PJN_43_2_194-207

Introduction

The root-knot nematode, Meloidogyne incognita is a sedentary endoparasite species, causing major economic damage to different crops worldwide (Rodríguez et al., 2009). The reniform nematode, Rotylenchulus reniformis is a semi-endoparasitic species, becoming a serious pest of multiple crops (Robinson, 2007). Also, the spiral nematode, Helicotylenchus indicus is an important and widely distributed ectoparasitic nematode, causing root damage and consequently limiting crop yield (Siddiqi, 2000; Diab et al., 2019). These nematode species were found the most dominant nematodes associated with the rhizosphere of different pomegranate cultivars in different regions in global survey studies (Ibrahim and Mokbel, 2009; Khan and Shaukat, 2010; Nayba et al., 2012; Korayem et al., 2014). Many research workers reported the damage of such nematode species to pomegranates (Shelke and Darekar, 2001; Khan and Shaukat, 2010; Nour El-Deen et al., 2016; El-Qurashi et al., 2017).

The incompatible resistant interactions to nematode infection may include many physiological defense actions, production of H2O2 (Peltzer et al., 2002), the formation of reactive oxygen species, ROS (Neill et al., 2002; Montes et al., 2004; Murgia et al., 2004), different enzymatic and non-enzymatic ascorbate (Yoshimura et al., 2004), increase in peroxidase and polyphenol oxidase level (Sarowar et al., 2005), antioxidant properties (Decker, 1997), polyphenols (Metodiewa et al., 1999).

Oxidative stress is an abnormal status associated with different organism infections; human diseases (Favier, 2006), phytoparasitic nematodes (Zhang et al., 2019), or even plant abiotic stress (Gechev and Petrov, 2020). In fact, inside the organism, there is an accurate balance between the ROS (free radicals) produced as byproducts of different metabolic pathways and the antioxidant scavenging system under homeostasis conditions (Reddy et al., 2005). However, the oxidative stress phenomenon is initiated when an excess of free radicals starts serial reactions damaging the essential molecules in the organism, for example, DNA and proteins. In this case, the antioxidant scavenging system is unable to suppress or scavenge all the serious free radicals produced (Halliwell and Gutteridge, 1999; Favier 2006). ROS reduced forms of atmospheric oxygen (O2). The information includes the excitation of O2 to form singlet oxygen (O2) or form gaining one, two, or three electrons for O2 to form, superoxide radical (O2−), H2O2, or a hydroxyl radical (HO−), respectively. ROS plays two very different roles; serial damage or signaling the activation of a defensive reaction. The key factor controlling these different roles is the cellular levels of ROS (Dat et al., 2000). Antioxidants are a major milestone that enhances the ability of plants to overcome oxidative stress. The regulation of ROS in plants is governed by an antioxidant defense system composed of antioxidant enzymes and non-enzymatic antioxidants (Hasanuzzaman et al., 2017).

Results of Afify et al. (2014) revealed that different hosts reacted similarly to different nematode species; in which oxidants such as MDA, H2O2, and antioxidants such as GSH, AA, SOD, CAT, and APX were all increased. The enhancement of oxidative stress inside nematode bodies dramatically could increase the pathogenicity of Bursaphelenchus xylophilus on pine. The EU-Bx3 nematode isolates with H2O2 enhancement were the most pathogenic, causing 70% death of pine saplings. Furthermore, H2O2 (20 mM) significantly improved the fecundity of Bu. xylophilus (Zhang et al., 2019). On the contrary, Karajeh (2008) reported that hydrogen peroxide soil drench application affects M. javanica reproduction in tomatoes. Noureldeen et al. (2021) suggested that the bioprotective nature of Pseudomonas fluorescens for tomato plants against M. incognita is due to the increased expression of phenol content and defensive enzymes such as peroxidase and superoxide dismutase, and the significant reduction in MDA and H2O2 contents.

Growing plants supplemented with fertilizers containing humic acid improved their resistance to nematode infection (Kesba and Al-Shalaby, 2008). Growing resistant pomegranate varieties supplemented with humic acid were supposed to result in better nematode management. Kesba and El-Beltagi (2012) reported that humic acid application reduced MDA and H2O2 in grapes while increasing GSH, ASA, TPH, APX, SOD, CAT, and PPO, which, in turn, reduced M. incognita and R. reniformis reproduction. Some studies pointed to the effect of humic substance’s application on different plant hosts infected with the root-knot nematode. However, few studies investigated the antioxidant status associated with such application. Further, there are contradictory results regarding the point, making it still unclear. This study was carried out to investigate the effect of type parasitism of three nematode species on nematode reproductivity, and oxidative and antioxidative enzymes in three pomegranate cultivars. Also, evaluate four commercial products of humic acid against the root-knot nematode, M. incognita, and pomegranate enzyme content under greenhouse conditions.

