Ipomoea carnea associated phytochemicals and their in silico investigation towards Meloidogyne incognita

ABSTRACT

Root-knot nematodes (Meloidogyne spp.) are sedentary endo-parasite that causes severe yield loss in carrot. Chemical nematicides currently used to manage Meloidogyne incognita are being phased out because of rising health and environmental issues. This study aimed to evaluate nematicidal effect of various concentrations, viz., 250, 500, 750, 1000ppm of leaf extract of Ipomoea carnea against M. incognita infecting carrot under in vitro and in pots assays. In our result, all tested concentrations displayed J2s mortality and egg hatching inhibition along with improving growth of carrot and reduced J2s population and root-knot index. Molecular docking performed predicts binding interactions of two major compounds, viz., neophytadiene and 2-amino-2-methyl-1-propanol as shown by GC-MS analysis with targeted protein, odorant response gene-1 of M. incognita, to confirm nematicidal action of I. carnea leaf extract. The obtained results also suggested that neophytadiene interacted more and strongly bound with odorant response gene-3 than 2-amino-2-methyl-1-propanol. The biochemical ligand-target protein interaction described in the present work will be helpful in the logical selection of biomolecules and essential proteins. Therefore, plant extract may be used the best alternative to chemical nematicides to control root-knot nematodes and caused longitudinal growth of the plant as well as reduce environmental risks.

KEYWORDS:

• Botanicals
• GC–MS analysis
• in silico study
• plant parasitic nematodes
• molecular docking

Introduction

Root-knot nematode (RKN), Meloidogyne incognita (Kofoid & White) Chitwood, a sedentary endo-parasite (Almutairi et al. 2022), is one of the most dreadful parasite of agricultural crops affecting a large spectrum of cultivated plants globally (Jones et al. 2013). Numerous severe infections can cause the formation of knot-like structure due to the chain-of-beads appearance to roots. Such aberrations completely deform the vascular tissues hampering the translocation of nutrient and water by roots; thereby, resulting in stunted growth, leaf yellowing, wilting and even death of the plant (Anwar et al. 2021), ultimately leading to an annual global setback of $US80 billion (Warnock et al. 2017). A plethora of approaches has been practiced to manage Meloidogyne spp., including organic soil amendments (Choudhary et al. 2022), anaerobic soil disinfestations (Panth et al. 2020), organic additives (Ansari et al. 2020), crop rotation using non-hosts (Xie et al. 2016), sometimes crop rotation could not be used for economical reasons, use of synthetic nematicides (Oka 2020), biological control agents (Patil et al. 2021) and resistant cultivars (Mihajlović et al. 2017). Crop rotation is among the oldest and most important methods for managing RKNs in annual crops, but the development of crop rotation programmes is often constrained by specialised cropping practices, equipment requirements, local climate and market value of the crops (Thomason and Caswell 1987). Chemical nematicides still remain a key element in controlling RKNs (Grabau et al. 2021). However, they pose severe environmental concerns and display detrimental effects on human health; restricting their usage worldwide (Pathak et al. 2022). Therefore, there is an urgent need or necessity to seek out novel and alternative approaches to manage RKNs. As such, plant-based extracts (botanicals) are garnering interest as nematode control agents due to their easy accessibility, low price, pollution-free and environmentally acceptable features (Dutta et al. 2019; Khan et al. 2021). Aqueous extracts of Azadirachta indica (Panpatte et al. 2021), Taxus baccata (Zaidat et al. 2020) and Xanthium strumarium (Akhter and Khan 2018) cause high mortality in M. incognita. More recently, granular or emulsifiable formulations of garlic extract rich in polysulphides have been proven as an effective control of the RKN, M. incognita on tomato and other vegetable crops (Ladurner et al. 2014; Eder et al. 2021).

Presently, molecular docking is an excellent technique for creating and providing details about ligand and receptor communication that are supportive of forecasting the binding course of ligands to their aim protein or DNA (Lee and Kim 2020). The odorant response gene-1 (ODR-1) protein selected for molecular docking and computational molecular simulation was explored for analysis of potentially bioactive compounds, viz., neophytadiene and 2-amino-2-methyl-1-propanol for their nematicidal activity represent the novelty of this study. ODR-1 is a protein found in M. incognita (Kundu et al. 2021) and is a chemosensory gene with a role in host recognition, enabling nematodes to sense volatile compounds and host root exudates (Shivakumara et al. 2019). The molecular docking technique to understand the bioactivity of the compounds is a novel approach to logic-driven selection of natural products and the detection of biopesticidal leads. In a preliminary report, we investigated the nematicidal potential of the aqueous extract of several weeds leaves including I. carnea (Khan et al. 2017). Taking a lead from our previous study, in vitro and in vivo trials were conducted to find out the nematicidal potential of leaf extract of I. carnea for the management of M. incognita on carrot. In addition, we employed gas chromatography-mass spectroscopy (GC–MS) to identify compounds present in the leaf extract of I. carnea. Subsequently, in silico molecular docking was explored to evaluate the binding interaction of two main compounds, namely, neophytadiene and 2-amino-2-methyl-1-propanol with a target protein, ODR-1, a M. incognita protein involved in host recognition. Using plant extract is a safe and cost-effective control option for the management of RKNs under integrated pest management (IPM). As a result, our strategies of using plant extract in agricultural approaches may boost the physiochemical properties of the soil and provide a method for controlling RKNs.

