Radiation Synthesis of Silver Nanoparticles/Chitosan for Controlling Leaf Fall Disease on Rubber Trees Causing by Corynespora cassiicola

Silver nanoparticles (AgNPs) were successfully prepared by γ-rays irradiation of solution containing 1.0–10mM of silver nitrate and 1% chitosan. The optical characteristics and particles sizes of AuNPs were determined by UV-Vis spectra and TEM images, respectively. The size of AgNPs increased by the increase of silver concentration or the decrease of chitosan molecular weight in irradiated solution.The in vitro test showed that AgNPs inhibited the growth ofCorynespora cassiicola on rubber-leaf extract media with the inhibitory efficiency of 52.1–100% by treatment of AgNPs with particle size from 15 to 5 nm, respectively. In addition, antifungal activity was found to reach ∼100% by the addition of 90 ppm AgNPs. The in vivo foliar treatment of AgNPs on 9month-old rubber plants showed that the treatment with 2.5–12.5 ppmAgNPs on tested plants after inoculation by spraying with C. cassiicola spores enhanced the rate of non-disease-infected plants from6.0 to 93.3%, respectively, compared to the untreated control. The inhibition effect of AgNPs on fungal growth of C. cassiicolamycelial was also elucidated via SEM images. The AgNPs/chitosan synthesized by γ-irradiation is potentially promising to use as a fungicidal product for treating C. cassiicola, a serious pathogen fungus on rubber trees.


Introduction
Rubbers (Hevea brasiliensis) are long day-industrial trees and provide raw materials for many industrial sectors.They possessed very high economic values and have already brought many profits for agriculture.According to General Statistics Office of Vietnam in 2017, Vietnam is now having the third ranking with rubber tree plantation area reaching 971.6 thousand hectares and the total production of rubber latex reached 1,086 thousand tons.However, rubber growers are now facing many difficulties due to the outspread of many diseases caused by microorganisms, among which, leaf fall disease caused by C. cassiicola is now severely affecting rubbers growth and yield [1]. C. cassiicola disease was first observed on rubber trees in Sierra Leone (Affrica) in 1936.More cases were reported in India and Malaysia in 1961; Nigeria in 1968; Thailand, Srilanca, and Indonesia in 1985; Brazil and Bangladesh in 1988 [2,3].Though leaf fall disease in rubbers was only observed in Vietnam from August 1999, the disease was spread rapidly and widely in many provinces in Southeast, Central Highlands, and Central Coast of Vietnam.Prevention of leaf fall disease is now still a challenging problem due to the lacking of specific fungicides while growers are now using many chemical derived products that would cause various negative impacts on the environment as well as rubber quality.Chitosan at low molecular weight has been proved as a natural, safe, and effective product for agriculture [4].Many researches have been already reported that besides having growth enhancing effects on plants, chitosan also provides plants with the ability to prevent many pathogenic infections by boosting the immunity system of plant cells, so called phytoalexin effects [5][6][7].In addition, silver nanoparticles (AgNPs) were extensively studied and widely used for a long time due to their unique properties such as bacterial and fungal inhibition and odor removal at low concentration and safe to human and the environment.The antifungal effect of AgNPs stabilized in chitosan has been studied on pathogen fungi such as Colletotrichum [8] and Corticium salmonicolor [9].Moreover, irradiation with -rays using a Co-60 source was considered as an effective method for the synthesis of noble metal nanoparticles [10].The advantages of irradiation method were energy, premises, and materials saving, environmentally friendly, and so forth.In particular, the process can be conducted at ambient temperature and favorably scaled up to mass production with reasonable price [10][11][12].This research aimed to synthesize AgNPs/chitosan product that is safe to human and effective in elimination of leaf fall disease caused by C. cassiicola on rubber trees.

