Chitosan Nanomaterials for Plant Nutrient Delivery PDF

Chapter 11 Chitosan nanomaterials for delivery of micronutrients in plants Garima Sharma1, Damyanti Prajapati1, Khaidem Aruna Devi1, Ajay Pal2 and Vinod Saharan1 1 Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Te...

Chapter 11 Chitosan nanomaterials for delivery of micronutrients in plants Garima Sharma1, Damyanti Prajapati1, Khaidem Aruna Devi1, Ajay Pal2 and Vinod Saharan1 1 Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India; 2 Department of Biochemistry, College of Basic Sciences and Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana, India 1. Introduction The rising global population and its amalgamation with improved income and dietary changes are operating an ever-increasing food demand that is expected to rise by 70% in 2050. Agriculture is of utmost importance in producing and providing the raw materials for the food and feed industries. The growth of the agriculture sector is defined by an important factor, which is crop yield. Micronutrients have a prominent role in plant development and crop yield. Sufficient nutrient supply improves the source activity that leads to a high rate of photosynthesis/energy translation, and translation of photo-assimilates [2e5]. Hence it is crucial to maintain the nutrient supply to strengthen the source activity and sink strength in plants that ultimately contribute to plant yield. Conventional fertilizers serve very low (40%) micronutrient use effi- ciency. To compensate for this effect excessive use of fertilizer is the only option left that contributes to undesirable effects on the environment as well as on crop yield [6,7]. Imprudent agrochemical consumption in agricultural fields for intensified crop production and protection has created immense environ- mental and health hazards. This consequence has raised the issue of global warming and resistance development in pathogenic microbes. To address all these issues there is a need for some alternative strategy that can deal with them together by improving plant immunity and strengthening plant growth [10e12]. The recent upswing in the synthesis of various nanomaterials in various sizes, shapes and functions has represented nanotechnology as an essential technology for agriculture [13,14]. The unique properties of Role of Chitosan and Chitosan-Based Nanomaterials in Plant Sciences https://doi.org/10.1016/B978-0-323-85391-0.00004-6 Copyright © 2022 Elsevier Inc. All rights reserved. 239 240 Role of Chitosan and Chitosan-Based Nanomaterials in Plant Sciences nanomaterials have a vital role in agriculture specifically in plant growth and protection. Among various nanomaterials, metal-based nanomaterials are profoundly used in agriculture as revealed by available literature [15e17]. Many reports of phytotoxicity of metal nanoparticles have raised serious concerns about their use [13,18e21]. This has accelerated the interest of re- searchers in some biopolymers for their use in crop protection by nano- technological approach [14,22]. Especially, chitosan-based biodegradable nanomaterials in the form of nanoparticles, nanogels and nanocomposites have made their way as prime choices in agriculture due to their quirky properties like antimicrobial and plant growth-promoting activity. Precision agri- cultural practices that promote the minimal input of pesticides and fertilizers per unit while improving crop productivity are of high need to substitute for the excessive use of agrochemicals. Metal-chitosan nanomaterial provides the way for slow and sustained release of encapsulated metal and hence lessens their toxic effect on plant and have a long-lasting phenomenon. Additionally, chitosan itself has many interesting properties viz. plant immune booster and antimicrobial activity [24e34]. So, it is most favorable to use chitosan in nanoagri-inputs specifically nanofertilizers to target the nutrient deficiency over the conventional fertilizer. In this chapter, we discuss the state-of-art facts exploring the usage of chitosan nanomaterials in the management of plant nutrition. 