Green Synthesized Silver Nanoparticles Mitigate Biotic Stress Induced by Meloidogyne incognita in Trachyspermum ammi (L.) by Improving Growth, Biochemical, and Antioxidant Enzyme Activities.

Meloidogyne incognita is an important plant-parasitic nematode that causes significant crop losses all over the world. The primary control strategy for this pathogen is still based on nematicides, which are hazardous to human health and the environment. Considering these problems, this study aimed to determine the efficacy of different concentrations (25, 50, and 100 ppm) of silver nanoparticles against M. incognita on Trachyspermum ammi. Silver nanoparticles synthesized from Senna siamea were thoroughly characterized using various physicochemical techniques, viz., UV-visible spectrophotometer, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray analyzer (EDX). Results revealed that plants treated with 50 ppm silver nanoparticles one week before M. incognita inoculation (T2) exhibited maximum and significant (p ≤ 0.05) increases in plant growth, biochemical characteristics, and activities of defense enzymes such as peroxidase, catalase, superoxide dismutase, and ascorbate peroxidase over the inoculated control (IC) plants. Furthermore, the maximum reduction in the number of galls, egg masses, and root-knot indices was recorded in plants treated with 100 ppm silver nanoparticles (T3) followed by plants treated with 50 ppm silver nanoparticles before nematode inoculation (T2), over inoculated plants (IC). Anatomical studies showed accumulation of lignin in the transverse section (TS) of roots treated with 50 ppm silver nanoparticles. As a result, the present finding strongly suggests that silver nanoparticles synthesized from S. siamea had nematicidal activity, and it could be an efficient, safe, cost-effective, and affordable alternative to chemical nematicide.

Introduction

Herbs have always been used to promote healing in all cultures. Plants continue to be the foundation for the growth of modern medicines, and medicinal plants have been used in everyday life for years to treat illnesses all over the world.[1] Natural medicine derived from plant parts or extracts has sparked renewed interest in recent years. In this regard, Trachyspermum ammi (L.) is an annual herbaceous plant commonly known as ajwain having a place with the exceptionally esteemed restoratively significant family, Apiaceae.[2] It is extensively distributed and cultivated in different countries such as Afghanistan, Iran, and India as well as Europe, while it originated in Egypt.[3] Grayish-brown seeds of T. ammi are usually utilized for medicinal and alimentary purposes, and they work as a powerful therapeutic agent in treating hemorrhoids, abdominal tumors, and abdominal pain.[4] Essential oil produced from T. ammi seeds contains about 50% thymol, which is a highly esteemed fungicide and strongly antiseptic.[5] Thymol is used in the preparation of toothpaste and perfumery.[6] The crop yield is influenced negatively due to the development of diseases caused by several pathogens such as Alternaria blight (Alternaria alternata), collar rot (Sclerotium rolfsii), powdery mildew (Eryiphe polygoni),[7] and root-knot disease caused by Meloidogyne incognita.[8]

Meloidogyne spp., are obligate sedentary endoparasites and infect more than 3000 plant species throughout the world.[9] Because they infect the root system, which absorbs water and minerals, the entire plant gets damaged. Their population is rapidly increased due to the completion of two or more life cycles in a single growing season, combined with high female fecundity. Hence, crop yield decline leads to huge monetary misfortunes of a few huge numbers of dollars around the world.[10]

Chemical nematicides have been restricted to be used for controlling this pathogen due to harmful effects on the environment and health hazards.[11,12] Such biologically disturbing forces pave the way for unacceptably poor well-being, stability, and a slew of environmental problems.[13] The potential risk to nontarget organisms has led researchers to search for novel and eco-friendly or less toxic methods for nematode management. To mitigate this issue, eco-friendly methods and sustainable crop protection technologies need to be employed in the current agriculture system, which can be easily used for protecting crop damage caused by nematodes to maintain growth and yield of the plant.

In recent years, nanotechnological science has been visualized to have the potential to transform agriculture systems.[14] Disease management and agriculture are the most significant areas of nanotechnology.[15,16] Nanoparticles with a small size and large surface area of 1–100 nm have potential application in agriculture systems. Several studies have observed that the nonmaterial is an alternative to chemical pesticide, making it an excellent option for new pesticide development. Silver nanoparticles are known for their broad spectrum of antimicrobial activities and plant disease management.[17,18] Silver nanoparticles synthesized from plants are less toxic in comparison to silver ions, while their antimicrobial properties increase markedly.[19,20] Due to the different modes of inhibitory properties against phytopathogens, they can be used for the control of various plant diseases sustainably as compared to chemical fungicides.[21] Examinations on the appropriateness of nanosilver for controlling plant diseases have been very few, to date.[22]

Although chemical and physical techniques are available for the synthesis of silver nanoparticles, all of these are associated with hazardous chemicals and require high amounts of energy.[23] Plant-mediated synthesis of silver nanoparticles is gaining popularity due to its low toxicity, cost efficiency, environmental friendliness, and less time requirement. Plants with antimicrobial properties, in particular, have a high potential for green synthesis of silver nanoparticles due to their complex phytochemical composition.[24] Further, plants are an easily available and rich source of secondary metabolites such as protein, polyphenols, flavonoids, and alkaloids, which act as a reducing agent and can cap the desired synthesized nanoparticles. In this context, Senna siamea has the vast potential of antimicrobial activity due to the presence of secondary metabolites such as phenolics, alkaloids, saponins, tannins, glycosides, and flavonoids.[25] For this reason, this study aimed to investigate the nemato-toxic properties of biosynthesized silver nanoparticles against M. incognita, which cause root-knot disease in T. ammi. The plant growth, yield, and biochemical characteristics were measured in response to silver nanoparticles under the stress of M. incognita.

Results

Characterization of Silver Nanoparticles Synthesized From Senna seamia Leaf Extract

The primary characterization of silver nanoparticles was performed using UV–visible spectroscopy. The morphology and size of bio-blended silver nanoparticles were selected based on an examination performed by electron microscopy (SEM), energy-dispersive X-beam spectroscopy (EDX), and transmission electron microscopy (TEM). The chemical characterization was performed using Fourier transform infrared (FTIR) spectroscopy.

