Improvement of Resistance of Forage Beans to Pathogens by Compositing Them with SiO2 and Chitosan Nanoparticles

Document Type : Research Paper


1 Department of Biotechnology and Microbiology, Belgorod State University, Belgorod, Russia

2 Department of Science and Technology of Quang Ngai province, Vietnam



Nanotechnology can offer advantages to pesticides, like reducing toxicity, improving the shelf-life, and increasing the solubility of poorly water-soluble pesticides, all of which could have positive environmental impacts. Vicia faba L. is a high-protein forage and vegetable crop with a high productivity potential limited by pathogenic mycoflora. Fusariomycoses are dominant in the bean mycoses complex. Fusariosis are widely known phytomycoses globally with a wide range of mycotoxins, and beans are used for food, including in dietetics, and livestock feed, all over the world. This work aimed to select vegetable and forage beans with different degrees of resistance to Fusarium seedlings. For several years (1999-2020) in the soil and climatic conditions of the city of Belgorod (south of the central black earth region of Russia) on the territory of the National Research University “BelGU”, a collection of beans for fodder and vegetable use was studied against a natural infectious background. Fusariosis appeared annually at all stages of the growing season of broad beans. Severe damage to the seeds led to the death of seedlings. In some years, the prevalence of fusarium disease reached 72%, and the loss of grain yield - 68%. Fusarium sporotrichioides and Fusarium oxysporum were isolated and identified from the affected seedlings. Moreover, the former predominated on seedlings, the latter - on adult plants. Most of the tested varieties of beans (69%) were characterized by low resistance. No immune and fusarium-resistant bean varieties have been identified.


Nanotechnology has led to the development of new concepts and agricultural products with immense potential to manage the aforementioned problems. Nanotechnology has substantially advanced in medicine and pharmacology, but has received comparatively less interest for agricultural applications [1,2]. The use of nanotechnology in agriculture is currently being explored in plant hormone delivery, seed germination, water management, transfer of target genes, nanobarcoding, nanosensors, and controlled release of agrichemicals [3]. Material scientists have engineered nanoparticles with desired characteristics, like shape, pore size, and surface properties, so that they can then be used as protectants or for precise and targeted delivery via adsorption, encapsulation, and/or conjugation of an active, such as a pesticide [4]. As agricultural nanotechnology develops, the potential to provide a new generation of pesticides and other actives for plant disease management will greatly increase. Broad beans (Vicia faba L.) is a cultivated plant from the Legume family, which is currently not found in the wild. Beans are a high-protein forage and vegetable crop, an excellent precursor and honey plant. The productivity potential of beans is high, but it is largely limited by pathogenic mycoflora [5]. Fusarium plant infection is one of the most common and harmful in the world [6-11]. Diseases of fusarium etiology are also dominant in the complex of mycoses of fodder and vegetable beans [12,13]. 
A complex of Fusarium fungi participates in the defeat of plants, many of which are ecologically plastic and widespread in all regions of Russia [14]. Mycotoxins Fusarium belong to the priority contaminants of food raw materials and food products that pose a danger to humans and animals [11-19]. Fungi of the genus Fusarium are opportunistic, or potentially pathogenic for humans and animals [20-22], can cause necrosis and ulcers on nails, fingers [23]. There is evidence that fusariotoxins are carcinogens (cause cancer of the esophagus), can cause toxicosis, aleukia and gastrointestinal diseases in humans [24-26]. These substances cause the development of equine leukoencephalomalacia, pulmonary edema in pigs, hepatosis and dyschondroplasia in chickens, and “deterioration in egg quality” syndrome in chickens [27-31]. Fusariums are widely known phytomycoses in the world with a wide range of mycotoxins, and beans are used for food, including in dietetics, and for livestock feed, all over the world. At the moment, there are no varieties of beans completely resistant to fusarium. Therefore, the problem of resistance of forage beans to Fusarium pathogens is urgent. The purpose of this work was to select varieties of vegetable and forage beans with different degrees of resistance to fusarium seedlings.

