Abstract

Nanopesticides represent the formulations of the efficient ingredients of the pesticides in nanoforms which have slow degradations, target delivery, and control liberate of active ingredients for longer period, making them safe environmentally and less toxicity than classical chemical pesticides. This study was designed to produce nano biopolymer composite of nano chitosan entrapped spores of biocontrol Bacillus thuringienesis bacterium and coated with colloidal silver nanoparticles of 50 nm mean size (NC:Bt@AgNPs) and study the effect of UV- radiation at 320nm and 0 0.5, 1 and 5min of exposure, on the viability of spores in comparison with free spores. The results of exposure to ultraviolet radiation showed good protection for the spores in the nanostructures compared to the free spores. The death of all free spores was observed after 0.5min of exposure to the irradiation, while the number of spores remaining in the nanocomposite after five minutes was 1X 10² spore/ml in spite of using the radiation at 320nm

Keywords: Bacillus thuringiensis, Biocontrol, Chitosan Nanoparticles

Introduction

Recently, nanotechnology has emerged as a promising tool for pest control. Biopesticides have long attracted worldwide attention as a safer plan over chemical pest controls, with less risks to human and  environment. Globally, the apply of biopesticide  increased steadily by (10%) each year. Nanotechnology  provided modern tools like nano pesticides ,and nanosensors for pest managements in agriculture with the concepts of minimal uses for the highest effect. At present, there is a growing need to develop environmentally benign nanoparticle synthesis without using any toxic chemicals and the preparation of uniform nanosized particles with specific requirements in terms of size, shape, and physical-chemical properties is of great interest in the formulation of new microbial biocontrol products. Nanotechnology refers broadly to a field of applied science and technology whose unifying theme is controlling matter on the atomic and molecular scale (Camara et al.,2019).

The most widely used microbial insecticides are those based on the bacterial pathogen Bacillus thuringiensis (Bt) used as a prosperous biological insecticide and an alternative to chemical pesticides for many years. It has been applied widely in pest management in forestry, agriculture and public health because of its safety to humans, animals, and the environment (Jozani et al., 2005).

Different environmental agents, like rain, the ultra violet radiation, and also temperature, lead to the crystal proteins’ microbial degradations or inactivation. Recently, many methods and studies have been underway to evolve various formulations for the entomopathogens that can progrss this action and keep microorganisms from the solar disruption and others harmful impacts of environment. So, to conquer these limitations, like susceptibility to the light, short lifetime, and other of conditions,  Bt toxins formulation optimization via nanoparticles is an main parameter for its trade productions (Maghsoudi and Jalali ,2017). Typically, the formulations should supply highest protective effects to active factor, whereas it has  no antibacterial effect or less antibacterial impact (Zhang et al., 2016).

The study aims to evaluate the vitality and stability of the B. thuringeinsis spores loaded in nano chitosan, synthesized in the laboratory and coated with silver nanoparticles (NC:Spores@ AgNPs) , under the influence of ultraviolet radiation compared to free spores.

Material and methods

Microorganisms

Bacillus thuringiensis BT (ASF-1) isolate was supplied by laboratory of Biotechnology College /Al-Nahrian University. It was re-purified isolates by  nutrient broth activation and streaked on nutrient agar at 37 ^ºC for 24 hr. The pure colony was diagnosed  via gram stain and microscope. It was maintained isolates in the slant and the nutrient agar plate in the refrigerator.

Bt spores suspension preparation

Pure colonies of Bt were spread on NA and incubated for 48 – 72hr at 37 ºC. The formation of spores was monitored every 24 hours by taking a loop from the surface of the culture and examined under a microscope. After the formation of the high percentage of spores, the surface of culture plates was harvested and transferred into sterile normal saline in screw-capped tubes and mixed well by vortex for 5min. The tubes were heated at 80 ºC for 10min to kill the vegetative cells. The spores count was made by serial dilution count of 10 folded and cultured of 0.1ml from dilutions on NA by spreading using L- shape glass rod. The plates were incubated at 37 ºC for 24hr. The suitable count for the subsequent experiments was determined. It also made the count using a Neubauer hemocytometer.

