The objective of this work was to evaluate the microbiological effects of silver nanoparticles (AgNPs) on poisonous fungi capable of affecting cocoa trees (Theobroma cocoa). These fungi, isolated from diseased cocoa pods, are characterized in terms of phenotype and genotype. The bactericidal effect was assessed by measuring the growth of afferent mycelia, in synthetic cultures and at different nano silver concentrations in plant tissues. Inhibitory effects were observed in Petri dishes, and changes in fungal structure were observed through scanning electron microscopy. Two very common poisonous fungi: Aspergillus flavus and Fusarium solani. Inhibition tests, performed in synthetic liquid and solid cultures, showed that AgNPs did not significantly affect the release. growth of these fungi, even at the highest concentrations (100 ppm). In contrast, they showed a positive inhibitory effect in plant tissues, especially in the shells when infected with A. flavus, where a dose of 80 ppm completely inhibited fungal growth. However, once the fungus gets inside the pods, their growth is inevitable, and the effects of AgNP are reduced. On F. solani, the nanomaterials studied induce only some changes in texture and pigmentation. The microbiological effect of chemically synthesized silver nanoparticles in plant tissue is greater than in the culture medium.
The global cocoa agenda calls for a knowledge creation strategy that combines productivity, technological innovation and sustainability in the cocoa value chain. World cocoa production (Theobroma cocoa L.) reached five million tons in 2012 (Ortiz-Rodrigues et al., 2015). Currently, Colombia is the 4th cocoa producer in Latin America, after Brazil, Ecuador and the Dominican Republic. In 2012, the cocoa cultivated area in Colombia was about 158,000 ha, with a yield of 460 kg ha-1, which is certainly low compared to Côte d’Ivoire, the first producer in the world, with 700 kg ha-1. (Ortiz et al, 2014; Martínez-Ángel et al., 2015).
Cocoa production is significantly affected by fungal diseases as pod fermentation is favored not only by specific pH and humidity conditions, but also by the positive feedback effect of organic acids. that it creates. Furthermore, the main problem associated with the presence of mycelium in cocoa is the risk of mycotoxin production, potentially affecting the health of consumers (Copetti et al., 2014). Chocolate has been reported to contribute with about 6% of total dietary exposure to ochratoxin A, but the levels of mycotoxins in cocoa and chocolate are often very low. However, because its detection methods are costly and labor intensive, the Codex Alimentarius Commission (2013) recommends keeping cocoa plantations as free of mold contamination as possible.
Conventional control mechanisms are associated with good farming practices, sometimes associated with the use of fungicides. The first approach is easy to apply, but besides being labor intensive, its economic viability depends on enhanced cocoa market awards (Hanada et al., 2009). Although the fungicides protect plants from pathogens, they can have harmful effects on the Forcipomyia sp., Cocoa flower pollinator (FAO, 2012). Biological control is currently being used as an easy to apply and environmentally friendly control tool for phytopathogens (Cuervo-Parra et al., 2011). However, it poses certain difficulties, such as the high specificity between the biological control agents and the pathogen (Krauss et al., 2013), the need for the controller to adapt to the soil conditions and climate of the crop, and even resistance to pathogens (Mbarga et al., 2014). This made it essential to explore non-conventional control alternatives.
Nanotechnology is such an important tool in modern agriculture, that agri-food nanotechnology oriented sustainable production of food for humans and animals has the potential to become one of the most beneficial fields. highest profitability in this sector in the future, especially in developing countries (Ranjan et al., 2014). Nano silver (AgNPs) are one of the most frequently used pesticide nanomaterials. They have received special attention for their low volatility, high stability and extensive antimicrobial activity (Pulit et al., 2013). These materials have been used successfully to inhibit the growth of phytopathogenic fungi on grasses and on cucumbers, rice (Krishnaraj et al., 2012), and timber trees (Nasrollahi et al., 2011).
This effort is framed in exploring more broadly new alternatives to controlling phytopathogens that interfere with the crop’s sustainability.
The objective of this work was to evaluate the microbiological effects of silver nanoparticles (AgNPs) on poisonous fungi capable of affecting cocoa trees (Theobroma cocoa).
Nano silver materials and synthesis methods
Biopure-AgNP nano silver (NanoComposix, San Diego, CA, USA) at initial concentration of 1,000 ppm (1.8×1014 mL-1 particles), atomic molar concentration 9.27 mmol L-1, morphology bridge and 10 nm in diameter were used as inhibitory material. This colloidal AgNP is diluted in sterile distilled water at room temperature (22 ° C), to obtain a suspension at concentrations between 50 and 100 ppm. All suspension was stored at 4 ° C, in the dark, until application.
