MOLECULAR DIAGNOSIS OF Guignardia citricarpa IN ASYMPTOMATIC SWEET ORANGE TISSUE

Abstract

RESUMO -A pinta preta ou mancha preta dos citros, causada pelo fungo Guignardia citricarpa, é considerada uma doença quarentenária, que impõe restrições ao transporte de frutas frescas para países da União Europeia. A ocorrência de infecções latentes e o tempo para o diagnóstico por métodos convencionais levam à necessidade de validar protocolos moleculares rápidos, eficientes e reprodutíveis para detecção do patógeno em tecidos assintomáticos. Assim, este trabalho visou detectar G. citricarpa em tecidos de frutos sintomáticos e em folhas assintomáticas de laranja Pêra por PCR convencional e por PCR em tempo real.A especificidade e o limite de detecção foram avaliados em amostras de tecidos de lesões em frutos e em folhas assintomáticas. Em folhas assintomáticas a presença do fungo foi detectada em baixas concentrações, nessas condições, a PCR em tempo real demonstrou ser viável, reprodutível e altamente sensível para a detecção do patógeno. Termos para indexação: Citricultura, diagnose molecular, Phyllosticta citricarpa. DETECÇÃO MOLECULAR DE Guignardia citricarpa EM TECIDOS ASSINTOMÁTICOS DE LARANJA-PERAABSTRACT -Citrus black spot, a fungal disease caused by the quarantine fungus Guignardia citricarpa, restricts the exportation of fresh fruit to countries in the European Union. The occurrence of latent infections and the time required for diagnosis using conventional methods have brought about the need to validate fast, efficient and reproducible molecular techniques to detect the pathogen in asymptomatic tissue. As such, this study aims to detect G. citricarpa in the symptomatic fruit and asymptomatic leaf tissue of sweet oranges by conventional and real-time polymerase chain reaction (PCR). Specificity and limit of detection (LOD) were assessed in tissue samples of fruit lesions and asymptomatic leaves. Low concentrations of the fungus were found in asymptomatic leaves. Under these conditions, real-time PCR proved to be viable, reproducible and highly sensitive to detection of the pathogen.

Detection of sugarcane leaf scald from latent infections

“…This founded bacterial population is considered very critical population densities for molecular detection by PCR. For G. citricarpa conventional PCR was suitable for detecting DNA with 90% repeatability of detection at a concentration of 10 ng and 70% at a minimum of 0.01 ng according to the work of Faganello et al (2017).…”

Isolation, Identification and Characterization of Phyllosticta citricarpa Causing Citrus Black Spot Disease in Benin

“…These results confirm those of Van Gent-Pelzer et al [21] who reported that realtime PCR with primers GcF1 and GcR1, is a rapid method for the specific detection of P. citricarpa generating much smaller amplicons of 69 bp. Faganello et al [30] reported that real-time PCR is a robust and more sensitive method than conventional PCR and allows detection of P. citricarpa in asymptomatic plant tissues. According to Schirmacher et al [31], this method can detect and distinguish P. citricarpa from other pathogens without the need for sequencing.…”