Evaluation of Resistance in Indian Rice to Root-Knot Nematode (Meloidogyne graminicola): Insights from Field and Histopathological Studies

Abstract

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Rice (Oryza sativa L.) is a staple crop for over half of the global population, yet its cultivation faces significant threats from biotic stresses, particularly root-knot nematodes (Meloidogyne spp.). Among these, M. graminicola poses a major challenge in rice-growing regions, leading to substantial yield losses. This study evaluated the resistance of 348 rice varieties to M. graminicola through controlled pot and field experiments over two years (2023 and 2024). Varieties were classified based on gall index, revealing a spectrum of susceptibility from highly susceptible to highly resistant. Notably, varieties such as JR-1124 and JR-403 exhibited high gall index, while others like RP-5219-9-7-3-2-1-1, NPT-10, MTU 1390 (IR17M1172), Kushiari, RP 6750-RMS-2-23-67-91, Sonkharchi, Sugandha-3, HRT-183, and HR-12 demonstrated significant resistance. Resistant rice genotypes exhibited significantly higher PAL, POX, and total phenolic content at all intervals, indicating a strong biochemical defense response against Meloidogyne graminicola. Advanced techniques, including confocal microscopy, revealed distinct histopathological responses to M. graminicola infection, with susceptible rice varieties exhibiting extensive giant cell formation and root tissue degradation, while a resistant variety displayed restricted giant cell development, enhanced callose deposition, and maintained vascular integrity—highlighting robust defense mechanisms against nematode invasion. The findings underscore the potential for breeding programs to enhance resistance traits in rice, contributing to sustainable agricultural practices and improved food security. As nematode populations evolve, ongoing research is essential to adapt breeding strategies and maintain effective management of this significant pest in rice production systems.

Background

Rice (Oryza sativa L.) is a vital staple crop, feeding over half of the global population and playing a critical role in food security. Grown across diverse agro-climatic zones, rice production is increasingly threatened by biotic stresses, particularly from root-knot nematodes (Meloidogyne spp.). Among these, Meloidogyne graminicola is a major pest that significantly reduces rice yields and challenges sustainable cultivation.

M. graminicola is a sedentary endoparasite that thrives in flooded rice ecosystems. It infects over 100 plant species, causing characteristic hook-shaped galls on rice roots. These galls disrupt nutrient and water uptake, leading to stunted growth, chlorosis, and reduced plant vigor (Jain et al. 2012; Mantelin et al. 2017). The nematode’s life cycle, lasting 15 to 51 days depending on temperature that facilitates rapid population growth (Upadhyay and Bhardwaj 2014). Even at low densities of 500 juveniles per plant, significant reductions in plant height, panicle growth, and photosynthetic efficiency are observed (Chen et al. 2022). Yield losses due to M. graminicola range from 28% to over 80%, depending on environmental conditions and host susceptibility (Mantelin et al. 2017).

The nematode’s global distribution spans tropical and subtropical rice-growing regions, underscoring its threat to food security. Its reproductive efficiency enables second-stage juveniles (J₂) to invade roots soon after hatching, with females maturing in two weeks (Huang et al. 2015). The ability to persist in soil without a host complicates management, particularly in systems with limited crop rotation. While the use of chemical nematicides possess environmental and health concerns, emphasizing the need for sustainable alternatives.

Developing resistant rice varieties is a promising strategy to combat M. graminicola. Resistance varies among rice genotypes, ranging from high susceptibility to robust resistance. Resistant varieties impede nematode reproduction and minimize damage. For example, Oryza glaberrima genotypes exhibit lower juvenile penetration rates, while resistant genotypes like Zhonghua 11 display hypersensitive responses (HR) characterized by rapid reactive oxygen species (ROS) accumulation and necrosis of infected cells (Nguyen et al. 2022). Environmental factors such as temperature also influence resistance; for instance, Zhenshan 97 B exhibits strong resistance under elevated temperatures, forming significantly fewer galls compared to susceptible varieties (Devaraja et al. 2022). Gall indices are widely used to evaluate resistance, categorizing rice cultivars from immune to highly susceptible. Resistant varieties like Zhenshan 97 B show minimal gall formation, whereas susceptible ones like IR 64 Sub 1 exhibit high gall indices, reflecting their vulnerability (Devaraja et al. 2022; Pandey 2020). Marker-assisted selection (MAS) accelerates the breeding of resistant cultivars by targeting specific resistance traits. Seasonal variations in resistance highlight the need for repeated evaluations under diverse conditions to ensure stability.

Physiological and biochemical responses differentiate resistant and susceptible varieties. Resistant genotypes often have higher levels of defense-related proteins and phenolic compounds, contributing to nematode resistance (Pandey 2020). Advances in metabolomics have identified key pathways and compounds involved in these defense mechanisms (Gautam et al. 2024a, b). Histopathological studies further reveal differences in feeding site development. Resistant varieties develop fewer and smaller feeding sites, limiting nematode development, while susceptible varieties exhibit extensive site formation that disrupts root architecture (Levin et al. 2021).

Molecular studies have advanced our understanding of plant-nematode interactions. Identifying nematode effectors and host defense activations has clarified resistance mechanisms (Kaloshian and Teixeira 2019a, b). However, breeding for durable resistance is challenging due to the nematode’s adaptability. Integrating molecular, histopathological, and phenotypic approaches is essential for sustainable resistance strategies.

Diverse genetic resources for resistance have been identified, including accessions of Oryza glaberrima, O. eichingeri, and O. grandiglumis and these accessions hold significant potential for breeding programs (Kaur et al. 2023). The identification of quantitative trait loci (QTLs) linked to resistance traits has enhanced MAS, facilitating the development of nematode-resistant rice cultivars. Advances in genomics, transcriptomics, and high-throughput screening further strengthen MAS applications (Das et al. 2017; Mapari and Mehandi 2024).

