Presence of non-regulated Elsinoë species on citrus fruits and their impact on regulatory plant health diagnostics

Abstract Citrus fruits such as oranges, lemons, limes, grapefruits, and tangerines are economically significant crops worldwide. The EU is a net importer of citrus fruits to meet consumer demand, with the Netherlands functioning as main distribution hub. Citrus fruit imports may pose a risk to EU production, particularly through the introduction of regulated plant pathogens. Considering that only the three regulated Elsinoë species were known to occur on citrus fruits, the diagnosis of the regulated Elsinoë species in the Netherlands was based on a genus-level real-time PCR detection combined with symptom analysis, host plant species, and origin of the fruits. Import samples originating from countries where none of the regulated species are known to occur, as well as samples exhibiting late Elsinoë spp. Cq values, raised questions regarding the presumed host specificity of Elsinoë species. Metabarcoding was performed in an attempt to determine presence of the (regulated) Elsinoë species in symptomatic citrus samples from Asia, Africa, Europe and South America, representing nine countries and five hosts. Surprisingly, the ITS1 barcode revealed the presence of non-regulated Elsinoë species on symptomatic fruits. In some consignments presence of several different species was observed. Symptoms typically associated with E. fawcettii and E. australis were observed on fruits in which only non-regulated Elsinoë spp. were detected. This further underlines the limitations to distinguish the regulated Elsinoë species from other organisms based on symptomology. The occurrence of non-regulated Elsinoë species on citrus fruit prevents accurate diagnosis of regulated pests following a positive genus-level test. Culturing and identification of the non-regulated species on citrus fruits is needed. Additionally, broader validation of alternative diagnostic tests is required to demonstrate their ability to distinguish regulated pathogens from non-regulated Elsinoë species on citrus fruits.