Materials and Methods

Inoculum preparation.

Single egg mass isolates of M. incognita race 3, M. arenaria, M. javanica, M. floridensis, and M. enterolobii were obtained from the Entomology and Nematology Department, University of Florida. The identity of each species was confirmed via esterase and malate dehydrogenase enzyme profiles (Dickson, et al., 1971) and host differential tests (Taylor and Sasser, 1978). All Meloidogyne spp. were increased on tomato (Solanum lycopersicum L. cv. BHN 589) and maintained in the greenhouse at 25 ± 5 °C. Eggs were extracted from the infected tomato roots by dissolving the gelatinous egg sacs with 0.25% (w/v) sodium hypochlorite solution (Hussey and Barker, 1973), and eggs were captured on a sieve with 25-μm-pore openings. The eggs were allowed to hatch at room temperature using a modified Baermann technique with the eggs placed on a wire mesh lined with moistened tissue paper inside a petri dish. Only 24-h-old J2s were used for the experiments.

Pepper genotypes.

Seeds of ‘California Wonder’ bell pepper, ‘Round of Hungary’ sweet pepper, and ‘Jalapeño’ chili pepper were purchased from Park Seeds (Greenwood, SC), and ‘Charleston Belle’ bell pepper and ‘Datil’ chili pepper were purchased from Baker Creek Heirloom Seeds (Mansfield, MO). ‘California Wonder’ is a known susceptible variety (Thies et al., 1998). Resistance to M. incognita is relatively well-described in ‘Charleston Belle’ (Fery et al., 1998), whereas resistances in ‘Round of Hungary’, ‘Jalapeño’, and ‘Datil’—a hot pepper variety commercially grown predominantly in Saint Augustine, FL—have not been formally tested. The advanced inbred line ‘UFRJ107(6)A3’ originated from the controlled crossing of ‘Round of Hungary’ with pollen from ‘Jalapeno’ (Fig. 1). ‘UFRJ107(6)A3’ is an F₇ breeding line derived from an F₂ plant grown in the greenhouse and selected based on broad-mite tolerance. Superior F₃ lines selected for their general field performance and fruit quality were advanced through F₇. Therefore, the pedigree of ‘UFRJ107(6)A3’ combines the desirable characteristics of ‘Jalapeño’ (for pungency) and ‘Round of Hungary’ (for fruit color, high brix, and fleshy pericarp), as well as novel traits resulting from selections including broad-mite tolerance and adaptability to subtropical climate.

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Preparation of plant materials.

Seeds were surface-treated with 10% (v/v) commercial bleach for 20 min, rinsed with sterile water five times, and allowed to germinate in a 125-mL erlenmeyer flask containing 25 mL 0.1% (w/v) potassium nitrate solution. The flask was incubated on an orbital shaker (80 rpm at 28 °C). The germinated seeds were planted in 128-cell plug trays with autoclaved (121 °C for 30 min) peat-based soil mix (Fafard 3B; Sun Gro Horticulture, Agawam, MA). Each plant was watered daily with 50 mL water and fertilized every 3 weeks with 50 mL 0.21% (w/v) fertilizer solution (24N–8P–16K, Miracle-Gro; Scotts Miracle-Gro Products, Inc., Marysville, OH). The plants were maintained on lighted shelves at 25 ± 2 °C for 8 weeks before they were transferred to environmental growth chambers. Four- to five-leaf-stage seedlings were transplanted individually, unless otherwise mentioned, to autoclaved all-purpose sand (Quikrete, Atlanta, GA) contained in 251-mL Styrofoam cups with needle-punched drainage holes in the bottom.

Growth chamber conditions.

Experiments to assess reproduction, root penetration, and host preference of RKN were performed in plant growth chambers (Percival Scientific, Perry, IA) set at a constant temperature of 28 °C or 30 °C with a 12-h photoperiod (150 µmol·m⁻²·s⁻¹ provided by fluorescent lamps). Plants were watered and fertilized as described.

Assessment of nematode reproduction.

