Enhalus acoroides is a large Pacific seagrass species, and the most abundant seagrass found in Guam. Seagrass in Guam has recently declined by 22%, and drivers of decline are poorly understood. (LaRoche et al. 2018). Labyrinthula, a fungus like protist, causes seagrass wasting disease (SWD), which was associated with major die offs of Zostera marina in the Atlantic (Muehlstein et al. 1991). Dark lesions are common in Guam’s various E. acoroides beds, suggesting SWD may be one potential cause of decline. This study observed, cultured, and tested pathogenicity of Labyrinthula spp. in lesions on E. acoroides collected in September 2024 from Hagåtña and Achang Bays, Guam. Twelve Hagåtña Bay seagrass blades exhibited irregular, dark to necrotic lesions (2.25 cm 2 ± 0.32 cm 2 , n=18) characteristic of SWD. Microscopy showed spindle-shaped cells inside E. acoroides cells traveling along exuded slime nets characteristic of Labyrinthula spp. Small (< 1cm 2 ) sections of tissue cut from the lesions’ edge were incubated on seawater agar (SWA) plates (1% agar, 1% horse sera, 50 mg/mL rifampicin, Hagåtña Bay seawater) at 28°C. Within 24 hours Labyrinthula spp. colonies expanded into agar. Two isolates (HG4 from Hagåtña Bay and AC12C from Achang Bay) were subcultured in liquid media at room temperature for approximately 2 weeks. To fulfill Koch’s postulates, reinfection was tested on eighteen asymptomatic E. acoroides blades from Hagåtña Bay. Six were used as negative controls, and twelve were inoculated with 10 µL of Labyrinthula spp. in liquid media, pressing a pipette tip on the surface of the leaf to simulate a small entry wound. Labyrinthula spp. HG4 and AC12C were tested, with 6 inoculated samples and 3 controls each. Leaves were kept in petri dishes with seawater at room temperature and observed over 3 days. Areas of water-soaked damaged tissue expanded from the inoculation point in all inoculated leaves. Microscopy of inoculated samples showed Labyrinthula spp. traveling along slime nets and channels between E. acoroides cells. Infected plant cells appeared damaged compared to healthy cells, with chloroplasts clumping to one side of the cell. Control leaves did not show Labyrinthula spp. infection. After 3 days, inoculated and control leaf sections were placed on SWA to reisolate the pathogen. Plates with inoculated leaves grew Labyrinthula spp., and control leaves did not result in any Labyrinthula spp. growth. DNA for sequencing was extracted from liquid cultured Labyrinthula spp. using a Qiagen DNeasy Blood and Tissue kit. PCR using pathogenic Labyrinthula spp. specific ITS1 region primers: LabPathITS1-3F and LabPathITS1-3R (Duffin et al. 2020) produced diagnostic 250bp fragments from both isolates (PX057181 and PX057182) with 87.40% and 84.25% sequence identity determined using BLASTn to a Labyrinthula sp. isolate 178b3-ZC from Australia (KU559373.1). Additionally, a 1500bp region of the 18S rRNA gene was amplified using the universal primers: primer A,18S forward and primer B,18S reverse (Medline et al., 1988). Samples were sequenced at Genewiz (South Plainfield, NJ). The Guam Labyrinthula spp. HG4 and AC12C 18s rDNA sequences (PV826276 and PV826279) showed similarity to other Labyrinthula spp., with highest sequence identity (97.88% and 97.40%, respectively) to the same Labyrinthula sp. isolate 178b3-ZC. This first confirmation of pathogenic Labyrinthula spp. in Micronesia and in E. acoroides as a host may be an important factor in understanding and managing seagrass ecosystems that provide critical ecosystem services to coastal communities.
Lin et al. (Sat,) studied this question.