UV–Vis Spectroscopic Analysis

UV–visible spectroscopy was deployed for preliminary characterization of silver nanoparticles. The initial color of the reaction mixture was light yellow, which turned to dark brown, indicating the formation of silver nanoparticles. The reaction mixture initially showed a weak absorbance in the UV–vis region. When the color started to change, the absorption band at 430 nm was recorded, as shown in Figure A.

Figure 1

Characterization of silver nanoparticles synthesized from Senna seamia leaf extract. UV–vis spectra of silver nanoparticles (AgNPs) (A). FTIR spectrum of silver nanoparticles (B). Transmission electron micrographs of silver nanoparticles (AgNPs) (C). Scanning electron micrographs of silver nanoparticles (AgNPs) (D). EDX spectrum of silver nanoparticles (AgNPs) (E). Histogram of wt % of major elements in silver nanoparticles (AgNPs) (F).

FTIR Spectroscopic Analysis

FTIR analysis was performed to decipher the role of phytoconstituents of the plant extract, responsible for the capping and stabilization of silver nanoparticles (Figure B). The FTIR spectrum of silver nanoparticles showed a number of bands, especially from 500 to 3500 cm–1, indicating the presence of various functional groups on the silver nanoparticles.

Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) Analyses

Transmission electron microscopy was performed to provide further understanding of the size, shape, and morphology of the blended silver nanoparticles (Figure C). The TEM images show that the shape of the incorporated silver nanoparticles was round and polydisperse with widths in the range of 05–60 nm. Scanning electron microscopy (SEM) investigation was performed to assess the surface morphology of synthesized silver nanoparticles. The SEM image of silver nanoparticles is shown in Figure D. As is evident from the figure, the biosynthesized nanoparticles varied in shape and size, indicating the nonuniform size distribution. The energy-dispersive X-ray (EDX) spectrum of silver nanoparticles deciphered the presence of silver, oxygen, carbon, and chlorine as the most abundant elements, as presented in Figure E. The weight percentages of silver, oxygen, and carbon were found to be 58.69, 24.14, and 16.19%, respectively (Figure F).

Green Synthesized Silver Nanoparticles Suppressed Phytonematodes and Improved Growth Features

Growth and Yield Parameters

The length of the root and shoot of IC plants decreased significantly when compared with uninoculated plants (control). There was a significant increase in the length of the plants T1, T4, T5, T6, T7, T8, and T9 in comparison to plants inoculated with M. incognita. Moreover, reduction in the T2 plants was nonsignificant when compared with uninoculated untreated plants. There was a nonsignificant increase in the length of T3 plants, which was treated with 100 ppm silver nanoparticles, 1 week prior to nematode inoculation, when compared with IC plants. The length of T3 plants, which were treated with 100 ppm silver nanoparticles, was higher than that of nematode-inoculated plants (IC) (Table ).

Table 1

Effect of Different Concentrations (25, 50, and 100 ppm) of Green Synthesized Silver Nanoparticles (AgNPs) on Plant Height, Fresh Weight, and Dry Biomass of T. ammi Plants Inoculated with M. incognita under the Greenhouse Conditiona

treatment		shoot length (cm)	root length (cm)	shoot fresh weight (g)	root fresh weight (g)	shoot dry weight (g)	root dry weight (g)
control (C)		68.17 ± 0.60a	12.76 ± 0.14a	52.50 ± 0.28a	7.00 ± 0.11a	12.8 ± 0.11a	0.95 ± 0.01a
inoculated control (IC)		46.40 ± 0.49i	6.00 ± 0.17g	32.64 ± 0.47i	4.00 ± 0.15g	7.65 ± 0.08e	0.55 ± 0.01g
silver nanoparticles 1 week prior to M. incognita	25 ppm (T1)	53.50 ± 0.47d	7.43 ± 0.23d	38.58 ± 0.34d	4.85 ± 0.16d	8.81 ± 0.16c	0.72 ± 0.00d
	50 ppm (T2)	67.26 ± 0.72a	12.60 ± 0.20ab	51.87 ± 0.74ab	6.93 ± 0.23a	12.62 ± 0.16a	0.94 ± 0.01a
	100 ppm (T3)	47.30 ± 0.47hi	6.28 ± 0.23fg	33.86 ± 0.63h	4.01 ± 0.09g	7.83 ± 0.22e	0.56 ± 0.00g
simultaneous inoculation of M. incognita and silver nanoparticles	25 ppm (T4)	51.50 ± 0.63e	6.90 ± 0.14ef	37.00 ± 0.50e	4.72 ± 0.19de	8.61 ± 0.14c	0.70 ± 0.01de
	50 ppm (T5)	66.20 ± 0.41ab	12.06 ± 0.23b	51.17 ± 0.25b	6.39 ± 0.18b	12.51 ± 0.07a	0.88 ± 0.01b
	100 ppm (T6)	49.20 ± 0.58fg	6.40 ± 0.11efg	35.18 ± 0.10fg	4.24 ± 0.16efg	8.06 ± 0.13de	0.66 ± 0.01e
M. incognita 1 week prior to silver nanoparticles	25 ppm (T7)	50.50 ± 0.57ef	6.73 ± 0.20de	35.46 ± 0.20f	4.60 ± 0.12def	8.51 ± 0.18cd	0.69 ± 0.00de
	50 ppm (T8)	65.10 ± 0.49c	11.50 ± 0.11c	49.07 ± 0.22c	5.81 ± 0.18c	11.98 ± 0.22b	0.82 ± 0.02c
	100 ppm (T9)	48.50 ± 0.28gh	6.43 ± 0.13efg	34.02 ± 0.14gh	4.16± 0.11fg	7.86 ± 0.13e	0.62 ± 0.01f
least-square mean		55.78	8.64	41.03	5.15	9.75	0.73
LSD@≤0.5		1.57	0.53	1.19	0.47	0.46	0.04
F-value		275.1	248.9	404.5	51.03	195.1	100.20

Each value is a mean (mean ± SE) of five replicates (n = 5). Different alphabets denote significant difference from each other and with respective treatments (at p ≤ 0.05) according to Duncan’s multiple range test (DMRT).

