2025 Volume 94 Issue 2 Pages 243-254
Soilborne pest and disease management is one of the most important challenges in the cultivation of sweet bell pepper, sweet pepper, and chili pepper (Capsicum annuum L.). In Japan, the nematode Meloidogyne incognita is a soilborne pest responsible for considerable economic losses. Unfortunately, although F1 rootstock cultivars resistant to M. incognita infestation have been developed, resistance-breaking populations of M. incognita have emerged that can attack these cultivars. To address this issue through crossbreeding, breeding materials that are resistant to the new populations of resistance-breaking nematodes are needed. Here, we obtained 288 Capsicum germplasms from the NARO Genebank (Japan) and screened them for resistance to infestation by normal nematodes (strain Mi-Nishigoshi), resistance-breaking nematodes (‘LS 2341’-derived strain), and nematodes collected from 14 sweet pepper fields in Miyazaki Prefecture, Japan. Two of the germplasms, ‘NuMex Bailey Piquin’ and ‘PM 217-1-3’, showed strong resistance to all of the M. incognita tested and were used for progeny testing. ‘NuMex Bailey Piquin’ was selected as the parental line for a new F1 rootstock with resistance to a wide range of nematodes, including resistance-breaking nematodes. Since segregation of resistance was observed in ‘NuMex Bailey Piquin’, we selected one line named ‘J159’ among the selfing population of ‘NuMex Bailey Piquin’ to fix the resistance to the resistance-breaking nematode. We then evaluated the nematode resistance of ‘J159’ F1s crossed with the doubled haploid line ‘KLDH89’, which is resistant to bacterial blight, normal nematodes, and pepper mild mottle virus (pathotype P1,2), but not to resistance-breaking nematodes. The ‘KLDH89 × J159’ cross not only showed high resistance to resistance-breaking nematodes, but also high yield when used as a rootstock. It is registered in Japan under the rootstock cultivar name ‘Dai-Hinata’.
Sweet bell peppers, sweet peppers, and chili peppers (Capsicum annuum L.) are widely grown around the world and are used in various cuisines and as spices; they are some of the most important vegetables that characterize the food culture of various regions around the world. In 2021, the global cultivated area of sweet pepper was 2.06 million hectares and production amounted to 36.29 million tonnes. Two-thirds were produced in Asia, mainly in China and Indonesia (FAOSTAT 2021, http://faostat.fao.org/). However, in cultivation areas, the damage caused by the nematode Meloidogyne incognita, bacterial wilt, Phytophthora blight, and pepper mild mottle virus (PMMoV; genus, Tobamovirus) infection are becoming more severe (Parisi et al., 2020). In the past, these soilborne pests and diseases were effectively controlled by the chemical fumigant methyl bromide; however, this chemical was designated as an ozone-depleting substance under the Montreal Protocol of 1992, and its production and use in developed countries, except where deemed essential, was prohibited in 2005. Subsequent decreases in the use of methyl bromide for essential purposes ultimately led to its being phased out of production. Following the 23rd Meeting of the Parties to the Montreal Protocol on Substances that Deplete the Ozone Layer in 2011, Japan stopped using methyl bromide after 2014. Since 2014, methyl bromide has not been used for soil disinfection in Japan.
The lack of an effective alternative to methyl bromide has resulted in inadequate control of soilborne pests and diseases in sweet pepper production areas in Japan. As an alternative to chemical-based measures to control soilborne pests and diseases, sweet pepper scions with resistant traits have been bred, with the initial focus on the control of Phytophthora blight (Kobayashi et al., 1984; Yanokuchi et al., 1993). However, resistance to individual soilborne pests and diseases is often found only in genetically distant germplasms; these are difficult to breed for the coexistence of resistance, yield, and quality. This has led to the breeding of rootstock cultivars for grafting instead, several of which have been reported, including ‘Dai-Power’ (Saito et al., 2011), which was bred from the bacterial wilt–resistant, Japan-originated sweet pepper cultivar ‘Mie-Midori’ (Matsunaga and Monma, 1999) and the Phytophthora blight–resistant, Mexican breeding material ‘SCM-334’ (Ortega et al., 1991). In addition, F1 hybrid rootstock cultivars that possess multiple dominant resistance genes inherited from their parental lines have been developed; examples include ‘Miyazaki Daigi 3gou’ (Semi et al., 2013), ‘Murasaki L4 Daisuke’ (Genda et al., 2017), and ‘Dai-Pawer Z’ (Matsunaga and Saito, 2018), which were bred from the bacterial wilt–resistant Malaysian breeding material ‘LS 2341’ (Mimura et al., 2009). The two rootstock breeding materials ‘LS 2341’ and ‘SCM-334’ (‘CM334’) are reported to possess resistance not only to bacterial wilt and Phytophthora blight, but also to M. incognita infestation (Iwahori et al., 2015; Pegard et al., 2005), suggesting that use of these two rootstock materials could lead to cultivars with high M. incognita resistance. Unfortunately, however, it was found that M. incognita can still infest rootstock cultivars derived from ‘LS 2341’ and ‘SCM-334’ (‘CM334’) (Iwahori et al., 2015).