2. Chitosan nonmaterial and their application Chitosan is the deacetylated form of chitin and linear copolymer of 2- acetamido-2-deoxy-b-D- glucopyranose and 2-amino-2-deoxy-b-D-glucopyr- anose. It occurs in the cell walls of fungi, insect cuticles, and the exoskeleton of crustaceans. Chitosan has some unique properties such as biocom- patibility, biodegradability, antimicrobial activity and nontoxicity. These properties render this biopolymer most suitable for a number of applications including antifungal activity , antibacterial activity [37e39], and plant growth-promoting activity [40e42] (Fig. 11.1) and nano-fertilizers [43,44]. In this chapter, the discussion will be focused on the application of chitosan- based nanomaterial in nutrient supply to plants. 2.1 Use of chitosan nanomaterial for nutrient supply in the plant Higher plant growth via vigorous seedling establishment and efficient nutrient utilization is the most crucial approach to determining the final crop yield. To cope with the various biotic and abiotic stresses at the early and late stages of growth, higher germination, appreciable shoot-root length, root number, and higher biomass are pivotal. There is a range of reports available indicating the application of chitosan-based nanomaterials in vigorous seedling growth and significant enhancement in food mobilizing enzymes (amylase and protease)  Chitosan nanomaterials for delivery Chapter | 11 241 FIGURE 11.1 Cu-chitosan NPs boost the de-novo synthesis and enhance the activity of a amylase and protease enzymes. Increased a amylase and protease activity up-regulate the rapid degradation and mobilization of stored food which enhanced the germination and seedling vigor index in maize. Courtesy Saharan et al. (2016) , copyright permission from American chemical society Elsevier. [40,44e46]. Exquisite properties of chitosan and extensive research regarding chitosan application in plant growth have recently attracted much attention from researchers in the agricultural application of chitosan-based nano- materials to enhance nutrient use efficiency. The bioavailability of nutrients is the most targeted phenomenon to achieve plant growth and quality. Nano- technology has explored two ways to maneuver the nutrients- (1) conversion of nutrients to the nanoscale to provide an easy passage in plant cells and (2) encapsulation of nutrients in nano-matrix for their slow and sustained release. Chitosan biopolymer is a popular encapsulating agent for nano delivery of nutrients and agrochemicals. Chitosan has an affinity for metals and this property can be utilized for the encapsulation of micronutrients (Cu, Zn, Fe, Si)and macronutrients (N, P and K) for crop improvement and better nutrient use efficiency. Chitosan itself serves as a nitrogen source to plants due to the presence of nitrogen (w8.9%e9.5%) in its amino group. Application of chitosan to the plant increases the uptake of many mineral nutrients including nitrogen, potassium, phosphorous, calcium and magnesium. Chitosan triggers the molecular signal to induce the growth and developmental pro- cesses in plants. Nano chitosan has significantly improved the seedling growth in Robusta coffee by higher nutrient uptake. Cu encapsulating chitosan nanoparticles provided the slow and sustained release of Cu and enhanced the plant growth in maize and tomato [40,41]. Foliar spray of nano chitosan-NPK has increased the growth of wheat. 242 Role of Chitosan and Chitosan-Based Nanomaterials in Plant Sciences 2.2 Various chitosan-based nanomaterials The abundance of literature indicates that chitosan has immense antimicrobial activity against plant and animal microbes [50e53]. Although bulk chitosan has limited use as an antimicrobial agent in agriculture due to its aqueous insolu- bility. Further chitosan is used commonly in acidic aqueous medium for better dispersion and homogenous applicability. But the acidic aqueous medium has a toxic effect on the target organisms and bulk chitosan has a lower growth inhibitory effect on microbes [36,41,54]. Therefore practices are initiated to improve its bioactivity by modification in the physicochemical properties for enhanced dispersion in an aqueous medium [36,55]. Technically, chitosan is more feasible to manipulate without interfering with its innate activity as compared to other biopolymers like chitin, starch, gelatin, cellulose and glucans [56e58]. Chitosan can easily be tuned for various applications by modification of physicochemical and biophysical properties due to the presence of amino (-NH 2) functional group, high responsiveness to pH changes, and amenability to size changes [22,54,59e62]. Various engineered chitosan-based nanomaterial have been synthesized and studied to