Resistant Pepper Carrying N, Me1, and Me3 have Different Effects on Penetration and Reproduction of Four Major Meloidogyne species

Pure cultures of M. incognita race 3 and M. arenaria race 1 were obtained from P. Timper and R. Davis (USDA–ARS, Tifton, GA), culture of M. javanica was obtained from J. Noe (University of Georgia, Athens), and culture of M. haplanaria was obtained from T. Mengistu (Formerly at University of Florida, Gainesville). These nematode isolates were multiplied separately on coleus (Plectranthus sp.) plant in the greenhouse for 15 weeks. Second-stage juveniles (J2) of each Meloidogyne spp. isolate were recovered from the infected coleus roots by incubating chopped roots in a mist chamber for five days.

The pepper cultivars/lines used were Mellow Star (susceptible control pepper; Johnny’s selected seeds, Maine), Yolo Wonder B, Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687. Yolo Wonder B is resistant to M. arenaria and its resistance is conferred by the Me5 gene. Charleston Belle is resistant to M. incognita, M. arenaria races 1 and 2, and M. javanica and its resistance is conditioned by the N gene (Fery et al., 1998; Thies and Fery, 2000). HDA-149 and HDA-330 are double haploid lines that harbored the Me3 and Me1 genes, respectively and conferred resistance to M. incognita, M. arenaria, and M. javanica. PM-217 and PM-687 have the Me1 and Me3 genes, respectively for resistance to M. incognita, M. arenaria, M. javanica (Hendy et al., 1985; Berthou et al., 2003).

Pepper seeds were planted in 128-cell plug trays (Speedling Incorporated, Ruskin, FL) filled with Miracle-Gro Moisture Control potting mix (The Scotts Miracle-Gro Company, Marysville, OH) two to three weeks before nematode inoculation. Pepper seedlings, at two true leaf stage and approximately 6 cm tall, were transplanted into Deepot D40L cell containers (6.9-cm-dia. × 25.4-cm deep, vol. 410 mL; Stuewe & Sons, Inc., Tangent, Oregon) containing pasteurized field soil: washed sand (2:1 v/v). Before transplanting, potted soils were watered and seedlings were transplanted individually in cell containers, and then inoculated with 300 J2 in 1 ml water pipetted into two holes (3 cm deep) made in the soil around the plant base. The plants were arranged in a completely randomized design with four replicates on support trays (Stuewe & Sons, Inc., Tangent, Oregon) in a greenhouse at 25±3 ° C. Enough plants were inoculated with M. incognita race 3, M. arenaria race 1, M. javanica, and M. haplanaria to allow destructive sampling of three plants every other day up to five days. The pots were watered lightly each day. On each sampling day, three seedlings were randomly taken and harvested to recover the root system. The root systems were washed gently with tap water to remove soil, soaked in 1.5% NaOCL (wt/vol) for 3 min and a final rinse of tap water. The nematode J2 in intact roots were stained by boiling for 30 sec in red food color (Thies et al., 2002). After staining, the roots were rinsed with tap water, destained in lactophenol for 48 hr (Hajihassani et al., 2017). Visualization of nematode J2 in root tissues was done by pressing each root system between two glass slides and examination with a stereomicroscope at ×20 to ×90 magnification. Nematode penetration was assessed by the enumeration of the J2 stained inside the roots. This experiment was repeated once.

Pepper seedlings were transplanted into the Deepot D40L cell containers filled with pasteurized field soil: washed sand (2:1 v/v). At transplanting, 1,000 J2 in 1 ml water were pipetted into two holes (2-3-cm deep) made in the soil around the plant base. The plants were arranged in a completely randomized design with five replicates on support trays in the greenhouse. Plants were watered once a day with equal amounts of water, fertilized once during the experiment with 10-g Osmocote smart-release fertilizer (15-9-12, The Scotts, Marysville, OH), grown at 28 ± 3°C for eight weeks, at which time root systems were harvested, washed gently with running water, air dried briefly on paper towels, and weighed. The root systems were stained as described previously and then rated for nematode reproduction with a gall index (GI) using a 0 to 5 scale as follow: 0 = no gall; 1 = 1 to 2 galls on root system, 2 = 3 to 10 galls, 3 = 11 to 30 galls, 4 = 31 to 100 galls, and 5 =>100 galls (Taylor and Sasser, 1978). Eggs were extracted from root systems separately using the NaOCl method (Hussey and Barker, 1973) and counted under an inverted microscope. Nematode reproduction was also measured by calculating the reproduction factor (RF: final number of nematodes/initial number inoculated). The GI and RF are important measures of resistance of a plant species to Meloidogyne spp. (Sasser et al., 1984). These measurements were selected as the primary parameters in determining resistance/susceptibility in pepper cultivars/lines against Meloidogyne spp. The experiment was repeated once.

