Chitosan Nanoparticles-Based Ionic Gelation Method: A Promising Candidate for Plant Disease Management

By 2050, population growth and climate change will lead to increased demand for food and water. Nanoparticles (NPs), an advanced technology, can be applied to many areas of agriculture, including crop protection and growth enhancement, to build sustainable agricultural production. Ionic gelation method is a synthesis of microparticles or NPs, based on an electrostatic interaction between opposite charge types that contains at least one polymer under mechanical stirring conditions. NPs, which are commonly based on chitosan (CS), have been applied to many agricultural fields, including nanopesticides, nanofertilizers, and nanoherbicides. The CS-NP or CS-NPs-loaded active ingredients (Cu, saponin, harpin, Zn, hexaconazole, salicylic acid (SA), NPK, thiamine, silicon, and silver (Ag)) are effective in controlling plant diseases and enhancing plant growth, depending on the concentration and application method by direct and indirect mechanisms, and have attracted much attention in the last five years. Many crops have been evaluated in in vivo or in greenhouse conditions but only maize (CS-NP-loaded Cu, Zn, SA, and silicon) and soybean (CS-NP-loaded Cu) were tested for manage post flowering stalk rot, Curvularia leaf spot, and bacterial pustule disease in field condition. Since 2019, five of eight studies have been performed in field conditions that have shown interest in CS-NPs synthesized by the ionic gelation method. In this review, we summarized the current state of research and provided a forward-looking view of the use of CS-NPs in plant disease management.


Introduction
The world population is predicted to reach 9.8 billion by 2050. Demand for food and water will increase, especially in developing countries, where incomes are projected to increase dramatically [1]. Over the period 2010-2050, the total food demand is projected to increase by 35 to 56% and the population at risk of hunger to change from −91 to +8%. Food production is annually affected by climate change as well as pest and disease damage. When affected by climate change, the total food demand and the population at risk of hunger increases, namely, from 30 to 62% and −91 to +30% [2]. Moreover, food production is severely affected by pests and phytopathogens. Plant diseases caused a 16% loss in global crop production between 2001 and 2003 [3]. According to an assessment of [4], phytopathogens cause a 25% yield loss in developing countries. Among them, fungi are the most common (42%), followed by bacteria (27%), viruses (18%), and nematodes (13%). Phytopathogens cause both direct and indirect effects. The consequences of harmful effects stirring conditions leads to hydrogel formation. The reaction efficiency or properties of the NPs (size, polydispersity indexes determined) differ depending on the ratio of CS and TPP. The process consists of three phases which are solution, aggregation, and opalescent suspension. The materials and instruments for this method can be easily found in conventional laboratories [32][33][34][35][36][37]. Previously, this CS-NPs method was attended in pharmacy to control bacteria on people. The authors of [32] have synthesized CS-NPs-loaded with various metals, including Ag, Cu, Zn, Mn, or Fe, by ionic gelation method. Antimicrobial activity test showed that NPs except Fe could inhibit Escherichia coli, Salmonella choleraesuis, and Staphylococcus aureus at low concentrations, from 3-85 µg/mL, among them, the minimum inhibitory concentration of CS-NPs-loaded Ag and Cu from 3-9 and 9-21 µg/mL, respectively. In addition, the study of [38] showed that CS-NP loaded Ag could inhibit S. aureus, E. coli, and Klebsiella pneumonia with minimum concentrations of 1.69, 1.69, and 3.38 µg Ag/mL, respectively. Moreover, there have been many studies applying NPs synthesized by this method to control plant diseases and enhancing plant growth, most of which are related to CS (shown below). The present article provides a general review of CS-NPs synthesized by the ionic gelation method and their application in plant disease management.