The nematode inocula (24-h-old J2s) were pipetted into three holes (depth, 3 cm) made in the soil around the plant base. During the first growth chamber experiment at 28 °C that examined M. incognita resistance in five pepper genotypes, the applied inoculum of freshly hatched J2 ranged from 60 to 240; the level of inoculum was adjusted based on the plant size variation. During subsequent growth chamber experiments at 30 °C and tests with five RKN species (M. incognita, M. arenaria, M. javanica, M. enterolobii, and M. floridensis), the level of nematode inoculum was 240 J2s per pot. Five plants of each pepper genotype were maintained in the growth chamber for 30 d; after which, the plants were removed from containers and the roots were washed gently with running water to remove sand particles. Root fresh weights were recorded to estimate the number of egg masses and eggs per gram of root before soaking the roots in red food color (McCormick and Co. Inc., Hunt Valley, MD) to aid with the observation of egg masses (Thies et al., 2002). Nematode reproduction was assessed by counting the stained egg masses. No data were collected for plants that were damaged by wilt disease during the course of the experiment. The stained egg masses on roots were counted under the stereomicroscope equipped with an overhead illuminator. Each plant was rated based on the number of egg masses using a scale of 0 to 5: 0 = no egg mass; 1 = 1 to 2 egg masses per whole root system; 2 = 3 to 10 egg masses; 3 = 11 to 30; 4 = 31 to 100; and 5 = more than 100 (Taylor and Sasser 1978). After counting the egg masses, eggs were extracted from whole root systems using the sodium-hypochlorite method (Hussey and Barker, 1973). Nematode reproduction was measured by calculating the reproduction factor (RF = ratio of the number of nematode eggs at 30 d postinoculation to the initial number of J2s with which the plants were inoculated). Egg and egg mass densities, expressed as number per gram of fresh root to account for variations in the size of roots from different pepper genotypes, were also compared.

Root penetration of M. incognita race 3.

Pepper seeds were germinated and seedlings were maintained as mentioned. At transplanting, 500 J2s suspended in 3 mL water were pipetted into three holes (3-cm deep) made in the soil around the plant base. Six replicates each of ‘Datil’ and ‘UFRJ107(6)A’ were maintained in a growth chamber at 30 °C with a 12-h photoperiod. At 7 d after inoculation, plants were removed from containers and washed gently with running water to remove soil particles. Roots were soaked in 1.5% sodium hypochlorite solution for 2 min and stained with acid fuchsin (Byrd et al., 1983) before examination under a stereomicroscope.

Single-pot choice test.

In the growth chamber set at 30 °C, 500 M. incognita race 3 J2s were inoculated in each pot containing two plants. Each plant in the pot was inoculated with 250 J2s suspended in 3 mL water and distributed to three holes around the stem base of each plant, for a total of 6 mL (500 J2s) per pot when two plants were grown in the same container. In this experiment, a total of 12 plants were distributed to the following treatments, each with two replicate pots: 1) two ‘California Wonder’ plants per pot; 2) two ‘UFRJ107(6)A3’ plants per pot; and 3) one plant of each genotype together in the same pot. The preference of RKN was evaluated at 7 d after inoculation by root-staining with acid fuchsin (Byrd et al., 1983) and examining root tissues under a stereomicroscope for the presence of J2s.

Statistical treatment of data.

During all experiments, a completely randomized design was used. Quantitative data were used to calculate means and the se. Data were normalized using the square-root (x + 1) transformation for RF and egg mass index and log (x + 1) transformation for egg or egg mass numbers per gram of root before being subjected to one-way analysis of variance (ANOVA) using the SAS statistical package (SAS Institute, Cary, NC). For nematode reproduction parameters that differed significantly (P ≤ 0.05) based on the ANOVA, Tukey’s honestly significant difference test (P ≤ 0.05) was used to compare means across pepper genotypes. The statistical significance of the difference in nematode root penetration between two genotypes was tested using Student’s t test (P ≤ 0.05).

Results and Discussion

We used multiple methods to characterize RKN resistance of pepper using an inbred line developed by our program and several commercially available varieties. Because the nematode reproductive ability in a host is directly related to the host’s resistance (Taylor and Sasser, 1978), the egg mass index and RF were used as key parameters to assess the level of nematode resistance in ‘Datil’, ‘Round of Hungary’, ‘Jalapeño’, and ‘UFRJ107(6)A3’, along with known resistant (‘Charleston Belle’) and susceptible (‘California Wonder’) cultivars. The RKN penetration in host roots and resistance to multiple species of Meloidogyne were performed to gain a better understanding of the nature and spectrum of resistance in ‘UFRJ107(6)A3’.

The reproductive potential of M. incognita race 3 was affected by the genotype of its host (Table 1). At 28 °C, the mean egg masses per gram of root ranged from 0.2 to 58, with ‘California Wonder’ and ‘Datil’ being most susceptible, ‘Charleston Belle’ being moderately susceptible, and ‘Jalapeno’ and ‘UFRJ107(6)A3’ being highly resistant. The RFs reflected these gradations in M. incognita resistance, with the highest mean RF for ‘California Wonder’, followed by ‘Datil’, ‘Charleston Belle’, ‘Jalapeño’, and ‘UFRJ107(6)A3’ in decreasing order.

