An Insight into Occurrence, Biology, and Pathogenesis of Rice Root-Knot Nematode Meloidogyne graminicola

Simple Summary Rice root-knot nematode is a plant-parasitic nematode that infects the roots of rice plants. It is also known as Meloidogyne graminicola, and is one of the most damaging pests of rice crops worldwide. The nematode infects the roots of rice plants and causes the formation of characteristic knots or galls. Both morphological and molecular characterization can be used in combination to provide a more complete understanding of nematodes. Molecular analysis can be used to identify new nematode species, while morphological analysis can be used to describe their physical features and provide a more complete picture of their biology and ecology. The current study aimed at utilizing both morphological and molecular characterization, and life stage as well, as molecular aspects of interaction between the rice root-knot nematode Meloidogyne graminicola and its host plants. Abstract Rice (Oryza sativa L.) is one of the most widely grown crops in the world, and is a staple food for more than half of the global total population. Root-knot nematodes (RKNs), Meloidogyne spp., and especially M. graminicola, seem to be significant rice pests, which makes them the most economically important plant-parasitic nematode in this crop. RKNs develop a feeding site in galls by causing host cells to differentiate into hypertrophied, multinucleate, metabolically active cells known as giant cells. This grazing framework gives the nematode a constant food source, permitting it to develop into a fecund female and complete its life cycle inside the host root. M. graminicola effector proteins involved in nematode parasitism, including pioneer genes, were functionally characterized in earlier studies. Molecular modelling and docking studies were performed on Meloidogyne graminicola protein targets, such as β-1,4-endoglucanase, pectate lyase, phospholipase B-like protein, and G protein-coupled receptor kinase, to understand the binding affinity of Beta-D-Galacturonic Acid, 2,6,10,15,19,23-hexamethyltetracosane, (2S)-2-amino-3-phenylpropanoic acid, and 4-O-Beta-D-Galactopyranosyl-Alpha-D-Glucopyranose against ligand molecules of rice. This study discovered important molecular aspects of plant–nematode interaction and candidate effector proteins that were regulated by M. graminicola-infected rice plants. To the best of our knowledge, this is the first study to describe M. graminicola’s molecular adaptation to host parasitism.


Introduction
Rice (Oryza sativa L.) is among India's foremost staple food crops, providing calories to over sixty percent of the country's population, and influencing the livelihoods and economics of several billion people, primarily in Asia, Latin America, the Middle East, and the West Indies. Rice has influenced Asian societies and cultures for ages. Many Asian societies have a relationship with rice that goes beyond the fulfilment of fundamental

Survey and Sampling
A routine investigation for the existence of root-knot nematode infestations was made in rice fields in Tamil Nadu, India throughout 2021 and 2022 (Table 1). Based on the crops' aboveground abnormalities, such as wilting, stunted growth, yellowing of the leaves, and root galls, nematode-infested fields were detected (Figure 1). At a depth of 10-15 cm, midseason soil and root samples were taken from the paddy rhizospheres.
Five plants were randomly chosen from each locality, and three samples were taken from each plant. The soil samples were carefully combined, and a 200 cc composite sample was taken in polythene bags and labelled appropriately for examination. Additionally, galled or infested roots were gathered ( Figure 2, Table 2). The samples were transported to the lab for nematode extraction while being maintained in plastic bags in an ice box. leaves, and root galls, nematode-infested fields were detected (Figure 1). At a depth of 10-15 cm, midseason soil and root samples were taken from the paddy rhizospheres.

Survey and Sampling
A routine investigation for the existence of root-knot nematode infestations was made in rice fields in Tamil Nadu, India throughout 2021 and 2022 (Table 1). Based on the  (Table 1)).

