Analysis of the fungal diversity in citrus leaves with greasy spot disease symptoms

Citrus greasy spot (CGS) is a disease of citrus with worldwide distribution and recent surveys have revealed a high level of incidence and severity of symptoms of the disease in Sicily, southern Italy. Although Mycosphaerella citri (anamorph Zasmidium citri-griseum) and other related species are generally considered as causal agents, the etiology of CGS is still unclear. Here, we report the use of an amplicon metagenomic approach to investigate the fungal communities on citrus leaves symptomatic or asymptomatic for CGS from an orchard in Sicily showing typical CGS symptoms. A total of 35,537 high-quality chimeric free reads were obtained and assigned to 176 operational taxonomic units (OTUs), clustered at 99 % similarity threshold. Data revealed a dominating presence of the phylum Ascomycota (92.6 %) over other fungal phyla. No significant difference was observed between symptomatic and asymptomatic leaves according to both alpha and beta diversity analyses. The family Mycosphaerellaceae was the most abundant and was represented by the genera Ramularia, Mycosphaerella, and Septoria with 44.8, 2.4, and 1.7 % of the total detected sequences, respectively. However, none of the species currently reported as causal agents of CGS was detected in the present study. The most abundant sequence type (ST) was associated to Ramularia brunnea, a species originally described to cause leaf spot in a perennial herbaceous plant of the family Asteraceae. Results exclude that CGS symptoms observed in Sicily are caused by Z. citri-griseum and, moreover, they indicate that a considerable part of the fungal diversity in citrus leaves is still unknown.