Miyazaki Prefecture is one of the largest sweet pepper production areas in Japan, with a cultivated area of 304 ha, a shipment volume of 26,400 tons (Ministry of Agriculture, Forestry, and Fisheries, 2023a), and a production value of 11 billion yen (Ministry of Agriculture, Forestry, and Fisheries, 2023b). However nematode damage has been increasing since 2010 in sweet pepper cultivation fields planted with traditional nematode-resistant rootstock cultivars (Akagi and Chishaki, 2014). To reduce the damage caused by these populations of resistance-breaking nematodes (Rb-nematodes), new resistant rootstock cultivars are needed. However, no breeding materials with such resistance have been reported. Here, we screened 288 pepper germplasms obtained from the NARO Genebank (Tsukuba, Japan) for resistance to Rb-nematodes. The results confirmed that the F1 progeny of the resistant material was also resistant to the Rb-nematode, and that the resistance was inherited with a high dominant effect. We then bred a practical rootstock cultivar from this material.
Tables 1 and S1 provide details of the plant materials used in this study. To identify breeding materials with resistance to the Rb-nematode, we screened 288 germplasms of the genus Capsicum obtained from the NARO Genebank. In addition, we tested two lines that showed strong nematode resistance in previous phenotypic observations at Miyazaki Agricultural Research Institute (Miyazaki, Japan): ‘Jal A’, a screened line derived from ‘Jalapeño’ (NARO Genebank), and ‘Dul B’, a screened line derived from ‘Dulce Ruby King’ (NARO Genebank). As control cultivars that are susceptible to both normal and Rb-nematodes, we used ‘K9-11’ (Miyazaki Agricultural Research Institute; Sugita et al., 2004), ‘Kyo-Suzu’ (Takii & Co., Ltd., Kyoto, Japan), and ‘Beruhomare’ (Nagano Chushin Agricultural Experiment Station, Nagano, Japan; Kobayashi et al., 1984). As control cultivars that are resistant to normal nematodes, but susceptible to the Rb-nematode, we used ‘LS 2341’ (NARO Genebank), ‘SCM-334’ (Institute of Vegetable and Floriculture Science, NARO), ‘PI 322719’ (Institute of Vegetable and Floriculture Science, NARO), ‘Dai-Suke’ (Institute for Horticultural Plant Breeding, Matsudo, Japan), ‘Miyazaki Daigi 3gou’ (Miyazaki Agricultural Research Institute), and ‘Miyazaki Daigi 5gou’ (Miyazaki Agricultural Research Institute; Takeda, 2018).
Summary of plant materials tested.
In progeny tests of the screened germplasms, we used the doubled haploid lines ‘KLDH89’, ‘KLDH126’, and ‘KLDH165’, which are resistant to normal nematodes, bacterial wilt, and PMMoV (pathotype P1,2). Their resistance was confirmed in previous resistance tests. These doubled haploid lines were derived from F1 hybrids that themselves were derived from a cross between the PMMoV-resistant (L3) doubled haploid lines ‘K9-11’ and ‘LS 2341’ bred by the Miyazaki Agricultural Research Institute (Sugita et al., 2013).
As control cultivars for evaluation of bacterial wilt resistance, we used ‘Miyazaki Daigi 3gou’, ‘Miyazaki Daigi 5gou’, ‘Miyazaki L1 Daigi 1gou’ (Miyazaki Agricultural Research Institute), and ‘Dai-Suke’, which are resistant to bacterial wilt (Matsunaga et al., 2021), and ‘Kyo-Suzu’ and ‘Miogi’ (Institute for Horticultural Plant Breeding, Matsudo, Japan), which are susceptible to bacterial wilt (Semi et al., 2013).
As control cultivars for evaluation of Phytophthora blight resistance, we used the resistant line ‘SCM-334’, the moderately resistant cultivar ‘Bellmasari’ (Nagano Chushin Agricultural Experiment Station, Nagano, Japan; Yanokuchi et al., 1993), and the susceptible cultivars ‘Miyazaki L1 Daigi 1gou’, ‘Dai-Suke’ (Matsunaga et al., 2021), and ‘Ace’ (Takii & Co., Ltd.; Saito et al., 2011).