Morphological Characterization of Meloidogyne spp. Infesting Rice
Nematodes were extracted by using Cobb's sieving and decanting method with a modified Baermann's funnel method. Then, they were killed in hot formalin at 65 • C and fixed in a formalin:acetic acid fixative (FA 4:1) [17]. The nematode specimens were dehydrated through the rapid glycerin (Seinhorst's) method and mounted in pure glycerin on glass slides supported by glass rods of a diameter slightly larger than that of the nematodes [17]. Adult females were extracted, cleaned of dirt from tip-galled roots, and thoroughly separated from galls, from which posterior cuticular patterns (PCPs) were acquired for species diagnosis. A Nikon eclipse Ti2-U inverted microscope was used for obtaining microphotographs of posterior cuticular patterns. The morphometrics were analyzed by using NIS-Elements Denoise.ai software. Galled roots were also diagnosed for the presence of any egg mass. The rice root-knot nematode was examined using the criteria of Ye and Hunt [18] for species diagnosis.  [19]. In the PCR investigations, three sets of primers (synthesised by Eurofins, Bangalore, India) were employed to amplify the ITS D2-D3 expansion portions of 28S rRNA, as well as the coxII region. ITS-F (5 -GTT TCC GTA GGT GGT GAA CCT GC-3 ) and IT-S R (5 -ATA TGC TTA AGT TCA GCG GGT-3 ) primers were used to amplify the ITS [20]. The forward D2A (5 -ACA AGT ACC GTG AGG GAA AGT TG-3 ) and reverse D3B (5 -TCG GAA GGA ACC TAC TA-3 ) primers were used for amplification of the D2-D3 28S rRNA gene [21]. The mitochondrial region between the partial coxII and the partial 16S was amplified using the forward primer COI-F (5 -TTT TTT GGG CAT CCT GAG-3 ) and the reverse primer COI-R (5 -AGC ACC TAA CTT AAA C-3 ) [22]. The PCR conditions were followed as specified by Ye and Hunt [18].
All of the PCR amplifications were performed separately in 25 µL of reaction volumes containing 2.0 µL of DNA, 1.0 µL of each 10 µM primer (forward and reverse), 2.5 µL of 10X buffer, 1.5 µL of 200 mM of each dNTP, and 2 units of Taq polymerase enzyme, and the final volume was prepared with approximately 25 µL MilliQ nuclease-free water. Amplified PCR products were sequenced by the Sanger dideoxy method (Eurofins, Bengaluru, India). The derived sequences were blasted with NCBI BLASTN for phylogenetic analysis. The phylogenetic tree was constructed by using MEGA7 software. An appropriate model, as established by MEGA7, was utilized to generate the evolutionary origins using maximum likelihood analysis. One thousand replications were used to construct the bootstrap consensus tree. Branches associated with partitioning that were only replicated in 70% or fewer bootstrap replicates were collapsed. Initial trees for the heuristic search were automatically generated using the neighbor-joining and BioNJ algorithms on a pairwise distance matrix calculated using the JTT model, and the topology with the highest log likelihood value. Regions with gaps and incomplete data were removed.

Life Cycle Analysis
Under glasshouse conditions, the nematode culture was kept in potted plants of the rice variety TN 1. The aforementioned kind of seed was immersed in tap water for 24 to 48 h. The sprouting seeds were planted on 18 October 2022, in soil that was taken from a paddy field that had been infected with M. graminicola, and contained one second-stage juvenile (J2) per gram of soil. On 23 October 2022, a nutrient solution containing N, P, K, and Zn was added. Three plants were involved, and they were periodically uprooted so that life cycle research could be conducted. Under a stereo binocular microscope, the roots were stained with 0.1% acid fuchsin lactophenol to reveal the nematode's various developmental phases [23]. A binocular microscope was used to observe the nematode at various stages of development.

Histopathological Studies of Roots Infested by M. graminicola
Root samples from rice plants that had been severely infested with root galls caused by the root-knot nematode M. graminicola were used in this study. Roots were cleaned to remove any remaining soil, cut into 0.3 cm pieces with a sharp blade, fixed in FAA (formalin acetic acid ethanol) for 36 h, dehydrated using a graded ethanol-xylol series, embedded in paraffin wax (melting point: 56-58 • C), and sectioned at a thickness of 8 µm using a hand rotary microtome. The sections were mounted in DPX mountant, and the sections were stained with safranin and counterstained with fast green [23]. The sectioned tissues were visualised using a Nikon eclipse Ti2-U inverted microscope.

