Nano silver treats anthracnose on pepper caused by the fungus Colletotrichum
Pepper anthracnose caused by the Colletotrichum species is one of the most important limiting factors for pepper production in Korea, the management of pepper depends heavily on chemicals. The purpose of this work is to evaluate the use of silver nanoparticles in place of commercial fungicides. In this study, we evaluated the effects of nano-silver on anthracnose on pepper plants under different growing conditions. Silver nanoparticles (WA-PR-WB13R) were applied at different concentrations to determine antifungal activities in vitro and in the field. The application of nano silver with concentration of 100 ppm produced maximum inhibition of mycelium growth as well as spore germination compared to in vitro control. In field trials, the ability to inhibit fungi was significantly high when silver nanoparticles were applied prior to disease outbreaks in plants. Results of scanning electron microscopy showed that silver nanoparticles had a negative effect on the growth of mycelium of Colletotrichum species.
Anthracnose is caused by the fungus Colletotrichum, a serious disease that occurs in many commercial crops such as beans, mulberry, perilla and other crops . To control various plant fungi, including Colletotrichum species, agrochemicals have long been used. The widespread use of agrochemicals has undoubtedly reduced fungal outbreaks, but also contributes to the development of resistant pathogens [2, 3]. Furthermore, such chemicals can kill beneficial microorganisms in the biosphere and beneficial insects in the soil, and they can also enter the food chain and accumulate in the human body below. form of unwanted chemical residues . With the emergence and proliferation of antibiotic-resistant microorganisms, and the continued emphasis on healthcare costs, many researchers have attempted to develop new and effective antimicrobial reagents. , does not stimulate resistance and is less expensive.
Nanoscale materials have emerged as a new antimicrobial agent thanks to their high surface area to volume ratio and unique physical and chemical properties, increasing their exposure to bacteria. and cell permeability [5, 6]. In addition, nanotechnology has amplified the effectiveness of silver particles as an antimicrobial agent.
Silver is known to attack a wide range of biological processes in microorganisms including membrane structure and function [7-9]. Silver also inhibits the expression of proteins involved in ATP production , although its specific antibacterial mechanisms are not fully understood. The use of nano-sized silver particles as an antimicrobial agent has become more common as technological advances have made their production more economical. One of the potential uses for silver is in crop disease management.
Since silver displays a variety of modes of inhibitory action , it can be used to control various plant pathogens in a relatively safer manner than synthetic fungicides [12 ]. To date, limited studies have provided some evidence of silver’s applicability to crop disease control . Reducing the particle size of materials is an effective and reliable tool for improving their biocompatibility. In fact, nanotechnology helps overcome the limitations of size and can change the world’s view of science . Furthermore, nanomaterials can be modified for better efficiency in order to facilitate their applications in various fields such as biological sciences and medicine.
This simple size comparison gives the idea of using nanoparticles as very small probes that allow to elucidate the cell apparatus without causing too much interference . Understanding biological processes at the nano level is a powerful driving force behind the development of nanotechnology .
In this study, we demonstrate the inhibitory effects of silver nanoparticles on the Colletotrichum and anthracnose species on pepper plants in vitro and under field trials, respectively. In vitro studies have mentioned the effect of silver nanoparticles on Colletotrichum species at different concentrations and growth media as well as the control mechanism of silver nanoparticles against Colletotrichum in real trials. geography.
Materials and methods
Silver nanoparticles and fungicides
Nano silver (WA-PR-WB13R [PR]) provided by Bio-Plus Co. Ltd. (Pohang, Korea) at an initial concentration of 1000 ppm. Colloidal silver nanoparticle solutions were diluted to different concentrations of 10 ppm, 30 ppm, 50 ppm and 100 ppm using room temperature distilled water (25 ℃). These nanoparticles have colloidal shape with average size from 4 ~ 8 nm. All these solutions are stored at 40 ℃ for further use. Distilled water is used for control and two different fungicides are used for active control: NSS-F consists of nano silver and titanium (Dongbangagro Co., Seoul, Korea) and Fenari (Dongbu HiTek, Seoul , Korea).
