Spice Defense: Resistance, Capsaicin, and Photosynthesis in Diverse Capsicum Genotypes Under Root-Knot Nematode Stress

Abstract

Meloidogyne enterolobii is an aggressive root-knot nematode that poses a significant threat to global chili (Capsicum spp.) production. This study evaluated the resistance levels, physiological responses, and capsaicin accumulation patterns of diverse Capsicum genotypes—including C. annuum, C. chinense, C. frutescens, and C. baccatum—under nematode-infested and non-infested conditions. Resistance was assessed using the gall index (GI), egg per g of root, and reproductive factor (Rf). Among these evaluated parameters, Rf and egg count consistently reflected nematode reproductive success, whereas the GI proved less reliable for resistance classification. Several genotypes—notably from C. chinense and C. frutescens—exhibited strong resistance (Rf < 1), suggesting their potential for nematode-infection cultivar development. Physiological assessments revealed variable photosynthetic responses, with some genotypes showing increased photosynthetic rates of post-infection, indicating potential compensatory mechanisms. In contrast, capsaicin accumulation was influenced by nematode stress and genetic background, indicating their roles in capsaicin biosynthesis. These findings highlight the genotype-specific biochemical and physiological responses of Capsicum species to M. enterolobii infection and underscore the value of integrating physiological, biochemical, and molecular data in breeding programs. Future research should focus on dissecting hormonal signaling pathways and post-infection metabolic shifts to accelerate the development of robust, high-yielding cultivars with durable resistance.

1. Introduction

Peppers (Capsicum spp.) are one of Thailand’s most important economic crops. They are integral to Thai cuisine, where their distinctive pungency [1]—derived from the compound capsaicin—is a key flavor element. Beyond its culinary applications, capsaicin possesses various bioactive properties which are beneficial to human health, such as antioxidant activity and pain relief [2]. These properties have driven the growing demand for chili-derived products in the functional food, pharmaceutical, cosmetic, beverage, and pest control industries. Given current global health trends and the increasing awareness of human nutrition and medicinal phytochemicals, peppers are expected to play an even more vital role in future health-related industries [3].

Despite their economic and nutritional significance, pepper production faces numerous challenges, especially from plant pests and diseases [4,5]. Among these, root-knot disease caused by root-knot nematodes (Meloidogyne spp.) remains a major unresolved issue —particularly in Ubon Ratchathani Province—causing yield losses of 50–100% and resulting in economic damage amounting to thousands of USD [6]. Due to their climatic suitability, the four major pepper species cultivated in Thailand are Capsicum annuum L., C. chinense Jacq., C. baccatum L., and C. frutescens L., each comprising various cultivars. However, limited research exists regarding the resistance of these cultivars to root-knot nematodes, particularly Meloidogyne incognita and M. enterolobii, which are commonly reported in chili-growing regions [7,8]. Meloidogyne enterolobii continues to pose a serious threat in Thailand, where its distribution has expanded across a variety of economically important crops, including guava (Psidium guajava) [9], rain tree (Samanea saman) [10], shallot (Allium cepa) [11], and chili (Capsicum spp.) [7,8]. Several reports have described the variable susceptibility or resistance to root-knot nematodes in Capsicum spp. In particular, these plants exhibit more susceptibility to M. enterolobii than other species of root-knot nematodes [12,13,14]. Meloidogyne enterolobii, recognized as one of the most aggressive root-knot nematode species, has few known resistance sources. However, previous studies have indicated that Capsicum chinense and C. frutescens generally exhibit greater resistance than other species [15,16]. Given the substantial threat posed by M. enterolobii to pepper cultivation, the identification and characterization of resistant genotypes are critical. These genotypes can be used in breeding programs to develop new cultivars or hybrids that combine nematode resistance with other essential agronomic traits.

In the pepper production process, yield is regarded as a crucial component, followed by the yield quality, which includes the capsaicin content. These attributes directly influence the market value of peppers. Exposure to both biotic and abiotic stress frequently leads to physiological and biochemical changes during pepper production. This stress exposure may result in reduced yield and fluctuating capsaicin levels, contributing to variability in production. It is essential to examine the potential alterations caused by such stressors to provide critical insights for future pepper production. Interestingly, capsaicin has demonstrated nematocidal properties, such as reducing egg hatching and larval survival in root-knot nematodes. However, few studies have explored the roles of naturally occurring capsaicin in different chili cultivars in suppressing nematode development or how nematode infection might influence capsaicin levels within the plant. Moreover, there is a lack of data on cultivar-specific resistance and biochemical responses in Thai-grown peppers when challenged by M. enterolobii.

At present, limited data are available on the extent of yield loss caused by nematode infection and its impact on capsaicin content in infected plants. Therefore, this study aimed to evaluate the resistance levels of various Capsicum genotypes against M. enterolobii infection, assess associated physiological responses, and investigate the changes in capsaicin levels under root-knot nematode infection. The findings from this research may help to identify and select chili cultivars with resistance to root-knot nematodes, thus providing valuable insights for future breeding programs focused on enhancing disease resistance and economic quality in pepper production.

2. Materials and Methods

2.1. Seedling Preparation of Pepper

Seeds from four chili species—Capsicum annuum, C. baccatum, C. chinense, and C. frutescens—totaling 105 accessions, were obtained from the Plant Breeding Research Center for Sustainable Agriculture, Khon Kaen University. The seeds were primed by soaking them in warm water (cold water–hot water ratio of 1:0.5) for 30 min. After washing, the seeds were placed on moist germination paper in seedling boxes. Once radicles emerged and reached approximately 1–2 mm in length, the germinated seeds were transferred to peat moss in seedling trays (Figure 1A). The seedlings were watered twice daily and fertilized at 20 g/20 L (15-15-15) every other day after emergence from the substrate. Fungicide was applied once per week, and insecticide was sprayed every 5–7 days, following the protocol described by Techawongstien [17]. The seedlings were grown under greenhouse conditions at the Vegetable Section, Faculty of Agriculture, Khon Kaen University, until they were 35 days old or had developed two pairs of true leaves. Insecticide application ceased two weeks before transplanting for further experiments.

2.2. Meloidogyne enterolbiii Preparation

Meloidogyne enterolobii (Me) specimens were obtained from the Department of Plant Pathology, Faculty of Agriculture, Khon Kaen University, Thailand. The nematode inoculum was maintained and propagated by inoculating 3-week-old okra (Abelmoschus esculentus) plants, which were kept in a greenhouse for 2 months. After this period, infected plants were uprooted and thoroughly washed with running tap water. Before use, M. enterolobii was confirmed using the C2F3/1108 primer set, which amplifies the ITS region between the ribosomal DNA and the cytochrome oxidase II genes [18,19]. The PCR product was approximately 700 bp. The egg masses of M. enterolobii were carefully extracted from the infected okra roots into sterile water via the egg extraction method [20]. Following a 7-day incubation period, second-stage juveniles (J2) that hatched from the egg masses were collected, washed with sterilized water, and counted to prepare concentrations of 500 J2/mL and 1000 J2/mL. These concentrations were used for subsequent inoculation in resistance screening experimental trials and capsaicin experimental trials, respectively.