Ionic Gelation Method
In 1997, two publications by Calvo et al. about the synthesis of NPs by a method called ionic (ionotropic) gelation. There, TPP solution is added to CS and/or diblock copolymer of ethylene oxide and propylene oxide under stirring conditions, leading to the formation of CS-NP particles, with a size 200-1000 nm and zeta potential of 20-60 mV, depending on mass ratio CS/TPP or molecular weight of CS. This hydrogel formation is known due to the electrostatic interactions of the amino group of CS and the polyanion group of TPP. In addition, in these studies, bovine serum albumin (protein), tetanus, and diphtheria toxoid (vaccine) were also successfully loaded into NPs [30,31]. Since then (2021), approximately 11,700 research and review articles related to this method have been published according to the statistics of Google Scholar (Figure 1) [39]. Interestingly, the number of articles has increased year by year, reaching a peak in 2021 with 2090 publications. This shows the researchers' interest in the ionic gelation method and NPs.
Polymers 2022, 14, x FOR PEER REVIEW 3 of 25 [30,31]. This technique requires polymeric, usually CS and alginate. The cation of CS (R-NH3 + ) crosslink with polyanion of sodium triphosphate (TPP) (phosphoric ion) under constant stirring conditions leads to hydrogel formation. The reaction efficiency or properties of the NPs (size, polydispersity indexes determined) differ depending on the ratio of CS and TPP. The process consists of three phases which are solution, aggregation, and opalescent suspension. The materials and instruments for this method can be easily found in conventional laboratories [32][33][34][35][36][37]. Previously, this CS-NPs method was attended in pharmacy to control bacteria on people. The authors of [32] have synthesized CS-NPs-loaded with various metals, including Ag, Cu, Zn, Mn, or Fe, by ionic gelation method. Antimicrobial activity test showed that NPs except Fe could inhibit Escherichia coli, Salmonella choleraesuis, and Staphylococcus aureus at low concentrations, from 3-85 µg/mL, among them, the minimum inhibitory concentration of CS-NPs-loaded Ag and Cu from 3-9 and 9-21 µg/mL, respectively. In addition, the study of [38] showed that CS-NP loaded Ag could inhibit S. aureus, E. coli, and Klebsiella pneumonia with minimum concentrations of 1.69, 1.69, and 3.38 µg Ag/mL, respectively. Moreover, there have been many studies applying NPs synthesized by this method to control plant diseases and enhancing plant growth, most of which are related to CS (shown below). The present article provides a general review of CS-NPs synthesized by the ionic gelation method and their application in plant disease management.

Ionic Gelation Method
In 1997, two publications by Calvo et al. about the synthesis of NPs by a method called ionic (ionotropic) gelation. There, TPP solution is added to CS and/or diblock copolymer of ethylene oxide and propylene oxide under stirring conditions, leading to the formation of CS-NP particles, with a size 200-1000 nm and zeta potential of 20-60 mV, depending on mass ratio CS/TPP or molecular weight of CS. This hydrogel formation is known due to the electrostatic interactions of the amino group of CS and the polyanion group of TPP. In addition, in these studies, bovine serum albumin (protein), tetanus, and diphtheria toxoid (vaccine) were also successfully loaded into NPs [30,31]. Since then (2021), approximately 11,700 research and review articles related to this method have been published according to the statistics of Google Scholar (Figure 1) [39]. Interestingly, the number of articles has increased year by year, reaching a peak in 2021 with 2090 publications. This shows the researchers' interest in the ionic gelation method and NPs. Figure 1. The number of articles in Google Scholar searched by key word "ionic gelation" + "nanoparticles" [39]. Figure 1. The number of articles in Google Scholar searched by key word "ionic gelation" + "nanoparticles" [39].
CS is a natural polysaccharide, produced by the alkaline deacetylation of chitin, which possesses excellent characteristics, including low toxicity, low cost, biodegradability, biocompatibility, environmental non-toxicity, and adsorption abilities [18,42,43]. With its superiority, CS is used in wastewater treatment, cosmetics, toiletries, food, beverages, agrochemicals, and pharmaceuticals, and its production in the South-East Asian region reaches 1.5 million tons per year [44,45]. The properties of CS can be modified by chemical and/or mechanical processes by hydroxide and/or amide groups, respectively [46]. TPP is also a safe material, commonly used in the synthesis of NPs by the ionic gelation method as a crosslinking agent [37,47].
