Chitosan-Based Agronanochemicals

Chitosan-Based Agronanochemicals: Comparison

Please note this is a comparison between Version 2 by Peter Tang and Version 1 by Farhatun Najat Maluin.

Chitosan is established as a non-toxic, biodegradable, and biocompatible compound. It offers fascinating properties; antimicrobial, antiviral, antifungal, antioxidant, anti-inflammatory, bio-adhesion, adsorption enhancer, etc. Chitosan coupled with nanotechnology could offer a sustainable alternative to the use of conventional agrochemicals towards a safer agriculture industry.

sustainable agriculture
chitosan-based agronanochemicals
crop protection

1. Introduction

The increased usage of agrochemicals due to the increase in the World’s food demand urges the need for more research on sustainable agricultural production systems, hence, a heightened drive in agriculture and food security [1,2]^[1][2]. However, the excessive use of agrochemicals represents a significant hindrance in achieving global agriculture security as it poses a negative impact on human health and environmental wellbeing. During the last decade, the global annual agrochemical consumption was approximately 2 million tonnes, where China is the major consumer with 1.8 million tonnes/year, followed by the United States (USA) and Argentina with 500,000 and 236,000 tonnes/year, respectively, compared to only 49,199 tonnes/year used in Malaysia ^[3]. Moreover, the global usage of agrochemicals has been estimated to rise by 3.5 million tonnes in 2020 ^[4]. The term agrochemicals covers a wide range of substances, including fungicides, insecticides, herbicides, rodenticides, fertilizers, plant growth stimulants, etc. ^[5]. In general, agrochemicals are used in crop management to enhance crop productivity and yield, reduce crop losses, combat plant-related diseases and increase food quality ^[6]. Alongside their benefits, agrochemicals are also known for their toxic properties, hence posing a threat to living organisms in the soil and rivers due to losses during their application via degradation, volatilization, photolysis and leaching.

2. Chitosan-Based Agronanochemicals

According to the United Stated (USA) Food and Drug Association (FDA), chitosan is established as a non-toxic, biodegradable, and biocompatible compound [13]^[7]. It offers fascinating properties; antimicrobial, antiviral, antifungal, antioxidant, anti-inflammatory, bio-adhesion, adsorption enhancer, etc. [14]^[8]. Chitosan is soluble at acidic pHs due to the protonation of its amino group. It is derived from chitin via chemical deacetylation under alkaline conditions, where chitin is the second most abundant natural biopolymer and can be found in the shell of crustaceans, insect cuticles and fungal cell walls [15]^[9]. Besides, the production of chitosan is one of the ways to utilize the bio-waste that comes from the crustacean production industries, where its global production are approximately 6–8 million tonnes/year with 1.5 million being produced by Southeast Asian countries [16]^[10]. This is an effort towards achieving a “zero-waste” food industry, hence benefiting to both the economy and the environment [17]^[11]. Nevertheless, it is worth noting that the production of 1 kg of chitosan consumes over than 1 tonne of water. Therefore, the utilization of chitosan-based agronanochemicals as a sustainable alternative in crops management has raised a debate among researchers. However, wthe researchers believe that the advantages of chitosan production to convert waste to wealth, together with the positive outcomes of chitosan nanoformulations in crops management; especially the synergistic effect, toxic-protection abilities, minimization of agrochemical leaching and runoff to the soil and water body, high potency, high efficiency etc., outweigh the need for a huge amount of water during the production of chitosan. The controlled release formulation and high bioavailability could overcome environmental and health issues such as run-off and accumulation of agrochemicals, as well as helping in reducing the labor cost in the agricultural industry. The low toxicity properties make them harmless to the farmers and the person who will be applying it. Again, all in all, the benefits of chitosan-based agronanochemicals outweigh the huge water consumption required for the production of chitosan and therefore it is a way forward, especially for crop management. In agriculture, chitosan nanoparticles by themselves can act as growth enhancers and potent antimicrobial agents against pathogenic fungi and bacteria [18]^[12]. Alternatively, they can also act as a nanocarriers for existing agrochemicals, which hence are referred to as chitosan-based agronanochemicals [19,20,21]^[13][14][15]. The nanocarrier system enables the agriculturally active ingredient to be encapsulated via ionic or covalent inter/intramolecular bonds or entrapped in a polymeric matrix of chitosan to develop an effective nanodelivery system formulation [21]^[15]. Chitosan-based agronanochemicals can be prepared using several methods, including ionic gelation, emulsion cross-linking, spray drying, precipitation, reverse micellar and sieving methods [22]^[16]. Out of these methods, the sieving method is the simplest and direct method. However, the method has been reported to produce nanoparticles of irregular shape and size. On the other hand, the ionic gelation method is the subject of intense research in the formulation of chitosan nanoparticulate systems due to its simplicity and relatively cheap cost. The method does not require many chemicals, hence reducing the possible toxic side effects. It also employs the use of polyanions with a negative charge (e.g., tripolyphosphate) to bond with the positive charge of the protonated amino group of chitosan under acidic conditions. The emulsion cross-linking method produces stable nanoparticulate systems, however, the process is quite tedious and requires crosslinking agents such as glutaraldehyde, formaldehyde, alginate, etc. which might cause complications due to their incompatibility with the active ingredients. The resulting particle size mainly depends on the emulsion droplet size which in turn depends on the crosslinking degree, molecular weight of the chitosan, surfactant type, and the speed of stirring. Nanoformulations aim to enhance the benefits of chitosan and agrochemicals while simultaneously reducing the adverse outcomes. Due to its amphiphilic properties, the encapsulation of chitosan could overcome the poor solubility of many agrochemicals in water, providing an alternative use of inert chemicals in conventional agrochemicals, hence, subsequently reducing theirs toxicity level [19]^[13]. The bioadhesive properties in chitosan provide excellent protection to the encapsulated agrochemicals, thus, increasing the stability and bioavailability in the plant [23]^[17].