Fungi and means of growth
Six species of Colletotrichum were taken from the Korean Agricultural Culture Collection (KACC), Suwon, Korea (Table 1). These fungi were grown on potato dextrose agar (PDA) and incubated at 28 ± 2 ℃. To differentiate the antifungal activities of silver nanoparticles in culture media, three different agar media were used: PDA, malt extract agar (MEA) and corn starch agar (CMA).
Table 1 Colletotrichum species and their main host plants were used in this study
Test in vitro
In vitro tests were performed on PDA, MEA and CMA treated with silver nanoparticles WA-PR-WB13R 10, 30, 50 and 100 ppm. 5 mL of WA-PR-WB13R were poured into the medium prior to plating 90 × 15 mm Petri dishes. The medium containing silver nanoparticles was incubated at room temperature. After 48 h of incubation, a stopper of agar 8 mm diameter containing fungi was simultaneously inoculated in the center of each Petri dish and incubated at 28 ± 2 ° C. After 2 weeks of incubation, inhibitory areas were measured. The test was repeated twice and each treatment was repeated three times. The inhibition rate (%) is calculated by the following formula:
Inhibition rate (%) = (R-r) * 100 / R
where R is the radial growth of the fungus in the control plate and r is the radial growth of the fungus in the silver nanoparticle treated plate. Data from the experiments were analyzed by analysis of variance (ANOVA) using SAS ver. 9.2 (SAS Institute Inc., Cary, NC, USA) and mean values were compared using Duncan multi-band test (DMRT) at p = 0.05.
Field testing and data analysis
To determine the effect of silver nanoparticles on anthracnose on pepper, an experiment was carried out in Chuncheon, Kangwon-do, Korea before and after the pepper was naturally infected. Silver nanoparticles are used simultaneously at 10, 30, 50 and 100 ppm. Silver nanoparticles were used on the leaves of plants 3 ~ 4 weeks before disease outbreak and after disease outbreak. Silver nanoparticles were used every week for four weeks. Results were obtained 4 weeks after the last treatment of the treatment before the outbreak and one week after the last treatment of the treatment after the outbreak. The commercial fungicide ‘NSS-F’, containing nano silver and platinum, and the chemical fungicide ‘Fenari’ was used for active control. Distilled water was used as a negative control. The incidence (%) was calculated by counting the number of infected leaves out of 150 leaves of the plants treated in field trials. Each experiment was repeated three times.
Analysis by scanning electron microscopy (SEM)
Approximately 5 mL of solution with four different concentrations, 10 ppm, 30 ppm, 50 ppm and 100 ppm, of the silver nanoparticles WA-PR-WB13R were applied on fully grown C. gloeosporioides mycelium. grown on PDA by spray machine. Application was performed at intervals of 1, 3, 5, 10 and 15 days and the untreated mycelium was cultured as controls. Each treatment was incubated at room temperature. The whole sample was fixed in 4% glutaraldehyde for 3 hours and treated with 0.1M cacodylate buffer for 1 hour. After washing with distilled water, the test sample is dehydrated in a series of classified ethanol to 100%, dried at the critical point, and covered with gold using an ion diffraction booster. The specimen is observed on SEM (S-3500N; Hitachi Company, Tokyo, Japan) at an accelerating voltage of 10 kV.
Inhibition of Colletotrichum species on different culture media
Six Colletotrichum species were selected to analyze the inhibitory effects of the silver nanoparticles WA-PR-WB13B on PDA, MEA and CMA. Fungal isolates treated with distilled water were used as controls. The silver nanoparticles showed different levels of inhibition on the mycelium growth of Colletotrichum species (Table 2). Apparently a significant inhibition of Colletotrichum mycelium growth was provided with 100 ppm silver nanoparticles on PDA. Full inhibition was observed on PDAs treated with 100 ppm silver nanoparticles against isolates C-3 and C-5. Strains C-7 and C-8 also showed more than 90% inhibition of PDAs treated with 100 ppm silver nanoparticles. Lowest inhibition was observed for C-6 isolates on PDA. In all treatments using 10 ppm silver nanoparticles, the inhibition rate was lower than that of the other treatments.