Materials and Methods

Nematode species sources

Pure cultures of the endoparasitic root-knot nematode (RKN), M. incognita (Chitwood, 1949), the semi-endo parasitic reniform nematode (RN), R. reniformis (Adam et al., 2018), and the ectoparasitic spiral nematode (SN), Helicotylenchus indicus (Siddiqi, 1963) were obtained from isolates belonging to the Nematology Research Center, Faculty of Agriculture, Cairo University. Nematode species have been propagated separately, M. incognita on eggplant cv. Classic, R. reniformis on pigeon peas, and H. indicus on Italian cypress, plants were grown in 20 cm diameter clay pots filled with sterilized loamy soil. To avoid contamination, cultures of each nematode species were arranged separately, checked, and replenished periodically to ensure a continuous supply of inocula for the experimental work.

Glasshouse experiments

Three-month-old seedlings of 3 pomegranate cultivars (Assuity, Manfalouty, and Wonderful) with uniform size were obtained from Horticulture Research Institute, Agriculture Research Center and cultivated singly in 20 cm diameter clay pots filled with steam-sterilized sandy loam soil (1:1, v/v). After a week, 5 seedlings of each cultivar were inoculated separately with 5000 infective stages of M. incognita, R. reniformis, and H. indicus by pipetting the nematode water suspension into 4 holes around the root system which was immediately covered with soil. Pots were labeled and arranged randomly on a glasshouse clean bench, receiving similar horticulture treatments. Seedlings were left out after 3 months from inoculation. Soil population was extracted using (Hooper et al., 2005) and counted. The nematode-embedded stages of each species were also counted.

For testing the effect of commercial products of humic acid on the root-knot nematode development and reproduction, another 5 seedlings of each cultivar were inoculated with 5000 newly hatched J2 of M. incognita/plant/pot. Two weeks after inoculation, four commercial products of humic acid were applied (recommended dose and method of application) as mentioned in (Table 1). All treatments were arranged in a fully randomized design on a clean bench in the glasshouse at 32±5oC receiving similar horticultural treatments. After 3 months, nematode soil populations were extracted and counted using a Hawksley counting slide, under a binocular microscope. A subsample (5 g) of roots from each plant was stained and gall numbers, embedded stages (developmental stages + eggmasses) per root were calculated, final population (embedded stages + nematodes in soil), nematode reproduction factor (Pf/Pi), an average of eggs/egg-mass were estimated.

Plant chemical analysis

Six subsamples of the fresh root of each treatment were chemically analyzed at the Central Chemistry Lab, Faculty of Agriculture Research Park (FARP), Faculty of Agriculture, Cairo University as follows:

Preparation of enzyme extracts: A sample of 1 g was homogenized in 3 ml of 50 mM phosphate buffer pH 7.0 containing 1.0 N NaCl, 1% PVP (Sigma), and 1 mM ascorbate (Sigma) at 4ºC. After centrifugation at 15,000×g for 15 min, the supernatant was collected.

Assay of protein content: The protein was estimated by (Bradford, 1976) with standard curves prepared using bovine serum albumin.

Table 1: Structure, dose, and application method of Humic commercial products.

Trade name	Structure	Recommended
Trade name	Structure	Dose/plant	Application method
Actosol®	(Humic acid and Fulvic acid) 20% + K 6%	5 ml	Soil drench
Decka™	Humic acid 10% + Fulvic acid 8% + Amino acids 8% + N 2% + P 4.2% + K 5%	5 ml	Soil drench
Humic acid™	Potassium humate 85% + Dissolved potassium 8% + Fulvic acid 3%	2.5 g	Foliar spray
Humo plus 40™	Humic acid 40% + N 1% + P 3% + K 5% + Fe 5000 ppm + Zn 1000 ppm + Mn1000 ppm + Cu 200 ppm + Mo 50 ppm	2.5 ml	Foliar spray

Determination of oxidative burst

Lipid Peroxide, MDA contents: The thiobarbituric acid (TBA) reaction is described by Heath and Packer (1968). Fresh mass (200 mg) from the culture was homogenized in 2 ml of 0.1% (w/v) trichloroacetic acid (TCA), followed by centrifugation at 12,000×g for 20 min. The obtained supernatant (1 ml) was mixed with an equal volume of TCA (10%) containing 0.5% (w/v) TBA or without TBA as the blank, and heated at 95°C for 30 min and then cooled in ice. The reaction product was centrifuged at 12,000×g for 15 min and the absorbance of the supernatant was measured at 400, 532, and 600 nm. The MDA equivalent was derived from the absorbance according to (Hodges et al., 1999).

