Biopolymers are naturally occurring materials formed during the life cycle of plants, animals, bacteria, and fungi (Yadav et al., 2015). Biopolymers are easily biodegradable because they include oxygen and nitrogen atoms. Through biological processes, the biopolymers are naturally recycled. Worldwide, chitosan is the second most abundant biopolymer, second only to cellulose. Through enzymatic and chemical deacetylation processes, chitin is converted to chitosan (Kafetzopoulos et al., 1993). Chitosan was first discovered in mushrooms by Henri Braconnot in 1811 (Periayah et al., 2016). It is obtained from crustaceous shells, e.g., from the exoskeletons of crabs, shrimps, prawns, fungi, and insects. Chitosan consists of N-actetyl D-glucosamine and β-(1-4) D-glucosamine. Glucosamine (GlcN) is product of the decomposition of chitosan by the chitosanase enzyme (Jung and Park, 2014). Chitosan is considered a cationic polymer, and due to its biocompatibility, nontoxicity, and bio-degradability properties, it is used in agricultural, medical, biotechnological, environmental, and industrial applications (Naveed et al., 2019). It is also known to possess antifungal, antibacterial, antiviral and antinematicidal properties (Goy et al., 2016).
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Nanospheres are nanostructures formed by a dense polymeric matrix. The drugs are dispersed in the matrix-type structure of the nanospheres. Nanospheres are prepared using several polymers like cellulose, chitosan, and Poly (d, l-lactic acid). The size range of nanospheres is between 10 and 200 nm in diameter. Nanospheres are amorphous or crystalline in nature (Singh and Sharma, 2010). Embedding the desired compounds in nanospheres protects the drugs from enzymatic and chemical degradation. Chitosan nanoparticles are used as nanopesticides and carriers of fungicides, insecticides, herbicides, plant hormones, elicitors, and nucleic acids (Zhang et al., 2003; Dei Lam et al., 2006). Because of their small size and high contact area, chitosan nanoparticles can easily penetrate and permeate into the membrane of phytopathogens or plant tissues, resulting in increased control or defense response activity.
Alfy et al. (2020) reported that a chitosan nanoparticle at 2000 ppm was the most efficient biopolymer in controlling the root-knot nematode,
Human health concerns and environmental considerations call for environmentally-friendly alternatives that can be integrated into existing practices. With this background in mind, a study was undertaken to assess the efficacy of chitosan nanospheres against root knot nematode,
Commercial-grade high-molecular-weight chitosan was used in the study (obtained from Panvo pvt ltd. Chennai). Chitosan nanospheres were prepared using the ionic gelation method (Youssef and Masry, 2018). 1g of crude chitosan was dissolved in 1% glacial acetic acid and stirred for 24 hr in a magnetic stirrer at 500 rpm (Spinit TM Digital Magnetic stirrer). After 24 hr of stirring, one part 0.7 mg/ml Sodium Tripolyphosphate (TPP) was added dropwise to 3 parts of 1% chitosan acidic solution and again allowed for 2 hr, stirring at 500 rpm. The pH of the prepared solution was checked using a pH meter and later sonicated in an Ultraprobe sonicator (Ultra Sonics instruments) with an amplitude of 35%, pulse 10 seconds, and temperature of 35ºC, for seven minutes.
In a second experiment, 5 ml of surfactant 2% Tween 80 was slowly added to 3 parts of 1% chitosan acidic solution at 45ºC. The mix was stirred for 2 hr in a magnetic stirrer at 500 rpm. One part of TPP was added to three parts of 1% chitosan-acetic acid solution. The sample was then homogenized in a pressurized homogenizer (Homeostat instrument) for 5 minutes and ultrasonicated for 15 min.
The particle size and polydispersity index (PI) of the synthesized chitosan nanoparticles were measured in a particle size analyzer (HORIBA SZ-100, Japan). Their stability was determined using zeta potential. The size and shape of the chitosan nanospheres were characterized using transmission electron microscopy (TEM). The functional groups of biomolecules present in the chitosan nanospheres were identified using Fourier infrared spectroscopy (FTIR).