CS and TPP can be seen as a "legendary" pair of counter ions in the ionic gelation method because of their popularity in studies. Typically, cations and polyanions are released from dissolving CS and TPP in acetic acid and distilled water, respectively. When TPP is dripped into a CS solution, the polyanion (negative charge) bonds to an amino group (positive charge) by electrostatic interaction, which causes CS to undergo a gel ionization process, leading to the formation of NPs that are usually collected by centrifuge [18,48,49]. The primary interactions in ionic crosslinking configuration are H-link and T-link. The H-link is interaction of O − and NH 3 + in the same plane, while the T-link is interaction of nonbrinding oxygen atom and NH 3 + in different plane ( Figure 2) [34,37]. The formation of NPs is influenced by CS concentration, CS molecular weight, CS/TPP ratio, drug or bioactive molecules concentration, pH, stirring, and centrifuge (time, speed) [48]. In general, this is a method to form microparticles or NPs which based on electrostatic interaction between opposite charge types that contain at least one polymer under mechanical stirring conditions [37]. On the records of [37,40,41], polymers including CS, carboxymethyl cellulose, collagen, dextran, fibrin, gelatin, gellan gum, hyaluronic acid, sodium alginate, pectin, and anions, including chloride salts (Ba, Ca, Mg, Cu, Zn, and Co), sulfate salts (Na, Mg, or octyl-, lauryl-, hexadecyl-, cetostearyl-), polyphosphate salts (pyro-, tri-, tetra-, octa-, and hexameta-), ferrocyanide, and ferricyanide salts were used for synthesis of NPs. Among them, CS (polymer-cation) and TPP (anion) are most commonly used. Furthermore, drugs or bioactive molecules can be encapsulated into matrix of NPs to increase their efficacy.
CS is a natural polysaccharide, produced by the alkaline deacetylation of chitin, which possesses excellent characteristics, including low toxicity, low cost, biodegradability, biocompatibility, environmental non-toxicity, and adsorption abilities [18,42,43]. With its superiority, CS is used in wastewater treatment, cosmetics, toiletries, food, beverages, agrochemicals, and pharmaceuticals, and its production in the South-East Asian region reaches 1.5 million tons per year [44,45]. The properties of CS can be modified by chemical and/or mechanical processes by hydroxide and/or amide groups, respectively [46]. TPP is also a safe material, commonly used in the synthesis of NPs by the ionic gelation method as a crosslinking agent [37,47].
CS and TPP can be seen as a "legendary" pair of counter ions in the ionic gelation method because of their popularity in studies. Typically, cations and polyanions are released from dissolving CS and TPP in acetic acid and distilled water, respectively. When TPP is dripped into a CS solution, the polyanion (negative charge) bonds to an amino group (positive charge) by electrostatic interaction, which causes CS to undergo a gel ionization process, leading to the formation of NPs that are usually collected by centrifuge [18,48,49]. The primary interactions in ionic crosslinking configuration are H-link and Tlink. The H-link is interaction of Oand NH3 + in the same plane, while the T-link is interaction of nonbrinding oxygen atom and NH3 + in different plane ( Figure 2) [34,37]. The formation of NPs is influenced by CS concentration, CS molecular weight, CS/TPP ratio, drug or bioactive molecules concentration, pH, stirring, and centrifuge (time, speed) [48]. After the synthesis of NPs, their popular features include hydrodynamic diameter, zeta potential, polydispersity index (PDI), morphology, dry state diameter, interaction confirms, encapsulation efficiency (EE), loading capacity (LC), and crystal phase, defined by dynamic light scattering (DLS), scanning electron microscope (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), inductively After the synthesis of NPs, their popular features include hydrodynamic diameter, zeta potential, polydispersity index (PDI), morphology, dry state diameter, interaction confirms, encapsulation efficiency (EE), loading capacity (LC), and crystal phase, defined by dynamic light scattering (DLS), scanning electron microscope (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), inductively coupled plasma atomic emission spectroscopy (ICP-OES), atomic absorption spectrometry (AAS), ultraviolet visible (UV-Vis), and X-ray diffraction techniques. These features will vary depending on the synthesis condition, see Tables 1 and 2. NPs include material with at least one dimension in 1-100 nanometers (nm) range [50]. However, the size of the NPs synthesized by the ionic gelation method is usually greater than 100 nm [51][52][53][54][55][56]. The characteristics of CS-NPs synthesized according to the ionic gelation method are presented in Table 1. In the synthesis, CS-NPs, mass ratios, and volume ratios between CS and TPP vary from 1:10 to 15:1 and 1:10 to 25:1 [32,51,53,57]. CS-NPs smaller than 100 nm are synthesized with CS and TPP mass-to-volume ratios reported in studies of [57] with 2:5 and 1:10 (50 nm), [58] with 5:4 and 1:1 (2.3-7.5 nm), [59]  . CS molecular weight also influences NPs formation. As reported by [52], CS-NPs were synthesized from CS low molecular weight (161 kDa) of 180.9 nm in size and were smaller than medium (300 kDa) and high (810 kDa) molecular weights of 309.9 and 339.4 nm, respectively. The authors of [56] had various mass (10:1 and 8:1) and volume (5:1 and 4:1) ratios between CS and TPP with time stirring (60 and 30 min). Results showed that CS-NPs with longer stirring would have smaller sizes in the same mass ratios of 126.2, 167.1, 493.3, and 573.1 nm for 10:1 and 5:1, 8:1, and 4:1 (volume ratio), respectively. This is slightly different from the study of [53] when mass ratio CS:TPP was increased from 5:1 to 20:1 with the same volume ratio, the NPs' size increased from 238.17 to 1315.37 nm. The authors of [55] conducted research with various sonicate times for 3, 5, 10, and 20 min, and the results showed that the NPs' size decreased (344.6, 472.1, 261.3, and 204.8 nm, respectively). In addition to centrifuge, the authors of [52] adjusted the pH of the CS:TPP mixture to 4.5-5 to collect NPs. The results showed that the NPs size was greater than that of the centrifugation method for low (180.9 and 225.7 nm) and high (339.4 and 595.7 nm) CS molecular weight and similar to medium molecular weight (309.9 and 301.5 nm), respectively. The PDI of CS-NPs ranged from 0.195 [55] to 1.0 [32]. A low PDI shows a high uniform dispersion of the particles in the solution and vice versa [61]. CS-NPs have PDIs of 0.31-0.52 [52], 0.6 [51], 0.44-0.69 [56], and 0.195-0.57 [55]. Zeta potential is an effective electric charge on NPs' surface, from −100 mV to 100 mV, representing only NPs' stability [62] [56], and 51.37 mV [32]. CS-NPs were mostly spherical when observed under SEM or TEM [51,55,[57][58][59][60]63], or sphere-like [54,56]. Furthermore, sizes under TEM (dry state) that were smaller than DLS (hydrodynamic size) were shown in the study of [58] with 1.5 nm, [59] with 20-50 nm, [63] with 10-30 nm, and [54] with 100 nm, while DLS was 2.3-7.5 nm, 83.32 nm, 89.8 nm, and 100-1000 nm, respectively. In CS-NPs, the interaction between ammonium group of CS and polyphosphoric of TPP was determined by FTIR with peaks (cm −1 ) of 3428, 1580 [57], 3288, 1647 [58], 1636, 3410 [51], 1648.84, 1527.35 [59], 1563 [60], and 3421.2 [53]. The crystal phase of CS-NPs is amorphous and has been identified by X-ray diffraction [58,59]. UV-Vis is not common in determining properties of CS-NPs synthesized according to the ionic gelation method. As reported by the authors of [59] and [54], CS-NPs absorb at wavelengths of 295 and 320 nm, respectively. Interestingly, the authors of [63] synthesized CS-NPs by CS and anionic protein of Penicillium oxalicum by mass and volume ratio by 7.5:0.108 and 5:2 under stirring for 30 min. Hydrodynamic size, dry state size, PDI, and zeta potential were 89.8 nm, 10-30 nm (spherical), 0.225, and −37 mV, respectively, and the peaks of 1602.8, 1564.18, and 1403.5 cm −1 characterized the binding of proteins and CS. These NPs absorb at wavelength of 285 nm and are amorphous.