Preparation of nano chitosan loaded with BT spores and coated with colloidal nanosilver (NC:Bt@ AgNPs)

Preparation of chitosan nanoparticles and loaded with Bt spores then coated with coloideal sliver nanoparticles was conducted by following the methods of  Lifeng et al., (2004) and Tang (2007). The modification in methods was done as following: Chitosan nanoparticles prepared by the ionic gelation method. The chitosan (0.5%) mg) was weighed and dissolved in 100ml of glacial acetic acid (2%) in dH2O, then mixing for 30 min at 4000 rpm using a heat magnetic stirrer. A volume of 30 ml of aqueous Tripolyphosphate (TPP) (0.25%) with mixing. At once, 5 ml spore suspension ( 1X10 8 spores/ml) was added under mixing at the same speed for 1hr. After that, 2ml of colloidal silver nanoparticles (<50nm) was added and mixing was continued for 30min. Mixing was stopped and the mixture was left to settle for the next day in the refrigerator. The mixture was centrifuged at 14,000 rpm for a period of 20 min at 4 ° C. The supernatant was discarded and the deposit was washed twice with deionized water and repeating the centrifugation. The precipitate was dispersed in sterile petridishes and drying in vacuum oven for 6hr. The product was crashed and weighted ,then collected in sterile screw cupped tubes and stored in refrigerator until used.

Characterization of AgNPs and NC:Bt@ AgNPs nanopolymer composite

The characterization of the was done using UV-Visble spectrophotometer (Metertech SP 8001), in the range of (190-1100); FTIR, Shimadzu launched the AIM-9000 FTIR microscope; XRD, XRD-7000 Shimadzu Maxima-a, 40 kV voltage devices at AL-Nahrain University/ College of Science/ Department of Chemistry and Physics; AFM, IBM, New York in the Baghdad University, College of Science, Department of Chemistry and SEM model (TESCAN-VEGA/USA) in Iran.

UV-Irradiation

Weight of 0.5gm of the dry product was resuspended in 4.5ml dH2O and spread in sterile Petri dish. The count was calculated by dilution method and spreading of 0.1ml in the NA plate . Two duplicates were prepared.Five ml of diluted spores suspension were prepared to obtain 1X10⁶ spores/ml and poured in sterile Petri dish. Two duplicates were prepared.The plates were exposure to UV irradiation at 320nm in UV-radiation instrument and 0.1ml sample taken at 0.5, 1 and 5 min from each plate and ten folded dilutions were made. 0.1ml from each dilution was cultured over NA plate by spreading and incubated at 37 ºC for 24-48hr.A count was made for each plate and the average of the count recoded.

Production of Bt sporesFigure(1) shows the microscopic high power filed of B. theuringensis spores suspension producing in NA culture. As showed, the spores are mature and free of vegetative cells and consisted of 100% spores in the fields examination. The number of spores was made by total viable count (TVB) using serial dilution after heating the harvested spores at 80 ºC for 10min and using neubauer hemocytometer.

Figure 1: Spores of Bacillus thuringiensis under microscope (high power filed 40X)

Production of nanopolymer composite NC:Bt@ AgNPs

The synthesis of nanopolymer composite consists of nanochitosan loaded with spores of B. thuringiensis and coated with colloidal silver nanoparticles was made by combination of two methods ( Lifeng et al.,2004 and Tang, 2007) with modification. The idea of loading depends on filling the spores inside chitosan nanoparticles during their preparation by adding TPP, which is function on cutting the chitosan chain and creating cross-links to form nanoparticles.   This is followed by the addition of colloidal silver nanoparticles of less than 50nm size, which will begin to come into contact and cluster in the form of an envelope around chitosan nanoparticles, where the formation of the coat matures after leaving the mixture with cooling for the next day.

Characterization of AgNPs and NC:Bt@ AgNPs

UV-Visible scanning

Figure (2A,B) illustrates the scanning UV-Vis. Spectroscopy of AgNPs and NC:Bt@ AgNPs nanopolymer, respectively. The spectrum of the of scanning wavelength extended from 100- 1100nm. The Ag absorbance realized between 400 -420 nm and this range was reflected the presence of silver nanoparticles within the range of 50nm for Ag alone (figure 3-2a) and more than 50nm (figure 3-2b) and low density because of the interaction of silver with nano chitosan.

