Resistance analysis of the Tobacco Variety SA1214 against M. incognita based on physiological and biochemical assays

Abstract

Background

Meloidogyne incognita is a major soil-borne pathogen in tobacco production, which severely restricts the sustainable development of the Chinese tobacco industry. At present, problems including the diminished disease resistance of mainstream tobacco cultivars and the scarcity of resistant germplasm resources have become increasingly pronounced, and thus the breeding of new tobacco varieties with high resistance to M. incognita has become an urgent priority. Previous studies have revealed that the tobacco germplasm SA1214 originating from South Africa displays extremely high resistance to root-knot nematodes in field trials, yet its specific resistance grade and the systematic physiological and biochemical mechanisms underlying this resistance remain unclear.

Results

The results showed that SA1214 displayed strong resistance to M. incognita, with all its disease indices being significantly lower than those of susceptible cultivars such as Yunyan 87. This cultivar effectively inhibited the invasion by M. incognita and significantly delayed the late developmental process of the nematode post-infection. The giant cells at the infection sites exhibited characteristics of early vacuolization and disintegration, accompanied by the induction of plant defense responses including a prominent reactive oxygen species (ROS) burst and programmed cell death (PCD).

Conclusion

In summary, SA1214 is a tobacco cultivar with favorable application prospects for resistance to M. incognita. This study provides technical support for in-depth investigations into the molecular mechanisms of tobacco nematode resistance and the breeding of disease-resistant tobacco cultivars, and also contributes to the green and sustainable development of the tobacco industry.

Introduction

Tobacco (Nicotiana tabacum L.), a vital raw material for the global tobacco industry, not only underpins agricultural economies in producing areas but also safeguards local employment [1, 2]. In recent years, tobacco root-knot nematode (RKN)disease, caused by Meloidogyne spp., has spread rapidly throughout China [3]. Owing to broad host range, high pathogenicity, short reproductive cycle and efficient dispersal capacity, this nematode inflicts substantial losses to tobacco leaf yield and quality in major tobacco-growing regions worldwide. Consequently, it has emerged as a key limiting factor for the sustainable development of the Chinese tobacco industry [4, 5]. RKNs primarily infect tobacco root systems, inducing galls that severely impair nutrient and water uptake and thereby disrupt normal plant growth and development [6]. Furthermore, the feeding wounds created by RKNs provide entry points for secondary fungal and bacterial pathogens, leading to complex co-infections [7]. This disease complex results in intricate field symptoms, which not only complicates accurate diagnosis but also imposes stringent demands on the development of effective control strategies. Therefore, advancing research into the epidemiology and integrated management of tobacco RKN disease is a critical priority for ensuring sustainable tobacco production.

In tobacco production, the management of RKNs relies primarily on chemical control measures. While such methods provide moderate efficacy, prolonged reliance on chemical nematicides can drive the development of nematode resistance and cause adverse environmental impacts, including soil and groundwater pollution [8]. Furthermore, chemical residues may enter the food chain, posing potential risks to human and animal health [9]. Given these drawbacks, numerous countries have explicitly prohibited the use of specific chemical nematicides, such as methyl bromide [10, 11]. The development and utilization of disease-resistant tobacco varieties represents a more economical, eco-friendly, and sustainable management strategy. This approach not only improves tobacco yield and quality but also effectively prevents and controls RKN disease outbreaks [12, 13]. In recent years, significant progress has been achieved in the research on RKN-resistant germplasm resources across multiple crop species. For instance, Dimkpa et al. and Zhan et al. independently identified rice varieties resistant to Meloidogyne graminicola [14, 15]; Pinheiro et al. and Carrillo-Fasio et al. screened pepper germplasm for accessions with stable resistance to Meloidogyne enterolobii [16, 17]. These studies provide a crucial theoretical basis and practical guidance for breeding RKN-resistant crops. Presently, in Yunnan Province, China, M. incognita is an important pathogen responsible for tobacco RKN disease. Its infestation not only severely impairs local tobacco production but also causes substantial economic losses to the tobacco industry of China [18]. Given the widespread loss of resistance to M. incognita in currently cultivated tobacco varieties, accelerating the development of new M. incognita-resistant tobacco cultivars is imperative to mitigate this increasingly severe disease threat.

In this study, we characterized a resistant tobacco cultivar, SA1214, against M. incognita. Using the susceptible cultivar Yunyan 87 as a control, we conducted a comparative analysis of the post-infection developmental progression of the nematode, the infection rates of its different life stages over time, and the associated host responses, including callose deposition, reactive oxygen species (ROS) accumulation, and programmed cell death (PCD). This research lays a theoretical foundation for the efficient breeding of flue-cured tobacco cultivars resistant to M. incognita.

Materials and methods

Testing of tobacco cultivation and M. incognita culture

The tobacco varieties used in this experiment were Yunyan 87, Changbohuang, NC95, Honghua Dajinyuan, K326, and SA1214, all of which were provided by the Yunnan Academy of Tobacco Agricultural Sciences. Seeds of these six varieties were sown in seedling trays for seedling cultivation. After the seedlings developed four true leaves, they were transplanted.

The RKN used in this study was M. incognita, which was provided by the Nematology Laboratory, College of Plant Protection, Yunnan Agricultural University. After purification and identification, the nematodes were cultured on the root tips of the susceptible tomato cultivar ‘Zhongshu No.4’ and subcultured every 45–60 days. The nematode eggs were extracted using a modified sodium hypochlorite (NaOCl) method [19]. Briefly, infected roots were washed, cut into 1–2 cm segments, and immersed in a 1:14 (v/v) sodium hypochlorite solution before being vigorously stirred for 4 min. The mixture was filtered through a double-layer sieve stack (a 200 mesh sieve over a 500 mesh sieve). The root segments were rinsed with running water until the odor of NaOCl was no longer detectable, and the eggs retained on the 500 mesh sieve were collected. The egg suspension was incubated at 26 °C for 3–5 days using the shallow dish method to obtain second-stage juveniles (J2s) for inoculation.

Evaluation of resistance of tobacco to M. incognita

To identify tobacco germplasm resistant to M. incognita, roots of the six tobacco cultivars were separately inoculated with 1500 J2s per plant. Each treatment was arranged with three biological replications, with a fixed number of uniform tobacco seedlings per replication, and an equal volume of sterile deionized water served as the blank control. At 45 days after inoculation (DAI), plant height and root length were measured. Additionally, the fresh weight and dry weight of shoots and roots were determined. Meanwhile, root symptoms were assessed: the number of root galls, the number of eggs, and the percentage of roots infected by root galls were recorded, and photographs of the roots were taken. Following the root infection rating scales of Fan et al. and Li et al. disease severity was graded on a 0–9 scale [20, 21]. Additionally, the gall index (GI), egg mass index (EI), disease index (DI) and reproductive factor (RF) were calculated following the formulas reported by Li et al., Sun et al., Huang et al. and Feng et al. [21–24]. Resistance evaluation was performed according to the disease index-based criteria proposed by Li et al. where the experiment and its subsequent evaluation were deemed valid only if the DI of the susceptible control was no less than 60 [21]. The resistance classification standards were defined as follows: immune (I), disease index = 0; resistant (R), disease index = 0.1–20; moderately resistant (MR), disease index = 20.1–40; moderately susceptible (MS), disease index = 40.1–60; susceptible (S), disease index = 60.1–80; highly susceptible (HS), disease index = 80.1–100.

M. incognita penetration, development, and reproduction assays

Using the susceptible tobacco cultivar Yunyan 87 as the control, the roots of Yunyan 87 and SA1214 plants were separately inoculated with 500 J2s of M. incognita per plant. Root samples were collected at 1, 3, 5, 8, 12, 16, 24, and 30 DAI and subjected to NaOCl–acid fuchsin–glycerol staining. The staining protocol followed Zhou et al. with minor modifications: the washed tobacco roots were immersed in a 10% NaOCl solution for 4 min, rinsed in distilled water for 5 min to remove residual NaOCl, boiled for 2 min in an acid fuchsin solution (30 mL distilled water plus 1 mL acid fuchsin stock solution), cooled to room temperature, and transferred to 100% glycerol [25]. The glycerol containing the roots was heated until partial destaining was achieved. After cooling to room temperature again, the roots were mounted on microscope slides and examined under an Olympus BX53 microscope.

Histopathological study on the tissues of tobacco

The J2s of M. incognita were inoculated into Yunyan 87 and SA1214 tobacco plants, and root tips or regions with the largest root galls were collected at 6, 14, and 25 DAI. Galled and ungalled roots of Yunyan 87 and SA1214 tobacco plants were processed for histopathological studies using the method described by Basavaraj et al [26]. The root galls were thoroughly cleaned and fixed in FAA fixative (90 mL 70% ethanol, 5 mL glacial acetic acid, 5 mL formalin) at 4 °C for 3 days, rinsed three times with 70% ethanol, and dehydrated through a graded ethanol series until no bubbles or residual fluid remained. This was followed by xylene clearing, paraffin infiltration, and final embedding in paraffin blocks. Paraffin Sect. (8 μm thick) were prepared using a rotary microtome to obtain continuous sections. After dewaxing, clearing, and rehydration (via xylene and graded ethanol), cell structures were stained using the safranin and fast green staining method. Finally, the sections were subjected to gradient dehydration and clearing, mounted with neutral balsam to prepare permanent specimens, and observed under an Olympus BX53 microscope. Five of the largest giant cells per section were selected, and cell counting as well as area measurement were performed using ImageJ software.

Callose staining of tobacco

Following inoculation with J2s of M. incognita into the roots of Yunyan 87 and SA1214 tobacco plants, root tips measuring 1–2 cm in length were harvested at 12, 24, 48, and 72 h post-inoculation (hpi). Uninoculated plants at 0 hpi served as the untreated control. Callose observation was performed following the method described by Yang et al. employing aniline blue fluorescent staining for callose visualization [27]. Quantitative analysis was conducted using ImageJ software to calculate the relative callose content in the roots of Yunyan 87 and SA1214 at different time points following M. incognita infection.

ROS staining and Hydrogen Peroxide (H₂O₂) content determination

After inoculation of J2s of M. incognita into Yunyan 87 and SA1214 tobacco plants, root samples of the two cultivars were collected at 12, 24, 48, and 72 hpi. Uninoculated plants at 0 hpi served as the blank control. ROS staining was performed following the method described by Melillo et al. After DCF fluorescent staining of root tip tissues, the fluorescence intensity was observed and photographed under a Leica fluorescence microscope with a 10×objective lens (excitation wavelength of blue light: 488 nm; emission wavelength: 525 nm) [28]. H₂O₂ content was determined following the instructions provided with the ADS-W-YH015 Hydrogen Peroxide (H₂O₂) Assay Kit (Jiangsu Aidisheng Biological Technology Co.,Ltd). The H₂O₂ concentration (µ mol/g fresh weight) was calculated using the formula: , which is simplified to: .

In the above equation, V represents the volume of extraction solution added (1 mL); V₁ represents the volume of sample added to the reaction system (0.1 mL); W represents the sample mass (g); D represents the dilution factor (D = 1 for undiluted samples).

PCD detection

After inoculation of J2s of M. incognita into Yunyan 87 and SA1214 tobacco plants, root tip tissues (1–2 cm in length) were sampled at 12, 24, 48, and 72 hpi. Meanwhile, non-inoculated plants sampled at 0 hpi were used as the blank control. For PCD detection, root tip segments of samples were stained using the trypan blue (TB) method following the protocol described by Phillips and Hayman [29]. After staining, the occurrence of PCD was observed under an Olympus BX53 microscope, and images were captured for documentation.

Results

Evaluation of resistance of tobacco to M. incognita

Resistance evaluation showed that SA1214 exhibited the highest resistance, with few or no root galls formed. The gall number, egg mass number, GI, EI, DI and RF of SA1214 were significantly lower than those of Changbohuang, Yunyan 87, and Honghua Dajinyuan, measuring 26, 1560, 1.62, 96.57, 11.11 and 1.04, respectively (Fig. 1). NC95 and K326 displayed moderate resistance, whereas Changbohuang, Yunyan 87, and Honghua Dajinyuan were highly susceptible, their roots being densely covered with galls.

Fig. 1.

Effects of M. incognita inoculation on six tobacco cultivars at 45 Days After Inoculation (DAI). A: Root symptoms of Changbohuang; B: Root symptoms of Yunyan 87; C: Root symptoms of Honghua Dajinyuan; D: Root symptoms of K326; E: Root symptoms of NC95; F: Root symptoms of SA1214; G: The number of root galls; H: The root Gall Index (GI); I: The Disease Index (DI); J: The number of eggs; K: The Egg Index (EI); L: The Reproductive Factor (RF). G-L: Data are presented as mean ± standard error (SE). Error bars above each column indicate the SE. Significant differences between means were determined using Tukey’s HSD test (P ≤ 0.05). Values sharing the same letter are not significantly different

Analysis of growth parameters indicated that the aboveground growth of all tested cultivars was significantly inhibited. Changbohuang was the most sensitive to M. incognita stress, with plant height and aboveground dry weight significantly reduced compared with the non‑inoculated control (CK). Yunyan 87 and Honghua Dajinyuan also showed significant reductions in plant height and aboveground dry weight relative to CK (Table 1). Although SA1214 remained uninfected, its growth was still slightly inhibited. According to the DI‑based classification, SA1214 was rated as R, K326 and NC95 as MR, and Yunyan 87 and Honghua Dajinyuan as HS.

Table 1.

Effects of M. incognita on growth indices and biomass of six tobacco cultivars

Tobacco variety	Plant height (cm)			Root length (cm)			Fresh Shoot fresh weight (g)			Root fresh weight (g)			Shoot dry weight(g)				Root dry weight (g)
	CK	Inoculated		CK	Inoculated		CK	Inoculated		CK	Inoculated		CK	Inoculated		CK		Inoculated
Changbohuang	50.14 ± 0.92	27.03 ± 0.60	***	13.59 ± 0.30	13.93 ± 0.40	NS	87.26 ± 2.61	52.22 ± 3.07	***	9.42 ± 0.34	17.02 ± 0.46	***	8.22 ± 0.21	5.35 ± 0.31	***	0.88 ± 0.03		1.02 ± 0.04	**
Yunyan 87	59.89 ± 1.06	40.77 ± 0.71	***	15.97 ± 0.37	14.44 ± 0.31	**	94.03 ± 1.57	55.06 ± 1.80	***	14.29 ± 0.39	14.59 ± 0.33	NS	9.13 ± 0.19	6.08 ± 0.10	***	1.38 ± 0.11		1.23 ± 0.05	NS
Honghua Dajinyuan	35 ± 0.90	29.91 ± 1.37	**	12.31 ± 0.30	13.67 ± 0.20	**	56 ± 2.52	46.81 ± 2.03	*	12.04 ± 0.57	9.24 ± 0.41	**	7.32 ± 0.19	5.73 ± 0.17	***	0.95 ± 0.04		0.77 ± 0.04	**
K326	19.20 ± 0.55	15.88 ± 0.54	**	15.46 ± 0.26	15.86 ± 0.22	NS	46.13 ± 0.95	18.02 ± 1.08	***	9.19 ± 0.26	6.92 ± 0.40	***	5.37 ± 0.06	2.91 ± 0.09	***	0.84 ± 0.04		0.52 ± 0.03	***
NC95	28.00 ± 1.61	25.42 ± 1.01	NS	11.32 ± 0.26	11.60 ± 0.23	NS	72.07 ± 1.80	35.62 ± 2.27	***	13.16 ± 0.50	9.87 ± 0.63	**	7.17 ± 0.19	5.60 ± 0.14	***	0.97 ± 0.04		0.87 ± 0.03	NS
SA1214	29.46 ± 0.50	23.58 ± 0.67	***	16.32 ± 0.25	14.94 ± 0.14	***	65.53 ± 2.36	50.02 ± 1.03	***	20.50 ± 0.56	16.18 ± 0.25	***	7.48 ± 0.25	6.18 ± 0.06	**	2.38 ± 0.06		1.27 ± 0.05	***

Values are presented as mean ± standard error of three biological replicates. For each tobacco variety, significant differences between control and inoculated plants were indicated at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***); NS indicates no significant difference (*p*>0.05)

Attraction and penetration of M. incognita in tobacco

Based on the resistance evaluation, the highly resistant tobacco cultivar SA1214 was selected in this study, while Yunyan 87, a widely cultivated susceptible tobacco cultivar in Yunnan Province, was used as the control. The invasion rate and developmental stages of M. incognita in tobacco roots were determined using the NaClO-fuchsin acid-glycerol staining method. The results demonstrated that, relative to Yunyan 87, both nematode invasion and development in SA1214 roots were significantly suppressed throughout the experimental period. Specifically, at 24 DAI, nematodes in Yunyan 87 roots had developed into adult females, whereas those in SA1214 roots were arrested at the J4 stage (Fig. 2).