Hydrogen peroxide concentration check

Hydrogen peroxide was measured by the method described by Capaldi and Taylor (1983), with a slight modification. The callus was ground in 5% TCA (2.5 ml per 0.5 g callus) with 50 mg active charcoal at 0°C, and centrifuged for 10 min at 15,000 × g. The supernatant was collected, neutralized with 4 N KOH to pH 3.6, and used for the H2O2 assay. The reaction mixture contained 200 µl of leaf extract and 100 µl of 3.4 mM 3-methyl benzothiazoline hydrazone (MBTH). The reaction was started by adding 500 µl of horseradish peroxidase solution (90 units per 100 ml) in 0.2 μl sodium acetate (pH 3.6). After two minutes 1400 µl of 1 N HCl was added. The absorbance was read at 630 nm after 15 min.

Determination of total glutathione

The level of total acid-soluble SH (glutathione GSH) was determined using Ellman’s reagent (De Vos et al., 1992). The buffer solution was mixed with 630 µl of 0.5 M K2HPO4 and 25 µl of 5 mM, 5’–dithiobis (2-nitrobenzoic acid) (final pH 7). The absorbance was read at 412 nm after 2 minutes. GSH was used as a standard.

Determination of ascorbic acid

Ascorbate content was measured using 2,4-dinitrophenol indophenol. The absorbance was measured at 520 nm according to Omaye et al. (1979).

Phenol check

The phenolic assay was performed following the method of Zieslin and Ben-Zaken (1993). The samples were homogenized at the rate of 0.1 g per 1 ml of 80% methanol and the methanolic extract was kept in a water bath at 70ºC for 15 min with frequent stirring. One ml of methanolic extract was added to 5 ml of distilled water and 250 ml of Folin-Ciocalteau reagent (1 N) and the solution was kept at 25oC for 30 min. Finally, 1 ml of a saturated solution of Na2CO3 and 1 ml of distilled water was added and the reaction mixture was incubated for 1 h at 25oC. After the blue color was developed, the absorbance was recorded at 725 nm. The contents of the total dissolved phenols were calculated according to a standard curve obtained from the reaction of Folin-Ciocalteau with a catechol solution. The phenol content was expressed as phenol equivalents in mg/g fresh weight of callus tissues.

Determination of the activity of antioxidant defense enzymes

SOD activity check: The activity of SOD was evaluated by measuring its ability to inhibit the photochemical reduction of NBT using a method (Beauchamp and Fridovich, 1971). The 3 ml reaction mixture contained 50 mM phosphate buffer pH 7.8, 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 1.0 mM EDTA, and 20 µl enzyme extract. Riboflavin was finally added and the reaction started by placing 30 cm tubes under 15 W fluorescent lamps. The reaction was started by turning on the light and allowed to work for 10 min. Turning off the light stopped the reaction and the tubes were covered with a black cloth. Unilluminated tubes were used as a control. The absorbance was read at 560 nm. The volume of enzyme extract corresponding to 50% reaction inhibition was taken as one enzyme unit.

Ascorbate peroxidase (APX) activity assay

The activity of ascorbate peroxidase was determined spectrophotometrically by decreasing the absorption at 265 nm (e = 13.7mM–1 cm–1) using the (Nakano et al., 1981) method. The reaction mixture contains 50 mM potassium phosphate solution pH 7.0, 5 mM ascorbate, 0.5 mM H2O2, and enzyme extract. Started adding the reaction. The non-enzymatic oxidation rates of ascorbate were corrected by including a reaction mixture without the enzymatic extract.

Catalase activity assay

Catalase activity was determined by H2O2 consumption using the method of Dhindsa et al. (1981). The reaction mixture contains 50 mM potassium phosphate pH 7.0, 15 mM H2O2, and an enzyme extract. The consumption of H2O2 was monitored spectrophotometrically at 240 nm (e= 45.2mM–1 cm–1). Enzyme activity was expressed in µM H2O2 min–1.

Polyphenol oxidase activity assay

Polyphenol oxidase was tested using the photochemical method as described by Coseteng and Lee (1987). The reaction mixture contains 50 mM potassium phosphate solution pH 6.2, 250 mM catechol, and enzyme extract. The increase in absorbance was measured at 420 nm. One unit of enzyme activity is defined as the amount of enzyme that causes an increase of 0.001 absorption units per minute at 25ºC.

Statistical analysis

Data were compared by Duncan’s Multiple Range Test (DMRT) at a 5% level of probability using MSTAT version 4 (1987).

Results

The primary pathogenicity test for the three pomegranate cultivars against M. incognita, R. reniformis, and H. indicus (Table 2) revealed that all three cultivars were susceptible to the infection of the three nematode species. Nematodes were successfully able to penetrate, develop, reproduce, and consequently fold many times (at least 5) on the three cultivars during the experimental period. Assuity cultivar was the most susceptible host, recording RF of 6.7, 11.8, and 12.1 for M. incognita, R. reniformis, and H. indicus, respectively. The three cultivars almost reacted the same to the endoparasitic nematode, M. incognita. Yet Manfalouty achieved a low significance to the semi-endoparasitic nematode, R. reniformis parameters when compared with the other two cultivars (Assuity and Wonderful) which were similar in susceptibility. The ectoparasitic nematode, H. indicus severely infected the three cultivars, and the highest nematode numbers (embedded, in soil or final population) were recorded on Assutiy in particular.

