Characterization and Application of Nanomaterials (2022) Volume 5 Issue 2 doi:10.24294/can.v5i2.1769 111 Review Article Biofertilization and nanotechnology in alfalfa (Medicago sativa L.) as alternatives for a sustainable crop Carmen María Ramos-Ulate, Sandra Pérez-Álvarez*, Sergio Guerrero-Morales, Abdon Palacios-Monarrez Facultad de Ciencias Agrícolas and Forestales, Universidad Autónoma de Chihuahua, Campus Delicias, Chihuahua 33000, Mexico. E-mail: spalvarez@uach.mx ABSTRACT Alfalfa is considered the most used forage crop in the world, its main use is for cattle feeding, due to its high nutri- tional value, specifically in protein and digestible fiber. Currently, the trend in agriculture is to reduce the application of chemicals and among them are fertilizers that pollute soil and water, so the adoption of new technologies and other not so new is becoming a good habit among farmers. Nanotechnology in the plant system allows the development of new fertilizers to improve agricultural productivity and the release of mineral nutrients in nanoforms, which has a wide vari- ety of benefits, including the timing and direct release of nutrients, as well as synchronizing or specifying the environ- mental response. Biofertilizers are important components of integrated nutrient management and play a key role in soil productivity and sustainability. While protecting the environment, they are a cost-effective, environmentally friendly and renewable source of plant nutrients to supplement chemical fertilizers in the sustainable agricultural system. Nano- technology and biofertilization allow in a practical way the reduction in the application of chemicals, contributing to the sustainability of agriculture, so this work aims to review the relevant results on biofertilization, the use of nanotechnol- ogy and the evaluation of the nutritional composition of alfalfa when grown with the application of biofertilizers. Keywords: Agriculture; Biofertilizers; Nanomaterials; Nanomaterials ARTICLE INFO Received: 19 August 2022 Accepted: 9 October 2022 Available online: 21 October 2022 COPYRIGHT Copyright © 2022 Carmen María Ra- mos-Ulate, et al. EnPress Publisher LLC. This work is li- censed under the Creative Commons At- tribution-NonCommercial 4.0 International License (CC BY-NC 4.0). https://creativecommons.org/licenses/by-nc/ 4.0/ 1. Introduction The demand for food in the world is increasing exponentially, more so in developing countries where land and agricultural resources hardly contribute to efficient crop production needed to meet such an urgent demand for food. There is a need to intensify agricultural pro- duction in a sustainable manner through efficient use of resources con- sidering the full biochemical diversity of the agroecosystem and its po- tential to mitigate the adverse impacts of low soil fertility, abiotic stresses, pathogens and pests[1]. Nutrients are essential for plant growth and development and some of them are not available in the soil, due to many factors such as leach- ing, degradation by protolysis, hydrolysis and decomposition, so it is necessary to reduce the loss of these nutrients during fertilization and increase crop production through new technologies[2]. One of these technologies is nanotechnology and nanomaterials (NMs), because nanofertilizers could have effective qualities for crops, such as being able to release nutrients according to demand, controlled release of chemical fertilizers that regulate plant growth and development and improve target activity[2]. Another technology is the application of biofertilization. Bioferti- 112 lizers are used to supplement chemical fertilizers mainly to maintain soil fertility. These fertilizers are organic, biodegradable, contain microorganisms, provide nutrients, antibiotics, hormones such as auxins, cytokinin, vitamins that enrich the root rhi- zosphere[3]. Legumes contribute to the sustainability of ag- riculture: they reduce mineral fertilizers, thus de- creasing N2O production and increasing N2 fixation, renew and enrich soil fertility due to their deep rooting systems, rapidly decompose their root bio- mass and accumulate in the soil[4]. Alfalfa (Medi- cago sativa L.) has the ability to accumulate signif- icantly greater amounts of nitrogen than other legumes through its deep rooting system and, in addition, fixes atmospheric N2 by 40 to 80% through biological fixation of this element[5]. Therefore, the objective of this work is to re- view the relevant results on biofertilization and the use of nanotechnology in alfalfa cultivation, illus- trating how these technologies can lead to a reduc- tion in the application of chemical fertilizers. 