Characterization of Citrus-Associated Alternaria Species in Mediterranean Areas

Abstract

Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. Recently, outbreaks in Mediterranean areas related to susceptible cultivars, refocused attention on the disease. Twenty representatives were selected from a collection of 180 isolates of Alternaria spp. from citrus leaves and fruit. They were characterized along with reference strains of Alternaria spp. Micro- and macroscopic characteristics separated most Alternaria isolates into six morphotypes referable to A. alternata (5) and A. arborescens (1). Phylogenetic analyses, based on endopolygalacturonase (endopg) and internal transcribed spacer (ITS), confirmed this finding. Moreover, a five-gene phylogeny including two anonymous genomics regions (OPA 1–3 and OPA 2–1), and the beta-tubulin gene (ß-tub), produced a further clustering of A. alternata into three clades. This analysis suggested the existence of intra-species molecular variability. Investigated isolates showed different levels of virulence on leaves and fruit. In particular, the pathogenicity on fruit seemed to be correlated with the tissue of isolation and the clade. The toxigenic behavior of Alternaria isolates was also investigated, with tenuazonic acid (TeA) being the most abundant mycotoxin (0.2–20 mg/L). Isolates also synthesized the mycotoxins alternariol (AOH), its derivate alternariol monomethyl ether (AME), and altenuene (ALT), although to a lesser extent. AME production significantly varied among the six morphotypes. The expression of pksJ/pksH, biosynthetic genes of AOH/AME, was not correlated with actual toxin production, but it was significantly different between the two genotypes and among the four clades. Finally, ten isolates proved to express the biosynthetic genes of ACTT1 phytotoxin, and thus to be included in the Alternaria pathotype tangerine. A significant correlation between pathogenicity on leaves and ACTT1 gene expression was recorded. The latter was significantly dependent on geographical origin. The widespread occurrence of Alternaria spp. on citrus fruit and their ability to produce mycotoxins might represent a serious concern for producers and consumers.

A new high‐resolution melting assay for genotyping Alternaria species causing citrus brown spot

“…were not taken into consideration. Moreover, the variability of endopolygalacturonase gene has been reported to not be sufficient to discriminate accurately the different morphotypes . Indeed, authors identified a lower number of discriminating SNPs as compared to the present investigation.…”

“…Concerning the first one, several genetic regions with higher sequence variation, compared to more classical barcoding regions such as ITS or endopolygalacturonase, were identified in Alternaria genus, and used to estimate phylogenies among small‐spored species . In particular, the SCAR marker OPA1–3 proved to be highly efficient in generating discriminant sequences . In the present study, the alignment of the OPA1–3 sequences, belonging to Alternaria reference strains of the species/morphotypes, more commonly reported on citrus, showed evidence of a high degree of variability.…”

“…Thus, the HRM assay herein was extended to the whole collection of 180 Alternaria isolates from citrus fruit and leaves, allowing separation of isolates between a species ( A. alternata ) and a species complex ( A. arborescens ). Most of the isolates were identified as A. alternata – a finding which is not surprising since it is the species most frequently responsible for brown spot in citrus . Moreover, within the species alternata , two clusters were identified, corresponding to the morphotypes alternata and limoniasperae , pathogenic on tangerine and hybrids and producers of the host‐specific ACT‐toxin .…”

“…The OPA1–3 region of the six CBS type strains was amplified and purified as reported by Garganese et al . and sequenced by Macrogen Europe (Amsterdam, the Netherlands).…”

“…Based on morphological and phylogenetic studies, the cosmopolitan fungal genus Alternaria is divided into 26 sections. Among them, the Alternaria section Alternaria contains most of the small‐spored species with concatenated conidia, including important plant and human pathogens . Recently, species within this section have been redefined with the aid of advanced molecular techniques, so that currently the section consists of 11 species, of which A. alternata is the most frequently encountered, and one species complex ( A. arborescens ) .…”