In view of the host response to nematode infection, the root content of MDA, H2O2, GSH, TAA, and TPH was very much higher than in the healthy (nematode-free) plants. Obviously, under all three nematode species infections, the oxidative stress on the most susceptible cultivar ‘Assuity’ represented in MDA and H2O2 contents had the highest and the most significant values, while it was not for the antioxidants GSH, TAA, and TPH. The response of the cultivars (as measured by the antioxidant content) for the three nematode species was significantly increased and different from each other in the three pomegranate cultivars. Also, significant differences were noticed among the cultivars as a response to the same nematode species (Table 3). Likewise, in Table 4, the enzymatic antioxidants response due to the nematode infection followed the same awarding trend. The enzymatic antioxidant content due to H. indicus infection stress was the highest in all cultivars. It is also clear from Tables 3 and 4 that the biochemical response in the three cultivars due to the same nematode species infection was significantly different.

Table 2: Reproductivity of M. incognita, R. reniformis, and H. indicus on pomegranate cultivars.

Cultivar	Galls/Root	Embedded stages/Root	Soil population	Final population (Pf)	RF (Pf/Pi)
M. incognita
Assuity	1588 b	2108 a	31410 a	33518 a	6.7 a
Std.D.	96.1	114.0	1561.0	1569.5	0.3
Manfalouty	1086 c	1499 b	27500 b	28999 b	5.8 b
Std.D.	102.2	91.3	1731.6	1735.9	0.3
Wonderful	1802 a	2262 a	31650 a	33912 a	6.8 a
Std.D.	93.8	169.1	1401.9	1454.5	0.2
R. reniformis
Assuity	-	2419 a	56475 a	58894 a	11.8 a
Std.D.		146.0	3180.2	3258.7	0.6
Manfalouty	-	1474 c	33500 c	34974 c	7.0 c
Std.D.		165.9	4038.7	4088.8	0.8
Wonderful	-	1854 b	51650 b	53504 b	10.7 b
Std.D.		163.4	385.0	389.7	0.1
H. indicus
Assuity	-	2263 a	58475 a	60738 a	12.1 a
Std.D.		178.2	1788.8	1681.8	0.3
Manfalouty	-	1484 b	32000 c	33484 c	6.7 c
Std.D.		201.1	685.9	877.8	0.1
Wonderful	-	2058 a	41650 b	43708 b	8.7 b
Std.D.		138.5	921.6	951.3	0.1

Means followed by the same letter(s) within a column of each nematode species are not significantly different (P ≤ 0.05) according to Duncans’ multiple range test. Final population (PF) = Embedded stages + Soil population, Rf (Reproduction factor) = Pf (Final population)/Pi (Initial population), Std.D.= Standard deviation.

Data in Table 5 showed the effects of humic acid application on the reproduction of M. incognita on pomegranate. It is clear that application of all humic acid commercial products significantly reduced nematode criteria in terms of galls, final population, and reproduction factor, whether it was applied as a soil drench (Actosol® and Decka™) or foliar spray (Humic acid™ and Humo plus™), with many exceptions in numbers of eggs per egg mass which showed non-significant differences in several treatments when compared to control. Drench application showed more superiority in reducing nematode parameters (Actosol® and Decka™) than that of foliar spray products (Humic acid™ and Humo plus™), regardless that the products Humic acid™ and Humo plus™ were richer in such ratio than the other two drench products (Table 1). This, in turn, reflects the higher efficacy of humic acid against nematode when applied as a drench application. However, the reductions in nematode numbers were significant compared to control, the nematode final population was still high in all treatments.

Table 3: Effect of M. incognita, R. reniformis, and H. indicus on pomegranate cultivars root contents of non-enzymatic antioxidants.

