Plant-parasitic nematodes associated with the root zone of hop cultivars planted in a Florida field soil

The studies were conducted at the Gulf Coast Research and Education Center research farm in Wimauma, Florida, United States. Soil at the site is an Arredondo fine sand (95% sand, 3% silt, 2% clay). The field had been fallow for over 10 years under natural grass cover prior to conducting the following trials.

In 2016, a new hop yard was established at the research farm to evaluate plant-parasitic nematode population development on 10 hop cultivars from two planting material sources. Planting rows were 82-m long and were spaced 4.88-m apart, with a 0.91-m wide strip of black geotextile mulch covering the center of each row. Hills were spaced 0.91-m apart within the planting row. A 6.1-m tall trellis wire was installed above each row. Plants were irrigated with two drip tapes (30-cm emitter spacing), each located 0.46-m feet away from the row center along both sides of the planting row. Plants were fertilized with all-purpose fertilizer (20:20:20) at a rate of 149 kg N/ha/season. Ground cover between rows consisted of perennial ryegrass (Lolium perenne) that was mowed periodically throughout the growing season.

The experimental design was a randomized complete block with three blocks and 12 cultivar treatments. In total, 36 plots were overlaid on to three planting rows within the hop yard. Plots consisted of four hills (3.66-m long) and were planted at a density of two rhizomes per hill. A 0.91-m buffer zone was located between plots to minimize edge effects. On April 12, 2016, triplicate plots were planted with rhizomes from each of the following cultivars: (i) ‘CTZ’, (ii) ‘Cascade’ (source: Oregon), (iii) ‘Cascade’ (source: Washington), (iv) ‘Centennial’ (source: Oregon), (v) ‘Centennial’ (source: Washington), (vi) ‘Chinook’, (vii) ‘Fuggle’ (source: Oregon), (viii) ‘Magnum’ (source: Washington), (ix) ‘Nugget’ (source: Washington), (x) ‘Sorachi Ace’ (source: Washington), (xi) ‘Triple Perle’ (source: Washington), or (xii) ‘Willamette’ (source: Washington).

Plants were harvested in August 2016 and December 2016 by cutting the bines by hand at ground level. During the growing season, a number of the cultivars displayed symptoms of a viral disease, which was subsequently confirmed as Apple Mosaic Virus (ApMV). Due to the distribution and severity of the symptoms the hop yard was scheduled to be renovated using clean planting material in 2017.

In December 2016, plants were uprooted from the soil after harvest using a shovel and assessed for root galling. Entire root systems were assessed for galling and given a root gall index rating on a scale from 0 to 10 (Bridge and Page, 1980), with 0 indicating no visible galling and 10 indicating 100% galled. The abundance of plant-parasitic nematodes in soil was also determined for each plot. Eight soil cores (20 cm in length, 2.5 cm in diameter) were obtained from each plot directly from the previous rooting zone (two soil cores per hill). Soil samples were placed into plastic bags and were stored at 4°C for a maximum of 14 days prior to subsequent processing. Nematodes were extracted from a 200-mL subsample of soil from each plot using the Baermann pan technique (Forge and Kimpinski, 2007), with a two-day incubation period. After collecting the nematodes over a 25-µm sieve, the nematodes were transferred in water into plastic scintillation vials and store at 4°C for a maximum of 14 days prior counting on an inverted compound microscope.

In 2017, a new hop planting was established in the previously renovated hop yard. On August 15, 2017, metam potassium (K-Pam^® HL; AMVAC Chemical Corporation, Los Angeles, CA) was drip applied to the previous planting rows at a rate of 584 L/ha. The experimental design was a randomized complete block with three blocks and 12 cultivar treatments overlaid on the previous plots from the first field experiment in 2016. Each hill within a plot was planted with two transplants. On September 13, 2017, triplicate plots were planted with tissue-cultured transplants from each of the following cultivars: (i) ‘Cascade’ (source: Washington), (ii) ‘Cascade’ (source: Florida), (iii) ‘Cashmere’, (iv) ‘Centennial’ (source: Washington), (v) ‘Centennial’ (source: Florida), (vi) ‘Chinook’ (source: Washington), (vii) ‘Chinook’ (source: Florida), (viii) ‘Comet’ (source: Washington), (ix) ‘Magnum’ (source: Washington), (x) ‘Nugget’ (source: Washington), (xi) ‘Triple Perle’ (source: Washington), or (xii) ‘Zeus’ (source: Washington).

In February 2018, artificial lights (Philips Flowering Lamp 2.0; Philips, Amsterdam, the Netherlands) were installed every 2.74 m along the trellis wire to extend the daylight length by 5 hr from March to May and from September to November. Plants were harvested by hand in December 2017, July 2018, December 2018, and July 2019 by cutting the bines at ground level.

The abundance of plant-parasitic nematodes in soil was determined on four sampling dates (December 2017, July 2018, December 2018, and July 2019). Soil was sampled and nematodes were quantified as described previously.