Materials and methods

Chemicals

Methanol (HPLC grade) was obtained from Fisher Scientific (Illkirch, France) for sample preparation.

Collection and extraction of I. carnea

Fresh leaves of I. carnea were collected from Aligarh Muslim University Campus, Aligarh (India), and air dried at ambient temperature for a period of four weeks. The dried leaves were crushed with the help of a mortar and pestle to form a fine powder. About 0.5 g of leaves powder was taken and mixed in 20 mL methanol (HPLC grade). The mixture was vortexed and positioned in an ultrasonicator bath for 15 min, centrifuged at 500 rpm for 15 min. Afterward, the leaf extract was filtered using Whatman filter paper No.1 and was subjected to GC–MS analysis. For hatching and mortality experiments, 1.0 g of dry leaves powder of I. carnea was dissolved in 1000 mL of double distilled water (DDW) and filtered by Whatman filter paper No.1. This mixture called as 1000 ppm solution (stock solution) was used to prepare different concentrations (250, 500 and 750 ppm) by the addition of the requisite quantity of DDW.

GC–MS analysis of leaf extract of I. carnea

GC–MS (Shimadzu QP2010 Plus) outfitted with a capillary column of Rtx-5MS (30 m length, 0.25 mm diameter) was utilised for the identification of bioactive compounds present in methanolic leaf extracts. Using helium as a carrier gas, a sample volume of 250 mL was placed into GC glass vials, and 1 mL was injected into the column at a rate of 1.21 mL per minute. The oven temperature was set at 100°C, then raised to 250°C with a 5°C per minute increase and lastly to 280°C with a 10°C/min increment. Using an MS detector in full scan mode, distinct peak fragmentation patterns of metabolites were observed. The data were examined with GC–MS solutions (Lab Solutions) program, peaks manually incorporated and chromatograms were deconvoluted with the same software. The retention periods were utilised to identify metabolites. Various metabolites were quantified by their particular peak regions and molecular masses were determined. The discovered compounds were validated through comparing peak spectra to reference mass spectra from three library databases (National Institute of Standards and Technology, NIST 14, NIST 14s and Wiley 8). The metabolite chemicals were also compared to internal standards.

Maintenance of inoculum of M. incognita

The inoculum of M. incognita (Kofoid and White, 1919, Chitwood 1949) was maintained in a glasshouse on brinjal plant at the Department of Botany, Aligarh Muslim University, Aligarh (India). We harvested egg masses from M. incognita-infested brinjal roots using sterile forceps. Collected egg masses were carefully washed with DDW before being loaded into sieves with a mesh size of 25 μm aperture sieves and a tissue paper cross-layer placed in a petri dish containing DDW. For the J2s hatch, these Petri dishes were placed in a BOD (biological oxygen demand) incubator (temperature-30 ± 2°C; Period-3 days). The mesh retained egg masses, whereas hatched J2s passed through a sieve and sunk to the bottom of the Petri dish. Freshly emerged J2s were collected in a clean beaker for mortality, hatching and pot bioassays.

Scanning electron microscopy analysis for Meloidogyne species identification

For identifying M. incognita species, SEM analysis was utilised. A matured female M. incognita was isolated from an infested eggplant root, and a perineal pattern was created utilising the procedure provided earlier (Abrantes and Santos 1989). The perineal pattern was coated with 14 nm of gold and SEM images were taken using SEM (JSM 6510 LV Jeol-Japan). The appearance of M. incognita’s perineal pattern was examined in order to identify the Meloidogyne species (Figure 1). The perineal pattern was distinguished by an angularly oblong shape featuring prominent dorsal arches in a characteristic pyriform shape. Striae formed distinct waves that curved towards lateral lines and were not interrupted. Striae appeared straighter and more oval in ventral areas.

Figure 1. Scanning electron microscopy showing the perineal pattern of M. incognita. The high squared dorsal arch, wavy striae are key features of M. incognita.

In vitro analysis

Hatching bioassay

The inhibitory capacity of diverse concentrations (250, 500, 750 and 1000 ppm) of I. carnea leaf extract on J2s hatching of M. incognita was analysed by means of the egg mass dipping method. Fresh egg masses handpicked from galled roots of the brinjal plant were rinsed 3–4 times with DDW. Six freshly picked egg masses were then transferred into Petri dishes having 10 mL of various concentrations of leaf extract as stated earlier. Each Petri dish was covered with parafilm to prevent evaporation and incubated at 28°C. Egg masses dipped in DDW were taken as control. Each experiment has six replicates, excluding control. The work was repeated twice to validate the results. After 4 days, the number of hatched J2s from egg masses was counted in each replicate by means of a binocular microscope, and percent inhibition in J2s hatching was calculated from mean value employing subsequent formula (Almutairi et al. 2022) (1) $\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{a}\mathrm{t}\mathrm{c}\mathrm{h}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left({\displaystyle \frac{C_0-T_{\propto }}{C_0}}\right)\times 100$(1) where

C₀ = number of hatched J2s in DDW (control)