2. Alfalfa, generalities, uses and ap- plications Alfalfa is a perennial legume, representative of temperate regions and is mainly used as livestock feed, and is universally considered one of the high- est quality forages. It is a valuable crop because among its many agronomic and environmental ad- vantages are the preservation of soil fertility and biodiversity, protection against soil erosion, mitigation of climate change impacts, reduction of nitrate contamination of groundwater, reduction of fossil fuel consumption, reduction of greenhouse gas emissions, among others[6–9]. This legume (alfalfa) has a set of variable morphological and physiological characteristics of importance in world agriculture and contributes with its high and stable performance as a nutritious grass[10]. Its economic importance is based on its high biomass production potential, exceeding 80 t∙ha−1 green and about 20 t∙ha−1 dry matter[11]. Al- falfa forages are characterized by a high crude pro- tein content[12], well balanced with respect to amino acid. It is enriched with vitally important vitamins and several microelements essential for normal animal growth and development. Alfalfa is the basic component in the feeding program for dairy cattle, as well as for cattle, horses, sheep and other live- stock[13]. Alfalfa has also become interesting as a poten- tial source of secondary metabolites, because it is considered an alternative of phytoestrogens useful in health (human food ingredient and supplements), so its growth has become widespread in different continents, due to its high adaptability to different types of soils, pH values and environmental condi- tions, as well as the possibility of sustainable and ecological production[14,15]. In Mexico[16], 385,992 ha of green Alfalfa have been planted, of which 384,693 ha have been harvested for a production of 15,360,646 and a yield of 39,930 t∙ha−1. In this country, the main use of alfalfa is to feed dairy cattle in arid, semi-arid and temperate regions. The crop is cut at medium intervals to harvest the highest forage yield per year per unit area, as well as for its good crude protein content, digestibility and degree of acceptance by cattle[17,18]. This plant can be used as fodder in dif- ferent ways, fresh, hayed and ensiled in mixture with one or more grasses[19,20]. 3. Biofertilization in alfalfa The development of a country is directly pro- portional to the amount of food or nutrients availa- ble to the population. The growing increase in world population creates an ever-increasing demand for food and to supply it, fertilizers are used which are defined as any substance used to increase the productivity of the soil, promoting its fertility by adding nutrients, which aids in plant growth. Ferti- lizers that are composed of crude chemicals in solid or liquid form made in factories targeted to the nu- tritional requirements of plants are, by definition, called a chemical fertilizer. Nitrogen (N), phospho- rus (P) and potassium (K), called NPK, are normal- ly present in these chemical fertilizers along with other nutrients[21]. The excessive use of chemical fertilizers has generated several problems in nature such as, for example, water acidification; damage to the ozone 113 layer; the greenhouse effect; using them for a long time can change the pH of the soil, eutrophication of the water where the nutritional content in these environments increases, causing algae proliferation and, consequently, the reduction of oxygen in the water, which damages marine life[22]. A current so- lution to decrease the use of these fertilizers in ag- riculture is the use of biofertilizers[23]. Biofertilizers are microbial inoculants con- taining live or dormant cells of efficient strains of nitrogen fixing, phosphate solubilizing and cellu- lose decomposing microorganisms[3]. These are in- tended to be applied primarily to soils to improve soil fertility and plant growth by increasing the number and biological activity of beneficial micro- organisms[3]. Some of the advantages of biofertilizers are that they are cost-effective and environmentally friendly, gradually improving soil quality. The mi- croorganisms contained in the biofertilizer promote the supply of nutrients to the plants, thus ensuring their development, growth and physiological regu- lation. In addition, crop yields can increase by 10 to 25% and plants are less prone to soil diseases. Among the main limitations of biofertilizers are that they act more slowly than chemical fertilizers; they are difficult to store due to their high sensitivity to changes in temperature and humidity; they cannot replace other fertilizers completely; and the scarcity of particular or local strains of microorganisms re- duces their availability[24]. The types of biofertiliz- ers available[22] are: 1. Nitrogen fixing biofertilizer: Rhizobium, Azoto- bacter, Azospirillium, Bradyrhizobium. 