Pure cultures of root-knot nematode,
Egg masses of
Freshly hatched second-stage juveniles (J2) were used for juvenile mortality studies. For each different concentration of the chitosan nanospheres, 1ml of the suspension was placed in a 5-cm diameter petri dish, to which 100 infective juveniles (J2) were added (the 1ml suspension containing nematodes were counted with the help of a counting dish under a stereo zoom microscope). The five treatments were replicated four times and arranged in a randomized complete block design. All 100 juveniles were examined, and the number of dead juveniles were recorded after 24 hr, 48 hr and 72 hr.
A greenhouse experiment in pots (3 kg of soil; pot size 20.7 × 11 × 16.4 cm) was conducted to study the effect of different concentrations of chitosan nanospheres against
The treatments included:
Chitosan nanospheres (1%) 1ml/plant. Chitosan nanospheres (1%) 2ml/plant. Chitosan nanospheres (1%) 3ml/plant. Chitosan (1%) 1ml/plant. Chitosan (1%) 2ml/plant. Chitosan (1%) 3ml/plant. Velum prime 1ml/plant-34.6% Fluopyran Purpureocillium lilacinum 1g/plant. Chitosan nanospheres (1%) 3ml without nematodes. Untreated control.
A
The following treatments were used:
T1- Chitosan nanospheres 1% (5ml/plant). T2- Chitosan nanospheres 2% (5ml/plant). T3- Chitosan 1% (5ml/plant). T4- Chitosan 2% (5ml/plant). T5- Velum prime (34.46% fluopyran)- 500 ml/acre.- 0.0005 metric T6- Carbofuran 3G 1kg a.i/ha. T7- Untreated control.
The data obtained from the above-mentioned experiments were subjected to statistical analysis following the method formulated by Panse and Sukhatme (1967). The obtained data were run in Aggress software with single-factor analysis to obtain the results.
1g chitosan was dissolved in 1% acetic acid in the presence of Tween 80 and TPP. The prepared solution was homogenized in a pressurized homogenizer for 5 minutes. The homogenized solution yielded a size of 380.2 nm with a PI of 0.4 (Fig. 1). The present study proved that synthesized chitosan nanospheres using sodium tripolyphosphate and Tween 80 were highly stable, as measured by their zeta potential value of +49.7 mV (Fig. 1b). The zeta potential of the chitosan nano formulation obtained in this study was well above +30 mV, indicating high stability.
TEM micrography was used to study the morphology, shape, and size of the synthesized chitosan nanospheres. The nanospheres were predominately spherical, with no agglomerates. The average size of each nanosphere was 89.0 nm (Fig. 2).
The chitosan nanospheres obtained with the above method were analyzed using Fourier Transform Infra–red Spectroscopy (FTIR), which was used to study the chemical interaction between chitosan and sodium tripolyphosphate molecules. The peak at 3334 cm−1 showed a stretching of the OH and NH group. Peaks at 2925 cm−1 and 2856 cm−1 show the stretching of the CH group. A peak of 2285 cm−1 represented N=C=O stretching. A peak at 2114 cm−1 showed N=C=S stretching and a peak at 1635 cm−1 explained the stretching of C=N. The peak at 1412 cm−1 showed bending of the O-H group and the peak at 1004 cm−1 revealed the stretching of C-F group (Fig. 3).
To assess the efficacy of chitosan nanospheres on the hatching of
Effect of chitosan nanospheres on the egg hatching of
Chitosan nanospheres at 100 ppm | 12.50b (3.31) | 16.50b (4.84) | 41.50b (6.29) |
Chitosan nanospheres at 500 ppm | 10.50b (3.09) | 14.16b (3.61) | 26.50b (4.79) |
Chitosan nanospheres at 1000 ppm | 0.00a (0.70) | 0.00a (0.70) | 0.00a (0.70) |
Chitosan nanospheres at 5000 ppm | 0.00a (0.70) | 0.00a (0.70) | 0.00a (0.70) |
Control- tap water | 38.83c (6.21) | 61.66c (7.85) | 84.80c (17.27) |
SEd | 0.72 | 0.46 | 1.12 |
CD (p=0.01%) | 2.29 | 1.48 | 3.88 |
Figures in parentheses are square root transformed value. In a column, means followed by common different from each other at 1% level by DMRT.