T_α = number of hatched J2s in each concentration of I. carnea leaf extract

Mortality bioassay

To evaluate the anti-nematode impact of various concentrations (250, 500, 750 and 1000 ppm) of I. carnea leaf extract on J2s of M. incognita, 1 mL of nematode suspension (containing 70 J2s) was added to 9 mL of the leaf extract of above-mentioned concentrations in Petri dishes. Petri dishes containing DDW were designated as control. To minimise drying, Petri dishes were suitably covered with parafilm and then incubated at 28°C for a specified period. After 12, 24, 48 and 72 h of exposure, the number of live and dead J2s was counted using a binocular microscope. The second stage juveniles exhibiting any mobility or seen as having snaky shape were regarded as alive (El-Rokiek and El-Nagdi 2011), whereas J2s with no movement as well as straight body shape and appearance were considered dead (Aissani et al. 2015). The test was performed with six replicates of each concentration and LC50 values were obtained (Behreus and Karbeur 1953). The work was repeated twice to validate the results. The second stage juvenile’s per cent mortality was estimated according to the formula given below: (2) $\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{m}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{J}2\mathrm{s}=\left({\displaystyle \frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{J}2\mathrm{s}-\mathrm{D}\mathrm{e}\mathrm{a}\mathrm{d}\mathrm{J}2\mathrm{s}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{J}2\mathrm{s}}}\right)\times 100$(2)

Infectivity bioassay and experimental design (Pot study)

The pots designed investigation was carried out in a glasshouse to ascertain the effectiveness of I. carnea leaf extract against M. incognita. Carrot cv. Gourav seeds were obtained from the local market. Carrot seeds were first sanitised by shaking with 0.02% mercuric chloride for 5 min before immediately washing 2–3 times under running tap water. Clay pots (15 cm in diameter) were filled with 1 kg of autoclaved mixed soil of loam with farmyard manure in a 3:1 ratio. In each pot, five sterilised carrot seeds were sown. After germination, undesirable seedlings were carefully removed, leaving the healthiest one in each pot. Around 2–3 holes were drilled near the seedling’s root, and 2500 J2s were injected into the soil. After two days of J2s inoculation, 10 mL of various concentrations (250, 500, 750, and 1000 ppm) of I. carnea leaf extract were pipette-added around the seedling’s root. Throughout the experiment, plants received adequate watering and irrigation, and the study was meticulously tracked to weed out flaws. The bioassay was performed with six replicates of each treatment. The work was repeated twice to validate the results. The treatments include (a) control (no leaf extract of I. carnea and J2s); (b) untreated inoculated control (J2s only); (c) ten millilitre of 250 ppm leaf extract of I. carnea + 2500 freshly hatched J2s; (d) ten millilitre of 500 ppm leaf extract of I. carnea + 2500 freshly hatched J2s and (e) ten millilitre of 750 ppm leaf extract of I. carnea.

Estimation of growth and physiological attributes

After 90 days of sowing, plants reached maturity; they were picked, and cleaned under running water to remove clingy soil particles and the physiological and growth characteristics were determined. Photosynthetic pigments, viz., chlorophyll content (mg/g) and carotenoid content (mg/g) were calculated using the procedure given by Mackinney (1941) and MacLachlan and Zalik (1963), respectively.

Determination of pathological parameters

Nematode population in soil

A garden trowel was used to collect soil samples from each treated pot at the end of the experiment, which was then thoroughly mixed. A total of 200 g of well-mixed soil from each treatment was then weighed in order to estimate the nematode population. Cobb’s sifting and decanting procedure (Cobb 1918), followed by a modified Baermann's funnel technique, were used to evaluate the J2s population (Southey 1986) and a stereoscope microscope (Motic SMZ 168 series) was used for counting J2s.

Root-knot index (RKI)

Taylor and Sasser’s 0–5 scale was used to calculate the RKI (1978), where 0 means no galls, 1 means 1–2 galls, 2 means 3–10 galls, 3 means 11–30 galls, 4 means 31–100 galls and 5 means more than 100 galls.

Molecular docking

Ligand preparation

The 3-D structure of the concerned compound was acquired from the PubMed database (https://pubmed.ncbi.nlm.nih.gov/) in SDF format with their respective CID. The ligand energy was reduced using a conjugate gradient optimisation approach and MM2 force field.

Receptor preparation

The protein sequences were retrieved from NCBI and UNIPORT. The sequences were searched for their molecular and biological functions and were annotated using BLAST servers (http://blast.ncbi.nlm.nih.gov and https://parasite.wormbase.org//Tools/Blast). The template for modelling was obtained from the PDB database. The homology modelling of ODR-1 was accomplished by Modeller version 9.24. The protein was prepared by removing the natural inhibitor and other heteroatoms, including water molecules. After that the protein was stored in a PDB format. The protein was assigned polar hydrogen atoms using the Auto Dock Tool (Trott and Olson 2010), Kollman charges were calculated and partial charges were also allocated.

Molecular docking

The prepared files of ligand and receptor were saved in PDBQT format. The docking of all concerned ligands into binding pocket of ODR-1 was accomplished using Auto Dock Vina software (Trott and Olson 2010) on Intel (R) Core^TM i5-4200U CPU-2.10 GHz, 64-bit processor. This computational tool is comprised of a program for estimating the conformation of ligands as well as their orientation with respect to the active site of the receptor. The grid box parametric dimension values were adjusted as X = 52.84, Y = 31.608 and Z = 91.047 (size; X = 50, Y = 50 and Z = 50) for ODR-1. To ensure that the protein–ligand combination was in an effective binding shape, the exhaustiveness parameter was set to 8. Following docking, Auto Dock Vina created a docked complex for each ligand with a variety of conformations and affinity scores (in kcal/mol), and it ranked them according to theory of docked complexes with the lowest binding energy (kcal/mol) (whereby more negative values meant greater binding affinity). Discovery Studio (DS) version 4.1 Client assessed and graphically represented the top protein–ligand complex docked poses.