Likewise, inhibition caused by silver nanoparticles against mycelium growth of Colletotrichum species was also observed on MEA medium; however, the inhibition rate is not significant compared with PDA medium. The highest inhibition rate observed for C-6 treated with 50 ppm silver nanoparticles (84.56%) and treated against C-6 on 30 ppm silver nanoparticles also showed significant inhibition. for MEA (84.50%). The lowest inhibition rate was observed for C-7 treated with 10 ppm silver nanoparticles on MEA (11.33%).
However, on CMA medium, all concentrations showed more than 45% inhibition but the results were not as significant as on PDA. The highest inhibition observed on CMA was against C-7 over 100 ppm concentration of silver nanoparticles (93.50%) and the lowest inhibition was observed for C-8 treated with 100 ppm silver nanoparticles. Therefore, the results showed that the inhibition rate varied according to the choice of growth medium and concentration of silver nanoparticles. In this experiment, PDA was the most favorable environment for the containment of pathogens in vitro. The results also showed different inhibition rates in Colletotrichum species when used in different growing media with different silver nanoparticle concentrations.
Table 2. Inhibition rate (%) of WA-PR-WB13R silver nanoparticles against Colletotrichum species on different growing media and concentration (ppm)
Data followed by the same letter (s) in the same column did not differ significantly according to the Duncan multi-range test (DMRT) at p = 0.05.
a The inhibition rate (%) was determined based on the 3 repetitive methods.
Effects of nano-silver on anthracnose in field trials
The inhibitory effect of WA-PR-WB13B silver nanoparticles was analyzed for anthracnose on pepper plants near Chuncheon, Korea (Figure 1). Average disease rate was the maximum (84.1%) in the control plants. Results showed that, in all treatments, a higher incidence of disease was observed in the plants treated after an outbreak. The commercial fungicides, NSS-F and Fenari, showed disease rates of 35.2% and 25.3%, respectively, on plants treated before disease outbreaks.
However, in the treatment after onset of the disease, the incidence was significantly higher than the pre-onset treatment with NSS-F and Fenari (72.1% and 63.4%, respectively). Of all treatments, the lowest disease incidence was observed on plants treated with 50 ppm silver nanoparticles before disease outbreak (9.7%), and the highest rate of disease was observed on plants treated with NSS-F after disease outbreak (72.1%). The results showed that the silver nanoparticle treatment prevented the onset of the pathogen when applied before an outbreak in the crop. In addition, results also indicated that nano silver WA-PR-WB13B treatments were more effective than commercial fungicides NSS-F and Fenari in field trials.
Figure 1. The effects of nano silver WA-PR-WA13B prevent anthracnose from pepper in the field. Results obtained one week after the last treatment to treat after flare-up (three treatments for three weeks at one week apart) and results obtained at 4 weeks for previous treatment when the disease flares (treated three times for 3 weeks at one week intervals). The commercial fungicides NSS-F and Fenari were used as active controls. Distilled water was used as a negative control. Data are obtained from the triple tests and presented as mean ± SD.
SEM observed the mycelium C. gloeosporioides
Different concentrations of silver nanoparticles and inhibition of germination of C. gloeosporioides were analyzed via SEM. To elucidate the effect of silver nanoparticles on fungal growth, healthy mycelia grown on PDA plates were sprayed with a solution of 30, 50 or 100 ppm silver nanoparticles and observed with SEM. This microscopic observation showed that the silver nanoparticles clearly damaged the mycelium (Fig. 2B ~ 2D), while the water-treated mycelium (control) appeared to remain intact. (Figure 2A). Mycelium lesions increase with increasing concentration and time. Mycelium is sunken and damaged by the effect of silver nanoparticles (Figure 2B ~ 2D). The mycelium observed 3 days after treatment of the 100 ppm silver nanoparticles showed defects in mycelium growth and the shape of the mycelium walls. The layers of the mycelium wall were also torn on damaged mycelium after 3 days (Figure 2D) and many mycelium was broken after 5 days. Similar results were observed on the spores of C. gloeosporioides (not shown). Therefore, the results suggest that concentration and duration of treatment play an important role in inhibiting the pathogen.
