Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis)

Abstract

The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment. Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (<2-mm diameter) without pycnidia. These observations support the historic distinction between slowly growing pathogenic isolates and morphologically similar fast-growing, nonpathogenic isolates of G. citricarpa. The latter proved to belong to G. mangiferae (P. capitalensis), a ubiquitous endophyte of woody plants with numerous probable synonyms including G. endophyllicola, G. psidii, P. anacardiacearum, and P. theacearum. G. mangiferae occurs in the European Union and the United States on many host species including citrus, and does not cause symptoms of citrus black spot, justifying its exclusion from quarantine measures.

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

“…The fragments were then placed on potato dextrose agar (PDA) and incubated at 25ºC until colonies were found that were characteristic for Phyllosticta species. Subcultures were transferred to oatmeal agar (OA) and colonies were checked for a yellow halo to distinguish P. citricarpa from P. capitalensis (Baayen et al, 2002). Morphology and size of conidia were checked microscopically (Wikee et al, 2011).…”

“…However, strains that have been described as G. mangiferae do not always have P. capitalensis as anamorph (Glienke et al, 2011). 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. Phyllosticta capitalensis was sometimes misidentified as P. citricarpa before Baayen et al (2002) classified it as P. capitalensis (Everett &Rees-George, 2006;Glienke-Blanco et al, 2002).…”

“…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).…”

“…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). The variation in these markers was greater among nonpathogenic isolates than among pathogenic isolates (Baayen et al, 2002;Baldassari et al, 2008;Glienke et al, 2011;Stringari et al, 2009).…”