Despite these advancements, managing M. graminicola remains a challenge. The genetic basis of resistance in rice is complex, often involving multiple genes with variable effects across environments. Additionally, the nematode’s capacity to adapt necessitates continuous monitoring and breeding efforts to counter evolving populations. Incorporating resistant cultivars into cropping systems can reduce reliance on chemical controls while maintaining productivity. These cultivars also provide valuable genetic resources for future breeding programs.

In conclusion, effective management of Meloidogyne graminicola in rice requires understanding the interaction between nematodes and host plants. Resistance diversity among rice varieties presents opportunities for breeding programs to enhance resistance traits. Continued research integrating molecular, histopathological, and field-based approaches will contribute to sustainable rice production and global food security.

Materials and Methods

Rice Seed Collection

A total of 348 rice varieties were employed to elucidate the response of plants to M. graminicola, including one local variety HUR917 and one international variety TN1 serving as highly susceptible controls. Among the 348 varieties, 235 were sourced from the ICAR-Indian Institute of Rice Research (IIRR), Hyderabad, 100 were obtained from Jawaharlal Nehru Krishi Vishwavidyalaya, Jabalpur and 12 local varieties from the College of Agriculture, Rewa.

Nematode Culture and Inoculation

M. graminicola was initially isolated from an infested agricultural field at BHU farm in Varanasi, India, and was subsequently maintained on O. sativa cv. HUR-917 within a net-house at a controlled temperature of 26 °C (Gautam et al. 2024a). The nematode eggs were harvested from root galls and incubated using a 200-µm sieve. Juvenile stage J₂ nematodes were extracted utilizing a modified flotation-sieving technique and subsequently collected with a 25-µm sieve, as delineated in Huang et al. (2015). The nematode population was quantified under microscopic examination and adjusted to 150 ± 5 juveniles per ml for forthcoming inoculation.

Field Experiment

The field experiment was conducted from August to September in a continuously cultivated field at BHU farm, under irrigated conditions during the years 2023 and 2024. This particular field had experienced severe infestation by M. graminicola for a minimum of five years, exhibiting a density of 154 ± 22 nematodes per gram of soil. Prior to seeding, the soils in this field were thoroughly mixed using a rotary cultivator. The management protocols remained consistent across the various tested varieties. Seeds were directly sown into the plot, ensuring the maintenance of 20 ± 2 plants per line.

The dimensions of the plot was approximately 60 m² (30 m × 2 m), wherein all 348 varieties in both years were organized according to a randomized complete block design with three replicates. To mitigate water loss and limit the dispersion of nematodes, an earthen levee measuring 20 cm in height and 20 cm in width was constructed around the plot (Khanam et al. 2016). At 30 days post seeding, five plants from each line were uprooted, and the roots were meticulously washed to remove soil for the purpose of quantifying the root galls.

Pot Experiments

Cultivation of plants occurred in earthen pots measuring 15 × 7.5 cm (H × D). Four resistant and six highly susceptible rice varieties, based on field data, were selected for the experiment. These varieties were germinated at a temperature of 30 ± 2 °C for 5 days and one seed from each variety was sown in pots filled with a soil and sand mixture (3:1). Each plant, at an age of 10 days, was inoculated with approximately 150 ± 5 nematode juveniles, as per the methodology outlined by Huang et al. (2015). The experiment was replicated twice, with three replicates for each trial. The plants received irrigation three times per week. At 14 days post-inoculation (dpi), five plants were uprooted, and the roots were thoroughly washed to facilitate the calculation of root galls. All roots underwent further clarification in 0.6% NaOCl for 5 min and were subsequently boiled in a solution of 0.8% acetic acid and 0.013% acid fuchsin for 3 min, following the procedures established by Nahar et al. (2011). After destaining in a 4% acidified glycerol solution for a period of 3 to 4 days, nematodes at various developmental stages within the roots were examined using stereomicroscope. The reproductive factor, [RF = Pf/Pi; where Pf = total number of eggs and second-stage juveniles ( J₂) extracted from roots and soil at harvest, and Pi = number of J₂ at initiation of the experiment], was calculated for each experimental unit.

Resistance/susceptibility Scoring Methods

Five plants per line were randomly pulled out at 14 dpi in the pot experiment or 30 d after sowing in the field experiment. Resistance/susceptibility was assessed using the gall index of Pederson and Windham (1989) with slight modifications. Root galling was rated on a scale of 0 to 5, where level 0 = no galls, level 1 = 1–2, level 2 = 3–10, level 3 = 11–20, level 4 = 21–30, level 5 ≥ 30 galls per root system. The susceptible varieties TN1 and HUR-917 were used as the controls. For each rice variety, five plants were evaluated, and the Gall Index (GI) was calculated using the following formula:

$$\:\frac{\left(\sum\:Gall\:score\:of\:all\:plants\right)\times\:100}{Total\:no.\:\:of\:plants\times\:5}$$

where, ∑ gall scores is the sum of individual gall ratings per plant, the total number of plants refers to the number of replications per treatment (n = 3), and the maximum scale value is 5 based on the rating scale. GI was used to score resistance/susceptibility as follows: immune (I) GI = 0; highly resistant (HR) 0.1 ≤ GI ≤ 5.0; resistant (R) 5.1 ≤ GI ≤ 25.0; moderately susceptible (MS) 25.1 ≤ GI ≤ 50.0; susceptible (S) 50.1 ≤ GI ≤ 75.0; highly susceptible (HS) GI > 75.0.