Different concentrations of 1% chitosan nanospheres
Effect of chitosan nanospheres on
Chitosan nanospheres at 100 ppm | 13.00b (5.40) | 21.5c (3.47) | 26.5c (5.10) |
Chitosan nanospheres at 500 ppm | 27.25 b (5.14) | 31.25b (5.67) | 28.25c (5.26) |
Chitosan nanospheres at 1000 ppm | 29.00 b (5.85) | 36.00b (5.96) | 46.25b (6.70) |
Chitosan nanospheres at 5000 ppm | 100.00a (10.02) | 10.00a (10.02) | 100.00a (10.02) |
Control | 0.00c (0.707) | 0.00d (0.70) | 0.00d (0.70) |
SEd | 1.52 | 0.72 | 0.55 |
CD (p=0.01%) | 4.48 | 2.14 | 1.64 |
Figures in parentheses are square root transformed value. In a column, means followed by common alphabet are significantly different from each other at 1% level by DMRT.
In-vitro results revealed that chitosan nanospheres reduced nematode population in roots and soil. Application of chitosan nanospheres at 2ml/plant decreased root galls by 83.68%. Application of chitosan nano formulation at 2ml/plant decreased the number of egg masses by 83.85%. The chitosan nanosphere formulation at 2ml/plant registered the lowest number of adult females, with the highest percent reduction of 66.56 % (Table 3). The highest reduction of infective juveniles (73.20%) was recorded with the 2ml/plant formulation (Fig. 5). In field experiments, the chitosan nanosphere formulation (2%) at 5ml/plant decreased galls by 92.47%. Furthermore, the fruit yield was higher by 18.75% in plots treated with chitosan nanospheres (Table 4).
Effect chitosan nanospheres against root knot nematode,
Chitosan nanospheres 1ml/plant+ |
13.30a (3.51) | 11.67bcd (3.32) | 17.67cd (4.25) | 48.60bc (12.06) |
Chitosan nanospheres 2ml/plant+ |
10.00a (3.12) | 6.00b (2.53) | 14.60b (3.87) | 28.30ab (5.36) |
Chitosan nanospheres 3ml/plant+ |
15.00a (3.87) | 7.33bc (2.73) | 17.60cd (4.25) | 40.30bc (6.38) |
Chitosan 1ml/plant+ |
18.00a (4.28) | 19.00c (4.41) | 22.30f (4.76) | 62.00bcd (7.90) |
Chitosan 2ml/plant + |
17.00a (4.25) | 10.00bcd (3.22) | 16.67c (4.13) | 46.70bc (6.86) |
Chitosan 3ml/plant + |
17.60a (3.34) | 10.66bcd (3.30) | 17.90d (4.19) | 44.70bc (6.66) |
Velum prime 0.5ml/plant + |
25.67a (4.87) | 15.67de (3.95) | 19.30e (4.44) | 32.67bc (5.75) |
17.67a (4.25) | 13.39cde (1.37) | 18.67e (4.44) | 50.60bc (7.14) | |
Chitosan nanospheres without nematode Inoculums | 0.00a (0.70) | 0.00a (0.70) | 0.00a (0.70) | 0.00a (0.70) |
Untreated control | 61.30c (7.83) | 37.16f (6.04) | 43.67g (6.63) | 105.60cd (10.21) |
SEd | 0.96 | 0.47 | 0.06 | 2.26 |
CD (p=0.01%) | 2.73 | 1.36 | 0.18 | 6.44 |
Figures in parentheses are square root transformed value. In a column, means followed by common alphabet are significantly different from each other at 1% level by DMRT.
