US banana producers are looking for the organic banana market in the southeastern US including Florida and the coastal region of Georgia (Schupska, 2008). In December of 2020, a 6-hand bunched banana (cv. Pisang Awak, belongs to tetraploid AABB genome) with nearly 50% infection (with 15-20% disease severity, <1% of the total harvest) was received from the UGA Banana Research Plot, Tifton, GA with typical stem end rot symptoms of softened and water-soaked flesh. To identify the pathogen, the infected tissues were separated with a sterilized blade, surface disinfested with 10% bleach solution for 1 min, and subsequently washed in three changes of sterile distilled water. The sterilized tissues were aseptically placed on potato dextrose agar (PDA) medium and incubated at 25°C in the dark for 5-10 days. Two isolates of the pathogen with similar colony morphology were obtained and initially identified morphologically using a Botryosphaeriaceae taxonomic key (Phillips et al., 2013). The first growth phase for the isolates documented on PDA, gave rise to white colonies, followed by a dense, black mycelium. The mycelium was fast-spreading, immersed, branched, and septate. The shiny black pycnidia were viewed on the PDA surface after 8-10 days of incubation. Initially, the morphological features of the isolates were identified as Lasiodiplodia spp. (Phillips et al., 2013). To identify to species level, genomic DNA was extracted from two isolates (SW1 & SW2) and amplified by PCR for sequencing using ITS1/ITS4 (White et al., 1990), EF1- 688F/ EF1- 1251R (Alves et al., 2008), Bt2a/Bt2b (Glass & Donaldson, 1995) and rpb2-LasF/ rpb2-LasR (Cruywagen et al., 2017). The ITS (MZ293097 and MZ293114), EF1(OL657173 and OL657174) and rpb2 (OL704860 and OL704861) sequences showed 100% identity and Bt (OL657175 and OL657176) sequences showed 99.5% and 99.7% identity to the corresponding sequences of Lasiodiplodia brasiliensis type strain CMW35884 in GenBank (ITS: KU887094, EF1: KU886972, Bt: KU887466 and rpb2: KU696345). To further affirm the identity, a concatenated phylogenetic analysis was executed with ITS, EF1, Bt, and rpb2 sequences of both isolates and 31 reference strains using Geneious Prime 2019.2.3 Tamura-Nei Neighbor-joining method with 1,000 bootstrap replications, and the outcome was consistent with the conclusion above. To fulfill Koch's postulates, a pathogenicity test was performed with bunched bananas. Two whole bunched bananas surface sterilized with 10% bleach solutions and subsequent washing with sterilized water were cut into 3 bananas per brunch. The inoculum was prepared with 105 spores/ml. The conidial suspension was inoculated on the on-cut surface of the banana crown (300 µl per crown) using a micropipette. Sterile distilled water was applied as a control. The fruit was then packed and sealed in plastic bags and incubated at 25°C. Stem end rot symptoms were first appeared at 5 dpi and increased 7 days later. Two weeks post-inoculation, typical blackened and softened rot tissues were observed, and control fruits remained asymptomatic. To the best of our knowledge, this is the first report of Lasiodiplodia brasiliensis causing stem-end rot of bananas in the USA. This report would be valuable to the banana growers in the southeastern US by taking suitable control measures to confront this fungal disease.
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Jan 1, 2023
Mh Shete + 3
Open Access