2. Phosphorus solubilizing biofertilizer: Bacillus, Pseudomonas, Aspergillus. 3. Phosphorus mobilizing biofertilizer - Mycorrhi- za. 4. Biofertilizer plant growth promoters: Psue- domonas, Trichoderma. The effects of the above mentioned biofertiliz- ers in terms of nitrogen fixation in the soil is carried out through the root nodules of the leguminous crop, making N2 available to the plant. Other microor- ganisms that can be used as biofertilizers are: Azolla which is a heterogeneous fern with seven species that are endosymbionts with Anabaena azollae, a nitrogen fixing cyanobacterium[25] and blue green algae can fix nitrogen in the anaerobic environment due to a specialized cell called heterocyst[26]. Phosphate-solubilizing bacteria produce or- ganic and inorganic acids such as gulconic acid and ketogulconic acid that solubilize phosphorus[27]. Gluconic acid produces a carboxyl and hydroxyl group, this group will function as a chelator of Fe2+, Al3+ and Ca2+, which will reduce soil pH. It is also important to mention that there is a positive interac- tion between Gluconacetobacter spp and Burklderia spp to increase dehydrogenase activity in soil. Dehydrogenases are involved in the soil ox- idation process and are used as an indicator of soil microbial activity[28]. In Alfalfa, some studies have been conducted using organic cultivation, which includes the use of biofertilizers. The application of liquid microbial inoculants to legume seeds is a sustainable agricul- tural practice that can improve plant nutrient uptake and increase crop productivity. After application to legume seeds the inoculants should provide long-term survival of rhizobia in the final product and to study the survival of Sinorhizobium (Ensifer) meliloti L3 Si, ten different media formulations of microbial inoculants (yeast mannitol broth with the addition of agar, sodium alginate, calcium chloride, glycerol or ferric chloride and combinations thereof) were examined. For survival of L3 Si, for a storage time of 150 days, the medium formulation contain- ing glycerol in combination with agar or sodium alginate was applied, which was used as a liquid inoculant. Alfalfa seeds were pre-inoculated with four formulations (mannitol yeast broth (YMB), YMB with agar (1 g·L−1), YMB with 1 or 5 g·L−1 sodium alginate) for three months. Seeds pre-inoculated and stored for one month produced successful alfalfa plants. Nitrogen content in alfalfa obtained from seeds pre-inoculated one month be- fore sowing increased between 3.72%–4.19%[29]. The ability of 17 rhizobacterial strains to im- prove physiology, nutrient uptake, growth and yield of alfalfa plants grown under desert agricultural conditions in Saudi Arabia was studied by some 114 authors[30]. The 17 rhizobacterial isolates were con- firmed as plant growth promoting rhizobacteria by classical biochemical tests and using 16S rDNA gene sequence analysis, the strains were identified as Bacillus, Acinetobacter and Enterobacter. Inocu- lation of alfalfa with any of these 17 strains im- proved relative water content; chlorophyll a; chlo- rophyll b; carotenoid content; N, P and K content; plant height; leaf-to-stem ratio; fresh and dry mass. Acinetobacter pittiiJD-14 was more effective in in- creasing alfalfa fresh and dry mass by 41 and 34%, respectively, compared to uninoculated control plants. However, all strains improved crop charac- teristics compared to control plants, indicating that these desert rhizobacterial strains could be used to develop an environmentally friendly biofertilizer for alfalfa and possibly other crop plants to improve sustainable production in arid regions. 