In 2017, an additional new hop planting was established on a single planting row adjacent to the previous hop yard from 2016. On August 15, 2017, metam potassium was drip applied to the planting row, as described above. Planting rows were covered with a 0.91-m wide strip of metallic plastic mulch. The experimental design was a randomized complete block with four blocks and five cultivars overlaid on the previous plots from the first field experiment in 2016. Each hill within a plot was planted with two transplants. On September 15, 2017, plots were planted with tissue-cultured hop transplants of each of the following cultivars sourced from Washington: (i) ‘Canadian Red Vine’, (ii) ‘Galena’, (iii) ‘Mt. Rainier’, (iv) ‘Tahoma’, or (v) ‘Willamette’.

Artificial lighting was installed every 2.74 m along the trellis wire to extend the daylight length during March to May and September to November. Plants were harvested by hand in December 2017, July 2018, December 2018, and July 2019 by cutting the bines at ground level.

The abundance of plant-parasitic nematodes in soil was determined on three sampling dates (December 2017, July 2018, December 2018, and July 2019). Six soil cores were obtained from each plot directly from the previous planting holes (two cores per hill). Soil samples were processed, and nematodes were quantified as described previously.

In 2017, a greenhouse experiment was performed to evaluate the susceptibility of 14 hop cultivars to M. javanica. The experimental design was a randomized complete block with six blocks and 14 cultivar treatments. In total, 84 20-cm diameter circular pots were filled with 3.5 L of soil mix (three parts steam sterilized field soil: two parts potting mix). The nematode inoculum was prepared by extracting eggs from a population of M. javanica maintained in a greenhouse on tomato roots using the sodium hypochlorite technique (Hussey and Barker, 1973), followed by washing the eggs in water over a 25-µm sieve. The eggs were inoculated as 1-mL of nematode egg suspension (2,500 eggs/mL) into each of four 1-cm deep holes made in the top of the soil in each pot (10,000 eggs/pot). After nematode inoculation, six replicate pots were planted on September 18, 2017 with a single tissue-cultured hop transplant from one of the following cultivars sourced from Washington: (i) ‘Canadian Red Vine’, (ii) ‘Cascade’, (iii) ‘Cashmere’, (iv) ‘Centennial’, (v) ‘Chinook’, (vi) ‘Comet’, (vii) ‘Galena’, (viii) ‘Magnum’, (ix) ‘Mt. Rainier’, (x) ‘Nugget’, (xi) ‘Tahoma’, (xii) ‘Triple Perle’, (xiii) ‘Willamette’, or (xiv) ‘Zeus’. The pots were irrigated as required with a drip irrigation system. Pots were fertilized biweekly with all-purpose fertilizer (20:20:20), with a cumulative application of 0.45 g of mineral N supplied to each plant. The plants were grown in a greenhouse for 12 weeks prior to subsequent analysis.

At harvest, plants were carefully uprooted from the soil and assessed for root galling, as described above. The abundance of eggs on each root system was determined by extracting eggs from entire root systems using the sodium hypochlorite technique, as described above. Eggs were collected over a 25-µm sieve, transferred in water into plastic scintillation vials, and store at 4°C for a maximum of 14 days prior counting on an inverted compound microscope.

In July 2019, dagger (Xiphinema sp.), lesion (Pratylenchus sp.), ring (Mesocriconema sp.), spiral (Helicotylenchus sp.), and stubby-root (Paratrichodorus sp.) nematodes extracted from the root zone of hop plants were identified to the species level by sequencing a partial fragment of the 28S ribosomal RNA gene region and the 18S ribosomal RNA gene region. For each genus, one to seven vermiform nematodes were plucked from the July 2019 sample nematode extractions and combined into a single sample for DNA extraction. Total genomic DNA was extracted using the sodium hydroxide nematode digestion protocol (Floyd et al., 2002). The D2-D3 expansion segments located within the 28S ribosomal RNA gene region was amplified using the primers D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTAGAT-3′). The 18S ribosomal RNA gene region was amplified using the 1813/2646 primer set (Holterman et al., 2006). Amplification reactions contained 2.5 μL of 10X ThermoPol^® Buffer (New England Biolabs, Beverly, MA, USA), 0.5 μL of 10 mM dNTPs, 0.5 μL of 25 mM MgCl₂, 0.5 μL of 10 μM forward and reverse primers, 1.0 μL of nematode digestion solution, and 0.125 μL of Taq DNA Polymerase (New England Biolabs, Beverly, MA, USA), brought to a volume of 25 μL. The 28S ribosomal RNA gene region was amplified using the following temperature profile: 5 min at 95°C, 35 cycles (30 sec at 94°C, 45 sec at 55°C, and 1 min at 68°C), followed by a final extension of 5 min at 68°C. The 18S ribosomal RNA gene region was amplified using the following temperature profile: 5 min at 95°C, 5 cycles (30 sec at 94°C, 30 sec at 45°C, and 70 sec at 68°C), 40 cycles (30 sec at 94°C, 30 sec at 54°C, 70 sec at 68°C) followed by a final extension of 5 min at 68°C. All amplification reactions were carried out on a T100 Bio-Rad Thermal Cycler (Bio-Rad, CA, USA), sequenced in both directions by GENEWIZ DNA sequencing services (GENEWIZ Inc., South Plainfield, NJ, USA), and assembled using Geneious Prime v. 2019.2.1 (Biomatters, Auckland, New Zealand). Consensus sequences were compared for similarities within the National Center for Biotechnology Information GenBank database using the Basic Local Alignment Search Tool.

Nematode data from the field and greenhouse experiments were subjected to a one-way ANOVA in SAS Studio (SAS University Edition; version 3.3; SAS Institute Inc., Cary, NC, USA) using the PROC GLM procedure. Differences among treatment means were examined using Tukey’s HSD test (p < 0.05).

Plant-parasitic nematode species recovered from the root zone of hop plants grown in a Florida field soil during the three-year trial period included M. javanica, Pratylenchus brachyurus Filipjev & Schuurmans-Stekhoven, Paratrichodorus minor Siddiqi, Belonolaimus longicaudatus Rau, Xiphinema setariae/vulgare complex Luc, Mesocriconema xenoplax Loof & De Grisse, and Helicotylenchus dihystera Sher (Table 1). Throughout the study, soil population densities of P. minor, B. longicaudatus, X. setariae/vulgare complex, M. xenoplax, and H. dihystera remained low.

Soil population densities of M. javanica did not differ significantly among the hop cultivars (Table 2); however, populations were large (> 200 nematodes per 200 mL of soil) in the ‘Chinook’, ‘Centennial’ (WA), ‘Centennial’ (OR), and ‘Cascade’ (OR) cultivars. Population densities of M. javanica remained low (< 50 nematodes per 200 mL soil) in the ‘CTZ’, ‘Magnum’, and ‘Triple Perle’ cultivars. Soil population densities of P. brachyurus did not differ among the hop cultivars; however, populations were largest in the ‘Magnum’ cultivar (69 nematodes per 200 mL of soil).

Root galling varied considerably among the hop cultivars in Hop Yard Planting No. 1. The most severe root galling was observed on ‘Chinook’, ‘Centennial’ (OR), ‘Fuggle’, and ‘Centennial’ (WA). The lowest amount of root galling was observed on ‘Magnum’, ‘Triple Perle’, ‘CTZ’, and ‘Nugget’.

In December 2017, M. javanica population densities in soil did not differ significantly among the hop cultivars; however, populations were largest in the ‘Cascade’ (FL), ‘Centennial’ (WA), ‘Centennial’ (FL), ‘Chinook’ (FL), and ‘Comet’ cultivars (Table 3). In July 2018, soil population densities of M. javanica were significantly larger in the ‘Centennial’ (WA) cultivar relative to that of the ‘Cascade’ (FL), ‘Chinook’ (FL), ‘Comet’, ‘Magnum’, ‘Nugget’, and ‘Triple Perle’ cultivars. In December 2018 and July 2019, M. javanica populations in soil did not differ significantly among the hop cultivars; however, population densities were consistently large in the ‘Centennial’ (WA), ‘Centennial’ (FL), and ‘Comet’ cultivars.

Soil population densities of P. brachyurus did not differ significantly among the hop cultivars throughout the first two years of plant establishment in Hop Yard Planting No. 2 (Table 4). Populations of P. brachyurus remained numerically low in soil planted with ‘Cascade’ (WA), ‘Cashmere’, ‘Centennial’ (WA), ‘Nugget’, and ‘Triple Perle’.

Soil population densities of M. javanica did not differ significantly among the hop cultivars (Table 5) but in July 2018 and December 2018 soil population densities were consistently large (> 100 M. javanica per 200 mL soil) in the ‘Canadian Red Vine’, ‘Mt. Ranier’, ‘Tahoma’, and ‘Willamette’ cultivars. Similarly, soil population densities of P. brachyurus did not differ among the hop cultivars throughout the first two years of plant establishment (Table 6) however, population densities were consistently large in the ‘Galena’ and ‘Mt. Rainier’ cultivars.

Root galling differed significantly among the hop cultivars evaluated in the greenhouse experiment (Table 7). The most severe root galling was observed on ‘Comet’, ‘Cashmere’, ‘Canadian Red Vine’, and ‘Chinook’. The lowest amount of root galling was observed on ‘Galena’, ‘Willamette’, and ‘Cascade’. The number of M. javanica eggs extracted per plant also differed significantly among the various hop cultivars. The greatest number of eggs was obtained from the ‘Comet’, ‘Cashmere’, ‘Centennial’, ‘Cascade’, and ‘Chinook’ cultivars. The fewest number of eggs was obtained from the ‘Magnum’, ‘Willamette’, ‘Galena’, and ‘Canadian Red Vine’ cultivars.