Nematicidal action of Clonostachys rosea against Meloidogyne incognita: in-vitro and in-silico analyses

ABSTRACT

Clonostachys rosea is a biocontrol agent against a wide range of pests and diseases. In the present study, an in-vitro nematicidal activity of C. rosea has been documented by observing the behavioural response of Meloidogyne incognita (Mi) towards the culture filtrate of C. rosea. Eleven different biomolecules observed in GC-MS analysis were tested against seven potential target proteins of Mi through molecular modelling using PyRx 0.8 software. Among the biomolecules tested, Dihydro-4-hydroxy-2(3H)-furanone had the highest binding affinity for Acetylcholine esterase (AchE) (−7.5 kcal/mol), Cytochrome c oxidase subunit 1 (COX) (−7.8 kcal/mol), Heat shock protein (Hsp90) (−8.4 kcal/mol), Odorant response gene 1 (ODR 3) (−6.5 kcal/mol), Neuropeptide G-protein coupled receptor (nGPCR) (−7.6 kcal/mol) and Cathepsin L-protease (cpl-1) (−6.8 kcal/mol) protein targets compared to synthetic pesticide, carbofuran. The biomolecule 2,3-dihydroxypropanal has shown the highest binding affinity for Odorant response gene 1 (ODR 1) (−6.6 kcal/mol). Understanding the interactions between the target protein-ligands docked complexes displayed additional contacts such as hydrophobic interactions, van der Waals, pi-pi stacking, alkyl and pi-alkyl. The present investigation demonstrated that C. rosea is a hub of potential biomolecules that could be explored for nematicidal activities.

KEYWORDS:

• Clonostachys rosea
• nematicide
• Meloidogyne incognita
• molecular modeling
• dihydro-4-hydroxy-2(3H)-furanone
• 2,3-dihydroxy propanal

1. Introduction

Nematodes are microscopic worms and are known to be serious parasites on crops. Depending on the vulnerability of the host, they could cause significant yield loss directly affecting the crop or facilitating the entry of soil-borne fungal and bacterial pathogens [1]. Among different types of nematodes, root-knot nematodes are reported as serious plant parasites that could cause 30% of crop losses directly in vegetables [2]. Of the root-knot nematodes, Meloidogyne incognita is the most prevalent in tropical soils, rarely sparing any crop family. Management of nematodes in the present growing interest in sustainable cropping systems requires safer alternatives to regular synthetic pesticides (carbofuran and fluopyram), which have exhibited negative environmental effects either eliminating non-target species or polluting different spheres of ecosystems [3]. In addition, many synthetic pesticides have been facing de-registration due to their ill effects on environment at the global level [4,5]. This poses a significant challenge to the pest management efforts [6]. As a result, the Integrated Pest Management (IPM) paradigm is preferred, which emphasizes the use of biopesticides as a key component for pest management [7]. Researchers have demonstrated the potential use of beneficial microorganisms in the suppression of plant pathogens including nematodes [8,9]. One such soil-borne fungus extensively studied with specificity to suppress plant parasitic nematodes is Clonostachys rosea. Walker et al. [10] investigated C. rosea as a mycoparasite against Phomopsis sclerotioides on cucumber and sclerotia of Botrytis allii. After it was determined to be a harmful mycoparasite of Botrytis species, several additional researchers began investigating its possible use as a commercial biological control for diseases caused by several pathogens [11–15].

Recent research has shown that various Clonostachys strains directly parasitize a variety of plant parasitic nematodes [16–18]. Furthermore, it has been characterized that C. rosea produces nematicidal compounds and enzymes viz., leptosins, chetracin A, chaetocin and gliocladines A, B, C, D and E and extracellular serine proteases and chitinases attributing to the antagonistic activity against plant parasitic nematodes [19–21]. It is imperative to isolate, screen and test the efficacy of native isolates against specific plant pathogens to identify the elite strains. To investigate their nematicidal activities, a thorough roving survey was carried out to isolate the fungus C. rosea, followed by a culture characterization. It was also tested in-vitro against the M. incognita using efficient native isolates. There are no studies on the possible bioactivity of secondary metabolites from C. rosea on plant parasitic nematodes, despite several periodic attempts to isolate and identify these compounds. Therefore, an attempt was made earlier to isolate the crude metabolites from the effective native isolate of C. rosea followed by GC–MS analysis to identify potential biomolecules. Nevertheless, little information was known on the interaction of these biomolecules with the target sites of the nematodes. Hence, the present study aimed to understand the nematicidal activity of underexplored biocontrol agent C. rosea through in-silico analysis using molecular docking.

