Host status and susceptibility of Cannabis sativa cultivars to root-knot nematodes

The host status and susceptibility of 13 different hemp cultivars to root-knot nematode species was evaluated at the University of Florida Gulf Coast Research and Education Center (GCREC) in Wimauma, FL, in a series of five greenhouse studies between April 2019 and March 2021. Hemp cultivars used included fiber, grain, CBD, and CBG cultivars from different geographic regions (Europe, China, US; Table 1). Trial 1 included six European fiber or dual-use cultivars (both with and without nematode inoculation), trial 2 included the same six cultivars plus three Chinese fiber/dual-use and two US CBD cultivars (only with nematode inoculation), and trial 3 included the three Chinese fiber/dual-use and two US CBD cultivars (both with and without nematode inoculation). In trial 4, two US CBG cultivars were tested in a highly infested local field soil (M. javanica and M. incognita) with and without nematicide or soil pasteurization. In trial 5, we evaluated a CBD cultivar and a fiber cultivar against a tropical RKN M. enterolobii and a temperate RKN M. hapla.

Cultivars were screened (1) in pasteurized soil that was inoculated with a natural field population of RKN containing a mixture of M. incognita and M. javanica (trials 1,2,3); (2) in non-pasteurized, naturally infested soil (mixture of M. incognita and M. javanica) with and without a nematicide (trial 4); and (3) in pasteurized soil that was inoculated with either M. enterolobii or M. hapla (trial 5).

For the greenhouse experiments pertaining to the natural field population (trials 1,2,3), RKN eggs were obtained from infected tomato roots from the GCREC farm. Eggs were extracted from galled tomato roots using the NaOCl method as described by Hussey and Barker (1973). Root-knot nematode populations were identified as M. incognita (Mi) and M javanica (Mj) via PCR/RFLP, as outlined by Baidoo et al. (2016), where nematode DNA was digested with NaOH and the mitochondrial DNA was amplified by PCR primers TRNAH/MRH106 and restriction enzymes HinfI dig and MnlI dig.

For the naturally infested soil experiment (trial 4), soil containing M. javanica and M. incognita was collected from an infested cucumber field. For the selected species experiment (trial 5) with two different species of RKN, Meloidogyne enterolobii (Me) and M. hapla (Mh) were obtained from pure cultures that were maintained, respectively, on tomato and strawberry in the greenhouse at the GCREC.

For each trial, hemp seeds were presoaked in distilled water for one hour before being placed in a moisture chamber consisting of paper and plastic zipper-sealed bags where they were kept for four days. Four-day-old, germinated seeds were then planted in 20 cm wide × 19 cm tall clay pots that were filled with steamed field soil. The soil used in the experiment was Myakka fine sand (96% sand, 3% silt, and 1% clay, with a pH of 7.6 and 0.8% organic matter) which was pasteurized at 70°C for 12 h in an SST-15 120v Soil Sterilizer (Pro-Grow Supply Corp., Brooksville, WI, USA). Nematode inoculation was done three days after planting by adding 10,000 RKN eggs in 1-mL aliquots, each containing 2,500 RKN eggs, into four small, 2.5 cm-deep holes surrounding the germinated seed. The cucumber cultivar “Dasher II” was used in all trials as a positive control.

The length of each trial, unless otherwise stated, was 60 days from the day of inoculation, allowing multiple generations of RKN to develop within the roots. Pots were arranged on greenhouse benches in a complete randomized design, watered as needed, and fertilizer was applied once a week with approximately 200 mL of 20-20-20 (NPK) per pot.

Throughout the 60-day duration, height measurements were taken bi-weekly. At the completion of the trial, roots were rated for root galls, root-knot eggs were extracted from the whole root system, root-knot juveniles were extracted from the soil, and plant biomass measurements were taken including dry root and shoot weights for each plant. Plant material was dried for one week at 60° Celsius. Gall ratings were completed by following the RKN rating chart, as outlined by Bridge and Page (1980), where zero indicates no galls and ten indicates severe galling on all roots. Root-knot nematode eggs were extracted using the Hussey and Barker (1973) bleach method from the entire root system, and juveniles (J2) were extracted from a 200-cm³ soil sample from each pot by means of a modified Baermann funnel method (Viglierchio and Schmitt, 1983). For trials 3 and 5, a reproduction factor (Rf) was calculated for each cultivar after two months by dividing the final population (Pf), the total number of eggs and juveniles after 60 days, by the initial population (Pi) of 10,000 RKN eggs (Rf=Pf/Pi).

Early infection of J2 within the roots was evaluated in trials 1 and 2 by planting two germinated seeds in the nematode-inoculated pots. One seedling was removed one week after inoculation, and nematodes within the roots were differentially stained with a food dye method (Thies et al., 2002). Plant roots were gently washed free of soil and debris, heated for thirty seconds in 12% red food dye, and then de-stained by exposure to warmed acidified glycerin, leaving the stained juvenile inside the root visible. Juveniles were visually observed and quantified using a stereoscope at 40x magnification. Because of the plant removal, no reproduction factor was calculated for trials 1 and 2. Also, for trial 4, no reproduction factor was calculated as naturally infested field soil was used, and the initial egg inoculum could not be quantified.

