Nano silver treats white powder on cucumbers (cucumbers), pumpkins
Powdery mildew is one of the diseases that seriously harms cucurbits. Crop yield may decrease as disease severity increases. In this study, we have evaluated the effects of silver nanoparticles on white chalk disease under different in vitro and in vivo cultivation conditions. Silver nanoparticles (WA-CV-WA13B) at different concentrations were used before and after an outbreak in plants to determine antifungal activity. In field trials, the use of 100 ppm nano-silver showed the highest inhibition rates both before and after disease outbreaks on cucumbers and pumpkins. In addition, the application of 100 ppm silver nanoparticles showed maximum inhibition of mycelium growth and spore germination in in vivo tests. Scanning electron microscopy results showed that silver nanoparticles exerted adverse effects on mycelium growth and germ cell germination.
Powdery mildew is one of the most destructive diseases of the cucumber family tree. In cucurbits, the disease can be caused by two fungal species, Golovinomyces cichoracearum or Sphaerotheca fusca, which are obligatory parasites that cause the same symptoms but can be easily distinguished under a light microscope. [ first ].
Many vegetable crops are affected by powdery mildew, including artichokes, beans, beetroot, carrots, cucumbers, eggplant, lettuce, melons, peas, peppers, pumpkins, radicchio, radishes, squash, tomato and turnip. Powdery mildew is a group of pathogens that can cause disease in a wide range of environmental conditions. However, a number of environmental factors that can directly affect the development of this disease in cucurbits include temperature, relative humidity and light. Temperature and humidity have to be checked together because the water vapor pressure deficit has the greatest effect on host-parasitic interactions . For example, a temperature of 75 ~ 85 ℉ and a relatively high humidity (80 ~ 95%) in the absence of rain will promote the development of this disease.
Heavily infected white powdery mildew before flowering can reduce cucumber yield by 20 ~ 40%. Leaf damage by this pathogen interferes with photosynthesis and respiration, leading to reduced fruiting, inadequate ripening and poor flavor development . It causes talcum powder, like talc, that develops on the surface of leaves, petiole, and stems. Infected leaves often wither and die, and the plant will age prematurely . The disease is a major production problem in many regions of the world, and the reduction in fruit quality and crop yield are the most prominent aspects of white loss.
Genetic resistance is widely used as a control on cucumbers and pumpkins, and it is being introduced into other cucurbits crops. The yield potential should be considered when selecting varieties because some resistant varieties produce less fruit than susceptible varieties that have not been treated with pesticides. Resistant and winter squash varieties are being developed. The next planting of cucurbits should be physically segregated as older plants may act as a source of spores.
Silver ions are very reactive. They inhibit the respiration and metabolism of microorganisms and they cause physical damage [5, 6]. Furthermore, silver ions can interfere with the bacterial DNA when they enter the cell, preventing further growth of the pathogen . Silver has been used to treat the disease for over 100 years due to its natural antibacterial and antifungal properties. It is also used in many applications as a pure free metal or as a compound because it has antibacterial activity against pathogens but is not toxic to humans.
Recently, nanotechnology has amplified the effectiveness of silver particles as an antimicrobial agent. Nano silver are typically 25 nm in size. 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 . The silver nanoparticles have a relatively large surface area that increases their exposure to bacteria or fungi, greatly improving its bactericidal and fungicidal efficiency. The larger ratio of surface area to volume of silver nanoparticles increases their contact with microorganisms and their ability to permeate cells. When exposed to bacteria and fungi, they adversely affect the cell’s metabolism and inhibit cell growth. Silver inhibits respiration, the basic metabolism of the electronic transmission system, and the transport of the substrate in the cell membrane of microorganisms. Silver nano inhibits the multiplication and growth of bacteria and fungi that cause infections, odors, itching, and sores. The development of nanoparticles has restored interest in the antibacterial effects of metals, which have decreased after modern synthetic antibiotics are widely applied. However, studies on the antibacterial activity of silver nanoparticles have been performed mostly against animal pathogens .
Here, we report that silver nanoparticles can be effectively used in chalk control and preventing harmful infections. Our results support the hypothesis that silver nanoparticles can be prepared in a simple and cost-effective way and suitable for the creation of new types of fungicidal materials.
Materials and methods
Nano silver solution and fungicide
AgNPs used in this study (WA-CV-WA13B, CV) were obtained from Bio-Plus Co. Ltd (Pohang, Korea) at an initial concentration of 1,000 ppm (Table 1). Different working concentrations of silver nanoparticles (10 ppm, 30 ppm, 50 ppm and 100 ppm) were prepared by diluting the original stock solution. All solutions were stored at 4 ℃ until use. Two different fungicides such as NSS-F (Dongbangagro, Co., Seoul, Korea) and Fenari (Dongbu HiTek, Seoul, Korea) were used as active controls.