2. Materials and methods

2.1. Fungal and nematode cultures

Six C. rosea isolates isolated from different agroecosystems in Tamil Nadu, India were tested against M. incognita (Mi) to study their nematicidal properties. C. rosea cultures were maintained in a PDA medium at 25 ± 2°C with regular sub-culturing throughout the study period. The Mi culture was maintained by infecting tomato (Var. PKM1) plants grown under semi-controlled greenhouse conditions. Mi was injected into the 4-week-old tomato seedlings. After 45 days, egg masses were harnessed from the galled roots of the inoculated plants and used to assess the efficacy of C. rosea against the hatching of eggs and mortality of juveniles at various stages. Egg masses were placed in a Petri dish with fresh tap water for 5 days at 27°C for hatching.

2.2. Preparation of conidial suspension of C. rosea

C. rosea isolates (9 mm disc) were inoculated separately onto a conical flask containing 250 ml Potato Dextrose Broth (PDB) and incubated at 25°C for 15 days. The fully grown culture with mycelial mat was strained through two layers of Whatman filter paper to separate the filtrate from the mycelial mat. The conidial concentration was assessed using the hemocytometer and adjusted to 6 × 10⁷ conidiaml⁻¹ using sterile distilled water.

2.3. Testing the efficacy of conidial suspension of six isolates of C. rosea in parasitizing Mi eggs

The conidial suspension (6 × 10⁷ conidiaml⁻¹) of six C. rosea isolates was prepared from concentration. One millilitre conidial suspension was added onto a microfuge tube and inoculated with an egg mass at the rate of one per tube. As a control, sterile distilled water was used. The egg mass-filled tubes were incubated for 96 hrs. The parasitized and non-parasitized eggs were counted under a microscope to assess the percentage of eggs at 24 hrs intervals. Infected eggs with direct hyphal penetrations and internal contents that had disintegrated were counted as parasitized eggs, but eggs with live or newly born juveniles were counted as healthy eggs. The following formula was used to determine the per cent egg parasitism: Egg parasitism (%) = (Total number of parasitized eggs/Total number of eggs) × 100.

2.4. Study on the efficacy of C. rosea TNAU CR 01against Mi hatching

Four different conidial suspensions (25, 50, 75 and 100%) of C. rosea TNAU CR01 were prepared from 60 × 10⁶ conidiaml⁻¹. A 45 mm Petri plate was amended separately with five ml conidial suspension of C. rosea at 25, 50, 75 and 100%concentrations. An egg mass of identical size (± 5.71 µm length and ± 2.83 µm width) harnessed from the Mi-infected plants was placed in centre of a Petri dish (one egg mass/dish) as an inoculum. The egg mass placed in the Petri dish with PDB and sterile distilled water served as controls. The inoculated Petri plates were incubated for 96 hrs at 25°C and hatching was recorded using a stereo microscope at 24 hr interval. Three replications were maintained for each treatment. The following formula was used to compute the per cent egg hatching inhibition: Egg hatching inhibition (%) = (Total number of unhatched eggs/Total number of eggs) × 100.

2.5. Testing the efficacy of C. rosea TNAU CR01 against the survival of Mi juveniles

A 45 mm Petri plate was added separately with five ml conidial suspension of C. rosea TNAU CR01 at 25, 50, 75 and 100% concentrations. Each Petri dish was inoculated with 100 s stage juveniles (J2) of Mi. Juveniles inoculated in Petri dishes containing PDB and sterile distilled water served as controls. At 24, 48, 72 and 96 hrs after incubation, the influence of culture filtrate and PDB on the mortality of juveniles was counted using microscopic observations. After being exposed to each treatment for 24, 48 and 72 hrs, the immobilized nematodes with straightened bodies were transferred to new Petri plates containing 1 ml of distilled water for a revival test, respectively. The Petri plates housing the immobilized juveniles were then treated with a few drops of a 1M sodium hydroxide (NaOH) solution. After the revival test, the average number of dead nematodes and the total number of nematodes utilized for each of the three replicates in each treatment were determined [22]. The adjusted mortality rate was calculated as previously mentioned, and the juvenile death rate was then calculated using the following formula: Mortality (%) = (Total number of deal juveniles/Total number of juveniles inoculated) × 100.

2.6. Molecular docking of biomolecules of C. rosea with Mi protein targets

The biomolecules of the C. rosea isolate TNAU CR 01 identified from earlier studies using GC–MS were used for ligand analysis with target proteins (Table S1). Among 40 therapeutic biomolecules, twelve biomolecules with the highest peak areas were chosen for docking:-(1) 2-4-hydroxy-2,5-bis(hydroxymethyl)-2-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxolan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol, (2) 2,3-dihydroxypropanal, (3) 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, (4) Dihydro-4-hydroxy-2(3H)-furanone, (5) Tetrahydro-5-methyl-2-furanmethanol, (6) 1,2,3-Propanetriol-acetate, (7) 2-Hexyl tiglate, (8) 2-Nitro-2-(hydroxymethyl)-1,3-propanediol, (9) 3,5-dihydroxy-6-(hydroxymethyl)oxan-2-one, (10)-3-(2-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one, (11) Tetratetracontane and (12) 9-Octadecenamide. The commercial nematicide carbofuran 3G was utilized as a reference ligand molecule to evaluate the performance of test compounds. Thus, fourteen chemicals were downloaded in SDF format from the PubChem database (http://pubchem.ncbi.nlm.nih.gov) and transformed into PDB files using the Open Babel program.