Plant Fresh Weight and Dry Biomass

A nonsignificant reduction was observed in both shoot and root fresh weight of T2 plants when compared with control plants. The maximum and highest reduction was noticed in IC plants, which were treated with nematodes only. The value of T3 plants was higher but was nonsignificantly different in comparison to IC plants. Significant increases in the values of T1, T4, T5, T6, T7, T8, and T9 plants were observed when compared with the IC plants (Table ).

The most noteworthy and huge decline in the dry weight of shoot and root was seen in IC plants that were treated with nematode only, compared with the uninoculated plants. There was a significant increase in the values of T1, T4, T5, T6, T7, T8, and T9 plants when compared with the values of IC plants. Maximum and significant increases in the values of shoot and root dry weight were observed in T2 plants compared with the IC plants. The value of T2 plants was nonsignificant when compared with the value of control plants (Table ).

Numbers of Branches and Numbers of Umbels per Plant

Maximum and significant reductions in the number of branches and number of umbels per plant were encountered in IC plants inoculated with only nematodes, in comparison with the control plants. The values of T2 plants treated with 50 ppm silver nanoparticles 1 week prior to nematode inoculation decreased nonsignificantly over the control plants. Further significant increases in T1, T4, T5, T6, T7, T8, and T9 plants were also observed when compared with IC plants. Nonsignificant increase was observed when the values of T3 and IC plants were compared (Table ).

Table 2

Effect of Different Concentrations (25, 50, and 100 ppm) of Green Synthesized Silver Nanoparticles (AgNPs) on the Number of Branches, Number of Umbel, Seed Yield, and Seed Oil Content of T. ammi Plants Inoculated with M. incognita under the Greenhouse Conditiona

treatment		number of branches per plant	number of umbels per plant	seed yield per plant (g)	seed oil content (%)
control (C)		15.6 ± 0.24a	75.6 ± 0.92a	6.46 ± 0.11a	2.73 ± 0.015a
inoculated control (IC)		8.8 ± 0.37g	39.4 ± 0.50j	3.65 ± 0.12h	1.60 ± 0.012i
AgNP 1 week prior to M. incognita	25 ppm (T1)	11.00 ± 0.37c	61.6 ± 0.67d	5.25 ± 0.11d	2.24 ± 0.01d
	50 ppm (T2)	15.4 ± 0.24a	74.0 ± 0.70a	6.20 ± 0.15ab	2.71 ± 0.015a
	100 ppm (T3)	9.4 ± 0.24fg	43.0 ± 0.83i	3.85 ± 0.14gh	1.62 ± 0.014hi
simultaneous inoculation of M. incognita and AgNP	25 ppm (T4)	10.6 ± 0.24cd	58.2 ± 0.86e	5.15 ± 0.11d	2.02 ± 0.014e
	50 ppm (T5)	14.4 ± 0.24b	70.0 ± 0.54b	6.02 ± 0.13bc	2.65 ± 0.012b
	100 ppm (T6)	9.8 ± 0.20def	49.2 ± 0.37g	4.26 ± 0.12f	1.72 ± 0.015g
M. incognita 1 week prior to AgNP	25 ppm (T7)	10.4 ± 0.24cde	53.6 ± 0.60f	4.72 ± 0.12e	1.87 ± 0.012f
	50 ppm (T8)	13.8 ± 0.37b	67.2 ± 0.58c	5.68 ± 0.11c	2.57 ± 0.014c
	100 ppm (T9)	9.6 ± 0.24efg	46.0 ± 0.54h	4.16 ± 0.11fg	1.65 ± 0.014h
least-square mean		11.70	57.98	5.03	2.12
LSD@≤0.5		0.80	1.91	0.37	0.04
F- value		82.54	359.1	60.29	1099

Each value is a mean (mean ± SE) of five replicates (n = 5). Different alphabets denote significant difference from each other and with respective treatments (at p ≤ 0.05) according to DMRT.

Seed Oil Content

There was an enhancement in the seed oil content in T2 plants when compared with IC plants. The values of seed oil content in T2 plants were at par with the values of uninoculated and untreated plants (control). The highest and significant reduction was observed in IC plants compared with the control plants. Significant increases in T1, T4, T5, T6, T7, T8, and T9 plants were observed when compared with IC plants. Maximum and significant increases were noticed in T2 plants, in comparison with IC plants. The values of seed oil content in the IC and T3 plants were at par, although higher than in T3 plants (Table ).

Effect of Green Synthesized Silver Nanoparticles on Photosynthetic Pigments and Biochemical Characteristics of T. ammi Infected with M. incognita

Photosynthetic Pigments

Chlorophyll a

The chlorophyll content of IC plants decreased significantly when compared with the control plant. Compared with IC plants, the amount of chlorophyll a was found significantly increased in T2 plants and was at par with the control plants. There was a significant increase in the amount of chlorophyll a in the T1, T4, T5, T6, T7, T8, and T9 plants in comparison with IC plants. Nonsignificant and lowest increase in the chlorophyll a content of T3 plants was observed when compared with IC plants (Figure A).

Figure 2

Effect of different concentrations (25, 50, and 100 ppm) of S. siamea-synthesized silver nanoparticles (AgNPs) on chl a (A), chl b (B), and carotenoid content (C) of T. ammi plants inoculated with M. incognita. In these figures, bar diagrams represent the mean values (mean ± SE) of five replicates (n = 5). Error bars represent standard errors (±SE). Different alphabets denote significant difference between the control and with respective treatments (at p ≤ 0.05) according to Duncan’s multiple range test (DMRT). C, control; IC, inoculated control.

Chlorophyll b

The amount of chlorophyll b was significantly reduced in IC plants compared with the control plants. Nonsignificant reduction in the chlorophyll b content was noticed in T2 and T5 plants when compared with the control plants. The values were nonsignificant by difference among T1, T4, and T7 plants and were on the order of T1 > T4 > T7, respectively, but increased significantly compared with the IC plants. The value of chlorophyll b was nonsignificant when compared between IC and T9, although a slight increase was noticed in T3 plants followed by T6 plants (Figure B).

Carotenoid Content

The highest and significant reduction in the carotenoid content was noticed in IC plants when compared with control plants. The values between T3 and T9 plants were nonsignificant, but they were significantly higher when compared with IC plants, which were inoculated with nematode only. Highest and significant increases in T2 and T5 plants were observed when compared with IC plants, and at par with the control plants, although the values of T2 plants were more than those of T5 plants (Figure C).

Biochemical Features

Nitrate Reductase and Carbonic Anhydrase Activity

Nitrate reductase and carbonic anhydrase activities were found decreased in IC plants, which were inoculated with nematode only. Both enzymatic activities increased significantly in all of the treated plants when compared with the IC plants. The values of nitrate reductase (Figure A) and carbonic anhydrase activity (Figure B) were increased and the highest in T2 plants and the lowest in T9 plants compared with IC plants. The T2 plants, which were treated with 50 ppm silver nanoparticles 1 week prior to Meloidogyne inculcation, proved to be the best concentration, and both enzymatic values were reduced nonsignificantly when compared with the values of uninoculated untreated plants (control).

Figure 3

Effect of different concentrations (25, 50, and 100 ppm) of S. siamea-synthesized silver nanoparticles (AgNPs) on nitrate reductase activity (A) and carbonic anhydrase activity (B), leaf nitrogen content (C), and leaf protein content (D) of T. ammi plants inoculated with M. incognita. In these figures, bar diagrams represent the mean values (mean ± SE) of five replicates (n = 5). Error bars represent standard errors (±SE). Different alphabets show significant difference between the control and with respective treatments (at p ≤ 0.05) according to Duncan’s multiple range test (DMRT). C, control; IC, inoculated control.

Leaf Nitrogen Content

Leaf nitrogen content was found increased significantly in all of the treatments when compared with IC plants (Figure ). The enhancement in the leaf nitrogen content was the maximum and significant in T2 plants followed by T5 and T8 plants, in comparison to the values of IC plants, but the values of T2 were at par with the values of control plants. The highest and significant decrease was observed in IC plants when compared with control plants. The results of T3, T6, and T9 plants were nonsignificant when compared with the values of IC plants (Figure C).

Figure 4

Effect of different concentrations (25, 50, and 100 ppm) of S. siamea-synthesized silver nanoparticles (AgNPs) on the number of galls formed by nematodes ( A), number of egg masses (B), nematode populations in the soil (C), nematode population in the root (D), the root-knot index (E), and the egg mass index (F) in T. ammi plants inoculated with M. incognita. In these figures, bars, lines, and scatter diagrams represent the mean values (mean ± SE) of five replicates (n = 5). Error bars represent standard errors (±SE). Different alphabets show significant difference between the control and with respective treatments (at p ≤ 0.05) according to Duncan’s multiple range test (DMRT). C, control; IC, inoculated control.

Leaf Protein Content

The protein content in the leaves significantly decreased in all of the plants when compared with the healthy plants. Maximum and significant decreases in protein content were noticed in IC plants inoculated with M. incognita alone compared with the values of uninoculated plants (control). The least but significant reduction was observed in T2 plants in comparison to control plants. The values were nonsignificant between T2 and T5 plants but increased significantly compared with IC plants. The protein content was higher in T2 plants than in T5 plants (Figure D).

Activities of the Defense Enzyme

With the increase in concentration of silver nanoparticles, all of the enzyme activities like peroxidase (POS), catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) increased considerably (Figure A–D). The maximum and significant (p ≤ 0.05) enhancement was found in T3 plants treated with 100 ppm silver nanoparticles 1 week prior to M. incognita inoculation. Significant (p ≤ 0.05) and lowest increases were obtained in T1 plants treated with 25 ppm silver nanoparticles 1 week after the inoculation of M. incognita. Additionally, plants (IC) inoculated with M. incognita only exhibited a significant increase over control plants (C).

Figure 5

Effect of different concentrations (25, 50, and 100 ppm) of S. siamea-synthesized silver nanoparticles (AgNPs) on the activity of the defense antioxidant enzyme (A) peroxidase, POD; (B) catalase, CAT; (C) superoxide dismutase, SOD; and (D) ascorbate peroxidase, APX in T. ammi plants inoculated with M. incognita. In these figures, bars, lines, and scatter diagrams represent the mean values (mean ± SE) of five replicates (n = 5). Error bars represent standard errors (±SE). Different alphabets show significant difference between the control and with respective treatments (at p ≤ 0.05) according to Duncan’s multiple range test (DMRT). C, control; IC, inoculated control.

Number of Galls and Number of Egg Masses

Gall formation was observed in all M. incognita-treated plants. However, the number of galls and number of egg masses were higher in IC plants, in comparison to other plants that were treated with silver nanoparticles. In all of the treatments, the number of galls and number of egg masses were found reduced significantly compared with IC plants. The highest and significant reduction in gall formation and egg mass production was noticed in T3 plants followed by T2 plants when compared with IC plants. The lowest but significant decrease was observed in T1 plants followed by T4 and T7 plants in comparison to IC plants (Figure A,B).

Nematode Population (Root and Soil)

The nematode population was the highest in IC plants that were inoculated with M. incognita only. Among the treated plants from T1 to T9, significant reductions in the nematode population were observed when compared with the plants inoculated with nematodes only. Maximum and significant reductions were observed in T3 plants followed by T6 plants in comparison to T1, T2, T4, T5, T7, T8, and T9 plants (Figure B,C).

Principal Component Analysis (PCA) and Pearson Correlation Analysis

Biplot results of principal component analysis (PCA) accounted for a total variance of 94.37% (PIC = 57.87%; PC2 = 36.50%) in plants treated with different concentrations of silver nanoparticles (25, 50, 100 ppm) and inoculated with M. incognita (Figure A). Growth, yield, and biochemical parameters (shoot and root dry weight, seed yield per plant, seed oil content, chlorophyll a, nitrate reductase activity, and carbonic anhydrase) are clustered together to explain the negative correlation with the nematode disease parameters like number of gall and number of egg masses. Similarly, increases in the activities of defense enzymes like POD, CAT, SOD, and APX were also found to be negatively correlated with the number of galls and number of egg masses. Significant correlations between each of the two variables (p-value 0.05) are shown in Figure B (for the Pearson correlation matrix, see the Supporting Information Table S1).

Figure 6

Relationships of some studied parameters of T. ammi at different concentrations of silver nanoparticles (AgNPs) (25, 50, 100 ppm) before, simultaneously, and after 1 week of M. incognita inoculation. (A) Biplot results of principal component analysis (PCA) of various growth, yield, biochemical, and antioxidant enzyme and nematode parameters. (B) Pearson correlation results. SDW, shoot dry weight; RDW, root dry weight; SYPP, seed yield per plant (g); SOC, seed oil content (%); Chl a, chlorophyll a; NRA, nitrate reductase activity; CA, anhydrase activity; POD, peroxidase; CAT, catalase; SOD, superoxide dismutase; APX, ascorbate peroxidase; NOG, number of galls per root system; and NOE, number of egg masses per root system. Control, untreated and uninoculated; IC, inoculated with 2000 J2 of M. incognita only; T1, 25 ppm silver nanoparticles 1 week prior to 2000 J2 of M. incognita; T2, 50 ppm silver nanoparticles 1 week prior to 2000 J2 of M. incognita; T3, 100 ppm silver nanoparticles 1 week prior to 2000 J2 of M. incognita; T4, simultaneous inoculation of 2000 J2of M. inocgnita and 25 ppm silver nanoparticles; T5, simultaneous inoculation of 2000 J2 of M. inocgnita and 50 ppm silver nanoparticles; T6, simultaneous inoculation of 2000 J20of M. inocgnita and 100 ppm silver nanoparticles; T7, 2000 J2 of M. inocgnita inoculation 1 week prior to 25 ppm silver nanoparticles; T8, 2000 J2of M. inocgnita inoculation 1 week prior to 50 ppm silver nanoparticles; and T9, 2000 J2 of M. inocgnita inoculation 1 week prior to 100 ppm silver nanoparticles.

Anatomical Observation

Histopathological observation in the transverse section of the roots of T. ammi of different treatments showed a marked defense strategy adopted by plants against M. incognita infection. The plants that were inoculated with M. incognita only (IC) showed maximum damage including an abnormal xylem and phloem and 5–8 multinucleate giant cells at the site of feeding. The juveniles were clearly observed in the infected roots, which showed minimum accumulation of lignin (Figure B). Remarkable variations in lignin deposition of vascular bundles were observed among different treatments. Lignin was highly deposited in T2 plants that were treated with 50 ppm silver nanoparticles prior to M. incognita inoculation (Figure C). Xylem shape, phloem cells, and cortical cell size were reduced and got damaged in plants that were treated with 100 ppm silver nanoparticles (Figure D).

Figure 7

Effect of different concentrations of green synthesized silver nanoparticles (AgNPs) on lignification in T. ammi roots. Transverse section of control root (A), M. incognita-infected root (B), 50 ppm silver-nanoparticle-treated root + M. incognita inoculation (C), and 100 ppm silver-nanoparticle-treated root + M. incognita inoculation (D). Abbreviations: NX, normal xylem; NP, normal phloem; NCC, normal cortical cell; LD, lignin deposition; AX, abnormal xylem; AP, abnormal phloem; GC, giant cell; N, nematode; DCC, distorted cortical cell; RP, reduced phloem; RX, reduced xylem.

Discussion

As the aqueous leaf extract of S. siamea was added to silver nitrate solution, the color of the solution changed from yellow to brown because of the reduction of silver nitrate solution to silver ions (Ag+), and this indicates the formation of silver nanoparticles.[26] The absorption band in this range (300–350 nm) is attributed to the surface plasmon band of silver nanoparticles. Silver nanoparticles exhibit brown color in aqueous suspensions, which is due to the oscillation modes arising from the electromagnetic field in the visible range and also due to the collective oscillations of conduction electrons.[27] The absorption peak of biosynthesized silver nanoparticles was found to be broad, which can be attributed to the polydispersed nature of the nanoparticles.[28,29] Moreover, the asymmetrical absorption spectrum of silver nanoparticles indicates the size variation and anisotropy of nanoparticles.[30]

The absorption band at 2924 cm–1 is assigned to the vibrations of secondary amines.[31] The broad band at 3432 cm–1 is due to the stretching of O–H groups.[32] Moreover, the peak at 824 cm–1 is characteristic of the aromatic ring.[33] This indicates the role of aromatic compounds in the stabilization or surface coating of silver nanoparticles. The FTIR bands near 2390 cm–1 confirm the presence of alkynes on the surface of silver nanoparticles.[34] The presence of carboxylic acids was also confirmed by the band at 1762 cm–1.[35] The very sharp band at 1382 cm–1 may be attributed to the −C–O stretching mode.[36] It has been documented in the literature that the size distribution of nanoparticles depends on the relative rate of nucleation as well as on the extent of agglomeration.[37]

