Discerning the global phylogeographic distribution of Phyllosticta citricarpa by means of whole genome sequencing.

Phyllosticta citricarpa is a fungal pathogen causing citrus black spot (CBS). As a regulated pest in some countries, the presence of the pathogen limits the export of fruit and is therefore of agricultural and economic importance. In this study, we used high throughput sequencing data to infer the global phylogeographic distribution of this pathogen, including 71 isolates from eight countries, Argentina, Australia, Brazil, China, Cuba, Eswatini, South Africa and the United States of America. We assembled draft genomes and used a pairwise read mapping approach for the detection and enumeration of variants between isolates. We performed SSR marker discovery based on the assembled genome with the best assembly statistics, and generated genotype profiles for all isolates with 1987 SSR markers in silico. Furthermore, we identified 32,560 SNPs relative to a reference sequence followed by population genetic analyses based on the three datasets; pairwise variant counts, SSR genotypes and SNP genotypes. All three analysis approaches gave similar overall results. Possible pathways of dissemination among the populations from China, Australia, southern Africa and the Americas are postulated. The Chinese population is the most diverse, and is genetically the furthest removed from all other populations, and is therefore considered the closest to the origin of the pathogen. Isolates from Australia, Eswatini and the South African province Mpumalanga are closely associated and clustered together with those from Argentina and Brazil. The Eastern Cape, North West, and KwaZulu-Natal populations in South Africa grouped in another cluster, while isolates from Limpopo are distributed between the two aforementioned clusters. Southern African populations showed a close relationship to populations in North America, and could be a possible source of P. citricarpa populations that are now found in North America. This study represents the largest whole genome sequencing survey of P. citricarpa to date and provides a more comprehensive assessment of the population genetic diversity and connectivity of P. citricarpa from different geographic origins. This information could further assist in a better understanding of the epidemiology of the CBS pathogen, its long-distance dispersal and dissemination pathways, and can be used to refine phytosanitary regulations and management programmes for the disease.

Genetic Diversity and Population Structure of Phyllosticta citriasiana in China.

Phyllosticta citriasiana is the causal agent of citrus tan spot, an important pomelo disease in Asia. At present, there is little or no information on the epidemiology or population structure of P. citriasiana. By using simple sequence repeat markers, we analyzed 94 isolates from three pomelo production regions in southern and southeastern China. The analyses showed high genetic diversity in each of the three geographic populations. A STRUCTURE analysis revealed two genetic clusters among the 94 isolates; one geographic population was dominated by genotypes in one cluster, and the other two geographic populations were dominated by genotypes of the second cluster. P. citriasiana has a heterothallic mating system with two idiomorphs, MAT1-1 and MAT1-2. Analyses using mating type-specific primers revealed that both mating types were present in all three geographic populations, and in all three populations the mating type ratios were in equilibrium. Although the sexual stage of the fungus has not been discovered yet, analyses of allelic associations indicated evidence for sexual and asexual reproduction within and between populations. Despite the observed genetic differentiation between the three geographic populations, evidence for long-distance gene flow was found.

Spatial and Temporal Genetic Analyses of Phyllosticta citricarpa in Two Lemon Orchards in South Africa Reveal a Role of Asexual Reproduction Within Sexually Reproducing Populations.

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is a disease that affects citrus worldwide. In different regions of the world where both mating types occur, reports differ as to whether asexually produced pycnidiospores play an important role in the epidemiology of CBS and fruit infections. Therefore, we investigated the potential role of pycnidiospores in two lemon orchards in South Africa by using microsatellite-based analysis of fruit populations over time (two seasons) and space (distance). The two orchards were situated in the semiarid North West province (NW) and subtropical Mpumalanga province (MP). Each population contained both mating types in 1:1 ratios, and linkage disequilibrium analysis indicated a random mating population. A total of 109 and 94 multilocus genotypes (MLGs) were detected across the two seasons in the NW and MP orchards, respectively. Psex analyses indicated that most MLGs probably resulted from sexual reproduction, but there were six predominant MLGs in each orchard that were probably replicated via asexual reproduction. Each of the predominant MLGs was monomorphic for mating type. In the NW, five predominant and widespread MLGs caused 46 and 44% of the fruit infections in the two seasons, whereas in MP, three MLGs caused 34 and 48% of the infections. Asexual reproduction in both orchards was supported by low MLG evenness values in all populations. In both orchards, distance was not a reliable predictor of population genetic substructuring or season. Populations of P. citricarpa in the MP and NW orchards were significantly genetically differentiated from each other.