Nematode species	MDA (μ mol/g FW)	H2O2 (μ mol/g FW)	GSH (μ mol/g FW)	TAA (mg/g FW)	TPH (mg/g FW)
Assuity
M. incognita	8.61 a	235.12 a	6.29 i	10.89 f	5.36 g
Std.D.	0.07	0.85	0.10	0.07	0.08
R. reniformis	8.19 b	229.49 b	8.78 f	14.42 c	7.76 d
Std.D.	0.03	1.43	0.05	0.67	0.13
H. indicus	8.08 c	223.06 c	11.45 c	17.59 a	9.98 b
Std.D.	0.06	1.06	0.07	0.30	0.07
Healthy	2.95 h	90.46 k	2.98 k	3.97 gh	1.30 i
Std.D.	0.06	0.52	0.08	0.13	0.03
Manfalouty
M. incognita	7.54 d	208.10 d	7.58 g	12.85 d	6.09 f
Std.D.	0.07	0.84	0.08	0.52	0.24
R. reniformis	7.46 d	196.46 e	10.81 d	14.92 c	7.35 e
Std.D.	0.06	0.78	0.06	0.21	0.14
H. indicus	7.56 d	181.07 g	12.78 a	15.72 b	8.97 c
Std.D.	0.04	1.03	0.03	0.11	0.32
Healthy	2.40 i	96.24 j	2.84 l	4.37 g	1.69 h
Std.D.	0.03	0.88	0.15	0.06	0.07
Wonderful
M. incognita	6.93 e	188.04 f	7.33 h	12.24 e	5.97 f
Std.D.	0.06	1.26	0.07	0.21	0.06
R. reniformis	6.77 f	163.42 h	9.08 e	14.96 c	8.94 c
Std.D.	0.10	1.25	0.05	0.21	0.07
H. indicus	6.08 g	153.02 i	12.28 b	17.15 a	11.01 a
Std.D.	0.08	1.07	0.05	0.25	0.03
Healthy	2.14 j	96.73 j	3.28 j	3.76 h	1.61 h
Std.D.	0.03	1.12	0.04	0.04	0.12

Means followed by the same letter(s) within a column are not significantly different (P ≤ 0.05) according to Duncans׳ multiple range test. MDA=Lipid peroxidation, H2O2=Hydrogen peroxide, GSH = Glutathione, TAA = Total ascorbic acid, TPH = Total phenols, Std.D.= Standard division.

Table 4: Effect of M. incognita, R. reniformis, or H. indicus on pomegranate cultivars root contents of antioxidant enzymes.

Nematode species	APX (unit/mg protein)	CAT (unit/mg protein)	PPO (unit/mg protein)	SOD (unit/mg protein)
Assuity
M. incognita	16.49 h	52.93 e	14.26 h	217.99 f
Std.D.	0.17	0.59	0.24	1.48
R. reniformis	26.41 e	68.78 c	28.86 e	247.35 d
Std.D.	0.11	2.09	0.13	8.73
H. indicus	35.79 b	84.82 a	43.11 b	277.08 b
Std.D.	0.25	1.02	3.32	8.35
Healthy	9.88 j	24.86 i	3.11 i	127.46 j
Std.D.	0.11	0.91	0.16	0.42
Manfalouty
M. incognita	15.83 i	42.64 g	13.11 h	199.82 g
Std.D.	0.22	2.53	0.19	0.24
R. reniformis	24.73 f	61.06 d	26.84 f	228.32 e
Std.D.	0.29	1.21	0.17	2.86
H. indicus	32.90 c	80.57 b	39.85 c	256.63 c
Std.D.	0.22	0.37	0.88	3.24
Healthy	9.66 j	24.72 i	3.75 i	144.46 i
Std.D.	0.06	0.42	0.15	0.13
Wonderful
M. incognita	17.54 g	46.94 f	16.22 g	225.89 e
Std.D.	0.04	1.31	0.70	1.50
R. reniformis	30.47 d	66.80 c	31.05 d	281.30 b
Std.D.	0.09	0.50	1.32	1.37
H. indicus	43.94 a	85.23 a	46.78 a	336.18 a
Std.D.	0.15	1.56	0.25	1.86
Healthy	8.92 k	30.64 h	2.80 i	154.15 h
Std.D.	0.20	0.67	0.12	0.49

Means followed by the same letter(s) within a column are not significantly different (P ≤ 0.05) according to Duncans multiple range test.APX = Ascorbat peroxidase, CAT = Catalase, PPO = Polyphenol oxidase, SOD = Superoxide dismutase, Std.D.= Standard division.

Data in Tables 5, 6, and 7 indicated that nematode reproduction had a prominent positive relationship with MDA and H2O2 contents and a negative relationship with GSH, TAA, TPH, APX, CAT, PPO, and SOD contents of root tissues when compared with the untreated inoculated plants. Drench application treatments of Actosol® and Decka™ products caused the highest significant increases in all antioxidants activities (GSH, TAA, TPH, APX, CAT, PPO, and SOD). These increments were the most in the Manfaloutycultivar. Simultaneously, oxidative stress (MDA, H2O2) was lower due to humic acid product application. The lowest values of MDA and H2O2 were achieved in the Manfalouty cultivar. Significant differences were achieved in root contents of oxidant and antioxidant enzymes either between cultivars or nematode species, treated or not treated.

Discussion

In this study, the three pomegranate cultivars (Assuity, Manfalouty, and Wonderful) were suitable hosts for parasitism of M. incognita, R. reniformis, and H. indicus, which was evidenced by the measured high reproduction factor for all nematode species.

Table 5: Reproductivity of M. incognita on pomegranate cultivars as influenced by the addition of commercial products of humic acid.

