First report of<i>Phyllosticta citricarpa</i>and description of two new species,<i>P. paracapitalensis</i>and<i>P. paracitricarpa</i>, from citrus in Europe

Abstract

The genus <i>Phyllosticta</i> occurs worldwide, and contains numerous plant pathogenic, endophytic and saprobic species. <i>Phyllosticta citricarpa</i> is the causal agent of Citrus Black Spot disease (CBS), affecting fruits and leaves of several citrus hosts (<i>Rutaceae</i>), and can also be isolated from asymptomatic citrus tissues. Citrus Black Spot occurs in citrus-growing regions with warm summer rainfall climates, but is absent in countries of the European Union (EU). <i>Phyllosticta capitalensis</i> is morphologically similar to <i>P. citricarpa</i>, but is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts, and is known to occur in Europe. To determine which <i>Phyllosticta</i> spp. occur within citrus growing regions of EU countries, several surveys were conducted (2015-2017) in the major citrus production areas of Greece, Italy, Malta, Portugal and Spain to collect both living plant material and leaf litter in commercial nurseries, orchards, gardens, backyards and plant collections. A total of 64 <i>Phyllosticta</i> isolates were obtained from citrus in Europe, of which 52 were included in a multi-locus (ITS, <i>actA</i>, <i>tef1</i>, <i>gapdh</i>, LSU and <i>rpb2</i> genes) DNA dataset. Two isolates from Florida (USA), three isolates from China, and several reference strains from Australia, South Africa and South America were included in the overall 99 isolate dataset. Based on the data obtained, two known species were identified, namely <i>P. capitalensis</i> (from asymptomatic living leaves of <i>Citrus</i> spp.) in Greece, Italy, Malta, Portugal and Spain, and <i>P. citricarpa</i> (from leaf litter of <i>C</i>. <i>sinensis</i> and <i>C</i>. <i>limon</i>) in Italy, Malta and Portugal. Moreover, two new species were described, namely <i>P. paracapitalensis</i> (from asymptomatic living leaves of <i>Citrus</i> spp.) in Italy and Spain, and <i>P. paracitricarpa</i> (from leaf litter of <i>C</i>. <i>limon</i>) in Greece. On a genotypic level, isolates of <i>P</i>. <i>citricarpa</i> populations from Italy and Malta (MAT1-2-1) represented a single clone, and those from Portugal (MAT1-1-1) another. Isolates of <i>P. citricarpa</i> and <i>P. paracitricarpa</i> were able to induce atypical lesions (necrosis) in artificially inoculated mature sweet orange fruit, while <i>P. capitalensis</i> and <i>P. paracapitalensis</i> induced no lesions. The <i>Phyllosticta</i> species recovered were not found to be widespread, and were not associated with disease symptoms, indicating that the fungi persisted over time, but did not cause disease.