To improve application efficiency, CS-NPs can load drugs and bioactive molecules (active ingredients), depending on the purpose. These NPs were synthesized by adding drugs and bioactive molecules solution to CS and TPP during the gelation process (incorporation) or after that (incubation) [18,42,49,50]. This is shown in Table 2. The active ingredient can be metal ion (Cu, Ag, Zn, Mn, and Fe) [32,51], drug (gentamicin-salicylic acid (SA) complex and Ag-Furosemide complex) [55,64], protein (Harpin from Pseudomonas syringae pv. syringae), agrochemical (Hexaconazole) [58], hormone (SA) [65], or other bio-molecules (saponin, thiamine, or Achillea millefolium extract) [51,66,67]. The mass ratio between the active ingredient and the CS or TPP can be smaller or larger, which affects the characters of the NPs. Metal ions (Ag, Cu, Zn, Mn, and Fe) are added to CS: TPP (1:10 or 15:2) at a rate such that the final ion concentration reaches 0.012% [32,51]. Ag-Furosemide complex was added to CS:TPP, with ratios of 0.005:10:2 and 0.01:10:2. The DLS of the two NPs was 210.5 nm, PDI 0.232, and 41.5 mV; and 197.1 nm, PDI 0.234, and 36.7 mV, respectively [55]. When changing the mass ratio TPP:CS by 1:3, 1:4, 1:5, 1:6, and 1:7 (keeping the gentamicinsalicylic complex ratio), the DLS of NPs changed with decreasing size and increasing zeta potential (343. 3 [64]. On the other hand, the results of various mass ratios between TPP and CS:Hexaconazole by 1:5:10, 2:5:10, 4:5:10, and 8:5:10 showed that the sizes of the NPs decreased with respect to their ratios (220.2, 164.2, 68.1, and 6.5-18.1 nm, respectively) [58]. The Harpin protein (P. syringae pv. syringae) was also loaded into CS-NPs, with size of 133.7 nm and zeta potential at 48.6 mV, with an initial mass ratio of 1: 100: 20 [60]. When adding SA to CS: TPP at the ratio 1: 4: 2, the DLS was 368.7 nm, PDI 0.1, and +34.1 mV [65]. The authors of [51] and [66] added Saponin and Thiamine to CS:TPP at the ratio 1:2:20 and 25:24:4, and DLS of the two NPs was 373.9 nm (2 peaks), PDI 1.0, +31 mV and 596 nm, 37.7 mV, respectively. In a study by [67], CS-NP was loaded with A. millefolium extract by mix the extract (semi-solid form) with 0.1% CS solution to obtain 20% before adding 1% TPP solution. This led to the formation of NPs with a size of 118 nm but containing 3 peaks (10, 122, and 712 nm). CS-NPs-loaded mental ions have a compact polyhedron shape, while CS-NPs-loaded saponin, SA, and gentamicin-SA complex were spherical when observed under SEM and TEM [51,64,65]. The size of CS-NPs-loaded active ingredient when recorded under TEM is sometimes larger than the size specified by DSL. CS-NPs-loaded hexaconazole with initial CS:TPP:hexaconazole ratios of 5:1:10 and 5:2:10 had dried state sizes at 271.4 nm and 168.5 nm, while DLS is 220.2 and 164.2 nm, respectively. However, at the ratio of 5:4:10 and 5: 8:10, the TEM sizes were 32.3 and 8.1 nm, compared with DLS with 68.1 and 6.5-18.1 nm (2 peaks), respectively [58]. In contrast, CS-NPs-loaded thiamine was 596 nm by DLS but 10-60 nm by TEM [66]. Not only using TPP as anions, the authors of [68] also synthesized a novel conductive bio-composite membrane by combining CS and phosphotungstate anions on an aluminum substrate using the the ionic gelation method. Interaction in these NPs can also be determined by FTIR. CS-NPs-loaded Cu is characterized by peaks of 1631 and 1536 cm −1 for amide (-CONH 2 ) and primary amide (-NH 2 ), respectively [51]. Peaks of 1345 and 1095 cm −1 feature Harpin protein assigned to C-N and C-O stretch [60]. Peaks of 3218 and 3430 cm −1 characterize a hydrogen bonding between three chemicals in CS-NPs-loaded hexaconazole [58]. CS-NPs-loaded saponin was characterized by peak 3430 cm −1 for the hydrogen