   A                                                                                      B

Figure 2: Scanning UV-Vis. Spectroscopy of  A: AgNPs and  B: NC:Bt@ AgNPs nano polymer

X-Ray Diffraction (XRD)Figure (3 A and B) shows that the silver has 111 facets and has a purity of 90% compared to 40% in the mixture, due to the bond of silver on the surface of the polymer and its formation in a new range, thus the purity decreased to 40% because the X-ray beam does not penetrate the sample more than 5 nm and the particle interferes with Some of them.

Figure 3: XRD analysis of  A: AgNPs, B: NC-Bt@AgNPs

FTIR analysis

Figures (4A,B) shows to the FTIR analysis of AgNPs (a) and NC- Bt@AgNPs (b). The differences were absolutely clear to refer the integration and formation

A                                                                             B

Figure4: FTIR analysis of A: AgNPs, B: NC-Bt@AgNPs.

the nanocomposite. The appearance of deep peaks in AgNPs was retained to the capping agent used for dispersion the colloidal nanoparticles.

Scanning Electron Microscope (SEM)

Figure (5) shows the SEM of NC-Bt@AgNPs which was done in Iran. It clearly shows the nanoparticle sizes of less than 200 nm for the nanoparticle mixture and the consistency of the nanoparticles.

Figure 5: SEM image of NC-Bt@AgNPs.

Atomic Force Microscope (AFM)

Figure (6A and B) shows the AFM Images and granular distribution of AgNPs and NC-Bt@AgNPs . The average diameter for AgNPs was 62nm meanwhile for NC-Bt@AgNPs was 82nm.These results illustrated the complete loading of nano chitosan with spore and surrounding AgNPs.

Figure 6: AFM granular distribution of AgNPs

Figure 7: AFM granular distribution of NC- Bt@AgNPs

Effect UV-Radiation on the viability of Bt spores and NC:Bt@ AgNPs nanopolymer

Table (3-1) shows the effect of UV-radiation on the viability of Bt spores as free spores and loaded within nanochitosan surrounding with AgNPs. All free spores were deactivated in 0.5min of radiation, while in nanocomposite structure are still viable in count of 1X 105 spore/ml. After 5min, the remaining spores count in nanocomposite was 1X102 spore/ml. These results reflect the efficiency of nanocomposite to protect the spores from the direct effect of radiation in spite of using UV-radiation at wavelength 320nm which is represent killing or mutated wavelength if the exposure exceeded many seconds.

Table 1: Effect UV-radiation on the viability of Bt spore

Discussion

This study was designed to produce nanobiopolymer composite of nanochitosan entrapped spores of biocontrol Bacillus thuringienesis bacterium and coated with colloidal silver nanoparticles of 50 nm mean size (NC:Bt@AgNPs) and studying the effect of UV- radiation at 320nm at different time of exposure, on the viability of spores in comparison with free spores. The results of exposure to ultraviolet radiation showed a good protection for the spores in the nanostructures compared to the free spores. The death of all free spores was observed ,while the number of viable spores remaining in the nancomposite after five minutes. These results support the opportunity to use bio-nanomixtures to protect the biological agents against insect infestations as well as their specialization and perpetuate their effectiveness.This study was approved by many scientists when the silver nanoparticles interacted with polymers. (Jeevan and Rena , 2012; Dugal and Chakraborty,2013).

Previous studies  showed the effect of Bacillus thuringiensis (Bt) as  insecticide  used in pest-control products in agriculture, and forestry due to  the safety to  humans, and the environment. The insecticidal crystal of proteins produce via  Bt is a toxic to f insect including :Lepidoptera, ,Coleoptera, Diptera, Hemiptera, and Nematoda. Bt stability  is an important agent in determining the effect of Bt  as a pest-control factor (Federici, 2022 ; Ortiz and  Sansinenea, 2023).

Another study showed importance of using nano chitosan as a foliar spray (0.1%–1%) successfully to protection of staple crops including wheat, maize ,rice(Karamchandani et al.,2022 ).

Conclusions

A biological nanocomposite was produced consisting of nanochitosan, loaded with Bt spores and coated with silver nanoparticles.The mixture has proven its efficiency in protecting the spores from the influence of ultraviolet radiation, with a very effective wavelength compared to the death of all free spores.

Acknowledgment

None .

Conflicts of Interest

None.

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