2.1. Controlled Release Formulations

Chitosan-based agronanochemicals exhibit highly controlled release behavior that subsequently increases its bioavailability with high circulation and retention time in the plant tissue (higher half-lives, t_1/2). Thus, the controlled release of active ingredients in agrochemicals aims to address the problems associated with the excessive usage of agrochemicals by reducing the quantiies and frequency application in the field. The agrochemical release from the chitosan matrix can be triggered by two types of stimuli: (1) biotic stress, such as the presence of plant pathogens (fungi and bacteria), nematodes, insects, pest and weeds; and (2) abiotic stress factors, such as pH, temperature, salinity, flooding, drought and other environmental factors [7,24]^[18][19]. The release mechanism upon stimulus-response is through pore diffusion, surface desorption, capsule swelling and degradation, as illustrated in Figure 1 [7,25]^[18][20]. The diffusion-controlled mechanism relies on a diffusion rate gradient while the surface desorption refers to the active ingredient adsorbed on the surface of the nanoformulation. Upon hydration, the release of agrochemicals depends on the swelling of the chitosan capsule. Moreover, enzymatic reactions or other environmental factors might result in the rupture or degradation of the capsule matrix. Hence, the controlled release based on the stimuli response in nanoformulations enables the release of the agrochemicals effectively and efficiently at the target site of interest. A pH-dependent release of Cu was observed upon its encapsulation into chitosan nanoparticles, in which the decrease from pH 3 to pH 1 leads to the increased release of Cu from 21.5% to 44.1%, respectively [26]^[21]. This is due to the protonation of the chitosan’s amino group. At higher pH of 6 and 7, a drastic decrease of Cu release was observed (6.1% and 4.9%, respectively), due to the deprotonation of the chitosan’s amino group. Moreover, the sustained release of Cu for up to 96 h was obtained at pH 4.5. A stimulus-response release mechanism was observed for chitosan-Zn nanoparticles, in which the Zn release was mainly due to the stomatal uptake, followed by diffusion and swelling of polymers upon water penetration [27]^[22]. The slightly acidic environment of the intracellular medium is also reported to be able to help release Zn from chitosan nanoparticles. Chitosan-hexaconazole nanoparticles and chitosan-dazomet nanoparticles demonstrated diffusion-controlled release of the fungicides at pH 5.5 with half release times (t_1/2) of 42 and 11 h, respectively [28,29]^[23][24]. Moreover, the co-release of hexaconazole and dazomet from the chitosan-hexaconazole-dazomet nanoparticles prolonged the release time with t_1/2 of 53 and 15 h, respectively [30]^[25]. The diffusion-controlled release of methomyl at pH 6.0 with t_1/2 of 36–70 h has been obtained by its encapsulation into carboxymethyl chitosan and azidobenzaldehyde [31]^[26]. The release of the insecticide acetamiprid from nanocapsules of chitosan-alginate was reported to be pH-dependent, in which half of the acetamiprid was released after 36 h at pH 7.0 and 4.0 compared to only 24 h needed to release the same amount at pH 10 [32]^[27].