Statistical analysis

Data were statistically analysed using R (version 2.14.1) with the Duncan Multiple Range Test (DMRT) and the standard error (±SE) of the mean was calculated. ANOVA and LC₅₀ value of all treatments was calculated using OPSTAT (Sheoran et al. 1998).

Results

GC–MS analysis

Firstly, we carried out GC–MS analysis of the leaf extract of I. carnea and identified 16 compounds (Figure 2). Table 1 depicts the list of compounds along with their molecular weight, molecular formula, retention time, area percent and area. The main compounds identified were neophytadiene, stigmast-5-en-3-ol, 2-amino-2-methyl-1-propanol, phytol, silane and diethyldi (3-phenylpropoxy). We speculated that all the characterised substances could be harmful to M. incognita to a varying extent, either alone or in combination. Based on the study, some of the constituents were shown by GC–MS as biologically active compounds. They were proven to possess nematicidal properties, which may contribute to managing RKNs for sustainable agriculture. GC–MS is a combined analytical technique to determine and identify compounds in a liquid sample. GC–MS plays an essential role in the active compound analysis and chemotaxonomic study of organic materials containing biologically active components. Taking this into account, we investigated the nematicidal potential of I. carnea leaf extract against M. incognita.

Figure 2. GC–MS chromatograms of methanolic leaf extract of I. carnea. Values given in parentheses represent the retention time of each compound; Values given without parentheses represent the peak number of each compound.

Table 1. Catalog of identified compounds present in methanolic leaf extract of I. carnea determined by GC–MS analysis.

Peak Number	Retention time	Area	Area %	Name of compound	Molecular weight	Molecular formula
1	4.62	1,279,396	3.17	Silane, diethyldi(3-phenylpropoxy)-	356.57	C22H32O2Si
2	7.88	42,439	1.05	1-Methyl-1-n-pentyloxy-1-silacyclobutane	172.34	C9H20OSi
3	8.11	541,090	1.34	Adipic acid, octyl trans-hex-3-enyl ester	340.49	C20H36O4
4	8.80	261,571	0.65	Fumaric acid, cis-hex-3-enyl eicosyl ester	478.74	C30H54O4
5	8.94	363,658	0.90	1-Isopropyl-4,7-dimethyl-1,2,3,5,6,8a-hexahydronaphthalen	204.35	C15H24
6	9.29	5,183,170	12.84	2-Amino-2-methyl-1-propanol	89.14	C4H11NO
7	12.46	11,819,240	29.29	Neophytadiene	278.5	C20H38
8	13.43	150,604	0.37	Pentadecanoic acid	242.40	C15H30O2
9	14.41	421,869	1.05	Cyclohexane	84.16	C6H12
10	15.18	315,486	0.78	1-Dodecanol	186.33	C12H26O
11	16.09	979,940	2.43	Phytol	296.50	C20H40O
12	23.41	183,244	0.45	Squalene	410.70	C30H50
13	24.11	218,194	0.54	5-7A-Isopropenyl-4,5-Dimethyl-Octahydro-In	290.50	C20H34O
14	24.41	299,593	0.74	Stigmasta-5,22-dien-3-ol, acetate	454.70	C31H50O2
15	25.08	824,823	2.04	Stigmast-5-En-3-Ol, (3Beta)	414.70	C29H50O
16	26.77	167,236	0.41	Stigmast-5-En-3-Ol, Oleate	679.20	C47H82O2

I. carnea leaf extract inhibit J2s hatching

The suppression of M. incognita J2s hatching in various concentrations (250, 500, 750 and 1000 ppm) of I. carnea leaf extract revealed noteworthy differences (Figure 3). All concentrations were effective in reducing J2s hatching in comparison with the control (water). On raising the concentration from 250 to 1000 ppm, a considerable upsurge in the inhibition of J2s hatching was detected. After 4 days of exposure, the highest percentage of J2s’ hatching inhibition (80.83%) was seen at 1000 ppm, followed by 750 ppm (73.10%) and 500 ppm (64.14%), while 250 ppm exhibited the least hatching inhibition (53.65%). The individual inhibitory effect of various concentrations (250, 500, 750 and 1000 ppm) of I. carnea leaf extract on J2s hatching after 4 days of the incubation period is given in Figure 3.

Figure 3. Effect of several concentrations of the leaf extract of I. carnea on J2s hatching of M. incognita over 4 days of incubation period under in vitro. Each value is an average of six replicates. Each bar followed by the same letter is not significantly different according to Duncan’s multiple-range test (p ≤ 0.05). [DW – distilled water; (Control); J2s – second stage juveniles; ppm – parts per million].

Increasing concentration of I. carnea leaf extract affect J2s mortality

A direct contact approach was used to estimate the mortality of J2s. At every tested concentration, the extract was found harmful to M. incognita J2s to some extent, exhibiting J2s mortality ranging from 45% to 88% at 72 h of the exposure period (Table 2). The extract used at 1000 ppm was proven to be extremely lethal against J2s after 72 h of incubation. Typically, the mortality of J2s increased with an increment in the incubation period as well as concentration. The lowest LC50 (lethal concentration caused by 50% mortality at 95% confidence limits) value was obtained at 1000 ppm after 72 h of incubation (Table 3). LC50 value of treatment decreases with incubation time; suggesting treatment with high LC50 values was less toxic to J2s of M. incognita.

Table 2. Effect of various concentrations of the leaf extract of I. carnea on mortality of J2s of M. incognita over 12, 24, 48 and 72 h of exposure period under in vitro.

Treatment	Concentration (ppm)	Number of dead J2s (Mean ± SE) at different time interval (h)
		12	24	48	72
I. carnea	250	12^c ± 1.52 (17.14%)	18^d ± 1.73 (25.71%)	25^d ± 1.73 (35.71%)	32^d ± 2.09 (45.71%)
	500	17^b ± 1.52 (24.28%)	26^c ± 1 (37.14%)	34^c ± 1.73 (48.57%)	44^c ± 2.08 (62.85%)
	750	21^b ± 1.73 (32.00%)	33^b ± 1.52 (47.14%)	42^b ± 2.08 (60.00%)	53^b ± 2.31 (75.71%)
	1000	28^a ± 1.52 (40.00%)	40^a ± 2.08 (57.14%)	52^a ± 1.73 (74.28%)	62^a ± 2.30 (88.57%)
	DW	0^d ± 0 (0.00%)	0^e ± 0 (0.00%)	0^e ± 0 (0.00%)	0^e ± 0 (0.00%)
Degrees of freedom		3	3	3	3
Sum of Squares		411	825.25	1190.25	1478.25
Mean Squares		137	266.75	396.75	492.75
F-Calculated		18.26	33.34	39.67	33.98
Significance		0.0006	0.00007	0.00004	0.00007

Each value is an average of six replicates. DW = double water (control). SE = standard error. J2s = second stage juveniles. Values given in parentheses represent the per cent J2 mortality over control. Values given without parentheses represent the number of dead J2s of M. incognita in different concentrations. Values followed by same letter within a column are not significantly different according to Duncan’s multiple-range test (p ≤ 0.05).

Table 3. Nematicidal activity of leaf extract of I. carnea against J2s of M. incognita.

Treatment	Exposure Period (hours)	LC50^1 value in mg/L (95% CL^2)
I. carnea	12	3,411.65
	24	1,068.72
	48	522.83
	72	304.32

¹ LC₅₀: lethal concentration caused by 50% mortality after 8, 16 and 24 h at 95% confidence limits.

² CL: confidence limit.

Effect of I. carnea leaf extract on growth parameters and pigment content

Under pots conditions, leaf extract of I. carnea exhibited substantial progress in growth metrics. All concentrations were successful in promoting plant development in contrast to nematode-infected plants (J2s only). When treated with 1000 ppm of the leaf extract, the highest plant length and fresh weight (p ≤ 0.05) were recorded, followed by 750, 500, 250 ppm (Figure 4). Similarly, at 1000 ppm, substantial gains in chlorophyll and carotenoid content were found (Figure 5). Other concentrations such as 250, 500, and 750 ppm also increased chlorophyll and carotenoid content relative to pot infected with J2s only. Plants inoculated with J2s only displayed a maximum decrease in chlorophyll and carotenoid content.

Figure 4. Nematicidal effect of different concentrations of (250, 500, 750 and 1000 ppm) of leaf extract of I. carnea on the growth attributes of J2s inoculated carrot plants under pot condition. Each bar followed by same letter is not significantly different according to Duncan’s multiple-range test (p ≤ 0.05). (J2s only – second stage juveniles only; ppm – parts per million).

Figure 5. Nematicidal effect of different concentrations of (250, 500, 750 and 1000 ppm) of leaf extract of I. carnea on the physiological attributes of J2s inoculated carrot plants under pot condition. Each bar followed by the same letter is not significantly different according to Duncan’s multiple-range test (p ≤ 0.05). (J2s only – second stage juveniles only; ppm – parts per million).

I. carnea leaf extract reduces nematode population in soil

As shown in Figure 6, different concentrations of I. carnea leaf extract display significant differences with regard to J2s density and RKI. On increasing concentration of the extract, a substantial decline in J2s population in soil as well as RKI was observed. The highest reduction in J2s population as well as the lowest RKI was observed at a concentration of 1000 ppm, followed by 750, 500 and 250 ppm when contrasted with nematode-infected plants. The results of principal component analysis (PCA) revealed further that the frequency of RKNs in pots and root galls/plant was profoundly linked to key attributes of carrots. Scatter biplot analysis revealed the efficacy of different dosages of I. carnea leaf extract in diminishing M. incognita infection and improving carrot growth parameters (Figure 7).

Figure 6. Nematicidal effect of different concentrations (250, 500, 750 and 1000 ppm) of leaf extract of I. carnea on the pathological attributes of J2s inoculated carrot plants under pot condition. Each bar followed by the same letter is not significantly different according to Duncan’s multiple-range test (p ≤ 0.05). (J2s only – second stage juveniles only; ppm – parts per million).

