Nematodes, especially root-knot nematodes (
Florida growers are increasingly interested in new alternative crops as many of the traditional crops in Florida, such as citrus, fruiting vegetables, and strawberries, are facing progressively more pressure due to disease issues and increasing competition from abroad. With the removal of hemp (
There are multiple types of hemp for various uses, including fiber, grain, dual-use, and cannabinoid extracts (Cherney and Small, 2016; Fike, 2016). Different parts of the hemp plant are harvested for specific purposes. Fiber hemp varieties are known to have tall, preferably slender stalks and can grow anywhere from 1 to 5 m in height. Clothing, rope, housing materials, compost, and paper are a few items that can be made from just the stalk of the fiber hemp plant (Cherney and Small, 2016). There is great potential for fiber hemp products to be used as eco-friendly alternatives or replacements for construction materials and plastics in the future. Grain hemp cultivars are plants that produce a high density of seed from which a multitude of products can be made, including food items, animal feed, and cosmetics (Cherney and Small, 2016). These plants do not grow as tall as fiber plants, usually averaging 1 to 2 m in height. Dual-use varieties tend to produce a large number of seeds to use for hemp grain products, but they also have a stalk that is suitable for use in fiber hemp production. Producers harvesting hemp grain would also harvest the remaining stems for fiber, and this is the most common example of a dual-use hemp crop (Williams and Mundell, 2015).
Hemp that is cultivated and bred to process flower parts for cannabinoid extracts are usually shorter and have a more brachiate or bushy appearance as compared to fiber and grain. CBD, or cannabidiol, is the non-psychoactive compound in marijuana and hemp, and it closely resembles THC or tetrahydrocannabinol (Atakan, 2012). CBD can be obtained from the plant by extracting the oil from the flower or by burning or vaporizing the dried flower material. CBD products include topicals, edible forms, and smokable products and are used medicinally to curb anxiety, pain, cognitive disorders, and to aid in sleeping (Grinspoon, 2018). Cannabinoids are highly concentrated in the trichomes of the bracts of unfertilized female flowers and are much lower in the root and plant tissue, and at even lower concentrations in hemp pollen and seeds (Williams and Mundell, 2015). CBG, or cannabigerol, is another non-psychoactive cannabinoid found in cannabis. Just like CBD hemp, CBG hemp is bred and cultivated for the unpollinated female flower.
Due to legal constraints, only recently has research on hemp been able to begin. Thus, there is a lack of information and knowledge on cultivars that are more tolerant to environmental stressors, including nematodes. Recently, we reported that hops (
The main focus of this research was to explore the host status and susceptibility of hemp cultivars of different geographical locations and uses to different species of RKN and potentially identify cultivars that are less susceptible to any of the species of root-knot nematode.
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 (
Hemp cultivars evaluated with their given use and geographical origin.
Cherry Blossom | USA | CBD |
Cherry Blossom × T1 | USA | CBD |
Gold | USA | CBG |
Panacea | USA | CBG |
Bama | South China | DUAL |
Carmagnola | Italy | DUAL |
Carmagnola Selezionata | Italy | DUAL |
Helena | Serbia | DUAL |
Tygra | Poland | DUAL |
Yuma-2 | South China | DUAL |
Eletta Campana | Italy | FIBER |
Fibranova | Italy | FIBER |
Puma-3 | South China | FIBER |
Use: Fiber (tall, slender varieties used for materials and textiles), Dual (produce a large number of seed to use for grain products but also have a stalk that is used in fiber hemp production), CBD (cannabidiol, a cannabinoid extract), and CBG (cannabigerol, a cannabinoid extract).
Cultivars were screened (1) in pasteurized soil that was inoculated with a natural field population of RKN containing a mixture of
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
For the naturally infested soil experiment (trial 4), soil containing
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
Trial 1 was conducted from April to June 2019 to evaluate the host status of six European hemp cultivars (Helena, Tygra, Fibranova, Eletta Campana, Carmagnola, Carmagnola Selezionata, Table 1) to a mixed field population of
Trial 2 was conducted from June to August 2019 to evaluate the host status of 11 different European, Chinese, and US cultivars (the same six European cultivars as in trial 1, plus the Chinese fiber/dual cultivars Yuma-2, Puma-3, Bama, and two US CBD cultivars Cherry Blossom and Cherry Blossom × T1, Table 1). Cultivars were evaluated against the same mixed population of RKN as in trial 1. The average greenhouse temperature was 26° C ± 4° C. Nematode inoculation was done as explained previously, and there were no uninoculated pots. There were five replicates of each cultivar.
Trial 3 was conducted from January to March 2021 to evaluate the host status of three Chinese hemp cultivars (Yuma-2, Bama, Puma-3) and two US CBD cultivars (Cherry Blossom × T1, Cherry Blossom) to the same mixed population of RKN as in trial 1, with ten pots for each cultivar, five inoculated with 10,000 RKN eggs and five uninoculated. The average greenhouse temperature was 20° C ± 2° C.