For evaluation of yield in F1 rootstock candidates, ‘Miyazaki Daigi 5gou’ was used as the control rootstock, and ‘Kyo-Suzu’ was used for comparison with non-grafted cultures.
PestsThree strains of M. incognita were used in the nematode resistance test: ‘Mi-Nishigoshi’ (National Agricultural Research Center for Kyushu Okinawa Region, Koshi, Japan) was used as a normal nematode, and ‘Mi-RBS1’ (M. incognita resistance-breaking strain No. 1; derived from a single egg mass from the roots of ‘Miyazaki Daigi 3gou’ in 2011), ‘Mi-RBS2’ (M. incognita resistance-breaking strain No. 2; derived from a single egg mass from the roots of ‘LS 2341’ in 2011), were used as Rb-nematodes.
For evaluation of resistance using nematodes collected from local fields (1 to 14 in Fig. 1), single egg masses were collected from the soil from 14 sweet pepper fields in Miyazaki Prefecture in 2011. To maintain the nematode strains, they were reared cumulatively on the susceptible tomato cultivar ‘Prits’ (Kaneko Seed & Seedling Co., Ltd., Maebashi, Japan) that was planted in a greenhouse in contaminated soil collected from each of the fields.
Collection sites for root-knot nematodes in Miyazaki Prefecture, Japan. To evaluate resistance to local field nematode populations, single egg masses of Meloidogyne incognita were collected from 14 sweet pepper fields in Miyazaki Prefecture in 2011. Site 15 is where planting experiments were conducted in nematode-contaminated fields.
The screening was conducted by laboratory assay using contaminated soil, as reported elsewhere (Uesugi, 2014a). In short, the test germplasms were sown and reared under standard conditions. Then, at three to four weeks after sowing, three seedlings of each were transplanted into a nematode-contaminated seedling bed. The seedling bed was created by using a 200-L polypropylene container (Tafubune 200; 1290 mm × 828 mm × 192 mm; Sanko Co., Ltd., Tokyo, Japan) filled with 150 L of nematode-contaminated soil in which the nematodes were reared cumulatively on the susceptible tomato cultivar ‘Prits’. To improve drainage, five holes (3 cm diameter) were drilled in the four corners and in the center of the container. At around 10 weeks after transplanting, the roots of the seedlings were inspected for root gall formation and assigned a number on a root gall index: (0, no galls; 1, ≤ 5 galls; 2, 5–20 galls; 3, > 20 galls and sparse gall formation; and 4, > 20 galls and dense gall formation). Resistant materials were defined as those for which the average root gall index for three individuals was ≤ 1.0. This method was used in the first, second, and third screenings of the materials. In the second screening, to confirm reproducibility, inoculation tests were conducted twice for normal nematodes and three times for the RB-nematode.
2) Resistance evaluation in test-crossed F1 plantsTest-crossed F1 plants were planted directly in a farmer’s field in which damage by Rb-nematodes had been confirmed by observation of root-knots on the normal-nematode-resistant rootstock cultivar ‘Dai-Suke’ (field 15 in Fig. 1). Sowing, transplanting, and evaluation were conducted as in Subsection 1). Resistant materials were defined as those for which the average root gall index for three individuals was ≤ 1.5.
3) Resistance evaluation in candidate F1 rootstockThis evaluation was conducted by laboratory assay using inoculation with second-stage nematode juveniles (J2) (Uesugi, 2014b). At around 30 days after sowing in 6-cm pots, each seedling was inoculated with approximately 300 J2 nematodes. Six plants of each line were inoculated with normal nematodes and 15 with the Rb-nematode. Once the effective accumulated temperature after inoculation had reached 500–600°C at the nematode developmental zero temperature of 13.2°C, the seedling roots were stained with a 0.5% aqueous solution of Phloxine B (Nacalai Tesque, Inc., Kyoto, Japan) and observed for egg masses. Each plant was then assigned an egg mass index value (0, no masses; 1, 1–5 egg masses; 2, > 5–20 egg masses; 3, > 20–100 egg masses; and 4, > 100 egg masses). The average egg mass index value was calculated for each plant (n = 6–15), and resistance and susceptibility were assigned as follows: ≤ 1, strong resistance; > 1–2, moderate resistance; > 2–4, susceptibility.
Bacterial wilt resistance testStrain 2-d (collected in 2000 at a sweet pepper field in Kunitomi, Miyazaki Prefecture, Japan) was used as the pathogen. Samples of the bacteria were stored at −80°C and thawed before use.