Nematode Development in Resistant Cultivars

To examine the development of nematodes, seeds of one resistant cultivar and three highly susceptible cultivars were sown in plastic pots within a controlled net house environment, followed by the inoculation of 150 ± 5 nematodes. The experiment was executed in triplicate, utilizing 15 pots from each cultivar for histopathological analyses via confocal microscopy. After a period of 10 days, each plant was inoculated with approximately 150 juvenile nematodes. At predetermined intervals, three plants from each cultivar were uprooted, the roots were thoroughly washed, and the number of root galls as well as the nematode population within the roots were determined as previously described. At 3 dpi, 7 dpi, 11 dpi, and 14 dpi, additional plants were uprooted, and the various developmental stages of nematodes along with their feeding activities within the roots were scrutinized under confocal microscopy. This experiment was replicated three times for each treatment.

Sample Preparation, Microscopy, and Imaging

Roots from inoculated rice genotypes i.e., JR-1124, TN1, and HUR-917 (all highly susceptible, HS), and RP-5219-9-7-3-2-1-1 (resistant, R) were harvested at specific intervals. Sampling was done at 7 and 14 days post-inoculation (dpi) for JR-1124, TN1, RP-5219-9-7-3-2-1-1, and at 3, 7, 11, and 14 dpi for the susceptible local control HUR-917. Root regions (2–4 mm) showing swelling were excised, stained with acid fuchsin as per Bybd et al. (1983), and embedded in 4% agarose. Transverse Sect. (120 μm thick) were prepared using a Leica VT1200 vibratome (Leica Biosystems, Nussloch, Germany).

To visualize nematode-induced cytological changes, dual-fluorescence staining was performed using a 1:1 mixture of 0.1% calcofluor white (in 20% ethanol) and 10 µg mL⁻¹ propidium iodide (in water). Calcofluor white binds to cellulose and callose, enabling detection of polysaccharide deposition and structural reinforcement. Propidium iodide intercalates with nucleic acids and stains compromised cell walls, providing insights into cellular viability and nematode-induced damage. This dual-staining strategy facilitates concurrent visualization of host defense responses (e.g., cell wall thickening) and the extent of tissue disruption.

Stained sections were rinsed thrice with sterile water or 1× phosphate-buffered saline and mounted in three-well Teflon slides using 50% glycerol, then covered with cover slips. Imaging was performed on a Leica SP8 super-resolution confocal microscope (Leica Microsystems, Nussloch, Germany) using DAPI (405 nm) and TRITC (561 nm) laser channels, following the protocol of Levin et al. (2020).

Biochemical Assays for Defense Response

To evaluate defense-associated biochemical responses, phenylalanine ammonia-lyase (PAL), peroxidase (PO) activity, and total phenolic content (TPC) were quantified in root tissues of six rice varieties identified through preliminary field and pot screening. The three resistant varieties RP-5219-9-7-3-2-1-1, RP 6750-RMS-2-23-67-91 and MTU 1390 and three highly susceptible varieties JR-1124, TN1, and HUR-917 were selected for biochemical analyses.

PAL Activity

Root galls and tips from five biological replicates (each pooled from three plants) were immediately frozen in liquid nitrogen, ground, and assayed following Camacho-Cristóbal et al. (2002). A 100 mg sample was homogenized in 800 µL of 50 mM sodium phosphate buffer (pH 7.0) containing 2% polyvinylpolypyrrolidone, 2 mM EDTA, 18 mM β-mercaptoethanol, and 0.1% Triton X-100. After centrifugation (8,000 rpm, 4 °C, 10 min), 20 µL supernatant was mixed with 135 µL reaction buffer and 50 µL of 5 mM L-phenylalanine. Absorbance was measured at 290 nm before and after incubation at 40 °C for 30 min. One unit (U) of PAL activity corresponds to 1 nmol trans-cinnamic acid produced per hour.

Peroxidase (PO) Activity

PO activity was measured as per Fernando and Soysa (2015) with minor modifications. Root tissue (0.5 g) was homogenized in 5 mL of 0.1 M phosphate buffer (pH 7.0), and volume adjusted to 10 mL. After centrifugation (16,000 rpm, 4 °C, 15 min), 50 µL supernatant was reacted with 1.5 mL pyrogallol and 500 µL H₂O₂. Absorbance at 420 nm was recorded every 30 s for 3 min. Activity was expressed as U mg⁻¹ fresh weight (FW).

Total Phenolic Content (TPC)

Following Zheng and Shetty (2000), 0.1 g fresh root tissue was extracted in 5 mL 95% ethanol at 0 °C for 48 h. After centrifugation (13,000 × g, 10 min), 1 mL supernatant was mixed with 1 mL 95% ethanol, 5 mL sterile distilled water, 0.5 mL Folin–Ciocalteu reagent, and 1 mL of 5% Na₂CO₃. Absorbance was recorded at 725 nm after 60 min. Gallic acid was used as the standard, and results were expressed in mM gallic acid equivalents per gram FW (mM GAE g⁻¹ FW). All spectrophotometric readings were performed using a double-beam UV-VIS spectrophotometer (Model LT-2700).

Results

Developmental Stages of Meloidogyne graminicola

The life cycle of Meloidogyne graminicola was observed and documented over 14 days under a bright-field microscope using the rice variety HUR-917. Figure 1 illustrates the different developmental stages, starting from eggs to mature adult females. The eggs of M. graminicola (Fig. 1A) were elliptical and transparent, measuring approximately 90–110 μm in length. They exhibited uniformity in shape and size, indicating successful reproduction. The second-stage juveniles (J₂) emerged within 48–72 h (Fig. 1B). These slender, vermiform juveniles measured about 400–500 μm in length and were actively motile, representing the infective stage of the nematode. Their rapid movement signified their readiness to invade the host tissue.