2.3. Resistance Screening of Pepper Cultivars to M. enterolobii

Thirty-five-day-old pepper seedlings were inoculated with 500 J2s M. enterolobii—confirmed by molecular identification—planted in peat moss 4-inch-diameter plastic pots, and harvested at 45 days after inoculation (DAI). This study comprised 105 accessions by 71 accessions of Capsicum annuum L., 19 accessions of C. chinense Jacq, 5 accessions of C. baccatum L., and 10 accessions of C. frutescens L. The experiment was arranged in a randomized complete block design (RCBD) with 4 replicates per accession. Data on accessions were assessed for root gall index, following the method outlined by Barker [21]: 0 = no galls; 1 = 1–25% galls in the root system; 2 = 26–50% galls in the root system; 3 = 51–75% galls in the root system; 4 = more than 75% galls in the root system. After counting, the roots were chopped into small pieces (~1–2 cm long) and shaken with 0.6% sodium hypochlorite (Clorox, Kuala Lumpur, Malaysia) for 4 min using a homogenizer. The egg suspension was poured into a series of sieves with 150 μm and 25 μm apertures and rinsed with running tap water [20]. Nematode eggs retained on the 25 μm sieve were collected, counted under an inverted microscope (Olympus CKX53, Tokyo, Japan), and reported as the number of eggs per g of root. The resistance level was evaluated from reproductive factors (Rfs) based on the following formula, with the resistance levels immune (Rf = 0); highly resistant, HR (Rf < 0.1); very resistant, VR (1 > Rf ≥ 0.1); resistant, R (2 > Rf ≥ 1); very susceptible, VS (3 > Rf ≥ 2); susceptible, S (4 > Rf ≥ 3); moderately susceptible, MS (7> Rf ≥ 4); highly susceptible, HS (Rf ≥ 7) (modified from Hajihassani et al. [22]):

$$Reproductive factors \left(Rf\right)={\displaystyle \frac{final population \left(Pf\right)}{initial population \left(Pi\right)}}$$

2.4. Changes in Capsaicin Content in Peppers in Response to M. enterolobii Infection

Nine accessions were selected from the results in Section 2.3 to represent Capsaicum species—Capsicum annuum L., C. chinense Jacq., C. baccatum L., and C. frutescens L.—as resistant or susceptible plants. A thirty-five-day-old seedling was inoculated with 1000 J2 M. enterolobii and planted in peat moss in a 10-inch-diameter plastic pot. The seedlings were watered twice daily and fertilized with 10 g (15-15-15) every 15 days after planting, and fully ripened red chili fruits were harvested at 4 months old. Capsaicinoid content was analyzed in fully ripened red chili fruits dried at 60 °C for 72 h. After drying, the fruits were finely ground. The ground material was then sieved through a 20-mesh screen before being subjected to capsaicinoid extraction. The capsaicinoids were extracted and quantified according to the ‘short run’ method using High-Performance Liquid Chromatography (HPLC), following the procedure described by Collin et al. [23] Capsaicinoids were extracted from 1 g of hot pepper powder using 10 mL of acetonitrile at 80 °C for 4 h. The prepared solution was filtered with a 0.45 µL polyamide syringe, and 10 µL of the filtered extract was injected for HPLC analysis. The separation was performed on a µ-Bondapak C18 column (5 µm, 4.6 mm × 250 mm, Inertsil) coupled with a guard column (µ-Bondapak Guard-Pak, Water, Milford, MA, USA). Absorbance of the analyte was detected at 284 nm with a UV detector. The solvent was a mixture of MeOH and H₂O (80:20 v/v) with a flow rate of 1.0 mL/min used for HPLC analysis (Shimadzu-Model, 10AT-VP series, Nakagyo-ku, Kyoto, Japan). Capsaicinoid content was calculated as the sum of capsaicin and dihydrocapsaicin.

2.5. Photosynthetic Performance of Pepper Accessions with M. enterolobii Infection

Leaf gas exchange parameters including net photosynthesis (Pn), stomatal conductance (gs), transpiration rate (Tr), and water use efficiency (WUE) were measured on the same leaves using an infrared gas analyzer (IRGA) model Li-cor 6400xt, equipped with an LED light source and a standard 2 cm × 3 cm leaf chamber (Li-Cor Inc., Lincoln, NE, USA). The measurement conditions were controlled as follows: light intensity at 1000 µmol (photon) m⁻² s⁻¹, CO₂ concentration at 400 µmol mol⁻¹, and temperature at 30 ± 2 °C, following the method described by Erwin et al. [24]. Measurements were conducted on 9 April 2025 between 09:00 and 11:00 AM under ambient environmental conditions to minimize diurnal variation in photosynthetic performance. The assessment was performed on four-month-old pepper (Capsicum spp.) plants at the post-anthesis (flowering) stage. For each accession, fully expanded mature leaves were selected from both root-knot nematode-infected and non-infected plants to evaluate the impact of infection on photosynthetic efficiency. Data were collected from at least three biological replicates per treatment group to ensure statistical reliability.

2.6. Statistical Analysis

As no significant differences were observed between repeated experiments for each treatment—based on a homogeneity of variance analysis using SPSS software (version 28.0.1.0, licensed to Khon Kaen University), the data were pooled for further analysis. One-way analysis of variance (ANOVA) was used to determine significant differences at p < 0.05. Mean differences in the number of eggs, gall index, and reproductive factor of root-knot nematodes (RKNs) were evaluated using Duncan’s Multiple Range Test (DMRT) and analyzed for correlation by Pearson correlation coefficients. Additionally, differences in capsaicinoid content and photosynthetic rate between resistant and susceptible pepper accessions were analyzed using DMRT at a significance level of p < 0.05.

3. Results

3.1. Resistance Screening of Pepper Accessions to M. enterolobii

This study provides valuable insight into the resistance variability of 105 Capsicum accessions against M. enterolobii. Four hundred and twenty individual peppers from four Capsicum species were screened at 45 DAI. All C. annuum were scored with a gall index of 0–5 (Figure 2). Among the susceptible accessions, gall index (GI) values ranged from 0.25 to 2.50, while resistant accessions ranged from 0.00 to 2.25. In addition, the egg per g of root ranged from 89.90 to 1762.45 in susceptible accessions and from 4.88 to 119.52 in resistant accessions. Resistance levels were determined using reproductive factor (Rf), classified as follows: Among the 71 C. annuum accessions evaluated, 55% were classified as susceptible. By contrast, 45% were resistant, yielding a resistant-to-susceptible ratio of approximately 1:2. Therefore, KKU-11013 exhibited the highest susceptibility (Rf = 36.18), and seven other accessions were also classified as highly susceptible (Rf ≥ 7). Conversely, KKU-34004 revealed the greatest resistance (Rf = 0.11), with 19 accessions categorized as very resistant (1 > Rf ≥ 0.1), as shown in

Table 1. Average resistance levels of C. annuum accessions in 45 days after inoculation by M. enterolobii.

No	Code	Capsicum Species	Gall Index	Egg/g Root	Rf	Resistance Level
1	KKU-P11013	C. annuum	1.25 ± 0.25	1762.45 ± 1115.21	36.18 ± 27.47	HS
2	KKU-P11216	C. annuum	1.25 ± 0.25	1170.55 ± 478.86	22.42 ± 12.91	HS
3	KKU-P11027	C. annuum	0.75 ± 0.25	1319.40 ± 301.14	16.23 ± 2.71	HS
4	KKU-P31075	C. annuum	1.5 ± 0.29	732.04 ± 250.51	15.34 ± 5.48	HS
5	KKU-P21004	C. annuum	0.75 ± 0.25	1890.40 ± 517.82	13.72 ± 13.72	HS
6	KKU-P21003	C. annuum	1.25 ± 0.25	984.03 ± 289.92	8.88 ± 2.75	HS
7	KKU-P21009	C. annuum	0.75 ± 0.25	427.33 ± 239.83	7.86 ± 3.55	HS
8	KKU-P31117	C. annuum	0.75 ± 0.48	773.74 ± 255.97	7.09 ± 1.84	HS
9	KKU-P11037	C. annuum	1.25 ± 0.25	635.61 ± 880.36	6.64 ± 2.04	MS
10	KKU-P28004	C. annuum	2.50 ± 0.29	658.51 ± 223.33	6.40 ± 1.71	MS
11	KKU-P28013	C. annuum	0.25 ± 0.25	650.86 ± 68.78	6.35 ± 0.45	MS
12	KKU-P11032	C. annuum	0.75 ± 0.25	317.89 ± 170.52	6.04 ± 4.06	MS
13	KKU-P31078	C. annuum	0.50 ± 0.50	488.48 ± 164.44	6.00 ± 2.55	MS
14	KKU-P21002	C. annuum	1.75 ± 0.25	468.32 ± 86.85	5.93 ± 1.26	MS
15	KKU-P41012	C. annuum	2.00 ± 0.41	691.62 ± 100.44	5.82 ± 0.71	MS
16	KKU-P21036	C. annuum	0.75 ± 0.25	368.17 ± 102.65	5.82 ± 1.97	MS
17	KKU-P28003	C. annuum	1.00 ± 0.00	445.39 ± 219.28	5.43 ± 1.76	MS
18	KKU-P11241	C. annuum	1.25 ± 0.25	486.39 ± 180.86	5.38 ± 1.67	MS
19	local	C. annuum	1.25 ± 0.73	330.58 ± 49.13	5.30 ± 0.25	MS
20	KKU-P28010	C. annuum	1.00 ± 0.41	439.52 ± 198.3	4.89 ± 1.67	MS
21	KKU-P28009	C. annuum	1.00 ± 0.41	233.07 ± 74.32	4.86 ± 2.17	MS
22	Huysiton	C. annuum	0.5 ± 0.29	216.13 ± 40.60	4.85 ± 4.85	MS
23	JD16	C. annuum	1.00 ± 0.00	263.15 ± 55.06	4.50 ± 1.18	MS
24	KKU-P11043	C. annuum	0.75 ± 0.25	341.92 ± 127.05	4.25 ± 1.45	MS
25	KKU-P28007	C. annuum	0.75 ± 0.25	420.04 ± 86.65	4.18 ± 4.18	MS
26	KKU-P24001	C. annuum	1.00 ± 0.00	222.74 ± 100.58	4.10 ± 1.58	MS
27	KKU-P21010	C. annuum	0.75 ± 0.25	618.27 ± 97.43	4.04 ± 0.84	MS
28	Mundam KKU	C. annuum	0.25 ± 0.25	377.78 ± 78.61	3.60 ± 1.45	S
29	Bushy hot	C. annuum	0.75 ± 0.25	302.33 ± 113.44	3.51 ± 1.27	S
30	KKU-P31115	C. annuum	1.75 ± 0.25	264.23 ± 64.69	3.29 ± 0.76	S
31	KKU-P31105	C. annuum	0.25 ± 0.25	411.74 ± 106.48	3.27 ± 0.76	S
32	KKU-P11015	C. annuum	1.00 ± 0.00	187.67 ± 27.64	3.22 ± 0.40	S
33	KKU-P28011	C. annuum	1.25 ± 0.25	165.20 ± 41.54	3.15 ± 0.71	S
34	KKU-P31032	C. annuum	1.00 ± 0.00	180.67 ± 64.35	2.64 ± 0.86	VS
35	KKU-P10003	C. annuum	0.50 ± 0.29	237.70 ± 108.57	2.32 ± 1.13	VS
36	KKU-P11036	C. annuum	1.75 ± 0.75	90.15 ± 23.53	2.18 ± 2.18	VS
37	KKU-P11010	C. annuum	0.75 ± 0.25	109.15 ± 12.96	2.11 ± 0.72	VS
38	KKU-P11051	C. annuum	1.25 ± 0.25	157.46 ± 61.28	2.10 ± 1.27	VS
39	KKU-P28002	C. annuum	1.25 ± 0.25	89.90 ± 28.62	2.03 ± 0.48	VS
40	KKU-P21005	C. annuum	1.50 ± 0.50	119.52 ± 54.04	1.99 ± 1.26	R
41	KKU-P11012	C. annuum	1.50 ± 0.29	106.43 ± 35.1	1.97 ± 0.62	R
42	KKU-P31069	C. annuum	0.75 ± 0.25	151.43 ± 21.77	1.88 ± 0.37	R
43	KKU-P11045	C. annuum	2.25 ± 0.75	174.97 ± 77.9	1.83 ± 0.42	R
44	KKU-P31048	C. annuum	0.00 ± 0.00	442.55 ± 73.62	1.60 ± 0.70	R
45	KKU-P11217	C. annuum	1.00 ± 0.00	106.53 ± 31.43	1.48 ± 0.49	R
46	KKU-P28005	C. annuum	0.00 ± 0.00	222.74 ± 63.78	1.31 ± 0.32	R
47	KKU-P31118	C. annuum	2.00 ± 0.41	98.42 ± 19.79	1.21 ± 0.40	R
48	KKU-P21056	C. annuum	0.75 ± 0.25	102.86 ± 21.83	1.20 ± 0.2	R
49	KKU-P21080	C. annuum	1.00 ± 0.00	74.08 ± 17.56	1.15 ± 0.37	R
50	KKU-P28008	C. annuum	1.75 ± 0.48	119.08 ± 75	1.11 ± 0.69	R
51	KKU-P31082	C. annuum	1.75 ± 0.48	37.26 ± 10.82	1.00 ± 0.30	R
52	KKU-P11135	C. annuum	0.75 ± 0.25	68.81 ± 17.32	0.98 ± 0.3	VR
53	KKU-P11007	C. annuum	1.00 ± 0.26	50.18 ± 12.19	0.97 ± 0	VR
54	Red sun-Esan	C. annuum	0.50 ± 0.29	84.29 ± 14.38	0.97 ± 0.97	VR
55	KKU-P21006	C. annuum	1.25 ± 0.25	175.85 ± 49.50	0.93 ± 0.33	VR
56	KKU-P11024	C. annuum	0.5 ± 0.29	132.70 ± 16.80	0.90 ± 0.20	VR
57	KKU-P21008	C. annuum	1.00 ± 0.00	60.87 ± 31.56	0.87 ± 0.35	VR
58	KKU-P18037	C. annuum	0.75 ± 0.25	69.71 ± 11.16	0.86 ± 0.17	VR
59	KKU-P11231	C. annuum	1.00 ± 0.41	59.64 ± 35.11	0.80 ± 0.33	VR
60	KKU-P28012	C. annuum	1.25 ± 0.25	53.86 ± 15.95	0.70 ± 0.17	VR
61	KKU-P21055	C. annuum	0.5 ± 0.29	38.68 ± 4.97	0.61 ± 0.24	VR
62	KKU-P21041	C. annuum	2.00 ± 0.41	42.14 ± 20.5	0.54 ± 0.54	VR
63	KKU-P11084	C. annuum	0.25 ± 0.25	33.13 ± 2.35	0.49 ± 0.49	VR
64	KKU-P18005	C. annuum	1.00 ± 0.00	75.98 ± 52.93	0.47 ± 0.47	VR
65	Tepin	C. annuum	1.50 ± 0.00	51.36 ± 8.55	0.45 ± 0.84	VR
66	KKU-P31135	C. annuum	0.00 ± 0.00	48.43 ± 13.59	0.45 ± 0.11	VR
67	KKU-P13004	C. annuum	0.00 ± 0.00	38.73 ± 9.56	0.35 ± 0.01	VR
68	KKU-P11044	C. annuum	1.00 ± 0.00	50.42 ± 12.20	0.26 ± 0.06	VR
69	KKU-P11034	C. annuum	1.00 ± 0.00	10.07 ± 1.26	0.11 ± 0.01	VR
70	KKU-P11095	C. annuum	0.50 ± 0.29	11.64 ± 2.99	0.11 ± 0.04	VR
71	KKU-P34004	C. annuum	1.00 ± 0.00	4.88 ± 1.62	0.11 ± 0.03	VR