A two-way analysis of variance (ANOVA) using PROC Mixed within SAS (v. 9.2, SAS Institute, Cary, NC) was performed on data obtained in the penetration and reproduction studies. Since no significant differences in the fresh root weight, egg counts, GI, and RF were observed between two trials in both penetration (p > F = 0.08) and reproduction (p > F = 0.1) studies, data were grouped for statistical analysis. Means were separated with Tukey’s adjustment for multiple comparisons test. The confidence interval for statistical significance was 95%.

Nematode penetration as evident from enumeration of the J2 inside the roots was affected by pepper cultivar/lines among all Meloidogyne spp. The nematodes penetrated roots of all susceptible and resistant peppers, but a significant effect of plant genotype (p < 0.0001), DAI (p < 0.0001) and line/cultivar × DAI interaction (p < 0.0001) was observed.

By 1 DAI, the number of J2 in the roots of susceptible control cultivar Mellow Star and Yolo Wonder B did not differ for M. incognita and M. arenaria but differed for M. javanica and M. haplanaria. A significantly greater number of M. incognita and M. arenaria J2 entered roots of the susceptible cultivars Mellow Star and Yolo Wonder B compared to the resistant lines Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687. For M. javanica and M. haplanaria, however, greater numbers of J2 were present in roots of Mellow Star than in roots of other cultivars/lines. No significant difference between numbers of Meloidogyne J2 was found among Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687 (Table 1). Similar results in the nematode penetration, except observing a significant difference in the number of M. incognita J2 between Mellow Star and Yolo Wonder B, were found at 3 DAI (Table 1). At 5 DAI, although, fewer J2 were present in roots of HDA-149 than in Charleston Belle, HDA-330, PM-217, and PM-687, no significant difference in M. arenaria penetration was observed among Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687 (Table 1). At 5 DAI, significantly more J2 of M. javanica and M. haplanaria penetrated Mellow Star than other cultivars/lines. Also, the number of M. javanica J2 in roots of Charleston Belle was numerically lowest, but no significant difference in the nematode penetration was found between this cultivar with other lines of HDA-149, HDA-330, and PM-687.

There was no significant difference in fresh root weight among the pepper lines infected with all Meloidogyne spp. In contrast, significant differences were observed for GI (p < 0.0001), egg counts/g fresh root (p < 0.0001), and RF (p < 0.0001) (Table 2).

While the roots of PM-217 showed lowest GI (GI = 0.2) caused by M. incognita, no significant difference for egg counts and RF was observed among the resistant peppers Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687 (Table 2). All isogenic lines had no visible root galling in contrast to Mellow Star and Yolo Wonder B which exhibited high root galling (GI = 3.7 and 4.0, respectively). The number of M. incognita eggs produced per gram of root mass was greatest in Mellow Star and Yolo Wonder B compared to all cultivars/lines examined. The RF value of M. incognita for Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687 were 0.6, 0.1, 0.9, 0.1, and 0.3, respectively (Table 2). All isogenic lines, except Yolo Wonder B and HDA-330, were highly resistant to M. incognita (Table 3). Meloidogyne arenaria produced significantly fewer galls and eggs in the roots of Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687 than in Mellow Star and Yolo Wonder B which reproduced aggressively, having an RF of 34.7 and 30.2, respectively (Table 2). Among isogenic lines examined, only Yolo Wonder B was found highly susceptible to M. arenaria (Table 3). Meloidogyne javanica was highly virulent on Mellow Star producing over 2,000 egg per g root. This cultivar had the highest GI (3.7) and RF value (23.5) as compared to other cultivars/lines that had a RF value < 0.1. No significant difference for the RF was observed among Charleston Belle and four isogenic lines. The RF value of M. javanica was higher (RF = 23.5) in Mellow Star than in other pepper cultivars/lines, with the RF values ranging from 0.01 to 0.24 (Table 2). Meloidogyne haplanaria reproduced (GI = 2.7, RF = 8.1) well on Mellow Star only, but it was not able to reproduce on the other pepper lines (Table 2). All isogenic lines examined were highly resistant to both M. javanica and M. haplanaria (Table 3).