analyze their effects on plant growth and protection [22,54]. Calvo and coworkers described the ionic gelation method that involves the ionic interaction between the positively charged amino (-NH 2) groups of chitosan and negatively charged tripolyphosphate that allow the chitosan molecules to bring them into nanoscale size. TPP has been reported as the first choice as a crosslinker in chitosan nanomaterial synthesis as it has no innate biological activity and no effect on the bioactivity of chitosan [64e66]. Cross-linking of TPP with chitosan changes the physicochemical properties of chitosan like particle size, size distribution/polydispersity index (PDI), and surface charge (zeta-potential). Chitosan has a wide spectrum of antimicrobial and bio-regulatory activity. Chitosan TPP cross-links also serve as an efficient encapsulating agent. Further, many reports show the chelating properties of chitosan for various organic and inorganic compounds including transition metals Cu, Zn [40,41,63], Fe and Si and signaling molecule salicylic acid (SA) [44,45]. These facts facilitated the major interest of researchers in manufacturing chitosan-based nanomaterials and their use in plant protection and growth over traditional methods. Cu-Chitosan-based nanomaterials have been synthesized and have already been tested on tomato and maize plants (Saharan et al., 2015; Choudhary et al., 2017) [41,67]. Chitosan-based nano- materials encapsulating Zn were reported to enhance plant protection and growth in maize. Below we will discuss various chitosan-based nanoparticles specifically metal chitosan nanoparticles, salicylic acid chitosan nanoparticles, chitosan nanoparticles as coencapsulating agents. 2.2.1 Copper-chitosan nanomaterial Copper is a prevalent micronutrient in the plant system and an essential component of many important enzymes of plant metabolism. It plays an  Chitosan nanomaterials for delivery Chapter | 11 243 exclusive role as a cofactor of several enzymes in the electron transport chain and redox reaction [68,69]. Additionally, copper is well known antimicrobial agent. United States Environmental Protection Agency (USEPA) recognized Cu as the first metallic antimicrobial agent. Recently several studies have come up with chitosan nanoparticles encapsulating copper. Chitosan has some affinity toward copper as compared to other transition metals. This distinctive ability is the reason for the availability of various reports claiming successful synthesis and application of Cu-chitosan nanoparticles and their application in various fields [40,44,71e73]. Use of Cu encapsulated nano-chitosan (360.3 nm mean hydrodynamic diameter, 0.48 PDI, and þ32.6 mV zeta potential) (Fig. 11.2) provided effective control of tomato fungal pathogen. Further, successive studies on chitosan NPs encapsulating Cu confirmed the enhanced growth of seedlings by mobilizing the reserve food in maize. In maize plants, Cu- chitosan NPs raised defense response against CLS disease via increased activ- ity of antioxidant and defense enzymes in addition to increased plant growth. Additionally, studies on Cu-chitosan NPs showed the slow and sustained release of Cu from NPs which was the pinpoint for its effectiveness [40,44,67]. Chitosan nanoparticles facilitate the slow and sustained release of copper and mitigate its toxic effect on the plant. Additionally, nanoencapsulation of copper in chitosan nanoshell improves the seedling growth in a plant by enhancing the food mobilizing enzymes namely amylase and protease. Defense and antioxi- dant enzyme activity is also improved by Cu-chitosan nanoparticles. These nanoparticles mimic the natural elicitation of plant defense and antioxidant FIGURE 11.2 SEM-EDS elemental analysis of Cuechitosan nanoparticles: (A) spectra of a nonporous surface, and (B) spectra of porous surface. Courtesy Saharan et al. (2015) , copyright permission from Elsevier. 244 Role of Chitosan and Chitosan-Based Nanomaterials in Plant Sciences systems for sustainable growth. Healthy seedlings and improved innate immune response render the plant protected from various abiotic and biotic stresses. As reported by Choudhary et al., 2017 Cu-chitosan nanoparticles treatment has a four to six fold increase in SOD activity. The activity of Phenylalanine ammonia lyase (PAL) and Polyphenol oxidase (PPO) was persuaded from 46.15 to 66.66%and 3.05%e16.39%. Further Cu and SA co-encapsulated chitosan nanoparticles also reported significant improvement in antioxidant and defense enzyme activity. The reproducible method of ionic gelation facilitates the use of Cu-chitosan nanoparticles in plant growth and protection. 