Figure 1. The release mechanism of active agents from chitosan-based agronanochemicals (reproduced based on [21] and [33]).

The release mechanism of active agents from chitosan-based agronanochemicals ^[15][28].

2.2. Plant Growth Promoter

The use of nanoformulations of chitosan itself as a plant growth promoter has been extensively researched. The protonated chitosan, rich in positive charges, shows increased affinity towards plant cell membranes, resulting in enhanced reactivity in the plant system. Also, 9–10% nitrogen, which is the main component of chitosan, serves as a macronutrient in a plant [22]^[16]. Alternatively, chitosan can be incorporated with plant macronutrients (nitrogen [N], phosphorus [P], potassium [K], magnesium [Mg], calcium [Ca] and sulfur [S]) and micronutrient (copper [Cu], manganese [Mn], nickel [Ni], zinc [Zn], boron [B], iron [Fe] and chlorine [Cl]). The summary of some of the recent works on the use of nanochitosan and macro/micronutrient nanocarriers as a plant growth promoter are listed in Table 1. As shown in Table 1, chitosan nanoformulations have been widely used as an alternative method in seed treatment to promote seed germination and increase biomass accumulation. Moreover, chitosan nanoformulations have also been used as growth promoters by enhancing the nutrient uptake, chlorophyll content and photosynthesis rate.

Table 1.

Some recent works on the use of chitosan nanoformulations as a plant growth promoters.

Nanoformulations, Molecular Weight (MW), Deacetylation Degree and Final pH of the Product	Plant and Application Type	Average Size * and Zeta Potential	Findings	Ref.

* ^a hydrodynamic mean size, ^b high-resolution transmission electron microscopy (HRTEM) mean diameter size and ^c field emission electron microscopy (FESEM) diameter size. In addition, the chitosan nanodelivery system was loaded with agrochemicals as the active agent, for the formation of chitosan-agrochemical nanoparticles that offer controlled release properties with high efficacy and potency, as the active ingredient can reach the target cell or plant parts more effectively within a defined time [12]^[54]. Some of the recent works on chitosan-agrochemical nanoparticles are listed in Table 3. The crucial parameters in the design and preparation of chitosan-agrochemicals nanoparticles include loading content of active agent, encapsulation efficiency of active agent, the release profile of active agent, their particle size and morphology. There are several works reporting on the design and preparation of these nanoformulations by focusing on these parameters. Nanocarrier system of herbicides (diuron) as a photosynthetic inhibitor for the weed control was developed by crosslinking carboxymethyl chitosan and 2-nitro benzyl (140 nm, average HRTEM diameter size). The nanoformulations were developed with a photo-controlled release mechanism [46]^[55]. In another work, a smart formulation of chitosan-alginate nanocapsules (30–40 nm diameter size of HRTEM) was developed for the controlled release of acetamiprid, in which the controlled release properties was achieved at three different pHs, where a 50% insecticide release was found after 24 h at pH 10 and after 24 h at pH 7 and 4, compared to only about 6 h for the conventional insecticide release at all pHs [32]^[27]. Carboxymethyl chitosan incorporating ricinoleic acid was developed for a 200–500 nm (hydrodynamic size) nanoemulsion of azadirachtin. The nanoformulations enhanced the solubility and stability with a slow and stable release of the insecticides [47]^[56].

Table 3.

Some of the recent works on the use of chitosan (CS) nanocarriers for existing agrochemicals as the active ingredient (AI.).