Twenty diseased pods were selected from farms in the cities of Chinácota and El Zulia, in the Norte de Santander Ministry, Colombia, at an altitude of 220 to 1,200 m, temperature between 22 and 30 ° C and degrees. relative humidity from 63 to 86%. Farms were selected from the list of associates of the National Federation of Cocoa Growers of the Norte de Santander. Various biological materials – clones and hybrids – were sampled from cocoa pods with symptoms such as: deformation; golden halo; and well-contoured chocolate stains or cream-colored powders (Phillips-Mora et al., 2007). The pods are wrapped in a plastic sheet, labeled and transported in a box to the laboratory for testing.
The toxic fungus is obtained from the pods of the cocoa. The spores contained in the outer pod are plated directly on potato agar (PDA), modified by cocoa pod extract and chloramphenicol (PDA-CC). Pure cultures are obtained from heterogeneous samples, which are morphologically and molecularly characterized.
Morphological characterization follows a modified methodology from Ab Majid et al. (2015). Reproductive structure (spore), mycelium type and the presence of septum were observed by lactophenol blue staining. Image record obtained by Eclipse 80i phase contrast optical microscope (Nikon, Japan).
DNA was isolated using ultraclean microbial DNA isolation kit (Mo Bio Laboratory, Carlsbad, CA, USA) and prepared to manufacturer specifications. The isolated DNA was amplified using two molecular markers, corresponding to the ITS region and the β-tubulin gene (Ab Majid et al., 2015) (Table 1). Then, the amplified DNA was sequenced by Macrogen (Seoul, Korea) and analyzed using Blast database.
Tests for inhibition of nano silver were performed in different media: liquid and solid synthetic media, and cocoa pulp and pods. The first test was performed on AgNPs modified malt broth broth at 80, 85, 90, 95 and 100 ppm. Serum microfiltration plates (Sterile, Corning Costar Corporation, New York, NY, USA) were inoculated with 200 µL of modified medium and 100 µL of each fungus isolates (107 conidia mL-1). Five iterations of each test were performed (CLSI, 2012).
The second test was performed on Petri dishes (60×15 mm) containing PDA-CC modified with AgNPs at 50, 70, 90 and 100 ppm. Each Petri dish was inoculated with agar stoppers 6 mm in diameter, each containing a preterm culture of isolated phytopathogens. Radial growth of mycelium cultured at 25 ° C in PDA-CC was measured over a period of 18 days. Petri dishes free of AgNP and inoculated with pathogens were used as active controls, following a modified methodology from Nasrollahi et al. (2011). All tests were repeated three times.
The final test is performed directly on the cocoa pulp and on the pods. A sterile, 24-well, lidded TC sheet (Cellstar, Sigma Aldrich, St. Louis, MO, USA) was used to place 0.5×0.5 cm cocoa pods and pulp, previously disinfected. in moist heat treatment (121 ° C, pressure 15 lb, and 20 min) and artificially enriched with 100 µL of 107 ml-1 conidia spore suspension. The plates are incubated at 25 ° C for 18 days. Three wells containing cocoa residue and cerebral cortex contaminated with phytopathogens respectively were used as active controls, as modified by Nasrollahi et al. (2011). The uninfected cocoa pulp and peel were used as negative controls. The inhibitory effect was determined macroscopically as the lowest AgNP dilution at which no phytopathogens tested were observed developing after an 18-day incubation period (Monteiro et al., 2011). . The structural changes of the fungus after treatment with 50 ppm AgNP were monitored by scanning electron microscopy (SEM 08 30 SEI, Korea).
Results and discussion of nano-silver efficiency
It is likely that my gener cotoxigenic such as Aspergillus and Fusarium are among the most commonly found pathogens in the diseased fruit that have been studied. Mounjouenpou et al. (2008) analyzed the growth of mycotoxins in cocoa trees, in Cameroon, and identified the same genus, including A. carbonarius and A. niger, which are capable of producing ochratoxin A. The The macroscopic and microscopic characteristics of the phytopathogens that coincided with the previous report via Suárez Contreras & Rangel Riaño (2013) are shown in Figure 1.
The molecular characterization allows the identification of isolated strains of Aspergillus flavus and Fusarium solani. These two species are secondary pathogens that cause completely opportunistic infection and fruit degradation, by taking advantage of the spoilage of cocoa pods caused by major pathogens with strong enzyme activity (Cuervo-Parra et al. the, 2011). However, the presence of these fungi is of great significance to public health because of their toxin-causing potential (Villamizar et al., 2011) .Copetti et al. (2014) reported that the frequent isolation of potentially toxic fungi in cocoa is a cause for concern as they can induce aflatoxin and type A ochratoxin – both metabolites are effective. Carcinogenic – and has been found in cocoa beans and in manufactured and processed chocolate.
