Evaluating the In vivo Efficacy of Copper-Chitosan Nanocomposition for Treating Vascular Wilt Disease in Date Palm

Date palm, Phoenix dactylifera, as one of the most important fruit crops in Egypt and many other countries, can be affected by many fungal diseases, among which the vascular wilt disease, caused by the fungal pathogen Fusarium oxysporum, is considered the most deteriorating one. This study aims at evaluating the efficiency of Copper-Chitosan Nanopcomposition for treating the vascular wilt disease in date palm. The study relies mainly on beleaguering the disease via the double-role functionality of copper-chitosan nanocomposition, i.e. its potential antifungal effect on the fungal pathogen, besides its capability to enhance the immune responses of the infected plant. In this regard, chitosan nanoparticles were prepared according to the ionic gelation method, whereas copper nanoparticles were prepared according to the chemical reduction method. Physicochemical characterization of both chitosan and copper nanoparticles was performed using dynamic light scattering (DLS), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR) and x-ray diffraction (XRD). Copper-chitosan nanocomposition could significantly reduce the vascular wilt disease severity; this means that the nanocomposition can be used in the future for developing new nano-fungicides to control such pathogens.


INTRODUCTION
Date palm, Phoenix dactylifera, is considered one of the most important fruit crops in many arid regions including the Middle East; This is due to its versatility, as it has a variety of uses includes eating fresh fruit and benefiting from its high nutritional value and its richness of carotenoids, citric acid, folic acid, and provitamins; this in addition to the antiviral, antibacterial, antifungal, antiulcer, antitumor and immunomodulatory properties of phenolic compounds detected in dates. Moreover, this crop has a great potential as a source of renewable energy by producing biofuel due to the high carbohydrates content in the fruits. Also, seeds are used in animal feeding, cosmetics, source of oxalic acid and charcoal, besides using them as a paste to relieve ague (Al-Shahib and Marshall, 2003). The worldwide production of dates reached 7600315 tons annually. In this context, Egypt is considered the largest producer of dates all over the world with annual production of 1465030 tons (FAO, 2014). This massive production of dates makes date palm a promising potential source of national income in Egypt and other large producers. But, this important crop, like all other crops, is threatened with many fungal and bacterial phytopathogens. Generally speaking, more than 70% of the crop diseases result from fungal pathogens (Agrios, G.N., 2005), this indicates to the importance of controlling such virulent phytopathogens. In this regard, surveys showed that among these fungal pathogens, the fungal pathogen Fusarium oxysporum, which causes vascular wilt disease (also known as fusarium wilt disease), is considered the most common and most virulent one (Flood, 2006). In this context, there are many traditional chemicals that are used to control such pathogenic fungi. But many phytopathogens have exhibited resistance against many traditional chemicals that are used to control them On the other hand, chitosan has gained much attention in many different applications, including but not limited to pharmaceutical, medical, agricultural, nutritional and industrial applications, this is due to its superior characteristics such as non-toxicity, biodegradability and biocompatibility (Harish P. and Tharanathan, 2007). Also, chitosan nanoparticles (CsNPs) have a potential ability to enhance the immune responses of plants (Chandra, S. et al., 2015), this makes CsNPs a potential competitor in formulating the protective compositions directed toward enhancing the plant immunity. In this work, the In vivo efficacy of copper-chitosan nanocomposition (CuCs) will be evaluated to treat the vascular wilt disease in date palm. The idea relies upon exploiting the dual functionality of this nanocomposition, i.e. its ability to induce and augment the innate immune responses in the plant and its potential antifungal activity against the fungal pathogen itself. This can hinder the fungal growth inside the vascular system of the infected plant and hence beleaguering the vascular wilt disease.

II.
MATERIALS AND METHODS All chemicals used were analytical grade of purity, and were used as received without any further purification.  2 g of chitosan powder (Degree of Deactylation ≥ 90%; water content ≤ 8.0%; Carl Roth, Germany) were dissolved into 1 liter of 1% acetic acid solution.  0.5 g of sodium tripolyphosphate (TPP) (Sigma Aldrich) was dissolved into 1 liter of distilled water.  TPP solution was added dropwise under stirring to the chitosan solution in 1:10 volumetric ratio.  Temperature was kept at 25 °C and pH was adjusted at 4.

