Fusarium wilt of tomato seedling is of great economic importance worldwide due to the considerable damage it causes in the crop. Advances in nanotechnology provide alternatives that can be applied to control pathogens. Chitosan-stabilized Nano silver (AgNP) (Cs) are now widely used to control plant pathogens. The objective of this investigation was to determine the effect of application of Ag-Cs nanoparticles on tomato plant tolerance and control of Fusarium oxysporum wilt disease. Applying nanoparticles did not show negative effects on normal vegetative growth of tomato seedlings (up to 2000 ppm). The nanostructured was significantly effective in inhibiting mycelium growth by more than 70% and in addition, the treatment was effective to reduce disease severity in the following Fusarium oxysporum seedlings. 14 days after transplanting.
Plant disease control is the most challenging aspect of crop production. In recent years, resistance to pesticides available on the market has increased and has become a serious problem (Chattopadhyay et al., 2017; Goffeau, 2008). Among the microorganisms involved in plant diseases, fungi induce higher rates in agricultural systems. Fusarium wilt is a fatal vascular wilt syndrome in plants of the Solanaceae family (Omar & Ahmed, 2014).
The fungus F. oxysporum includes more than 120 known strains or specialties, each specific to a single host plant in which it causes disease. Fusarium oxysporum f. sp. Lycopersici causes wilt on tomatoes. Fusarium wilt is a disease that destroys tomatoes in several countries around the world, and is of great concern to the producers due to the high yield loss, the persistence of fungi in the soil and the development of resistant strains. The disease can be alleviated to a certain extent using chemically and drug resistant cultivars. However, the development of new pathogenic strains is a continuing problem, and the use of chemical products is expensive and not always effective (Amini & Sidovich, 2010; Malandrakis et al. , 2018). Therefore, the control mechanism for this disease, which is neither harmful to the crop nor the environment, is of great interest for researchers working in the field of disease management.
Therefore, finding alternative means of disease control that are safe for the environment is an immediate necessity. In recent years, the use of nanomaterials is increasingly expanding and is seen as an alternative to control plant pathogens. Several metal nanoparticles have been studied and tested for their antifungal properties (Al-Huqail et al., 2018; Janaki et al., 2015; Medda et al., 2015; Shaikh et al., 2019. ). Several studies have demonstrated the effectiveness of Nano silver against plant pathogenic microorganisms. Jo et al. (2009) demonstrated that AgNP was effective in reducing the growth of Bipolaris sorokiniana and Magnaporthe grisea fungi in vitro and reducing disease severity in plants by applying nanomaterials 3 hours after transplanting. with spores, but their effectiveness was significantly reduced when applied 24 hours after transplantation. Likewise, nano-silver was effective against various phytopathogens such as Alternaria, Botrytis cinerea, F. oxysporum and Pythium spinosum (Kim et al., 2012). Mishra et al. (2017) showed antifungal activity of AgNPs against phytopathogens from leaves and soil. The inhibitory effects of different silver concentrations (2, 4, 10 μg / mL) on spore germination were noted under in vitro conditions. In another study, Karimi and Sadeghi (2019) isolated 3 different phytopathogens (Alternaria Alternata, Alternaria citri and Penicillium digitatum) from citrus samples. In vitro tests were performed on PDA media treated with 50, 100 and 150 ppm silver nanoparticles and two common fungicides. The results showed that Ag-NPs showed stronger antifungal activity against isolate fungi that are conventional fungicides.
However, these authors did not evaluate the nanoparticles in vivo. There is a lot of information in the literature on the antifungal effects of AgNP; however, there is a lack of information on the effect on the crop and on the development of the disease at the field level, as well as the association with the literature. Other materials or polymers make the nanostructure more stable and improve its properties. Therefore, the antifungal effects of NP Ag-Cs on Fusarium wilt in tomato seedlings were investigated in this study under greenhouse and in vitro conditions.
MATERIALS AND METHODS
All reagents are of recognized analytical grade and used without further purification. Silver nitrate (AgNO3), sodium borohydride and Chitosan of LMW were purchased from Sigma-Aldrich with purity ≥99.5%. Fusarium oxysporum wild fungus was isolated from infected plants in Costa de Hermosillo, this strain was subcultured in potato dextrose agar (scientifically ThermoFisher) to determine morphology.