bonding between saponin and CS, and 1536 cm −1 for amide linkage between saponin and CS-NPs [51]. The peak at 1317 cm −1 featured an interaction between -COOH of SA and primary amide (-NH 2 ) of CS in a CS-NPs-loaded SA [65]. In CS-NPs-loaded gentamicin-SA complex, a peak at 3423 cm −1 characterizes the hydrogen bonding between -OH group bending of gentamicin and CS, two peaks of 1542 and 1637 cm −1 for interaction between NH 3 + of CS and TPP, and 1300 cm −1 for CN bending between COOH of SA and primary amide of CS [64]. Additionally, the peak of 1657 cm −1 characterizes the binding of thiamine and CS in these NPs [66]. The crystal phase of the NPs can also be identified by the X-ray diffraction technique. CS-NPs-loaded Ag-Furosemide complex was amorphous, while the crystalline peak of hexaconazole was clearly embedded in the amorphous phase of CS [58]. In addition, peak 2θ of 10 • -20 • and 18 • -30 • was recognized for SA and CS, respectively [65]. UV-Vis is seldom used, and only CS-NPs-loaded with 267 nm absorption thiamine was reported by [66]. In general, the main steps for synthesizing and building the CS-NPs-loaded active ingredients using the ionic gelation method are shown in Figure 3. Parameters can be optimized to suit each laboratory's conditions. used, and only CS-NPs-loaded with 267 nm absorption thiamine was reported by [66]. In general, the main steps for synthesizing and building the CS-NPs-loaded active ingredients using the ionic gelation method are shown in Figure 3. Parameters can be optimized to suit each laboratory's conditions. The ionic gelation method requires simple, easy-to-find, and expensive materials and equipment, so it can be done easily, mildly, and quickly in normal laboratories. In addition, the mechanism based on electrostatic interaction instead of chemical reaction leads to no need to use organic solvents, thus avoiding potential toxicity of chemicals or reagents. However, the disadvantage of this method is that it is not easy to produce uniformly sized NPs, and research on other polymers (not CS) is limited [18,37,40,49].  The ionic gelation method requires simple, easy-to-find, and expensive materials and equipment, so it can be done easily, mildly, and quickly in normal laboratories. In addition, the mechanism based on electrostatic interaction instead of chemical reaction leads to no need to use organic solvents, thus avoiding potential toxicity of chemicals or reagents. However, the disadvantage of this method is that it is not easy to produce uniformly sized NPs, and research on other polymers (not CS) is limited [18,37,40,49].

Application of CS-NPs-Based Ionic Gelation Method in Plant Disease Management
With its advantages, CS-NPs synthesized according to the ionic gelation method has been applied in many fields, including pharmaceuticals, new materials, and agriculture (nanopesticides, nanofertilizers, and nanoherbicides) [18,42,49,55,64,68].
For the management of plant diseases, CS-NPs can be applied as protectants (nano pesticides) and carriers (fungicides, insecticides, herbicides, plant hormones, elicitors, and nucleic acids) [18,23,70]. In particular, using CS-NPs as a delivery system is of special interest because it can load and protect the ingredients surrounding the environment and release them to the target site uptake of the plants [18]. In addition, with the basic properties of NPs having a small size and high contact area, CS-NPs or CS-NPs-loaded active ingredients can be easily penetrated and permeated into the membrane of phytopathogens or enhanced plant tissues uptake, resulting in an increased control or defense response activity, respectively [49]. Therefore, these NPs can be used directly and indirectly to manage plant diseases.