Inhibition tests using AgNPs in a liquid medium showed F. solani and A. flavus developed at all tested concentrations, subsequently demonstrating the highest resistance to the effects of AgNPs ( Table 2). The inhibitory effect of AgNP on these phytopathogens was also tested in Petri dishes with solid medium (PDA-CC) (Figure 2), covering a wider AgNP concentration range (50 to 100 ppm).
The antibacterial properties of AgNP depend on several aspects, such as morphology, size, and concentration. In principle, spherical nanoparticles less than 10 nm in diameter used in this test can facilitate interaction with the target by electronic effects at the cellular level (Kim et al., 2007). ). However, only changes in texture and pigmentation were caused in the phytopathogens tested.
Mycelium growth, measured on Petri dishes, allowed the effect of nano silver on phytopathogens to be quantified. Figure 3 shows the behavior of the two fungi studied when treated with or without AgNPs. The inhibitory effects were 5.32 and 3.29% for F. solani and A. flavus, respectively. Consequently, no significant changes can be attributed to AgNPs.
The fungal resistance to silver nanoparticles is due to their ability to form secondary metabolites (Pulit et al., 2013). The main secondary metabolites produced by the genus Aspergillu s include polyketides (PK), ribosomal and non-ribosome peptides (NRPs), and terpenoids (Andersen et al., 2013). However, this specific aspect needs to be studied in more detail. Another explanation is based on the ability of some strains of F. solani and A. flavus to conduct extracellular synthesis of silver nanoparticles. This implies the presence of silver deionizing enzymes and, therefore, some resistance to this metal (Alghuthaymi et al., 2015).
Tests performed directly on cocoa pod tissues (pulp and skin) showed that A. flavus grew slightly on the bark. Therefore, although an inhibitory effect is observed at 50 ppm, a minimum AgNP concentration of 80 ppm is required to completely inhibit fungal growth. This may be due to a cortical component, which interferes with fungal penetration. In contrast, fungi can grow in any concentration in the pulp. This can be explained by the ability of some species to hydrolyze this tissue, thus generating acids that facilitate fungal growth through a decrease in pH (Copetti et al., 2014). In this tissue, even the highest concentration (90-100 ppm) cannot significantly affect fungal growth.
Fusarium solani was observed to be more resistant to AgNPs (Figure 4). Kasprowicz et al. (2010) reported that a nutrient-poor PDA medium modified with silver nanoparticles increased spore production. This phenomenon is probably caused by F. solani produced polyphoric polyps, by expressing a complex multicellular structure (Harris, 2005), which could interfere with the internal transport of silver nanoparticles, thus reduce their fungicidal effect.
The antibacterial effect of nanoparticles is related to the concentration used and the rate at which they are released. In the present study, this effect on culture media differs from the effect observed in the fruit tissues (skin and pulp). This can be explained by the fact that the synthetic culture medium contains all the nutrients required for fungal growth, while plant tissues – especially the bark – provide a natural barrier. to penetrate the pod of the fruit. Therefore, the silver nanoparticles are more likely to affect the natural reproduction of the fungus. This is consistent with previous findings reported by Lamsal et al. (2011), who reported growth inhibition of Colletotrichum sp., Pepper anthracnose agent, by AgNP at 100 ppm in PDA culture; while a concentration below 50 ppm is sufficient to obtain the same results in the field.
While metallic silver is inert, nano silver are highly reactive due to the formation of Ag + ions (Morones et al., 2005). In fungi, silver nanoparticles can cause structural changes in the mycelium, cell wall deformation, membrane damage, and significant changes in spore form and germination, depending on the concentration. fungicides (Lamsal et al., 2011). In this study, the 50 ppm treatment caused small changes in mycelium structure, characterized by mycelium deformation in F. solani, and by shrinking the reproductive structure, such as conidia, in A. flavus (Figure 5). However, these morphological changes did not affect the life cycle of the fungus studied.
Future research perspectives include exploring the use of biotech-synthesized silver nanoparticles or organic nanomaterials, whose superficial biological ligands can enhance their effects. kill their fungus.
Reference source: Fungicidal effect of silver nanoparticles on toxigenic fungi in cocoa Raquel Villamizar-GallardoJohann Faccelo Osma CruzOscar Orlando Ortíz-Rodriguez
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