Agrochemicals Type and Its Active Ingredient	Nanocarrier Formulations, Loading Content % (LC), Loading Efficiency % (LE), Encapsulation Efficiency % (EE), and its Average Size *	Plant Pathogen	In Vitro/In Vivo	Findings	Ref.
Nano-chitosan, 600 kDa, 85%, pH 6.0	Alternaria solani, Fusarium oxysporum, and Robusta coffee (Coffea canephora), foliar spray	420, 750 and 970 nm ^c	Pyricularia grisea,
Fungicide,		Increase chlorophyll content (30–50%), enhance nutrient uptake (10–27% N, 17–30% P, 30–45% K) and photosynthesis rate (30%).			Dazomet Nano-CS, 10-30 nm ^b, –37 mV (fungicides)	CS nanoparticles, ^[1] 276 nm ^b, 28% (LC), 78% (EE); ^[2] 32 nm ^b, 48% (LC), 98% (EE); ^[3] 31 nm ^b, 35% (LC), 85% (EE); ^[4] 7 nm ^b, 33% (LC), 83% (EE) In vitro		Ganoderma boninense High inhibition on mycelial growth with the percentage of inhibition rate recorded at 92%, 87%, and 72% for P. grisea, F. oxysporum and A. solani, respectively. [34]^[29]
		[	43	]^[33]	In vitro	Controlled release with saturation release of 97.9% and half release time (t_1/2) of 11 h at pH 5.5. Increase fungicidal activity up to 30-fold compared to their counterparts.	Nano-chitosan, 110 kDa, 85%–90%, pH 4.0
	[	29	]	^[	^24]	Aphis gossypii
Fungicides, Hexaconazole and Dazomet	Chilli (Capsicum annuum), seed treatment	CS-polyacrylic acid nanoparticles, 50 nm ^a (insecticides)	CS nanoparticles, ^[1] 157 nm ^b, 17% (LC), 67% (EE); ^[2] 58 nm ^b, 17% (LC), 67% (EE); ^[3 163 nm ^a, +60.4 mV		^] 31 nm ^b, 17% (LC), 67% (EE); ^[⁴^] 5 nm In vivo, reared on castor leaves Enhance in total root and leaf fresh mass up to 77% and 28%, respectively upon application of 1 mg/L of nano-chitosan. The increase of leaf catalase (33%) and peroxidase activities (23%) also been observed.	^b, 13% (LC), 64% (EE)	Ganoderma boninense The mean number of eggs/females reduce significantly under the laboratory conditions and field conditions with 76% and 61%, respectively.	In vitro [48]^[30]
			[	41	]^[42]	Controlled release with half release time (t_1/2) up to 66 and 19 h for hexaconazole and dazomet, respectively, at pH 5.5. Increase fungicidal activity up to 40-fold compared to their counterparts.	[30]^[25]	Nano-chitosan, 100–399 kDa,	Bean (Phaseolus vulgaris), seed treatment
Fungicide, Hexaconazole			46 nm ^a		CS nanoparticles, 100 nm ^b, 73% (EE) Promote seed germination (123% after 72 h) and radical length (231% after 72 h) under salinity stress.	Rhizoctonia solani	[35]^[31]
Callosobruchus chinensis	In vivo, reared on castor leaves	The mean number of eggs/females reduce significantly under the laboratory conditions and store conditions with 74% and 70%, respectively.	[41]^[42]	In vitro	Controlled release with prolongs the release time of hexaconazole up to 14 days at pH 8.3 while the conventional pesticides only last up to 5 days. Significant higher antifungal activity compared to the conventional counterpart.	[62]^[57]	Nano-chitosan, pH 7.0–9.0
Fungicide,	Maize (Zea mays), seed treatment	Hexaconazole	CS nanoparticles, ^[1 80–100 nm ^d
Callosobruchus maculatus:	In vivo, reared on soybean ^]		272 nm ^b, 11% (LC), 56% (EE); ^[^2] 169 nm ^b, 17% (LC), 67% (EE); ^[3] 32 nm ^b, 15% (LC), 65% (EE); ^[4] 18 nm ^b The mean number of eggs/females reduce significantly under the laboratory condition and store condition with 84% and 74%, respectively. Promote seed germination (37%), plant height (1.5-fold increase) and leaf area (2-fold increase).	, 15% (LC), 65% (EE) [41]^[	Ganoderma boninense ^42]	In vitro [49]^[32]
	Controlled release with saturation release of 99.9% and half release time (t	_1/2	) of 42 h at pH 5.5. Increase fungicidal activity up to 3-fold compared to their counterparts.		[28]^[23]	Nano-chitosan, pH 4.8
Colletotrichum Gloeosporioides and Alternaria spp.
Fungicide, Pyraclostrobin	Chickpea (Cicer arietinum), seed treatment	Nano-CS, 406 nm ^a, –4.9 to –7.9 mV (fungicides)	CS-lactide nanoparticles, ^[1] 128 nm ^a, 18% (LC), 45% (EE); ^[2] 90 nm ^a, 11% (LC), 85% (EE); ^[3] 10–30 nm ^b, −37 mV	Enhance germination (100%), seedling vigor index (57%) and vegetative biomass of seedlings (3-fold).		77 nm ^a, 2% (LC), 91% (EE); In vitro [43	Colletotrichum gossypii ]^[33]
	Higher inhibition on mycelial (up to 82%) and sporulation of fungus, compared to the counterpart. Enhance seeds germination.			[44]^[43]	In vitro	Better stability of AI under light stress with 81% compared to the counterpart with 41%. Controlled release (75%) of AI up to 10 h at pH 8.3. High fungicidal activity with up to 85% inhibition rate at day 7 of incubation.	[63]^[58]	Chitosan-polymethacrylic acid-NPK nanoparticles	Wheat (Triticumaestivum), foliar spray	26 and 31 nm ^b
Curvularia lunata									Wheat (Triticumaestivum), foliar spray
Fungicide,	CS-Cu nanoparticles, 361 nm ^a, +22.1 mV (fungicides)	Pyraclostrobin	Quarternized CS-silica nanoparticles, 110 nm ^b, 27%–42% (LC) In vitro and In vivo (Maize, Zea mays)	Enhance harvest index (24%), crop yield (59%), and mobilization index (42%).	Phomopsis asparagi [36]^[34]
	Induce more defense response: 1.5–2 fold of peroxidase, a significant amount of superoxide dismutase, 2–3 fold of phenylalanine ammonia-lyase, and a significant amount of polyphenol oxidase.			[	In vitro 26]^[21]	Controlled release (72%) with prolongs release time up to 13 h. Inhibition percentage of fungi up to 95%	[46]^[55		20 nm ^b
^]		Fusarium oxysporum Enhance polysaccharides (10%) and total saccharides (11%).
Fungicides, Tricyclazole and Hexaconazole	CS-CuO, 350 nm ^b, –26.8 mV; CS-ZnO, 441 nm ^b, –24.5 mV; and CS-Ag, 348 nm ^b, –49.1 mV (fungicides)		CS-Ag nanoparticles, 17 nm [50]^[35]
	^b	In vitro and In vivo (chickpea, Cicer arietinum)	In vitro results showed that the antifungal activity follows: CS-ZnO > CS-CuO > CS-Ag, while in vivo results showed that the wilt disease reduction follows: CS-CuO (47%) > CS-ZnO (40%) > CS-Ag (33%).		Pyricularia oryzae [	In vitro 53]^[44]	Significantly increased the inhibition zone by 2-fold compared to the counterpart	[64]^[59]	French bean (Phaseolus vulgaris), foliar spray	20 nm ^b
Fungicide, Avermectin		Enhance plant growth, nutrient uptake, and biomass accumulation. The nanoformulations was found on the leaf phloem via HRTEM image	[51
Fusarium graminearum	Nano-CS, 181 nm ^a, +45.6 mV (fungicides)	]	^[36]
		In vitro and in vivo (wheat)		CS-lanthanum-nanoparticles, 333 nm ^a, 46% (LE), 65% (EE) 85% inhibition of mycelial growth in plate treated with 5000 mg/mL of CS nanoparticles (in vitro) and 53% reduction in disease severity on wheat (in vivo). Deformation and dehydration of fungus mycelial growth also can be seen.	[54	Magnaporthe grisea	In vitro and In vivo ]^[45]	Rapid release on the first 36 h followed by sustained release until day-10. No inhibitory of fungus was observed in the in vitro study. However, significant disease reduction was observed in the in vivo study (Rice, Oryza sativa).	[65]^[60]	Pea (Pisum sativum), seed treatment
	Nano-CS, ^[1] 181 nm ^a, +45.6 mV; ^[2] 310 nm^a, +33.2 mV; ^[3] 340 nm ^a, +21.7 mV (fungicides)
Fungicide, Tebuconazole	20 nm ^b		In vitro and in vivo (wheat)	CS-porphyrinic-pectin nanoparticles, 100 nm ^c, 30% (LE) Induce mitotic cell division (1.5 fold) and enhance of total soluble protein (i.e., legumin β, vicilin 1, 2 and 3, and convicilin)	[52]^[37]
	Inhibition rate (%) at 1000 mg/mL follows: (1) Nano-CS (71.1%) > (3) Nano-CS (17.7%) > (2) Nano-CS (14.1%)		Xanthomonas campestris, Pseudomonas syringae, and Alternaria alternate [45]^[46]	In vitro	Metal-organic frameworks (MOFs) capsule comprise of chitosan, porous porhpyrinic, and pectin demonstrated a stimuli-responsive sustained release of AI with prolonged-release time up to 174 h at pH 7. The nanocapsule exhibited high antimicrobials activities and no phytotoxic effect on Chinese cabbage.	Chitosan-Cu nanoparticles, low MW, 80%	Maize (
	[	66	]	^[	^61]	CS-Cu nanoparticles, 220 nm ^a, +40.0 mV (fungicides)Surya local), seed treatment	150 nm ^b, +22.6 mV	Increase α-amylase and protease activity as well as promote seedling growth.		In vitro
Herbicides, Imazapic, and Imazapyr		CS-alginate nanoparticles, 378 nm ^a, 62% (EE) of imazapic, 71% (EE) of imazapyr;CS-tripolyphosphate nanoparticles, 479 nm ^a, 59% (EE) of imazapic, 70% (EE) of imazapyr Minimum inhibitory concentration after one week incubation follows: Cu (250 µg/mL) > CS-Cu nanoparticles (17.5 mg/mL) > chitosan (10 mg/mL).		[55]^[47]	Bidens pilosa [37]^[38]
	In vivo			After 300 min under gentle agitation, 30% (imazapic) and 20% (imazapyr) were released in CS-alginate nanoparticles, while 59% (imazapic) and 9% (imazapyr) were released in CS-tripolyphosphate nanoparticles. Meanwhile, free imazapic and imazapyr were released up to 55% and 97%, respectively, hence, showing the slow-release formulation of the nanoparticulate system. The encapsulation of herbicides also reduced the toxicity of herbicides against the nontarget organism while maintaining its herbicidal activity on the tested weeds.	Chitosan-Cu nanoparticles, 50–190 kDa, 80%
	[	67	]	^[	^62]	Fusarium verticillioids Maize (Zea mays), foliar spray	CS-Cu nanoparticles, 296 nm ^a, +19.6 mV (fungicides)
Herbicide, Paraquat		CS-Ag nanoparticles, 100 nm ^c, 90% (EE) 361 nm ^a,+22.1 mV	In vivo (Maize, Zea mays pH-responsive sustained release of Cu was obtained. Promote seedling growth (significant increase in plant height, stem diameter, and root length).	)	Eichhornia crassipes [26]^[	At 4 and 8 h after treatment, the disease has been reduced by 48% and 50%, respectively.^21]
		In vivo		[56]^[48]	Improved herbicidal activity on the tested weed with a 90% release of paraquat was observed for up to 24 h. Improved the microbial population, bacteria, and yeast compared to its free herbicide.	[68]^[63]	Chitosan-Zn nanoparticles, 60 kDa, 85%	Wheat (Triticum durum), foliar spray
Pyricularia grisea		325 nm ^a, +42.3 mV	Nano-CS, 83 nm ^a, –28.0 mV (fungicides)	Stomatal localization of nanoparticles was observed. Increase grain zinc content by up to 42%.
	Nematicide, Avermectin	In vitro and In vivo (rice,	Nano-CS, 83 nm ^a, –28.0 mV (fungicides)	Oryza sativa)		[27]^[22]
		No inhibitory activity was observed in the in vitro. However, in vivo results revealed its ability in suppressing the disease with zero percent disease incidence at 10 days after infection, where 100% disease incidence was observed in control.			[57	CS-γ-polyglutamic acid nanoparticles, 61 and 56 nm ^b, 31% (LC), 35% (EE)	Caenorhabditis elegans	In vitro ]^[49]	The controlled release rate governed by pH. The mortality rate of nematodes was significantly increased by 29%, compared to its counterpart.	[69]^[64	Chitosan-γ-polyglutamic acid-gibberellic acid nanoparticles, 290 kDa, 75%–85%, pH 4.5	French bean (Phaseolus vulgaris), seed treatment	134 nm ^a, −29.0 mV	61% of the encapsulation efficiency of hormone in the nanoformulation. Offer sustained-release with 58% after 48 h. Exhibited high biological activity with 50–70% enhance of seed germination, leaf area, and root development compared to counterpart.	^[39]
^]		Chitosan-gibberellic acid nanoparticles, 27 kDa, 75%–85%, pH 4.5	French bean (Phaseolus vulgaris), seed treatment
In vitro and In vivo (finger millet, Eleusine coracana)	In the in vitro evaluation, 65% of radial growth inhibition was obtained. Meanwhile, delayed disease symptom (25 days) and low disease infection (23%) was observed in the in vivo evaluation, while for control, the symptoms started appear in 15 days and 100% disease infection was recorded. Enhance in peroxidase activity level (reached maximum on day 50) also been observed.	[58]^[50]
CS-Cu nanoparticles, 88 nm ^a, –29.0 mV (fungicides)		450 nm ^a, +27.0 mV	90% of the encapsulation efficiency of hormone in the nanoformulation. Offer stability up to 60 days with pH and temperature-controlled release mechanism. Upon treatment, the seedlings showed an increase of leaf area, chlorophyll and carotenoids amount.	In vitro and In vivo (finger millet, Eleusine coracana) [38]^[40]
	Induce resistance against the pathogen attack: a 2-fold increase in chitinase and chitosanase and produce more protease inhibitors, peroxidase, β-1,3 glucanase, and polyphenol oxidase compared to the untreated plant.		[59]^[51]	Chitosan-thiamine nanoparticles, 27 kDa, 85%	Chickpea (Cicer arietinum), seed treatment	596 nm ^a, +37.7 mV	99% of the encapsulation efficiency of hormone in the nanoformulation. Enhance seeds germination and induce more defense enzymes (peroxidase, glucanase, chitinase, polyphenol oxidase, protease, and chitosanase activity) and increase 10-fold auxins level compared to the untreated seeds.	[40]^[41]