The AutoDock vina module of PyRx 0.8 was utilized for the molecular docking process. The software PyRx’s Make macromolecule option was used to prepare the proteins. All ligand structures were reduced in 200 steps of conjugate gradient optimization using commercial molecular mechanic parameters and a unified force field (UFF). To find pockets at the target binding position, CASTp3.0 was utilized. AutoDock4 and AutoGrid4 were used to configure the ligands for grid and docking. The target proteins’ and their ligands’ 3D structures were converted to PDBQT format. To visualize the docked protein–ligand complex interactions, BIOVIA discovery studio client 2021 was used.

2.7. Mi protein targets used in the study for molecular modelling

The Acetylcholine esterase (AchE), Cytochrome c oxidase subunit 1 (COX), Heat shock protein (Hsp 90), odorant response gene 1 (ODR1), odorant response gene 3 (ODR3), the neuropeptide G-protein coupled receptor (nGPCR) and the putative cathepsin L-protease (cpl-1) of Mi were chosen as potential Mi target proteins for modelling based on the desktop research [23]. The Protein Data Bank Database (PDB) (https://www.rcsb.org/) does not contain experimentally or computationally solved structures for the chosen target protein. To carry out homology modelling, the SWISS-MODEL (Method: Rigid-body assembly) was employed. The Ramachandra plot of the PROCHECK tool from the Structural Analysis and Verification Server (SAVES, Meta server) (http://saves.mbi.ucla.edu/) was used to validate the protein target to ensure the accuracy of the protein model. Using SWISS PDB Viewer (http://www.expasy.org/spdbv/), energy minimization of the modelled protein was carried out.

2.8. Statistical analysis

The data were statistically analysed using the SPSS version 16.0 (Statistical Package for the Social Sciences) developed by Norman. H. Nie, Dale H. Bent and C. Hadlai Hull in 1968. The data were subjected to the analysis of variance (ANOVA) at P < 0.05 and means were compared by Duncan’s Multiple Range Test (DMRT).

3. Results

3.1. Parasitization potential of C. rosea against Mi

The parasitization ability of six isolates of C. rosea was tested by the microfuge method. Among six isolates tested, the highest egg parasitization capacity of C. rosea was observed in egg mass treated with TNAU CR 01 isolate by exhibiting 69.38% parasitization over control. This was followed by isolate TNAU CR 02 which showed 60.97% parasitization over control. The low egg parasitization potential was recorded with TNAU CR 04 isolate (44.73% inhibition over control) (Table 1). A colonization of Mi eggs by the chlamydospores and conidia of C. rosea was observed under a microscope (Figure 1). Based on the high parasitization potential of TNAU CR01 isolate in this experiment, further studies were progressed using only TNAU CR01 isolate.

Figure 1. Effect of C. rosea on M. incognita egg parasitization. (A) Penetration of C. rosea TNAU CR 01 mycelium. (B) Infected eggs with spores of C. rosea TNAU CR 01.

Table 1. Effect of C. rosea on M. incognita egg parasitization.

Treatments	Percentage of eggs parasitized (%)
C. rosea TNAU CR 01	69.38^a
	(56.40)
C. rosea TNAU CR 02	60.97^b
	(51.34)
C. rosea TNAU CR 03	56.09^c
	(48.50)
C. rosea TNAU CR 04	44.73^e
	(41.97)
C. rosea TNAU CR 05	53.12^d
	(46.79)
C. rosea TNAU CR 06	52.91^d
	(46.67)
Sterile water (Control)	0^f
	(0.00)
CD	1.8
SEd	0.86

*Values are mean of three replications.

Figures in parentheses of Percentage of eggs parasitized column are arcsine transformed values.

Means in a column followed by same superscript letters are not significantly different according to DMRT test at P ≤ 0.05.

3.2. C. rosea against egg hatching of Mi in-vitro

The hatching test results showed that Mi’s capacity to hatch was reduced after being exposed to TNAU CR 01 conidial suspension at 25% concentration. In comparison to control (368.21 eggs) and PDB (395.65 eggs), the number of eggs hatched per egg mass was significantly reduced in egg masses treated with C. rosea (98.75 eggs) at a 25% conidial suspension within 96 hrs. A high suppression of egg hatching was recorded in all three concentrations (50, 75 and 100%) of conidial suspension (Table 2). This demonstrated unequivocally that the nematicidal characteristics of C. rosea suppressed the hatching ability of Mi to decline with increasing concentration of conidia and incubation period. The gelatin matrix of the infected Mi eggs was darker and displayed substantial deformation when examined under a microscope. Bulging of internal content and egg disintegration was also evident from the nematicidal properties of C.rosea (Figure 2).

