First Report of Neofusicoccum parvum Causing Shoot Blight and Stem Cankers on Pomegranate in Florida

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Abstract
A survey on prevalence of pomegranate (Punica granatum L.) diseases in Central Florida was conducted during the spring and summer of 2014 and 2015. Approximately 30% of the trees had blighted shoots and 10% had stem canker leading to death of entire tree. Symptomatic tissues were collected, surface-disinfected with 1% chlorine for 1 min, and placed on V8 juice agar amended with rifampicin (0.01 mg L-1) and ampicillin (0.25 mg L-1). After 3 days of incubation at 21o C and 12 hrs continuous light, fast-growing white colonies with abundant aerial mycelia were observed. The reverse sides of the plates were initially white but turned dark to olive green after 1 week of incubation. Observed conidiomata were globose and lined with conidiogenous cells. Conidia were hyaline, unicellular, ellipsoidal with flat base and round apex, and ranged from 10 to 14 x 3 to 5 µm (average of 50 conidia, 12 x 4 µm). The causal pathogen was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (Crous et al. 2006; Palavouzis et al. 2015) based on morphological characteristics. For further confirmation, the rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF-1α) gene, and β-tubulin gene were amplified using the respective primer pairs ITS1 and ITS4, EF-AF and EF-BR, and T1 and T2, and subjected to Sanger sequencing. The sequence data were deposited in the gene bank (Accession Nos. KU530156, KU530155, and KU530157 for ITS, EF-1α, and β-tubulin, respectively). A BLAST analysis with corresponding GenBank sequences found that the ITS, EF-1α and β-tubulin sequences exhibited 100% (522/522), 99% (318/320), and 99% (603/605) identity with N. parvum (CMW37263 and GenBank KF432951; CBS 111524 and GenBank FJ150709; CCF216 and GenBank KC507808), respectively. Pathogenicity tests were conducted on three replicated sets of 2-year-old, containerized trees by applying a 1 x 105 spores/ml conidial suspension of an N. parvum isolate with a hand-held sprayer, and covering branches with plastic bags for 48 hrs to maintain high humidity. Three additional trees sprayed with sterile distilled water served as controls. Branches of inoculated trees started to blight three months after inoculation and exhibited abundant black conidiomata on the surface of infected stems similar to the symptoms and signs observed on stems in the field. After six months, cankers developed on the main stem. No symptoms developed on any control treatments. The fungus, N. parvum was re-isolated from all symptomatic tissues, satisfying Koch’s postulates. Neofusicoccum parvum has been reported to cause shoot blight of pomegranate in Northern Greece causing 5% yield loss with 2% disease incidence (Palavouzis et al., 2015). A similar species, N. mediterraneum, has been reported to cause cankers on the trunks and branches of pomegranate in California (Morgan and Michilides, 2012). However, N. parvum is morphologically and phylogenetically distinct from N. mediterraneum (Crous et al. 2006). To our knowledge, this is the first report of N. parvum causing shoot blight and stem canker on pomegranate in Florida. The pathogen is of significant importance due to its ability to cause severe losses on pomegranate, an emerging alternative crop in Southeastern U.S.

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