Treatment	Dose/Plant	Galls/Root	Embedded stages/Root	Final population (Pf)	Rf (Pf/Pi)	Eggs/ Egg-mass
Assuity
Actosol®	5ml (SD)	714 h	895 f	17815 g	3.6 g	339 abc
Std.D.		25.35	34.82	725.06	0.15	30.70
Decka™	5ml (SD)	826 fg	1156 ef	19596 f	3.9 f	321 bc
Std.D.		39.27	108.82	614.20	0.12	40.52
Humic acid™	2.5g (FS)	847 f	1103 ef	20703 f	4.1 f	357 abc
Std.D.		45.66	118.17	615.19	0.13	31.38
Humo plus 40™	2.5 ml (FS)	867 ef	1099 ef	22499 e	4.5 e	351 abc
Std.D.		46.72	97.69	905.84	0.18	36.64
Inoculated only		1588 b	2108 ab	33518 a	6.7 a	367 ab
Std.D.		67.69	1168.62	566.00	0.11	25.88
Manfalouty
Actosol®	5ml (SD)	734 gh	979 f	17699 g	3.5 g	314 c
Std.D.		61.68	32.29	721.69	0.14	26.55
Decka™	5ml (SD)	923 ef	1200 def	18360 g	3.7 g	326 bc
Std.D.		31.74	91.79	878.59	0.18	41.59
Humic acid™	2.5g (FS)	930 ef	1230 def	19850 f	4.0 f	346 abc
Std.D.		31.61	264.19	953.10	0.19	16.73
Humo plus 40™	2.5 ml (FS)	1051 cd	1325 cdef	20425 f	4.1 f	349 abc
Std.D.		79.87	79.06	1069.46	0.21	7.42
Inoculated only		1086 c	1499 cde	28999 b	5.8 b	336 abc
Std.D.		87.85	90.56	1278.46	0.25	33.81
Wonderful
Actosol®	5ml (SD)	848 f	1554 cde	26934 d	5.4 d	324 cd
Std.D.		42.51	134.60	1041.07	0.21	48.40
Decka™	5ml (SD)	916 ef	1572 cde	27052 cd	5.4 cd	330 abc
Std.D.		42.49	122.86	1334.05	0.27	57.01
Humic acid™	2.5g (FS)	969 de	1672 bcd	28092 bcd	5.6 bcd	379 a
Std.D.		29.03	155.99	575.53	0.12	6.36
Humo plus 40™	2.5 ml (FS)	1059 cd	1730 bc	28310 bc	5.7 bc	344 abc
Std.D.		111.77	166.25	1184.33	0.24	31.10
Inoculated only		1802 a	2262 a	33912 a	6.8 a	341 abc
Std.D.		215.13	218.65	1518.49	0.30	34.89

Means followed by the same letter(s) within a column are not significantly different (P ≤ 0.05) according to Duncans’ multiple range test. Final population (PF) = Embedded stages + Soil population, Rf (Reproduction factor) = Pf (Final population)/Pi (Initial population), Std.D.= Standard division, SD = Soil drench, FS = Foliar spray.

Table 6: Effect of commercial products of humic acid on infected pomegranate cultivars root contents of non-enzymatic antioxidants.

Treatment	Dose / Plant	MDA (μ mol/g FW)	H2O2 (μ mol/g FW)	GSH (μ mol/g FW)	TAA (mg/g FW)	TPH (mg/g FW)
Assuity
Actosol®	5ml (SD)	2.33 l	133.44 f	17.12 b	21.68 b	12.57 e
Std.D.		0.02	0.05	0.46	0.08	0.03
Decka™	5ml (SD)	3.52 f	146.37 e	17.04 b	21.47 b	12.91 d
Std.D.		0.04	0.06	0.65	0.02	0.06
Humic acid™	2.5g (FS)	3.66 e	148.60 d	11.18 e	16.97 cd	9.73 i
Std.D.		0.04	0.01	0.13	0.06	0.01
Humo plus 40™	2.5 ml (FS)	4.35 d	152.39 c	11.16 e	17.33 c	9.85 h
Std.D.		0.03	0.29	0.24	0.17	0.07
Inoculated only		8.31 a	226.80 a	5.57 h	9.65 j	4.75 m
Std.D.		0.02	0.03	0.06	0.06	0.05
Manfalouty
Actosol®	5ml (SD)	2.47 k	104.24 k	18.36 a	23.55 a	15.64 a
Std.D.		0.03	1.45	0.13	0.51	0.10
Decka™	5ml (SD)	2.54 j	104.79 k	18.24 a	23.48 a	15.71 a
Std.D.		0.03	0.14	0.92	0.26	0.09
Humic acid™	2.5g (FS)	3.30 g	116.55 i	12.36 cd	14.60 f	8.80 j
Std.D.		0.03	0.03	0.04	0.12	0.05
Humo plus 40™	2.5 ml (FS)	3.35 g	119.44 h	12.54 c	15.41 e	8.53 k
Std.D.		0.02	0.05	0.12	0.02	0.03
Inoculated only		7.90 b	221.37 b	6.71 fg	11.39 h	5.40 l
Std.D.		0.04	0.88	0.05	0.03	0.03
Wonderful
Actosol®	5ml (SD)	2.54 j	114.16 j	11.86 d	16.64 d	13.60 b
Std.D.		0.02	0.06	0.23	0.49	0.10
Decka™	5ml (SD)	2.74 i	117.27 i	12.08 cd	16.68 d	13.33 c
Std.D.		0.02	0.10	0.08	0.04	0.10
Humic acid™	2.5g (FS)	2.99 h	119.01 h	7.06 fg	11.76 gh	10.78 f
Std.D.		0.01	0.08	0.31	0.09	0.06
Humo plus 40™	2.5 ml (FS)	3.31 g	125.71 g	7.28 f	12.13 g	10.58 g
Std.D.		0.03	0.05	0.08	0.06	0.03
Inoculated only		7.19 c	215.17 c	6.50 g	10.85 i	5.29 l
Std.D.		0.02	0.04	0.07	0.05	0.05

