Development and characterisation of microsatellite markers for the fungus Lasiodiplodia theobromae

Abstract

Lasiodiplodia theobromae is an important fungal pathogen of higher plants from tropical and sub-tropical regions. The fungus infects divergent hosts in a wide range of environmental conditions, suggesting that it is highly variable. The aim of this study was to develop new polymorphic microsatellite markers from a Brazilian isolate of L. theobromae that can be used in population studies of this Brazil. This fungus is both ubiquitous and plurivorous and is able to infect over 500 plant species; causing symptoms ranging from seed rot to the discolouration of timber (7, 8). Infection is generally limited to wounded or stressed-weakened plants (1, 4, 5, 6). The species reproduces mainly by asexual mitospores, with the sexual stage rarely being observed under field conditions (7).The current taxonomic treatment of L. theobromae is based entirely on culture characteristics and asexual reproductive structures (e. g. pycnidium, conidiophore, and conidiospores) (7). The extensive list of synonyms that apply to the species illustrates the confusion that exists over its taxonomic and phylogenetic status. Therefore, it is important to determine whether different genetic variants have evolved to specialise towards the infection of particular hosts or if the entire aggregate is characterised by genotypes that all possess the capacity for a broad host range. In order to distinguish between these alternative scenarios, it is first important to develop the capability to discriminate between closely related pathotypes.Simple Sequence Repeat (SSR) or microsatellite analysis is widely acknowledged as the method of choice for molecular studies of population genetic structure, kinship, genotype diagnosis and genetic evolution. Whilst some SSR markers have recently been developed for species closely related to L. theobromae from South Africa (2, 3, 9), the aim of this study was to develop polymorphic microsatellite markers from Brazilian isolates that can be used in population studies of L. theobromae and related fungi.A mycelial mat (200mg) was cultured from an isolate (Ao1) from a gummosis-infected cashew plant from northeastern Brazil (Piauí state) and grown in liquid still culture of potato dextrose broth (PDB) for 15 days. The culture was then macerated and its DNA extracted using the DNeasy kit (Qiagen R ) according to the manufacturer's protocol. A genomic library was constructed by first double-digesting and related fungi. The nine microsatellite markers developed included six that revealed allelic polymorphisms among nine isolates of the disease collected from infected plants in Brazil. Preliminary evaluation of the markers suggested substantial genetic variability among Brazilian L. theobromae populations. These markers have potential utility for evolutionary and epidemiologic studies of this fungus.