Interaction between M. graminicola Effector Proteins and the Rice Plants
To understand the pathogenicity pattern of Meloidogyne graminicola parasitic proteins against rice cultivars, molecular modelling and docking studies were performed on protein targets such as pectate lyase [13], phospholipase B-like protein [14], G protein-coupled receptor kinase [15], and β-1,4-endoglucanase [16].

Protein Target Identification and Molecular Modelling
Based on a review of the literature, the proteins pectate lyase, phospholipase B-like protein, G protein-coupled receptor kinase, and β-1,4-endoglucanase were identified as potential targets of the rice root-knot nematode M. graminicola. Based on literature mining, the protein sequences of selected targets for the rice root-knot nematode M. graminicola were retrieved from the UniProt database. The rice root-knot nematode's chosen virulent target, M. graminicola, lacks experimentally and computationally solved structures. Therefore, SWISS-MODEL (method: rigid-body assembly), Phyre2 (method: profile-based alignment), and ROBETTA were used for molecular modelling (Metaserver). First, a model of each target sequence was created using the SWISS-MODEL server. Based on query coverage performance, SWISS-MODEL and Phyre2/ROBETTA were used for structure modelling in the absence of templates. The homology modelling for the target's pectate lyase, phospholipase B-like protein, and G protein-coupled receptor kinase was created using the software SWISS-MODEL. Phyre2 was additionally used to model β-1,4-endoglucanase. Pectate lyase, phospholipase B-like protein, G protein-coupled receptor kinase, and model β-1,4-endoglucanase were among the protein targets in the homology modelling protocol that underwent BLAST search followed by HHblitsin SUZUKI-MODEL [24]. Maximum query coverage and a global mean quality estimation (GMQE) score of close to 1 were used as the parameters to ensure the high quality of modelled structures (Supplementary File S1). Using comparative modelling domains, the ROBETTA server http://robetta.bakerlab.org/ (accessed on 11 April 2022) models multichain complexes using RoseTTAFold.

Testing of the Protein Model
To assure model quality based on the residues residing in preferred and permitted regions, modelled protein targets were checked using the Ramachandran plot of the PROCHECK tool from the structural analysis and verification service (SAVES, Meta server) Biology 2023, 12, 987 7 of 18 https://saves.mbi.ucla.edu/ (accessed on 18 April 2022). The Swiss PDB viewer was used to generate loops for residues in forbidden regions, and to minimise energy in proteins that were modelled "http://www.expasy.org/spdbv/" (accessed on 20 April 2022).

Preparation and Analysis of Ligands
Three substances, Beta-D-Galacturonic Acid, 2,6,10,15,19,23-hexamethyltetracosane, and (2S)-2-amino-3-phenylpropanoic acid were gathered from the Pubchem database. The Pubchem database was accessed in SDF format to obtain 4-O-Beta-D-Galactopyranosyl-Alpha-D-Glucopyranose. Compounds were converted from the SDF file format to the PDB file format using the programme Open Babel v 2.3.1.

Small Molecule Similarity Analysis
The similarity score between the compound pairs was calculated using the ChemMine online software. Tanimoto coefficients such as atom pair Tanimoto (AP), maximum common substructure Tanimoto (MCS), MCS size, MCS min/max, and SMILES were used to investigate the structural similarity of small molecules. Tanimoto is defined as c/(a + b + c), where c represents the number of features in a compound pair, a represents features unique to one compound, and b represents features unique to another. For atom pairs, the Tanimoto coefficient ranges from 0 to 1, with a larger coefficient indicating greater similarity (similar structural descriptors). Compounds with large Tanimoto differences (0.20) and the MCS make obtaining the most accurate and sensitive similarity measure possible. Since it is likely that similar molecules will share a large MCS size (9), the similarity analysis was established.

Molecular Docking and Virtual Screening
PyRx 0.8's AutoDock vina module was used to perform molecular docking [9]. The make macromolecule option in PyRx software was used to prepare the protein. The conjugate gradient, first-order derivatives of an optimization process with 200 steps, and commercial molecular mechanics parameters unified force field were used to minimise all ligand structures (UFF). To find binding site pockets for the targets, the Computed Atlas Topography of Proteins CASTp 3.0 server was used [25]. AutoDock4 and autogrid4 parameter files were used to set the grid and dock. During the docking protocol execution, ligands were allowed to generate flexible conformations and orientations with a value of 8 exhaustiveness. BIOVIA Discovery studio client 2021 https://www.3ds.com/productsservices/biovia/ (accessed on 23 May 2022) was used to visualise interactions of docked conformations of protein-ligand complexes. To distinguish the receptor, ligand, and interacting atoms, different colours were assigned to them.

Incidence and Dispersion of Meloidogyne graminicola in Tamil Nadu
Among the major rice-growing regions of Tamil Nadu, India surveyed for the occurrence of rice root-knot nematode M. graminicola, Coimbatore, Villupuram, Ariyalur, and Krishnigiri districts of Tamil Nadu were encountered with nematode infestation symptoms. Patches in the crop canopy and poor plant growth, including a stunted look and chlorotic leaves, were M. graminicola's aboveground symptoms. Belowground symptoms were swollen and hooked root tip gall ends on infested root systems ( Figure 2). Galled roots were examined, and it was discovered that they included both males and females. Pear-shaped females were revealed entrenched in the cortical layer of the galled root. The gall index [9] for rice root-knot nematode ranged from 3 to 4 where the districts encountered root-knot nematode symptoms (Table 3).

Morphometrics
Posterior cuticular pattern (PCP): the female's perineal patterns were oblong-shaped, with fine striae, dorsal semicircular arches, occasionally very few lines converged at each end of the vulva, and unclear or nonexistent lateral fields ( Figure 3). Since 1949, morphological diagnosis based on the perineal pattern is one of the traditional methods for classifying Meloidogyne species [9]. The perineal pattern of isolates from Tamil Nadu, as determined by light as well as phase contrast microscopy, are somewhat different from previously reported patterns of M. graminicola [26,27], and overlap with both the patterns of M. trifoliophila and M. oryzae. gall index [9] for rice root-knot nematode ranged from 3 to 4 where the districts encountered root-knot nematode symptoms (Table 3).

Morphometrics
Posterior cuticular pattern (PCP): the female's perineal patterns were oblongshaped, with fine striae, dorsal semicircular arches, occasionally very few lines converged at each end of the vulva, and unclear or nonexistent lateral fields ( Figure 3). Since 1949, morphological diagnosis based on the perineal pattern is one of the traditional methods for classifying Meloidogyne species [9]. The perineal pattern of isolates from Tamil Nadu, as determined by light as well as phase contrast microscopy, are somewhat different from previously reported patterns of M. graminicola [26,27], and overlap with both the patterns of M. trifoliophila and M. oryzae. Female: the body is pearly white, pear-shaped, and has a short neck. The cuticle of the body has been annulated. The head is not clearly separated from the neck. The cephalic framework is weakly sclerotized. Small and delicate stylet with rounded knobs sloping backward. The oesophagus is substantially developed, with an elongate cylindrical procorpus and a broad, rounded metacorpus with a strongly sclerotized valve. Dorsal oesophageal gland aperture is located 3.9 µ (3.6-5.9) posterior to the base of the stylet ( Table  3). Excretory pore distinct, one and a half stylet lengths from the stylet's base. Two prodelphic convoluted ovaries. Vulva and anus are both terminally placed.    Female: the body is pearly white, pear-shaped, and has a short neck. The cuticle of the body has been annulated. The head is not clearly separated from the neck. The cephalic framework is weakly sclerotized. Small and delicate stylet with rounded knobs sloping backward. The oesophagus is substantially developed, with an elongate cylindrical procorpus and a broad, rounded metacorpus with a strongly sclerotized valve. Dorsal oesophageal gland aperture is located 3.9 µ (3.6-5.9) posterior to the base of the stylet (Table 3). Excretory pore distinct, one and a half stylet lengths from the stylet's base. Two prodelphic convoluted ovaries. Vulva and anus are both terminally placed.
Male: body cylindroid, vermiform, tapering gradually at both ends. Body width: 34.6 µ (26.5-34.7). The head and body are not clearly separated. The cephalic framework is prominent. Cuticular annulation is extremely apparent. Blunt lips with a pronounced annulus and a longer spear conus than the shaft. Knobs are obvious and prominent.  Figure 4G).    2) in length and lacks a regular and conspicuous annulation. Tail end that is rounded and slightly clavate (Table 5, Figure 4A-F and Figure 5).

