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

Abstract Root-knot nematode (Meloidogyne spp.) exhibits a substantial problem in pepper production, causing reduction in yield throughout the world. Continued assessment for root-knot resistance is important for developing new resistance cultivars. In this study, the effect of Me and N genes on the penetration and reproduction of M. incognita race 3, M. arenaria race 1, M. javanica, and M. haplanaria was examined under controlled greenhouse conditions using susceptible and resistant pepper lines/cultivars (Mellow Star, Yolo Wonder B, Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687) differing in the presence or absence of resistant genes. The penetration and resistance responses of these pepper lines differed depending on the nematode species. More second-stage juveniles penetrated roots of susceptible control cultivar Mellow Star than roots of resistant cultivars/lines. Although, there was no significant difference in the nematode penetration among resistant lines 1 and 3 days after inoculation (DAI), variability in the penetration of M. incognita, M. javanica, and M. haplanaria was observed 5 DAI. This demonstrates the variability among different nematode resistance genes to invasion by Meloidogyne spp. Based on nematode gall index (GI) and reproduction factor (RF), Charleston Belle, HDA-149, PM-217 and PM-687 showed very high resistance (GI < 1 and RF < 0.1) to M. incognita, M. arenaria, and M. javanica. Although, all the Meloidogyne-resistant pepper lines evaluated were resistant to M. javanica and M. haplanaria, the susceptible cultivar Mellow Star was a good host for all nematode species having an RF ranging from 8.1 to 34.7. The N, Me1, and Me3 genes controlled resistance to reproduction of all species of Meloidogyne examined.

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 nonhost 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  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, 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 Meloidogyneresistant cultivars, Yolo Wonder B and Charleston Belle (Fery et al., 1998).

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 cells (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 2 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 cells 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 (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%.

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 (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.

Reproduction study
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) 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).

Discussion
The first purpose of this research was to examine the variability in penetration of four species of root-knot nematodes in pepper. We found that M. incognita race 3, M. arenaria race 1, M. javanica, and M. haplanaria were capable of entering the root systems of susceptible and resistance cultivar/lines, but the penetration rates were considerably reduced in the resistant cultivars/lines at 1, 3 and 5 DAI. These results are consistent with the results obtained by Pegard et al. (2005) and Bleve-Zaccheo et al. (1998). In our study, between 13 and 30% more M. incognita J2s were present in roots of susceptible cultivars Mellow Star and Yolo Wonder B than in roots of all resistant lines Charleston Belle, HDA-149, HDA-
330, PM-217, and PM-687 at 5 DAI. There were also significant differences between the resistant lines. Fewer M. incognita J2 penetrated the root system of Charleston Belle, HDA-149, and PM-687, than in HDA-330 and PM-217 at 5 DAI. This is consistent with Bleve-Zaccheo et al. (1998) which reported that fewer M. incognita J2 were present in root of the line HDA-149 (carrying Me3) than in HDA-330 (carrying Me1) at 4 DAI and which reported that lines carrying Me1 exhibit a delayed hypersensitive response compared to those carrying Me3. Interestingly, we did not see these same differences in the penetration of the other three Meloidogyne species tested against these same resistant lines. This could be the result of other resistance loci outside of the well characterized Me genes which may specifically contribute to resistance against these other Meloidogyne species. We found that 17 to 29% greater J2 of M. arenaria penetrated Mellow Star and Yolo Wonder B root systems than in Charleston Belle, HDA-149, HDA-330, PM-217, and PM-687, respectively at 5 DAI. The numbers of M. arenaria penetrated the pepper roots were consistent among all resistant cultivars/lines examined, suggesting that the nematode infection did not affect plant defense response. The numbers of M. javanica and M. haplanaria J2 present in the root of Mellow Star were greater than the roots of all other cultivars/lines examined. Fewer M. javanica J2 penetrated the root system of Charleston Belle, . The total number of M. haplanaria J2 in roots of Charleston Belle 5 DAI was 42 to 63% fewer than in Yolo Wonder B, HDA-149, HDA-330, PM-217, and PM-687, respectively. Our results demonstrate that J2 penetration on the plant vary with species of root-knot nematode and pepper cultivars/lines carrying resistance genes.
Resistance to root-knot nematode infection is established as an inhibition or reduction of nematode reproduction (Trudgill, 1991) or prevention of feeding site establishment (Williamson and Kumar, 2006) in host plants. In the present study, resistance responses of the pepper cultivars/lines differed depending on Meloidogyne spp. This variability in resistance to the nematode species was found based on the GI and RF among pepper lines tested. Resistance to Meloidogyne spp. in pepper is conferred by several single dominant genes. Some of these genes induce resistance to only one nematode species, while others are effective to a wide range of Meloidogyne spp. (Djian-Caporalino et al., 1999). Meloidogyne incognita race 3 was highly pathogenic on Mellow Star and Yolo Wonder B producing the GI ³ 3 and RF ³ 10, respectively. Other lines including Charleston Belle, HDA-149, PM-217, and PM-687 showed high resistance to M. incognita. The low RF (Table 3) of M. incognita in the resistant pepper lines is thought to be related to the presence of phenolic compounds and chlorogenic acid which reduce nematode penetration and affect adversely nematode development and reproduction in pepper roots (Bleve-Zaccheo et al., 1998;Pegard et al., 2005).
The isogenic line HDA-330 exhibited a GI < 1 and RF £ 1 in response to M. incognita, and was found to  Fery, 2000, 2001;Djian-Caporalino et al., 2007). While Mellow Star was highly susceptible to M. javanica, the other six pepper cultivar/lines tested were highly resistant to the nematode, which is consistent with the finding that the Me5 resistance gene, carried by Yolo Wonder, is specific against M. javanica (Hendy et al., 1985).
To our knowledge this was the first time M. haplanaria has been tested against any root-knot nematode resistant pepper lines. Interestingly, we found that all the resistant pepper lines, including Yolo Wonder B (Me5) were significantly more resistant to M. haplanaria compared to our susceptible control Mellow Star (GI = 2.7 2 and RF = 8.1). Meloidogyne haplanaria is able to overcome the resistance in tomato mediated by the Mi gene (Bendezu et al., 2004). The lack of ability of M. haplanaria to damage commercial resistant cultivars of peppers is important for their use in rotation with other vegetable crops for managing this nematode pest in infested fields in the southern USA.
In summary, fewer root-knot nematode J2 penetrated the roots of the resistant peppers than susceptible peppers. This study showed a correlation between the fullness of resistance and lines specificity during the examination of pepper lines carrying different root-knot nematode resistance genes. Our data supports previously observed virulence/ avirulence trends for M. incognita, M. javanica, and M. arenaria against pepper lines carrying the N, Me1,Me3(=Me7), and Me5 resistance genes, and has provided evidence that all four of these genes are also likely to be effective against M. haplanaria. Identification of the genes and molecular pathways involved in pepper responses and development of genetic markers for resistance will facilitate the breeding programs for the development of peppers with broad and durable Meloidogyne resistance.