Abstract
Root Knot Nematode (Meloidogyne incognita) is a major agricultural pest that significantly reduces crop yield. This study investigates the nematicidal potential of Bradyrhizobium japonicum strain 11477 against M. incognita to regulate its pathogenicity in Solanum lycopersicum. Tomato seeds were treated with bacterial cells and supernatant, grown under controlled conditions and later infested with nematode juveniles (5J2/seedling). After 10 days, nematode infestation led to reduced seedling growth, lower root and shoot biomass and decreased photosynthetic pigments. It also triggered oxidative stress, as indicated by elevated stress markers. Enzymatic and non-enzymatic antioxidants along with phenolic compounds showed increased activity in response to nematode-induced stress. However, B. japonicum treatment significantly reduced gall formation, improved plant growth and enhanced biochemical and histochemical attributes. Rhizobacteria also alleviated stress indices, strengthened antioxidant defenses and increased metabolite production. Confocal microscopy revealed hydrogen peroxide localization, glutathione content and nuclear and membrane damage in root apices, correlating with plant defense responses. This study highlights B. japonicum as a potent biocontrol agent that enhances plant growth and resilience against M. incognita. Notably, this is the first report on the impact of a leguminous rhizobacterium on a non-leguminous tomato plant, providing new insights into its potential for sustainable pest management.
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Introduction
Within ecosystems, plants engage with diverse biotic and abiotic factors. These interactions shape their growth, survival and ecological impact. Microorganisms, particularly plant-parasitic nematodes are critical biotic stressors due to their reciprocal interactions with plants (such as carrot, potato, tomato, brinjal, guava etc.) and significant impact on agricultural crop productivity. Root-knot nematodes (RKNs), are pivotal in both economic and scientific domains due to their intricate host plant interactions, broad host range and destructive capabilities facilitated by their stylet. Meloidogyne spp. are among the most harmful plant parasites which penetrates root epidermal tissue and inducing gall formation1. During the infective second juvenile (J2) stage, they secrete hydrolytic enzymes to invade roots, disrupting water and nutrient uptake, nitrogen fixation and mineral absorption. Infestation leads to stunted growth, reduced photosynthesis and oxidative stress due to reactive oxygen species (ROS) accumulation. ROS play a crucial role in host–pathogen interactions, influencing signaling pathways and plant defense responses2.
Tomato (Solanum lycopersicum), a widely cultivated and nutritionally rich (contains various vitamins and minerals, carbohydrates, flavonoids and antioxidants) crop in India is highly consumed. Economically, its production is severely affected by Meloidogyne spp. which pose a significant threat to its morphology, physiology and yield3. Their infestations accounts for 30–50% of yield losses in India, contributing to an estimated global economic loss of $358 billion4. Such infestations pose a significant challenge to farming, particularly in organic systems. While synthetic nematicides were once the primary control method, concerns over their environmental and health risks have led to their phased withdrawal. This shift has intensified the search for cost-effective sustainable alternatives. Globally it comprises of 19% of vegetable consumption. So, addressing RKN-induced losses is critical for sustainable agriculture. Biological agents, including microbes have emerged as promising eco-friendly solutions, offering both nematicidal effects and plant growth benefits in modern agriculture5.
Microscopic organisms, including mycorrhizal fungi and plant growth-promoting rhizobacteria (PGPR), play a crucial role in suppressing RKNs while promoting plant health6,7. Various PGPRs, such as Serratia, Enterobacter, Azotobacter, Pseudomonas and Bacillus spp. improve root growth conditions and reduce nematode populations in the rhizosphere8,9. These bacteria produce bio-metabolites with nematicidal properties, enhancing the host plant’s defensive and antioxidative mechanisms against nematode infection10. Additionally, PGPR improve soil performance and increase plant tolerance to biotic and abiotic stresses by upregulating stress-related genes.
On account of this, the occurrent study was aimed to decrease the disease progression of M. incognita in S. lycopersicum seedlings by employing the least explored rhizobium strain i.e. Bradyrhizobium japonicum 11477 with MTCC accession number 120T. This study uniquely explores its nematicidal potential. Limited research has examined its role in enhancing antioxidative capacity, secondary metabolite production and live-cell imaging under various treatments in vitro. This study intends to investigate the dual effects of rhizobacteria via cell pellets and its supernatant on tomato growth by assessing key physiological, biochemical and structural parameters under controlled conditions. The primary hypothesizes of the study are: (i) B. japonicum exhibits potent nematicidal activity against M. incognita. (ii) Dual contribution of bacterium via cell pellets and its supernatant suppress nematode galls (iii) Seeds treated with B. japonicum enhance photosynthesis, reduce oxidative stress and strengthen antioxidative defenses leading to improved plant growth and development.
Results
Mortality
Percentage J2 mortality was assessed using different treatments of B. japonicum against M. incognita juveniles. While nematodes treated with rhizobium exhibited rigid and straight nematode posture and did not react when contacted by a small probe were deemed dead and those nematodes which exhibited relaxed and undulating nematode posture and responded to touch by a small probe were considered alive. The percentage mortality of M. incognita juveniles increased with increase in exposure time from day 1 to day 7 in each treatment of at p < 0.01. ANOVA (one-way analysis of variance with p = 0.000 revealed that the maximum mortality of 100% was observed in supernatant at day 5 and remaining there through day 7. It was 1.76 (F = 52.357), 1.19 (F = 110.263), 1.86 (F = 141.262), 1.95 (F = 219.176), 2.26 (F = 537.523), 1.86 (F = 227.431) and 2.31-folds (F = 153.919) higher than control from day 1 to day 7, respectively. Bacterial cell pellets showed a steady increase in mortality, surpassing control significantly by day 3 and reaching over 62% by day 7. It was 5.15, 3.96, 4.53, 4.59, 5.17, 3.74 and 3.74-folds higher than control from day 1 to day 7. So, the further studies on growth and biochemical parameters were carried out with these treatments of bacterium (Fig. 1).