Preparation of CuNPs
CuNPs were prepared according to the chemical reduction method (Mustafa B. and Ilkay S., 2010), in which Lascorbic acid (0.11M) (Future Modern Co., Egypt) was added into aqueous solution of CTAB (0.09M) (Sigma-Aldrich, Egypt). NaOH solution was used to adjust pH at 6.8; temperature was kept at 85 °C. During stirring of the previous mixture of CTAB and L-ascorbic acid, Copper sulfate pentahydrate (0.03M) (Elnasr Pharmacuticals Co., Egypt) solution was introduced. The reaction was continued until reddish brown color was developed. Then, CuNPs were collected by centrifugation for further characterization and application.

Characterization of CsNPs and CuNPs 2.2.1. Fourier Transform Infrared Spectroscopy (FTIR) of CsNPs
Fourier Transform Infrared Spectroscopy (FTIR) was performed to confirm the successful ionic gelation between chitosan and TPP. In this step, solutions of chitosan and CsNPs were firstly lyophilized, using the lyophilizer (EDWARDS Freeze Dryer), then FTIR analysis was performed on the lyophilized chitosan and CsNPs using FTIR spectrophotometer (Model Jasco 4100, Japan; 400 -4000 cm -1 ).

UV-Vis spectroscopy of CuNPs
The characteristic surface plasmon resonance of CuNPs was detected using Helios Gamma Spectrophotometer.

Determining Particle Size Distribution of CsNPs and CuNPs
Dynamic light scattering (DLS) (Zetasizer nano series (Nano ZS), Malvern, UK) was used to measure the particles size of the synthesized CsNPs and CuNPs.

Transmission Electron Microscopy (TEM) of CsNPs and CuNPs
Transmission Electron Microscopy (Tecnai G20, Super twin, double tilt, FEI, Netherland) was used to figure out the shape of the synthesized CsNPs and CuNPs.

Evaluating the Antifungal Activity of CuCs
Spores` inoculum of the fungus was prepared; then the microscopic enumeration technique was used to adjust the fungal inoculum size at 1.0 × 10 6 spores/ml. After that, 100 ul of the inoculum suspension were used to inoculate each of potato dextrose broth (control) and potato dextrose broth containing different concentrations of the nanocomposition. Both the control and treatments were kept at 30 °C for 2 days. Then, the optical densities (OD) of the cultures were detected using Uv-Vis Spectrophotometer (ORION AQUAMATE 8000) at 530 nm (Eva Petrikkou et al., 2001). Percentage of inhibition at each concentration was calculated according to the following formula (Ling Yien Ing et al., 2012).

Evaluating the Immune responses of Date Palm Seedlings caused by CuCs
Four groups of date palm seedlings, Sewi cultivar, (10 months) were used. Each group consists of 20 healthy seedlings. Each group was irrigated with 50 ml of CuCs with the respective concentration (0.5, 1.0, 1.5 or 2.0 g/l) per seedling. The fifth group of seedlings was used as a control, in which seedlings were irrigated with 50 ml water per seedling. Leaves from each seedling were dried, then the total phenolics and enzymes were extracted (kâhkônen et al.,1999).  The level of total phenolics in the extracts was quantified by the modified Folin -Ciocateu method (Singelton and Rossi, I965). Gallic acid standard (5 g%) was used, and the total phenolic content was expressed as milligram Gallic Acid per gram dry weight of the original sample (mg GA/g dw).  Phenoloxidase activity was determined according to (Ishaaya, I971). The phenol oxidase activity was determined as optical density (OD405) units ×10 3 at an absorbance of 405 nm.  Peroxidase activity was determined according to (Vetter et al., I958). The enzyme activity was expressed as the change in absorbance at 430 nm (∆OD430)/minute/g fresh weight.

Evaluation of the In vivo Efficiency of CuCs in Treating Fusarium Wilted-Date Palm Seedlings In Comparison With Rizolex TM 2.7.1. Initiation of Fusarium Wilt Disease
Firstly, the fungal culture was raised on Richard's liquid medium and incubated at 26°C for 2 weeks. Thus, the fungal mat is formed (Riker and Riker, 1936). After that, the fungal inoculums were prepared through mixing of 10 g of the mycelial mat with 100 mL of distilled water in a blender (S . Ansari et al., 2012). Each seedling was inoculated through adding 50 mL of the fungal inoculum in its root zone.