Indirectly
Pre-treatment of CS-NPs at 0.1% reduced sheath blight and blast in rice caused by R. solani and P. grisea by 92.78% and 100% under detach leaves assay, respectively [59,74].

Indirectly
Previously, Harpin protein (from Erwinia amylovora) was known for its ability to induce systemic acquired resistance in plants [79]. With the same amount (20 µg), CS-NPsloaded Harpin protein (from P. syringae pv. syringae) enhanced cell death, necrotic lesions, and H 2 O 2 accumulation faster and stronger than Harpin protein only [60]. Furthermore, treatment of these NPs reduced fungal biomass (5-fold) and lesion diameter (12-fold) and caused failing colonization of R. solani in tomato leaves compared with the control. Peroxidase and phenylalanine ammonia-lyase activity also steadily increased up to 72 h. Interestingly, the transcriptome changes, including defense response, signal transduction, transport, transcription, photosynthesis, housekeeping, and aromatic biosynthesis, were enhanced more than 2-fold at 24, 48, and 72 h after spraying.
On the other hand, in the study of [80], CS-NPs-loaded Cu at 0.12-0.06% was treated before and after an infection of Xanthomonas axonopodis pv. glycine could reduce bacterial pustule disease in soybean by 40.6-49.7%, respectively. Interestingly, the low concentration is more effective. In addition, application of the mixture of CS-NPs (ionic gelation) and Cu-NPs (chemical reduction) to date palm root zone increased plant immunomodulatory, including total phenols (1.1-1.5 folds), phenoloxidases (1.1-2.0 folds), and peroxidase (1.6-3.0 folds), which led to a reduction in disease by 16.2-59.3% [57].

Plant Growth Promotion
A concern for any agrochemical is the safety of plants, environment, farmers, and consumers. In recent reviews, NP is a biosafety solution. However, nanotoxicology still remains to be noticed [81,82]. When applying NPs to plants, they will enter the tissues and cause positive and negative impacts depending on their size, shape, and concentration. NPs usually enhance shoot elongation, root elongation, seed germination at low concentration, and in contrast at high concentration [17]. The effective concentration varies between NPs and crops. In the study by the authors of [69], the CS-NP-loaded SA and silver were tested for phytotoxicity with the cassava by leaf disk assay method before being applied to cassava plants at net house condition. Results showed that these formulations did not cause damage in leaf disk up to 800 ppm. Then, researchers varied concentrations of 25-800 ppm for stalk-soaking and foliar spraying to enhance cassava growth and reduce leaf spot disease. This is an easy way to know what "safe" concentrations are for the plant. When applied to soil, NPs can cause negative impacts on soil microflora but will be less damaging than agrochemical applications [83]. On the other hand, the amount of agrochemical and fertilizer applied to agriculture is reduced if they are replaced by NPs, which leads to a reduction in their toxicity. Usually, the safety-by-design principle is applied to screen potential risks from materials and methods synthesis to NP formulation [84]. As mentioned above, ionic gelation method and CS-a natural polymer-are friendly, safe, and biodegradable solutions.
In addition to its ability to directly inhibit pathogens or induce plant defense system against diseases, CS-NPs or CS-NPs-loaded active ingredients have the ability to stimulate plant growth. At this time, they act as fertilizers or nutrients, affecting plant physiological processes, including nutrient uptake, cell division, cell elongation, enzymatic activation, and the synthesis of protein that leads to increase yield [43]. Efficiency depends on both the CS-NPs and the active ingredient, even when it releases all the active ingredients because the main component of CS is nitrogen, which takes 9-10% [46]. Furthermore, the rich positive charge of CS leads to increased affinity toward the plant cell membrane, which enhances reactivity in the plant system [49].
Several types of NPs presented in Table 3 have been shown to stimulate plant growth.

P. grisea /Blast
In vitro: Treat CS-NPs not cause inhibit mycelial and spore germination even 0.1%.