Synthesis of AgNPs/Chitosan by 𝛾-Ray Irradiation.
Silver nitrate solutions at different concentrations of 1.0; 2,5; 5.0; and 10 mM dissolved in 1% chitosan solution were stored in glass bottles and irradiated at different doses from 8 to 28 kGy using -rays from a Co-60 source (Gamma Chamber 5000, BRIT, India) with a dose rate of 3 kGy/h.AgNPs colloidal solutions synthesized by -ray irradiation were used for further experiments.

Characterization of Silver
Nanoparticles/Chitosan.The UV-Vis spectra of resultant AgNPs solution samples which were diluted to 0.1 mM calculated as Ag + concentration using deionized water were measured on an UV-2401PC, Shimadzu, Japan [9,11,13].The AgNPs size and size distribution were determined by TEM images on a JEM 1400, JEOL, Japan, followed the method described by Li et al. [11] and Phu et al. [9].

Antifungal Activity of AgNPs/Chitosan
. Rubber-leaf extract media were used for C. cassiicola culture.The rubberleaf extract was prepared by boiling 200 g of fresh rubber leaves (free disease) in 1000 ml distilled water for 45 minutes and followed by filtering to remove residue and adjusting pH ∼ 6.5.One liter of rubber-leaf extract was mixed with 20 g agar and supplemented with AgNPs at different particle sizes 5; 10; and 15 nm, and AgNPs concentration varied from 10 to 90 ppm before autoclaving at 121 ∘ C, 1 atm for 20 min.Fungal samples with diameter ∼4 mm were cultured on the center of rubber-leaf extract agar plates and incubated in dark condition at 28 ± 2 ∘ C. Five replicate plates were applied for each concentration or particle size text and all experiments were repeated in triplicate.The colony diameters of C. cassiicola in the medium were measured every 24 hours and the antifungal efficiency of AgNPs/chitosan was calculated using the following: where  (mm) and  (mm) are fungal colony diameters on the medium with or without supplemented with AgNPs, respectively.

Scanning Electron Microscopy (SEM)
Analysis.Approximately 5 ml solution containing 7.5 and 12.5 ppm of AgNPs was spread on fully developed C. cassiicola mycelia grown on rubber-leaf extract agar media by a sprayer.The control was applied by distilled water in the same volume.All applications were done with the time interval of four days and were incubated at room temperature.The SEM images of treated samples were observed on a FE-SEM S4800, Hitachi Co., Tokyo, Japan, at an accelerating voltage of 10 kV.

In Vivo Fungicidal Effect of AgNPs against C. cassiicola on Rubber
Plants.Nine-month-old rubber trees (diseasefree) were grown and maintained in a greenhouse before the experiment.For testing the elimination effect of AgNP on fungal pathogen C. cassiicola, leaves of 210 disease-free rubber trees (30 plants for each treatment) were first wounded before inoculating with 300 mL C. cassiicola solution containing 10 4 fungal spores/mL.The control ones were foliar sprayed with the same volume of distilled water.After infection about 7 days, the number of disease-infected plants reached to about 50% for all of the treatments; 50 mL AgNPs solution with concentrations of 2.5, 5, 7.5, 10, and 12.5 ppm was foliar sprayed every 3 days up to 3 times.The number of diseaseinfected plants and the number of disease-infected leaves in each treatment were observed after 7 days within 28 days of experiment.Plant and leaf disease incidence (DI) were expressed in percentage (%) and calculated by using the following [14]: ( Each experiment was carried out in triplicate and data were statistically analyzed using the analysis of variance (ANOVA) test.The mean values were compared using Duncan's multiple range test at a probability level at 5%.