To evaluate resistance to bacterial wilt, we used the cut-root inoculation method and juvenile seedlings, as reported elsewhere (Semi et al., 2013), but using a different seedling box and soil volume. The seeds were sown in type A seedling boxes (607 mm × 305 mm × 38 mm; Sanko Co., Ltd.) with 45 seeds per line (15 seeds per row, 3 rows per line) and grown under standard conditions. At around three weeks after sowing, a box cutter was used to injure the roots by cutting a 2-cm-deep line in the soil down each row and on both sides of the seedlings, 1-cm away from the seedlings’ base. Then, 10 mL of bacterial solution adjusted to a concentration of 2 × 108 colony-forming units/mL was poured into the lines in the soil to inoculate the seedlings’ roots with bacteria. At around two weeks after inoculation, each plant was assigned a value on a disease index (0, healthy; 1, 1 leaf partially wilted; 2, 2 or 3 leaves wilted; 3, all leaves wilted; 4, dead), and resistance was evaluated by calculating the average disease index (Σ (each disease index × no. of plants by each disease index)/(no. of test plants)) as follows: 0–0.5, strong resistance; > 0.5–1, resistance; > 1–1.5, moderate resistance; > 1.5, susceptible.
Phytophthora blight resistance testStrain PPh-k (Kochi Prefectural Agricultural Technology Center, Kochi, Japan) was used as the pathogen. The fungi were sub-cultured on potato dextrose agar medium and grown on V8 juice medium under standard conditions.
To evaluate resistance to Phytophthora blight, we used the root inoculation method and juvenile seedlings as reported elsewhere (Saito et al., 2011), but using a different seedling box and soil volume. A total of 7 or 15 seedlings of each line were grown under standard conditions. At about four weeks after sowing, the roots of each seedling were immersed for at least 1 min in a fungal solution adjusted to a concentration of 9 × 102 zoosporangia/mL, and then planted in type 51 seedling boxes (326 mm × 477 mm × 76 mm; Anzen Kougyou Co., Kobe, Japan). Electric heating mats (0.9 m × 1.8 m; Tsukuba Denki Co., Ltd., Joso, Japan) and an electric thermostat (single-phase 100 V; Tsukuba Denki Co., Ltd.) were used to maintain the soil temperature in the seedling boxes at 30°C to promote disease development. At around three weeks after planting, each plant was assigned a value on a disease index (0, healthy; 1, wilted; 2, dead), and resistance was evaluated by calculating the average disease index (Σ (each disease index × no. of plants by each disease index)/no. of test plants) as follows: 0–0.5, strong resistance; > 0.5–1, resistance; > 1, susceptible.
Evaluation of the cultivation characteristics of a candidate F1 rootstockThe scion cultivar ‘Kyo-Suzu’ was grafted on a candidate F1 rootstock cross, ‘KLDH89 × J159’, and the production characteristics were evaluated by soil cultivation in a greenhouse. 12 kg of Magnesium lime (Tsukumi Ryujyo Kudo Sekkai 1-gou, Tsukumi Dolomite Kogyo Co., Ltd., Tsukumi, Japan) per 100 m2 was applied to the soil in the greenhouse, followed by 40 kg of basal fertilizer (Kudoyuukiirikasei-ShintokuA801 [N:P2O5:K2O = 8:8:8]; JCAM AGRI. Co., Ltd., Tokyo, Japan) per 100 m2. The rootstock seeds were sown on 23 August and the scion seeds on 24 August 2018. The seedlings were grafted and potted up into 12-cm pots filled with potting soils (Ryoto engei baido pottoyo; Ryoto Hiryo Co., Ltd., Oita, Japan) on 18 September. In addition to evaluating the cultivation characteristics of the cross, comparisons were also made with own-rooted ‘Kyo-Suzu’. Seeds of these plants were sown on 6 September 2018 and potted up into 12-cm pots filled with the same potting soils on 28 September. Both grafted and self-rooted seedlings were planted in the greenhouse on 24 October. The greenhouse temperature was set at 18°C and irrigation was provided daily at approximately one to three liters per plant using Chapin Drip Tape (5 cm-pitch, JAIN Irrigation Inc., New York, USA). Additional fertilizer was applied at the same time as irrigation using a 700 × to 2000 × solution of additional fertilizer (OK-F-1 [N:P2O5:K2O = 15:8:17]; OAT Agrio Co., Ltd., Tokyo, Japan). Harvest surveys were conducted from 22 November 2018 to 30 May 2019 by measuring the total number of fruits, total yield (kg), number of saleable fruits, and yield of saleable fruits (kg), and were calculated on a per square meter basis.