Figure 7. The biplots of principal component analysis, comparing the effects of different concentrations (250, 500, 750 and 1000 ppm) of leaf extract of I. carnea on various studied parameters of J2s inoculated carrot plants (PFW = plant fresh weight; PL = plant length; CHL = chlorophyll content; CRT = carotenoid content; NP/200 g = nematode population /200 g soil; RKI = root-knot index).

Molecular docking analysis

Docking was carried out using Auto Dock Vina 4.2 to investigate the precise binding mechanism and binding location of 2-amino-2-methyl-1-propanol and neophytadiene with ODR-1. By evaluating the binding free energy (BFE), we were able to forecast ligand docking positions with receptors and sort them by least binding energies. The best docked poses concerned receptor–ligand complexes with the lowest binding energies have been depicted in Figure 8 (2-amino-2-methyl-1-propanol-ODR-1 complex) and Figure 9 (neophytadiene-ODR-1 complex). Table 4 displays the different interaction forces of interactions between involved ligands and proteins. The values of BFE are negative for all concerned systems (2-amino-2-methyl-1-propanol -ODR-1: −4.5 kcal/mol and neophytadiene -ODR-1: −5.7 kcal/mol), signifying the interaction mechanisms are spontaneous in nature. Finally, obtained results also suggested that neophytadiene interacted more strongly with the ODR-1 than 2-amino-2-methyl-1-propanol. On other hand, it can be said that ODR-1 behaves as a good receptor for both concerned ligands based on BFE. It was shown that –NH₂ group of 2-amino-2-methyl-1-propanol binds with the –COOH group of the Asp-405 residue of ODR-1 via hydrogen bond (Figure 8), while a majority of residues of ODR-1 interact with ligand through Van Der Waals interaction. Neophytadiene does not form any hydrogen bond with receptor ODR-1 (Figure 9). The several residues, such as Lys-208, Thr-212, Gln-216, Ala-219, Ser-221, Leu-231, Val-290, Glu-291, Gly-293, Thr-294 and Thr-352 of ODR-1 receptor, interact via van der forces with the neophytadiene ligand. These ligands share the majority of residues of both receptors, indicating that they both bind to functionally active residues of both receptors.

Figure 8. Representation of (a) docked 2-amino-2-methyl-1-propanol into ODR-1 at active sites, (inset) representation of an enlarged view of involved molecular interactions and (b) 2-D representation of involved molecular interactions.

Figure 9. Representation of (a) docked neophytadiene into ODR-1 at active sites, (inset) representation of an enlarged view of involved molecular interactions and (b) 2-D representation of involved molecular interactions.

Table 4. The receptor–ligand interactions result by Discovery.

Complex	Interactions
	Hydrogen bonding	Others
2-Amino-2-methyl-1-propanol + ODR-1	Asp-405	Gly-334, Met-335, Leu-336, Ile-375, Thr-383, Val-383, Ala-384, Asn-387, Gly-400, Gly-404, Tyr-407, Ser-408
Neophytadiene + ODR-1	-	Lys-208, Lys-210, Thr-212, Glu-216, Ala-219, Ser-221, Leu-231, His-287, Leu-289, Val-290, Glu-291, Gly-293, Thr-294, Leu-342, Thr-352, Asn-353

Discussion

During in vitro study, all tested concentrations, viz., 250, 500, 750, 1000 ppm of leaf extract of I. carnea were effectively inhibited J2s hatching and caused J2s mortality of M. incognita. Concentration of 1000 ppm was found to be highly effective in reducing J2s hatching and showed highest toxicity towards J2s of M. incognita, followed by 750, 500, 250 ppm (Figure 3; Table 2). However, contrary findings were also reported by Nikure and Lanjewar (1981). They found in their study that treatment with aqueous flower extracts at 5% or higher of I. carnea did not show significant nematicidal properties against second stage juveniles of M. incognita. Elbadri et al. (2008) reported that leaf extract of Calotropis procera is not much effective against M. incognita. A direct correlation between concentrations of extract as well as the duration of exposure with anti-nematode action of I. carnea leaf extract was observed. This nematicidal action of all tested concentrations might be due to chemical constituents dissolved in the leaf extract which act as a natural nematicide. The mechanisms of plant extracts are not well understood, but secondary metabolites in the leaf extract act as antagonistic to J2s and eggmasses of M. incognita, present as alkaloids, flavonoids, phenolics and saponins in botanical extracts (Chitwood 2002; Mousa et al. 2011). Applied leaf extract of I. carnea after 72 h exhibited a minimum LC50 value than the other exposure period like 12, 24 and 48 h (Table 3). From our study, it was observed that the lower LC50 value of a treatment, the greater its toxicity to J2s of M. incognita and treatment with a high LC50 value, least toxic to J2s (Table 3). Our results were similar to the finding of Khalil and Badawy (2012). They reported that chitosan possesses significantly prominent nematicidal activity with LC50 of 283.47 and 124.90 mg/l after 24 and 48 h, respectively.