Table 1.Reproductive potential of Meloidogyne incognita race 3 on six varieties of Capsicum species after 30 d from inoculation under two temperature regimes in the growth chamber.

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The trial was repeated at 30 °C and included ‘Round of Hungary’, which is a parental variety of ‘UFRJ107(6)A3’ (Fig. 1, Table 1). The mean egg masses per gram of root ranged from 0.3 to 62, with the highest value in ‘Round of Hungary’, followed by ‘Charleston Belle’, ‘California Wonder’, ‘Jalapeño’, and ‘UFRJ107(6)A3’ (Table 1) in decreasing order. Mean RF values followed the same order of ranking as mean egg masses per gram of root (Table 1). Although a full replication of ‘Datil’ was not included in the experiment at 30 °C, two plants that were inoculated were highly susceptible to M. incognita (data not shown). ‘Datil’ did not exhibit the high levels of M. incognita resistance found in some Scotch Bonnet-type peppers​, thereby reflecting the intraspecific variability of M. incognita resistance in C. chinense (Fery and Thies, 1997; Gisbert et al., 2013).

The resistance responses of ‘UFRJ107(6)A3’ from the two growth chamber experiments at constant temperatures of 28 and 30 °C were in good agreement, suggesting stability of its resistance at a higher temperature. ‘UFRJ107(6)A3’ was highly resistant to M. incognita in both trials (egg mass index and RF <1) (Table 1). Because the mean RF value for ‘Jalapeño’ was less than that of ‘Round of Hungary’, we surmised that ‘UFRJ107(6)A3’ may have inherited M. incognita resistance from the ‘Jalapeño’ parent. Nematode reproduction in ‘UFRJ107(6)A3’ was minimal and less than that of its ‘Jalapeño’ parent; hence, additional genetic factors may be involved in the resistance of ‘UFRJ107(6)A3’, which is not uncommon in C. annuum (Fery and Dukes, 1996).

The susceptibility of ‘California Wonder’ to M. incognita race 3 in this study confirmed previous observations by Thies et al. (1998). ‘Charleston Belle’, homozygous for the N gene, was reported to be resistant to M. incognita race 3 under field and greenhouse conditions (Fery et al., 1998; Hajihassani et al., 2019); however, under the conditions used in the present study, this cultivar was susceptible (RF = 24) when compared with ‘UFRJ107(6)A3’ (RF = 0.2–0.8) (Table 1). Results have been inconsistent with regard to resistance in ‘Charleston Belle’ during field, growth chamber, and greenhouse tests. Thies and Fery (1998) reported that the expression of N gene is modified at high temperatures (28 and 32 °C) and further confirmed partial loss of resistance (based on eggs per gram of fresh root and reproductive index) conferred by the N gene in C. annuum and an allelic gene in C. chinense at these higher temperatures in growth chambers (Thies and Fery, 2000). However, in field trials set in a subtropical environment with soil temperatures ranging widely from 17 to 37 °C in spring and 20 to 36 °C in fall, minimal galling (less than 3% root system galled) on ‘Charleston Belle’ was observed (Thies, et al., 2008). Under greenhouse conditions, Aguiar et al. (2014) observed higher infestation levels of M. incognita J2 in ‘Charleston Belle’ than in ‘Carolina Wonder’, which is also homozygous for the N gene, after 3 months from inoculation. Although we do not know the underlying cause for the instability of resistance in ‘Charleston Belle’, the different results obtained in the present study may be related to the assay conditions, including seedling age, evaluation period, continuous exposure to high temperatures in the growth chamber, and the nature of the nematode population used.

We compared ‘UFRJ107(6)A3’ with ‘California Wonder’ to determine resistance to five different species of Meloidogyne using similar bioassays at 28 °C, as described. Based on egg masses per gram of root, eggs per gram of root, egg mass index, and RF, ‘California Wonder’ was highly susceptible to M. incognita race 3, whereas ‘UFRJ107(6)A3’ was resistant (Table 2). Both genotypes were resistant to M. arenaria and M. floridensis but susceptible to M. enterolobii and M. javanica (Table 2). The RF of Meloidogyne enterolobii was ≈10-fold higher than that of M. javanica on both genotypes. Meloidogyne arenaria failed to reproduce on ‘UFRJ107(6)A3’, as did M. floridensis on both ‘UFRJ107(6)A3’ and ‘California Wonder’ (Table 2).

Table 2.Degree of resistance in Capsicum annuum ‘UFRJ107(6)A3’ to five Meloidogyne species in comparison with M. incognita-susceptible cultivar California Wonder after 30 d from inoculation.