Data were analyzed via one-way ANOVA through JMP 15 (SAS Institute Inc., 1988–2021) and differences among treatment means were examined by using Tukey’s HSD at P ≤ 0.05.

All hemp cultivars were good hosts for RKN based on root gall symptoms and nematode reproduction. Root gall ratings were generally less than those noted on the cucumber or tomato controls, but reproduction was often similarly high. The mixed population of M. javanica and M. incognita (trials 1, 2, and 4) reproduced rapidly on all tested cultivars (fiber, dual, CBD, and CBG). The same population showed fewer root galls and lower reproduction in trial 3. This effect probably was related to the time of the year the experiment was conducted (winter), as cucumber control plants in these trials also had fewer root galls and lower nematode reproduction. Lower greenhouse temperatures during winter (20–21 °C as compared to 25–26 °C in spring-summer) and shorter day length are probably the main reason for this. The same was noted for trial 5 (pure populations of M. enterolobii and M. hapla), which was also done during winter. Trials 1, 2, and 4 were done during spring-summer, and all hemp cultivars in those trials showed high root-knot infection and reproduction. Tyler (1933) reported an optimum temperature for thermophilic root-knot nematodes of 28 °C, and several other authors have reported slower development of M. incognita and M. javanica at lower temperatures, such as those observed in our winter trials (Davide and Triantaphyllou, 1967; Tzortzakakis and Trudgill, 2005; Davila-Negron and Dickson, 2013). In the case of M. hapla, the northern root-knot nematode (trial 5), which is better adapted to cooler temperatures (Vrain et al., 1978), lower temperatures would not explain the low reproduction. Possibly, the specific population of M. hapla, which originates from a local strawberry field, was not particularly virulent. Previous greenhouse trials in our lab with M. hapla also often failed to show high nematode reproduction on crops other than strawberries (personal observation). In addition to lower temperatures, slower plant growth in winter because of shorter day lengths could also have affected nematode infectivity and reproduction. Especially for daylength-sensitive plants like hemp, plant growth is significantly impacted by photoperiod, which was clearly shown by reduced hemp plant weights when trials were done during winter.

Our results confirm several recent reports from the US and China showing that hemp can be a good host for various RKN, including M. incognita, M. hapla, and M. enterolobii (Song et al., 2017; Kotcon et al., 2018; Ren et al., 2020; Lawaju et al., 2021; Bernard et al., 2022; Coburn et al., 2022).

No clear differences among hemp cultivars in nematode symptoms and host status were seen, except in experiment 2, where cultivar Cherr y Blossom had less visible root galls than other cultivars, although reproduction was similar. Differences in nematode reproduction have been reported on hemp cultivars with M. incognita and M. hapla (Kotcon et al., 2018), and resistance against M. incognita was recently reported in the CBD cultivar Wife (Hansen et al., 2020). Root-knot resistance would be a very useful trait if hemp is to become a major crop in Florida and other southeastern US states, where root-knot nematodes are among the most widespread and damaging soilborne pathogens.

The main differences observed in plant height and biomass between cultivars were due to the physiology of the plants. The Chinese fiber cultivars are naturally tall and slender, the European fiber and dual-use cultivars are slightly shorter than the fiber hemp cultivars, and CBD plants are short and bushy. In most trials, no negative effects of root knot on hemp shoot growth were observed, except for a negative effect on dry root weight from the mixed population of M. javanica and M. incognita for the European fiber/dual-use cultivars Helena, Fibranova, and Eletta Campana, the Chinese fiber cultivar Puma-3, and the US CBD cultivar Cherry Blossom × T1. The exception was in trial 4, where naturally infested soil (from a local cucumber field) was used. In this trial, growth of two CBG cultivars, Gold and Panacea, was significantly reduced, as was shown by visibly improved growth when a nematicide was applied, and especially when soil was pasteurized. This shows that under natural conditions in the field, hemp growth could be greatly reduced by RKN. Most greenhouse trials are conducted by inoculating nematodes into pasteurized soil, which is a good method to evaluate nematode host status, but it may underestimate actual nematode damage potential under natural conditions.

Considering the importance of RKN in Florida, it is essential to evaluate any new crop that is introduced in the state for its susceptibility and host status to this nematode. Hemp or Cannabis sativa is one of these new crops, and all tested hemp cultivars in our trials, regardless of use (fiber, dual, CBD) or origin (Europe, China, US), were good hosts for local RKN populations (M. incognita + M. javanica, M. enterolobii, and to a lesser extent M. hapla). All these Meloidogyne species are common in Florida, and often, fields are infested with multiple species (Riva et al., 2022).

Our study provides a first assessment of the effect of different root-knot nematodes on a range of C. sativa cultivars. This knowledge is important for first-time farmers who want to grow hemp, as well as for established farmers who are considering using hemp in their current production system as a rotation crop.