Impact of B. japonicum on mortality of M. incognita. Different treatments in figure are represented as C (control@1 ml distilled water/100J2 of M. incognita), BC (B. japonicum cell pellets @109/ml/100J2 of M. incognita), S (Supernatant @1 ml/100J2 of nematode). Results are given in the form of Mean ± SE (standard error). **Indicates significance at p ≤ 0.01. Different letters a, b and c in graph represents significant difference whereas, same letters represent no significant difference.
Morphological parameters
The impact of treatment with B. japonicum was evaluated on a number of morphological attributes of tomato plants infected with M. incognita, including the percentage germination, the length of roots (RL) and shoots (SL), weight of roots (RW) and shoots (SW) and number of galls on root (Table 1). ANOVA indicated signifcant variations in these parameters across different treatments and inoculation methods.
Percentage germination was decreased by 9.30% in nematode infected tomato seedlings as compared to non-infested tomato seedlings whereas application with B. japonicum cells and supernatant significantly enhanced the percentage germination by 11.63 and 27.91% respectively when compared to control seeds treated with distilled water (p = 0.001; F = 9.910). However, nematode infected, B. japonicum cells and supernatant treated seeds significantly enhanced the percent germination by 20.51% and 30.77% as compared to the nematode infected and untreated seedlings. Similar notable decrease was observed with nematode infection at p = 0.000 in RL (F = 19.425), SL (F = 27.367), RW (F = 119.751) and weights (F = 684.505) of S. lycopersicum seedlings by 21.87, 19.73, 15.02 and 14.28% as compared to non-infected control seedlings. But treatment with B. japonicum cells markedly enhanced RL, SL, RW and SW by 17.08, 18.98, 9.04 and 31.01% whereas supernatant treatment markedly elevated the parameters by 24.81, 37.25, 29.86 and 69.80% respectively as compared to control seedlings. However, nematode infected B. japonicum cells and supernatant treated plants showed significantly higher values by 24.82, 32.61, 12.85, 18.27% and 34.31, 52.52, 27.51, 85.39% respectively as compared to the nematode infected and untreated seedlings. Additionally, it was shown that formation of galls in B. japonicum cells treated and infected seedlings and supernatanttreated seedlings reduced significantly by (F = 142.800) 22.22 and 55.55% respectively as compared to tomato seedlings that were both untreated but nematode infected.
Photosynthetic pigments
At p = 0.000, ANOVA results indicated slight decrease in photosynthetic pigments chlorophyll a (Chl a) (F = 13.143) chlorophyll b (Chl b) (F = 21.703), total chlorophyll (F = 29.982) and carotenoid (F = 191.038) by 23.78, 23.60, 23.70 and 15.12% in seedlings infected with nematodes when compared to non-infected tomato seedlings. Upsurge in Chl a, Chl b, total chlorophyll and carotenoid by 26.93, 83.63, 49.45 and 20.60% respectively was observed when seedlings were treated with bacterial cells whereas significant elevation in Chl a, Chl b, total chlorophyll and carotenoid content by 34.88, 91.61, 57.51 and 23.25% was observed when the seeds were treated with supernatant respectively as compared to non-infected control seedlings. Similar enhancement in Chl a, Chl b, total chlorophyll and carotenoid content by 74.79, 207.05, 127.59, 12.92% and 91.60, 212.17, 139.74, 13.58% was observed in bacterial cell and supernatant treated and nematode infected as compared to nematode infected untreated seedlings (Fig. 2a–d).
(a–d) Effect of B. japonicum on chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content in tomato plants 10 days after M. incognita infection. Different treatments in figure are represented as C (control), N (Nematode inoculated), T1 (B. japonicum cells), T1 + N (B. japonicum cells + Nematode inoculated), T2 (Supernatant) and T2 + N (Supernatant + Nemato de inoculated). Results are given in the form of Mean ± SE. **Indicates significance at p ≤ 0.01. Different letters a, b and c in graph represents significant difference whereas, same letters represent no significant difference.
Stress indices
Oxidative stress indices faced evident modifications in the bioassay tests. With nematode stress, malondialdehyde (MDA) and hydrogen peroxide (H2O2) content observed a substantial rise by 231.10% (F = 140.247) and 29.11% (F = 42.243) respectively in S. lycopersicum seedlings when compared to control seedlings as indicated by ANOVA (p = 0.000). However, B. japonicum cells and supernatant treated tomato seedlings showed significantly lowered levels of MDA content by 15.02% and 24.98% (p < 0.01) respectively whereas H2O2 content by 3.74% and 7.90% respectively in contrast to control seedlings. However, combined effect of PGPRs’ cells and supernatant treated and nematode infected seedlings showed reduced levels of MDA by 31.82% and 54.55% respectively and H2O2 content by 15.46% and 19.97% respectively, when compared to untreated and nematode infected seedlings (Fig. 3a,b).
(a,b) Effect of B. japonicum on MDA and H2O2 in tomato plants 10 days after M. incognita infection. Different treatments in figure are represented as C (control), N (Nematode inoculated), T1 (B. japonicum cells), T1 + N (B. japonicum cells + Nematode inoculated), T2 (Supernatant) and T2 + N (Supernatant + Nematode inoculated). Results are given in the form of Mean ± SE. **Indicates significance at p ≤ 0.01. Different letters a, b, c and d in graph represents significant difference, whereas same letters represent no significant difference.
Enzymatic antioxidants
Using ANOVA, the activities of various antioxidative enzymes like CAT (Catalase), SOD (Superoxide dismutase), APOX (Ascorbate peroxidase), PPO (Polyphenol oxidase), GST (Glutathione-S-transferase) and GuPOX (Guaiacol peroxidase) were found to be appraised in nematode infested S. lycopersicum seedlings by 118.18 (F = 257.447), 88.88 (F = 31.435), 62.87 (F = 54.300), 73.37 (67.370), 81.35 (F = 18.100) and 47.69% (F = 25.400) with p = 0.000 respectively, when compared to control seedlings. Application of B. japonicum cells further enhanced the activity of antioxidative enzymes by 161.70, 44.44, 41.04, 100, 54.24 and 27.69% respectively whereas treatment with supernatant boosted the activities by 412.12, 18.52, 56.35, 87.84, 8.47 and 30.77% respectively in relation to control seedlings. Similarly, the treatment of PGPR cells and supernatant to nematode treated seedlings showed the increment in the activities by 72.22, 1.96, 28, 26.48, 19.63, 6.25% and 158.33, 13.72, 12, 14.63, 14.95 and 11.46% (p < 0.01) respectively, in comparison to nematode infected and untreated seedlings (Table 2).
Non-enzymatic antioxidants
Significant difference by ANOVA (p = 0.000) in the elevation of glutathione (GC), tocopherol (TC), proline (PRC) and ascorbic acid content (AAC) was seen in the nematode infected tomato seedlings by 70.55 (F = 48.392), 53.91 (F = 89.512), 73.97 (F = 19.415) and 40.65% (F = 97.562) respectively as compared to control seedlings. Application of B. japonicum cells further increased the activities of these biometabolites significantly by 29.00, 26.30, 63.01 and 29.64% respectively and notable augmentation in supernatant treated seedlings by 52.93, 32.98, 9.59 and 20.55% respectively was observed as compared to control seedlings. Similarly, GC, TC, PRC and AAC were appreciably enhanced in PGPR cells and supernatant treated nematode infected seedlings by 53.60, 17.14, 14.17 and 27.73% and 68.18, 19.10, 4.72 and 51.49% respectively as compared to untreated and nematode infected S. lycopersicum seedlings (Fig. 4a–d).
(a–d) Effect of B. japonicum on glutathione, tocopherol, proline and ascorbic acid content in tomato plants 10 days after M. incognita infection. Different treatments in figure are represented as C (control), N (Nematode inoculated), T1 (B. japonicum cells), T1 + N (B. japonicum cells + Nematode inoculated), T2 (Supernatant) and T2 + N (Supernatant + Nemato de inoculated). Results are given in the form of Mean ± SE. **Indicates significance at p ≤ 0.01. Different letters a, b, c, d and e in graph represents significant difference, whereas same letters represent no significant difference.
Phenolic compounds
The ANOVA indicated significant variation across various treatments at p = 0.000. Nematode infection markedly enhanced the total phenol (F = 14.431), flavonoid (F = 40.700) and anthocyanin (F = 53.916) content by 210.18, 32.03 and 93.94% respectively in S. lycopersicum seedlings as compared to control seedlings. However, application of B. japonicum cells considerably enhanced the total phenol content by 480.84% whereas flavonoid and anthocyanin content was appraised by 38.09 and 218.18% as compared to control seedlings. Similar pattern was observed in the levels of phenol, flavonoid and anthocyanin content that were further elevated (p < 0.01) by 376.65, 79.22 and 227.27% respectively, in case of seedlings subjected to pre-treatment with supernatant compared untreated control seedlings. The treatment of PGPR cells to nematode infected seedlings noticeably increased the above phenolic compounds by 70.08, 47.21 and 31.25% and by supernatant, it was enhanced by 62.16, 17.37 and 34.37% respectively, as compared to untreated and nematode infected seedlings (Fig. 5a–c).
(a–c) Effect of B. japonicum on total phenolic, flavonoid and anthocyanin content in tomato plants 10 days after M. incognita infection. Different treatments in figure are represented as C (control), N (Nematode inoculated), T1 (B. japonicum cells), T1 + N (B. japonicum cells + Nematode inoculated), T2 (Supernatant) and T2 + N (Supernatant + Nemato de inoculated). Results are given in the form of Mean ± SE. **Indicates significance at p ≤ 0.01. Different letters a, b, c and d in graph represents significant difference, whereas same letters represent no significant difference.
Confocal microscopy
The microscopic staining of roots of S. lycopersicum seedlings with DCF-DA indicated a substantial green color in nematode-stressed roots, bacterium treated roots had reduced fluorescence as compared to control seedlings whereas combined treatment viz., cell and supernatant with nematode respectively showed lesser intensity when compared to untreated and nematode infected seedlings (Fig. 6a). Glutathione tagging evaluated through blue color intensity. It was highest in nematode inoculated seedlings which further amplified in nematode-infected seedlings treated with bacterial cells and supernatant respectively, compared to untreated and nematode infested tomato seedlings (Fig. 6b). Nuclear membrane damage was observed through high intensity of blue color. It was found that the maximum DAPI intensity was seen in nematode infected seedlings while bacterium treated seedlings showed less blue color compared to untreated and nematode infected seedlings. (Fig. 6c). Cell membrane integrity was evaluated based on red color intensity. Maximum coloration of PI dye was seen in nematode infested seedlings compared to uninfected tomato seedlings. However, treated seedlings showed decreased intensity (Fig. 6d).
(a–d) Effect of soaking treatment of DCF-DA, MCB, DAPI and PI on accumulation of (a) Hydrogen peroxide (b) glutathione localization (c) nuclear membrane and (d) cell membrane damage in S. lycopersicum seedlings after 10 days of nematode inoculation.
Correlation analysis
It revealed significant relationships between physiological and biochemical parameters of the tomato plant upon being treated with bacteria in certain treatments. The strength and degree of association is shown by the Pearson’s correlation coefficient, which is represented by the intensity of hues (r values from − 1 to 1) (Fig. 7). The stronger the association between the relevant parameters, the intenser the color (as the correlation coefficient travels away from 0 on either side). An eclipse without a cross indicates a substantial (p < 0.05) correlation between the corresponding parameters, whereas an eclipse with a cross indicates a non-significant (p < 0.05) association. Correlation analysis indicated that strong positive correlation (r ≥ 0.7) exists between growth parameters viz., RL and SL, RL and RW, SL and RW, Chl a and Chl b, total chlorophyll and % germination. Whereas, moderate positive correlation (0.5 ≤ r ≤ 0.7) exists between antioxidative enzymes (SOD, CAT, APOX and PPO). Stress markers (MDA and H2O2) are also moderately positively correlated with each other and gall number at p < 0.05. Likewise, morphological parameters are positively correlated with photosynthetic pigments. Parameters such as stress markers and Chl a have strong negative correlations (r ≤ − 0.7) with each other. Non-enzymatic antioxidants showed a positive correlation with glutathione and tocopherol. Phenolic compounds were positively associated with growth parameters and photosynthetic pigments (0.5 ≤ r ≤ 0.7).
The impact of treatment with bacterium cells and supernatant was evaluated using correlation analysis among several parameters, after 10 days of nematode inoculation in S. lycopersicum seedlings. The various hue variations are represented by the scale on the right, where the positive correlation is represented by the blue scale and the negative correlation by the red scale. (Abbreviations: % GM, Percentage Germination; RL, Root Length; SL, Shoot Length; RW, Root Weight; SW, Shoot Weight; Chl a, Chlorophyll a; Chl b, Chlorophyll b; Tot Chl, Total Chlorophyll; CR, Carotenoid; MDA, Malondialdehyde Content; H2O2, Hydrogen Peroxide Content; CAT, Catalase activity; SOD, Superoxide Dismutase activity; APOX, Ascorbate Peroxidase activity; PPO, Polyphenol oxidase activity; GST, Glutathione-S-Transferase activity; GuPOX, Guaiacol Peroxidase activity; GC, Glutathione content; TC, Tocopherol content; PRC, Proline content; AAC, Ascorbic acid content; PHC, Phenolic content; FC, Flavonoid content).
Discussion
In the current investigation, it is seen that B. japonicum strain 11477 had promising impact on alleviating nematode stress in S. lycopersicum seedlings. Juvenile mortality was enhanced which is similar to a study by Abbas et al.8. The morphological traits such as height and biomass also dropped. This study aligns with research on Ocimum basilicum affected by M. incognita, which showed a reduction in FW. The researchers suggested that reduction was impacted by inability of injured roots to absorb essential nutrients and transport it to shoot system during nematode invasion11. Another study noted a substantial drop in morphological traits including RL, SL, RW and SW, in nematode-infested tomato and cucumber plants that are consistent with our investigations10,12. It was deduced that nematodes obstruct the pathways associated with mineral acquisition that inhibited the growth rate of plants. Prior research on tomato revealed identical results with lowered biomass where the supplementation with Pasteuria isolates increased growth attributes in plants that were infected with nematodes13. Similar enhancement was noted Pseudomonas aeruginosa BHU B13-398 and Bacillus subtilis BHU M treated mung bean9. They revealed that microbes helped in phosphate solubilisation and produced metabolites that enhanced the parameters.
Photosynthetic pigments were also lowered in the current investigation. Comparable decrease was seen in the concentration of pigments such as chlorophyll, carotenoid and beta-carotene in cowpea during nematode stress14. Carotenoids play a crucial role in protecting plants from free radicals and their reduction leads to the disruption of Photosystem II (PSII) through degradation of the D1 protein, which in turn inhibited the synthesis of chlorophyll pigments15. Additionally, tomato plants with galls that disrupted the water movement led to disturbed photosynthetic pigments16. Our research also found that rhizobium treated tomato infected with nematode had elevation in these photosynthetic pigments. Our findings are corroborated by earlier research on tomato plants where B. megaterium, Azotobacter chroococcum, A. brasilense and P. fluorescens improved the activities of photosynthetic pigments17. This is likely due to siderophore production, phosphate solubilization and nitrogen fixation by certain PGPR strains that increase pigments by improving iron and essential nutrient availability to plants18,19. Observations also revealed that the stimulatory effects on photosynthetic apparatus have been caused by microbe-mediated upregulation of the pigment synthesis enzymes such as porphobilinogen deaminase and magnesium chelatase, Chl a and Chl b synthases which are essential for the formation of chlorophyll molecules. PGPRs potentially modulated phytohormone levels that helps in cross-talks between signaling pathways that enhanced photosynthesis and plant health even during stress16.
High levels of oxidative stress indicators (MDA and H2O2) was also determined by our research in nematode afflicted seedlings. In Pinus pinaster plants treated with nematodes, a comparable rise in MDA level was seen20. Increased MDA levels are brought on by the oxidative breakdown of lipid molecules in the cell membranes. Elevated ROS within stressed plants is a consequence of the pathogenicity of worms and is accountable for the problem. A study evaluated the H2O2 concentration in sweet potato plants where they proposed that nematode invasion caused host plants to produce ROS21. The current study also evaluated B. japonicum’s impact on oxidative damage in plants with M. incognita infestations. They were lowered after treatment with bacterial cells and supernatant. Our results were consistent with studies of researchers who found that B. japonicum administration reduced MDA and lipid peroxidation levels in plants under salinity stress22. Through its relationship with plants, B. japonicum enhances the plant’s resistance to ROS through systemic acquired resistance (SAR) or induced systemic resistance (ISR). This triggers plant signaling pathways that activate antioxidant defenses, increasing the production of ROS-scavenging enzymes and metabolites23. Also, MDA and lipid peroxidation levels were decreased in soybean exposed to salt stress on treating with Streptomyces lasalocidi Jcm 337324. It was suggested that the reason for this drop is increased defense enzyme activity in plants that were treated with microbes. More importantly, since microbes may emit chelating compounds like organic acids, iron-chelating compounds and other secondary metabolites, microorganisms have the potential to scavenge the ROS generated by plants under stressful conditions25.
The current pursuit found that the activity of several enzymes (CAT, APOX, GuPOX and GST) were elevated in infected tomato seedlings. Parallel findings were recently disclosed where it was found that nematode infection elevated the performance of SOD, POD, CAT, GPOX and other enzymes in pea and tomato plants, respectively12,26. The antioxidant defense mechanism and ROS buildup sustain a steady-state equilibrium in plant cells27. Management of various key plant functions, including the development and growth of the plant is made possible by maintaining ideal ROS levels in the cell28. Additionally, it was shown that beetroot plants exposed to nematodes had increased SOD, POD and CAT activity which primarily relied on the foundation of making adjustment in the defense pathways occurring in plants. Our research tailored an inclination in enzymatic activity on being treated with B. japonicum. Rise in the activity of CAT and SOD was supported by PGPR treated, water stressed Cymbopogon citratus plants29. Also, an upsurge in PPO activity was reported in tomato plants under nematode infections infused with Streptomyces sp.30. Similarly, augmented activities of APOX, CAT, GST and GuPOX were resulted by endophytic Micrococcus luteus and Enterobacter cloacae during Ni and Cd toxicity31. They speculated that elevated enzymatic activity during microbial inoculation likely results from increased protein synthesis that upregulates defense-related genes enhancing resistance mechanisms against pathogens at the infection site. Glutathione and ascorbic acid together produce antioxidant enzymes and upregulates defense related genes which detoxify harmful ROS (such as superoxide anion (\({\text{O}}_{2}^{ - }\)), hydroxyl radicals (\({\text{OH}} \cdot\)) and \({\text{H}}_{{2}} {\text{O}}_{{2}}\)) and over-accumulate proline whereas tocopherol scavenges lipid peroxyl radicals under stressed conditions26. These non-enzymatic antioxidants were also increased in nematode-infected tomato seedlings. It is likely due to increased metabolite production in response to nematode attack, enhancing scavenging properties32. Infected plants like pea, sargassum, eggplant, papaya and grapevine had similar higher levels of these non-enzymatic antioxidants due to the prevalence of nematode infection1. They discovered a strong correlation between this improvement and plants’ nematode defense capabilities33. On top of that, it is shown that tomato seedlings treated with bacterium had also elevated antioxidants. PGPR is believed to secrete these bio-metabolites and nematicidal compounds which can directly neutralize ROS, improve antioxidant synthesis and prevents oxidative damage in plant cells under nematode stress32. In one of the comparative studies, it was observed that tolerant strain of Bradyrhizobium (NLH25) reduced Cd accumulation by reducing ROS production which modulated nitrogen fixation, siderophore production and phosphate solubilization and thereby, improved plant’s overall health as compared to sensitive strain of Bradyrhizobium (SEMIA6144) under Cd stress in peanut plants18. Similarly, PGPR formulations (particularly made from Serratia marcescens, P. putida and P. fluorescens) enhanced eggplant growth and stimulated ROS scavenging by improving antioxidant activity, despite nematode reinfestation35. It is speculated that microbes activate antioxidants which act as redox buffers which blocked nematode invasion2. Also, defense related gene expression related to scavenging of ROS viz., CAT3 (catalase 3) and MDAR2 (monodehydroascorbate reductase 2) and RD20 (response to desiccation) were upregulated in shoots of B. japonicum inoculated Arabidopsis plants36. Similar study conducted on tomato by Maqsood et al.37, also found increased levels of tocopherol, on being inoculated with Bacillus spp. contended that Bacillus treatment may have enhanced the expression of antioxidative enzymes, boosting tocopherol production where it reduced H2O2 to H2O and protected cellular membranes by quenching singlet oxygen species32.
The current investigation also accounted for a rise in the overall phenol and flavonoid contents in S. lycopersicum seedlings infected with M. incognita. Present study coincided with surveys which have shown that levels of various phenolic compounds in tomato and brinjal plants increased to protect them against nematode assault and to offset the reactions10,38. It has been recognized that phenols also serve as crucial buffers in regulating the stress responses within plants where an array of phenolic chemical groups serves as nematicidal agents39. Elevated levels are linked to upregulated expression of phenylalanine ammonia lyase activity and phenol metabolism in Vigna radiata40. Increased nematicidal phenolic compounds enhance nematode resistance by inhibiting key enzymes for feeding and reproduction. PGPRs also trigger signaling pathways where phytohormones like and jasmonic acid (ISR triggered by PGPRs) and salicylic acid (SAR triggered by pathogens) disrupts the nematode invasion. Other hormones (i.e. auxins, cytokinins etc.) integrate with these signaling molecules. This signaling crosstalk regulates antioxidant activity, redox homeostasis and bio-metabolite (such as lignin) production whilst enhancing plant resilience32,36. The observations we recorded also showed that these chemicals were present in higher concentrations in seedlings pretreated with B. japonicum cells and supernatant. Likewise, Pogostemon cablin plants with nematode infestations that had received microbial therapy also had increased amounts of phenolic compounds, flavonoids, anthocyanins and polyphenols41. The chelating activity of anthocyanins and flavonoids against pathogenic reactions also may be the cause of an upsurge. Flavonoids resist nematodes by inducing juvenile quiescence, repelling migration or directly killing them. Their effects vary across nematode species due to differences in chemosensory receptors, receptor binding affinities and cuticle solute permeability.