4. Evaluation of the nutritional composition of alfalfa (M. sativa) when grown with the application of biofertilizers Six biofertilizer doses of cattle manure fer- mented in a biodigester (0, 25, 50, 100, 200 and 400 m3∙ha−1) and five replicates were used. The chemi- cal characteristics of the biofertilizer were: 0.300 g N (Nitrogen) L−1; 0.057 g P (Phosphorus) L−1; 0.188 g K (Potassium) L−1; 0.105 g Ca (calcium) L−1; 0.057 g Mg (magnesium) L−1, 1 mg Mn (manganese) L−1; 1 mg Fe (iron) L−1, and 1 mg Zn (zinc) L−1. As a result, the best absorption of N, K, Ca and Mg was obtained with the dose of 400 m3∙ha−1. In the case of N, it was 22% more than in the control and was linear with the increase in biomass. The levels of the micronutrients Cu, Mn and Zn did not differ significantly among the doses applied, as did the crude protein concentrations[31]. On the other hand, the effect of S. meliloti strain ENRRI A12 and chicken manure (0, 2, 4, 6, 6, 8 and 10 t∙ha−1) on alfalfa cultivar (M. sativa) “He- gazi” was studied under pot and field conditions. In the pot experiment, S. melioti inoculation and chicken manure levels significantly increased plant height, root fresh and dry mass, and nodule number and dry weight. In the field experiment, both S. me- lioti and chicken manure significantly increased plant density, fresh forage yield and protein content, and significantly decreased crude fiber percentage. Fresh forage yield and chicken manure level were highly correlated (r > 0.99)[32]. 5. Nanotechnology in alfalfa Nanotechnology is one of the latest technolog- ical innovations. The term “nanotechnology” was first coined by Norio Taniguichi, a professor at To- kyo University of Science, in 1974[33]. Although the term “nanotechnology” has long been introduced in multiple disciplines, the idea that nanoparticles (NPs) could be of interest in agricultural develop- ment is a recent technological innovation and is still under progressive development[34]. NPs are organic, inorganic or hybrid materials with at least one of their dimensions ranging from 1 to 100 nm (nanoscale). NPs that exist in the natural world can be produced from photochemical reaction processes, volcanic eruptions, forest fires, erosion, plants and animals or even by microorganisms[35]. The production of NPs derived from plants and mi- croorganisms has become an efficient biological source of green NPs attracting additional attention from scientists in recent times due to their environ- mentally friendly nature and the simplicity of the production process compared to the other routes[36]. NPs, depending on their properties, interact with plants causing various morphological and physiological changes. The efficiency of NPs is de- termined by their chemical composition, size, sur- face coverage, reactivity, and most importantly, the dose at which they are effective[37]. Researchers re- port both positive and negative effects on plant growth and development when using NPs and the impact of NPs depends on the composition, con- centration, size, chemical and physical properties, as well as the plant species[38]. For the exploitation of green nanotechnology, a number of plant species and microorganisms, in- cluding bacteria, algae and fungi, are currently be- ing used for the synthesis of NPs. For example, the plant species M. sativa and Sesbania are used to formulate gold nanoparticles. Similarly, inorganic nanomaterials, made of silver (Ag), nickel (Ni), 115 cobalt (Co), zinc (Zn) and copper (Cu), can be syn- thesized within living plants, such as Bras- sicqjuncea, M. sativa and Heleanthusannus[36]. Synthesized nanofertilizers have a specific use to regulate nutrient release according to crop re- quirements, while minimizing differential losses. For example, conventional nitrogen fertilizers are characterized by large losses to the soil through leaching, evaporation or even degradation of up to 50%–70%, which ultimately reduces fertilizer effi- ciency and raises the cost of production[39]. On the other hand, nitrogen fertilizer nanoformulations synchronize the release of N-fertilizer with its up- take demand by crops. Consequently, nanoformula- tions prevent undesirable losses of nutrients through direct internalization by crops and thus avoid nutri- ent interaction with soil, water, air and microorgan- isms[36]. Micronutrient deficiency decreases not only crop productivity, but also affects human health through the consumption of micronutrient-deficient foods. For example, iron deficiency causes anemia, impaired growth, reproductive health problems, and even decreased cognitive and physical performance in humans[40]. In this regard, the use of nano-formulated micronutrients for slow or con- trolled release of nutrients would stimulate the pro- cess of plant uptake, promote crop growth and productivity, and also contribute to maintaining soil health[41]. For example, in zinc deficient soils, ap- plication of nano zinc oxide at low doses positively influences growth and physiological responses such as shoot and root elongation, fresh dry weight and photosynthesis in many plant species compared to control[42,43]. 