Journal of Plant Pathology (2004) 86, p. 313 (Catara et al.)

V. Catara, G. Licciardello, V. Coco, V. Grimaldi and S. Grasso (2004)
Real-time PCR for the diagnosis of citrus mal secco disease
Journal of Plant Pathology 86 (4), 313-313
XI Meeting, Italian Society for Plant Pathology, Milan, 29/9 - 1/10, 2004 (poster)
Abstract: Real-time PCR was used to detect Phoma tracheiphila, the causal agent of mal secco disease, in citrus samples. Specific primers (GR70 and GL1) and a dual-labeled fluorogenic probe were designed. Reactions were performed with OmniMix HS lyophilised beads (TaKaRa Bio Inc). Both wood samples from naturally infected lemon and artificially inoculated leaves and stems from sour orange seedlings were analysed. DNAs were extracted with Puregene DNA isolation kit (Gentry System) and with a fast extraction method based on alkaline lysis. Standard curves showed a linear relation between log values of fungal genomic DNA and real-time PCR threshold cycles. The sensitivity of the real-time PCR assay, evaluated by testing P. tracheiphila DNA dilutions, was ten fold higher than that of the previous published PCR method performed on the same samples. The analysis of citrus tissues showed that the best results were obtained when woody samples were ground in liquid nitrogen and extracted with the DNA extraction kit, rather than with the NaOH extraction method. A preliminary enrichment step of the fungal mass in potato dextrose broth for 24 h at 24°C favoured the detection in samples hard to grind. A higher number of citrus samples was positive by real-time PCR than by the conventional PCR assay. In symptomless leaves of dour orange the fungus was detected after three days at the point of inoculation, and after one week along the midvein, more than one week before the appearance of the first symptom. The Smart-Cycler TD II System (Cepheid Europe, SA) characterised by rapid cycling times allowed detection of positive samples within 20 min.
Database assignments for author(s): Vittoria Catara, Virgilio Grimaldi

Research topic(s) for pests/diseases/weeds:
identification/taxonomy
surveys/sampling/distribution

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Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Plenodomus tracheiphilus	Citrus (genus)	Italy