Means followed by the same letter(s) within a column are not significantly different (P ≤ 0.05) according to Duncans multiple range test. MDA=Lipid peroxidation, H2O2=Hydrogen peroxide, GSH = Glutathione, TAA = Total ascorbic acid, TPH = Total phenols, Std.D.= Standard division, SD = Soil drench, FS = Foliar spray.

As a result of the three nematode species parasitism, contents of lipid peroxidation and hydrogen peroxide were raised in all tested cultivars. Also, non-enzymatic antioxidants; Glutathione (GSH), Total ascorbic acid (TAA), and Total phenols (TPH) were sharply increased. Regarding MDA and H2O2, the over-production of reactive oxygen species due to nematode penetration wounds and/or secretions could interpret the higher amounts of MDA and H2O2 (Davis et al., 2000; Huang et al., 2004). It maybe considered a defensive tactic leading to programmed cell death (Borden and Higgins, 2002; Mellersh et al., 2002) in a trial of the plants to overcome nematode infection. From another point of view, the overproduction of

Table 7: Effect of commercial products of humic acid on infected pomegranate cultivars root contents of antioxidants enzymes.

Treatment	Dose / Plant	APX (unit/mg protein)	CAT (unit/mg protein)	PPO (unit/mg protein)	SOD (unit/mg protein)
Assuity
Actosol®	5ml (SD)	34.91 e	84.16 cd	37.28 e	300.26 d
Std.D.		0.11	1.65	1.48	2.45
Decka™	5ml (SD)	33.62 f	85.23 c	45.73 c	308.31 c
Std.D.		0.07	3.12	2.50	1.55
Humic acid™	2.5g (FS)	16.16 j	41.89 hi	15.73 gh	231.63 gh
Std.D.		0.14	0.77	1.08	9.56
Humo plus 40™	2.5 ml (FS)	15.73 k	49.30 f	12.60 ij	228.32 h
Std.D.		0.19	2.52	0.73	1.70
Inoculated only		14.62 l	46.90 fg	12.63 ij	210.27 j
Std.D.		0.11	4.01	0.13	5.24
Manfalouty
Actosol®	5ml (SD)	45.70 a	97.38 a	48.49 b	381.40 a
Std.D.		0.20	1.71	0.62	4.96
Decka™	5ml (SD)	40.90 d	92.57 b	50.60 a	375.93 a
Std.D.		0.12	1.65	2.39	4.74
Humic acid™	2.5g (FS)	16.40 i	54.43 e	17.70 fg	244.97 e
Std.D.		0.13	5.01	0.54	2.48
Humo plus 40™	2.5 ml (FS)	17.63 g	50.69 ef	18.44 f	236.81 fg
Std.D.		0.32	1.66	1.32	1.68
Inoculated only		14.03 m	37.79 i	11.62 j	192.74 k
Std.D.		0.06	0.64	1.03	2.22
Wonderful
Actosol®	5ml (SD)	44.50 b	79.90 d	43.31 d	359.72 b
Std.D.		0.09	4.19	0.63	6.24
Decka™	5ml (SD)	42.11 c	82.94 cd	46.97 bc	358.66 b
Std.D.		0.09	0.83	0.87	5.89
Humic acid™	2.5g (FS)	17.00 h	47.97 f	16.83 fg	239.99 ef
Std.D.		0.11	1.83	1.28	3.06
Humo plus 40™	2.5 ml (FS)	17.13 h	43.53 gh	15.75 gh	236.36 fg
Std.D.		0.05	1.26	0.33	1.30
Inoculated only		15.54 k	41.59 hi	14.38 hi	217.89 i
Std.D.		0.13	1.65	0.65	2.18

ROS may be a signal for defensive reaction activation. Such a dual role, according to (Dat et al., 2000), is controlled by the cellular level of the ROS. Suggesting the hypothesis of ROS signaling, we can conclude that the significant increase of non-enzymatic antioxidants (GSH, TAA, and TPH) may have resulted from the enhancement of MDA and H2O2 production after nematode infection.

