AFLP markers differentiate isolates of Colletotrichum gossypii from C. gossypii var. cephalosporioides

Abstract

Fungal diseases in cotton (Gossypium hirsutum), such as anthracnose caused by Colletotrichum gossypii and ramulose caused by C. gossypii var. cephalosporioides, are responsible for large yield losses. These pathogens are seed borne and morphologically similar although they induce different symptoms, which can lead to misdiagnosis using the blotter testing method. The present study was carried out to assess the viability of using Amplified Fragment Length Polymorphism (AFLP) markers to differentiate these pathogens. Five isolates, for each pathogen, were classified according to pathogenicity on cotton plants, and mycelial growth morphology. Conidial suspensions were sprayed on 30-day-old cotton plants and the symptoms assessed ten and 40 days after inoculation. For growth morphology 200 cottonseeds were inoculated with seven-day-old pure cultures, and the mycelial traits observed under a stereoscopic microscope seven days after inoculation. The DNA for AFLP analysis was obtained from seven-day-old fungal mycelia grown in liquid medium, using the Dneasy Qiagen protocol. Using the AFLP technique 318 polymorphic bands were selected to estimate similarities using Dice's Coefficient. The results clearly distinguished between ramulose and anthracnose isolates, which agreed with morphological and pathogenicity testing.Additional keywords: molecular markers, anthracnose, ramulose, cotton diseases, seed borne pathogens. RESUMOMarcadores AFLP diferenciam Colletotrichum gossypii de C. gossypii var. cephalosporioides Doenças fúngicas em algodoeiro (Gossypium hirsutum), tais como antracnose causada por Colletotrichum gossypii e ramulose causada por C. gossypii var. cephalosporioides, levam a grandes perdas em produtividade. Estes microrganismos são patógenos veiculados por sementes e morfologicamente similares apesar deles induzirem sintomas diferentes, que podem levar a um erro no diagnóstico usando o teste de sanidade com sementes em rolo de papel. O presente estudo foi realizado para acessar a viabilidade do uso de marcadores Amplified Fragment Length Polymorphism (AFLP) para diferenciar estes patógenos. Cinco isolados, para cada patógeno, foram classificados de acordo com a patogenicidade e morfologia de crescimento micelial. As suspensões de conídios foram pulverizadas em plantas de algodoeiro com 30 dias e os sintomas avaliados aos dez e 40 dias após a inoculação. Para a morfologia de crescimento micelial, 200 sementes foram inoculadas com culturas puras com sete dias, e as características miceliais observadas sob microscópio esterioscópico sete dias após a inoculação. O DNA para a análise de AFLP foi obtido de micélio fúngico com sete dias, crescido em meio líquido, usando o protocolo Dneasy Qiagen. A técnica AFLP produziu 318 bandas polimórficas que foram selecionadas para calcular as similaridades usando o Coeficiente de Dice. Os resultados claramente distinguiram os isolados causadores de ramulose dos de antracnose, os quais concordam, com dados morfológicos e de patogenicidade.

The etiological agent of cotton ramulosis represents a single phylogenetic lineage within the Colletotrichum gloeosporioides species complex

“…In Brazil, ramulosis and anthracnose appear to be caused by two distinct pathogens that belong to the Colletotrichum gloeosporioides species complex (Bailey et al, 1996;Silva-Mann et al, 2005). This complex is composed of species that share common morphological traits and form a strongly supported monophyletic group, as determined by independent studies (Cai et al, 2011;Cannon et al, 2012;Weir et al, 2012).…”

“…Ramulosis is one of the most important diseases of cotton (Gossypium hirsutum) in Brazil, and occurs in all major producing areas of the country (Silva-Mann et al, 2005). The disease is also known in Paraguay and Venezuela (Malaguti, 1955;Mathieson & Mangano, 1985), and may be present elsewhere in South America.…”

“…In 1946, Viegas validated the name of this variety, providing a Latin description and denoting a type (Viegas, 1946). Ever since, this name has been widely applied to the pathogen causing ramulosis of cotton in Brazil (Kirkpatrick & Rothrock, 2001;Monteiro et al, 2009;Silva-Mann et al, 2005).…”

“…In Brazil, the anthracnose pathogen is mainly regarded as a damping-off agent and a minor foliar pathogen in the end of the vegetative cycle (Silva-Mann et al, 2005). The ramulosis and anthracnose pathogens are supposed to be seed transmitted (Lima et al, 1985).…”

Variabilidade genética entre isolados de Colletotrichum gossypii do algodoeiro

“…Num trabalho preliminar, Mehta et al (9), utilizando os critérios morfológicos sugeridos por Pizzinatto & Tanaka (12) e Tanaka et al (16), e também utilizando a técnica molecular de ERIC-e REP-PCR, não distinguiram os isolados de CG e CGC. Posteriormente, 15), estudaram a variabilidade genética entre três isolados de CG e oito isolados de CGC utilizando a técnica de RAPD e AFLP e indicaram a potencialidade desses métodos para a distinção entre esses patógenos. Entretanto, devido à existência de variação entre os isolados de CG e CGC, a utilização de um número maior de isolados se faz necessária.…”