MOLECULAR DIAGNOSIS OF Guignardia citricarpa IN ASYMPTOMATIC SWEET ORANGE TISSUE

ABSTRACT 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.

The Effects of Postharvest Hot Water and Fungicide Treatments on Guignardia citricarpa Growth and the Development of Citrus Black Spot Symptoms on ‘Valencia’ Orange Fruit

Citrus black spot (CBS), caused by Guignardia citricarpa, is a fungal disease that was first described in Australia in the 1890s and has since been discovered in Southwest Florida in 2010. The current study evaluated the effects of hot water treatments on mycelial growth of G. citricarpa in vitro and also evaluated postharvest hot-water dips and fungicide treatments on CBS development on ‘Valencia’ oranges. In vitro exposure to 56 °C for 120 seconds, 59 °C for 60 seconds, or 62 °C for 30 seconds suppressed mycelial growth of all three G. citricarpa isolates by >30%. These treatments did not significantly reduce disease incidence or severity of CBS lesion development on whole ‘Valencia’ oranges from CBS-infected trees when the fruit already had visible CBS symptoms before treatment. On asymptomatic fruit, while the treatments did not significantly reduce the incidence of CBS lesion development, fruit dipped in 56 °C water for 120 seconds significantly reduced disease severity after 2 weeks of storage compared with the control. None of the treatments caused peel scalding or fruit quality deterioration. Postharvest application of azoxystrobin, imazalil, or thiabendazole significantly reduced CBS disease severity on fruit that were asymptomatic at harvest, but did not affect disease incidence. These fungicides were not effective on fruit harvested later in the season (April), possibly because most lesion expression had already occurred before harvest, with little left to develop after harvest. On fruit showing CBS symptoms at harvest, postharvest fungicide treatments did not significantly affect disease incidence or severity after storage. Heating the fungicide solutions did not significantly improve fungicide effectiveness. These results demonstrated that fungicide azoxystrobin, imazalil, or thiabendazole could reduce CBS severity, but not incidence, on orange fruit that are still asymptomatic at harvest.