Enzymatic antioxidants (APX, CAT, PPO, and SOD) activities showed an upwards trend in plants infected with the three nematode species. The excess superoxide anion (O2-) produced through plant-nematode interaction is scavenged by SOD in the susceptible cultivars. Similarly, ascorbate peroxidase and catalase scavenge hydrogen peroxide (Zacheo and Bleve-Zacheo, 1988). Chemically, the enzymatic detoxification process of ROS includes sequential steps; for example, superoxide dismutase transforms the superoxide anion into hydrogen peroxide, which is finally transformed into atmospheric oxygen and water by catalase (Zacheo et al., 1987). The fluctuations in antioxidant activities among cultivars due to the same commercial products may reflect the effect of the genetic variations among different cultivars. Herein, one could conclude that the biochemical response is nematode species-dependent.

The antioxidant response due to ectoparasitic nematode, H. indicus in all cultivars, recorded the highest significant values when compared to the other two nematode species regardless of the host type. This may be due to its feeding habits, where Helicotylenchus spp. used to feed externally on the root cortical layer producing small necrotic lesions (Bridge and Gowen 1993). In a single feed time, their stylet is repetitively inserted until having enough meal, then after being hungry, they return to the same actions, causing large numbers of wounds. These wounds may force the plant to produce much higher responsive molecules. Such a case could be supported by Sango et al. (2004) findings which found the high damage potential of H. multicinctus was achieved in relatively low populations.

When comparing the pomegranate cultivar’s reaction against the endoparasitic nematode, M. incognita infection, the highest oxidative stress (MDA, and H2O2 contents) was noticed in the most susceptible cultivar ‘Assuity’. This reflects the supporting role of oxidative stress in nematode pathogenicity.

The application of commercial humic acid products either through foliar spray or soil drench greatly enhanced the antioxidant activities (enzymatic and non-enzymatic) on the RKN-infected cultivars while reducing the MDA and hydrogen peroxide contents. These alterations were significant and sharply altered when compared to the untreated plants. In fact, ROS/antioxidant activities seem to be extremely dynamic, complex, and rather very correlative. Stimulation of the antioxidant system by humic acid products application increased the scavenging capacity inside plants which reduced the oxidative stress represented in MDA and H2O2. Decreasing ROS could hinder nematode pathogenicity and reproduction (Zhang et al., 2019), which is why RKN was partially suppressed due to humic acid treatments. In conclusion, we can implement humic acid products in the IPM programs as a secondary approach useful in managing the root-knot nematode.

Conclusion

The findings of this study have several important implications for the management of plant-parasitic nematodes in pomegranate orchards. The identification of susceptible cultivars can guide growers in selecting resistant or tolerant varieties. Monitoring the levels of antioxidant enzymes and oxidative stress markers can provide valuable insights into the plant’s defense response against nematode infection.

Furthermore, the study highlights the potential of humic acid as a natural nematicide for pomegranate. Future research should focus on optimizing the application rates and timing of humic acid for maximum efficacy against different phyto-nematode species. Additionally, investigations into the mechanisms by which humic acid enhances plant resistance to nematodes are warranted.

By integrating these findings into an integrated pest management (IPM) program, pomegranate growers can develop sustainable strategies to control plant-parasitic nematodes and improve crop health and productivity.

Acknowledgment

We would like to thank to the Faculty of Agriculture, Cairo University, for supporting this research work.

Novelty Statement

This study provides novel insights by comprehensively linking the type of nematode parasitism (endo, semi-endo, and ecto) to the specific oxidative and antioxidative responses in different pomegranate cultivars. Unlike previous research that often focuses on a single nematode species, we demonstrate that the ectoparasite Helicotylenchus indicus induces the most severe biochemical defense response, likely due to its feeding behavior. Furthermore, we present a comparative efficacy analysis of four distinct commercial humic acid products, revealing that soil drench applications are significantly more effective than foliar sprays in modulating the plant’s antioxidant defense system to suppress root-knot nematode reproduction. These findings offer a new, physiology-based strategy for integrating specific humic acid products into integrated pest management programs for pomegranate.

Author’s Contribution

All authors carried out the experiments, Kesba, H.H. and El-Ganainy, S.M. designed and performed the statistical analysis, Diab, S.F. prepared results tables, Abdel-rahman, A.A. prepared the first look of the manuiscript. All authors revised and approved the manuscript.

Funding

This research work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Grant No. KFUXXX).

Generative AI and AI-assisted technology statement

No generative AI or AI-assisted technologies were used in this research or manuscript writing.

Conflict of interest

The authors have declared no conflict of interest.

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