodia iranensis (syn: L. iraniensis). L. pseudotheobromae was recovered from root rot, stem canker, graft failure, and dieback; L. brasiliensis recovered from root rot and stem canker; and L. iranensis was recovered from stem canker samples. Further pathogenicity tests confirmed that these Lasiodiplodia isolates could induce disease in various avocado plant parts, regardless of their tissue of isolation. A loop-mediated isothermal amplification (LAMP) assay was developed for detecting L. pseudotheobromae targeting the tef1-α gene sequence. The comparative testing of LAMP products using a colorimetric dye (hydroxy naphthol blue) confirmed the presence of L. pseudotheobromae in symptomatic samples. This LAMP assay offers superior ease of use, cost-effectiveness, and rapid results for detecting L. pseudotheobromae in symptomatic plants. This study represents the first report of L. pseudotheobromae, L. brasiliensis, and L. iranensis infecting avocados in India.