Statistical analysesThe results in Figure 5A, C, and D were analyzed with ‘EZR’ (Easy R), which is based on R and R commander (Kanda, 2013). In Figure 5A, the Steel-Dwass test (P < 0.05) was used. In Figure 5C and D, Tukey-Kramer’s multiple range test (P < 0.05) was used. In Figure 5B, since there was an obvious difference between the groups, no statistical test was performed.
First, we screened the 288 germplasms by conducting resistance tests using two strains of Rb-nematode: ‘Mi-RBS1’ (Fig. 2A) and ‘Mi-RBS2’ (Fig. 2B). A total of 14 germplasms inoculated with ‘Mi-RBS1’ and 12 with ‘Mi-RBS2’ had an average root gall index of ≤ 1.0 and were considered to show resistance to the two Rb-strains. That is, 15 germplasms showed resistance in the first Rb-nematode tests, but 12 germplasms except ‘PI281423’, ‘Irabu 2’, and ‘Aji Pique’ were used for subsequent tests. ‘PI281423’ was omitted owing to its poor germination rate, which prevented us from obtaining sufficient data. ‘Irabu 2’ and ‘Aji Pique’ were omitted owing to their poor growth. A previously unpublished study suggested that ‘Jal A’ and ‘Dul B’ are resistant to Rb-nematodes, so a total of 14 germplasms (the above 12 plus ‘Jal A’ and ‘Dul B’) were used for the second screening. In the Rb-nematode resistance test, 11 germplasms (except ‘Wase Uemuki Piiman’, ‘Cayenne (Large Red Thick)’, ‘CS25’, and ‘Dul B’) had an average root gall index of ≤ 2.0 across two inoculations (Fig. 3A). Similarly, in the normal nematode resistance test, 13 germplasms (except ‘Wase Uemuki Piiman’ and ‘CS24’) had an average root gall index of ≤ 2.0 across three inoculations (Fig. 3B).
Frequency distribution of the average root gall index of 288 germplasms inoculated with Rb-nematodes. A: Inoculation with nematode strain ‘Mi-RBS1’ (n = 3). B: Inoculation with nematode strain ‘Mi-RBS2’ (n = 3). The callouts show the germplasms with an average root gall index of ≤ 1.
Evaluation of resistance to normal and resistance-breaking nematodes in candidate materials for Rb-nematode resistance. A: inoculation with the Rb-nematode strain ‘Mi-RBS2’, two replications performed (n = 3). B: inoculation with the normal nematode strain ‘Mi-Nishigoshi’, three replications performed (n = 3). Abbreviations: ‘W. U. P.’ = ‘Wase Uemuki Piiman’, ‘C. F. T.’ = ‘Capsicum Frutescens Tabasco’, ‘C. (L. R. T.)’ = ‘Cayenne (Large Red Thick)’, ‘N. C. P.’ = ‘NuMex Chile Piquin’, ‘N. B. P.’ = ‘NuMex Bailey Piquin’.
For the third screening, a total of 12 germplasms (except ‘Cayenne (Large Red Thick)’ and ‘Aji Picucho’, because of low germination rates) were tested for resistance to M. incognita collected at 14 sweet pepper cultivation fields in Miyazaki Prefecture (Fig. 1; Table 2). Two of the germplasms (‘NuMex Bailey Piquin’ and ‘PM 217-1-3’) had an average root gall severity of < 1.5 in all 14 fields, and five of them (‘CS24’, ‘CS25’, ‘Tabasco’, ‘Jal A’, and ‘Dul B‘) had an average root gall severity of < 1.5 in 13 fields. The control cultivars with known resistance to normal nematodes (‘LS 2341’, ‘SCM-334’, ‘PI 322719’, and ‘Dai-Suke’) showed resistance to the nematodes present in fields in which the non-nematode-resistant cultivar ‘Kyo-Yutaka’ is usually grown (fields 1–3), but not to the nematodes present in fields in which the normal-nematode-resistant cultivar ‘Dai-Suke’ is usually grown (fields 4–14).
Average root gall index of the identified breeding materials after inoculation with 14 strains of Meloidogyne incognita collected from sweet pepper cultivation fields.
Next, we conducted progeny testing to examine the resistance of the breeding materials identified in the previous screening tests to both normal and Rb-nematodes. ‘NuMex Bailey Piquin’, ‘PM 217-1-3’, and ‘Jal A’, which all showed a low average root gall severity when grown in all or most of the 14 nematode-contaminated fields, were used as the pollen parents. ‘KLDH89’, ‘KLDH126’, and ‘KLDH165’, which are resistant to normal nematodes, bacterial wilt, and PMMoV (P1,2), were used to produce F1 combinations, but only five F1 combinations could be produced owing to the difficulty in collecting seeds. The five F1 combinations were examined for resistance to bacterial wilt and the Rb-nematode (Fig. 4). Nematode resistance was evaluated by growing the crosses in a field contaminated with Rb-nematodes.