This study revealed that the growth, yield, and biochemical characteristics of T. ammi decreased in the presence of M. incognita (Tables and 2). Reductions were higher in the length and weight of roots than in shoots. The decrease in growth attribute might be due to the malfunctioning of tissues in the stellar region of the affected root. It has been observed that plants infected with M. incognita caused significant reductions in growth parameters[38,39] and biochemical characteristic[40] of the plant. Application of silver nanoparticles decreased the nematode population and improved the growth and yield parameters of T. ammi. These results were in accordance with the earlier findings of Abdellatif et al.,[41] in which the authors demonstrated that eggplants treated with green synthesized silver nanoparticles showed a significant increase in plant growth and decrease in gall number, egg mass number, and soil population of the nematode. Cromwell et al.[42] observed nematicidal effects of silver nanoparticles on Bermuda grass infected with the root-knot nematode. Silver nanoparticles are highly reactive and effective even under lower concentrations and act as antimicrobial agents.[43] From our studies, it was evident that the use of silver nanoparticles greatly influenced the plants when applied at appropriate times and concentrations. It was noticed that 50 ppm silver nanoparticles applied 1 week before M. incognita inoculation (T2 plants) caused the highest and maximum reduction in disease incidence followed by simultaneous inoculation (T5 plants). A higher concentration of 100 ppm silver nanoparticles (T3, T6, and T9 plants) applied before, simultaneously, or after M. incognita inoculation proved toxic to the plant. These findings support those of Lamsal et al.[22]

In the present study, it was found that growth and yield were increased due to the enhanced production of photosynthetic pigments (chl a, chl b, and carotenoids). The rate of photosynthesis depends on the quality and intensity of light.[44] UV-B radiation leads to aggregation of reactive oxygen species (ROS) in the chloroplast, which destroys PS II, leading to a reduced rate of electron transfer and damage of the thylakoid membrane. The nanoparticles alleviate the effects of UV-B radiation and improve the photosynthetic rate.[45−47] The present study revealed that nitrate reductase and carbonic anhydrase activity along with leaf protein content and leaf nitrogen content significantly increased in plants treated with silver nanoparticles. Application of 50 ppm silver nanoparticles, 1 week before M. incognita inoculation (T2 plants), showed the maximum increase in biochemical characteristics followed by simultaneous inoculation of 50 ppm silver nanoparticles and M. incognita (T5 plants). Significant increases were also recorded in other treatments (T1, T4, T6, T7, T8, and T9). Treatment of NP enhanced growth and biochemical parameters in Brassica juncea.[48] These findings are similar to our results in terms of chlorophyll and protein contents. In the present study, by comparing the activities of the defense enzyme such as catalase, peroxidase, superoxide dismutase, and ascorbate peroxidase (CAT, POD, SOD, and APX, respectively), it is evident that the accumulation of studied silver nanoparticles induced a strong antioxidant response in T. ammi against M. incognita by reducing the number of galls, number of egg masses, and the nematode population. Application of nanoparticles was responsible for enhancing the antioxidant enzyme activities like POD, CAT, SOD, and APX, but the degree of this increase depended on the dose and on the type of NPs.[49]

Reduction in gall formation and production of egg masses was the highest in T3 plants treated with 100 ppm silver nanoparticles compared with IC plants. It seems that this concentration was not favorable for plants as was evident from the physiological parameters of the treated plants. However, the values of T2, T3, and T5 plants were nonsignificant, although the number of gall and number of egg masses were the lowest in T3 plants. From the results, it is clear that 50 ppm concentration of silver nanoparticles before M. incognita inoculation (T2 plants) was the best dose followed by simultaneous inoculation (T5 plants). Decrease in the number of gall number and egg mass number was due to the activity of silver nanoparticles that enhances the growth and development of the plant. Similar findings were reported by Abdellatif et al.[41] in eggplants and Hassan et al.[50] in tomatoes. Antimicrobial properties of silver nanoparticles against fungal phytopathogens were also established.[51−53] This might be due to a reduction in enzyme and toxin production utilized by the pathogenic fungus for disease incidence.[54,55] The nematicidal effect of allicin (diallyl thio sulfinate), the key natural antimicrobial compound of garlic, has been observed under the greenhouse condition. Allicin effectively inhibited the juvenile activity and improved the yield of tomato plants.[56] Cinnamyl acetate purified from Cinnamomum aromaticum showed 100% mortality against juveniles of M. incognita.[57]

Silver nanoparticles might alter the behavior of soil-inhabiting organisms, which influence feeding and root penetration of the nematode. Lignin deposition in cells of vascular bundles of silver-nanoparticle-treated plants was confirmed by observing the transverse section of roots. The maximum lignin deposition occurred in T2 plants treated with 50 ppm silver nanoparticles before nematode inoculation. From the study, it is evident that lignin plays a crucial role in the development of resistance against M. incognita. The lignified cell wall acts as a physical barrier against pathogenic attack[58] and is the main resistance factor.[59] Works on the applicability of silver nanoparticles to control various kinds of phytopathogens have been extensively made by several researchers, for example, sclerotium-forming fungi,[53]Fusarium culmorum,[60] powdery mildew on pumpkin,[61]Pseudomonas syringae pv. on tomato,[62] and gray mold in strawberry.[63]

Conclusions

From the data of the results, it might be concluded that biosynthesized silver nanoparticles are less toxic and can be used effectively for the management of root-knot disease caused by M. incognita. Treatment of seedlings with 50 ppm silver nanoparticles before inoculation of M. incognita proved to be the best dose in comparison to simultaneous and postinoculation of M. incognita. The results indicated that the values of growth and yield parameters of plants treated with 100 ppm silver nanoparticles prior to nematode inoculation were at par with the values of IC plants. A higher concentration of silver nanoparticles was probably toxic to the plants. Thus, before recommending its dose in agricultural fields, an optimum concentration should be determined to check the efficiency of nanoparticles against the nematode and their toxicity toward plants. Further research is required to verify the effects of biosynthesized silver nanoparticles under field conditions. Competent delivery systems for the commercial application of silver nanoparticles could be developed based on soil conditions and crop type.

Experimental Methods

Collection and Preparation of Leaf Extract and Chemicals Used during the Study

Leaves of S. siamea were collected from the university campus. The leaves were thoroughly washed with double-distilled water (DDW) to remove debris from the surface and dried at room temperature. Dried leaves were ground in an electric grinder to make a fine powder. About 20 g of dried leaf powder was kept in a conical flask containing 200 mL of double-distilled water for 30 min with continuous stirring. The extract was allowed to pass through Whatman filter no. 1 and stored at 4 °C for further use. Silver nitrate (AgNO3) was purchased from Sigma-Aldrich (India). Leaf was used as the reducing agent to synthesize silver nanoparticles (AgNPs). Sterilized double-distilled water (DDW) was used throughout the experiments.