Table 1 Characteristics of silver nanoparticles used in this study
To determine the effect of nano-silver in field chalkiness, an experiment was performed in Gothan, Chuncheon, and Kangwon-do after a cucumber was naturally infected with this disease. Silver nanoparticle CVs are used at four different concentrations (10 ppm, 30 ppm, 50 ppm and 100 ppm). The above spray method was not used to apply silver nanoparticles around the shoots of entire plants 3 ~ 4 weeks before disease outbreak and after disease appeared. Fungicides NSS-F (Dongbangagro, Co.) and Fenari (Dongbu HiTek) were used as positive controls and distilled water was used as negative controls. Disease index was calculated by counting the number of infected leaves out of 150 leaves of the treated plants. In addition, to determine the efficacy of silver nanoparticles, the experiment was carried out on a pumpkin field in Sembat, Chuncheon, Kangwon-do with a similar procedure for cucumbers. Fungicides NSS-F and Fenari were used as positive controls and water was used as negative controls as described above. Each experiment was repeated three times and disease index was calculated by counting the number of infected leaves out of 150 leaves of the treated plants.
In vitro assay and scanning electron microscope (SEM) analysis
Infected leaves measuring about 5 cm x 5 cm were aseptically collected and submitted to the laboratory for in vitro analysis of chalkiness using SEM. The diseased portion is cut from the leaves and kept in a Petri dish (90 x 15 mm). Five mL with four different concentrations (ie, 10 ppm, 30 ppm, 50 ppm and 100 ppm) silver nanoparticles CV was applied on the surface of the leaves using an aerosol. Water was used for control. The treated leaves were incubated for four days at room temperature. The treated leaves were then observed by SEM supplied by SEM (LV-SEMS-3500N Hitachi, Korea Institute of Basic Science-Chuncheon).
Results are obtained one week after the last treatment session after an outbreak, and results are obtained four weeks after the last treatment for pre-outbreak treatment. The incidence (%) was determined by calculating the number of infected leaves out of 150 leaves of the treated plants. A plant with symptoms is considered infected.
The effect of nano silver on white powder on cucumber
The results for disease incidence (%) on silver-nanoparticles treated cucumbers before and after disease outbreaks are shown in Figure 1. Mean disease rates observed in the control plants were 82.0%. All plants treated with CV silver nanoparticles had disease reduction effects compared with the control. The disease incidence was significantly lower in plants treated with high concentrations of silver nanoparticles. Inhibition increases with increasing concentration of silver nanoparticles. The chemical fungicide “Fenari” showed the lowest rate of disease (3%). Crops treated with other commercial NSS-F fungicides showed a 25.5% disease incidence. A comparative analysis of disease rates when plants treated with different silver nanoparticle concentrations before and after disease outbreak were also evaluated. The incidence of the disease was higher in the plants treated after an outbreak in the plant. The incidence of disease was observed to be 57.8, 48.8, 40.2 and 20% at 10, 30, 50 and 100 ppm of silver nanoparticles treated after the outbreak of the crop. In a similar fashion, disease rates were observed to be 45, 40, 27 and 18% at 10, 30, 50 and 100 ppm of silver nanoparticles treated before disease outbreaks in crops. Therefore, the results showed that the application of silver nanoparticles was more effective when applied prior to any disease symptoms in plants. In addition, the 100 ppm concentration of silver nanoparticles was more effective than the commercial fungicide NSS-F under both pre- and post-outbreak conditions. Corresponding to concentrations of 50 and 100 ppm of silver nanoparticles treated before disease outbreak in plants. Therefore, the results showed that the application of silver nanoparticles was more effective when applied prior to any disease symptoms in plants. In addition, the 100 ppm concentration of silver nanoparticles was more effective than the commercial fungicide NSS-F under both pre- and post-outbreak conditions. Corresponding to concentrations of 50 and 100 ppm of silver nanoparticles treated before disease outbreak in plants. Therefore, the results showed that the application of silver nanoparticles was more effective when applied prior to any disease symptoms in plants. In addition, the 100 ppm concentration of silver nanoparticles was more effective than the commercial fungicide NSS-F under both pre- and post-outbreak conditions.
Figure 1 Effect of nano silver WA-CV-WA13B on white powder on cucumber. Results are obtained one week after the last treatment of the treatment after an outbreak and other results four weeks after the last treatment of the pre-outbreak treatment. Commercial fungicides NSS-F (Dongbangagro, Co., Seoul, Korea) and Fenari (Dongbu HiTek, Seoul, Korea) were used as an active control measure. Distilled water was used as a negative control. Data are obtained from the triple tests and presented as mean ± SD.
The effect of nano silver on white powder on pumpkins
Disease incidence of silver nanoparticle CV was analyzed against white powdery mildew disease on pumpkins (Figure 2). Average disease incidence was the maximum, ie 85% in the control plants. Active control “Fenari” showed the lowest disease rate (4%) for powdery mildew, and another commercial fungicide NSS-F showed a disease rate of 34.4%, which is higher than with the rate of 100 ppm nano silver fertilizing both before and after the disease. flare conditions. The efficiency of silver nanoparticles of 50 ppm was also higher than that of NSS-F when treated before disease outbreaks on plants. However, the morbidity rate was similar to that of NSS-F in treatment with 30 ppm silver nanoparticles. The highest prevalence of disease was observed in the case of silver nanoparticle treatment performed after an outbreak in a crop. The incidence of the disease is significantly low when treatment is carried out before the disease appears in the crop. Disease inhibition was significantly high at all four silver nanoparticle concentrations when it was applied prior to a crop outbreak. Disease rates were observed to be 60, 60, 65 and 25% at concentrations 10, 30, 50 and 100 ppm of silver nanoparticle treatment after outbreaks in plants.