Conclusions and Future Perspectives
Discovered since 1997, the studies on NPs synthesized by ionic gelation method have only received attention in the last ten years. The researches on using these NPs in plant disease management has only been interested in the last five years. With the advantage of being easy to implement, both CS-NP and CS-NP-loaded active ingredients (Cu, Saponin, Harpin protein, Zn, SA, Hexaconazole, NPK, Thiamine, Silicon, and Ag) are effective in plant disease management and enhancing plant growth depending on the concentration and application method by direct or indirect mechanisms. CS-NP-loaded active ingredients constitute the "drug delivery system" model. The effectiveness of disease management and enhanced plant growth of CS-NP or CS-NP depend on the mechanism of CS (carrier) and active ingredients (drug). At higher concentrations, CS-NP or CS-NP-loaded active ingredients are effective in directly inhibiting phytopathogens. This can be applied to control when the disease has broken out. In addition, CS-NP and CS-NP-loaded active ingredients at lower concentrations can indirectly reduce disease through activation of plant's innate immunity, including stimulating cell death, H 2 O 2 accumulation, oxidative burst (O 2 − ), enzymes (β-1,3-glucanase, catalase, chitinase, chitosanase, peroxidase, phenoloxidases, phenoloxidases, phenylalanine ammonia-lyase, polyphenol oxidase, protease, and superoxide dismutases), and secondary metabolites (total phenols, lignin). Moreover, their treatment can enhance transcriptome changes, including defense response, signal transduction, transport, transcription, photosynthesis, housekeeping, and aromatic biosyn-thesis. In nature, plant diseases often have seasonal outbreaks. Periodical pre-treat CS-NP or CS-NP-loaded active ingredients at sensitive periods can prevent disease and reduce the consequences of disease outbreaks. Furthermore, CS-NP and CS-NP-loaded active ingredients can enhance indole-3-acetic acid, α-amylase, protease, chlorophyll, carotenoid content, and photosynthesis rates, leading to increased plant growth, yield, and quality. When plants grow well, their health is enhanced and they can better tolerance diseases. In particular, CS-NP and CS-NP-loaded active ingredients are nano-sized, have a positive charge, and are able to easily penetrate cells or stick to plant surfaces. Moreover, the active ingredient can be slowly released into plant and easily absorbed with no waste. The CS (carrier) as a nitrogen source enhances cell division, cell elongation, enzymatic activation, and synthesis of protein. These preeminent characteristics lead to CS-NP or CS-NP-loaded active ingredients being more effective than CS or active ingredients alone. The CS-NP-loaded active ingredients are more interested in evaluating effectiveness in greenhouse and filed conditions. Most of the studies are more interested in fungal diseases (Alternaria spp., Aspergillus spp., B. bassiana, C. gloeosporioides, C. lunata, Fusarium spp., G. boninense, G. fujikuori, M. phaseolina, P. capsici, P. grisea, R. solani, S. sclerotiorum, and S. rolfsii) and bacteria (C. michiganensis, E. carotovora subsp. carotovora, and Xanthomonas spp.) than viruses, phytoplasma, viroid, and nematode. Many crops, including cassava, chickpea, chilli, date palm, fingermillet, maize, papaya, rice, soybean, tomato, and wheat, have been evaluated in in vivo or greenhouse conditions. However, field experiments are still limited as only maize (with CS-NP-loaded Cu, Zn, SA, and Silicon) and soybean (with CS-NP-loaded Cu) have been evaluated for managing post-flowering stalk rot, Curvularia leaf spot, and bacterial pustule disease and/or enhancing plant growth.
Nanotechnology is the trend of the future. Easy access and dissemination of nano pesticides are essential, especially in developing areas. Since 2019, five of eight studies performed in field conditions have shown interest in CS-NPs synthesized by the ionic gelation method. In the future, new active ingredients could be loaded into CS-NPs or new polymers with anions by ionic gelation methods and used to improve crop yields. A hypothesis is proposed that "mixing CS and TPP under stirring conditions will lead to CS-NPs formation"; then, character or not, they will still be NPs and possess the superiority of NPs. Therefore, the "legendary" pairs of counter ions, CS and TPP, can be studied for immediate application in fields in developing regions where advanced research facilities are limited to building sustainable agriculture.