Synthesis and Characterization of Silver Nanoparticles

Silver nanoparticles were synthesized using an aqueous extract of S. siamea leaves. In a typical reaction, 10 mL of aqueous leaf extracts was mixed in 90 mL of 0.1 M silver nitrate solution. The changes in the color of the mixture of the aqueous leaf extract and silver nitrate from light yellow to brown indicated the formation of nanoparticles.[64] After biosynthesis, the silver nanoparticles were separated by centrifugation of the solution at 15 000 rpm for 20 min. Silver nanoparticles were redispersed in distilled water and purified by repeated centrifugation three times to remove the plant extract. The pellet obtained was dried in a hot-air oven for characterization. Nanoparticle size and morphology were confirmed by transmission electron microscopy (TEM, Hitachi H-7500) and scanning electron microscopy (SEM, JEOL -JSM6100).

Plant Materials

The seeds of ajwain (Trachyspermumb ammiAA-1) were obtained from the National Research Centre on Seed Spices, Ajmer, India. Healthy seeds of uniform size were surface-sterilized with 1% sodium hypochlorite (NaOCl) solution for 15 min, followed by repeated washing with double-distilled water (DDW).

Source of M. incognita

Infected roots of eggplant (Solanum melongena) with the root-knot nematode (M. incognita) were collected from an eggplant field. Root-knot nematode species M. incognita was identified on the basis of the North Carolina differential host test and perennial pattern morphology.[65] A single egg mass was inoculated on an eggplant to maintain the M. incognita race-1 population. The egg masses were collected from the galled roots using sterilized forceps, transferred in a 20 μm sieve, and kept at room temperature for hatching of eggs following the Baermann funnel technique.[66] The second-stage juveniles (J2) were collected in distilled water in a beaker. The second-stage juveniles were counted in a counting dish under a stereomicroscope. The suspension was standardized to 1000 J2/10 mL of suspension.[67]

Experimental Design and Maintenance of T. ammi Plant

A pot experiment was conducted in the greenhouse of the Department of Botany, at Aligarh Muslim University, Aligarh (27°52′N latitude, 78°51′E longitude, and 187.45 m altitude). A homogenous mixture of 2.5 kg of steam-sterilized field soil and organic manure (3:1) was filled in autoclaved clay pots (38 cm) of 3 kg capacity. There were 55 pots arranged in a complete randomized block (CRD) design, and one treatment contained five pots. Two-week-old seedlings of 12 cm size were transplanted, and 5–7 cm deep holes were made about 2 cm deep from the base of the stem. The infective second-stage juveniles and silver nanoparticles were pipetted in these holes. Untreated plants were given water only and served as negative controls, while uninoculated and untreated plants serve as positive controls. The experiment was conducted in 2016–2017 and repeated in the 2017–2018 season. The concentration of silver nanoparticles and the treatment plan were based on a previous research by Kim et al.[68] and Lamsal et al.[22] with slight modifications, which are as follows:

Control = untreated and uninoculated

IC = inoculated with 2000 J2 of M. incognita only

T1 = 25 ppm silver nanoparticles 1 week prior to 2000 J2 of M. incognita

T2 = 50 ppm silver nanoparticles 1 week prior to 2000 J2 of M. incognita

T3 = 100 ppm silver nanoparticles 1 week prior to 2000 J2 of M. incognita

T4 = simultaneous inoculation of 2000 J2 of M. inocgnita and 25 ppm silver nanoparticles

T5 = simultaneous inoculation of 2000 J2 of M. inocgnita and 50 ppm silver nanoparticles

T6 = simultaneous inoculation of 2000 J2 of M. inocgnita and 100 ppm silver nanoparticles

T7 = 2000 J2 of M. inocgnita inoculation 1 week prior to 25 ppm silver nanoparticles

T8 = 2000 J2 of M. inocgnita inoculation 1 week prior to 50 ppm silver nanoparticles

T9 = 2000 J2 of M. inocgnita inoculation 1 week prior to 100 ppm silver nanoparticles

Estimation of Growth Biomarkers

After 4 months, the growth of silver-nanoparticle-treated and M. incognita-inoculated T. ammi plants was recorded. The length of plant organs (shoot and roots) was measured by means of a meter scale. Fresh weight and dry biomass of plant organs and seed yield per plant were measured using an electronic balance. Also, the numbers of branches and umbels per plant were counted visually.

Estimation of Essential Oil Content

The seeds of each treatment were collected and crushed in an electric grinder with DDW. The extraction of seed oil was carried out by following the method of Clevenger.[69] The oil percentage in seeds was calculated as follows

Estimation of Photosynthetic Pigments

Chlorophyll a, chlorophyll b, and carotenoid contents in the leaves of silver-nanoparticle-treated and M. incognita-inoculated T. ammi plants were measured by following the procedure of MacKinney.[70]

The following formula was usedV = total volume of the solution

W = weight of the leaves used for extraction of the pigment

D = optical density of the sample at 645 and 663 nm

Enzymatic Activity

Nitrate Reductase (NR, 1.6.6.1) Activity

The activity of nitrate reductase (NR, 1.6.6.1) in leaves detached from M. incognita-inoculated and silver-nanoparticle-treated T. ammi plants was estimated.[71] For the estimation, 100 mg of new leaves was slashed and taken into test tubes, containing 0.1 M phosphate buffer (pH 7.4), KNO3, and 5% isopropanol. This combination was hatched at 25 ± 2 °C for 2 h. After brooding, 0.2 mL of this hatched arrangement was moved into a discrete cylinder and 0.15 mL of each 1% sulfanilamide and 0.02% NED-HCL was blended and left for 20 min at room temperature for the greatest shading improvement. Tests were performed in a cuvette, and absorbance was perused at 540 nm by spectrophotometer against a blank. A standard curve was plotted utilizing the known convergence of sodium nitrite. The NR activity was expressed in nMNO2 g–1 F Wh–1 after comparing the OD of the sample with the standard curve.