Radiation Synthesis of Silver Nanoparticles/Chitosan by 𝛾-
Ray Irradiation.In this experiment, the solution containing silver ion concentrations of 1; 2.5; 5; and 10 mM stabilized in 1% chitosan with molecular weight (Mw) about 550 kDa was irradiated at 8, 12, 16, and 28 kGy, respectively.Huang et al. [15] reported that different AgNPs sizes would lead to various maximum absorbance wavelengths ( max ) and peak intensities.The value of  max shifted to the longer wavelength with the increase of AgNPs size [9,15,16].It can be seen from Figure 1 that the peak intensity values of irradiated samples in UV-Vis spectra decreased from 1.26 to 1.02 by the increase of the silver concentration in irradiated sample from 1 to 10 mM, while  max values of these sample shifted from 397 to 405.5 nm.In addition, the particle size estimated from TEM images of AgNPs samples after irradiation also increased from 5 to 10 nm when the sliver concentration increased from 1 to 10 mM, respectively.Many researchers reported that the antimicrobial activities of AgNPs increased by the decrease of particle size [17].Thus, in order to synthesize AgNPs with desired size for different applications, selection of the initial Ag + concentration to prepare AgNPs is very necessary.
On the other hand, the effect of Mw of chitosan on particles size of AgNPs was also investigated.The results in Figure 2 showed that Mw of chitosan affected the size of AgNPs formed in irradiated samples and the decrease of chitosan Mw from 550 to 100 kDa led to the increase of AgNPs size from 10 to 15 nm.Though the mechanism of these effects was not yet fully understood, the increase of solution viscosity corresponding to the increase of chitosan Mw might have reduced the dynamic of AgNPs and prevented them from agglomeration to form bigger nanocluster.

In Vitro Antifungal Activities of AgNPs/Chitosan against C. cassiicola.
For testing antifungal efficiency by particle size of AgNPs/chitosan sample, AgNPs with particles size of about 5, 10, and 15 nm were used.It can be seen in Figures 3 and 4 that the small particle size has a stronger effect on the growth of C. cassiicola on rubber-leaf extracted media after 192 hours of incubation compared to the big ones.Particularly, the supplementation of 50 ppm AgNPs with particles size 15 nm showed a strong inhibition effect on C. cassiicola with the diameter fungal colony only reaching 43.1 mm compared to 90 mm of the untreated control and the inhibition efficiency was calculated about 52.1%.The antifungal efficiency increased to 70.6% when the particle size decreased to 10 nm and reached to almost 100% for 5 nm particle size.Thus, the antifungal efficiency against C. cassiicola increased by the decrease of AgNPs size.The same tendency was also reported previously by Carlson et al. [17].In addition, Franci et al. [18] also pointed out that it is easier for AgNPs at smaller size to penetrate through cell walls and alter and inhibit the cell signaling pathways as well as DNA replication and damage of cell organs through oxidative reaction.
On the other hand, the antifungal activities of AgNPs have been proved to be increased by the increase of silver concentration [9,[19][20][21].In this study, AgNPs with size of 10 nm stabilized in 1% chitosan (Mw ∼ 550 kDa) were used to evaluate the in vitro antifungal activities against C. cassiicola at different AgNPs concentrations.Results from Figures 5  and 6 indicated that, after 10 days of incubation, the growth inhibition against C. cassiicola on cultural media plates supplemented with 10 and 30 ppm AgNPs with inhibition efficiencies from 6.3 to 18.5%, respectively, was rather low.Meanwhile, the growth of C. cassiicola was clearly inhibited by an addition of 50 ppm with the fungal colony diameter only 50 mm compared to that of 90 mm on the untreated control plates (inhibition efficiency ∼66.4%).However, the inhibition activity of AgNPs increased when an additive concentration increased to 70 ppm (inhibition efficiency ∼91.7%) and the growth of C. cassiicola was completely inhibited when the silver concentration reached 90 ppm with an inhibition efficiency of ∼100%.