Figure 2. Effect of C. rosea culture filtrate on M. incognita egghatching. (A) Unhatched infected eggs. (B) Infected eggs covered with chlamydospores of C. rosea TNAU CR 01. (C) Bulged eggs.

Table 2. Effect of different concentration of C. rosea culture filtrate on M. incognita egg hatching.

	Number of eggs hatched*
	25%				50%				75%				100%
Treatments	24 h	48 h	72 h	96 h	24 h	48 h	72 h	96 h	24 h	48 h	72 h	96 h	24 h	48 h	72 h	96 h
C. rosea	49.75^c	68^c	80.15^c	98.75^c	38.25^c	61.2^c	72.85^c	85.5^c	24.5^c	50.25^c	65.25^c	72.45^c	2.65^c	26.05^c	26.5^c	33.55^c
	(1.70)	(1.83)	(1.90)	(1.99)	(1.58)	(1.79)	(1.86)	(1.93)	(1.39)	(1.70)	(1.81)	(1.86)	(0.42)	(1.42)	(1.42)	(1.53)
Potato Dextrose Broth	75.52^b	121.52^b	285.12^b	395.65^b	72.25^b	110.71^b	235.45^b	335.45^b	95.2^b	100.2^b	195.25^b	238.5^b	61.25^b	87.55^b	180.25^b	286.75^b
	(1.88)	(2.08)	(2.46)	(2.60)	(1.86)	(2.04)	(2.37)	(2.53)	(1.98)	(2.00)	(2.29)	(2.38)	(1.79)	(1.94)	(2.26)	(2.46)
Sterile Water (Control)	87.21^a	152.33^a	274.65^a	368.21^a	89.56^a	145.27^a	280.5^a	355.5^a	86.25^a	142.55^a	282.2^a	360.25^a	85.2^a	135.45^a	289.65^a	365.52^a
	(1.94)	(2.18)	(2.44)	(2.57)	(1.95)	(2.16)	(2.45)	(2.55)	(1.94)	(2.15)	(2.45)	(2.56)	(1.93)	(2.13)	(2.46)	(2.56)
CD	1.30	2.75	12.36	10.07	1.63	2.91	10.30	8.23	3.63	4.12	13.76	15.16	1.53	6.13	9.86	15.16
SEd	0.62	1.32	5.35	4.82	0.78	1.39	4.46	3.42	1.58	1.79	5.97	6.55	3.46	1.98	4.23	5.35

*Values are mean of three replications.

Figures in parentheses are log transformed values.

Means in a column followed by same superscript letters are not significantly different according to DMRT test at P ≤ 0.05.

3.3. C. rosea against the survival of Mi juveniles in-vitro

The mortality test showed that Mi juvenile deaths substantially increased at higher concentrations of TNAU CR 01 conidial suspension up to 96 hrs of incubation. At a 25% conidial suspension of TNAU CR 01, the highest number of dead juveniles (65.45 dead juveniles) was observed after 96 hrs of incubation. A similar suppression against the survival of Mi juveniles was recorded in the three 50, 75 and 100% conidial suspensions. However, there was a higher survival rate of Mi juveniles at 96 hrs of incubation in the PDB (15.5 dead juveniles) treatment compared to the control (10.01 dead juveniles) (Table 3). This result demonstrated that owing to the presence of anti-nematodal chemicals, the ability of Mi juveniles to survive declines with increasing C. rosea conidial suspension and incubation period. The juveniles with the infection had a distorted body and bulged internal contents. The juveniles that died had upright posture (Figure 3).

Figure 3. Effect of C. rosea culture filtrate on M. incognita juvenile mortality. (A) C. rosea treated juveniles. (B) Disintegrated dead juvenile.

Table 3. Effect of different concentration of C. rosea culture filtrate on M. incognita juvenile mortality.