2.2.2 Zinc-chitosan nanomaterials Zinc is an important micronutrient that is involved in the expression of genes encoding antioxidantenzymes such as superoxide dismutase (SOD), peroxi- dase and glutathionereductase (GR) [74e77]. Many studies reported zinc- mediated generation of O2 free radicals and NADPH oxidase activation are associated with lignin deposition in the plant cell walls that impart resistance against invading plant pathogen [77e80]. Zinc is crucial during the repro- ductive and grain filling stages of the plant. Zinc deficiency is a major issue in cereal grain production. Cultivation of plants in zinc-deficient alkaline soil results in lower grain yield, plant growth depression, and high disease incidence. Slow and sustained release of Zn for improved plant growth, grain yield, and disease resistance can be the most promising solution to address these problems. Bulk zinc alone and its combination with humic acid and/or chitosan were tested in dry beans for growth, nutrient content, and yield. In wheat zinc complexed chitosan/tripolyphosphate nanocarrier was tested for zinc enrichment. Zinc encapsulated chitosan-based nanomaterial have been synthesized and evaluated for slow and sustained release of Zn and their role in plant defense against Curvularialeaf spot (CLS) disease in wheat. This study reported increased grain yield from 20.5% to 39.8% and there was 41.27e62.21 mg/g dry weight Zn enrichment found in wheat. Zn-chitosan nanoparticle synthesized in this study was of high stability with a zeta po- tential of þ34 mV and 82% Zn was encapsulated in chitosan nanoshell. The nanomaterials have shown significant antimicrobial activity in a laboratory (47.7%e65.2%) as well as field experiments (32.37%e42.93%) on treatment with nanoparticles in the range of 0.01%e0.16% concentration. Slow and targeted release of Zn from chitosan nanoshell has shown significant plant growth-promotion and plant protection activity. 2.2.3 Salicylic acid (SA)-chitosan nanomaterials In recent years, great attention has been attained to up-regulate the plant endogenous defense and growth regulatory system by various signaling mol- ecules. So Salicylic acid has attracted research interest due to its function as a local and systemic plant defense eliciting agent against pathogens as well as its  Chitosan nanomaterials for delivery Chapter | 11 245 role during plant response to abiotic stresses such as drought, chilling, heavy metal toxicity, heat and osmotic stress. Adding to its role in biotic and abiotic stresses, SA also plays an essential role in plant growth regulation. SA (ortho- hydroxybenzoic acid) belongs to plant phenolic compound, which has diverse effects on various physiological and biochemical processes like photosyn- thesis, ion uptake, various plant growth aspects, membrane permeability, de- fense and antioxidant enzyme activities related to biotic and abiotic stresses [83,84]. For disease control, strengthening the immune system, and promotion of plant growth, SA has been applied to plants via seed treatment and foliar application. Exogenous application of SA controlled disease caused by Rhizoctonia solani through rapid induction of PAL, PPO and POD enzymes in cowpea plant. Foliar application of SA on highly susceptible apple cultivar (Malus domesticaBorkh cv. “Gala”) induced disease resistance against Glomerella leaf spot (GST) caused by Glomereallacingulata.SA plays a key role in the plant signal transduction pathway for the commencement of systemic acquired resistance and is a naturally occurring plant phenolic compound [89,90]. Bioactivity of exogenously applied SA in a plant is affected by many factors like concentration, treatment duration, plant age, species and the targeted plant organ. So there is a high need to precisely optimize the application of SA on the plant for getting consistent results. Based on previous reports claiming sustainable exposure of micronutrients by chitosan nanoparticles that have shown a stable effect on plant growth and protection SA encapsulated chitosan nanoparticles have been prepared. SA functionalized chitosan nanoparticles are supposed to be having more potency in boosting plant immunity as compared