Following infection, the nematodes underwent successive developmental stages within the host tissue. Within 5–7 days post-infection, the nematodes transitioned into the third- and fourth-stage juveniles (Fig. 1C and E). These stages were characterized by gradual body swelling, transitioning from vermiform to saccate forms, signifying the beginning of their sedentary phase. By day 10, J₄ females began to appear (Fig. 1F). They displayed prominent body swelling with a distinct pear-shaped morphology. Further development was evident by day 12–14, as fully mature females with a spherical, robust body structure were observed (Fig. 1G and H). These mature females were approximately 25–300 μm in diameter and displayed prominent internal structures, indicative of reproductive readiness.

The progression from eggs to fully mature females within 14 days demonstrates the rapid life cycle of M. graminicola, highlighting its potential for fast population build-up under favorable conditions. The distinct morphological transitions at each stage provide critical insights into the biology of this pathogen, emphasizing the importance of timely intervention to mitigate its impact on rice crops.

Fig. 1

Morphological stages of nematode development. A Egg stage; B Second-stage juvenile (J2); C Migratory J2; D Sedentary J2; E Developing J3; F Juvenile J4; G Young adult female; H Fully developed sedentary female. Scale bars: 100 μm

Response of rice varieties to M. graminicola in field experiments

A total of 348 rice genotypes were evaluated under field conditions during the 2023 and 2024 kharif seasons to assess their response against Meloidogyne graminicola. The data were collected across three replications, each consisting of three plants per replicate, and were used to calculate the Gall Index (GI) for each genotype. The pooled GI values revealed significant genotypic variation in nematode resistance, with GI values ranging from 8.89 to 95.56.

Based on the GI-based classification, genotypes were grouped into six resistance categories. Among the tested genotypes, 9 entries (2.6%) were classified as resistant (R). These included RP-5219-9-7-3-2-1-1 (GI = 8.89 ± 2.22), NPT-10 (16.67 ± 1.93), MTU 1390 (17.78 ± 1.11), Kushiari (18.89 ± 1.11), RP 6750-RMS-2-23-67-91 (18.89 ± 2.22), Sonkharchi (20.00 ± 1.92), Sugandha-3 (21.11 ± 2.22), HRT-183 (23.33 ± 1.92), and HR-12 (24.44 ± 1.11).

On the other hand, 16 genotypes (4.6%) were found to be highly susceptible (HS), with GI values exceeding 75. These included AD 02207, JR-1101, CRHR-175, WGL 1533, JR-503, IR 8386-14-678-B, RTCNP-27, Mahamaya, NPT-15, UPR-2628-9-1-1, CBSN-168, Banko, CO-39, Bagari, JR-1124, and JR-403. GI values in this group ranged from 75.56 to 95.56, indicating extensive root galling. The remaining genotypes were distributed among the moderately susceptible (96 genotypes), susceptible (227 genotypes), and none were categorized as immune or highly resistant.

The resistant genotypes consistently recorded GI values significantly lower than both the international susceptible check TN1 (GI = 66.67) and the local check HUR-917 (GI = 73.33). The complete list of genotypes under each resistance category is presented in Supplementary Table 1, facilitating easy identification of candidates for future resistance breeding. Based on pooled Gall Index (GI) values obtained from field evaluations conducted during Kharif 2023 and 2024, the 348 rice genotypes were classified into different resistance categories as summarized in Table 1.

The genotypic effect was statistically significant (p < 0.001) based on ANOVA, indicating considerable genetic variability within the test panel (Supplementary File 3). Experimental reliability was supported by a coefficient of variation (CV) of 5.83%, a critical difference (CD) of 5.15, and a standard error of mean (SEm) of 1.86.

To ensure transparency and reproducibility, the complete raw dataset for all genotypes, including individual plant-level data across three replicates from 2023, is provided in Supplementary File 2. Year-wise compiled data for 2024 are presented in Supplementary File 3, while Supplementary File 4 contains the pooled dataset along with detailed statistical parameters including ANOVA, CD, SE, and CV.

Table 1 Distribution of 348 rice genotypes into resistance categories against Meloidogyne graminicola based on pooled gall index (GI) data from field evaluations during Kharif 2023 and 2024

Gall Development and Nematode Population Dynamics in Pot Experiments

The data, presented in Fig. 2, illustrates the time course of gall development and nematode populations within the roots of different varieties over three time points: 3 dpi, 7 dpi, and 14 dpi (days post-inoculation).

The highly susceptible (HS) genotypes JR-1124, JR-403, CO-39, RTCNP-28, TN1, and HUR-917 exhibited rapid and progressive increases in both gall index and nematode numbers over time. JR-1124 recorded the highest gall index (96.00 ± 1.634) at 14 dpi, closely followed by JR-403 (90.67 ± 1.631) and CO-39 (89.33 ± 1.631). Correspondingly, these genotypes also maintained consistently high nematode counts in the roots, with JR-1124 reaching 59.64 ± 0.355 nematodes per root system at 14 dpi.

In contrast, the resistant (R) genotypes—RP-5219-9-7-3-2-1-1, RP 6750-RMS-2-23-67-91, MTU 1390 (IR17M1172), and NPT-10—showed minimal gall development and significantly fewer nematodes in the roots. Notably, RP-5219-9-7-3-2-1-1 recorded a gall index of 0.00 at 3 dpi and only 8.00 ± 1.332 at 14 dpi, with a nematode count of 4.8 ± 0.063, highlighting its strong resistance. MTU 1390 and NPT-10 showed slightly higher but still moderate gall indices and nematode levels, with values of 20.00 ± 2.108 and 22.67 ± 1.633 in gall index and 9.28 ± 0.136 and 11.12 ± 0.185 in nematode counts, respectively.