Trial 4 was conducted from early-June to late-July 2020. Two-month-old CBG seedlings (cultivars Gold and Panacea) were evaluated and compared with one-month-old tomato seedlings (cultivar HM1823) in naturally infested soil (
Trial 5 evaluated the host potential of
One week after inoculation, RKN juveniles were found in all hemp cultivars with significantly more J2s in cultivar Carmagnola Selezionata (60 per root) as compared to cultivars Tygra (6) and Helena (15) (
Nematode reproduction on six European hemp cultivars after 60 days with a mixed population of
Helena | 15b | 2.4 | 292,020 | 53,600 | 600 |
Tygra | 6b | 2.6 | 523,300 | 138,700 | 1,500 |
Fibranova | 22ab | 2.0 | 333,600 | 49,010 | 1,550 |
Eletta Campana | 45ab | 2.6 | 468,960 | 56,250 | 990 |
Carmagnola | 41ab | 2.8 | 401,410 | 52,420 | 350 |
Carmagnola Selezionata | 60a | 3.0 | 498,940 | 71,710 | 430 |
Cucumber* | 45 | 7.0 | 460,800 | - | 225 |
0.008 | 0.630 | 0.557 | 0.501 | 0.114 |
J2s/stained root were evaluated after 7 days. Gall index scale: 0–10; 0= no galls, 10= 100% galls. Eggs and J2s rounded to nearest ten. Factor levels not connected by the same letter are significantly different according to Tukey’s HSD with
Plant height was similar for all cultivars, ranging between 140 and 170 cm after 60 days, and showed no significant interaction between inoculated and uninoculated plants (data not given). Shoot weight was not negatively affected by nematode inoculation, but root dry weight was slightly reduced in some cultivars such as Tygra, Helena, and Eletta Campana (
One week after inoculation, RKN juveniles were found in roots of the Chinese and US cultivars, ranging from 43 to 83 juveniles per root system (no staining was done for other cultivars) (
Nematode reproduction on 11 hemp cultivars (European, Chinese and American, fiber, dual-use and CBD) after 60 days with a mixed population of
Helena | - | 5.2a | 211,100 | 172,680 | 19,530ab |
Tygra | - | 5.8a | 107,530 | 62,640 | 18,930ab |
Fibranova | - | 3.8b | 271,710 | 136,540 | 14,310b |
Eletta Campana | - | 4.8a | 197,730 | 86,940 | 23,720a |
Carmagnola | - | 4.8a | 172,330 | 57,310 | 17,000b |
Carmagnola Selezionata | - | 4.8a | 101,680 | 34,650 | 15,220b |
Yuma-2 | 43 | 4.4b | 270,980 | 43,590 | 15,810b |
Bama | 45 | 3.4b | 153,540 | 29,740 | 8,600b |
Puma-3 | 48 | 3.8b | 180,480 | 31,580 | 7,420b |
Cherry Blossom × T1 | 83 | 4.2b | 187,900 | 51,740 | 11,490b |
Cherry Blossom | 74 | 1.8c | 68,160 | 21,320 | 4,000b |
Cucumber* | - | 7.2 | 218,260 | - | 9,702 |
0.01 | <0.001 | 0.599 | 0.093 | 0.014 |
J2s/stained root were evaluated seven days after inoculation. Gall index scale: 0–10; 0= no galls, 10= 100% galls. Eggs and J2s rounded to nearest ten. Factor levels not connected by the same letter are significantly different according to Tukey’s HSD with
The Chinese fiber and dual-use cultivars had the greatest plant heights, on average 1.5 m after sixty days, European fiber and dual-use cultivars averaged 1 m in height, and the CBD plants averaged just under 1 m (data not given). The same was noted for the plant weights, with cultivars that were of similar uses and geographical origins having similar overall plant biomass (Fig. 3).
Germination of cultivar Yuma-2 was very poor, and no further data were collected for this cultivar. There was no difference in root gall rating between cultivars (GI averaging 1.5 for hemp cultivars as compared to GI = 4.0 for cucumber). Overall, RKN reproduction in this trial was low (less than 1, as compared to 3.5 for cucumber) (Table 4). Just as in trial 1, there was no significant interaction in plant height between nematode-inoculated and uninoculated plants. Plant height was significantly greater for the fiber cultivars compared to the CBD cultivars (data not given). No difference in biomass weight was noted between cultivars, and while nematode-inoculated pots had numerically lower biomass weight for all cultivars, a significant reduction from nematodes was noted only for the root weight of cultivar Puma and cultivar Cherry Blossom T1 (Fig. 4).
Nematode reproduction on two Chinese fiber and two CBD hemp cultivars after 60 days with a mixed population of
Bama | 1.8 | 580 | 250 | 1,350 | 0.193 |
Puma-3 | 1.8 | 250 | 220 | 3,320 | 0.357 |
Cherry Blossom × T1 | 1.4 | 280 | 240 | 3,470 | 0.376 |
Cherry Blossom | 1.2 | 260 | 190 | 2,740 | 0.299 |
Cucumber* | 4.0 | 27,520 | - | 8,298 | 3.58 |
0.633 | 0.341 | 0.624 | 0.657 | 0.694 |
Gall index scale: 0–10; 0= no galls, 10= 100% galls. Eggs and J2s rounded to nearest ten. Reproduction factor (Rf = Pf/Pi) where Pf is total number of J2s and eggs after 60 days and Pi is 10,000 RKN eggs. Factor levels not connected by the same letter are significantly different according to Tukey’s HSD with
Both CBG hemp cultivars, cultivar Gold and cultivar Panacea, showed significant root galling and high nematode reproduction (Table 5), but these factors were lower in cultivar Panacea (GI 5.2) compared to cultivar Gold (GI 6.8). The tomato control averaged 8.2. The final population (Pf) was significantly (
Nematode reproduction on two cannabigerol (CBG) hemp cultivars after 60 days with a mixed natural field population of
Gold | 6.8b | 253,600 | 192,620a | 30,620b | 284,230ab |
Gold (V) | 3.4de | 82,980 | 68,710ab | 19,980b | 102,960b |
Panacea | 5.2c | 113,640 | 47,430b | 14,240b | 127,880b |
Panacea (V) | 2.3e | 47,100 | 24,400b | 9,040b | 44,910b |
Tomato | 8.2a | 235,600 | 122,800ab | 65,540a | 301,140a |
Tomato (V) | 4.4cd | 184,640 | 103,280ab | 23,270b | 207,910b |
< 0.0001 | 0.099 | 0.041 | < 0.0001 | 0.0002 |
Gall index scale: 0–10; 0= no galls, 10= 100% galls. Eggs and J2s rounded to nearest ten. Pf is total number of J2s and eggs after 60 days. V = Velum (a.i. fluopyram). Factor levels not connected by the same letter are significantly different according to Tukey’s HSD with
Overall, plants were short due to the transplants flowering only a few weeks after transplanting. Fluopyram increased plant height in cultivar Gold but not in cultivar Panacea (data not given). In terms of biomass weight, fluopyram had a greater positive impact on plant shoot weight of cultivar Panacea (50% increase) as compared to cultivar Gold, but root weights were not impacted by fluopyram (Fig. 5). Steaming soil prior to transplanting had a much greater effect than nematicide application on plant growth (Fig. 6). When planted in steamed field soil, plants of both cultivars nearly doubled in shoot weight as compared to when treated with the nematicide (Fig. 5). Dry root weights for both CBG cultivars in steamed soil increased four-fold when compared with fluopyram-treated plants (Fig. 5).
Nematode reproduction was low for both nematodes and for both hemp cultivars (Eletta Campana and Cherry Blossom × T1). Significant differences were noted between the two cultivars and nematode species (Table 6). In the case of
Nematode reproduction of
CBD - Cherry Blossom × T1 (+ Me) | 1.4b | 2,600 | 1,710 | 770b | 0.338 |
Fiber - Eletta Campana (+ Me) | 2.0b | 16,300 | 7,475 | 1,300b | 1.759 |
Cucumber (+ Me) | 3.8a | 27,030 | 16,850 | 4,860a | 3.189 |
0.004 | 0.188 | 0.187 | 0.046 | 0.158 | |
CBD - Cherry Blossom × T1 (+ Mh) | 1.8b | 3,470a | 1,490a | 4,400 | 0.787 |
Fiber - Eletta Campana (+ Mh) | 1.0b | 1,640b | 790ab | 5,860 | 0.750 |
Cucumber (+ Mh) | 3.8a | 500b | 130b | 5,880 | 0.638 |
< 0.0001 | 0.003 | 0.025 | 0.773 | 0.871 |
Gall index scale: 0–10; 0= no galls, 10= 100% galls. Eggs and J2s rounded to nearest ten. Reproduction factor (Rf = Pf/Pi) where Pf is total number of J2s and eggs after 60 days and Pi is 10,000 RKN eggs. Factor levels not connected by the same letter are significantly different according to Tukey’s HSD with
Plant height of cultivar Cherry Blossom × T1 was not negatively impacted by either RKN species with an average height of 50 cm, but for cultivar Eletta Campana, plant height was higher when inoculated with
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
Our results confirm several recent reports from the US and China showing that hemp can be a good host for various RKN, including
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
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
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
Our study provides a first assessment of the effect of different root-knot nematodes on a range of