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Worldwide Phylogeography of the Citrus Brown Spot Pathogen, Alternaria alternata

First author: Department of Plant Pathology, Washington State University, Pullman 99163; second and fourth authors: University of Florida, Citrus Research and Education Center, 700 Experiment Station Rd., Lake Alfred 33850; and third author: Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan

Sixty-five isolates of Alternaria alternata were sampled from brown spot lesions on tangerines and mandarins (Citrus reticulata) and tangerine × grapefruit (C. reticulata × C. paradisi) hybrids in the United States, Colombia, Australia, Turkey, South Africa, and Israel to investigate the worldwide phylogeography of the fungus. Genetic variation was scored at 15 putative random amplified polymorphic DNA (RAPD) loci and 465 bp of an endo-polygalacturonase (endo-PG) gene was sequenced for each isolate. Cluster analysis of RAPD genotypes revealed significant differentiation between United State and Colombia isolates and Turkey, South Africa, Israel, and Australia isolates. Sequencing of endo-PG revealed 21 variable sites when the outgroup A. gaisen (AK-toxin-producing pathogen of Japanese pear) was included and 13 variable sites among the sampled isolates. Nucleotide substitutions at 10 of 13 variable sites represented silent mutations when endo-PG was translated in frame. Eight distinct endo-PG haplotypes were found among the sampled isolates and estimation of a phylogeny with endo-PG sequence data revealed three clades, each with strong bootstrap support. The most basal clade (clade 1) was inferred based on its similarity to the outgroup A. gaisen and consisted exclusively of pathogenic isolates from the United States and Colombia. Clade 2 consisted of pathogenic and nonpathogenic isolates from the United States, Australia, South Africa, and Israel and clade 3 contained pathogenic and nonpathogenic isolates from Australia, South Africa, Israel, and Turkey. Quantitative estimates of virulence (disease incidence) were obtained for isolates from the United States, Colombia, South Africa, Israel, and Turkey by spray inoculating detached citrus leaves and counting the number of lesions 24 h after inoculation. Large differences in virulence were detected among isolates within each location and isolates from the United States were significantly more virulent than isolates from other locations. Several isolates from Colombia, South Africa, Israel, and Turkey had low virulence and 8% of all isolates were nonpathogenic. All but one of the nonpathogenic isolates were found in clade 2 of the endo-PG phylogeny, which also included the most highly virulent isolates sampled.

Additional keywords: Alternaria citri, Citrus, phylogenetic, polymerase chain reaction.