Statistical analysis indicated significant differences among genotypes at all time points. The critical difference (CD) for gall index ranged from 4.369 at 3 dpi to 4.981 at 14 dpi, and for nematode count from 0.578 to 0.595. The coefficient of variation (CV) was lowest for nematode counts (1.29–1.43%), reflecting reliable data, while gall index showed slightly higher variability, particularly at 7 dpi (CV = 18.498%).

These results clearly indicate that resistant genotypes restrict early nematode entry and gall formation, which remain limited even at later stages, in contrast to highly susceptible genotypes that support extensive nematode development and gall proliferation. This differential response underscores the potential of resistant lines for inclusion in breeding programs targeting nematode resistance.

The reproduction factor (Rf) of Meloidogyne graminicola showed significant variation among the tested rice varieties (Fig. 3A). Statistical analysis was performed using one-way ANOVA (p-value < 0.0001) followed by Tukey’s HSD test (p < 0.05) to determine significant differences among the treatments.

Among all the varieties, RP-5219-9-7-3-2-1-1 recorded the lowest Rf (≈ 0.3), which was significantly lower than all other varieties, indicating strong resistance to nematode multiplication. Similarly, RP 6750-RMS-2-23-67-91, MTU 1390(IR17M1172), and NPT-10 showed statistically lower Rf values (Rf < 1), and were grouped together with RP-5219-9-7-3-2-1-1, suggesting their resistant nature. In contrast, JR-1124, RTCNP-28, and JR-403 exhibited the highest Rf values (Rf > 3.0), forming a separate statistical group significantly different from the resistant varieties. These were followed by HUR-917, TN1 and CO-39, which had intermediate Rf values (≈ 1.8–2.8) and formed statistically distinct groups, indicating moderate susceptibility. A strong positive correlation was observed between Gall Index and the number of nematodes in roots across all the time points (Fig. 3B). As the Gall Index increased, nematode counts rose proportionally, with the steepest slope noted at 14 dpi, indicating enhanced nematode establishment over time. This linear relationship reinforces the reliability of the Gall Index as an effective indicator of nematode infestation severity and host susceptibility. The consistent trend across 3, 7, and 14 dpi highlights the progressive nature of root colonization and gall development in susceptible genotypes.

A comparative analysis of root galling progression in rice varieties revealed marked differences in their response to nematode infection over time (Fig. 4). In the highly susceptible variety JR 1124, gall formation initiated as early as 3 days post-inoculation (dpi) (Fig. 4A), with visibly swollen root tips. By 7 and 11 dpi, substantial gall proliferation and disorganization of root architecture were evident (Fig. 4B, C). Severe galling and extensive root damage peaked at 14 and 21 dpi, indicating aggressive nematode colonization (Fig. 4D, E). In contrast, the resistant genotype RP-5219-9-7-3-2-1-1 exhibited minimal signs of galling throughout the 21-day observation period (Fig. 4F–J). Roots remained structurally intact with negligible swelling, underscoring the genotype’s robust defense response and impaired nematode establishment. The temporal progression of root symptoms in JR 1124 and their near absence in RP-5219-9-7-3-2-1-1 highlight the contrasting susceptibility and resistance mechanisms, suggesting potential for the latter in integrated nematode management strategies.

Fig. 2

Temporal assessment of gall formation and nematode infestation in different rice varieties challenged with Meloidogyne graminicola. Bar plots represent the (A, C, E) Gall Index and (B, D, F) number of nematodes within roots at 3, 7, and 14 days post-inoculation (DPI), respectively. Each bar indicates the mean ± standard error (SE) of five replicates (n = 5). Different lowercase letters above bars indicate statistically significant differences among varieties according to Tukey’s HSD test at p < 0.05

Fig. 3

Evaluation of nematode reproduction and its correlation with root galling across rice genotypes. A Reproduction factor (Pf/Pi) of Meloidogyne graminicola on 10 rice genotypes, showing significant variation in nematode multiplication. Highly susceptible genotypes such as JR 1124, JR 409, and CO 39 recorded reproduction factors above 3.0, while resistant lines including RP-5219-9-7-3-2-1-1 and MTU 1390 (RMTW1172) exhibited minimal reproduction (Pf/Pi < 1.0), indicating effective nematode suppression. B Correlation between gall index and nematode count in roots at 3, 7, and 14 days post-inoculation (dpi).

Fig. 4

Progression of root galling in highly susceptible (JR 1124) and resistant (RP-5219-9-7-3-2-1-1) rice varieties over time. A–E Gall development in JR 1124 at 3, 7, 11, 14, and 21 days post-inoculation (dpi), showing extensive gall formation and root damage. F–J Minimal galling in RP-5219-9-7-3-2-1-1 at corresponding time points, indicating strong resistance to nematode infection. The contrasting root responses highlight susceptibility and resistance mechanisms

Histopathological Development of Meloidogyne graminicola in Rice Roots

Confocal microscopy analysis (Fig. 5) reveals contrasting cytological responses of rice varieties to Meloidogyne graminicola infection. Transverse sections of root tissues from susceptible (JR-1124 and TN1) and resistant (RP-5219-9-7-3-2-1-1) genotypes were examined at 7 and 14 days post-inoculation (dpi), following nematode challenge 10 days after germination. Dual-channel imaging with propidium iodide (red fluorescence) and calcofluor white (blue fluorescence) enabled visualization of nematode-induced structural alterations and defense-related polysaccharide deposition.