to metal-chitosan nano- particles due to the role of SA in plant signal transduction. SA e chitosan nanoparticles have significant antimicrobial activity against Fusarium verti- cillioides in a laboratory (62.2%e100%) and field (37.3%e49.5%) experi- ments. Slow and sustained exposure of SA to plants has significantly improved the antioxidant and defense response in the plant. SOD activity increases 2 fold after 2 days of SA e chitosan nanoparticles foliar application. Activities of POD, CAT, PAL and PPO were also enhanced by 7.7, 2.9, 2.3, 1.5 fold respectively after 2 days of foliar spray. Slow-release of SA from SA-chitosan NPs significantly amend physiological and biochemical response in plants for commendable disease control, plant growth, and yield as compared to sole SA application. 2.2.4 SA-Cu chitosan nanomaterial Successful encapsulation of Cu into chitosan NPs indicates that more than one valuable component can together be encapsulated for multiple activities. To explore the bioactivities of copper and SA together it is intended to compose a novel biodegradable nanofertilizer using chitosan as a base matrix. Coencapsulation is an emerging and well-adapted approach in 246 Role of Chitosan and Chitosan-Based Nanomaterials in Plant Sciences pharmaceuticals and food industries that increase the functionality of bioactive compounds for their higher bioactivities. In agriculture, this approach is explored with the recent study reporting chitosan nanofertilizer coencapsu- lating Cu and SA. SA and Cu coencapsulated chitosan nanofertilizer were tested for their subsequent application in maize via seed treatment and foliar application. The study proclaimed the improved source activity in maize by mobilization of reserve food toward maize grain. The slow and synergistic effect of Cu and SA boosts the source activity of plant cells by increasing the chlorophyll content and balancing the ROS. Cu and SA coencapsulated chi- tosan nanofertilizer significantly upregulated the stored food remobilization that ultimately aroused the sink strength. Higher source activity and simulta- neous higher sink strength contributed to higher yield under nanofertilizer treatments. 3. Chitosan nanomaterial in plant growth Chitosan is well known for its applicability in plant growth and protection as a nano encapsulating unit for agrochemicals [36,40,41,54,67,92e96]. Numerous reports explored the antimicrobial activity of chitosan nanomaterial. Seed priming and foliar application of chitosan nanomaterial improve the antioxi- dant (SOD, POD, and CAT) and defense enzyme activity(PAL, PPO) in the plant. Proper scavenging of ROS (reactive oxygen species) and optimum level of antioxidant enzyme build a healthy plant system. The application of chitosan-based nanomaterial also improves the defense response of plants by increasing defense enzyme activity. Chitosan nanoparticle-treated plants are reported to show higher lignin deposition as compared to control. Defense enzymes PAL and PPO have a prime role in the synthesis of cell wall strengthening material like lignin which provides resistance to cells against fungal invasion and other cell wall degrading enzymes. Chitosan NPs treatment benefits the plant in many ways. The improved plant growth under chitosan NPs treatment is the most fascinating feature to be understood. Below we summarize the role of chitosan nanomaterial in plant growth and nutrition. 3.1 Plant immune booster Chitosan triggers various physiological and biochemical responses in plants due to its natural eliciting property [60,96]. Increased accumulation of phenolic compounds, cell wall synthesis and generation of reactive oxygen species are some of the natural defense response that has been witnessed in cereals, ornamental, fruit, and medicinal plants under chitosan treatment [51,60,68,98,99]. Chitosan NPs are the positive modulator of innate immune response in plants. The study confirmed the twoefour fold increased defense enzyme activity (POD, PPO, PAL and b 1,3 glucanase) in tea leaves  Chitosan nanomaterials for delivery Chapter | 11 247 treated by chitosan NPs. In addition, chitosan NPs improve the level of anti- oxidant enzymes and secondary metabolites such as superoxide dismutase (SOD,41%), catalase (CAT, 49%), and phenol (24%) over control. Treated plants were further examined by transcript analysis has illustrated that the increased level of defense response