Resistance levels: immune (Rf = 0); highly resistant, HR (Rf < 0.1); very resistant, VR (1 > Rf ≥ 0.1); resistant, R (2 > Rf ≥ 1); very susceptible, VS (3 > Rf ≥ 2); susceptible, S (4 > Rf ≥ 3); moderately susceptible, MS (7 > Rf ≥ 4); highly susceptible, HS (Rf ≥ 7). Data show the average ± standard error.

.

Five C. baccatum accessions exhibited GI scores ranging from 0 to 5. At 45 DAI, the GI values ranged from 0.5 to 2.25 in susceptible accessions and 0.75 in resistant accessions. In addition, the number of eggs per g of root ranged from 108.98 to 229.82 in susceptible accessions and 80.26 in resistant ones. All accessions of C. baccatum were specifically evaluated to determine their resistance levels. Among them, 80% were susceptible, while only 20% were resistant, resulting in an estimated resistant-to-susceptible ratio of 1:4. These results identified KKU-34031 and KKU-34014 as the most resistant and susceptible accessions, respectively (

Table 2. Average resistance levels of C. baccatum accessions in 45 days after inoculation by M. enterolobii.

No	Accession Code	Capsicum Species	Gall Index	Egg/g Root	Rf	Resistance Level
1	KKU-P34014	C. baccatum	0.50± 0.29 b	163.98 ± 33.60 a	3.87 ± 1.23 a	S
2	KKU-P34011	C. baccatum	1.25 ± 0.25 b	229.82 ± 65.13 a	2.86 ± 0.66 a	VS
3	KKU-P24003	C. baccatum	2.25 ± 0.25 a	210.51 ± 110.16 a	2.21 ± 0.61 a	VS
4	KKU-P34012	C. baccatum	0.50 ± 0.29 b	108.98 ± 53.87 a	2.18 ± 0.76 a	VS
5	KKU-P34031	C. baccatum	0.75 ± 0.25 b	80.26 ± 23.55 a	1.96 ± 0.75 a	R

Resistance levels: immune (Rf = 0); highly resistant, HR (Rf < 0.1); very resistant, VR (1 > Rf ≥ 0.1); resistant, R (2 > Rf ≥ 1); very susceptible, VS (3 > Rf ≥ 2); susceptible, S (4 > Rf ≥ 3); moderately susceptible, MS (7 > Rf ≥ 4); highly susceptible, HS (Rf ≥ 7). Data show the average ± standard error. Duncan DMRT p < 0.05. Similar letters are not significantly different within a column.

).

Nineteen C. chinense accessions exhibited a GI with scores ranging from 0 to 5. At 45 DAI, the GI values ranged from 0.25 to 2.5 in susceptible accessions and from 0 to 2.25 in resistant accessions. In addition, the number of eggs per g of root ranged from 79.80 to 2001.98 in susceptible accessions and from 8.77 to 168.05 in resistant ones. All accessions of C. chinense were specifically evaluated to determine their resistance levels. Among them, 58% were classified as very susceptible to highly susceptible, while 42% were classified as resistant, resulting in an estimated resistant-to-susceptible ratio of 1:1.4. These results identified KKU-23018 and KKU-13008 as the most resistant and susceptible accessions, respectively (

Table 3. Average resistance levels of C. chinense accessions in 45 days after inoculation by M. enterolobii.