For a number of years (1999-2020), in the soil and climatic conditions of the city of Belgorod (south of the central chernozem region of Russia) on the territory of the National Research University «BelGU», a collection of beans (200 varieties) for fodder and vegetable use was studied against a natural infectious backgroundSowing of the faba bean’s varieties and care were carried out manually in accordance with the method of B.A. Dospekhov (1979) and the requirements of zonal agricultural technology without the use of fertilizers and pesticides. A wide-row sowing method was used, with a seeding rate of 0.3 million / ha. Crop care included post-sowing crust control, inter-row cultivation as the crops became clogged and after rains. The area of the accounting plot was 2 m2 with 2 replicates. Determination of plant diseases was carried out using keys Shkalikova et al. (2010), Gritsenko et al. (2008) and atlas by Booth (1971) and Gerlach (1982).
The prevalence of the disease was calculated using the formula: Р = (100×n) / N, where n – the number of infected plants in which at least one organ had a score of 1 or more, N – total number of plants in the sample, 100 – conversion of an indicator to a percentage. The shortfall, or losses, of the crop, was expressed as a percentage and was determined by the formula: Q = (А–а)×100/А, where А – harvest healthy plants, а – harvest of diseased plants (Shcherbakova, 2013). Isolated leaves of bean plants of 16 varieties were inoculated in the laboratory of mycology of the Department of Biotechnology and Microbiology of the National Research University “BelGU” with a suspension (5 ml) of pathogen spores in sterile water (1x106 conidia / ml). The leaves were incubated at a temperature of + 23 °C in a humid chamber. On the 4th day, the symptoms of the disease were described. In the control, the leaves were sprayed with water.
Bean leaf width varied by cultivars, therefore the size of spots after inoculation was evaluated in points according to a 4-point 5-step international scale, according to which 0 points (immune cultivars) are assigned when the leaf area is affected up to 10%, 1 point (resistant) - when 11-25%, 2 points (medium resistance) - at 26-50%, 3 points (weak resistance) - at 51-75% and 4 points (unstable varieties) - if 76-100% of the leaf is affected.