Fig. 2.

The sodium hypochlorite-acid fuchsin-glycerol staining results of M. incognita infecting Yunyan 87 and SA1214 tobacco cultivars. Scale bars: 100 μm

Statistical analysis showed the initial invasion rate of J2 in Yunyan 87 roots reached approximately 45.7%, declining to 37.2% by 30 DAI. In contrast, the nematode invasion rate in SA1214 roots remained consistently low, with only 21.3% detected at 30 DAI. Analysis of nematode population composition revealed a continuous decrease in the proportion of J2 in Yunyan 87 roots at 24 and 30 DAI, accompanied by a marked increase in adult females. However, substantial numbers of J2, J3, and J4 were still present in SA1214 roots, with the proportion of adult females at only approximately 56.1% (Fig. 3A, B).

Fig. 3.

Infection rate of Yunyan 87 and SA1214 tobacco inoculated by M. incognita in different stages. A: Infection rate of M. incognita different stages (%); B: Infection rate of stage of M. incognita in each period (%). Data are presented as mean ± standard error (SE). Error bars above each column indicate the SE. Significant differences between means were determined using Tukey’s HSD test (P ≤ 0.05). Values sharing the same letter are not significantly different

Histopathological changes in M. incognita after inoculation with tobacco

Paraffin section observation revealed that giant cells began to form in both tobacco cultivars at 6 DAI, with no significant differences in size and morphology, and all gradually enlarged as inoculation time prolonged (Fig. 4A). At 25 DAI, most giant cells in SA1214 exhibited cytoplasmic disintegration and cavitation, whereas those in Yunyan 87 maintained a compact structure without cytoplasmic disintegration. No significant difference in giant cell area was detected between the two cultivars at 6 DAI (Fig. 4B, C). At 14 DAI, Yunyan 87 had a significantly larger giant cell area than SA1214. By 25 DAI, the giant cell area in Yunyan 87 further increased to 23808.98 μm², while that in SA1214 was only 8293.53 μm² due to cellular structural disintegration. For giant cell number, Yunyan 87 contained 4–7 cells (average: ~5) and SA1214 contained 2–6 (average: ~4). Notably, Yunyan 87 had a significantly higher giant cell number than SA1214 at 14 and 25 DAI.

Fig. 4.

Paraffin section results of Yunyan 87 and SA1214 tobacco plants infected by M. incognita. A: Changes of giant cells in tobacco roots of Yunyan 87 and SA1214; black arrows indicate RNKs, and red arrows indicate giant cells. B: Area of giant cells in Yunyan 87 and SA1214. C: Giant cell number in Yunyan 87 and SA1214. Data are presented as mean ± standard error (SE). Error bars represent the standard error (SE). Significant differences were analyzed by Tukey’s HSD test (P ≤ 0.05). Values marked with the same letter are not significantly different. Scale bars: 100 μm

Callose deposition differences in tobacco roots following inoculation with M. incognita

Aniline blue staining indicated a gradual increase in callose deposition and a sustained enhancement of fluorescence intensity over the course of inoculation (Fig. 5). At 72 hpi, SA1214 root tips exhibited the highest callose accumulation and peak fluorescence intensity, while Yunyan 87 root tips showed much weaker fluorescent signals and less callose deposition. Fluorescence intensity measurements further confirmed that the callose fluorescence intensity increased in both tobacco cultivars with prolonged inoculation time.

Fig. 5.