	Number of juveniles dead*
	25%				50%				75%				100%
Treatments	24 h	48 h	72 h	96 h	24 h	48 h	72 h	96 h	24 h	48 h	72 h	96 h	24 h	48 h	72 h	96 h
C. rosea	28.5^a	38.65^a	55.65^a	65.45^a	39.25^a	58.85^a	69.5^a	78.5^a	54.65^a	75.25^a	89.55^a	89.65^a	65.85^a	98.55^a	100^a	100^a
	(5.34)	(6.22)	(7.46)	(8.09)	(6.26)	(7.67)	(8.34)	(8.86)	(7.39)	(8.67)	(9.46)	(9.47)	(8.11)	(9.93)	(10.00)	(10.00)
Potato Dextrose Broth	0^b	7.50^b	13.45^b	15.5^b	0^b	8.52^b	11.75^b	13.45^b	0^b	8.5^b	14.5^b	16.5^b	0^b	7.85^b	12.5^b	21.45^b
	(0.71)	(2.74)	(3.67)	(3.94)	(0.71)	(2.92)	(3.43)	(3.67)	(0.71)	(2.92)	(3.81)	(4.06)	(0.71)	(2.80)	(3.54)	(4.63)
Sterile Water (Control)	0^b	4.20^b	8.5^b	10.01^b	0^b	4.25^b	8.45^b	11.50^b	0^b	4.33^b	8.12^b	10.74^b	0^b	5.33^b	8.35^b	10.20^b
	(0.71)	(2.05)	(2.92)	(3.16)	(0.71)	(2.06)	(2.91)	(3.39)	(0.71)	(2.08)	(2.85)	(3.28)	(0.71)	(2.31)	(2.89)	(3.19)
CD =SEd =	0.62	1.03	0.72	1.15	0.76	0.55	2.01	1.51	0.65	1.25	0.99	1.15	1.13	1.62	1.57	1.43
	0.27	0.45	0.31	0.50	0.37	3.82	1.04	1.38	0.30	0.55	0.42	2.65	0.49	3.74	0.68	0.62

*Values are mean of three replicates.

Figures in parentheses are square transformed values.

Means in a column followed by same superscript letters are not significantly different according to DMRT test at P ≤ 0.05.

3.4. Molecular interaction between biomolecules of C. rosea and target proteins of Mi

Seven potential protein targets of Mi were modelled using the SWISS-MODEL. The protein sequence for the target AchE was retrieved using Uniprot ID O96529. The template for AchE was PDB ID 6EUC which had 44.13% identity, 86% coverage and a GMQE score of 0.67. The target protein COX sequence was retrieved using Uniprot ID A0A2Z5DTW9 and the template was PDB ID 6CI0, which showed 51.24% identity, 95% coverage and a GMQE score of 0.71. The protein sequence for the target Heat shock protein (Hsp 90) was retrieved using Uniprot IDB0LRB3. The template for Hsp90 was PDB ID6XLC had 63.31%identity, 95% coverage and a GMQE score of 0.59. The odorant response gene 1 (ODR1) protein sequence was retrieved using Uniprot ID A0A386R2C5. The template for ODR 1 was PDB ID 4IW0 which had 17.0% identity, 45% coverage and a GMQE score of 0.22. The odorant response gene 3 (ODR3) protein sequence was retrieved using Uniprot IDA0A386R2C9. The template for ODR 3 was PDB ID 1AGR with 50.30% identity, 99% coverage and a GMQE score of 0.76. The neuropeptide G-protein coupled receptor (nGPCR) protein sequence was retrieved using Uniprot IDB8XXG1. The template for nGPCR3 was PDB ID 7DDZ which had 27.39% identity, 83% coverage and a GMQE score of 0.49. The putative cathepsin L-protease (cpl-1) protein sequence was retrieved using Uniprot IDQ70P88. The template for cpl-1 was PDB ID 6JD0 which had 49.84% identity, 80% coverage and a GMQE score of 0.68.

3.5. Validation of the model used in the study

The target AchE contained 86.8% of its residues in the most favoured region or core region, 10.9% in additionally allowed regions and 1.9% in generously allowed regions, following the Ramachandran plot. COX contained 91.3% of its residues in the most favoured region or core region and 8.7% in additionally allowed regions. Hsp 90 contained 81.4% of its residues in the most favoured region or core region, 14.2% in additionally allowed regions and 3.3% in generously allowed regions. ODR1 contained 84.1% of its residues in the most favoured region or core region, 14.7% in additionally allowed regions and 0.9% in generously allowed regions. ODR3 contained 91.9% of its residues in the most favoured region or core region, 7.8% in additionally allowed regions and 0.3% in generously allowed regions. nGPCR contained 90.8% of its residues in the most favoured region or core region, 8.0% in additionally allowed regions and 1.1% in generously allowed regions and cpl-1 of Mi contained 88.9% of its residues in the most favoured region or core region, 10.0% in additionally allowed regions and 1.1% in generously allowed regions according to Ramachandra plot (Figures S1–S7). This verified the modelled structure’s acceptance and quality.

3.6. Sequence similarity analysis

To determine whether the tomato genome has any similar proteins, sequence similarity was carried out using the BLASTP program for the nematode target proteins as a query against the tomato genome proteins. In the similarity search, no single hit or comparable sequences were found. As a result, it showed that 2,3-dihydroxypropanal and Dihydro-4-hydroxy-2(3H)-furanone were specifically bound to the protein targets of Mi and did not bind to the protein targets of tomatoes.