No	Accession Code	Capsicum Species	Gall Index	Egg/g Root	Rf	Resistance Level
1	KKU-P13008	C. chinense	0.75 ± 0.25 cd	2001.98 ± 93.12 a	20.37 ± 1.6 a	HS
2	KKU-P33022	C. chinense	2.50 ± 0.29 a	761.34 ± 157.55 b	11.42 ± 2.53 ab	HS
3	KKU-P31066	C. chinense	1.00 ± 0.00 bcd	847.03 ± 247.03 b	8.16 ± 1.74 bc	HS
4	KKU-P33012	C. chinense	1.00 ± 0.00 bcd	845.42 ± 587.07 b	7.34 ± 4.14 bc	HS
5	KKU-P31137	C. chinense	0.75 ± 0.75 cd	377.15 ± 148.17 bcd	5.61 ± 1.71 bc	MS
6	KKU-P11124	C. chinense	2.25 ± 0.48 ab	385.42 ± 126.54 bcd	5.56 ± 0.11 bc	MS
7	Akanee pirote	C. chinense	2.25 ± 0.48 ab	205.11 ± 120.01 cd	5.20 ± 0.11 bc	MS
8	KKU-P34019	C. chinense	1.50 ± 0.50 abc	367.49 ± 141.58 bcd	5.20 ± 2.56 bc	MS
9	KKU-P37001	C. chinense	1.00 ± 0.00 bcd	213.01 ± 59.03 cd	2.87 ± 0.54 c	VS
10	KKU-P13004	C. chinense	1.00 ± 0.41 bcd	79.80 ± 55.26 d	2.63 ± 0.79 c	VS
11	Habanero	C. chinense	0.25 ± 0.25 cd	250.70 ± 57.23 cd	2.38 ± 0.64 c	VS
12	KKU-P23026	C. chinense	2.25 ± 0.25 ab	124.46 ± 35.13 d	1.85 ± 0.36 c	R
13	PBC932	C. chinense	0.75 ± 0.25 cd	168.05 ± 77.48 cd	1.70 ± 0.59 c	R
14	KKU-P33013	C. chinense	1.00 ± 0.00 bcd	121.57 ± 10.88 d	1.39 ± 0.33 c	R
15	KKU-P33030	C. chinense	1.25 ± 0.25 bcd	89.99 ± 28.62 d	1.15 ± 0.48 c	R
16	KKU-P23011	C. chinense	0.25 ± 0.25 cd	43.87 ± 10.09 d	0.43 ± 0.08 c	VR
17	KKU-P13001	C. chinense	0.00 ± 0.00 d	23.90 ± 4.65 d	0.33 ± 0.06 c	VR
18	KKU-P18021	C. chinense	1.00 ± 0.00 bcd	20.70 ± 3.90 d	0.23 ± 0.03 c	VR
19	KKU-P23018	C. chinense	0.50 ± 0.29 cd	8.77 ± 4.95 d	0.05 ± 0.02 c	HR

Resistance levels: immune (Rf = 0); highly resistant, HR (Rf < 0.1); very resistant, VR (1 > Rf ≥ 0.1); resistant, R (2 > Rf ≥ 1); very susceptible, VS (3 > Rf ≥ 2); susceptible, S (4 > Rf ≥ 3); moderately susceptible, MS (7 > Rf ≥ 4); highly susceptible, HS (Rf ≥ 7). Data show the average ± standard error. Duncan DMRT p < 0.05. Similar letters are not significantly different within a column.

).

Ten C. frutescens accessions exhibited a GI with scores ranging from 0 to 5. At 45 DAI, the GI values ranged from 1 to 2.75 in susceptible accessions and from 0.25 to 1.5 in resistant ones. In addition, the number of eggs per g of root ranged from 99.19 to 353.68 in susceptible accessions and from 26.03 to 80.35 in resistant ones. All accessions of C. frutescens were specifically evaluated to determine their resistance levels. Among them, 50% were classified as very susceptible to highly susceptible, while the remaining 50% were classified as resistant, resulting in an estimated resistant-to-susceptible ratio of 1:1. These results identified KKU-32012 and KKU-32024 as the most resistant and susceptible accessions, respectively (

Table 4. Average resistance levels of C. frutescens accessions in 45 days after inoculation by M. enterolobii.

No	Accession Code	Capsicum Species	Gall Index	Egg/g Root	Rf	Resistance Level
1	KKU-P32024	C. frutescens	2.75 ± 0.63 a	630.03 ± 179.90 a	8.61 ± 3.33 a	HS
2	KKU-P12010	C. frutescens	2.25 ± 0.25 ab	353.68 ± 139.42 bc	7.91 ± 2.75 ab	HS
3	KKU-P34021	C. frutescens	1.00 ± 0.00 bc	276.28 ± 95.77 bc	4.54 ± 1.11 abc	MS
4	KKU-P22006	C. frutescens	1.00 ± 0.00 bc	208.50 ± 75.43 bc	3.81 ± 0.15 abc	S
5	KKU-P11039	C. frutescens	1.00 ± 0.00 bc	99.19 ± 16.46 c	2.21 ± 0.53 abc	VS
6	KKU-P12005	C. frutescens	0.50 ± 0.29 c	80.35 ± 4.26 c	1.17 ± 0.37 bc	R
7	KKU-P32041	C. frutescens	1.00 ± 0.00 bc	52.89 ± 21.81 c	0.59 ± 0.15 c	VR
8	KKU-P22001	C. frutescens	0.25 ± 0.25 c	40.46 ± 8.33 c	0.50 ± 0.11 c	VR
9	KKU-P11174	C. frutescens	0.50 ± 0.29 c	23.80 ± 7.99 c	0.31 ± 0.04 c	VR
10	KKU-P32012	C. frutescens	1.50 ± 0.29 abc	26.03 ± 8.52 c	0.17 ± 0.04 c	VR

Resistance levels: immune (Rf = 0); highly resistant, HR (Rf < 0.1); very resistant, VR (1 > Rf ≥ 0.1); resistant, R (2 > Rf ≥ 1); very susceptible, VS (3 > Rf ≥ 2); susceptible, S (4 > Rf ≥ 3); moderately susceptible, MS (7 > Rf ≥ 4); highly susceptible, HS (Rf ≥ 7). Data show the average ± standard error. Duncan DMRT p < 0.05. Similar letters are not significantly different within a column.

).

This study provides critical insights into the interspecific variability in RKN resistance across four Capsicum species—C. annuum, C. baccatum, C. frutescens, and C. chinense—using quantitative parameters such as GI, eggs per g of root, and Rf. Resistance levels were classified across eight categories, ranging from highly resistant to highly susceptible. Our results revealed that the two most resistance Capsicum genotypes across species were KKU-P23018 (Rf = 0.05, C. chinense) and KKU-P32012 (Rf = 0.17, C. frutescens). While all accessions assessed for resistance, none exhibited complete immunity. Moreover, a strong and highly significant positive correlation was observed between reproductive factors (Rf) and eggs per g of root (r = 0.803, p < 0.001), with a narrow 95% confidence interval (0.763 to 0.836), indicating a reliable association. This finding suggests that accessions with higher eggs per g of root supported greater nematode reproduction. By contrast, correlations between GI and eggs per g of root (r = 0.0090, p = 0.080) and Rf (r = 0.071, p = 0.172) were weak and not statistically significant. These findings suggest that GI alone may not consistently represent root damage or nematode reproduction, supporting the use of eggs per g of root and reproductive potential (Rf) as more reliable indicators of resistance under the tested conditions (

Table 5. Pearson correlation coefficients and 95% confidence intervals between gall index (GI), eggs per g of root, and reproductive factor (Rf) in pepper accessions infected by M. enterolobii.

Variables	Pearson Correlation	Sig. (2-Tailed)	95% Confidence Interval (Lower–Upper)
Egg/g root vs. GI	0.090	0.080	–0.011 to 0.190
Rf vs. GI	0.071	0.172	–0.031 to 0.170
Egg/g root vs. GI	0.803	<0.001	0.763 to 0.836

Confidence intervals are estimated using Fisher’s r-to-z transformation.

).

3.2. Changes in Peppers’ Capsaicin Content in Response to M. enterolobii Infection

Three species of capsicum—namely, C. annuum, C. frutescens, and C. chinense—were chosen to represent two resistance accessions and one susceptibility accession, each based on their differing degrees of resistance to M. enterolobii infestation. For C. annuum, KKU-P11013 was classified as susceptible, while KKU-P11044 and KKU-P21055 were classified as resistant. In C. frutescens, KKU-P12010 was classified as susceptible, while KKU-P12005 and KKU-P11174 were classified as resistant. In C. chinense, KKU-P13008 was classified as susceptible, while KKU-P23011 and KKU-P23018 were classified as resistant. The levels of capsaicin, dihydrocapsaicin, and their combined total were measured in nematode-infected peppers and compared with control (non-infected) peppers. The results showed that capsaicinoid content varied significantly among species at the 99% confidence level.

Among highly susceptible genotypes, KKU-P12010 (C. frutescens) and KKU-P13008 (C. chinense) exhibited a reduction in combined capsaicin and dihydrocapsaicin content upon infection with M. enterolobii. However, the genotype KKU-P11013 from C. annuum displayed a slight increase in total capsaicinoid content following infection compared to the control. Among highly resistant and very resistant genotypes, two distinct patterns of capsaicinoid content accumulation were observed. In genotypes KKU-P21055 (C. annuum), KKU-P12005 (C. frutescens), and KKU-P23011 (C. chinense), the overall sum of capsaicin and dihydrocapsaicin content decreased upon infection compared to the control. By contrast, KKU-P11044 (C. annuum), KKU-P11174 (C. frutescens), and KKU-P23018 (C. chinense) showed increased capsaicinoid levels under the same conditions. The highest total capsaicinoid levels were recorded in KKU-P11174 (C. frutescens), reaching 89,206 SHU—an increase of 15.5% after infection. In comparison, KKU-P21055 (C. annuum) and KKU-P13008 (C. chinense) showed slight decreases with the lowest total capsaicinoid levels recorded at 45,736.7 SHU (a reduction of 14.67%) and 4523.5 SHU (a decrease of 48.62%), respectively (

Table 6. Capsaicin dihydrocapsaicin and sum of capsaicin and dihydrocapsaicin of nine pepper accessions with root-knot nematode infection vs. control.

Code	Reaction	Control			Root-Knot Nematode Infection
		Capsaicin (SHU)	Dihydrocapsaicin (SHU)	Sum of Capsaicin and Dihydrocapsaicin (SHU)	Capsaicin (SHU)	Dihydrocapsaicin (SHU)	Sum of Capsaicin and Dihydrocapsaicin (SHU)
C. annuum
KKU-P11013	Highly susceptible	11,676.0 g	6848.8 g	18,524.8 g	11,949.6 e	7071.6 d	19,021.2 e
KKU-P11044	Very resistant	15,505.4 f	9425.5 f	24,931.0 f	16,452.1 e	9704.2 d	26,156.4 e
KKU-P21055	Very resistant	30,632.6 d	22,973.7 b	53,606.2 d	25,981.2 d	19,755.5 b	45,736.7 cd
C. frutescens
KKU-P12010	Highly susceptible	37,644.4 c	20,852.1 d	58,496.6 c	26,807.8 d	15,504.2 c	42,312.0 d
KKU-P12005	Resistant	42,363.4 b	20,211.3 d	62,574.7 b	33,500.0 c	19,758.4 b	53,258.5 bc
KKU-P11174	Very resistant	51,016.2 a	26,211.4 a	77,227.6 a	54,128.4 a	35,077.7 a	89,206.0 a
C. chinense
KKU-P13008	Highly susceptible	4824.0 h	3976.8 h	8800.8 h	2567.8 f	1955.7 e	4523.5 f
KKU-P23011	Very resistant	20,575.2 e	13,066.2 e	33,641.3 e	14,396.4 e	7451.6 d	21,848.1 e
KKU-P23018	Highly resistant	37,106.7 c	21,878.1 c	58,984.8 c	39,864.6 b	19,934.7 b	59,799.3 b
F-test		**	**	**	**	**	**
CV (%)		2.1	3.5	2.3	11.0	11.1	10.9

Similar letters are not significantly different within a column; ** indicates significant differences among genotypes.

).

3.3. Photosynthetic Performance of Pepper Accessions with M. enterolobii Infection

The effects of RKN stress on the net photosynthesis rate (Pn), stomatal conductance (gs), transpiration rate (Tr), and water use efficiency (WUE) of nine pepper genotypes under different managements are shown in Figure 3. Under RKN stress, the average Pn values for the very resistant genotypes KKU-P21055 and KKU-P23011 (15.0 and 20.1 µmol CO₂ m⁻² s⁻¹, respectively) were higher than under control conditions (12.2 and 11.2 µmol CO₂ m⁻² s⁻¹, respectively). Conversely, genotypes KKU-P11044, KKU-P12005, KKU-P11174, and KKU-P23018 exhibited reduced Pn values (14.4, 9.5, 12.7, and 11.0 µmol CO₂ m⁻² s⁻¹, respectively) under RKN stress compared to their controls (15.6, 12.3, 19.5, and 15.5 µmol CO₂ m⁻² s⁻¹, respectively). Interestingly, the highly susceptible accessions, KKU-P11013, KKU-P12010, and KKU-P13008, showed higher average Pn values under RKN stress (19.6, 15.7, and 12.6 µmol CO₂ m⁻² s⁻¹, respectively) than under control conditions (18.5, 13.7, and 12.3 µmol CO₂ m⁻² s⁻¹, respectively) (Figure 3A,B).