Evaluation of five candidate F1 rootstock lines for resistance to bacterial wilt and the Rb-nematode. A: inoculation with bacterial wilt (n = 29 ~ 36); B: inoculation with Rb-nematode strain ‘Mi-RBS2’ (n = 3). The underline indicates that this cultivar was screened from five candidate rootstock lines. The other four cultivars were used as controls. Abbreviations: ‘89 × N.B.P.’ = ‘KLDH89 × NuMeX Bailey Piquin’, ‘89 × 217’ = ‘KLDH89 × PM217-2-3’, ‘126 × Jal A’ = ‘KLDH126 × Jal A’, ‘126 × 217’ = ‘KLDH126 × PM217-2-3’, ‘165 × Jal A’ = ‘KLDH165 × Jal A’, ‘Miya.D3’ = ‘Miyazaki Daigi 3gou’, ‘Miya.D5’ = ‘Miyazaki Daigi 5gou.
For resistance to bacterial wilt, ‘KLDH89’ × ‘NuMex Bailey Piquin’ showed the lowest average disease index value (0.9), which was similar to that of the highly resistant cultivars ‘Miyazaki Daigi 3gou’ (0.7) and ‘Miyazaki Daigi 5gou’ (0.6).
For resistance to the Rb-nematode, ‘KLDH165’ × ‘Jal A’ showed the lowest average root gall index (0.7 ± 0.3), which was much lower than those of the control cultivars ‘Miyazaki Daigi 3gou’, ‘Miyazaki Daigi 5gou’, ‘Dai-Suke’, and ‘Kyo-Suzu’. Overall, the average root gall index of the five candidate F1 hybrids (all ≤ 2.0) was much lower than those of the control cultivars (all ≥3.3).
Progeny testing of ‘J159’ as a parent for F1 root-stock cultivarsSome ‘NuMex Bailey Piquin’ susceptible seedlings were identified; following two generations of selfing to fix the resistance, one selected resistant line was named ‘J159’. To evaluate the suitability of using ‘J159’ as a parent for F1 rootstock cultivars, we evaluated the F1 hybrid ‘KLDH89’ × ‘J159’ for resistance and yield in grafted cultivation. In bacterial wilt resistance, it showed an average disease index of 0.5, which was comparable to that of the commercial cultivar ‘Dai-Suke’ (Fig. 5A). For Phytophthora blight resistance, it showed an average disease index of 2.0, and all of the plants died, the same as the susceptible commercial cultivar ‘Ace’ (Fig. 5B). In terms of normal nematode resistance, it showed an average egg mass of 0.0 per plant, which was comparable to that of the normal-nematode-resistant control material LS 2341 (Fig. 5C). For Rb-nematode resistance, it showed an average egg mass index of 32.4, which was comparable to that of the pollen parent ‘J159’ (3.1), but significantly lower than that of the susceptible control cultivar ‘Kyo-Suzu’ (103.7; Fig. 5D). For PMMoV resistance, it showed L3 type resistance.
Evaluation of F1 ‘KLDH89 × J159’ for resistance to bacterial wilt, Phytophthora blight and nematodes. A: inoculation with bacterial wilt (n = 14, 15); B: inoculation with Phytophthora blight (n = 7, 15). C: inoculation with the normal nematode strain ‘Mi-nisigoshi’ (n = 6). D: inoculation with the Rb-nematode strain ‘Mi-RBS2’ (n = 6 ~ 15). Different letters represent significantly different values (P < 0.05; (A) Steel–Dwass test (C, D) Tukey–Kramer multiple range test). The other lines and cultivars were used as controls. Abbreviations: ‘89 × 159’ = ‘KLDH89 × J159’, ‘Miya.D5’ = ‘Miyazaki Daigi 5gou’, ‘Miya.D3’ = ‘Miyazaki Daigi 3gou’, ‘Miya.L1’ = ‘Miyazaki L1 Daigi 1gou.
Next, we evaluated the yield performance of the scion ‘Kyo-Suzu’ when grafted onto ‘KLDH89’ × ‘J159’ as the rootstock and grown in an uncontaminated field (Fig. S1). This resulted in a higher total number of fruits, total yield, number of saleable fruits, and saleable yield than using the commercial rootstock cultivar ‘Miyazaki Daigi 5gou’. In addition, the total number of fruit and total yield, the number of saleable fruits, and saleable yield were similar to that of the own-rooted ‘Kyo-Suzu’.
