Sampling and analysis of asymptomatic Citrus fruits and leaf litter to detect the infection of Phyllosticta citricarpa


Inspection of Citrus fruits at the border for the detection of citrus black spot (CBS) is mostly based on visual examination followed by laboratory testing of fruits exhibiting suspicious symptoms. However, due to the long incubation period of Phyllosticta citricarpa, CBS symptoms on fruits are visible only at maturity, several months after infection. Furthermore, symptoms are usually non-specific and could be confused with other diseases such as (anthracnosis and alternariosis). Phyllosticta citricarpa has been reported in leaf litter in some EU citrus-growing countries although CBS symptoms were not observed on tree canopy. Recently, the pathogen has been reported for the first time in Tunisia. As a result, an EFSA project entitled “Reduce risk assessment uncertainty: suitability of Mediterranean citrus production areas for P. citricarpa (GP/EFSA/ALPHA/2019/04) was established, aiming at reducing the uncertainties related to the risk of introduction of P. citricarpa in the Mediterranean citrus-growing areas, by improving the knowledge on CBS epidemiology and climate suitability in the Mediterranean Basin.

Although surveys have been conducted in the EU Member States, more intensive surveys must be conducted by NPPOs in EPPO citrus-producing countries to evaluate the pest status in their territory. EFSA has developed a survey card to provide guidance on how to conduct the surveys to determine the pest status in EU citrus-producing Member States and raised the need to include asymptomatic fruits in the survey. Moreover, a new cryptic Phyllosticta species, namely P. paracitricarpa, detected in lemon leaf litter in Greece, has been reported to cause “atypical” CBS symptoms on citrus fruits following artificial inoculation. So far, this species has not been included in the EU quarantine list of harmful organisms.

In Argentina and Brazil (countries where CBS is present) exporters are performing an ethephon treatment to enhance symptom expression on fruits in order to avoid that asymptomatic CBS-infected fruits will be rejected upon arrival in Europe. Such treatment could be envisaged to be included as methodology in the CBS survey plan for fruits harvested in European citrus orchards. However, concerns have been raised on the efficacy of the proposed treatment and if it could be practically feasible to be extended to imported fruits.

In the framework of this Euphresco project, an evaluation of the efficacy of the ethephon treatment has been performed before considering including this methodology as an obligatory step in survey plans. 

Molecular tests, in particular real-time PCR, are already available to detect and identify P. citricarpa (Bonants et al., Eur. J. Plant Pathol. 109, 503–513, 2003; Meyer et al., Plant Dis. 90, 97–101, 2006; Peres et al., Plant Dis. 91, 525–531, 2007; van Gent-Pelzer et al., J. Phytopathol. 155, 357– 363, 2007) and some others have recently been published (Zajc et al., Plant Pathol. 72, 548–563, 2023). However, those tests have not been validated to detect the fungus on asymptomatic citrus tissues. In addition, a critical point is that, so far, these tests do not allow the distinction between P. citricarpa and P. paracitricarpa, thus, more specific detection assays need to be developed and validated.

The project included three main activities: 

Evaluation of methods, such as the ethephon treatment or other methods, that stimulate the early development of CBS symptoms and the production of P. citricarpa (and P. paracitricarpa) pycnidia on infected citrus fruit and/or leaves; the methods have been tested and validated on citrus matter in CBS-positive countries and imported fruit; Evaluation of the currently available DNA extraction methods from citrus matter and of the newly developed test to detect and identify P. citricarpa on asymptomatic infected citrus tissues and to discriminate it from the close-related P. paracitricarpa; the project included also the validation of the above-mentioned tests through a test performance study (TPS);Development of a sampling strategy to be used for the detection of P. citricarpa on asymptomatic infected citrus tissues both in the field and at the entry points.



Related resouces


  • Citrus sp.
  • Phyllosticta citricarpa


File Size
external link 125,67kB