Confocal microscopy allows for high-resolution, 3D- imaging, enabling precise localization and assessment of these parameters at a cellular level. This technique facilitates the detailed examination of oxidative stress markers and their effects on cellular integrity, providing a more comprehensive understanding of the biochemical and morphological changes occurring within the samples42. It confirmed that nematode infection induces oxidative stress, evident from increased H₂O₂, glutathione accumulation and nuclear and membrane damage whereas B. japonicum treatment alleviated these effects by reducing ROS levels and enhancing seedling resilience. The decline in cellular damage suggests a defensive role via antioxidant enzyme induction or antagonistic action. Mitigation in cell pellet and supernatant treatments highlights the potential of bacterial biocontrol against nematodes. The present study also found strong correlation associations among growth parameters, which means that the correlating parameters tend to increase and decrease together. Antioxidative enzymes sharing negative correlation with stress indices depicted that with higher enzymatic antioxidant activity, plants experience lower oxidative damage. Similarly, stress markers being negatively correlated with pigments depicted that as one increases, other decreases significantly. Non-enzymatic antioxidants which were positively correlated with each indicated that elevated levels could potentially result in heightened concentrations of GC and TC which might be capable of diminishing oxidative stress in nematode-infected seedlings. Our studies coincided with Tikoria et al.43. All of these findings showed how well B. japonicum helped M. incognita-infected host plants establish stress tolerance, making them ideal candidates for use as biocontrol agents against worm infections.
Material and methods
Preparation of Nematode inoculum
To extract M. incognita eggs and obtain J2, pure cultures were maintained on eggplant and tomato roots. Infected roots were removed, sterilized with a 1.5% sodium hypochlorite solution, washed using distilled water (DW). Eggs were gathered, incubated in petri dishes for egg hatching at 27 ± 3 °C for 72 h to obtain J2 stage of M. incognita for subsequent tests44.
Microbial strain
Bradyrhizobium japonicum strain 11477 was procured from IMTECH (MTCC 120T) Mohali, Punjab, India. The lyophilized culture was kept alive for 24–48 h in a BOD incubator at 28 °C in a flask with 50 ml Yeast Mannitol Broth (YMB) medium (HiMedia). Subsequently, sub-culturing in the agar media was done to preserve the culture stored at 4 °C for further use.
Preparation of bacterial culture filtrates
Following the time of incubation, bacterial broth was grown 2 days at 28 °C and centrifuged at 10,000 rpm for 15–20 min at 4 °C Supernatant solution or cell free broth underwent filtration for bioassay testing. Also, pellets were collected separately, cleaned and cells were put in DW and attained a state of concentration of 109 cells/ml DW.
Mortality test of Nematode Juveniles
An experiment was conducted to assess the nematicidal potential of B. japonicum using 100 J2 nematodes in 1 ml aqueous media in 35 mm petri dishes. The nematodes were treated with control (DW), bacterial cells (109/ml) and 100% bacterial supernatant. Mortality was observed daily at 28 ± 1 °C for 7 days, with dead nematodes identified by their lack of response to a probe. Mortality rate was calculated using formula as per Sun et al.45 viz.,
Invitro assay and treatments
Surface sterilized seeds of susceptible variety of S. lycopersicum cv. Pusa Ruby (PR) bought from a local seed market in Amritsar (Punjab) were allowed to germinate in petri plates with 90 mm diameter layered with autoclaved whatman filter paper grade 1. Individual petri plate had 25 seedlings with different treatments as control (C), Nematode inoculated (N), 109 cells’ of Bradyrhizobium japonicum (T1), cell pellets with nematode infestation (T1 + N), cell-free filtrates (supernatant as T2) and treated seeds with nematode inoculation (T2 + N), respectively. Germinated seedlings were infested @ 5 J2/seedling. Threefold replicates of every treatment (were sustained’ The B.O.D incubator “CALTAN” (Super Deluxe Automatic) was used to keep the petri plates at temperature of 28 ± 1 °C, humidity of 60 ± 10% for 10 days with 16h light cycle every day. The examination of morphological and biochemical parameters of tomato seedlings was concluded after 10 days following the inoculation of nematodes.
The results were compiled by comparing nematode infected seedlings with non-infected control seedlings, bacterium B. japonicum treatments with control and nematode infested B. japonicum treated seedlings with seedlings that were nematode infected and left untreated.
Morphological parameters
Morphological parameters including percentage seed germination, number of galls, root length, shoot length, root weight and shoot weight were examined.
Photosynthetic pigments
Fresh seedlings were homogenized with 80% acetone in 1:4 ratio (plant: 80% acetone). The homogenate was centrifuged at 10,000 rpm for 20 min at 4 °C. The absorbance of the supernatant was measured at 645 and 663 nm to estimate chlorophyll content. Additionally, absorbance readings at 480 and 510 nm were taken to quantify carotenoid content46.
