Differences in parasitism of root-knot nematodes (Meloidogyne spp.) on oilseed radish and oat

Pure cultures of M. javanica, M. incognita race 3, or M. arenaria race 1 were increased on eggplant (cv. Black Beauty) grown in steamed field soil in the greenhouse for two to three months. The nematodes were identified to species using species-specific primers and mitochondrial genes (Marquez et al., 2019). Inoculum for experiments was obtained by recovering second-stage juveniles (J2) of each nematode species from infected roots of eggplant by incubating chopped roots in a misting chamber for five to seven days. The J2 collected from a 38-µm-aperture sieve were stored at 5°C until use. The inoculation densities of J2 for the following experiments were determined from three replicates of 1 ml aliquots of the nematode suspension.

Nine cultivars of oilseed radish, and one cultivar each of white and black oat were evaluated. Seed of oilseed radish cv. Carwoodi, Cardinal, Final, and Image as well as Tachiibuki oat were obtained from Grassland Oregon, Salem, OR. Also, oilseed radish cv. Concorde, Control, Eco-Till, Karakter and Cannavaro as well as Pratex black oat were obtained from Allied Seed, LLC, Nampa, ID. Tomato cv. Rutgers, an excellent host for all root-knot nematode species, was included in the experiments as a standard host.

This study was conducted from June to October 2018. Seeds of the oilseed radish and oat cultivars were germinated in Miracle-Gro Moisture Control potting mix (The Scotts Miracle-Gro Company, Marysville, OH) in seed trays (Speedling Incorporated, Ruskin, FL). Two-week-old seedlings were transplanted individually into Deepot D40L cells (6.9-cm-diam. × 25.4-cm deep; Stuewe & Sons, Inc., Tangent, Oregon) filled with equal parts (v/v) of steamed field soil and washed sand. After two days, initial inoculum of M. javanica, M. incognita, and M. arenaria were applied to plants by pipetting 1,000 J2 into two depressions made in the soil at the base of the stems. The deepot cells were arranged in a completely randomized design with five replications on support trays (Stuewe & Sons, Inc., Tangent, OR), and plants were maintained for eight week, the time needed for the completion of two generations of Meloidogyne spp. Temperature in the greenhouse varied between 25 and 28°C. Plants were watered daily and fertilized once, at two-weeks-after planting with 2-g Osmocote smart-release fertilizer (15-9-12, The Scotts, Marysville, OH). Two separate experiments were conducted for each Meloidogyne species.

At the termination of each experiment, shoots of all plants were removed and root systems were washed free of soil. Root systems of each plant were weighed and assessed for severity of galling on a scale 0 to 5 where 0 = no galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls; and 5 = more than 100 galls (Taylor and Sasser, 1978). Root systems from each plant were cut into 2-cm pieces, and eggs were extracted with a 1.0% NaOCL solution (Hussey and Barker, 1973) followed by the standard centrifugal flotation technique. Eggs were counted using an inverted microscope and total number of eggs per gram of fresh root was determined. The reproductive factor (Rf) of Meloidogyne spp. was assessed by dividing the final nematode counts extracted from roots by initial number of J2 inoculated to plants at the beginning of experiment, and used as a measure for the host suitability of oilseed radish and oat cultivars to Meloidogyne spp. Host suitability was evaluated as follows: Rf < 0.1, nonhost (highly resistant); 0.1 ≤ Rf < 1, poor host (resistant); 1 ≤ Rf < 2, maintenance host (susceptible); and Rf ≥ 2, good host (highly susceptible).

This assay was conducted from March to May 2019 using Carwoodi and Eco-Till oilseed radish and Tachiibuki and Pratex oat based on their response to Meloidogyne spp. in the host suitability study (Table 1). The seeds were planted in 72-cell plug flats (Johnny’s Selected Seeds, Fairfield, Maine) filled with steamed Miracle-Gro potting mix two to three weeks prior to nematode inoculation. Each two-week-old seedling was inoculated with 200 J2 of M. incognita race 3, M. arenaria race 1, or M. javanica in 1 ml water pipetted into two holes (2-cm deep) made around the stem. The experiment was a completely randomized design with three replicates for each cultivar. It was conducted in a greenhouse at 25±3°C. The plants were watered lightly each day. The infected root systems were collected one, three, and five days after inoculation (DAI) to assess nematode penetration. Three seedlings were randomly selected, washed gently with tap water to remove the soil, soaked in 0.5% NaOCL for 30 sec and then rinse with tap water. The nematode J2 in intact roots were stained by boiling for 30 sec in 30 ml tap water containing 1 ml of 3.5% (wt/vol) acid fuchsin. After staining, the roots were rinsed with tap water and destained by boiling in acidified glycerol for about 1 min. Visualization of stained nematodes was done by placing the root systems in a Petri dish and examination under a stereomicroscope (Stemi 508, ZEISS, Hannover, Germany). The penetration of Meloidogyne spp. was examined by counting the total number of J2 stained inside the roots at each sampling day. To examine nematode development and reproduction, three root systems were collected at 28 DAI, stained as specified previously and 20 galls were randomly examined under a stereomicroscope to count females. Swollen females with no egg-mass in the root tissues were considered young females, whereas egg-laying females were considered adult females (Fig. 2). This experiment was conducted twice.