Synthesis and characterization of silver nanoparticles
The chitosan coated silver nanoparticles (Ag-CsNPs) were synthesized according to the method proposed by Bin Ahmad et al. (2011). The chitosan solution was prepared by dissolving 1,0 g of chitosan in 50 mL of acetic acid. Immediately thereafter, AgNO 3 (50 mL, 0.01 M) was added to the chitosan suspension under continuous stirring for 2 hours. Then, 20 mL of NaBH 4 (0.04 M) was added to AgNO3 – chitosan suspension, a change of color from light yellow to brown immediately observed indicating the formation of nanoparticles. Nanoparticles were characterized by evaluating surface charge using a zetaseizer device (Malvern Instruments) and morphology and size observed by transmission electron microscopy.
In vitro evaluation of the antifungal ability of NP Ag-Cs agaisnt Fusarium oxysporum
The antifungal effects of Ag-Cs NPs against the fungus F. oxysporum have been evaluated. To do this, the nanoparticles were dissolved at different concentrations (1000, 1500 and 2000 ppm) in a sterile PDA medium at 45 ° C and then the medium was added to Petri dishes to solidify. . Media without the addition of nanoparticles was considered a control treatment. Then, F. oxysporum was inoculated from 7-day old culture medium in the center of Petri dishes. They are incubated at 25 ° C for 7 days. Each treatment was carried out in three times. The diameters of the mycelium growing in vitro were determined, measured at 48, 96 and 168 hours after starting the test using vernier and results expressed in centimeters.
Evaluation of Ag-Cs NP in tomato seed development
To determine if NP Ag-Cs caused any toxic effects, different concentrations of the treatment method on tomato plants were evaluated. The tomato seedling used in this experiment was 20 days old after germination. On the substrate without or with 3% vermiculite, 50 mL of nanoparticles were applied at concentrations of 1000, 1500 and 2000 ppm every 7 days for 30 days. Plant height and diameter are measured as well as dry and fresh weight. Each treatment was carried out in three times.
Effect of NPs Ag-Cs on vascular wilt development in tomato cultured with Fusarium oxysporum
A biological assay was developed by preparing a fungal spore suspension at a concentration of 1 x 107 spores per mL with a 30% glucose solution. Then, a small incision in the root collar was made to facilitate infection, and seedlings 20 days after germination were soaked in a spore suspension for 4 hours. The seedlings were placed in Petri dishes filled with Peatmost® and incubated in the growth chamber for 14 days with 90% relative humidity at 25 ° C. The nanoparticles were applied on the soil at a concentration of 2000 ppm after 5 days. when transplanting. In disease-controlled experiments, non-disease control and treatment with nanoparticles. Each treatment is carried out with 10 repetitions.
The experiments were performed according to a completely randomized design. To determine the effect of the treatment on the response variables, an analysis of variance of one or two routes was performed, case by case, with a confidence index of 95%. Differences between treatments were compared using the Tukey-Kramer trial. The difference at P <0.05 is considered significant. All this using the 2007 NCSS statistics package.
RESULTS AND DISCUSSION
Synthesis and characterization of nanoparticles
Nanotechnology has created great interest in the development of agricultural products, as it presents an excellent opportunity to reduce the use of synthetic agrochemicals, with the potential to reduce environmental impacts and Enhances absorption of nutrients from the soil. Figure 1 shows TEM images of the chitosan-stabilized NPs and the size distribution of the nanoparticles refined, as morphology and size are extremely important for predicting whether the plant can capture and biological distribution of NPs or not. TEM images of the sample showed a spherical shape with a dispersion of less than 2%, with a mean diameter of 33.26 ± 6.15 nm (Figure 1b). The function and stability of chitosan can be clearly seen in Figure 1a with difference in contrast.
Figure 1 TEM image of NP Ag-Cs. a) approach 250,000 x nanoparticles, b) size distribution of AgNPs-CS.