Callose staining and relative amount of Yunyan 87 and SA1214 tobacco infected by M. incognita. A: Callose staining observation in tobacco roots of Yunyan 87 and SA1214. B: Mean fluorescence intensity of callose in tobacco roots of Yunyan 87 and SA1214. Data are presented as mean ± standard error (SE). Error bars represent the standard error (SE). Significant differences were analyzed by Tukey’s HSD test (P ≤ 0.05). Values marked with the same letter are not significantly different. Scale bars: 100 μm

ROS staining patterns and H₂O₂ content changes in tobacco roots inoculated with M. incognita

A prominent ROS burst was detected in the tobacco roots of Yunyan 87 and SA1214 following M. incognita inoculation (Fig. 6). At 48 hpi, SA1214 had a significantly higher ROS fluorescence intensity than Yunyan 87, whereas ROS levels in both cultivars decreased markedly by 72 hpi (Fig. 6A). Furthermore, H₂O₂ content assays demonstrated that SA1214 roots had a peak H₂O₂ concentration at 48 hpi, significantly higher than that in Yunyan 87, and this was consistent with ROS fluorescence staining results (Fig. 6B).

Fig. 6.

Reactive oxygen staining and changes of H₂O₂ content Yunyan 87 and SA1214 tobacco tobacco infected by M. incognita. A: ROS fluorescence staining observations in tobacco roots. B: Changes in H₂O₂ content in tobacco roots at different time points after inoculation. Data are presented as mean ± standard error (SE). Error bars represent the standard error (SE). Significant differences were analyzed by Tukey’s HSD test (P ≤ 0.05). Values marked with the same letter are not significantly different. Scale bars: 100 μm

PCD occurrence in inoculated tobacco roots with M. incognita

TB staining results revealed that after inoculation with M. incognita, Yunyan 87 exhibited a weak PCD response, with mild, sporadic and limited cell death. In contrast, SA1214 displayed an intense PCD response accompanied by severe and widespread cell death. No obvious cell death was observed in either cultivar at 12 h post inoculation (hpi). At 24 hpi, sporadic cell death was detected in SA1214, while it was barely visible in Yunyan 87. Obvious cell death occurred in SA1214 at 48 hpi, and a small number of dead cells also appeared in Yunyan 87. At 72 hpi, extensive cell death was found in SA1214, and the incidence of cell death in Yunyan 87 was significantly lower than that in SA1214. (Fig. 7).

Fig. 7.

PCD in root tips of Yunyan 87 and SA1214 tobacco infected by M. incognita. Scale bars: 100 μm

Discussion

The gall index (GI), egg mass index (EI), reproduction factor (RF), and disease index (DI) are the primary indicators for evaluating tobacco resistance to RKNs, yet most existing studies rely on only one or a few of these parameters [30–32]. In this study, M. incognita inoculation significantly reduced key disease indices (gall number, egg mass number, GI, EI, DI and RF) in SA1214 compared with susceptible tobacco controls Yunyan 87 and Changbohuang. This cultivar exerted its resistance mainly by inhibiting the growth and development of nematodes, yet it could not completely eliminate nematodes that have invaded plant tissues. Consequently, its reproduction factor was slightly above 1 (RF=1.04), while the overall disease index still met the evaluation criteria for resistant cultivars. Collectively, these findings confirm SA1214 as an elite germplasm with robust resistance to M. incognita, and further characterization of its resistance-associated physiological and biochemical mechanisms will establish a critical theoretical basis for nematode-resistant tobacco breeding.

Upon infection by RKNs, host plants activate a multi-layered defense system via the release of root-derived chemicals, activation of immune responses, and modulation of resistance gene expression [33, 34]. Here, the resistant cultivar SA1214 exhibited a markedly lower nematode infection rate than the susceptible control Yunyan 87; additionally, successfully invading nematodes showed arrested development and reduced relative abundance across all developmental stages. This pattern contrasts with the defense strategy reported by Phan et al. for the cultivar Zhonghua 11, which allowed extensive initial nematode penetration but conferred resistance through rapid post-invasion responses that immobilized nematodes, restricted their migration, and inhibited nematode development and giant cell formation [35]. Conversely, SA1214 has both pre-invasion and post-invasion resistance mechanisms against M. incognita, which are presumably mediated by two pathways. First, structural barriers formed in the root epidermis and cortex physically hinder nematode penetration [34]. Second, post-invasion defenses including giant cell disintegration and PCD inhibit the development of nematodes within root tissues.