A histopatological examination was also conducted to evaluate the response of Carwoodi and Eco-Till radish and Tachiibuki and Pratex oat to nematode infection. Two to three segments 1 to 2 cm long of galled roots from each infected plant were examined at 28 DAI. Root segments, including individual small galls and adjoining portions of non-galled tissue, were fixed in buffered formaldehyde solution for 24 to 48 hr at room temperature. The fixed segments were dehydrated in a graded ethanol series (70 to 100%) using a tissue processor (Excelsior AS, Thermo Scientific, Thermo Shandon Limited, Runcorn Cheshire, UK), and embedded in paraffin. Cross sections 4 to 5 µm thick were prepared using a rotary microtome (RM2125 RTS, Leica Biosystems, Nussloch, Germany), and then mounted on glass slides. The sections were stained with Hematoxylin-Eosin (HE), evaluated microscopically at 40 to 100 × magnifications (Olympus BX43, Olympus, Tokyo, Japan) and images captured using a digital camera (Olympus DP73, Olympus, Tokyo, Japan) (Hajihassani et al., 2020).

Galling severity, egg counts, and Rf from the host suitability test were subjected to one-way ANOVA, while data from the penetration and development assay were analyzed by two-way ANOVA using the PROC mixed models (SAS v. 9.4, SAS Institute, Cary, NC). Data were combined prior to ANOVA because the initial and repeat trials were not significantly different (P > 0.1). Plant cultivar and DAI (penetration and development assay) were considered fixed effects, and trial was a random effect. Means were compared using Tukey’s adjustment for multiple comparison test at P < 0.05. Least-squares means (LS-means) t-tests demonstrating if the mean was significantly different from 0 were used to determine whether the nematode Rf was different from 1 (the base 10 algorithm of 1 is 0). Therefore, single t-tests that showed the treatment mean was significantly different from 0 indicated that the Rf deviated significantly from 1 (Hajihassani et al., 2020).

Cultivar affected (P < 0.0001) all resistance characteristics including gall severity and nematode reproduction. Meloidogyne javanica, M. incognita, and M. arenaria produced varying numbers of galls and eggs in the roots of oilseed radish and oat cultivars (Fig. 1); however, significant differences among the cultivars were infrequent. Root galling severity on the oilseed radish and oat cultivars was in the range of 1.4 (Carwoodi oilseed radish) to 3.7 (Cannavaro radish) for M. javanica, 0.4 (Carwoodi) to 2.7 (Image radish and Pratex oat) for M. incognita, and 0.3 (Tachiibuki oat) to 3.3 (Eco-Till and Final) for M. arenaria. The numbers of M. javanica eggs per gram of fresh root ranged from 270 for Carwoodi to 4,946 for Cannavaro. Egg production by M. incognita varied from 21 for Carwoodi to 2,436 eggs per gram root for Image. For M. arenaria, Tachiibuki (32) and Eco-Till (3,780) had the minimum and maximum number of eggs per gram of root (Fig. 1). All three nematode species reproduced more on tomato than on the oat cultivars and most of the oilseed radish cultivars (Table 1). Meloidogyne javanica reproduced on eight cultivars of oilseed radish, and the white and black oats. Although the nematode had an Rf of 1.05 on Carwoodi, the Rf was not different from 1 based on the LS-means t-tests indicating that M. javanica failed to increase its population on Carwoodi.

Figure 1:

The Rf of the three nematode species did not often vary among the cultivars tested. The Rf of M. incognita was lower (P < 0.05) in Carwoodi and Control oilseed radish and Tachiibuki oat than all other cultivars examined. Meloidogyne javanica and M. incognita exhibited only slight reproduction on Karakter and Concorde, having Rf’s ranging from 1.0 to 1.9. The Rf of M. arenaria was significantly less than 1 on Karakter and Carwoodi oilseed radish and Tachiibuki oat compared to all other cultivars.

The penetration of J2 into roots of oilseed radish and oat was only affected (P < 0.0001) by DAI, showing that the susceptibility or resistance of the cultivars had no effect on nematode root penetration. At 1 DAI, J2 were observed in the roots of oilseed radish; however, no difference in the numbers of J2 was observed between susceptible Eco-Till and resistant Carwoodi cultivars for M. javanica, M. incognita, or M. arenaria (Table 2). A similar trend in the penetration of the nematodes in roots of susceptible Pratex and resistant Tachiibuki oats was also detected (Table 3). At 3 and 5 DAI, comparable numbers of J2 penetrated the roots of susceptible and resistant cultivars of oilseed radish and oat. Nematode development was studied by counting the numbers of young and adult (egg-laying) females of each nematode species within galled (swelled) roots at 28 DAI. For all three nematode species, greater numbers (P < 0.05) of young and adult females were observed in the root tissues of the susceptible cultivar Eco-Till than in resistant Carwoodi (Table 2). Similarly, the total number of young and adult females for M. incognita and M. arenaria was higher (P < 0.05) in the roots of susceptible Pratex (Fig. 2A and B) compared to resistant Tachiibuki oat (Table 3).

Figure 2:

Cross sections of the root system of oilseed radish and oat infected with all three root-knot nematode species showed morphological variations between susceptible and resistant cultivars. At 28 DAI, more than 96% of the root tissues of resistant Carwoodi (Fig. 3A) and Tachiibuki (not shown) inoculated with M. incognita or M. arenaria had no sign of feeding site or giant cell formation. In contrast, all the root tissues of susceptible Eco-Till (Fig. 3B) and Pratex (not shown) infected with M. arenaria or M. incognita had multiple giant cells. We noticed very limited hypertrophy in cortical cells and the vascular cylinder of Carwoodi and Tachiibuki infected with these two nematode species, suggesting that the nematode J2 penetrated the roots but failed to establish giant cells. Multinucleated giant cells were detected in 100% of the root tissues of Eco-Till infected with M. javanica (not shown). Cross sections of the root system of M. javanica-infected Carwoodi showed degraded giant cells in 5 to 10% of the roots (not shown). Multiple giant cells and severe hypertrophy in the vascular cylinder and cortical cells were detected in the root tissues of Tachiibuki and Pratex oats infected with M. javanica (Fig. 4A, B).

Figure 3:

Figure 4: