Root-knot nematodes, Meloidogyne spp., are one of the most yield-limiting parasites of peppers (Capsicum annuum L.) in the USA and worldwide (Sasser and Freckman, 1987; Thies and Fery, 2000). These parasites are widely distributed across the world and are adapted to develop and reproduce on peppers in tropical and subtropical climates. Infection of peppers by Meloidogyne spp. can cause changes in the plant physiology, fruit morphology and yield. Three species of M. incognita (Kofoid & White) Chitwood, M. arenaria (Neal) Chitwood, and M. javanica (Treub) Chitwood are particularly significant nematode pests of pepper (Fery et al., 1998; Castagnone-Sereno et al., 2001). Meloidogyne haplanaria, Eisenback, Bernard, Starr, Lee & Tomaszewski, a resistant (Mi gene)-breaking root-knot species of tomato, has also been reported to infect and reproduce on pepper (Eisenback et al., 2003; Bendezu et al., 2004; Joseph et al., 2016).
Successful management of Meloidogyne spp. in pepper includes one or combination of several tactics including rotation with non-host crops, chemical control using fumigant and non-fumigant nematicides, use of resistant cultivars, and other options. Soil fumigation with chemical products is widely used in pepper production in the Southern USA for the control of parasitic nematodes, soilborne pathogens, and weeds. Although some fumigant nematicides are currently available, they do not provide the level of control that was previously observed with methyl bromide and are more challenging to use by most growers (by including fumigant management plans and buffer zones). Additionally, use of non-fumigant nematicides is considered to be a short-term option for the control of root-knot nematodes on peppers grown on plasticulture systems with nematode population levels increasing at the end of the growing season. Since non-fumigant nematicides must be applied season after season, they are prohibitively costly for growers. Also, rotating pepper with non-host crops is not a long-term management option in limiting Meloidogyne spp. damage because of wide host range of the nematode (Trudgill and Blok, 2001). For these reasons, research efforts have been directed toward the development of sustainable and eco-friendly nematode management approaches.
In this context, the use of resistant cultivars with acceptable growth and yield characteristics appears to be an effective strategy to manage plant-parasitic nematodes, in particular root-knot nematodes (Hendy et al., 1985; Thies and Fery, 2000; Pegard et al., 2005). Resistance to root-knot nematode infection is established as an inhibition or decrease of nematode penetration and/or reproduction (Trudgill, 1991; Williamson and Kumar, 2006) and is characterized by a localized hypersensitive reaction in host plants (Pegard et al., 2005). The development of successful Meloidogyne-resistant pepper breeding programs is dependent on the characterization of new resistant pepper lines. Multiple dominant resistance genes effective against several species of root-knot nematodes have been discovered in the relative and wild species of peppers (Hendy et al., 1985). These genes are denoted as N, Me1, Me2, Me3 (=Me7), Me4, Me5, Me6, Mech1, and Mech2 (Hendy et al., 1985; Djian-Caporalino et al., 2001, 2007). Three of these genes (N, Me1, and Me3) are broadly effective against the three most widespread tropical root-knot nematode species (M. incognita, M. javanica, and M. arenaria). Pepper cultivars/lines carrying different resistant genes differ in their ability to withstand invasion and reproduction of different species or populations of root-knot nematodes (Bleve-Zaccheo et al., 1998; Pegard et al., 2005).
In the USA, investigation of resistance to root-knot nematodes in peppers has been restricted primarily to pepper lines carrying the N gene (Fery et al., 1998; Thies and Fery, 2000; Thies, 2011). Four isogenic lines (HDA-149, HDA-330, PM-217, and PM-687) were developed in France to be incorporated into pepper breeding programs. These lines carry additional resistance genes against Meloidogyne spp. and have been extensively evaluated against the M. incognita group species (MIG) (Hendy et al., 1985, Djian-Caporalino et al., 2001, 2007). However, the levels of resistance in these lines have not been assessed against populations of M. arenaria race 1 and M. javanica from the Southern USA, and to our knowledge these resistance genes have never been assessed for their efficacy against M. haplanaria. Meloidogyne haplanaria is a recently described species from the southern USA that is known to overcome the widely used Mi gene in tomato, but has not been evaluated against resistance genes from other solonacious crops (Eisenback et al., 2003, Bendezu et al., 2004, Joseph et al., 2016). Due to the well documented hyper variability of the MIG species, and their ability to break resistance in pepper lines carrying the Me3 and N genes (Castagnone-Sereno et al., 2001; Thies, 2011, Bucki et al., 2017), it is imperative to continue assessing the existing resistant pepper lines against different populations and species of Meloidogyne. The aim of this study was to examine the penetration and reproduction responses of HDA-149, HDA-330, PM-217, and PM-687 to M. incognita race 3, M. arenaria race 1, M. javanica, and M. haplanaria, and compared their responses to the Meloidogyne-resistant cultivars, Yolo Wonder B and Charleston Belle (Fery et al., 1998).
Materials and methods
Nematode inoculum
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.
Pepper cultivars/lines
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).
Penetration study
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.
Reproduction study
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.
Analysis of data
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%.
Results
Penetration study
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.
Table 1
Number of Meloidogyne incognita race 3, M. arenaria race 1, M. javanica, and M. haplanaria in roots of susceptible and resistant pepper lines at 1, 3, and 5 days after inoculation.
| Days after nematode inoculation | |||||
|---|---|---|---|---|---|
| Cultivar/line (resistant gene) | 1 | 3 | 5 | ||
| M. incognita race 3 | |||||
| Mellow StarX | 11.0±1.24 a | 21.8±2.47 a | 39.0±4.50 a | ||
| Yolo Wonder B (Me5)Y | 8.1±1.16 a | 15.0±1.74 b | 38.1±3.9 a | ||
| Charleston Belle (N)Y | 0.6±0.30 b | 3.8±0.72 c | 7.5±0.90 bc | ||
| HDA-149 (Me3) | 0.3±0.30 b | 3.0±0.60 c | 5.1±0.74 c | ||
| HDA-330 (Me1) | 0.8±0.45 b | 7.3±0.83 c | 12.0±1.16 b | ||
| PM-217 (Me1,Me2) | 0.5±0.30 b | 6.3±1.32 c | 10.6±2.60 b | ||
| PM-687 (Me3,Me4) | 0.5±0.30 b | 3.8±1.20 c | 6.8±1.30 bc | ||
| M. arenaria race 1 | |||||
| Mellow Star | 10.1±1.45 a | 17.1±2.93 a | 35.2±6.0 a | ||
| Yolo Wonder B | 7.8±1.85 a | 12.6±1.74 a | 37.0±5.8 a | ||
| Charleston Belle | 0.1±0.16 b | 2.3±0.77 b | 5.1±1.4 b | ||
| HDA-149 | 0.1±0.16 b | 3.0±0.77 b | 10.6±1.1 b | ||
| HDA-330 | 0.6±0.30 b | 5.0±0.78 b | 10.3±0.8 b | ||
| PM-217 | 0.3±0.30 b | 5.5±0.86 b | 9.5±1.1 b | ||
| PM-687 | 0.3±0.30 b | 4.6±1.67 b | 6.6±1.2 b | ||
| M. javanica | |||||
| Mellow Star | 9.6±0.9 a | 18.7±2.3 a | 30.7±4.5 a | ||
| Yolo Wonder B | 0.5±0.06 b | 3.8±1.0 b | 9.8±1.2 b | ||
| Charleston Belle | 0.1±0.02 b | 2.1±0.7 b | 4.6±1.3 c | ||
| HDA-149 | 0.5±0.03 b | 4.8±1.0 b | 9.0±2.6 bc | ||
| HDA-330 | 0.6±0.1 b | 3.6±1.3 b | 5.8±1.1 bc | ||
| PM-217 | 0.3±0.02 b | 4.8±1.4 b | 9.3±2.3 b | ||
| PM-687 | 0.3±0.02 b | 1.8±0.72 b | 6.3±0.94 bc | ||
| M. haplanaria | |||||
| Mellow Star | 7.2±1.04 a | 16.6±2.03 a | 21.1±2.1 a | ||
| Yolo Wonder B | 0.6±0.18 b | 4.5±0.8 b | 10.1±1.1 bc | ||
| Charleston Belle | 0.2±0.04 b | 2.5±0.8 b | 4.5±1.0 d | ||
| HDA-149 | 0.3±0.16 b | 3.9±0.7 b | 8.3±1.2 bc | ||
| HDA-330 | 0.3±0.06 b | 5.6±1.6 b | 9.6±2.1 bc | ||
| PM-217 | 0.3±0.06 b | 3.6±0.9 b | 10.8±1.1 b | ||
| PM-687 | 0.6±0.03 b | 4.3±1.3 b | 7.1±1.7 cd | ||
| Cultivar/line | Root weight | Gall indexY | Eggs/g root | Reproduction factorZ | |
|---|---|---|---|---|---|
| M. incognita race 3 | |||||
| Mellow StarX | 11.6±1.2 a | 3.7±0.6 a | 2,966.5±769.1 b | 33.9±7.37 b | |
| Yolo Wonder BW | 11.2±1.4 a | 4.0±0.5 a | 4,387.4±1,037.6 a | 48.1±9.87 a | |
| Charleston BelleW | 10.1±1.7 a | 0.5±0.3 b | 73.2±12.3 c | 0.6±0.09 c | |
| HDA-149 | 10.3±1.5 a | 0.3±0.3 b | 6.5±2.0 c | 0.1±0.01 c | |
| HDA-330 | 11.4±0.9 a | 0.7 ±0.2 b | 88.4±24.0 c | 0.9±0.27 c | |
| PM-217 | 11.3±1.5 a | 0.2±0.3 c | 6.7±1.7 c | 0.1±0.02 c | |
| PM-687 | 11.7±0.7 a | 0.3±0.3 b | 24.4±5.1 c | 0.3±0.06 c | |
| M. arenaria race 1 | |||||
| Mellow Star | 11.1±1.4 a | 3.6±0.7 a | 3,205.3±718.1 a | 34.7±6.07 a | |
| Yolo Wonder B | 10.0±1.5 a | 3.1±0.5 a | 3,163.4±904.2 a | 30.2±9.88 a | |
| Charleston Belle | 11.1±1.2 a | 0.4±0.4 b | 10.6±0.8 b | 0.05±0.01 b | |
| HDA-149 | 10.8±1.7 a | 0.4±0.3 b | 6.5±1.6 b | 0.07±0.02 b | |
| HDA-330 | 10.1±1.3 a | 0.7±0.3 b | 32.8±9.3 b | 0.31±0.07 b | |
| PM-217 | 10.7±1.6 a | 0.4±0.4 b | 8.4±1.7 b | 0.08±0.02 b | |
| PM-687 | 11.5±1.1 a | 0.5±0.3 b | 3.5±0.9 b | 0.04±0.01 b | |
| M. javanica | |||||
| Mellow Star | 11.7±1.3 a | 3.7±0.8 a | 2,059.5±454.0 a | 23.5±4.46 a | |
| Yolo Wonder B | 9.4±1.6 a | 0.9±0.4 b | 5.0± 1.4 b | 0.04±0.01 b | |
| Charleston Belle | 10.4±1.7 a | 0.4±0.4 b | 1.9±1.1 b | 0.01±0.01 b | |
| HDA-149 | 9.5±1.5 a | 0.4±0.3 b | 10.1±3.6 b | 0.09±0.02 b | |
| HDA-330 | 10.3±1.8 a | 0.5±0.4 b | 8.7±5.6 b | 0.06±0.03 b | |
| PM-217 | 10.1±1.5 a | 0.4±0.3 b | 24.6±8.9 b | 0.24±0.01 b | |
| PM-687 | 10.1±1.7 a | 0.7±0.3 b | 1.7±0.7 b | 0.01±0.00 b | |
| M. haplanaria | |||||
| Mellow Star | 12.4±1.4 a | 2.7±1.25 a | 655.0±57.0 a | 8.1±1.25 a | |
| Yolo Wonder B | 10.3±1.1 a | 0.3±0.02 b | 5.4±2.4 b | 0.06±0.02 b | |
| Charleston Belle | 9.3±1.3 a | 0.4±0.06 b | 20.2±8.9 b | 0.17±0.06 b | |
| HDA-149 | 10.7±1.1 a | 0.4±0.04 b | 12.7±4.9 b | 0.13±0.04 b | |
| HDA-330 | 10.5±1.1 a | 0.7±0.07 b | 25.9±7.3 b | 0.27±0.07 b | |
| PM-217 | 10.5±1.1 a | 0.4±0.01 b | 3.5±1.5 b | 0.03±0.01 b | |
| PM-687 | 12.0±1.2 a | 0.6±0.04 b | 13.3±2.9 b | 0.15±0.03 b | |
| Cultivar/Line | Nematode resistance genes | M. incognita race 3 | M. arenaria race 1 | M. javanica | M. haplanaria |
|---|---|---|---|---|---|
| Mellow Star | None | HS | HS | HS | S |
| Yolo Wonder B | Me5 | HS | HS | HR | HR |
| Charleston Belle | N | HR | HR | HR | HR |
| HDA-149 | Me3 (=Me7) | HR | HR | HR | HR |
| HDA-330 | Me1 | R | HR | HR | HR |
| PM-217 | Me1, Me2 | HR | HR | HR | HR |
| PM-687 | Me3 (=Me7), Me4 | HR | HR | HR | HR |