3.7. Virtual screening and molecular docking of target proteins with bioactive molecules

Modelled seven protein structures were docked with selected 13 compounds to understand their bioactivity by analyzing their binding site. Among the 13 compounds analyzed, 2,3-dihydroxy propanal, Dihydro-4-hydroxy-2(3H)-furanone were the best compounds with the highest binding affinity with low-binding energy (Table 4).

Table 4. Binding affinity of eleven different biomolecules of C. rosea and Carbofuran 3G with virulent targets.

		Binding affinity (kcal/mol) of biomolecules against target proteins
S. No.	Biomolecules	AchE	COX	Hsp90	ODR 1	ODR 3	nGPCR	Cpl-1
1	2,3-dihydroxypropanal	−7.3	−6.9	−6.5	−6.6	−6.4	−7.0	−6.1
2	Dihydro-4-hydroxy-2(3H)-furanone	−7.5	−7.8	−8.4	−6.1	−6.5	−7.6	−6.8
3	2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one	−6.2	−5.2	−5.1	−4.6	−4.4	−4.9	−5.2
4	2-4-hydroxy-2,5-bis(hydroxymethyl)-2-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxolan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol	−4.7	−4.4	−4.3	−4.0	−4.6	−4.4	−4.6
5	Tetrahydro-5-methyl-2-furanmethanol	−5.2	−4.1	−4.3	−4.5	−4.4	−4.4	−4.7
6	1,2,3-Propanetriol-acetate	−6.0	−4.9	−5.2	−4.8	−4.9	−5.1	−4.8
7	2-Hexyl tiglate	−5.8	−5.3	−5.1	−4.8	−4.7	−5.2	−4.7
8	2-Nitro-2-(hydroxymethyl)-1,3-propanediol	−4.3	−4.4	−4.4	−3.8	−4.7	−4.5	−4.2
9	3,5-dihydroxy-6-(hydroxymethyl)oxan-2-one	−4.6	−3.8	−4.0	−3.6	−4.1	−3.9	−3.7
10	3-(2-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one	−4.4	−4.3	−4.4	−4.6	−4.9	−4.7	−4.2
11	9-Octadecenamide	−4.4	−3.5	−4.4	−3.9	−5.1	−4.5	−4.2
12	Carbofuran 3G (Positive control)	−7.4	−6.1	−6.3	−5.9	−5.9	−6.7	−6.3

Values in bold indicate the best value for the respective compounds.

3.8. 2,3-dihydroxy propanal

2,3-dihydroxy propanal had the binding affinity value of −7.3 kcal/mol with the target AchE (H-bonds: TRP 164, TYR 380) (Figure 4), −6.9 kcal/mol (H-bonds: THR 61) for COX, for Hsp 90, −6.5 kcal/mol (H-bonds: ARG 278, ARG 249, TRP 208), with ODR 1, it had binding affinity of −6.6 kcal/mol (H-bonds: ARG 550, GLN 561, ASP564, GLN 524), ODR3 had −6.4 kcal/mol (H-bonds: ASN 149, SER 78), while nGPCR and cpl-1 had 7 kcal/mol (H-bonds: ARG 104, ARG 177, THR 371) and −6.1 kcal/mol (H-bonds: ASP 303, ASN 326) binding affinities, respectively. In the docked complexes, hydrogen bonds are formed with the backbone and side chain of binding site residues. The docked complex also displayed other types of contacts, including hydrophobic interactions, van der Waals, pi–pi stacking, alkyl and pi–alkyl (Figure S8).

Figure 4. Illustrative diagram representing the protein–ligand interaction between potential protein target AchEof Mi and 2,3-dihydroxypropanal.

3.9. Dihydro-4-hydroxy-2(3H)-furanone

Compound Dihydro-4-hydroxy-2(3H)-furanone the binding affinity value of −7.5 kcal/mol with the target AchE (H-bonds: SER 376, LEU 327), −7.8 kcal/mol with the target COX(H-bonds: GLY 162), −8.4 kcal/mol with the target Hsp 90 (H-bonds: TYR 275, ARG 277) (Figure 5), −6.1 kcal/mol with the target ODR 1 (H-bonds: LEU 103, HIS 35, HIS121), −6.5 kcal/mol with ODR 3 (H-bonds: MET 335), −7.6 kcal/mol with the target nGPCR(H-bonds: LEU 156) and −6.8 kcal/mol with the target cpl-1 (H-bonds: ARG 167). Hydrogen bonds were formed with the backbone and side chain of binding site residues. The docked complex also displayed other types of contacts, including hydrophobic interactions, van der Waals, pi–pi stacking, alkyl and pi–alkyl (Figure S9).

Figure 5. Illustrative diagram representing the protein–ligand interaction between potential protein target HSP of Mi and Dihydro-4-hydroxy-2(3H)-furanone.