* ^a hydrodynamic size, ^b high-resolution transmission electron microscopy (HRTEM) diameter size, ^c field emission electron microscopy (FESEM) diameter size and ^d unmentioned. Plant growth regulators can be encapsulated into chitosan nanocarriers for the development of an effective nanodelivery system of hormones in a slow-release manner and with high bioavailability. Plant growth regulators, also known as plant hormones, such as gibberellins, auxins, abscisic acid, cytokinin and ethylene are chemical substances responsible for regulating plant growth and plant cell development. Chitosan-gibberellic acid nanoparticles exhibited a 37% and 82% increase of root development and leaf area in French bean, respectively, compared to the free hormone, gibberellic acid [38]^[40]. Apart from that, more lateral roots were formed upon supplementation of chitosan-γ-polyglutamic acid-gibberellic acid nanoparticles on French bean seedlings compared to the free hormone ^[39], hence highlighting the benefits of the nanoparticulate systems. Chickpea seeds treated with chitosan-thiamine nanoparticles exhibited a higher germination percentage with 90% compared to the mixture of chitosan-thiamine and control (water) with 84% and 75%, respectively [40]^[41]. The seedlings treated with the nanoparticulate system also exhibited more defense enzymes and 10-fold higher auxin levels compared to the untreated seedlings.