Stress indices
In order to investigate the oxidative stress, hydrogen peroxide and malondialdehyde content were estimated.
H2O2
Following a homogenization process of 200 mg of plant tissue in 1% trichloroacetic acid (TCA) and 18 min centrifugation at 7000 rpm, 500 µl of potassium phosphate buffer (PPB) and 1 ml of potassium iodide (KI) were added in the supernatant to enable estimate at the optimal pH of 7. The Genesys 20 UV–VIS spectrophotometer was employed to detect the absorbance at 390 nm and a standard curve was created to assess the H2O2 concentration of the plant sample47.
MDA
Following the homogenization process in 200 µl of 0.1% of trichloroacetic acid, fresh plant tissue weighing 200 mg was centrifuged at 7000 rpm for 15 min at 4 °C. The supernatant portion was obtained and utilized for estimation. The reaction mixture for measuring the amount of MDA contained 400 µl of supernatant, 200 µl of 20% (w/v) TCA and 1 ml 0.5% (w/v) TBA, which were incubated in water bath at 95 ± 3 °C for 30 min. Samples were quickly chilled in an ice bath. At 532 nm and 600 nm, absorbance was measured48.
Antioxidative enzymes
Seedlings were grounded fine into a paste in 0.1 M PPB (pH 7.0) to produce homogenate, which was then centrifuged at 4 °C for 22 min at 13,000 rpm. Different antioxidative was tested using standard techniques.
Catalase (CAT; E.C. 1.11.16)
By combining 150 µl of supernatant with 3 ml of 0.1 M PPB at pH 7.0 and adding 800 µl of hydrogen peroxide (150 mM), the activity of CAT was measured. The mixture’s absorbance was then measured at 240 nm for a minute49.
Ascorbate peroxidase (APOX; E.C. 1.11.11.1)
In order to compute the activity of APOX, 1 ml of 0.1 M PPB with a pH of 7.0 was combined with 100 µl of supernatant. 400 µl of ascorbate (5 mM) and H2O2 (1 mM) were then added, respectively. The raised absorbance of the blended mix was measured at 290 nm for 1 min50.
Guaiacol peroxidase (GuPOX; E.C. 1.11.1.7)
To measure the GuPOX activity, 1 ml of 0.1 M PPB at a pH of 7.0 was combined with 50 µl of supernatant. 50 µl of guaiacol (20 mM) and 150 µl of hydrogen peroxide (12.3 mM) were then added. The increased absorbance of the homogenate mixture was measured for 1 min at 436 nm51.
Polyphenol oxidase (PPO; E.C. 1.10.3.1)
To estimate PPO, 200 µl of supernatant was blended with 2 ml of 0.1 M PPB at pH 7.0 and then 300 µl of catechol (0.01 M) was added. For 1 min, the mixture’s enhanced absorbance was measured at 412 nm52.
Glutathione-S-transferase (GST; E.C. 2.5.1.18)
GST activity was measured by adding 125 µl of reduced glutathione and cDNB (1-Chloro-2,4-dinitrobenzene), each with a molarity of 1 mM, to 50 µl of supernatant with 500 µl of 0.1 M PPB with a pH of 7.0. For 1 min, the mixture’s decreased absorbance was measured at 340 nm53.
Superoxide dismutase (SOD; E.C. 1.15.1.1)
The pulverization of planted seedlings (420 mg) was carried in 1.68 ml of ice-cold sodium carbonate (Na2CO3) and the consequential homogenate was prepared through centrifugation at 12,500 rpm for 25 min at 4 °C. The SOD activity was determined by mixing 600 µl of NBT (96 µM) and 240 µl of hydroxylamine hydrochloride (20 mM) with 1.2 ml of Na2CO3 buffer (50 mM), proceeded by the inclusion of 240 µl Triton-X 100 (0.6%). After a 2-min of incubation at room temperature (RT), 120 µl of the obtained supernatant was incorporated to halt the reaction and the absorbance rise at 540 nm was recorded with UV–VIS Spectrophotometer54.
Non-enzymatic antioxidants
Fresh plant material was homogenized in tris buffer under cold circumstances centrifuged for 20 min at 12,000 rpm maintained at 4 °C. The supernatant was collected to analyze non-enzymatic antioxidants.
Glutathione
Fresh seedlings (200 mg) were homogenized in 600 µl of cold tris buffer in ice cold conditions proceeded by centrifugation at 4 °C for 15 min at 13,000 rpm. The obtained supernatant was blended with DTNB, tris buffer and absolute methanol. After 15 min of incubation at RT, absorbance at 412 nm was recorded to measure glutathione concentration, utilizing a calibration curve using reduced glutathione of specified concentrations55.
Tocopherol
Fresh homogenate was prepared akin to the glutathione process. Vigorously mixed the resulting supernatant with 100 µl DW and 100 µl freshly prepared ethanolic ferric chloride, shaken succeeded by blending with 100 µl xylene and centrifuged at 5,000 rpm for 8 min at 4 °C. The top xylene layer, combined with 2,4,6-tripyridyl-s-triazine was used to measure the absorbance at 600 nm using UV–VIS spectrophotometer. Calibration curves were established using known tocopherol concentrations56.
Proline
Fresh homogenate was prepared using plant tissue (250 mg) crushed in 5 ml of 3% sulfosalicylic acid followed by centrifugation at 13,000 rpm for 12 min. The resulting supernatant was mixed and heated at 95 ± 5 °C with equivalent volume of Ninhydrin and glacial acetic acid. After termination, 2 ml of toluene was incorporated to the above mixture and vortexed for 1 min. The absorbance was measured at 520 nm on UV–VIS spectrophotometer. Calibration curves were established using known L-proline standard concentrations57.
Ascorbic acid
Fresh seedlings (240 mg) were homogenized in 720 µl of 2.5% metaphosphoric acid (HPO3) on ice, followed by ultra-centrifugation at 2500 rpm for 15 min at 4 °C for estimation of AAC. The resulting supernatant was unified with 100 µl of the solution and 200 µl of 5% HPO3, incubated for 30 min at optimum RT in the dark. Then, 320 µl of 2,6-dichlorophenolindophenol dye and 0.5 ml of n-Amyl alcohol were introduced followed by agitation of the mixture on a vortex shaker. The absorbance at 546 nm was measured in the top layer, using a calibration curve with known amounts of L-ascorbic acid58.
Phenolic compounds
Using standard protocols, phenolic compounds were investigated.
Phenol
Tomato seedlings (150 mg) were blended together uniformly in 450 µl of 80% ethanol (chilled) and centrifuged for 25 min at 13,000 rpm and 4 °C. 1.45 ml distilled water and 50 µl supernatant was combined. Subsequently, 250 µl Folin–Ciocalteu (FC) reagent and 1 ml of 20% Na2CO3 were added. After vortex mixing, absorbance was measured at 760 nm. A calibration curve was constructed using predetermined catechol concentrations59.
Flavonoid
Fresh homogenate was produced in a similar manner to phenol content. A 200 µl aliquot of supernatant was mixed with 5.8 ml methanol, followed by addition of 200 µl of 10% aluminum chloride, 200 µl of 5% sodium potassium tartrate and 1 ml DW to estimate flavonoid concentration. After 30 min at RT in dark, the mixture’s absorbance at 415 nm was observed. Rutin calibration curve was employed for assessment, using a specified concentration60.
Anthocyanin
Fresh homogenate of plant material was grounded in acidified methanol (made of methanol, distilled water and hydrochloride acid at a ratio of 79:20:1). After being stored at 4 °C overnight, the extract was centrifuged for 20 min at 10,000 rpm. Supernatant was employed in further estimation where absorbance was measured using a UV–VIS spectrophotometer at 530 and 657 nm61.
Confocal microscopy
Confocal microscopy was done to visualize and quantify oxidative burst through hydrogen peroxide levels, glutathione content, nuclear damage and cell membrane integrity damage in living samples. It was examined under Nikon AIR confocal laser scanning microscope (CLSM, New York, USA).
Hydrogen peroxide
H2O2 localization in fresh S. lycopersicum roots was visualized by staining them with 20, 70-dichlorofluorescin diacetate (DCF-DA) for 10–15 min and extra dye was removed with deionized water (excitation-488 nm and emission-530 nm)62.
Glutathione
Root tips of tomato seedlings were dipped in 25 mM monochlorobimane (MCB) solution and were kept in dark conditions for 20 min and extra stain was then washed with deionized water (excitation-351–364 nm and emission-477 nm)63.
Nuclear membrane damage
For nuclear membrane damage, the roots were treated with 4,6-diamino-2-phenylindole (DAPI) (0.1 mg in 100 mL PBS) and incubated in the dark for 30 min followed by washing with PBS and mounted slides were used for further examination (excitation-358 nm, emission-461 nm)64.
Cell membrane damage
For cell membrane damage, roots of tomato seedlings were dipped in propidium iodide (PI) (50 µM, Sigma-Aldrich, Mumbai, India). Afterwards, they were washed with dH2O and finally mounted on water for examination under microscope (excitation-543 nm, emission-617 nm)65.
Examining data with statistics
The studies were performed with the results presented as the mean of three independent sets, expressed with standard error of the mean (Mean ± S.E.M.) in Microsoft Office Excel. Data analysis was conducted using IBM SPSS 16.0 software, where one-way ANOVA was applied to assess the overall differences between treatments. Tukey’s post-hoc test was used to determine the significance between specific treatments, with a significance threshold set at p ≤ 0.01 and p ≤ 0.05. Pearson’s correlation analysis was performed to examine relationships between different variables (parameters), utilizing Past 4.03 software (p < 0.05).
Conclusion
The findings of this study highlight the possibility for potential role of PGPR Bradyrhizobium japonicum against plant-parasitic nematodes. Following nematode infection, plant growth was found suppressed and antioxidative defense system was found modulated along with increase in the level of stress markers. However, PGPR application to tomato plants have successfully overcome the nematode induced stress. Treating beneficial bacteria to diseased plants also boosts the host plant’s immune system and prevents nematodes from penetrating the plants. Further, all parameters of antioxidative defense system, photosynthetic pigments, secondary metabolites were found increased after application of bacterium. Therefore, a sustainable approach to combating these phytonematodes is to utilise Bradyrhizobium species as a biocontrol agent. The findings suggests that the intricate process behind the tritrophic interactions between microbial strains, plants and plant-parasitic nematodes can have substantial effects on agriculture.
Data availability
The original findings given in the study are provided in the article Material. Further inquiries, if any should be made to the corresponding authors.
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Acknowledgements
The authors are thankful to University Grants Commission, New Delhi, India (File Reference No. 191620133242), for providing financial support for completion of this research work.
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Sharma, R., Kapoor, N. & Ohri, P. Ameliorative effect of rhizobacteria Bradyrhizobium japonicum on antioxidant enzymes, cell viability and biochemistry in tomato plant under nematode stress. Sci Rep 15, 8017 (2025). https://doi.org/10.1038/s41598-025-92798-9
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DOI: https://doi.org/10.1038/s41598-025-92798-9
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