Nematicidal Properties of Chitosan Nanoformulation

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, M. incognita, were obtained at Department of Nematology, TNAU, Coimbatore. Concentrations of 100 ppm, 500 ppm, 1000 ppm and 5000 ppm were prepared from the 1% chitosan nanosphere suspensions in sterile (100 ml) water. Each concentration was sonicated in a water bath sonicator (POWERSONIC 510) for 20 minutes before testing them against M. incognita.

Egg masses of M. incognita were sterilized in 0.5% sodium hypochlorite solution for 1 minute. The culture was maintained at Department of Nematology, Glass house, TNAU, CBE. Egg masses were collected from nematode-infested root. The experiment was repeated 3 times. 2 ml of each concentration suspension was placed in a 5-cm diameter petri dish. One egg mass of M. incognita was placed into the liquid in the petri dish. The experiment was conducted in a randomized complete block design, with five treatments and four replications. Numbers of hatched juveniles were observed at different time intervals viz., 24 hr, 48 hr and 72 hr.

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 M. incognita. Seedlings purchased from the vegetable nursery at Thondamuthur (15 days old; Shivam hybrid) Coimbatore-03 were used for pot culture experiments. The 15-day-old seedlings were transplanted into a sterilized pot mixture, where they were inoculated with infective juvenile nematodes at a rate of 2/gm of soil. One week after inoculation, chitosan nanospheres were applied as a soil drench. On the 45th day after nematode inoculation, plants were uprooted, and plant shoot lengths and root weights were determined. The nematode populations were counted based on the gall numbers (Heald et al., 1989) and egg masses on the roots. In total, 10 treatments and three replications were arranged in a randomized complete block design.

The treatments included:

Chitosan nanospheres (1%) 1ml/plant.

A M. incognita-infested field was selected near Karadimadai Village, Coimbatore, India. Seven treatments were applied as soil drenches to one week-old tomato seedlings. On the 45th day after planting a second set of treatments were applied. Treatments were applied to three replicate plots with 40 plants each, arranged in a randomized block design. Plant growth parameters and nematode population density in soil and roots were observed 90 days after application. Fruit yields were measured for each treatment.

The following treatments were used:

T1- Chitosan nanospheres 1% (5ml/plant).

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.

Figure 1:

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).

Figure 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).

Figure 3:

To assess the efficacy of chitosan nanospheres on the hatching of M. incognita eggs, different concentrations of chitosan nanospheres were used. Among the concentrations tested, chitosan nanospheres at 1000 ppm and 5000 ppm completely inhibited egg hatching compared to the control. Exposure of M. incognita egg masses to chitosan nanospheres at 500 ppm concentration resulted in a 68.75% decrease in egg hatching compared to the untreated control 72 hr after treatment (Table 1). At this concentration, hatching of eggs was observed, but all the hatched juveniles from the eggs were found dead. A scanning electron microgram (x1000 magnification) of treated egg masses revealed degradation of the gelatinous matrix (Fig. 4). The first-stage juveniles found within the treated eggs were deformed.

Figure 4:

Different concentrations of 1% chitosan nanospheres viz., 100 ppm, 500 ppm, 1000 ppm and 5000 ppm were tested against infective juveniles (J2). Among the above-mentioned concentrations, chitosan nanospheres at 5000 ppm caused 100% juvenile mortality within 24 hr (Table 2). At the 1000-ppm concentration, the infective juvenile mortality was lower, with a maximum of 46.25% mortality observed after 72 hr of exposure.

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).

Figure 5: