Methods

Field work is being done at 7 locations in the northern Gulf of Mexico, focusing on MS and AL seagrass beds. Proposed locations were chosen based on where this parasite has been recorded previously (Walker and Campbell 2009) and include Grand Bay National Estuarine Research Reserve, Horn Island, Ship Island and Cat Island in MS Sound, the northern Chandeleur Islands (LA), Pointe aux Pines (AL) and Johnson Beach at Perdido Bay (FL). The latter two locations will be sampled once in late spring and once in late summer to determine whether seasonal conditions influence infection. These locations were chosen to the represent a range of environmental conditions along two gradients: (1) East to West, and (2) inshore to offshore.

At each location, 2-3 sites will be visited for intense sampling including obtaining seagrass cores and transect-based observations for infected H. wrightii plants. A schematic of the proposed sampling layout is shown in Figure 3, following the recommendations of Seagrass-Watch (http://www.seagrasswatch.org/manuals.html). Three seagrass cores (15 cm diameter, 30 cm deep) will be collected at 10 m intervals along each transect (total 6 cores/site). In addition to the seagrass core, water depth, seagrass percent cover in a 0.25 m2 quadrat, and prevalence of H. wrightii in mixed species beds will be noted for that station. Each core will be transferred to a floating sieve 850 μm (size 20) mesh to remove the sediments and the cleaned shoots placed in labelled sterile plastic bags and placed on ice. All samples will be enumerated in the lab on return. Additionally, visual observations and spatially “integrated” sampling for infected H. wrightii plants will be done by using a potato rake along 10 m segments between cores. The number of infected shoots observed along each segment will be recorded (total 6 rakes/site).

Core samples will be processed in the lab within 24 hours of collection. All shoots in a core will be counted and the number of infected shoots will be recorded to calculate the percent infection. Infected shoots will be frozen for later DNA analysis (see below). Notes on shoots attached to a common rhizome will be made where infected and non-infected shoots co-occur on the same individual. The core and rake data will be used to (a) calculate infection rate in the population for each location, and (b) provide information on spatial intensity of infection (is it one plant every once in a while, or is it strongly clustered within a short distance/small area) for each location. Infected shoot samples will be processed for DNA extraction and gene sequencing.

DNA will be extracted from infected H. wrightii tissue using an PowerSoil DNA Isolation Kit (Cat No. 12888, MoBio, Carlsbad, CA). The 18S region of ribosomal DNA will be amplified using Illustra Ready-To-Go™ PCR Beads (GE Healthcare, Waukesha, WI) with Plasmodiophorid-specific primers and thermocycler conditions (Neuhauser, pers.comm). 18S rDNA PCR amplification will be confirmed using gel electrophoresis and forward and reverse sequences will be obtained by the dideoxy chain-terminating method using Plasmodiophorid-specific sequencing primers (Neuhauser, pers.comm) and ABI PRISM 3100 or ABI PRISM 3130xl automated DNA sequencers (Applied Biosystems, Foster City, CA). Sequences will be edited and aligned using Geneious 6.0.4 software (Biomatters). Consensus sequences will searched against the reference NCBI sequence database GenBank using the BLAST search algorithm, as well as other relevant sequence databases. The top sequence matches will be subsequently downloaded and a maximum likelihood analysis will be performed in PHYML to place this species in a phylogenetic framework (Guindon & Gascuel 2003, Schmitt 2009).