At 7 dpi (Fig. 5A, C), susceptible varieties exhibited prominent giant cells within the vascular cylinder, characterized by hypertrophy, dense cytoplasm, and surrounding parenchyma proliferation. Thickened cell walls and disrupted vascular architecture were evident, along with enhanced cellulose deposition in cortical and vascular tissues. Developing nematodes were visible within feeding sites, confirming successful parasitism. By 14 dpi (Fig. 5B, D), mature females and egg masses were observed. Giant cells persisted with intensified metabolic activity, while vascular and cortical regions exhibited severe degradation and localized callose accumulation, indicative of a weak or ineffective defense response.

In contrast, the resistant variety RP-5219-9-7-3-2-1-1 (Fig. 5E, F) showed a markedly different response. At 7 dpi, localized root irregularities (LRIs) were observed with well-preserved vascular structure and minimal tissue disruption. Regions of nematode penetration exhibited limited damage (asterisk-marked), and dense blue fluorescence highlighted strong callose deposition—a potential barrier to nematode ingress. Notably, giant cell formation was aborted, with disrupted cytoplasmic content and absence of hypertrophy or multinucleation. Enhanced red fluorescence in infected zones suggested localized wall thickening or lignification, likely hindering nematode development.

Figure 6 illustrates the progression of Meloidogyne graminicola infection in transverse sections of rice roots from the highly susceptible variety HUR-917 at 3, 7, 11, and 14 dpi. Dual-channel confocal imaging, propidium iodide (red) for cell walls and calcofluor white (blue) for cellulose and callose revealed dynamic structural and cytological alterations in response to nematode infection.

At 3 dpi (Fig. 6A), early infection stages were marked by localized vascular irregularities and slight pericycle hypertrophy, with minimal callose deposition. By 7 dpi (Fig. 6B), giant cells formed within the vascular cylinder, displaying pronounced hypertrophy and multinucleation. Adjacent vascular tissues began to disorganize, and moderate cellulose and callose accumulation indicated early defense activation.

At 11 dpi (Fig. 6C), giant cells further enlarged with dense cytoplasm and prominent nuclei. Surrounding parenchyma proliferated, while cortical degradation and intensified callose deposition exhibited escalating damage. By 14 dpi (Fig. 6D), mature females and egg masses were present. Severe vascular disintegration and extensive cortical collapse were observed, with persistent, metabolically active giant cells and dense cell wall deposits reflecting a strong but ineffective host response.

Fig. 5

Confocal laser scanning microscopy of transverse root sections in rice varieties under nematode infection, highlighting differential anatomical responses. A Early infection stage in susceptible genotype (JR 1124): formation of giant cells (G), nematode feeding sites (F), proliferating vascular tissue, parenchyma, protoxylem, and metaxylem (pMF). Central metaxylem marked (cM-*). B Mature infection in highly susceptible genotype (JR 1124): well-developed multinucleated giant cells, feeding sites, nematode and associated eggs (Egg). C Close-up of a giant cell showing tissue hypertrophy and hyperplasia in TN1 D Advanced infection stage in TN1: multiple giant cells and eggs adjacent to the nematode. E Transverse section of the resistant genotype RP-5219-9-7-3-2-1-1 showing limited nematode-induced damage in vascular tissues and presence of lateral root initiation (LRI). F Resistant genotype showing outlined giant cells with restricted development, absence of hypertrophy, and lack of proliferative tissue changes. Fluorescent staining indicates maintained structural integrity. Scale bars: 100 μm (A, C, D, E, F), 250 μm (B)

Fig. 6

Confocal microscopy images of transverse sections of rice roots from the highly susceptible local control variety HUR-917 infected with Meloidogyne graminicola. The images illustrate structural and cytological changes at four time points: (A) 3 days post-inoculation (dpi), (B) 7 dpi, (C) 11 dpi, and (D) 14 dpi. Propidium iodide staining (red) highlights cell walls, while calcofluor white staining (blue) indicates cellulose and callose deposition. At 3 dpi (A), early stages of nematode feeding site formation are visible, with minimal root damage. At 7 dpi (B), giant cells exhibit pronounced hypertrophy and multinucleation, with increased cellulose deposition near the infection site. At 11 dpi (C), advanced giant cells and proliferating support cells are observed, accompanied by significant vascular disruption and partial cortical degradation. By 14 dpi (D), mature nematodes and egg masses are present, with severe tissue disorganization and extensive degradation of cortical layers. These images demonstrate the progression of nematode-induced damage and the ineffective defense response of the HUR-917. Scale bars: A, B, D = 100 μm; C = 50 μm

Biochemical defense responses in rice varieties against Meloidogyne graminicola

To assess the defense mechanisms activated in rice genotypes upon Meloidogyne graminicola infection, the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), and total phenolic content (TPC) were measured at 3,7 and 14 dpi. These results are visually represented through box plots in Fig. 7.

Among all 6 genotypes (3 Resistant and 3 highly susceptible) tested, resistant genotype RP-5219-9-7-3-2-1-1 recorded significantly higher PAL, POX, and TPC activities at all intervals. PAL activity peaked at 7.454 U/mg protein at 14 dpi, PO at 24.68 U/mg protein, and TPC also showed an early surge of 7.454 U/mg fresh weight at 3 dpi. The other two resistant genotypes RP 6750-RMS-2-23-67-91 and MTU 1390 showed comparable, though slightly lower, levels of biochemical activity. Conversely, the susceptible genotypes JR-1124 and TN1, and to a lesser extent HUR-917, demonstrated significantly lower activity across all biochemical parameters and time points.