For average gs, genotypes KKU-P21055, KKU-P12005, KKU-P11174, KKU-P13008, and KKU-P23018 showed a decline under RKN stress from 0.02, 0.04, 0.03, 0.02, and 0.03 mol H₂O m⁻² s⁻¹ to 0.02 mol H₂O m⁻² s⁻¹. Pepper plants subjected to stress displayed significantly lower mean gs values compared to the control plants. Interestingly, for the stressed plants, genotypes KKU-P11013, KKU-P11044, KKU-P12010, and KKU-P23011 (0.03, 0.03, 0.04, and 0.04 mol H₂O m⁻² s⁻¹, respectively) showed noticeably higher gs values than the control plants (0.02, 0.03, 0.04, and 0.01 mol H₂O m⁻² s⁻¹, respectively) (Figure 3C,D).

The mean Tr of the different RKN stress plants, KKU-P12010, KKU-P12005, KKU-P11174, KKU-P13008, and KKU-P23018, decreased from 1.5, 1.4, 1.3, 0.9, and 1.1 mmol H₂O m⁻² s⁻¹ to 1.4, 0.7, 1.0, 1.0, 0.5, and 0.7 mmol H₂O m⁻² s⁻¹, respectively. Pepper plants subjected to stress displayed significantly lower mean Tr values than the control plants. Interestingly, the mean Tr values across stressed genotypes, i.e., KKU-P11013, KKU-P11044, KKU-P21055, and KKU-P23011 (1.2, 1.2, 0.8, and 1.6 mmol H₂O m⁻² s⁻¹, respectively), were significantly higher than those of their corresponding control plants (0.9, 1.0, 0.7, and 0.5 mmol H₂O m⁻² s⁻¹, respectively) (Figure 3E,F).

The mean WUE for RKN stressed genotypes, KKU-P11013, KKU-P11044, KKU-P11174, KKU-P23011, and KKU-P23018, decreased from 22.9, 16.7, 16.1, 25.7, and 15.9 μmol CO₂ mmol H₂O⁻¹ to 16.8, 12.1, 12.9, 12.8, and 15.7 μmol CO₂ mmol H₂O⁻¹, respectively. Pepper plants subjected to stress displayed significantly lower mean WUE values than the control plants. On the other hand, the average WUE values for the stressed genotypes, KKU-P21055, KKU-P12010, KKU-P12005, and KKU-P13008 (18.8, 10.9, 13.9, and 24.2 μmol CO₂ mmol H₂O⁻¹, respectively), were notably higher than those of the control plants (16.6, 9.1, 9.6, and 11.3 μmol CO₂ mmol H₂O⁻¹, respectively) (Figure 3G,H).

4. Discussion

Meloidogyne enterolobii is one of the serious root-knot nematodes (RKNs) worldwide due to its growing pathogenicity, broad host range, and geographic dispersion in plants with resistance genes such as Mi-1, Mh, and Rk [25,26,27]. In Thailand, it was first found in guava in 2013 [9] and later reported in chili plants in Sisaket and Ubon Ratchathani Provinces in 2022 and 2024, respectively [7,8]. In addition, M. enterolobii has been continuously reported in several plants, including rain tree, ornamental plant, and shallot [10,11,28]. Infection by M. enterolobii is a major biotic stressor in chili pepper (Capsicum spp.) cultivation, affecting plant physiology, yield, and metabolic processes [29].

In this study, screening of 105 Capsicum accessions revealed a wide range of responses to M. enterolobii infection, highlighting genetic variability in resistance among and within species. We used multiple parameters when screening for M. enterolobii resistance: the reproductive factor (Rf), eggs per g of root, and gall index (GI)—to interpret plant response. Our results suggest the existence of valuable genetic resources for resistance breeding in Thailand. In particular, accessions such as KKU-P34004 (C. annuum) and KKU-23018 (C. chinense) demonstrated high resistance, indicating their potential as parental lines for nematode-resistant cultivar development. A correlation analysis revealed a strong and statistically significant relationship between eggs per g of root and Rf (r = 0.803, p < 0.001). Some pepper accessions showed minimal galling despite harboring considerable nematode populations such as KKU-P13008 (C. chinense) and KKU-P11027 (C. annuum), which had GI < 1 but Rf > 10 in. These findings are consistent with previous studies indicating that galling formation does not always reflect nematode reproduction [30,31,32]. This discrepancy underscores the importance of integrating multiple resistance parameters in screening programs, such as reproductive factor (Rf) and egg production. Certain genotypes may suppress egg production while still exhibiting visible galling, or vice versa [33]. By contrast, many reports suggest that increased gall formation is a reliable indicator of nematode reproduction levels [34,35]. In this study, the correlation between GI and both Rf and eggs per g of root were weak and not statistically significant. These findings suggest that gall formation does not consistently reflect nematode reproductive success, aligning with previous reports. Thus, our findings highlight the value of using Rf as a comprehensive resistance metric, as it integrates both nematode multiplication and plant response. Relying solely on GI may misrepresent true resistance levels, especially in accessions exhibiting partial resistance or tolerance. Such variations may result from peppers carrying different R genes and plants recognizing pathogen-associated molecular patterns (PAMPs), which trigger plant responses [22,36,37].

Moreover, a plant’s resistance to root-knot nematodes is influenced by multiple factors that affect how different genotypes cope with identical environmental conditions and nematode infestations [38]. The variation in resistance among Capsicum accessions may be driven by differential activation of plant hormonal signaling pathways and defense-related gene expression. Hormones such as jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) play central roles in modulating plant defense responses to nematode infection. Specifically, JA and ET have been associated with enhanced resistance to root-knot nematodes through their regulation of cell wall fortification, secondary metabolism, and activation of defense genes [39,40]. Although SA is typically involved in responses to biotrophic pathogens, it may also contribute to resistance via localized hypersensitive-like reactions [41].