Similarly, in the pot study, all tested concentrations, viz., 250, 500, 750, 1000 ppm of leaf extract of I. carnea significantly enhanced growth attributes of carrot and decreased disease severity caused by M. incognita to a varying degree. The 1000 ppm caused the highest reduction in root galling and nematode population (Figure 6) and also improves the growth and physiological attributes of carrots (Figures 4 and 5). However, contrary findings were also reported by Siddiqui and Husain (1990), who found that soil application of leaf extracts (5, 10 and 20 ml) of Cymbopogon citrates, Eichhornia crassipes and I. carnea had not significantly improved plant growth of chickpea. Patel et al. (1993) reported that water hyacinth and congress grass were moderately effective in reducing RKN population in tomatoes. The beneficial role of botanical application by soil treatment has contributed to improving root growth environment and soil nutrients. Furthermore, the addition of botanicals in the soil might promote nematode tolerance in crops and trigger the host defense system (Xu et al. 2019). Recent research has focused on plant-derived such as plant extracts in order to develop a safe alternative to conventionally utilised chemical nematicides for RKNs management (Marron 2019). Numerous plant-based materials, such as oil seed cake, dried powder of plant component, crude plant extract, are now being utilised for sustainable management of RKNs (Khan et al. 2017; Dutta et al. 2021). A diverse range of phytochemicals with anti-nematode activities against a variety of pests (Atolani and Fabiyi 2020) including Meloidogyne sp. (Lu et al. 2020) has been extensively documented. According to the literature, I. carnea contains substantial amounts of phytochemicals (Kar et al. 2018) that could contribute to its nematocidal action. Our data established that neophytadiene was more successful in inhibiting ODR-1 than 2-amino-2-methyl-1-propanol as exemplified by its high binding affinity (Figure 9). Previous ligand binding investigations revealed that plant-based molecules had a multi-modal antagonistic impact on several target proteins of M. incognita, including the ODR-1 gene (Keerthiraj et al. 2021). This study indicated that leaf extract of I. carnea is a potential source of natural nematicides, in addition to traditional uses of this plant. These new products should represent an additional response to farmers’ demand for effective and environment-safe nematode management tools in both organic and conventional cropping systems. The present investigation was also focused on the identification of various bioactive compounds in leaf extract of I. carnea by GC–MS analysis. The use of plant parts based products would be beneficial to the environment and serve as a more sustainable approach towards RKNs management. The present study employs analytical and molecular modelling tool to relate the nematicidal activity of two major compounds, namely neophytadiene and 2-amino-2-methyl-1-propanol with targeted protein ODR-1 of M. incognita. A molecular docking-based understanding of the bioactivity of pytocompounds is a novel attempt towards logic-driven selection of natural materials and the discovery of biopesticidal leads.

Acknowledgments

The authors are thankful to the Deanship of Scientific Research at Najran University for funding this work under the Research Groups Funding program grant code (NU/RG/SERC/11/19).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All the data is present in this manuscript.

Additional information

Notes on contributors

M. M. Abdullah

M. M. Abdullah, is a well-known academician and a young research scientist. He is currently an Associate Professor in the Department of Physics at Najran University in Saudi Arabia. In addition to editing a book and writing two book chapters, Dr. Abdullah has published more than 45 research articles. His scientific interests include the synthesis of materials and their applications to environmental sustainability, Sensors, and Energy in particular. His research findings were published in various peer-reviewed journals of international repute.

Amir Khan

Amir Khan, completed his Ph.D. in Botany with a specialization in Plant Pathology and Nematology from the Department of Botany, Aligarh Muslim University, Aligarh. He has done his B.Sc. and M.Sc. from the AMU, Aligarh, in 2012 and 2014, respectively. Besides, the thrust area of Dr. Amir and his group is to promote organic farming by utilizing organic matter, including plant parts, oil cakes, agricultural waste, etc., as well as bio-fertilizer and biocontrol agents for the sustainable management of nematodes. He is a Life Member of the Indian Phyto-pathological Society, New Delhi (India), and an Editor of the Journal of Ecology & Natural Resources.

Hasan B. Albargi

Hasan B. Albargi, is an Associate Professor of Solid State Physics in the Department of Physics, Faculty of Science and Arts at Najran University, Kingdom of Saudi Arabia. His research interests include the structural, electronic, optical and magnetic properties of advanced materials and the development of nanomaterials for various purposes, such as gas sensing and electronic devices applications. Dr. Hasan has published more than 40 research articles in different ISI journals.

Mohammad Zaki Ahmad

Mohammad Zaki Ahmad, is a well-known academician and a young research scientist. He was a university gold medalist for standing first in Master. He has been working in academics for more than 1 decade. Presently, he is working as a lecturer in the Department of Pharmaceutics, School of Pharmacy, Najran University, Najran, Saudi Arabia. Zaki has more than 100 publications, about 40 review articles, 15 books chapter to his credit. He has delivered 5 guest lectures. He has teaching experience at U.G. and P.G. level. His research interests are development and evaluation of various novel drug delivery systems, polyherbal drug formulations, cosmeceuticals, nutraceuticals, etc. At present, is intensively involved in the development of nano-sized drug delivery systems. Zaki is a member of various professional bodies such as IPA and IPGA and has published his research findings in various peer-reviewed journals of international repute.