Paraffin section observations in this study revealed that 25 DAI with M incognita, giant cells in the roots of SA1214 displayed distinct disintegration and cavitation, whereas those in Yunyan 87 roots developed normally with no detectable structural damage. Furthermore, both the number and area of giant cells in SA1214 roots were significantly reduced compared to those in Yunyan 87 roots. These observations are in line with previous studies, which have shown that giant cells in the roots of resistant plant cultivars are generally characterized by reduced quantity and smaller individual size, and tend to undergo cavitation, disintegration, and even necrosis during the late stage of nematode infection. Conversely, giant cells in susceptible cultivars develop robustly, featuring higher quantities, larger sizes, abundant nuclei, dense cytoplasm, and intact morphology [35–37]. It has been well documented that resistant cultivars trigger rapid post-invasion defense responses, such as an early hypersensitive response (HR), to suppress giant cell differentiation and block nematode development, ultimately resulting in nematode mortality [38]. Building on these findings, we propose that the premature breakdown of giant cells in SA1214 roots is a direct consequence of activated host plant defense responses, encompassing key physiological processes including ROS burst, PCD, and de novo lignin synthesis. The activation of these defense cascades impairs the nutritional supply niche essential for nematode survival, thereby inhibiting the development of invading nematodes and eventually causing their death due to nutrient starvation [34, 39].

Under normal physiological conditions, plants maintain homeostasis between ROS generation and scavenging. Pathogen infection triggers an oxidative burst, and the concomitant upregulation of ROS-scavenging enzymes alleviates oxidative damage [40]. H₂O₂, a pivotal signaling molecule among ROS, plays a central role in plant innate immunity by mediating early defense signaling and inducing PCD during incompatible interactions, thereby spatially confining pathogens [41, 42]. Accordingly, resistant plants respond to RNK invasion by rapidly initiating PCD at feeding sites, which disrupts the parasitic niche and arrests nematode growth and development [43]. Inoculation with M. incognita induced a marked oxidative burst in the roots of both susceptible Yunyan 87 and resistant SA1214 tobacco cultivars. Quantitative fluorescence staining indicated that ROS levels were significantly higher in SA1214 than in Yunyan 87 at 48 hpi, followed by a sharp decrease in both cultivars at 72 hpi. H₂O₂ quantification assays confirmed this temporal pattern. The resistant cultivar SA1214 converted this early oxidative signal into an extensive, HR-like, PCD response characterized by deep and widespread cell death. Conversely, the susceptible Yunyan 87 displayed only minimal and localized PCD, failing to establish an effective defense against nematode invasion. This is consistent with prior observations reported in Melillo et al. that incompatible interactions are characterized by sustained ROS/H₂O₂ accumulation within 12–48 hpi, whereas compatible interactions exhibit only a transient oxidative burst [28]. Therefore, the distinct temporal dynamics of the oxidative burst likely determine the efficacy of PCD-mediated defense and the resulting resistance outcome against nematode infection.

Conclusion

In conclusion, this study confirms that the tobacco cultivar SA1214 exhibits strong resistance to M incognita. Specifically, its disease index is significantly lower than that of the susceptible cultivar Yunyan 87 and other susceptible genotypes; it can effectively inhibit the nematode invasion rate and delay their late developmental process. Pathologically, the induced giant cells display early vacuolization and disintegration, accompanied by prominent defense responses such as ROS burst and PCD. As a tobacco germplasm resource with favorable application prospects for resistance to M incognita, SA1214 can provide material support for further disease-resistant breeding and mechanistic research on nematode resistance.

Authors’ contributions

CSM carried out data analysis and wrote the first draft of the manuscript. ZSF designed the experiments, conducted the trials and analyzed the data. GYZ and YH completed the relevant experiments and research. WY and CXJ provided experimental resources, assisted in identification experiments, and revised the manuscript. WWT conceived the overall experiments and critically revised the manuscript. All authors have read and approved the final version of the manuscript.

Funding

This work was supported by the Major Science and Technology Project of Yunnan Provincial Tobacco Company (2024530000241003), the Yunnan Provincial Basic Research Program (202501AU070032), and Yunnan Provincial Department of Education Scientific Research Fund Project (2025Y0429). The authors declare that there are no conflicts of interest between this manuscript and any institutions or companies.

Data availability

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Declarations

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Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.