Several variables, such as particle size, AgNP surface charge and soil physicochemical properties, largely govern the chemical properties, fate and transport of AgNP in the soil system and thus , its bioavailability and sequential bioaccumulation in plant tissues (Grün et al., 2019). The retention of silver nanoparticles has been shown in soil suspension to correlate mainly with the clay content of the soil, and the authors support the role of nano-silver heterogeneous with natural colloids to explain termites. This correlation (Grün et al., 2019; Tolaymat et al., 2010). Torrent et al. (2019) Evidence that cation exchange capacity and conductivity are the main parameters controlling the adsorption in soil of nano-silver of various sizes (45, 75, 200 and 200 nm) and coated by polymers with different charges (citrate, polyvi-nylpyrolidone, and polyethylnglycol (PEG))
It has been reported that some stabilizers alter the surface properties of AgNP where their mobility can be altered due to the electrostatic interactions of different soils. For example, positively charged soils can inhibit the long-haul travel of negatively charged silver nanoparticles. Conversely, negatively charged soils can make AgNP more mobile in such soils. In this study, we use Cs as a stabilizer and its surface charge is assessed to lead to a potential of about 50 (+), due to the ionization of the amino groups of the polymer chain. This promotes the uptake of the clay so that the plant will later incorporate by cation exchange (Thio et al., 2011).
Antifungal activity of nanoparticles
Figure 2 shows the in vitro antifungal ability of NP Ag-Cs against the plant fungus F. oxysporum. The results showed that the diameter of the fungal colony was larger at a longer incubation period, which was expected because of the ideal conditions for fungal growth. Highest fungal growth occurred in a control treatment with a final diameter of 50 mm. On the other hand, the nanostructures were significantly effective in inhibiting mycelium growth, compared with untreated (control) fungal growth (p <0.05). Among treatments, nanoparticles at a concentration of 1000 ppm reduced fungal growth by 64%, while at concentrations 1500 and 2000 ppm were more effective (p <0.05) as they could reduce fungal growth. fungal growth, 70% and 74% respectively, were not statistically different between them.
For decades, silver (Ag +) has been studied for use in disinfecting processes against a number of pathogenic microorganisms, for its environmentally friendly properties and strong antibacterial properties. Kim et al. (2012) evaluated the use of nano-silver at concentrations of 10, 25, 50 and 100 ppm against 18 phytopathogenic fungi on potato agar dextrose. The authors found a dose-dependent antifungal effect, observing 100% inhibition of the growth of fungi Alternaria brassicola, Cylindrocarpo Destutans, Fusarium sp, Pythium aphanidermatum and Pythium spinosum at the highest concentration (Kim and partners, 2012).
Figure 2 In vitro antifungal ability of chitosan coated silver nanoparticles against Fusarium oxysporum during an incubation period of 7 days at 25 ° C (p˃0.05).
In another study, the antifungal activity of nano silver, copper and the mixture of these NPs against two plant fungi, A. Alternata and Botrytis cinerea, was investigated (Ouda, 2014). In this study, metal nanoparticles were applied at different concentrations, found that at 15 ppm created maximum inhibition of mycelium growth. At this concentration, Nano silver inhibited the growth of A. alterta and B. cinerea by 59.3 and 52%, respectively, more efficiently than copper nanoparticles and their blends. It has been proposed that the antimicrobial effect of nano-silver differs according to the species of microorganism.
The mode of antifungal action of nano silver is possible through morphological, structural and physiological changes, altering the cell membranes, affecting mycelium and spores, producing reactive oxygen species ( ROS) damages proteins, lipids and nucleic acids (Ouda, 2014). Dasgupta and Ramalingam (2016) show that the main mechanism of the nano-silver formulation is ROS generation and increased membrane permeability. In addition, they may have an adverse effect on the sugars, proteins, nacetyl glucosamine and lipids of the cytostatics and the cell wall components of phytopathogens. They are also involved in binding and penetrating cell membranes to kill spores, although the mechanism is not fully understood (Pietrzak et al., 2015). Kumari et al. (2019) demonstrated that after membrane degradation, cell death due to loss of membrane structure and osmotic equilibrium in fungi treated with AgNP; In addition, they observed the collapse of redox homeostasis, antioxidant machinery, cytotoxicity, and damage to the cell walls and membranes by disrupting the osmotic equilibrium such as muscles. main institution.
Evaluation of Ag-Cs NP in tomato seed development
To ensure that the nanoparticles did not have any harm to the tomato plant, we evaluated their effect, at different concentrations, on plant size according to stem height and diameter. and the dry weight of leaves, stems and roots. In addition, this was done in the absence or presence of earthworm humus to improve the cation exchange of the substrate (Berilli et al., 2018).
Figure 3 shows results for tomato plant height after exposure to NPs Ag-Cs with and without 3% humus. Three different dosages of treatment were compared (1000, 1500 and 2000 ppm) and it can be observed that the heights of the control and the plants treated at 1000 and 1500 ppm with and without humus and 2000 ppm without humus. did not show any significant difference between them (p <0.05). On the other hand, the seedlings applied with the highest humus concentration showed stronger growth after 30 days from the date of application, showing significant differences with the control and the rest of the treatments with and without. humus (p <0.05).
Figure 3 Height of tomato plants exposed to different concentrations of chitosan-coated silver nanoparticles at 30 days post-fertilization.
Figure 4 shows results for the dry and fresh weights of tomato roots, stems and leaves 30 days after the first Ag-Cs application with and without vermicompost. It was found that the control treatment had the lowest dry and fresh weights while there was a significant difference due to the effects of the nanoparticles; although there was no clear trend in the concentrations of the treatments. Comparing the effects of earthworm humus, the presence of humus significantly increased the tomato plant biomass with the concentration of three nanoparticles. This may be because earthworm humus allows more cation exchange reactions to occur and this improves the uptake of silver particles in the soil (Anjumet al., 2013; De Farias et al., 2018) .
The results of our study show that humus increases plant height at high silver concentrations, as it plays a role in the root’s silver ion absorption, due to improved cation exchange capacity in the soil, this allows for greater mobility of Ag + ions and the retention of these ions is accomplished by the biosphere.
Figure 4 A) Dry weights and B) Fresh weights of tomato seedlings (stems, roots and leaves) exposed to different concentrations of silver nanoparticles
Several studies have evaluated the effect of nano-silver application on vegetative development in different plants. Raliya et al. (2015) applied zinc oxide and titanium nanoparticles on tomato plants, found a significant difference in vegetative growth with a 25% increase in best treatment. Similarly, AgNP-Cs (0.1% w / v) showed growth-promoting effects on mung bean germination, seedling length, fresh weight and dry weight (Anusuya & Banu, 2016). In addition, the treatment increases the chlorophyll content and the activity of the enzymes α, β-amylase, ascorbate peroxidase, peroxidase and catalase. The authors reported that the possible reason for the increased growth rate, is that the greater absorption of inorganic nutrients accelerates the decomposition of organic matter during photosynthesis, which accelerates photosynthesis. In addition, the key to increasing the seed germination rate is the penetration of nanomaterials in the seed (Anusuya & Banu, 2016).
However, some studies report side effects when applying nano silver. Song et al. (2013) found a decrease in biomass of tomato seedling at the lowest concentration was evaluated. In another study, Lupinus termis L. seedlings were exposed to 0.5 mg / L AgNP for ten days, with a significant decrease in growth parameters observed such as germination, root elongation, and weight gain. fresh quantity (Al-Huqail et al., 2018). In addition, the treatment also causes metabolic disorders such as decreased total chlorophyll, sugar and protein content. This can be attributed to the characteristics of the plant, the release of toxic ions, as well as the size and shape of the stress-stimulating particles. However, at lower concentrations, these effects were not observed.
In summary, the use of Ag-Cs NP at three concentrations was assessed to have no toxic effects after applying every 7 days for 30 days to tomato seedlings. In addition, earthworm humus with 3 nanoparticle concentrations is beneficial for the height, stem diameter and dry and fresh weight of the seedling biomass compared to the control plants, emphasizing this effect at the concentration of 2000 ppm.
Effect of NPs Ag-Cs on vascular wilt development in tomato cultured with Fusarium oxysporum
Tomato seedlings infected with F. oxysporum, wilting and yellowing symptoms were observed 14 days after transplanting (Figure 5). On the other hand, the control seedlings without vaccination and the seedlings treated only with Ag-Cs NP had a higher number of leaves and bright green color than the infected seedlings. The group treated with Ag-Cs NPs, had a reduction in disease severity with as-pect very similar to the control group, 5 days after inoculation.
Figure 5 Tomato seedlings after 14 days in incubation chamber at 25 ° C. A) seedlings not inoculated, B) seedlings inoculated with Fusarium oxysporum, C) inoculated seedlings treated with Ags-CS NPs, D) Seedlings were transplanted and treated with 2000 ppm AgNPs-CS.
Figure 6 shows the height and diameter of a tomato stalk with different treatments. Growth of Ag-Cs-treated seedlings after 5 days of transplanting did not affect stem height and diameter (p˃0.05), as there was no significant difference from the control plants. proof. Consequently, the treatment may be thought to not act as a growth promoter, even though it is not the goal of our study.