Carbonic Anhydrase (CA, 4.2.1.1) Activity

The carbonic anhydrase action (CA, 4.2.1.1) was resolved in fresh leaves of M. incognita-inoculated and silver-nanoparticle-treated T. ammi following the procedure of Dwivedi and Randhawa.[72] For this 100 mg of leaves was cut into little pieces and placed in a test tube containing 0.2 M cysteine hydrochloride solution. This blend was brooded at 4 °C for 15–20 min. To each test tube, 2 mL of phosphate support (pH 6.8) was blended, followed by 0.2 M sodium bicarbonate, bromothymol blue, and the methyl red marker. Each test tube was shaken appropriately and titrated against 0.5 N HCl. Readings were noted as and when the red-pink tone appeared. A control test, without leaf tissues, was likewise titrated against 0.05 N HCL. The CA activity was expressed in μM CO2 kg–1 leaf FW S–1.

Determination of Leaf Protein Content

The leaf protein content in silver-nanoparticle-treated and M. incognita-inoculated T. ammi plants was determined following the method of Lowry et al.[73] For the estimation, 20 mg of oven-dried leaves was thoroughly mixed with 5% trichloroacetic acid. The solution was incubated at room temperature for 19 min. The absorbance was read at 660 nm using a UV–visible spectrophotometer. A standard solution of a known concentration of bovine serum albumin (BSA) was prepared, and absorbance was read at 660 nm. A graph was plotted to compare the absorbance of each sample to calculate the percent total protein content.

Estimation of Leaf Nitrogen Content

Digestion of Leaves

Leaves collected from each treatment were dried in an oven at 80 °C using an electric grinder, and a fine powder was made. Fifty hundred milligram of leaf powder was digested in a digestion tube containing 2 mL of sulfuric acid followed by dropwise addition of 0.5 mL of 30% hydrogen peroxide. This sample was used to estimate the percent leaf nitrogen content on a dry-weight basis.

Leaf nitrogen content was estimated following the method previously described by Lindner.[74] A 10 mL aliquot of processed leaves was transferred into a 50 mL volumetric flask and 2 mL of 2.5 N sodium hydroxide was included into the reaction mixture followed by the addition of 1 mL of 10% sodium silicate so as to kill and evade the turbidity of the solution, separately. A 5 mL aliquot of this mixture was moved into another test tube followed by adding of 0.5 mL of Nessler’s reagent. To appraise the leaf nitrogen content, the solution was moved into a cuvette and the optical density (OD) was measured at 525 nm.

Activity of the Defense Enzyme Assay

Fresh leaf material (0.5 g) was ground to 10 mL chilled (50 mM phosphate buffer) for the extraction of antioxidant enzymes (pH 7.8). The mixture was then centrifuged at 10 000g for 20 min at 4 °C. The supernatant thus obtained was then used for the estimation of the activities of antioxidative enzymes.

The method assigned by Chance and Maehly[75] was used to test the activity of the peroxidase (POD) in the leaf buffer.

For the estimation of catalase (CAT) (EC 1.11.1.6) activity, a spectrophotometer was used after a decrease in absorption of H2O2 within 30 s at 240 nm.[76]

For the estimation of superoxide dismutase (SOD) (EC 1.15.1.1) activity, the method assigned by Giannopolitis and Ries[77] was used, and the ascorbate peroxidase (APX) (EC 1.11.1.11) activity was estimated using the method provided by Asada and Takahashi.[78]

The activity of these enzymes was estimated at a reduction in absorption of 240 and 290 nm, respectively, and expressed as units mg–1 protein.

Number of Galls, Egg Masses, and Root-Knot Index

The number of galls per plant was counted visually, and the size of gall was recorded by measuring its maximum length and width (in μm) using a micrometer.

The infected roots detached from silver-nanoparticle-treated and M. incognita-inoculated T. ammi plants were kept in the phloxin-B solution for 20 min. The roots were carefully washed in tap water, and the red-stained egg masses were counted as a per root system on infected roots.[79]

At harvest, roots were examined to count galls and egg masses on a 0–5 scale by the following method.[80]

0 = 0 gall/egg mass/root system.

1 = 1–2 galls/egg mass/root system.

2 = 3–10 galls/egg mass/root system.

3 = 11–30 galls/egg mass/root system.

4 = 31–100 galls/egg mass/root system.

5 = >100 galls/egg mass/root system.

Histopathological Studies

After 120 DAS of inoculation, roots of each treatment were gently washed with tap water and fixed in formalin and acetic acid alcohol solution (FAA). The anatomical studies were carried out by adopting the method of Johansen.[81] Preserved roots were dehydrated by a series of ethanol and xylene solutions and embedded in paraffin wax. Small-sized blocks of wax were prepared for sectioning of embedded root tissues. The 10 μm thick sections were cut into straight ribbons with the help of a rotary microtome. The ribbons were placed on a clean slide. These slides were further washed in a series of ethanol solutions and stained with safranin and fast green followed by dropwise washing with clove oil to remove the excess stain. Required amounts of Canada balsam were placed on the surface and covered by placing a rectangular coverslip to mount permanently. Slides were examined under a microscope (Nikon Eclipse E200, Japan, attached with a camera) and photographed.

Statistical Analysis

All data of both seasons were subjected to statistical analysis using R software. Means of five replicates were taken, and the least significant difference (LSD) was calculated at the probability level of p ≤ 0.05. Means were compared using Duncan’s multiple range test (DMRT) followed by the same letter within a treatment not significantly different at 0.05 levels. Principal component analysis (PCA) and Pearson correlation analyses were employed to determine the potential covariance between bean plant growth, yield, biochemical parameters, and nematode parameters like the number of galls and number of egg masses. All of the analyses were performed using XLSTAT version 2020.5.1.1075 of Addinsoft (2020).