was due to higher expression of defense- related genes. These studies justified the enhanced innate immunity of chitosan NPs treated plants. Researchers witnessed the antifungal and antibacterial activities of chitosan NPs. In a study, porous and stable Cu-chitosan NPs of 374 nm were synthesized and tested for their antifungal and plant growth promontory activity in tomatoes. Two fungal diseases namely early blight and Fusarium wilt were significantly controlled by the application of this Cu- chitosan NPs. Further, Choudhary et al. , demonstrated that Cu- chitosan NPs have improved the antioxidant and defense enzyme activity in maize plants to treat plants against CLS (Curvularia leaf spot) disease. 3.2 Plant growth promoter Chitosan NPs are frequently gaining popularity for their use in plant growth promotion due to their unique physicochemical characteristics viz. nanosize, more surface area, positive surface charge, and easy dispersibility in water. Various study reports revealed the successful application of metal encapsu- lating chitosan NPs in plant growth promotion by enhanced seed germination, seedling growth, plant height, leaf area, nutrient uptake, photosynthesis, early flowering, fruit set, and grain yield [36,40,41]. 3.2.1 Seedling growth, plant growth and yield Vigorous seedling growth is the first decisive phase of a plant that determines the final crop yield. High seed germination, significant shoot-root length, root number, and higher biomass are some of the basic criteria of vigorous seedling growth that furnish the plant to cope with biotic and abiotic stresses at the early and late stages of crop. Seed priming/seed treatment is performed by various means to achieve rapid seedling growth. Studies revealed the superi- ority of various chitosan NPs (Cu-chitosan, SA-chitosan, Zn- chitosan, SA-Cu chitosan NPs) over bulk chitosan in terms of seedling establishment, plant growth, and overall yield of the plant [40,41,44,45]. 3.2.2 Nutrient use efficiency The bioavailability of nutrients is the prime factor for appreciable plant growth. Nanotechnology is increasingly gaining attention in the agriculture sector to enhance nutrient use efficiency. There are two basic ways to exploit the nutrients by nanotechnological approach: (1) nutrients converted into nanoscale for easy passage in plant cells and (2) nutrients can be encapsulated in nano-matrix for their slow and sustained release. The unique property of chitosan to have an 248 Role of Chitosan and Chitosan-Based Nanomaterials in Plant Sciences affinity toward metals makes it an excellent tool for nanodelivery of micro- nutrients (Cu, Zn, Mn and Fe), macronutrients (N, P and K), and agrochemicals for crop improvement. Numerous researches reported the successful encapsulation of metals in chitosan NPs including Cu, Zn, Si [40,41,46,63]. Additionally, the presence of nitrogen (w8.9%e9.5%) in the amino group of chitosan also serves as a nitrogen source for plants. Nano-chitosan has significantly increased seedling growth through higher nutrient uptake in Robusta coffee. Cu-chitosan NPs have improved the plant growth in to- matoes and maize [40,41]. The slow and sustained release of Cu from chitosan NPs has proven their superiority over control and bulk chitosan in terms of plant growth and yield. Zinc deficiency or alkaline soil is the most negative factor in the cultivation of cereals in such soils that results in severe depression in plant growth, higher disease incidence, and lower yield. Zn encapsulated chitosan NPs provided an efficient way for the sustained supply of Zn. Signaling molecule SA and its co-encapsulation with Cu is also synthesized with chitosan NPs that have fostered the source activity in maize. Abdel-Aziz et al. reported that foliar spray of nano-chitosan-NPK increased the plant growth in wheat. Nano chitosan represents itself as a strong tool for efficient nutrient use. 4. Conclusion The abundance, biocompatibility and antimicrobial activity of chitosan represent this biomaterial as the most suitable alternative to the problems of excessive chemical use in agriculture. Additionally, chitosan has an immense potential to encapsulate the materials in it that has the potential to overcome the problems of leaching agrochemicals and their nontarget use. Two or more micronutrients can be encapsulated for their additive effect. 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