2.3. Biocides Against Plant Pathogens and Pests

Chitosan with or without the incorporation of macronutrients can act as an alternative sustainable potent biocidal agent against pathogenic fungi, viruses and bacteria. A summary of some of the recent works on the use of nanochitosan and its incorporation in plant management is provided in Table 2. As shown in the summary, chitosan with or without the incorporation of other active agents exhibited good potential as a sustainable alternative to the use of conventional fungicides against Fusarium head blight and wilt disease in wheat and chickpea, post-flowering stalk rot in maize, blast leaf in rice, blast disease in finger millet and leaf spot in maize, among others.

Table 2.

Some of the recent works on the use of chitosan nanoformulations as sustainable alternatives to conventional agrochemicals.

Plant Pathogen	Nanoformulations, Average Size *, Zeta Potential and its Application	In Vitro/In Vivo	Findings	Ref.


Pyricularia oryzae


Nano-CS, 28 nm
^b
, +49.0 to +53.0 mV and CS-protocatechuic acid, 33 nm
^b
, +11.0 mV (fungicides)

	In vitro		The diameter of inhibition zone follows: CS-protocatechuic acid nanoparticles > protocatechuic acid > chitosan nanoparticles. Up to a 3-fold increase of the inhibition zone compared to the counterpart.	[60]^[52]
Verticillium dahliae	Nano-oleoyl-CS, 297 nm ^c (fungicides)	In vitro	The nanoparticles internalized the fungal cell, hence leads to the deformation of spore and hyphae, thickened cell walls, cease of organelles and cytoplasmic vacuolation.	[61]^[53]

*^,a hydrodynamic mean size, ^b high-resolution transmission electron microscopy (HRTEM) mean diameter size and ^c field emission electron microscopy (FESEM) diameter size.

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