Therefore, we concluded that ‘KLDH89’ × ‘J159’ showed resistance to bacterial wilt, normal nematodes, the Rb-nematode, and PMMoV (P1,2) with no negative effect on yield, so therefore we applied for cultivar registration in Japan on 17 January 2022 (Application No. 35966) using the cultivar name ‘Dai-Hinata’.
We identified ‘NuMex Bailey Piquin’, a genetic resource originally from outside Japan, as a nematode-resistant breeding material from a catalog of 288 breeding materials. Phenotypic segregation was observed in the selected breeding material, but we were able to fix the resistance by selfing to develop line ‘J159’, which we then used to breed a new F1 rootstock cultivar. Other examples of the development of new disease-resistant breeding materials by screening using genetic resources from outside Japan include anthracnose resistance in cucumber (Matsuo et al., 2022) and Fusarium wilt race 1.2 resistance in melon (Ishikawa et al., 2023).
In F1 breeding, materials with genetically dominant traits can be used directly as parental lines to efficiently add the target trait to the F1 generation. Therefore, our discovery of a breeding material with dominant resistance to the Rb-nematode will be useful for F1 breeding. Practically, when searching for new characteristics not found in current cultivars, genetic resources that are distantly related to modern cultivars are the most sought after. However, even when resistant materials are found, they are often local varieties or wild species that are not distantly related to current varieties and are clearly inferior to commercial varieties in terms of fruit quality and other field characteristics. For this reason, F1 hybrids that use the resistant breeding material directly as a single parent often inherit other undesirable characteristics of the resistant parent and are not suitable as scion cultivars. Fortunately, in the case of sweet pepper, since the rootstock does not affect the fruit traits of the scion, F1 hybrids can be used for rootstock breeding, if resistance can be added. Therefore, screening for resistant breeding materials among a collection of foreign genetic resources with large diversity can be expected to be an effective approach for obtaining resistant F1 rootstock.
For more effective screening and efficient use of breeding materials, core collections are potentially useful. A core collection is a set of cultivars and lines that reflect the whole genetic diversity of a species (Frankel, 1984). By using core collections, the most useful materials can be selected from a smaller number of germplasms. In Japan, the National Institute of Agrobiological Resources has developed and distributed 14 core collections for various species, and these collections are freely available (https://www.gene.affrc.go.jp/databases-core_collections.php). However, they cover mainly rice and beans, and the only vegetable collection available is for eggplant (Miyatake et al., 2019; Wang et al., 2023). Although a core collection comprising 240 lines selected from among 3,821 pepper plant genetic resources has been reported (Lee et al., 2016), additional core collections for more efficient screening of characteristic strains are needed for pepper.
Mitigating normal nematode damage by using commercial rootstocks and outbreak of Rb-nematodesThe four cultivars with known resistance to normal nematodes (i.e., ‘LS 2341’, ‘SCM-334’, ‘PI 322719’, and ‘Dai-Suke’) showed resistance to infestation by nematodes collected from fields in which grafting of the non-resistant scion cultivar ‘Kyo-Yutaka’ had been used, but not to nematodes collected from fields in which grafting with the normal-nematode-resistant rootstock ‘Dai-Suke’ had been used (Table 2). This suggests that the use of existing rootstock cultivars such as ‘Dai-Suke’ may be sufficient to suppress infestation by normal nematodes in fields that have previously been planted under non-grafted conditions with cultivars such as ‘Kyo-Yutaka’ that are not resistant to the nematodes. However, this also suggests that in fields where the normal-nematode-resistant cultivar ‘Dai-Suke’ has been cultivated for many years, the normal nematode population has given way to the emergence of Rb-nematode populations. Furthermore, each of the locally collected nematode strains were able to infest a different subset of the identified breeding materials, indicating that the spread of Rb-nematodes has multiple origins.
Although the results indicate that using ‘NuMex Bailey Piquin’ and its selfed line ‘J159’, as well as our developed cultivar ‘Dai-Hinata’, may go some way to mitigating nematode damage, a breakdown of resistance is still possible. Therefore, despite the use of resistant rootstocks being extremely effective for achieving both high quality yields and resistance to soilborne pests and diseases, nematode control should not rely completely on the use of resistant rootstock cultivars, but should be combined with other control measures to delay resistance breakdown.