The use of nanoparticles to protect plants can occur via two different mechanisms: (a) nanoparticles themselves providing crop protection, or (b) nanoparticles as carriers for existing pesticides or other actives, such as double-stranded RNA (dsRNA), and can be applied by spray application or drenching/soaking onto seeds, foliar tissue, or roots. Nanoparticles, as carriers, can provide several benefits, like (i) enhanced shelf-life, (ii) improved solubility of poorly water-soluble pesticides, (iii) reduced toxicity, and (iv) boosting site-specific uptake into the target pest. Another possible nanocarrier benefit includes an increase in the efficacy of the activity and stability of the nanopesticides under environmental pressures (UV and rain), significantly reducing the number of applications, thereby decreasing toxicity and reducing their costs (Fig. 1).
The use of chitosan nanoparticles to manage rice blast disease pathogen, our findings showed that chitosan nanoparticles at a concentration of 350 ppb have also shown strong antifungal activity against the Pyricularia oryzae fungus, as presented in Fig. 2.
Chitosan nanocomposite-based chitosan hydrogels (Chit/NCs hydrogel) have been prepared using metal vapor synthesis (MVS). Also, SEM measurements revealed damage to A. flavus cell membranes. Current findings indicate that the antifungal activity of nanocomposites in vitro can be beneficial depending on the type of fungal strain and the concentration of nanocomposites (Fig. 3A). Chit/NCS hydrogel is a revolutionary nanobiopesticide developed by MVS used in food and feed to induce plant protection against mycotoxigenic fungi [32]. The fungicidal behavior of chitosan-silver nanocomposites (Chit-NCs) against Penicillium expansum from the feed samples was investigated. Chit-NCs < 10 nm in size have an important antifungal inhibitory effect against P. expansum, the causative agent of blue mold-contaminated dairy cattle feed [33]. P. expansum treated with Chit P. expansum treated with Chit NCs was investigated by HR-SEM, alterations in conidiophores, metulae, phialides, and mature conidia characteristics had been observed to obtain information about the mode of action of Chit-NCs (Fig. 3B). Therefore, nanocomposites can be utilized as viable alternatives to the already available arsenal of fungicides.
Fusariosis appeared annually at all stages of the growing season of beans. Affected seedlings quickly turned yellow (see Fig. 4), wither and die. Darkened vessels were observed on transverse sections of stems and roots (see Fig. 4).
Severe damage to the seeds led to the death of seedlings. In some years, the prevalence of fusarium disease reached 72%, and the loss of grain yield - 68%. From the affected seedlings were isolated and identified Fusarium sporotrichioides Sherbakoff (section Sporotrichiella) and Fusarium oxysporum Schlechtendahl (section Elegans). Moreover, the former prevailed on seedlings, the latter - on adult plants. It should be noted that fusarium wilting was accompanied by the colonization of weakened forage beans with Alternaria, and is consistent with the data of other authors, who noted the charred species of plants. According to [34,35] species F. oxysporum are relatively weak pathogens, while the species F. sporotrichioides causes widespread latent infection of grain [36,37]. Therefore, inoculation of leaves in the laboratory was carried out with a spore suspension F. sporotrichioides. 
Most of the tested varieties of beans (69%) were characterized by low resistance (3 points). Of all varieties only 25% (for example Russian Black, Alfred, Pirkkonen, Geo) are low-medium resistence (2.5 points) (see Fig. 5). And varieties like Ar-ban-cin-hu-dou (China), Josny (Poland), Chlumesky, Roschutjer Feldbohn и Stofil (Czech Republic) are medium resistance (2 points). No immune and fusarium-resistant bean varieties were identified (0-1 points).
Varieties with rapid growth at the initial stages of development may be of interest to breeders, due to which, in natural conditions, they will have a chance to “escape” from mass infection of seedlings with Fusarium. In our study, these are varieties Russian Black, Aquadul, Soving. 
It is known that the main factor in the spread of fusarium is the soil, and the additional factor is seed, air currents, and raindrops (Chulkina et al. 2008). The manifestation and development of fusariosis is also facilitated by the violation of the correct alternation of crops. Studies have shown that the weather conditions in May-June play an important role in the manifestation of Fusarium. So, for example, May in 2005 and 2019 was humid and warm (excess over the average annual values for temperature and precipitation in Figures 6 and 7), when diseased plants lost their turgor, turned yellow, turned black and shriveled. Such weather contributed to the spread of root rot on the crops of forage beans, when the white cobweb mycelium of the fungus was clearly visible in the area of the root collar. May and June 2017 were cool and dry (see Fig. 6 and Fig. 7), which favored fusariosis on the seedlings, which turned yellow and easily pulled out of the soil.
To protect plants from fusariosis, it is necessary to combine agrotechnical measures with the use of pesticides and the correct selection of varieties. For example, using a wide-row sowing method creates favorable conditions for the growth of beans, which increases the natural resistance of plants. Varieties with rapid growth at the initial stages of development may also be of particular interest to breeders, due to which, under natural conditions, they will have a chance to “escape” from mass infection of seedlings with Fusarium, for example, varieties Russian Black (Russia), Aquadul (Holland), Soving (Sweden), Survoy (France), Britz (Canada), Sinabe-Im (Peru).

Our findings have indicated that the control of toxigenic fungi and the detoxification of mycotoxins are not adequate for sustainable agricultural ergonomics. Therefore, novel treatment methods for improving the food safety and protection must be applied. Nanohybrid antifungals are thus, of primary importance for a synergistic approach to resolve diverse problems in the management of fungal pathogens causing agricultural/post-harvest diseases in the 21st century, with a focus on Green Nanotechnology, which is environmentally sustainable and provides a continuum for the plant, animal and human health. The nano-hybrid anti-fungals are anticipated to cater to the need of the growers, consumers as well as the environment activists through rapid, effective, and comparatively improved eco-safety attributes for controlling the yield and produce quality deterring potential of the fungal phytopathogens. Fusariosis appeared at all stages of the growing season of Vicia faba. The prevalence of fusarium disease reached 72%, and the loss of grain yield - 68%. Fusarium sporotrichioides (predominated on seedlings) and Fusarium oxysporum (on adult plants) were isolated and identified from the affected seedlings. And 69% of the tested varieties of Vicia faba were characterized by low resistance. The varieties Ar-ban-cin-hu-dou, Josny, Chlumesky, Roschutjer Feldbohn и Stofil are medium resistance and may be of interest for breeding for resistance to fusariosis in seedlings of broad beans. Also promising for select the varieties, as Akvadul, Russian Black, Soving, Survoy, Britz, Sinabe-Im, whose plants grow rapidly at the initial stages of development, so they have a chance to “escape” from the mass infection of seedlings with fusariomycosis.