Javed Ahmad

Javed Ahmad, is an Associate professor at College of Pharmacy, Najran University, Saudi Arabia. He obtained his Doctorate degree in pharmaceutical sciences from Jamia Hamdard, New Delhi (India). He is a recipient of CSIR-Senior Research Fellowship and International Travel Award from Department of Science & Technology, Govt. of India during his Ph.D. After his Ph.D, he joined NIPER Raebareli, an autonomous institute of Department of Pharmaceuticals, Govt. of India as a faculty position. He has published more than 100 high quality research and review articles in peer-reviewed journals of international repute. He also published various book chapters (>35) for edited book and book series. His current h-index is 32 with total number of citations of his publications near to 3000. He is reviewer of many peer-reviewed journals of International repute. He received many awards for his scientific findings and reviewing tasks. Recently, he enlisted among World's Top 2% Scientists for the year 2022 in the field of Pharmacology & Pharmacy, a list created by Stanford University, USA. His current research interest lies in design and characterization of nanomaterial to improve the efficacy of pharmaceuticals/phytopharmaceuticals.

Faheem Ahmad

Faheem Ahmad, Assistant Professor of Plant Pathology & Nematology in the Department of Botany at Aligarh Muslim University, India. Author of over 40 peer-reviewed publications. His research interests include plant-nematode interaction and nematode management based on nematicidal bioagents and plant natural product repertoire.

Mohammad Shabib Akhtar

Mohammad Shabib Akhtar, is a well-known academician and a young research scientist. Presently, he is working as a lecturer in the Department of Clinical Pharmacy, College of Pharmacy, Najran University, Saudi Arabia. From 2007 to 2009 he was the Senior research officer in Department of Neurology, All India Institute of Medical Science (AIIMS) New Delhi. He has published various high-quality research and review articles in peer-reviewed journals of international repute. He is a reviewer of many peer-reviewed journals of Clinical and Pharmaceutical Sciences. His current research interest lies in pharmacovigilance, Pharmacoeconomics and rationale use of antibiotics.

Nehal Mohsin

Nehal Mohsin, is a well-known academician and a young research scientist. He is working as a lecturer in the Department of Clinical pharmacy, College of Pharmacy, Najran University, Saudi Arabia. He has published various high-quality research and review articles in peer-reviewed journals of international repute. In addition, he contributed a book chapter in an edited book. His key researchare as are drug utilization, pharmacotherapy, clinical pharmacy, and nanomedicines related to various diseases.

Fuzail Ahmad

Fuzail Ahmad, is an accomplished academic and researcher currently serving as the Associate Professor and Assistant Dean of Academic and Research Excellence at the College of Applied Sciences, Almaarefa University in Diriya, Riyadh, Saudi Arabia. With a background in his area of expertise, Dr. Ahmad has made significant contributions to the field through his research, publications, and academic work. He is widely recognized for his research interests, which include but are not limited to, movement science, motor control & learning, neurological rehabilitation, and kinesiology. Dr. Ahmad's work has been influential in enhancing the understanding of these complex areas, and he continues to be an influential figure in the academic and research communities.

Mohammad Azhar Kamal

Mohammad Azhar Kamal, Assistant Professor at Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia and involves in teaching bachelor students. He graduated in Biochemistry from Bangalore University, India and his PhD from University of Brescia, Italy and Postdoctoral fellowship from Humanitas Clinical and Research Hospital, Milan. Later he joined as Assistant Professor at University of Jeddah where he was involved in teaching and he has been Visiting Research Scientist in Vaccine and Immunotherapy Unit at KFMRC, King Abdulaziz University, and T Cell Immunology, Infectious diseases, and diagnostic biomarkers from reputed international laboratory from Germany, Italy and India. He has published in highly prestigious peer reviewed international journals such as Cell, PLOS Pathogen, Blood, Oncoimmunology, Scientific Reports, BioMed Research International, Future Virology, Bioinformation etc. He has experience in research techniques including animal cell culture, animal studies, FACS, immunohistochemistry, MTT assay, ELISA, RT-PCR, Western Future Virology, Bioinformation etc. He has experience in research techniques including animal cell culture, animal studies, FACS, immunohistochemistry, MTT assay, ELISA, RT-PCR, Western blotting and basic molecular biology. His current research focuses on Host-Pathogen interaction, infectious diseases, and cancer immunology.

Yaser E. Alqurashi

Yaser E. Alqurashi, is a faculty member at Biology Department, College of Science, Majmaah University. His Current research interests include Cell Cancer and Molecular.

Hira Lal

Hira Lal, received his Doctorate degree in Chemistry from Aligrah Muslim University, Aligarh (U.P.), India in 2021. He completed his M.Sc. and B.Sc. (Hons.) in Chemistry from the same university. He qualified the Graduate Aptitude Test in Engineering (GATE) in 2015. His research work includes surfactant chemistry, protein binding and computational chemistry. He has published more than ten papers in international journals of repute.

Jari S. Algethami

Jari S. Algethami, is presently working as an Assistant Professor in the Department of Chemistry, College of Science and Arts, Najran University, Najran, Kingdom of Saudi Arabia. He obtained his MSc and PhD degree in Analytical Chemistry from the University of Hull, UK, in 2011 and 2017, respectively. He also teaches courses in general and environmental chemistry, quantitative chemistry, and instrumental analysis. His research focuses on environmental pollutants analysis and water treatment and purification. He has successfully run some research projects funded by the Deanship of Scientific Research at Najran University about the development of different types of nanocomposite materials for water decontamination. Currently, he is engaged in research to develop sustainable environmental remediation methods for pollution prevention and control.