‘NuMex Bailey Piquin’‘NuMex Bailey Piquin’ was bred from a single-plant selection of peppers collected in the Caribbean area of Mexico (Bosland and Ilesias, 1992). It was named for its breeder, Mr. Bailey, of New Mexico State University (NM, USA). It is a piquin pepper (C. annuum L. var. glabriusculum); its fruits are small, less than 2.5 cm in length and 1.5 cm in diameter. The fruits are deciduous like ‘Chiltepin’ (C. annuum L. var. glabriusculum) (Hu et al., 2023), so they can be harvested mechanically. Although ‘NuMex Bailey Piquin’ nematode resistance was previously unknown, our results indicate that it is resistant to the Rb-nematode.
The original population of ‘NuMex Bailey Piquin’ was reported to be a highly heterogeneous population with several fruit traits (Bosland and Ilesias, 1992). The breeding was based on three generations of selection that evaluated only the fruit-bearing traits, so it is possible that the nematode resistance was not fixed.
Piquin pepper (C. annuum L. var. glabriusculum) has been shown to be a distinct branch of C. annuum var. annuum in the phylogenetic tree (Liu et al., 2023). ‘NuMex Bailey Piquin’ is therefore considered a somewhat distant taxonomic relative, but it can be crossed with modern cultivars. Thus, this variety is highly valuable as a material that can be used to expand the gene pool of modern cultivars through crossbreeding.
Relationship between previously reported nematode-resistant cultivars and nematode resistance genes in ‘NuMex Bailey Piquin’Three genes that confer resistance to M. incognita have been reported in C. annuum: Me1 in ‘PI201234’ (Wang et al., 2018), Me3 in ‘PI 322719’ (Dijan-Caporalino et al., 2001), and Me7 in ‘SCM-334’ (‘CM334’) (Changkwian et al., 2019). Because the origins and taxonomic classifications of these previously reported nematode-resistant materials are different from ‘NuMex Bailey Piquin’ (Table 3), ‘J159’ is expected to be a new source of resistance genes.
List of pepper accessions and their resistance to M. incognita.
The three previously reported genes are all dominant genes. Me3 and Me7 are estimated to lie on the same locus, and Me1 and Me3/Me7 are closely linked with a distance of 9 cM between them on chromosome P9 according to linkage mapping (Fazari et al., 2012). Nematodes that can damage plants harboring Me3 are suppressed in plants harboring Me1 (Buchi et al., 2017), which is consistent with our findings. Further studies are required to elucidate whether our findings relate to Me1 and Me3.
Improvements when breeding future rootstock cultivarsTo test the resistance of ‘J159’ to Rb-nematodes, we produced the F1 hybrid ‘KLDH89’ × ‘J159’ and evaluated its resistance and fruit yield. It showed resistance to normal and Rb-nematodes, bacterial wilt, and PMMoV without any negative effects on yield. Therefore, we bred this F1 hybrid as ‘Dai-Hinata’ and began to use it in the field. However, the resistance of ‘Dai-Hinata’ to the Rb-nematode was weaker than that of ‘J159’ (Fig. 5D). This suggests that the resistance-conferring genes in ‘Dai-Hinata’ are incompletely dominant compared with those in ‘J159’, and that ‘Dai-Hinata’ has a heterozygous genotype. Therefore, to breed F1 cultivars with greater nematode resistance, it will be necessary to use parental lines that are both homozygous for the resistance gene.
Unfortunately, ‘Dai-Hinata’ did not show resistance to Phytophthora blight. To address this, an F1 rootstock breeding program is currently under way at the Miyazaki Agricultural Research Institute with the goal of producing a cultivar that has a homozygous genotype for the gene that confers resistance to Rb-nematodes and shows resistance to Phytophthora blight.
Since nematode resistance tests are time- and labor-consuming, we hope that in the mid to long term, marker-assisted selection for Rb-nematode resistance will become possible, which will contribute to increased breeding efficiency.
ConclusionBy screening a diverse set of genetic resources, we were able to identify a pepper breeding line with high Rb-nematode resistance. We then used it to develop a new rootstock F1 cultivar. This research is expected to contribute to future resistance breeding programs for F1 rootstock sweet bell pepper cultivars, sweet pepper, and chili pepper.
We are grateful to Dr. K. Miyatake (Institute of Vegetable and Floriculture Science, NARO, Tsu, Japan) and Prof. H. Iwahori (Ryukoku University, Kyoto, Japan) for their advice on this paper. Prof. T. Mitsunaga (Institute for Plant Protection, NARO, Tsukuba, Japan) advised us on statistical tests. We also thank the NARO Genebank and the Institute of Vegetable and Floriculture Science, NARO, for providing the pepper genetic resources.