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Although the ‘UFRJ107(6)A3’ line has shown resistance to four root-knot nematode species M. incognita, M. javanica, M. arenaria, and M. floridensis, additional sources of resistance to M. enterolobii are needed because this species has become highly pathogenic to economically important crops. It has been reported that M. enterolobii is able to overcome resistance conferred by the Mi-1 gene in tomato and N gene in pepper (Kiewnick et al., 2009). Elucidation of the mechanisms involved in plant–nematode interactions, specifically resistance to M. incognita but susceptibility to M. enterolobii in ‘UFRJ107(6)A3’, is an important area for further research to increase the efficacy and durability of plant resistance.

As a starting point for understanding the mechanism of resistance in ‘UFRJ107(6)A3’, we compared the root penetration response of M. incognita J2s in ‘UFRJ107(6)A3’ with that of ‘Datil’. The ability of the nematode to penetrate and migrate through the root cortex was affected by the host genotype (Table 3). Seven days after inoculation with 500 J2s, an average of 102 J2s were observed in roots of ‘Datil’ plants, whereas no J2s were found in roots of five out of six plants of ‘UFRJ107(6)A3’; the one plant had only one J2 inside the root (Table 3). During a single-pot choice test, when M. incognita-susceptible ‘California Wonder’ was planted together with ‘UFRJ107(6)A3’, ≈40 J2s had penetrated the roots of ‘California Wonder’, but none penetrated ‘UFRJ107(6)A3’ at 7 d after inoculation (Fig. 2, Table 4). Similar results were obtained when two of the same genotype were planted together in one pot because no nematodes penetrated the roots of ‘UFRJ107(6)A3’ (Table 4), confirming that resistance in ‘UFRJ107(6)A3’ is due to a lack of nematode penetration. This finding is consistent with those of previous reports that the nematode resistance mechanism in C. annuum genotypes ‘HDA149’ (Me1 and Me3 genes), ‘PM687’ (Me3 gene), and ‘CM334’ (Me7 gene) acted at the early stages of root invasion that limited penetration or blocked migration of the RKN (Bleve-Zacheo et al., 1998; Pegard et al., 2005). The nature of such an impediment in ‘UFRJ107(6)A3’ could be the presence of mechanical or biochemical barriers to RKN and needs further investigation.

Table 3.Number of M. incognita race 3 second-stage juveniles (J2s) observed in roots of Capsicum annuum ‘UFRJ107(6)A3’ and C. chinense ‘Datil’ peppers after 7 d from inoculation with 500 J2s.

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Table 4.Comparison of numbers of M. incognita race 3 second-stage juveniles (J2s) in roots of Capsicum annuum ‘UFRJ107(6)A3’ and ‘California Wonder’ after 7 d from inoculation with 500 J2s in a “choice test” where J2s had access to roots of either genotype.

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The primary purpose of our research was to examine the reproductive potential and penetration ability of M. incognita race 3 on a breeding line and open-pollinated varieties that were not previously evaluated for resistance. We found that the reproductive potential of M. incognita race 3 on the advanced breeding line ‘UFRJ107(6)A3’ was remarkably less than that of ‘Charleston Belle’. In addition, ‘UFRJ107(6)A3’ seemed to be able to prevent or limit the penetration and establishment of feeding sites and was broadly resistant against RKNs. During 7-d root penetration tests and choice tests, M. incognita race 3 J2s preferred the roots of susceptible varieties (‘Datil’ and ‘California Wonder’) over ‘UFRJ107(6)A3’. Future research is required to test whether inherent mechanisms of RKN resistance in ‘UFRJ107(6)A3’ are similar to that conferred by the Me3 gene in which the pepper line ‘PM687’ demonstrated immediate cellular necrosis in the root epidermis and inhibited penetration of M. incognita (Djian-Caporalino et al., 2001; Pegard et al., 2005). During Spring 2019, ‘UFRJ107(6)A3’ and two other breeding lines were grown in a field site infested with M. incognita. At the end of that season, roots of six plants per genotype were stained to evaluate egg masses. Although roots of two other lines contained egg masses, none was found in the roots of ‘UFRJ107(6)A3’ (data not shown). Altogether, ‘UFRJ107(6)A3’ has consistently shown a high degree of RKN resistance that would be useful for breeding programs and commercial pepper production. Its resistance is superior to that of the commercial cultivar Charleston Belle; therefore, it offers potential as a broad-spectrum RKN-resistant rootstock for grafting sweet bell peppers because the disease package in many of the modern hybrid pepper cultivars is lacking in RKN resistance.