Endophytic and pathogenic <I>Phyllosticta</I> species, with reference to those associated with Citrus Black Spot

Abstract

We investigated the identity and genetic diversity of more than 100 isolates belonging to Phyllosticta (teleomorph Guignardia), with particular emphasis on Phyllosticta citricarpa and Guignardia mangiferae s.l. occurring on Citrus. Phyllosticta citricarpa is the causal agent of Citrus Black Spot and is subject to phytosanitary legislation in the EU. This species is frequently confused with a taxon generally referred to as G. mangiferae, the presumed teleomorph of P. capitalensis, which is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts. DNA sequence analysis of the nrDNA internal transcribed spacer region (ITS1, 5.8S nrDNA, ITS2) and partial translation elongation factor 1-alpha (TEF1), actin and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes resolved nine clades correlating to seven known, and two apparently undescribed species. Phyllosticta citribraziliensis is newly described as an endophytic species occurring on Citrus in Brazil. An epitype is designated for P. citricarpa from material newly collected in Australia, which is distinct from P. citriasiana, presently only known on C. maxima from Asia. Phyllosticta bifrenariae is newly described for a species causing leaf and bulb spots on Bifrenaria harrisoniae (Orchidaceae) in Brazil. It is morphologically distinct from P. capitalensis, which was originally described from Stanhopea (Orchidaceae) in Brazil; an epitype is designated here. Guignardia mangiferae, which was originally described from Mangifera indica (Anacardiaceae) in India, is distinguished from the non-pathogenic endophyte, P. brazilianiae sp. nov., which is common on M. indica in Brazil. Furthermore, a combined phylogenetic tree revealed the P. capitalensis s.l. clade to be genetically distinct from the reference isolate of G. mangiferae. Several names are available for this clade, the oldest being P. capitalensis. These results suggest that endophytic, non-pathogenic isolates occurring on a wide host range would be more correctly referred to as P. capitalensis. However, more genes need to be analysed to fully resolve the morphological variation still observed within this clade.

Genetic variation among Phyllosticta strains isolated from citrus in Florida that are pathogenic or nonpathogenic to citrus

“…Uniformity in the ITS region was also found for many isolates of P. capitalensis from Japan (Okane et al, 2003) or P. capitalensis from Brazil (Rodrigues et al, 2004). Others found some variation in the ITS region of isolates of P. capitalensis (Wickert et al, 2012b) but high similarity in ITS sequences of P. citricarpa isolates from Brazil (Wickert et al, 2012a).When multi-locus analyses were carried out on a large number of Phyllosticta species more variation was detected (Glienke et al, 2011;Wang et al, 2012;Wulandariet al, 2009). Using three DNA regions, the rDNA internal transcribed spacer (ITS) region, the partial translation elongation factor 1-alpha (TEF1), and the actin gene (ACT), three Phyllosticta clades associated with citrus in Thailand were distinguished, namely P. capitalensis, P. citricarpa and Phyllosticta citriasiana Wulandari, Crous & Gruyter (Wulandari et al, 2009).…”

“…They were also able to distinguish two groups of P. citricarpa, one on mandarin and one on orange and lemon. When the number of sequenced DNA regions was increased to four, including the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene in addition to the three other regions used by Wulandari et al (2009) and Wang et al (2012), nine clades were distinguished (Glienke et al, 2011). Five of these contained isolates associated with citrus: P. citricarpa, P. citriasiana, Phyllosticta citribraziliensis C. Glienke & Crous, Phyllosticta brazilianiae D. Stringari, C. Glienke & Crous, and P. capitalensis.…”

“…P. capitalensis has been found on CBS affected citrus trees as well as healthy and asymptomatic trees, and has a wider host range than P. citricarpa extending beyond the Rutaceae, commonly as an endophyte (Baayen et al, 2002;Bezerraet al, 2012;Everett & Rees-George, 2006;Glienke et al, 2011;Johnston, 1998;Okane et al, 2001;Rakotoniriana et al, 2008;Rodrigues & Samuels, 1999;Rodrigues et al, 2004;Yuan et al, 2009). Guignardia mangiferae sensu stricto (not necessarily P. capitalensis) is a pathogen of mango (Mangifera indica L), but is nonpathogenic to other species (Baldassari et al, 2008;Glienke et al, 2011). Conversely, although P. citricarpa can be isolated from citrus fruit with CBS lesions, it has also been found in asymptomatic citrus tissues (Baldassari et al, 2008), but not in plant species outside of the Rutaceae.…”

“…Phyllostica citricarpa and P. capitalensis can be distinguished morphologically and physiologically based on colony type on oatmeal agar (yellow halo present around P. citricarpa but absent around colonies of P. capitalensis), growth rate (generally faster for P. capitalensis), conidia size (larger for P. capitalensis), and hydrolytic enzyme production (higher production by P. citricarpa) (Baayen et al, 2002;Glienke et al, 2011;Romão et al, 2011). In addition, the two groups were clearly distinct based on sequences of the rDNA internal transcribed spacer (ITS) region, while the ITS sequences of the isolates used were quite uniform within each group, identified as either P. citricarpa or P. capitalensis (Romão et al, 2011).…”

“…P. citriasiana is a pathogen causing tan spot on pummelo (Citrus maxima Merr. ), P. citrichinaensis is a very weak pathogen on various citrus species, P. citribraziliensis and P. brazilianae are nonpathogenic endophytes in citrus and mango, respectively, and P. capitalensis is the broad host range endophyte commonly referred to as G. mangiferae (Glienke et al, 2011). In addition to phylogenetic analyses based on DNA sequences, grouping of pathogenic and nonpathogenic Phyllosticta isolates from citrus was obtained using AFLP (Baldassari et al, 2008) and RAPD markers (Stringari et al, 2009).…”