In Vivo Antifungal Activities of AgNPs/Chitosan against C. cassiicola.
There are several reports suggesting the antimicrobial effect of AgNPs against bacteria and fungi [9,22,23].However, up to now, there are rare reports in the literature on the in vivo studies of the effect of AgNPs against fungi on plants.In the present study, the antifungal effect of AgNPs against C. cassiicola was investigated directly on rubber plants.The AgNPs with concentrations of 2.5 to 12.5 ppm were applied on the tested rubber plants via foliar application.Alternatively, the AgNP has been proved to be efficient in controlling the disease when sprayed after the infection caused by fungi.The results from Table 1 disclosed that the damage of rubber plants infected by C. cassiicola fungus could  be minimized by AgNP.In particular, plant DI decreased from 30 to 6.3% by treating AgNPs with the concentrations from 2.5 to 12.5 ppm, respectively.Furthermore, it can be seen from Table 1 and Figure 7 that the treatment of AgNPs also remarkably minimized the leave DI of infected plants and the rate of leave DI was inversely proportional to the treated concentration of AgNPs.These results are in agreement with the previous finding on Colletotrichum fungus species causing the anthracnose disease for pepper reported by Lamsal et al. [8].The reasons can be explained that, after spraying, AgNPs may attach and kill the mycelium of pathogen fungus in plant tissue [24,25].
The inhibition of AgNPs on C. cassiicola mycelial germination was analyzed via SEM in order to elucidate the effect of AgNPs on fungal growth.The SEM images in Figure 8 revealed that AgNPs clearly damaged the hyphae, while hyphae treated with water (control) appeared to remain intact.Fungal mycelia were sunken and damaged due to the effect of AgNPs.The fungal hyphae observing at the fourth day after the treatment of 7.5-12.5 ppm AgNPs showed  deformities in mycelial growth and the shape of hyphal walls.The layers of hyphal walls were also torn off on damaged hyphae at the fourth day and many hyphae were collapsed.

Conclusions
AgNPs with different particle sizes were successfully synthesized by gamma Co-60 irradiation method using chitosans with various Mw as stabilizers.The synthesized AgNPs/ chitosan products showed a strong inhibition effect against C. cassiicola on rubber-leaf extract media with the antifungal efficiency reaching ∼100% by the treatment of 50 ppm AgNPs with size of 5 nm or 90 ppm AgNPs with size of 10 nm.The foliar treatment with 5-12.5 ppm AgNPs solution strongly reduced the disease incidence of rubber trees as well as the disease incidence of rubber leaves infected by C. cassiicola fungus.Thus, AgNPs/chitosan synthesized by gamma Co-60 irradiation method that is considered as a green method could be used as a potentially and effectively fungicidal product for controlling of C. cassiicola, a serious pathogen fungus on rubber trees.

Figure 3 :Figure 4 :
Figure 3: The antifungal activity against C. cassiicola of AgNPs with different particle sizes.(a) Diametter of fungal colony on rubber-leaf extract media after 192 hours incubation and (b) inhibition efficiency.

Figure 5 :Figure 6 :
Figure 5: The antifungal activity against C. cassiicola of AgNPs (10 nm) at different concentration.(a) Diameter of fungal colony on rubber-leaf extract media after 192 hours of incubation and (b) inhibition efficiency.

Figure 7 :Figure 8 :
Figure 7: The rubber plants infected with C. cassiicola and treated with AgNPs (10 nm) at different concentration.Checking: plant without treating by fungal spore and AgNPs; control: plants infected by fungus spores before treating with only distilled water.
Silver nitrate powder (AgNO 3 ) in pure grade was supplied by Merck Co., Germany.Chitosan with degree of deacetylation of 80% was purchased from Loyou Chemical Co., Ltd., Japan.Pathogenesis fungus, namely, Corynespora cassiicola, was a gift from Rubber Research Institute of Vietnam. 2.1.Materials.

Table 1 :
Effect of AgNPs concentration on elimination of C. cassiicola after being inoculated in rubber plants.Checking: plant without treatment of fungal spore and AgNPs.* * Control: plants were infected by fungus spores before treating with only distilled water.* * * DAT: day after treatment; mean values followed by the same letter within a column are not statistically different according to Duncan's multiple range test at  < 0.05.