6. Nanotechnology applications in alfalfa cultivation Boron (B) is among the nutrients that are nec- essary for plant growth and yield production and can improve the nutritional properties of forage crops. However, at higher levels, it can be toxic and negatively affect plant growth and forage quality. The concentration of B in plants is affected by dif- ferent parameters, such as fertilization with this same micronutrient, soil, climate, plant species, etc. For all these reasons, the effects of different B treatments in alfalfa on B concentration and pig- ment content, including chlorophyll, b, total and carotenoids, were studied. Experimental treatments were: (1) six soil types (S1–S6); (2) B sources, in- cluding boricicide (B1) and nano boron (B2) ferti- lization; and (3) number of sprays (zero, one, two and three times). Results indicated that soil type, B source and number of sprays significantly (P ≤ 0.01) affected alfalfa B concentration and pigment con- tent. Spraying three times significantly increased B concentration as it resulted in 207.81% increase compared to the control treatment and equally in- creased pigment content (P ≤ 0.05) including chlo- rophyll, b, total and carotenoids compared to the other treatments[44]. A greenhouse study was conducted to explore the effect of various doses of potassium sulfate (K2SO4) NPs on alfalfa growth and physiological response under salt stress. A salt-tolerant genotype (Me-sa-Sirsa) and a salt-sensitive genotype (Bull- dog 505) were selected on the basis of germination under salt and planted in pots containing 2 kg of sand. The two genotypes were subjected to salt lev- els of 0 and 6 dS∙m-1 using CaCl2·2H2O:NaCl (2:1) mixed with Hoagland’s solution. Three treatments of K2SO4NPs consisting of 1/4, 1/8, and 1/10 of the K level in full-strength Hoagland solution (235 mg·L−1) were applied. The highest shoot dry weight, relative yield, root length and root dry weight in both genotypes were obtained when using K2SO4NPs at the 1/8 level. The different doses of K2SO4NPs significantly affected the Na/K ratio and Ca, P, Cu, Mn and Zn concentrations in plant tissue. Application of K2SO4 NPs at a rate of 1/8 improved plant physiological response to salt stress by reduc- ing electrolyte leakage, increasing catalase and pro- line content, and increasing antioxidant enzyme activity. These results suggest that the application of KNPs may have better efficiency than conventional K fertilizers in providing adequate plant nutrition and overcoming the negative effects of salt stress in alfalfa[45]. The toxicity of zinc oxide nanoparticles (ZnONPs) on seed germination/root elongation and uptake of ZnONPs and Zn2+ in alfalfa (M. sativa), 116 cucumber (Cucumis sativus L.) and tomato (Sola- num lycopersicum L.) seedlings was investigated by the literature[46]. Seeds were treated with ZnONPs at 0–1,600 mg·L−1 as well as at 0–250 mg·L−1 of Zn2+ for comparison purposes. The results showed that at 1,600 mg·L−1 of ZnONPs, germination in cucumber increased by 10% and germination of alfalfa and tomato was reduced by 40 and 20%, respectively. With 250 mg Zn2+ L−1, only tomato germination was reduced with respect to the controls. The high- est Zn content was 4,700 and 3,500 mg∙kg−1 dry weight (DW), for alfalfa seedlings germinated in 1,600 mg∙L−1 of ZnONPs and 250 mg∙L−1 of Zn2+, respectively. Alfalfa in nanotechnology has also been used to obtain NPs. Scientists have found a way to grow and harvest gold (Au) from crop plants. The NPs could be harvested industrially. For example, alfalfa plants grown in an environment rich in AuCl4 showed uptake of metallic gold. AuNPs can be me- chanically separated by dissolving the organic ma- terial (plant tissue) after harvest[47]. Alfalfa plants can also adsorb Ag from a solid medium rich in this element with subsequent formation of Ag NPs[48]. 7. Conclusions (1) The indiscriminate and unbalanced use of chemical fertilizers, especially urea, together with chemical pesticides and the lack of organic fertiliz- ers leads to a considerable reduction in soil health, so the use of biofertilizers is on the rise in various countries and crops. The cultivation of microbial communities induces high productivity with negli- gible energy investments and, therefore, signifi- cantly reduces the effects on the environment. (2) In sustainable agriculture and environmen- tal protection against pollution is critical, so the ap- plication of nanotechnology ensures better man- agement and conservation of inputs for agricultural food production. 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