Lasiodiplodia theobromae causes rachis blight on coconut palms (Cocos nucifera L.) in Florida

Lasiodiplodia, a genus in the family Botryosphaeriaceae, has a broad host range and causes dieback, root rot, fruit rot, leaf rot, and blights in many plant species across sub-tropical and tropical geographical areas (Alves et al., 2008). In palms, this fungal pathogen is known to cause fruit and heart rot, wood decay and leaf blight around the globe (Atallah et al., 2024; Santos et al., 2020). In field-grown coconut palms symptoms usually start on older fronds progressing to younger fronds. Symptoms including discoloration of internal tissue, profuse pathogen sporulation, fruiting body formation, and death of entire fronds, universally characterize rachis blight caused by Lasiodiplodia. In summer 2023, a disease incidence of 30% and severity ranging from 20% to 100% of the canopy was observed in Florida. Three diseased coconut palms were sampled from a five-acre plot and the symptomatic tissue (internal rachis discoloration) was excised at the margin of the advancing lesion, and surface sterilized (30s 75% EtOH, 1 min 3% sodium hypochlorite, 1 min rinse in sterile water). Six pieces were plated on one Potato Dextrose Agar (PDA, Difco) plate supplemented with three antibiotics, penicillin, streptomycin, and neomycin, each at 5mg/L (Gibco PSN), and a total of three PDA plates per sample were processed. Plates were incubated at 25°C under darkness and Lasiodiplodia-like colonies were consistently recovered from all 54 rachis pieces. Initially white in color, the fungal colony grew radially, turned gray, and eventually took on a darker shade, with an abundance of aerial mycelium and rare occurrence of conidiomata and conidia in older plates. Two morphologically similar isolates Las1A and Las1B were obtained from single spores and grown at 28°C in the dark for 7 days. DNA was extracted using the quick DNA extraction protocol (Liu et al., 2000) and regions from two fungal barcoding genes, nuclear ribosomal internal transcribed spacer (nrITS) and translation elongation factor 1-α (TEF1-α) were amplified using primers ITS1/ITS4 (White et al., 1990) and EF1-728F/EF1-986R (Carbone & Kohn, 1999), respectively, and sequenced. The ITS (PQ282128 and PQ282129) and TEF1-α sequences from both isolates (PQ278132 and PQ278133) were 100% identical to Lasiodiplodia theobromae isolate G112-8 (OR453211 and OR482955, respectively). One-year-old seedlings of coconut palm (Cocos nucifera L.) were used for pathogenicity tests. Agar plugs from margins of actively growing cultures of Las1A and Las1B were placed on cut end of petioles from fronds close to the spear leaf and covered with parafilm. The control palms were treated with distilled water. The onset of disease symptoms was observed about 14 days post inoculation. The inoculated petioles were soft, listless, lost turgidity, and could be easily bent without snapping about 1 month following inoculation. In contrast, the palm control seedlings that were inoculated with water remained turgid, healthy and alive. Fruiting body development was observed on inoculated petioles, the pathogen was reisolated, and examination of its colony and spore morphology as well as presence of paraphyses was used to verify its identity. This is the first report of Lasiodiplodia theobromae causing disease on Cocos nucifera in Florida. This pathogen is a potential threat to coconut palm production in Florida and further research on pathogen diversity, pathogenicity, and distribution is needed to develop options for disease management.