In vivo treatment
In this experiment, three groups of the inoculated date palm seedlings, Sewi cultivar, (10 months) were used. Each group consists of 20 inoculated seedlings. 10 days after the fungal inoculation, the treatment began as follow: The first group of inoculated seedlings was used as a treatment, in which 50 ml of CuCs with different concentrations was uniformly applied to root zone of each inoculated seedling. The second group of inoculated seedlings was used as a positive control. In which each inoculated seedling was treated with 50 ml of 3 g/l Rizolex TM fungicide. The third group of the inoculated seedlings was used as a negative control, in which 50 ml of water was added to the root zone of each seedling.

Assessment Disease Severity
Disease progression was observed through two successive weeks. Symptoms of Fusarium wilt disease on leaves (wilting and yellowing) of the date palm seedlings were used to measure the disease severity. In this regard, a standard rating (Campbell and Madden, 1990) was used to assess the disease severity for each group.

Confirming successful preparation of CsNPs by the Ionic Gelation Method
FTIR analysis showed that chitosan has two main characteristic peaks at 3433 cm -1 and 1644 cm -1 which correspond to stretching vibrations of the primary amine group (-NH2) and the amide group (-CONH2), respectively; as shown in figure (1). On the other hand, FTIR analysis of chitosan nanoparticles showed that both peaks were shifted to 3428 cm -1 and 1580 cm -1 , respectively; as shown in figure (2).

Characterization of CsNPs
Size distribution by number using dynamic light scattering revealed that the synthesized CsNPs have an average particle size about 50 nm as shown in figure (3). In addition, transmission electron microscopy showed spherical shape of the synthesized CsNPs, as shown in figure (4).

Confirming successful preparation of CuNPs
The Uv-vis spectroscopy of copper nanoparticles showed their characteristic resonance band at 572 nm, as shown in figure (5).

Characterization of CuNPs
Transmission electron microscopy showed that the synthesized CuNPs have spherical shape, as shown in figure (7). Moreover, dynamic light scattering revealed that the average size of CuNPs was about 100 nm, as shown in figure (8).

In vitro antifungal efficacy of copper-chitosan nanocomposition
All concentrations of CuCs had significant inhibition percentages, as shown in table (1); this demonstrates the potential antifungal efficacy of CuCs against the fungal pathogen, Fusarium oxysporum. Data express the average over triplicates.   Table (3) shows the summary of disease severity (DS) of the inoculated seedlings, which were treated with different concentrations of CuCs in comparison with those treated with water (-ve control) and with Rizolex TM (+ve control). Statistical analysis showed that the disease severity of the inoculated date palm seedlings treated with different concentrations of the nanocomposition was significantly lower than that of the inoculated date palm seedlings which were treated with Rizolex TM (+ve Control) and water (-ve Control), except the concentration of 0.50 g/l.

IV. CONCLUSION
To this point, it was clear that copper-chitosan nanocomposition may provide a competitive candidate for treating vascular wilt disease in date palm, which clearly manifested significantly higher efficiency than the commercially available fungicide at lower concentrations. This can be interpreted by virtue of two main axes: Firstly, the positive immunomodulatory effect of copperchitosan nanocomposition on date palm seedlings as shown from its ability to increase peroxidase, phenoloxidase and total phenols levels, which were considered main constitutes of the plant innate immune response against the invading fungal pathogen; Secondly, the antifungal effect of the nanocomposition on the fungal pathogen itself. Hence, this copperchitosan nanopcomposition with its dual complementary functionality may provide a potential approach to beleaguer the fusarium wilt disease in date palm via enhancing the plant immune responses at the same time of inhibiting the fungal growth. Thus, this nanocomposition can be used in developing new nanofungicides to control such pathogens. But, further research should be undertaken in order to investigate its potential toxicity on plant, human and environment; Thus, determining the optimal concentration that can be used in field without considerable toxicity consequences.