Histopathology
The presence of adult females and eggs was observed inside the cortical region of the root galls of susceptible rice plants with heavily infested galls, while groups of abnormally enlarged cells (giant cells) were common in the stele region ( Figure 6). Eggs were laid in a gelatinous matrix, with egg masses embedded in the cortical region. Eggs were freed from the old roots by breaking the epidermal layer. In young roots, eggs hatch within the root. The juveniles or immature females persisted in the maternal gall or migrated intercellularly through the aerenchymatous tissues of the cortex to other feeding sides within the same root. Cell division and hypertrophy occurred as a result of larval inter-and intracellular movement in the root cortex ( Figure 6). Juveniles gained access to the protophloem sites by destroying the pericycle cells. At larval establishment locations in the stele, aberrant xylem grew surrounding the large cell, producing expansion of the vascular cylinder. The histopathological analysis of nematode-infected rice roots revealed the development of "giant cells", or specialized feeding sites, which were modified procambial cells in the stele region of the roots.

In Silico Analysis of M. graminicola Effector Proteins
Pectate lyase, one of the rice root-knot nematode parasitic proteins, was modelled using SWISS-MODEL utilising a template protein with 46.59 percent identity, 91 percent coverage, and a 0.72 GMQE score that had previously been described by electron microscopy (PDB ID-1EGZ). When experimentally solved using the X-ray crystallography method, the template for the phospholipase B-like protein (PDB ID-KAF7) had a GMQE score of 0.78, 97.18 percent identity, and 79 percent coverage. Since there were no homologs or templates for another nematode parasitic protein target β-1,4-endoglucanase in the SWISS-MODEL database, Phyre2 (http://www. sbg.bio.ic.ac.uk/phyre2/) (accessed on 12 May 2022) was used. Using the UniProt ID A0A8S9ZFJ9, the target protein sequence for β-1,4-endoglucanase (467 residues) was found. The β-1,4-endoglucanase protein model

In Silico Analysis of M. graminicola Effector Proteins
Pectate lyase, one of the rice root-knot nematode parasitic proteins, was modelled using SWISS-MODEL utilising a template protein with 46.59 percent identity, 91 percent coverage, and a 0.72 GMQE score that had previously been described by electron microscopy (PDB ID-1EGZ). When experimentally solved using the X-ray crystallography method, the template for the phospholipase B-like protein (PDB ID-KAF7) had a GMQE score of 0.78, 97.18 percent identity, and 79 percent coverage. Since there were no homologs or templates for another nematode parasitic protein target β-1,4-endoglucanase in the SWISS-MODEL database, Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/) (accessed on 12 May 2022) was used. Using the UniProt ID A0A8S9ZFJ9, the target protein sequence for β-1,4-endoglucanase (467 residues) was found. The β-1,4-endoglucanase protein model had a 100% confidence score and 87 percent coverage, and was based on protein structures such as PDB ID-6GJF. A G protein-coupled receptor target was modelled using I-TASSER with a 60% confidence score.

Validation of Models
According to the Ramachandran plot, the target, phospholipase B-like protein, had 85.7 percent of its residues in the most favoured region or core region, 11.7 percent in additionally allowed regions, and 1.3 percent in generously allowed regions (Supplementary File S1). Residues in the G protein-coupled receptor targets allowed, additionally allowed, and generously allowed regions are 81.9 percent, 13.3 percent, and 2.9 percent, respectively (Supplementary File S1). The target β-1,4-endoglucanase had 75.2 percent of its residues in the most favoured region, 19.5 percent in additional allowed regions, and 3.5 percent in generously allowed regions (Supplementary File S1). The target pectate lyase contained 76.7 percent of its residues in the most favoured region, 17.8 percent of its residues in additional allowed regions, and 3.9 percent of its residues in the generously allowed region (Supplementary File S1).

Analysis of Sequence Similarity
To determine the presence of any similar proteins in rice plants, sequence similarity was performed using the BLASTP tool for the nematode target proteins as a query against the rice genome proteins. In the similarity search, no single hit or similar sequences were found. Thus, specific binding of Beta-D-Galacturonic Acid, 2,6,10,15,19,23hexamethyltetracosane, 4-O-Beta-D-Galactopyranosyl-Alpha-D-Glucopyranose, and (2S)-2amino-3-phenylpropanoic acid to nematode protein targets was observed, but no binding to rice proteins was observed.

Beta-D-Galacturonic Acid
Molecular docking and virtual screening modelled protein structures were docked with various compounds to determine their mode of binding (Figures 7 and 8, Table 6 Table 6).

Discussion
The general morphology and morphometrics of the M. graminicola population in Tamil Nadu, India is in satisfactory correlation with the original descriptions [5]. According to the results of the current study, M. graminicola females were compared to previous morphometric findings [7]. Adult female populations were slightly varied in body length (474-594 µm) and DOGO value (3.6-5.9 µm). The current findings show that the entire length (1096.1-1731.4 µm) of the male M. graminicola was identical to Golden and Birchfield's original description (1965) [5]. The highest body width (16.9 µm) of second-stage juveniles was encountered from Tamil Nadu populations when compared to earliest morphometric studies ( Table 4). The sequence size of ITS, coxII-16S rRNA, and D2-D3 region of 28S are 399, 790, and 764 bp, respectively. All of the amplified sequences were comparable, and shared 99-100% identity with NCBI GenBank sequences for M. graminicola. For the M. graminicola Tamil Nadu population, novel sequences were discovered and deposited in NCBI GenBank with the accession codes OP712502 for ITS, OP714360 for D2-D3 of 28S, and OP714470 for coxII-16S rRNA genes. Based on ITS-rDNA, D2-D3 of 28S, and coxII-16S rRNA sequences, molecular identification of Tamil Nadu populations of M. graminicola was performed in the current study. In contrast with using molecular diagnosis, it is thought that variations in morphological traits, such as the stylet length, spicule, and vulva, are important for diagnosing the presence rice root-knot nematode M. graminicola. Our findings concur with the findings of [17], showing that molecular studies are more trustworthy for identifying M. graminicola since minor morphometrical variances and observed morphological differences may be related to regional distribution. The entry of M. graminicola second-stage juveniles (J2) began on the fourth day after sprouting rice seeds. Juveniles (J2) can enter the rice root tip regions at 48-72 h after hatching, and their number in the roots increases with time. Invaded juveniles establish their feeding sites in vascular tissues and reach the J3 stage after 120-144 h of intensive feeding. After 72-96 h of the J3 stage, the J4 stage starts, and the Casper adult stage is formed after another 96-120 h. A fully developed adult stage begins 264-312 h into the preadult stage. The roots had records of all larval stages, as well as adult males and females. Thus, until the 14th day after seeding, the second-stage juveniles in the soil kept attacking the roots. There were no second-stage juveniles found after the 15th day, and only adult stages were discovered after the 16th day ( Figure 9). The histopathological studies revealed that rice root-knot nematode Meloidogyne graminicola-infested roots showed enlargements of giant cells with multiple nuclei at the stele region of the vascular system, and almost all of the females were mature, with a few being associated with egg masses. On the twentieth day, the maximum number of adults per root system (15-20 females and 2-3 males) was noted. Egg laying was noticed on day 20. Eggs were deposited both inside and outside of the gall system and wrapped in a viscous matrix. Six to seven members of the second-stage juveniles were encountered on the 24th day after sowing. M. graminicola's life span on rice lasted 26-28 days to complete. The current findings support those of [31], who observed egg-laying females on days 20 to 24 following M. graminicola inoculation on paddy seedlings. The authors of [31] showed that this nematode completes its life cycle in 26-51 days on paddys throughout the year in eastern areas of India, highlighting the influence of temperature on life cycle duration. The presence of a protractible stylet, which serves to withdraw nutrients from the giant cells, as well as to release molecules, including virulence effectors, into the apoplast or directly into the host cells, is a distinguishing feature of plant-parasitic nematodes. Plant-parasitic nematodes secrete effector molecules into plant tissues to facilitate infection, reconfigure the cellular metabolism, or dissuade the accomplishment of plant defence responses. Sedentary nematodes primarily release effectors produced in their esophageal glands into host tissues via their stylet, though other secretory organs may also play a role in the parasitic process [32,33]. Nematode management in large scale commercial rice cultivation was based on two to four nematicide uses, which are extremely harmful to soil microbial populations and biodiversity. It is, however, only a temporary solution because the nematode population grows vastly disproportionate after a few months, necessitating frequent applications of nematicides, which inevitably become toxic to the environment and uneconomical. Because of the negative effects of nematicides, scientists are eager to develop an alternative nematode management strategy. Virtual screening methods such Sedentary nematodes primarily release effectors produced in their esophageal glands into host tissues via their stylet, though other secretory organs may also play a role in the parasitic process [32,33]. Nematode management in large scale commercial rice cultivation was based on two to four nematicide uses, which are extremely harmful to soil microbial populations and biodiversity. It is, however, only a temporary solution because the nematode population grows vastly disproportionate after a few months, necessitating frequent applications of nematicides, which inevitably become toxic to the environment and uneconomical. Because of the negative effects of nematicides, scientists are eager to develop an alternative nematode management strategy. Virtual screening methods such as molecular docking had a significant impact in identifying a promising novel target site for the management of M. graminicola. We investigated potential protein target sites with rice biomolecules to use molecular docking to detect a molecule with the highest binding affinity on different M. graminicola target sites. The maximum binding (−7.7 kcal/ mol) of phospholipase B-like protein with the ligand 2,6,10,15,19,23-hexamethyltetracosane could promote nematode parasitic activity by regulating lipid catabolic processes in host plant cells, and may have increased nematode entry into plant tissues [14]. Similarly, the maximum binding energy of (2S)-2-amino-3-phenylpropanoic acid with G protein-coupled receptor kinase (−7.2 kcal/mol) could regulate cytological protein phosphorylation, nematode parasitism, nematode reproduction, and suppress the host defence response [34]. Furthermore, the maximum binding energy (−6.0 kcal/mol) for -1,4-endoglucanase with 2,6,10,15,19,23hexamethyltetracosane could promote nematode activity to degrade the b 1,4 linkage in cellulose polymer, and could have promoted entry into plant tissues [35]. Following that, the maximum binding energy of pectate lyase with 2,6,10,15,19,23-hexamethyltetracosane could promote nematode activity to cleave the 1,4 glycosidic bonds of polygalacturonic acid in plant tissues [13]. Docking interactions of nematicidal compounds with a b-tubulin protein from Brugia malayi were conducted, and reported albendazol sulfone as that of the ideal nematicidal (antifilarial) substance [36]. Common chokepoint reactions and enzymes in nematodes and prioritised drug targets suggest perhexiline as a nematicidal compound, considering its binding efficacy against Caenorhabditis elegans carnitine palmitoyl transferase 2 [37]. Thus, the current study is unique in that phospholipase, one of the most targeted virulent effector proteins of M. graminicola, had the highest binding affinity to rice ligand biomolecules. Furthermore, this study's findings highlighted the possibility of harnessing the potential of virulent effector protein phospholipase, which promotes parasitism with rice plants. However, more research is needed to confirm the pathogenicity of virulent effector phospholipase B-like protein in the wet lab using qRT-PCR and RNA interference techniques in rice plants challenged with rice root-knot nematode M. graminicola.

Conclusions
The current study provides a thorough molecular and morphological description of the M. graminicola population found in Tamil Nadu, India, as well as phase contrast microscopy analyses of adult females, second-stage juveniles, and posterior cuticular patterns. Furthermore, this study integrates M. graminicola's life span and provides detailed morphometric and molecular assessments of all Tamil Nadu populations. Further, the present study used an in silico approach to explain the multiple modes of action of M. graminicola's virulent proteins against various ligand biomolecules of rice targets. The virulent phospholipase B-like protein has a higher affinity for ligand biomolecules, which favours plant parasitism in rice.