Figure 2 Effect of nano silver WA-CV-WA13B on white powder on pumpkin. Results are obtained one week after the last treatment of the treatment after an outbreak and other results four weeks after the last treatment of the pre-outbreak treatment. Commercial fungicides NSS-F (Dongbangagro, Co., Seoul, Korea) and Fenari (Dongbu HiTek, Seoul, Korea) were used as an active control measure. Distilled water was used as a negative control. Data are obtained from the triple tests and presented as mean ± SD.
The inhibition of powdery mildew was observed to be significantly high in the case where treatment was performed prior to an outbreak on pumpkins. Disease rates observed in silver nanoparticles of 10 ppm, 30 ppm, 50 ppm and 100 ppm were 53.4, 34.4, 25 and 20%, respectively, treated before disease outbreaks in crops. The commercial fungicide NSS-F showed a lower disease suppression rate than treatment of 100 ppm on pumpkins under both pre- and post-outbreak conditions. The results showed that the inhibition of powdery mildew in both plants was dependent on the concentration of silver nanoparticles and the time of treatment. Pumpkin powdery mildew disease can be successfully reduced by using nano-silver according to dosage as well as before disease outbreak in host plants.
SEM analysis of silver nanoparticles against the germination of mycelium and spores
The leaves infected with chalk white were used for SEM analysis of the ability to inhibit the disease by silver nanoparticles. The control leaves treated with distilled water had slightly wrinkled spores and mycelium 2 days after treatment but regained their original shape 4 days after treatment (Figure 3). When powdery mildew was treated with 10 ppm silver nanoparticles, the spores and mycelium wrinkled and sunk (Figure 4). The situation worsened over time. Similar conditions were observed in the spores and mycelium treated with 30 ppm and 50 ppm silver nanoparticles (Figures 5 and 6). When powdery mildew was treated with 100 ppm silver nanoparticles, cell deformations occurred from day 2 after treatment and the condition worsened over time (Figure 7). In most cases, death and lysis of the spores and mycelium are observed.
Fig. 3 The spores and mycelium of powdery mildew were treated with distilled water (control) and observed with scanning electron microscopy for four days with a two-day period.
Fig. 4 The spores and mycelium of powdery mildew were treated with 10 ppm WA-CV-WA13B silver nanoparticles and observed by scanning electron microscopy for four days with a two-day period.
Fig. 5 The spores and mycelium of powdery mildew were treated with 30 ppm WA-CV-WA13B silver nanoparticles and were observed by scanning electron microscopy for four days with a two-day period.
Fig. 6 The spores and mycelium of powdery mildew were treated with 50 ppm WA-CV-WA13B silver nanoparticles and observed by scanning electron microscopy for four days with a two-day period.
Fig. 7 The spores and mycelium of powdery mildew were treated with 100 ppm WA-CV-WA13B silver nanoparticles and observed by scanning electron microscopy for four days with a two-day period.
Little is known about the effects of silver on phytopathogenic fungi, because most studies have focused on bacterial and viral pathogens in animals. In this study, we evaluated the inhibitory effects of silver nanoparticles on powdery mildew in cucumbers and pumpkins in the field. Our results clearly demonstrated that the silver nanoparticles inhibit the fungal pathogen white powder. Previous studies have suggested that nanometer-sized silver possesses different properties, possibly from morphological, structural and physiological changes . Silver nanoparticles are highly reactive because they produce Ag + ions, while metallic silver is relatively unreacted . Nanoparticles penetrate the microbial cell, which means that a lower concentration of nanoscale silver is 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 . The dysfunction of ionic flow can cause a rapid accumulation of silver ions, disrupting cellular processes such as metabolism and respiration by reacting with molecules. In addition, silver ions induce oxygen species that react through their reaction with oxygen, detrimental to cells, damaging proteins, lipids and nucleic acids [13, 14].
In this experiment, the 50 ppm and 100 ppm silver nanoparticles had a significant inhibitory effect on powdery mildew, in both field experiments. As we observed, a solution below 100 ppm showed a low inhibition rate when it was applied after an outbreak. But when silver nanoparticles were used 3 ~ 4 weeks before disease outbreak, even a concentration of 50 ppm silver nanoparticles could effectively inhibit chalk. This suggests that disease containment can be achieved with a low concentration of silver nanoparticles when it is applied prior to an outbreak in the field. Therefore, this study has demonstrated that silver nanoparticles can control powdery mildew under field conditions.
Inhibition Effects of Silver Nanoparticles against Powdery Mildews on Cucumber and Pumpkin