Effect of chitosan nanospheres against root knot nematode,
Chitosan nanospheres (1%)-5ml/plant | 5.61ab (2.29) | 3.34b (1.82) | 18.31b (4.26) | 50.63c (7.11) | 95a (18.75) | 14.25 |
Chitosan nanospheres (2%)-5ml/plant | 3.31a (1.81) | 0.99ab (1.14) | 17.38b (4.16) | 35.00b (5.90) | 89.5b (11.87) | 13.42 |
Chitosan (1%) - 5ml/plant | 11.25b (3.10) | 3.62b (1.93) | 20.90b (4.54) | 58.54e (7.61) | 86bc (7.5) | 12.90 |
Chitosan (2%)-5ml/plant | 4.91ab (2.30) | 1.88ab (1.52) | 17.79b (4.19) | 48.75b (6.92) | 83cd (3.75) | 12.45 |
Velum prime 500ml/acre | 2.83a (1.60) | 0.09a (0.75) | 11.42a (3.35) | 30.45a (5.47) | 99a (23.75) | 14.85 |
Carbofuran 3G- 1kg ai/ha | 7.32b (2.95) | 1.43ab (1.35) | 23.20b (4.78) | 54.23d (7.34) | 89b (11.25) | 13.35 |
Untreated control | 44.00c (0.62) | 22.3c (4.77) | 57.25c (7.57) | 165.80f (12.73) | 80d | 12.00 |
SEd | 0.46 | 0.37 | 0.37 | 0.10 | 2.28 | |
CD (p=0.05%) | 1.00 | 0.80 | 0.80 | 0.22 | 4.90 |
1 and 2-Figures in parentheses are square root transformed value and increased over control respectively. In a column, means followed by common alphabet are significantly different from each other at 1% level by DMRT.
Synthesis and characterization of chitosan nanoformulation and its potential antinemetic and antifungal effects are discussed. Per the observations of Budi et al. (2020), the average particle size of chitosan nanospheres was within the limit of 70 nm, with a Polydispersity Index of 0.3. In the present study, the particle size of chitosan nanospheres after using Tween 80 and sodium tripolyphosphate was found to be the 380.2 nm, with a PI of 0.4. Karava et al. (2020) determined that the optimum size of chitosan nanospheres was 150 nm, with a PI value below 0.6. A PI value between 0 and 0.5 indicates homogeneous particles and the PI value beyond 0.5 indicates particles in a heterogeneous condition (Danaei et al., 2018). The PI value of the nanoformulation obtained in our study was found to be within the range of 0.5, indicating nanoparticles of a homogenous nature.
High stability in synthesized chitosan nanospheres is considered to be a desirable characteristic. The present study proved that the chitosan nanospheres that were synthesized using sodium tripolyphosphate and Tween 80 were highly stable, as measured by their zeta potential value of +49.7 mV. The chemical interaction between chitosan and sodium tripolyphosphate molecules was studied using Fourier-transform infrared spectroscopy. The peak at 3334 cm−1 showed a stretching of the OH and NH groups. The findings of present study were similar to the observations of Youssef and Masry (2018), where observations made through transmission electron microscopy and field emission scanning electron microscopy revealed a spherical shape in the chitosan nanospheres with sizes of 89.0 nm - 187.0 nm and no agglomeration of particles. Youssef and Masry (2018) and Mohammadpour et al. (2011) also observed similar spherical shape of chitosan nanospheres.
Different concentrations
Adding chitosan to the soil increases the population of chitinolytic microogranisms, which produce enzymes that convert chitin (a polysaccharide) to chitobiose (a disaccharide). This destroys the eggs and cuticles of young juveniles, which contain chitin (Abd El-Aziz and Khalil, 2020). Because of chitosan's eliciting activity and ability to generate systemic resistance in the plant, as well as the release of different toxic chemical compounds during decomposition, it is lethal to
In the present study, the methodology of formulating chitosan nanospheres was standardized. The chitosan nanosphere formulations had both direct and indirect effect on root-knot nematodes. They degraded the chitin layer of nematode eggs and juveniles and caused death. Indirectly, they induced systemic resistance in plants, thereby reducing nematode infection. As chitosan nanospheres are synthesized from a biological source, the formulation is environmentally friendly and does not leave any toxic residues in the ecosystem.