The authors declare that there is no conflict of interests regarding the publication of this manuscript.


1.    Sinha K, Ghosh J, Sil PC. New pesticides: a cutting-edge view of contributions from nanotechnology for the development of sustainable agricultural pest control. New Pesticides and Soil Sensors: Elsevier; 2017. p. 47-79.
2.    Cătălin Balaure P, Gudovan D, Gudovan I. Nanopesticides: a new paradigm in crop protection. New Pesticides and Soil Sensors: Elsevier; 2017. p. 129-192.
3.    Hayles J, Johnson L, Worthley C, Losic D. Nanopesticides: a review of current research and perspectives. New Pesticides and Soil Sensors: Elsevier; 2017. p. 193-225.
4.    Khandelwal N, Barbole RS, Banerjee SS, Chate GP, Biradar AV, Khandare JJ, et al. Budding trends in integrated pest management using advanced micro- and nano-materials: Challenges and perspectives. J Environ Manage. 2016;184:157-169.
5.    Adeniji AA, Babalola OO. Tackling maize fusariosis: in search of Fusarium graminearum biosuppressors. Arch Microbiol. 2018;200(8):1239-1255.
6.    Alexandrova EN, Tarasov KL. Biology of causative agents of crayfish mycoses in connection with environmental protection in crayfish producing reservoirs. IOP Conference Series: Earth and Environmental Science. 2020;421(8):082027.
8.    Blacutt AA, Gold SE, Voss KA, Gao M, Glenn AE. <i>Fusarium verticillioides: Advancements in Understanding the Toxicity, Virulence, and Niche Adaptations of a Model Mycotoxigenic Pathogen of Maize. Phytopathology®. 2018;108(3):312-326.
9.    Gams W. Laboratory guide to the identification of the major species. Netherlands Journal of Plant Pathology. 1978;84(2):84-84.
10.    Chu Q, Cook ME, Wu W, Smalley EB. Immune and Bone Properties of Chicks Consuming Corn Contaminated with a Fusarium That Induces Dyschondroplasia. Avian Dis. 1988;32(1):132.
11.    Toropova EY, Vorob’eva IG, Chulkina VA, Marmuleva EY. ABOUT A ROLE OF BIOLOGICAL DIVERSITY IN THE PHYTOSANITARY OPTIMIZATION OF AGRARIAN LANDSCAPES. Sel’skokhozyaistvennaya Biologiya. 2013(3):12-17.
12.    Vasilenko AA, Kozulina NS, Shmeleva ZN. The assessment of the bioecological method use for spring barley cultivation in the Krasnoyarsk territory forest-steppe zone. IOP Conference Series: Earth and Environmental Science. 2019;315(2):022047.
13.    Konstantinov IS. International Scientific Conference of young scientists and specialists dedicated to the 160th anniversary of V. A. Mikhelson Collection of articles. Publishing house RGAU-MSHA; 2020.
14.    Osowski S. Voltage transfer function realisation using active R network: flow-graph technique. Electronics Letters. 1979;15(14):416.
15.    Gittich FW, Weik J, Wentz D. Streptomycinkontaktdermatitis. Klin Wochenschr. 1950;28(1-2):25-29.
16.    Foroud NA, Baines D, Gagkaeva TY, Thakor N, Badea A, Steiner B, et al. Trichothecenes in Cereal Grains – An Update. Toxins (Basel). 2019;11(11):634.
17.    Gagkaeva TY, Gavrilova OP, Orina AS. First Report of Fusarium globosum Associated with Barley Grain in the Southwestern Part of Siberia. Plant Dis. 2019;103(3):588-588.
18.    Gavrilova O, Skritnika A, Gagkaeva T. Identification and Characterization of Spontaneous Auxotrophic Mutants in Fusarium langsethiae. Microorganisms. 2017;5(2):14.
19.    Dropkin VH. Hackfruchtkrankheiten und Nematodenforschung. Festschrift anlasslich der Einweihung des Neubaues fur das Institut fur Hackfruchtkrankheiten und Nematodenforschung der Biologischen Bundesanstalt fur Landund Forstwirtschaft in Munster (Westf.). (Mitteilungen aus der Biologischen Bundesanstalt fur Landund Forstwirtschaft Berlin-Dahlem, vol. 99). Parey, Berlin, 1960. 119 pp. DM. 13.50. Science. 1961;133(3460):1247-1247.
