Plant-Parasitic Nematodes Associated with Cannabis sativa in Florida

Nematode soil samples were taken at the end of each crop/experiment, and roots were visually examined for nematode damage (no preplant samples were collected). Nematode soil samples were taken as four cores per cultivar plot with a locally made cone sampler (3.0 cm-diam. × 25 cm-deep). The four cores were composited for each plot. Soil samples were sealed in plastic bags and were stored at 4°C and nematodes extracted within two weeks. Nematodes were extracted at the GCREC nematology lab by taking a 200 cm³ soil sample from each composite sample. A modified Baermann method using a salad spinner (Henrik Preutz, IKEA® USA) was used (Viglierchio and Schmitt, 1983).

Plant-parasitic nematodes (PPN) were identified to genus or species level and by feeding group. Morphological identification was performed using a compound Zeiss AXIO Scope A1 microscope (Carl Zeiss, Göttingen, Germany). Plant-parasitic nematodes (PPN) were identified to genus, and depending on the morphological complexity of the species, the nematode was also identified into species level by using the pictorial key proposed by Mai and Mullin (1996). When root galls were observed on roots, Meloidogyne species identification was done by extracting DNA from female nematodes found inside root galls, and mitochondrial haplotype-based identification was done using primers, PCR and RFLP conditions as described by Pagan et al. (2014) and Baidoo et al. (2016). Non-plant-parasitic nematodes (NPPN) nematodes were also morphologically identified into trophic group (bacterivores, fungivores, omnivores).

Location 1: Central Florida (GCREC = Gulf Coast Research and Education Center)

Hemp cultivar trials at the Gulf Coast Research and Education Center farm in Wimauma, FL were conducted in October 2019 (season 1, fall) and February 2020 (season 2, spring) in the same location. The soil type in this area is classified as Myakka fine sand (95% sand, <1% organic matter). The experimental area was adjacent to an experimental hops (Humulus lupulus) field that had artificial lighting installed by poles with LED lights (GreenPower LED flowering DR/W, Philips) suspended 5.5 m above the ground, staggered 6 m apart (Agehara et al., 2020). Lights provided supplemental light (6 hrs/day) during the first two months of growth which is necessary to extend Florida’s natural day length (max. 14 hrs at GCREC) and create long day conditions required to produce meaningful vegetative growth of hops. Both hemp and hops belong to the Cannabaceae plant family, are dioecious and day-length-sensitive (DLS), with flowering being triggered by short days. The same supplemental lighting regime for hops was applied to hemp, meaning the lights started automatically illuminating an hour before sunset and then turned off at midnight for the first two months after planting. Also, the same fertigation and irrigation schedule was followed in the hemp trials as was done for hops (Desaeger et al., 2022). The experimental area measured 78 m long and 18 m wide and consisted of three rows 78 m long x 1 m wide. Rows were 6 m apart, with bahia grass in between, and were covered with landscape fabric with two lines of drip tape underneath. The drip tape used was Toro BlueLine® with 12″ emitter spacing (0.26 gph, 0.62″ internal diameter, 0.045″ wall thickness; Agehara et al., 2020).

Eight selected hemp cultivars (Table 2) were planted in a randomized block design consisting of four plants of the same cultivar per plot, and each plot was replicated six times (two replicates in each row), for a total of 192 hemp plants. Hemp seedlings were grown in a growth room for two months prior to transplanting in the field. Seeds were sown into 128-cell seedling trays filled with PRO-MIX HP growth medium (Premier Horticulture Inc., Quakertown, PA) on August 16, 2019, and December 29, 2019. Seedlings were grown under supplemental lighting (16-h light and 8-h dark) to maintain vegetative growth. Irrigation was supplied as needed using overhead irrigation. Uniform seedlings of each variety were transplanted to the field on 16 October 2019, and 23 February 2020. Plants measured on average 15–20 cm at the time of transplanting. Watering and fertilizing were administered via in-bed drip tape in both areas. Watering consisted of four one-hour cycles every five hours each day and a soluble fertilizer (N-P₂O₅-K₂O: 5-2-8) was applied with irrigation twice a week throughout the season, based on an accumulated rate of 103 kg N ha⁻¹.

Both trials were terminated after three to four months, depending on the cultivar.

Location 2: South Florida (TREC = Tropical Research and Education Center)

Hemp cultivar trials at TREC in Homestead, FL (25.4687° N, 80.5007° W) were conducted in summer 2019 and summer 2020 on two different fields that were adjacent to each other. The soil type is a shallow Krome, a gravelly loam soil series (loamy-skeletal, carbonatic, hyperthermic, Lithic Rendoll) containing 58% sand, 19% silt, 15% clay, and 8% gravel (Li and Zhang, 2002). Its plowed surface layer, largely crushed bedrock, is 15–20 cm deep with 34% to 76% of limestone fragments (≥ 2 mm in diameter; Bryan and Lance, 1991).

A total of 17 different hemp cultivars (fiber, dual, and CBD) were planted at this location: 13 in 2019 and six in 2020 (Table 2). All cultivars were direct-seeded in the field, and planting densities differed according to their usage. All fiber/dual cultivars were sown by hand in eight rows measuring 1.8 m long and were spaced 30 cm apart within a 5.58 m² (1.83 m × 3.05 m) rectangular plot. For dual cultivars, 900 seeds were planted per plot with an intended planting density of 161 plants/m², while 1500 seeds were planted for fiber cultivars to achieve 269 plants/m². CBD cultivars were planted to establish a density of 11 plants/m² by sowing 60 seeds in the experimental plot and after germination thinning to 11 plants. All plots were fertilized once before planting at 112, 56, and 300 kg/ha of N, P, and K, respectively, using a 6-3-13 slow-release granular fertilizer. Due to early indications of N deficiency, there was an additional N application in mid-July, which was top-dressed using 46-0-0 conventional urea at a rate of 56 kg/ha. Irrigation was provided as needed by overhead sprinklers.

Location 3: North Florida (NFREC = North Florida Research and Education Center)

Hemp cultivar trials at NFREC in Quincy, FL were conducted in summer 2019 and summer 2020. Soil type at NFREC is an Orangeburg loamy fine sand. The experimental design was a randomized complete block design with four replications. Three DLS cultivars, including Cherry Blossom (CBL), Cherry × T1 (CT1), and Cherry Wine (CW), and two day-length-neutral (DLN) cultivars, including Kayagene 9201 (KG9201) and Kayagene 9202 (KG9202) were evaluated in 2019. In 2020, cultivars evaluated were CBL, CW, Berry Blossom, Cinderella Story, Cherry Blonde, Cloud Berry, Hot Blonde, and Queens Dream (Table 2). Seeds were sown in the greenhouse using the same methodology as for Location 1, on 14 June 2019 and 2 May 2 2020. Uniform seedlings of each cultivar were transplanted to the field on 3 July 2019 and 12 June 2020.

Agronomic practices were similar between 2019 and 2020. The field setup was similar as for vegetable production in the area, which is plastic-mulch raised beds that were 20-cm high and 30-cm wide with a single drip tape underneath the plastic mulch. The spacing between rows and between plants within a row was 1.8 and 1.5 m, respectively. Therefore, the plant density was ~3600 plants per hectare. Irrigation was supplied daily through the drip tape. Fertilizer (N-P₂O₅-K₂O: 10-10-10) was applied at a rate of 112 kg ha⁻¹ immediately prior to transplanting and disked into soils. A soluble fertilizer (N-P₂O₅-K₂O: 4-0-8) was applied with irrigation as needed throughout the season based on an accumulated rate of 56 kg N ha⁻¹.