Identification of Alternaria Species Causing Heart Rot of Pomegranates in California

Alternaria heart rot of pomegranate fruit is generally considered a minor disease. However, the current listing of the causal pathogen in California as "Alternaria sp." or as "A. alternata and other Alternaria spp." restricts trade of the crop to some major export markets where quarantines are in place in an effort to prevent the spread of unknown or undescribed pathogens. Thus, species identification of the pathogen is critical in determining whether infected fruit should be regulated by quarantines. In this study, 86 isolates of Alternaria were collected from pomegranate fruit with Alternaria heart rot symptoms from major production areas in California. An unweighted pair group method with arithmetic means analysis based on amplified fragment length polymorphisms revealed two main clusters, each with a high degree of variability. One of the clusters contained 24 isolates from pomegranate and one reference isolate of Alternaria arborescens. Reference isolates of A. alternata and A. tenuissima were found among pomegranate isolates in the other cluster, and these two species could not be separated. In maximum-parsimony analysis of ribosomal DNA internal transcribed spacer sequence data, representative pomegranate isolates all clustered with reference sequences of species in section Alternaria. Single-nucleotide differences separated A. arborescens, A. gaisen, and two pomegranate isolates from the majority of the remaining isolates. Sequence comparisons of gapdh and OPA10-2 loci indicated that none of the pomegranate isolates were identical to A. gaisen, which is a quarantine pathogen. Due to high genetic similarity and variability of morphological characteristics, revisions of the taxonomy of small-spored Alternaria spp. have been proposed by others. Based on this recent taxonomic work and work herein by us, pomegranate isolates from California can be assigned to A. alternata and A. arborescens. Reassessment of export restrictions for California pomegranate due to the previous pathogen classification is warranted. Fruit injection inoculations with conidia of 12 representative isolates 3 months before harvest caused typical symptoms of Alternaria heart rot, whereas flower inoculations did not result in fruit disease.