3.10. Carbofuran 3G

The binding affinity between the biomolecules of C. rosea and Carbofuran 3G was compared since the latter is being used by the farming community as the commercial nematicide to manage root-knot nematode infection. The binding affinity of Carbofuran 3G with the target AchE (H-bonds: GLY 161, GLY 162, SER 242) was −7.4 kcal/mol (Figure 6), −6.1 kcal/mol for COX (H-bonds: HIS 35, HIS 113, ASP 109), for Hsp 90 it was −6.3 kcal/mol (H-bonds: SER 21, PHE 42, GLY 43) with ODR 1 (H-bonds: ALA 421) and ODR 3 (H-bonds: CYS 326) it was −5.9 kcal/mol, with the target nGPCR (H-bonds: ASN 390) it was −6.7 kcal/mol and with cpl-1 (H-bonds: GLN 310) it was – 6.3 kcal/mol. Hydrogen bonds were formed with the backbone and side chain of binding site residues. The docked complex also displayed other types of contacts, including hydrophobic interactions, van der Waals, pi–pi stacking, alkyl and pi–alkyl (Figure S10).

Figure 6. Illustrative diagram representing the protein–ligand interaction between potential protein target AchEof Mi and Carbofuran 3G.

4. Discussion

Root-knot nematodes (RKN) are sedentary obligate endoparasites. They can be found all over the world in different geographical regions but they are more prevalent in tropical and subtropical climate zones. They are considered the greatest threat to crops in the majority of developing countries. There is a growing need to establish sustainable management strategies and treatments for RKN control due to their significance to the economy. Despite being widely utilized, cultural controls are more constrained due to the wide host range of Meloidogyne species and the occurrence of mixed populations of many RKN species in the field [24,25]. Nematicide application has continued to be the most popular short-term management tactic against RKN. But in recent decades, several compounds, like methyl bromide and aldicarb, have been taken off the market because of risks to the environment, human health and non-target organisms [26,27]. In the current investigation, we tested the in-vitro nematicidal activities of native isolates of C. rosea against root-knot nematode Meloidogyne incognita (Mi) and employed the molecular modelling tools to understand the interaction of their small biomolecules against the seven potential target proteins of Mi.

Conidial suspension of effective isolate C. rosea TNAU CR 01 was tested for their bioefficacy against Mi through the reduction in the hatching of eggs and mortality of juveniles under in-vitro conditions. A significant reduction in the hatching ability of M. incognita was observed when treated with 25% conidial suspension of TNAU CR 01 isolate for about 96 hrs. According to Wang et al. [28], C. rosea ferment filtrate significantly reduced nematode egg hatching by 80.4% and improved juvenile mortality by 32.0%, respectively. The inhibition of nematode egg hatching and juvenile activity was lowered by diluting the ferment filtrate. Kassam et al. [29] demonstrated over 90% death of infectious second-stage juveniles after 24 hrs of exposure to the fungal filtrate Paecilomyces tenuis, along with 87% parasitization. Under both in-vitro and in-vivo settings, the fungal filtrate dramatically decreased the hatching of eggs and the penetration of Mi’s host roots. At an IC₅₀ of 2.85 ± 0.12 mg mL⁻¹ of the fungal filtrate, the isolate also exhibited acetylcholinesterase inhibition.

The mortality test revealed a significant increase in death of Mijuveniles at 6 × 10⁷ conidiaml⁻¹ of TNAU CR 01 culture filtrate incubated for 96 hrs. The highest number of dead juveniles (65.45 dead juveniles) was recorded at 25%conidial suspension of TNAU CR 01 incubated for 96 hrs. The findings of Rodríguez-Martínez et al. [30] provided evidence of C. rosea’s strong in-vitro predatory activity against five distinct nematodes (Haemonchuscontortus, Caenorhabditis elegans, Rhabditis sp., Panagrellus redivivus and Butlerius sp.). Butlerius sp. was the most vulnerable prey relative to the other evaluated nematodes and C. rosea’s antagonistic activity against various prey nematodes ranged from 71.9 to 100% following a five-day contact. Keerthiraj et al. [31] showed that essential oils hydro-distilled from Pogostemon cablin leaves may have a nematicidal impact on Mi juveniles. It was also proven that mortality rises with increasing time to 48 (LC₅₀ 33.6–71.6 µg mL⁻¹) and 72 hrs (LC₅₀ 27.7–61.2 µg mL⁻¹). Kundu et al. [32] extracted the volatile oil from the leaves of Eupatorium adenophorum and used gas chromatography–mass spectrometry (GC–MS) to analyze the substance. It was determined that the essential oil’s main component was sesquiterpene. Their antinemic activity ranged from LC₅₀ 133.7 to 189.2 µg mL⁻¹ when tested on Mi juveniles. All six isolates of C. rosea were tested for their ability to parasitize the eggs of Mi. Among the six isolates tested, the maximum egg parasitization capacity of C. rosea was observed in egg mass treated with TNAU CR 01 isolate by achieving 69.38% parasitization over control. Trifonova et al. [33] reported 7.6–23.5% of M. incognita egg infections because of the presence of Gliocladium roseum.