21.    Hassler A, Lieb A, Seidel D, Cesaro S, Greil J, Klimko N, et al. Disseminated Fusariosis in Immunocompromised Children—Analysis of Recent Cases Identified in the Global Fungiscope Registry. Pediatr Infect Dis J. 2017;36(2):230-231.
22.    Klingspor L, Saaedi B, Ljungman P, Szakos A. Epidemiology and outcomes of patients with invasive mould infections: a retrospective observational study from a single centre (2005-2009). Mycoses. 2015;58(8):470-477.
24.    Kurkina YN. Phenoloxidase activity of micromycetes strains isolated from the rhizosphere of vegetable leguminous crops. Vegetable crops of Russia. 2019(6):109-112.
25.    Sauviat M-P, Laurent D, Kohler F, Pellegrin F. Fumonisin, a toxin from the fungus Fusarium moniliforme sheld, blocks both the calcium current and the mechanical activity in frog atrial muscle. Toxicon. 1991;29(8):1025-1031.
26.    The Fusarium Laboratory Manual. Wiley; 2006.
27.    Lewis RE, Wurster S, Beyda ND, Albert ND, Kontoyiannis DP. Comparative in vitro pharmacodynamic analysis of isavuconazole, voriconazole, and posaconazole against clinical isolates of aspergillosis, mucormycosis, fusariosis, and phaeohyphomycosis. Diagnostic Microbiology and Infectious Disease. 2019;95(3):114861.
28.    Medentsev AG, Arinbasarova AY, Akimenko VK. Biosynthesis of Naphthoquinone Pigments by Fungi of the Genus Fusarium. Applied Biochemistry and Microbiology. 2005;41(5):503-507.
29.    Moreira BC, Prates Junior P, Jordão TC, de Cássia Soares da Silva M, Stürmer SL, Salomão LCC, et al. Effect of inoculation of symbiotic fungi on the growth and antioxidant enzymes activities in the presence of Fusarium subglutinans f. sp. ananas in pineapple plantlets. Acta Physiologiae Plantarum. 2016;38(10).
30.    Munkvold GP, Arias S, Taschl I, Gruber-Dorninger C. Mycotoxins in Corn: Occurrence, Impacts, and Management. Corn: Elsevier; 2019. p. 235-287.
31.    Orina A, Gavrilova OP, Gagkaeva T, Burkin A, Kononenko G. The contamination of Fabaceae plants with fungi and mycotoxins. Agricultural and Food Science. 2020;29(3).
32.    Alghuthaymi MA, Abd-Elsalam KA, Shami A, Said-Galive E, Shtykova EV, Naumkin AV. Silver/Chitosan Nanocomposites: Preparation and Characterization and Their Fungicidal Activity against Dairy Cattle Toxicosis Penicillium expansum. Journal of Fungi. 2020;6(2):51.
33.    Abd-Elsalam KA, Alghuthaymi MA, Shami A, Rubina MS, Abramchuk SS, Shtykova EV, et al. Copper-Chitosan Nanocomposite Hydrogels Against Aflatoxigenic Aspergillus flavus from Dairy Cattle Feed. Journal of Fungi. 2020;6(3):112.
34.    El-Adawi H. Protective effect of milk thistle and grape seed extracts on fumonisin B1 induced hepato- and nephro-toxicity in rats. Journal of Medicinal Plants Research. 2011;5(27).
35.    Sesquiterpenes with Phytopathogenic Fungi Inhibitory Activities from Fungus Trichoderma virens from Litchi chinensis Sonn. American Chemical Society (ACS).
36.    Chebanenko SI, Beloshapkina OО. Features of the development of test tasks for plant protection.  Russian State Agrarian University-Moscow State Agricultural Academy named after K A Timiryazev: Publishing house RGAU-MSHA; 2021.
37.    Tevell Åberg A, Solyakov A, Bondesson U. Development and in-house validation of an LC-MS/MS method for the quantification of the mycotoxins deoxynivalenol, zearalenone, T-2 and HT-2 toxin, ochratoxin A and fumonisin B1 and B2 in vegetable animal feed. Food Additives &amp; Contaminants: Part A. 2013;30(3):541-549.