Statistical analysis confirmed the significance of these variations. For PAL activity, the critical difference (C.D.) values at 3, 7, and 14 dpi were 0.166, 0.216, and 0.257, respectively, with corresponding standard error of mean [SE(m)] values of 0.056, 0.073, and 0.086. PO activity had C.D. values of 0.379, 0.35, and 0.461 at 3, 7, and 14 dpi, respectively, while SE(m) ranged from 0.118 to 0.155. TPC also showed significant differences with C.D. values of 0.257 (3 dpi), 0.072 (7 dpi), and 0.083 (14 dpi). The coefficient of variation (C.V.) was relatively low across all parameters viz., PAL (3.88–5.51%), PO (1.94–3.13%), and TPC (2.40–3.88%) indicating a high degree of reliability and reproducibility in the data.

These findings collectively highlight that enhanced biochemical responses, particularly increased PAL and PO activity, along with early accumulation of phenolics are closely associated with nematode resistance. The robust activation of these pathways in resistant genotypes suggests their crucial role in restricting nematode establishment and proliferation.

Fig. 7

Box plot representation of biochemical responses in six rice genotypes challenged with Meloidogyne graminicola at different time points post-inoculation. A–C Phenylalanine ammonia-lyase (PAL) activity, D–F Peroxidase (POX) activity, and G–I Total phenolic content (TPC) recorded at 3, 7 and 14 days post-inoculation (dpi), respectively. Each box represents the interquartile range (IQR), the line within the box indicates the median, and whiskers denote variability outside the upper and lower quartiles. Different lowercase letters above boxes indicate statistically significant differences among varieties according to Tukey’s HSD test at p < 0.05

Discussion

This study on Meloidogyne graminicola provides significant insights into its life cycle, impact on rice varieties, and biological mechanisms contributing to resistance and susceptibility. The nematode completes its life cycle from eggs to mature females in just 14 days, enabling rapid population increases under favorable conditions, which poses a serious threat to rice cultivation which aligns with the findings of Fernandez et al. (2014).

In the present investigation, a total of 348 rice cultivars were assessed under conditions of nematode infestation in the field experiments with the objective of identifying effective sources of resistance against M. graminicola in the Indian context. Pronounced variability in the susceptibility of Oryza sativa cultivars was documented, corroborating previous studies which indicated that rice varieties exhibit differential responses to nematode attack (Shrestha et al. 2007; Dimkpa et al. 2016). Importantly, the cultivars RP-5219-9-7-3-2-1-1, MTU 1390(IR17M1172), Kushiari, RP 6750-RMS-2-23-67-91, Sonkharchi, Sugandha-3, HRT-183, HR-12 and NPT-10 demonstrated substantial resistance to M. graminicola, exhibiting minimal gall formation, which suggests the presence of effective defensive strategies. The results obtained imply the existence of pre-infection resistance mechanisms within these resistant cultivars. Prior research has indicated that susceptible plants facilitate the feeding and successful development of root-knot nematodes, whereas resistant plants may permit penetration but inhibit maturation (Trudgill 1991). Waele et al. (2013) observed a delay in nematode reproduction in resistant O. glaberrima genotypes CG14, TOG5674, and TOG5675, characterized by reduced fecundity and smaller dimensions of M. graminicola females (Cabasan et al. 2012; Waele et al. 2013). Similarly, RP-5219-9-7-3-2-1-1 exhibited no gall formation after three days, aligning with findings by Dimkpa et al. (2016) who reported an absence of nematode penetration at two days post-inoculation (dpi) and no gall formation throughout a five-week evaluation for immune accessions LD24 (an aus from Thailand) and Khao Pahk Maw (an indica from Sri Lanka). Kumari et al. (2016) indicated that M. graminicola penetrated, developed, and reproduced at an accelerated rate in the susceptible rice variety Pusa 1121 in comparison to the resistant variety Vandana. Zhan et al. (2018) also found aus and hybrid aus varieties to be highly resistant under both pot and field conditions.

Evidence from analogous investigations suggests that the resistance exhibited by rice accessions to M. graminicola is correlated with diminished penetration (Cabasan et al. 2012). This resistance mechanism is distinct from that of the Mi gene in tomatoes, wherein M. incognita penetration prompts rapid and localized necrosis of host cells (Williamson and Hussey 1996). Resistant rice species may express pre-infection resistance factors at the root interface, such as epidermal barriers or biochemical secretions, which restrict nematode penetration (Huang 1985). Variations in root anatomy may also play a role in resistance mechanisms, as demonstrated in resistant cotton varieties against M. incognita (Anwar et al. 1994) and resistant grape rootstocks against M. arenaria (Anwar and McKenry 2000).

In this study, juvenile development was notably delayed in resistant rice varieties, with the majority of nematodes failing to mature into females by 14 dpi, aligning with findings observed in other resistant plant species (Cabasan et al. 2012). For instance, Dimkpa et al. (2016) noted that a reduced number of M. graminicola females developed in resistant genotypes TOG564 and Khao Pahk Maw when juxtaposed with susceptible genotypes. Furthermore, Priya and Subramanian (2013) documented an extension of the nematode life cycle in the moderately resistant rice variety ADT45, which took seven days longer than in the susceptible variety CO47.

Previous studies have shown significant variation in the susceptibility of rice cultivars to M. graminicola, with some exhibiting strong resistance. Resistant varieties, such as Abhishek and Huaidao 5, demonstrate reduced gall formation, suppressed nematode development, and potential genetic resistance (Mhatre et al. 2015; Feng et al. 2022). PTB-10 and TKM-6 have been reported to show resistance to different populations of M. graminicola and M. triticoryzae (Sabir and Gaur 2004). Prasad et al. (2006) identified drought-tolerant rice lines resistant to M. graminicola, such as Teqing, Type 3, Zihui 100, and Shwe Thwe Yin Hyv, while others like Binam and IR 64 were susceptible. Interestingly, progenies from Binam crossed with IR 64 or Teqing exhibited nematode resistance, indicating the potential for incorporating resistance through advanced backcross populations. Phan et al. (2017) observed that the resistant rice variety Zhonghua 11 exhibited a hypersensitivity-like response during early infection by M. graminicola, characterized by necrotic root cells and phenolic compound accumulation. This resistance was identified as qualitative, involving major resistance genes.