Transcriptomic studies in related solanaceous crops have identified the upregulation of defense-associated genes (e.g., PR1, LOX1, WRKY, and ACO) during incompatible interactions with root-knot nematodes [42,43]. These genes regulate downstream defense responses and may contribute to observed resistance phenotypes. Although transcriptomic data were not collected in the current study, future research should focus on comparative gene expression profiling in highly resistant (e.g., KKU-P34004) and highly susceptible (e.g., KKU-P11013) accessions at early stages of infection. Such analyses could clarify the molecular mechanisms underlying resistance and help identify functional markers for breeding nematode-resistant cultivars. Therefore, breeding programs aimed at developing new resistant cultivars could potentially use accessions demonstrating resistance to M. enterolobii (R-HR) in tropical climates. This information is crucial for breeding programs, as it highlights different resistance levels to root-knot nematodes within and among Capsicum species. Genetic resistance is an effective technique for disease management, especially against root-knot nematodes, due to its safety for both food and the environment as well as its commercial potential [44,45,46].

Capsaicin accumulation and photosynthetic rate are both critical indicators of stress response and plant performance. Capsaicin accumulation also varies by genotype. Some tolerant genotypes (e.g., KKU-P11174 and KKU-P23018) increased capsaicin production under stress, while others (e.g., KKU-P21055) did not—indicating that the capsaicin response is genotype-specific and influenced by internal signaling networks. Interestingly, a few susceptible genotypes (e.g., KKU-P11013) also showed increased capsaicin levels, suggesting early stress recognition and a partial defense response. Therefore, this study found that capsaicin accumulation patterns varied among chili pepper genotypes, likely reflecting their adaptive reactions to different stress levels caused by root-knot nematode infestation. Capsaicin is synthesized through the phenylpropanoid and branched-chain fatty acid pathways, both of which occur in the glandular cells of the fruit’s placenta and pericarp across all genotypes—however, the amount synthesized can differ depending on the plant’s genetic makeup and environmental conditions. Gene regulation also plays a significant role, with over 50 genes now known to participate in capsaicinoid biosynthesis [47,48]. As a result, different chili genotypes exhibit distinct patterns of capsaicinoid accumulation, particularly when exposed to environmental stress such as root-knot nematode infestation. While M. enterolobii may stimulate capsaicinoid production, these compounds can also be transported to roots under stress, suggesting a systemic defense role. In this study, genotypes such as KKU-P11174 and KKU-P23018, which exhibited higher capsaicin levels during nematode infestation, also demonstrated greater resistance (Rf < 1), implying a critical threshold of capsaicinoid accumulation for effective defense. Conversely, genotypes with low capsaicinoid levels were more susceptible. These results indicate that a minimum concentration of capsaicin may be necessary to hinder nematode development. Further investigation is needed to quantify this root threshold and clarify the relationship between capsaicinoid production and nematode resistance.

In a tropical climate such as that in Thailand, pepper can be planted throughout the year and harvested six months after planting. Environmental factors significantly affect its photosynthetic performance, assimilate partitioning, and ultimately overall plant growth and yields. Root-knot nematodes are located within the vascular cylinder, where they induce gall formation, thereby disrupting water flow and impairing photosynthesis in plants. Biotic and abiotic stress can increase the production of abscisic acid (ABA) and reactive oxygen species (ROS), triggering calcium waves in the cell fluid that lead to stomatal closure and reduced water loss [49]. Infected plants under RKN stress exhibited reduced photosynthetic rates in some genotypes, indicating stomatal closure attributable to RKNs. Thus, tolerant genotypes (KKU-P12005, KKU-P11174, and KKU-P23018) showed decreased photosynthetic rates, suggesting an adaptive response to RKN-induced stress. These findings suggest a stress-induced compensatory mechanism and align with previous studies that reported photosynthetic stimulation under mild biotic stress. Nematode exposure was found to decrease gaseous exchange parameters, including net photosynthetic rate, intercellular CO₂ concentration, stomatal conductance, and transpiration rate [50]. On the other hand, some pepper genotypes, such as KKU-P11013, KKU-P12010, KKU-P13008, KKU-P21055, and KKU-P23011, tolerated long-term exposure to RKN stress quite well. Although partially affected by RKN damage, they maintained strong growth well, as indicated by their net photosynthesis rates comparable to or significantly higher than the control group. The stress caused by root-knot nematodes appeared to have a negligible effect on these tolerant genotypes. During the experimental period, the greenhouse temperatures reached 40–42 °C, which may have contributed to additional heat stress. The peppers in this group kept their stomata open to transpire and dissipate heat from leaf surfaces, resulting in a high stomatal conductance. They could also exchange carbon dioxide and water with the atmosphere, allowing these pepper genotypes to sustain high photosynthesis rates despite stressful conditions [51]. These results align with the principle that photosynthesis plays a critical role in plant physiological and biochemical processes, influencing material conversion and energy metabolism. Photosynthesis is highly responsive to elevated temperatures [52] and under combined stress conditions of heat and root-knot nematodes infestation, pepper genotypes that demonstrate efficient photosynthesis show a significant capacity for adaptation. Therefore, they are ideal candidates for breeding programs aimed at developing resistance to RKNs in tropical regions.

This study reveals a broad range of Rf values both among and within Thai Capsicum species, reflecting differences in resistance levels that influence nematode reproduction. It also highlights the relationship between capsaicin accumulation, photosynthetic response, and plant genotypes under RKN stress conditions. While promising resistant accessions were identified under controlled conditions, further validation under more rigorous settings—using larger pots, higher inoculum levels, and extended evaluation periods—is needed to confirm resistance durability over multiple nematode generations and in long-term field resistance to M. enterolobii. These variations have the potential to reduce yield losses and offer valuable insights for future breeding strategies. Resistant genotypes may also help to reduce nematode soil populations, aligning with sustainable agriculture goals. Future research should focus on elucidating the hormonal signaling networks and metabolic reprogramming triggered by nematode infection. A deeper understanding of these physiological and molecular responses will be crucial for identifying key regulatory genes and biomarkers that can be used to accelerate the development of genetically enhanced cultivars with durable resistance and improved stress tolerance.

5. Conclusions

This study provided a comprehensive assessment of the physiological and biochemical responses of diverse Capsicum genotypes to M. enterolobii infestation. Significant variations in resistance levels were observed across and within Capsicum species, particularly in terms of reproductive factor (Rf), egg per root weight, and capsaicin accumulation. Several genotypes, notably from C. chinense and C. frutescens, exhibited strong resistance (Rf < 1), making them valuable candidates for breeding programs targeting nematode resilience. Moreover, the study highlights that physiological responses, such as changes in photosynthetic rate, do not always correlate directly with resistance levels, suggesting a complex interplay between stress perception, metabolic regulation, and genotype-specific adaptation mechanisms. The variability in capsaicin synthesis further emphasizes the influence of genetic background and environmental stressors on secondary metabolism. These findings emphasize the importance of integrated breeding strategies that account for not only nematode resistance but also physiological resilience and biochemical traits.