Enemy or ally: a genomic approach to elucidate the lifestyle of Phyllosticta citrichinaensis

Abstract

Members of the fungal genus Phyllosticta can colonize a variety of plant hosts, including several Citrus species such as Citrus sinensis (orange), Citrus limon (lemon), and Citrus maxima (pomelo). Some Phyllosticta species have the capacity to cause disease, such as Citrus Black Spot, while others have only been observed as endophytes. Thus far, genomic differences underlying lifestyle adaptations of Phyllosticta species have not yet been studied. Furthermore, the lifestyle of Phyllosticta citrichinaensis is ambiguous, as it has been described as a weak pathogen but Koch's postulates may not have been established and the presence of this species was never reported to cause any crop or economic losses. Here, we examined the genomic differences between pathogenic and endophytic Phyllosticta spp. colonizing Citrus and specifically aimed to elucidate the lifestyle of Phyllosticta citrichinaensis. We found several genomic differences between species of different lifestyles, including groups of genes that were only present in pathogens or endophytes. We also observed that species, based on their carbohydrate active enzymes, group independent of their phylogenetic association, and this clustering correlated with trophy prediction. Phyllosticta citrichinaensis shows an intermediate lifestyle, sharing genomic and phenotypic attributes of both pathogens and endophytes. We thus present the first genomic comparison of multiple citrus-colonizing pathogens and endophytes of the genus Phyllosticta, and therefore provide the basis for further comparative studies into the lifestyle adaptations within this genus.

Keywords: CAZymes; citrus; endophyte; fungal plant pathogens; genomics; lifestyle adaptations; pathogen.

Fig. 1.

Phyllosticta genomes are of high quality. The phylogenetic relationship of more than 100 fungal species (116 genomes) is shown. The phylogenetic tree was generated using OrthoFinder, and sub-trees were collapsed manually to enhance readability. Gray box indicates species outside the Dothideomycetes, red rectangle indicates the genus Phyllosticta. Red orbs = Phyllosticta pathogens, green orbs = Phyllosticta endophytes, blue orbs = P. citrichinaensis.

Fig. 2.

Phyllosticta citrichinaensis shares more unique OGs with endophytes than with pathogens, but pathogens share more OGs with each other. Yellow rectangles = P. citrichinaensis, red rectangles = pathogens, green rectangles = endophytes. Figure generated using UpSetR. Only the largest 35 groups are shown.

Fig. 3.

Species cluster independent of their taxonomic relationship based on presence effector proteins. a) The total number of proteins, b) the percentage of proteins that is predicted to be secreted, and c) the percentage of secreted proteins that is predicted to be an effector, in species of different lifestyles (as predicted by CATAstrophy, see Fig. 5). d) Clustered heatmap of the number of genes in OGs that were predicted to contain effectors. Lifestyles as predicted by CATAstrophy (see Fig. 5). Phyllosticta species are highlighted with green rectangle, Phyllosticta-unique OGs are highlighted in red rectangle.

Fig. 4.

Phyllosticta citrichinaensis clusters with pathogens based on growth on 35 carbon sources, but behaves like an endophyte in the presence of sugar beet pulp. a) Images of Phyllosticta species growing on a selection of different carbon sources. b) Clustering of Phyllosticta species based on their growth on different carbon sources. All species were grown on 35 different carbon sources, colony diameters were measured, and images takes on all sources when the biggest colony of a species reached the edge of its plate. All species grew fastest on wheat bran.

Fig. 5.

Separation of species into different trophy classes based on presence of CAZyme genes in their genomes. a) PCA plot of PC1 vs PC2. PC1 separates species of different trophy classes, PC2 separates species on phylogeny. The CATAstrophy dataset published by Hane et al. (2020) is shown in light gray for comparison. b) Number of CAZyme genes in each CAZy family per CATAstrophy class. c) Clustered heatmap of the number of CAZyme-gene-containing OGs per species. GH, glycosyl hydrolase; AA, auxiliary activity; CE, carbohydrate esterase; GT, glycosyl transferase; CBM, carbohydrate binding module; PL, pectin lyase; Po, polymertroph; Me, mesotroph; Sa, saprotroph; Mo, monomertroph; Va, vasculartroph.