Figure 6 A) Height and B) diameter of tomato seedling with different treatments for 14 days incubated at 25 ° C 90% relative humidity.
Figure 7 shows the dry weight of tomato plants 14 days after being inoculated with Fusarium oxysporum spore solution, and it can be seen that there was a significant difference at the end of the experiment for treatment without Ag- Cs NP (p ˃0.05). On the other hand, it did not show any difference in control, demonstrating that the application of NP Ag-Cs, to control phytopathogens such as F. oxysporum, could be an alternative solution to eradicate wilt. in the tomato plant and it can work. as a remedy for treatment.
Figure 7 Dry weight of tomato seedlings (stem, roots and leaves) treated with chitosan coated silver nanoparticles (2000 ppm). * indicates significant differences between all treatments (p <0.05).
Table 1 shows catalase activity in tomato seedlings with different treatment methods. Over time, catalase activity increased with the incubation days in most treatments. Compared to the control, the fungus F. oxysporum infects tomato plants and treated with Ag-Cs NPs 5 days after inoculation, showing higher catalase activity (130%), followed by tomato plants only. transplanting (128%). The control seedlings that were not infected and not exposed to the nanoparticles showed the lowest catalase activity. Catalase is an important antioxidant enzyme in plants’ defense systems against pathogens; therefore, these results indicate that both the transplanting and the nanoparticles induce the plant’s defense system.
Table 1 Tomato plant catalase activity 14 days after exposure to silver nanoparticles coated with chitosan.
Ag-Cs NPs showed significant antifungal activity in plant implantation experiments and silver nanoparticles were able to bind and penetrate directly into cell membranes to kill fungal spores, despite AgNP infiltration. into the cell membrane of microorganisms is not fully understood (Ouda, 2014). Because plants have a large canopy area and a stationary nature, they have a higher probability of being exposed to a variety of nanoparticles in their environment (Anjum et al., 2013). The main pathway of plants exposed to nanoparticles is through soil via leaching, direct application with intentional release or from other products (Rajput et al., 2018).
Plant roots are considered the main pathway of plant contact with NP, which may contain a significant portion of the nanoparticles that accumulate, which may or may not produce physical or chemical toxicity to plants (Anjum and partners, 2013). One study found that the Cucurbita pepo shoots had a silver concentration higher than 4.7 in silver nanoprocessed plants where the large solution-treated plants were at the same concentration, considering the release of silver more. Nanoparticles are the main cause (Stampoulis et al., 2009). Another study demonstrated AgNP uptake via Arabidopsis thaliana roots, where although most of the nanoparticles clung to the root boundary, their transport to the shoot was observed (Milewska-Hendel et al. ., 2019). The mechanism of absorption of nanoparticles from plants can be through primary or posterior roots. They are then transported from the base to the stem to the leaves. They can also be absorbed on the root surface (Tripathi et al., 2017). One study on silver and Ag + nanosorption in rice plants, observed a higher uptake of silver when it was in NP, compared with Ag +, which could be explained by the above AgCl formation. The root surface reduces Ag bioavailability and displacement. and the direct factory production of AgNPs (Yang et al., 2020).
There are very few studies evaluating the effects of metal nanoparticles on plants. It has been previously reported that nano-silver is effective in reducing the growth of phytopathogens Bipolaris sorokiniana and Magnaporthe grisea in perennial rye (Lolium perenne) by 50% (Jo et al., 2009). These authors concluded that the efficiency of the nanoparticles was higher with preventive application due to one application that promotes the direct contact of silver with the spores and germplasm to inhibit its viability. In our study, we first implant, and then apply a treatment, which is very interesting when evaluating the effects of nanoparticles in a preventive way. In a more recent study, Nejad et al. (2016) was able to control disease progression of Rhizotocnia solani-transplanted rice plants using concentration-dependent silver nanoparticles, which revealed that AgNP could replace chemical pesticides in control and Inhibits blight, a common rice disease.
In this study, chitosan coated silver nanoparticles were synthesized for the purpose of being used to control wilt caused by F. oxysporum in tomato plants. The NPs showed antifungal activity in vitro and did not affect normal plant growth or showed any toxic effects on tomato seedlings. In addition, the synthesized nanoparticles are effective in eradicating diseases caused by F. oxysporum. Our results indicate that nanotechnology is a promising strategy for controlling phytopathogens in agricultural crops.