Figure 2 An electron microscope scanned the mycelium of Colletotrichum gloeosporioides treated with silver nanoparticles. Mycelium grows on potato dextrose agar plates sprayed with water as a control (A) or a solution of 30, 50 and 100 ppm nano silver (B ~ D, respectively). The photo was taken 3 days after processing (graduations = 5 µm).
The results obtained on antifungal activity clearly showed that the growth of Colletotrichum species including C. gloeosporioides was inhibited at different concentrations of silver nanoparticles. Based on a comparison of results obtained before and after disease treatment, results indicate that fungal suppression can be achieved when applied before disease symptoms occur in the crop. The highest antifungal properties were observed in the case of treatment with 50 ppm silver nanoparticles in field tests and 100 ppm silver nanoparticles in vitro. Therefore, the results clearly demonstrate that silver nanoparticles are capable of inhibiting Colletotrichum pathogenic fungi under both field and controlled environment conditions.
Nanometer-sized silvers have different properties, possibly from morphological, structural and physiological changes . Indeed, some evidence supports the enhanced effect of silver nanoparticles on antimicrobial activity. Silver nanoparticles are highly reactive because they produce Ag + ions while metallic silver is relatively inactive . It was also shown that the nanoparticles effectively penetrated the microbial cells, which means that a lower nano size silver concentration would be sufficient for microbial control. This will be effective, especially for some organisms that are less susceptible to the antibiotic due to poor penetration of some antibiotics into cells .
A previous study observed that silver nanoparticles disrupt transport systems including ion flow . A dysfunction of ionic flow can cause a rapid accumulation of silver ions, disrupting cellular processes at their lower concentrations such as metabolism and respiration by reacting with molecules. In addition, silver ions are known to produce reactive oxygen species through their reaction with oxygen, detrimental to cells, causing damage to proteins, lipids and nucleic acids [18, 19].
Meanwhile, a previous study suggested that silver nanoparticles could significantly slow the growth of C. gloeosporioides mycelium in a dose-dependent manner in vitro . However, in this study, we focused on the inhibitory effect of silver nanoparticles on six Colletotrichum species on three different growth media in vitro as well as in field trials. Our previous studies also showed that the use of silver nanoparticles was effective against sclerosing fungi, powdery mildew and Raffaelea sp. [21 – 24].
Similarly, current research also shows that silver nanoparticles are effective in controlling anthracnose pathogens in Colletotrichum species including C. gloeosporioides. Therefore, the silver nanoparticles prepared by the cost effective method have great promise as an antimicrobial agent, and the antifungal activity of the silver nanoparticles has great potential for use in fungal control. plant spore pathogenesis. Field research also showed that, since silver’s effectiveness was greatly affected by long life, the preventive applications of silver nanoparticles performed better before the isolates of the fungus entered and migrated. resident in plant tissue.
In summary, the application of nano silver could lead to valuable discoveries in various areas such as pathogen control and antimicrobial systems. However, with the advent of silver nanoparticles and its primary use as an antimicrobial agent, more experimental trials are needed to understand toxicity. There are still some questions to be addressed, such as how exactly silver nanoparticles interact with fungal cells and how the nanoparticles’ surface area affects the lethal action. In addition, animal models and clinical studies will also need to be done to better understand the antifungal effects of silver nanoparticles.
Application of Silver Nanoparticles for the Control of Colletotrichum Species In Vitro and Pepper Anthracnose Disease in Field