In this study, in-silico molecular modelling was used to understand and identify the underlying causes of Mi’s behavioural reaction to C. rosea exposure. The favourable docking scores of 2,3-dihydroxypropanal and Dihydro-4-hydroxy-2(3H)-furanone biomolecules on each of the seven potential target proteins suggested that C. rosea may act in several inhibitory modes. Plant parasitic nematodes have neurosensitive receptor proteins that are important for neuronal processes and are commonly targeted for pharmacological blockade [34]. There have been reports of molecules’ modes of action, including their disruption and/or change of the permeability of nematode cell membranes and their suppression of neurosensitive receptor proteins like AChE [35]. They control synaptic transmission and movement mechanisms. Docking results confirmed the potential use of Dihydro-4-hydroxy-2(3H)-furanone biomolecules an alternative nematicide since it has the highest binding affinity with the lowest binding energy of −7.5 kcal/mol against AChE compared with commercial nematicide carbofuran 3G (−7.4 kcal/mol). To investigate the potential suppression of neurotransmission and chemosensing functions, Dutta et al. [36] conducted an in-silico study using allyl isothiocyanate (AITC), a major component of black mustard’s (Brassica nigra) essential oil against microorganisms. AITC had the highest binding affinity with the binding sites of acetyl cholinesterase (AChE: −10.5 kcal/mol), followed by odorant response gene-1 (ODR1: −8.9 kcal/mol) and neuropeptide G-protein coupled receptor (nGPCR: −6.5 kcal/mol). The oxidative phosphorylation pathway, a component of energy metabolism, uses cytochrome c oxidase subunit 1 [37]. The highest binding affinity with the lowest binding energy value against COX was recorded by Dihydro-4-hydroxy-2(3H)-furanone (−7.8 kcal/mol), followed by 2,3-dihydroxypropanal (−6.9 kcal/mol). Hsp90 develops its full functional activity in collaboration with other co-chaperones and is crucial for the folding of newly generated proteins as well as the stability and refolding of denatured proteins under stress [38]. The highest binding affinity with the lowest binding energy score of −8.4 kcal/mol was recorded by Dihydro-4-hydroxy-2(3H)-furanone against Hsp90 followed by 2,3-dihydroxy propanal (−6.5 kcal/mol) which is higher than the docking score of carbofuran 3G (−6.3 kcal/mol). Chemosensory processes are controlled by ODR1 and ODR3 [39]. The docking score of 2,3-dihydroxy propanal against ODR 1 and ODR 3 was −6.6 kcal/mol and −6.4 kcal/mol, respectively which was followed by Dihydro-4-hydroxy-2(3H)-furanone (−6.1 kcal/mol) against ODR 1 and −6.5 kcal/mol against ODR 3 which were considerably higher when compared with carbofuran 3G (−5.9kcal/mol) indicated that both the biomolecules are capable of affecting the chemosensory processes of Mi. The neuropeptide GPCR is involved in controlling the parasite’s migration toward (or inside of) its host [40]. The highest binding affinity with the lowest binding energy value of −7.6 kcal/mol was achieved by Dihydro-4-hydroxy-2(3H)-furanone followed by 2,3-dihydroxy propanal (−7.0 kcal/mol) against the target nGPCR indicated that they will play a major role in controlling the migration of Mi far better than carbofuran 3G (−6.7 kcal/mol). Dihydro-4-hydroxy-2(3H)-furanone compound with the maximum binding affinity (low-binding energy) of – 6.8 kcal/mol against the target cpl-1 protein will be the best choice among 2,3-dihydroxypropanal (−6.1 kcal/mol) and carbofuran 3G (−6.3 kcal/mol) as an alternate nematicide. As a result, the present study is distinctive in the way that 2,3-dihydroxy propanal and Dihydro-4-hydroxy-2(3H)-furanone biomolecules could be developed as a formulation for the control of the root-knot nematode. In addition, the research results highlighted the opportunity for utilizing both the biomolecules as special small molecules to regulate the nematodes that infects tomato plants. Nevertheless, additional research is necessary to validate the nematicidal characteristics of both the biomolecules in a wet lab by qRTPCR and transcriptome profiling of treated and untreated tomato plants challenged with Mi. Therefore, it would also provide new information about how to do 2,3-dihydroxy propanal and Dihydro-4-hydroxy-2(3H)-furanone generated by C. rosea work against the target nematodes.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Disclosure statement

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