Environmental and genetic factors can influence nematode interactions, as seen in the fluctuating gall formation of some varieties, such as CBSN-168. Environmental fluctuations can alter host-parasite interactions, suggesting their incorporation into studies of host-parasite coevolution (Wolinska and King 2009).

This study provides integrated biochemical and histopathological evidence elucidating the defense mechanisms employed by rice genotypes against Meloidogyne graminicola. Resistant varieties, particularly RP-5219-9-7-3-2-1-1, demonstrated strong and early activation of defense responses, as reflected by elevated phenylalanine ammonia-lyase (PAL), peroxidase (POX), and total phenolic content (TPC). These findings highlight the coordinated role of the phenylpropanoid pathway and oxidative enzymes in limiting nematode invasion and development.

PAL activity, which peaked at 14 dpi in resistant genotypes, plays a pivotal role in the biosynthesis of phenolics and lignin, both crucial for fortifying cell walls and mounting antimicrobial defenses. These results corroborate earlier findings in rice, sugarcane, and banana, where PAL was strongly induced in resistant cultivars following nematode infection (Mehta & Kathiresan 2005; Wuyts et al. 2006; Liu et al. 2024). Similarly, increased POX activity in resistant lines indicates active lignification and suberin deposition, which contribute to forming physical barriers that deter nematode migration—consistent with studies in tomato, cowpea, and black pepper (Sherin et al. 2024). The early surge in TPC, particularly at 3 dpi, suggests that phenolics may act as both structural and chemical defenses, inhibiting nematode development during initial infection stages (Galeng-Lawilao et al. 2018).

In contrast, susceptible genotypes such as TN1, JR-1124, and HUR-917 exhibited weak biochemical responses, which likely contributed to their failure in restricting nematode colonization. This aligns with previous studies where susceptible varieties showed limited or delayed induction of key defense enzymes (Wuyts et al. 2006), underscoring the importance of timing and magnitude of defense activation.

Histopathological analysis further reinforced these findings. Susceptible varieties exhibited pronounced giant cell formation, vascular disruption, and cortical degradation, facilitating nematode maturation by 14 dpi. The giant cells observed were hypertrophied, multinucleated, and metabolically active—typical of a compatible interaction (Williamson and Hussey 1996; Jena and Rao 1977). The absence of hypersensitive-like responses in these genotypes indicates a lack of effective post-penetration defenses. In contrast, resistant wild rice species such as Oryza glumaepatula and O. glaberrima are known to restrict giant cell formation through localized necrosis and impaired cell development (Mattos et al. 2019).

Although our staining results showed enhanced cellulose and callose deposition in susceptible roots, these responses appeared insufficient to prevent nematode advancement. This suggests that while such structural reinforcements are part of the host’s response, their efficacy is limited in the absence of early and robust signaling cascades. Previous studies have similarly indicated that mere biochemical accumulation without effective signaling or gene activation may not halt nematode progression (Khan et al. 2010; Mattos et al. 2019).

Additionally, susceptibility may also be influenced by root anatomical traits. Genotypes with larger root diameters and stele thickness have been reported to permit easier nematode entry and feeding site expansion (Jena & Rao 1977). While not examined here, future research should evaluate the role of root morphology in governing host susceptibility.

Collectively, our results underscore the importance of integrating both structural and biochemical responses in conferring resistance to M. graminicola. The observed upregulation of PAL, POX, and phenolic compounds in resistant genotypes serves as a strong indicator of enhanced defense capacity and offers potential biomarkers for resistance breeding. Moreover, these findings support the broader view that resistance is not determined by a single pathway but arises from a complex interplay of enzymatic activity, cellular responses, and anatomical features (Kaloshian and Teixeira 2019a, b).

Moving forward, elucidating the genetic basis of these responses through functional genomics and transcriptomics could greatly aid in the development of resistant cultivars. Marker-assisted selection and genome editing technologies offer promising avenues to introgress such traits into high-yielding rice backgrounds (Varshney et al. 2018). Incorporating these resistant lines into cropping systems could reduce reliance on nematicides, promoting environmentally sustainable and economically viable nematode management strategies.

Conclusion

This study provides critical insights into the resistance of various rice (Oryza sativa L.) varieties against the root-knot nematode Meloidogyne graminicola, a significant biotic stressor in rice cultivation. The evaluation of rice varieties over two years revealed a diverse spectrum of susceptibility, highlighting the potential for breeding programs to select and propagate resistant genotypes. Notably, the identification of varieties such as RP-5219-9-7-3-2-1-1, NPT-10, MTU 1390(IR17M1172), Kushiari, RP 6750-RMS-2-23-67-91, Sonkharchi, Sugandha-3, HRT-183 and HR-12, which demonstrated significant resistance, underscores the importance of integrating genetic diversity into breeding strategies.

Furthermore, advanced histopathological techniques, including confocal microscopy and biochemical assays have elucidated the complex interactions between rice plants and nematodes at the cellular level, revealing mechanisms that confer resistance. The findings advocate for a shift towards sustainable agricultural practices by reducing reliance on chemical controls through the deployment of resistant cultivars. As nematode populations continue to evolve, ongoing research is imperative to refine breeding strategies and ensure long-term management of this pest. Ultimately, enhancing resistance in rice not only contributes to improved crop yields but also plays a vital role in securing food resources amidst growing global challenges in agriculture.