1. Introduction
The cassava (
Manihot esculenta Crantz) plant and its tapioca are an important food source for world food security, especially in developing countries. Cassava is used in the production of food, industry, and animal feed [
1]. Thailand’s cassava acreage and production reached 1.34 million hectares and 30.84 million tons in 2017, which has increased 1.15-fold over the previous ten years. Although Thailand’s acreage and production account for only 5.45% and 11.04% of the world, respectively, the quantity and value of Thailand’s cassava crop are between 58.5–81.2% and 44.4–56.7% in the world export market, respectively. Furthermore, cassava is Thailand’s key export crop [
2]. Cassava can tolerate drought or nutrient-poor soil, so it also has a role in water-deficient farming areas. However, the biotic stress on a living organism including plant diseases, insects, and weeds, or abiotic stress, such as adverse environmental factors, could affect the growth and development of cassava, resulting in loss of yield. Currently, twenty-eight types of cassava diseases caused by fungi, viruses, or bacteria have been recorded [
3,
4]. Leaf spot disease is one of the most important cassava diseases. The disease causes a loss of up to 30% of cassava yield. However, the serious problem of cassava leaf spot disease has often been neglected, until a recent outbreak in Brazil [
5]. In 2019,
Alternaria sp. was reported as a pathogen causing leaf spot disease on cassava [
6]. It is now possible to implement cultural methods, chemical methods, and host resistance to achieve effective control over or manage diseases. Fungicides containing copper, benomyl, thiophanate, carbendazim, flutriafol, cyproconazole, pyraclostrobin, thiophanate-methyl, tebuconazole, and azoxystrobin can control pathogens with varying degrees of effectiveness. Hence, fungicides are a popular method to reduce the damage of cassava leaf spot. Also, the cassava cultivars Sri Prakash and Sri Visakam are recommended for their resistance to cassava brown leaf spot disease in India [
5,
7,
8]. However, this leaf spot disease-resistant cultivar is not present in Thailand. Therefore, it is necessary to look for a new method to increase the resistance of cassava. Elicitation by stimulating the secondary metabolites is one of the most effective tools to enhance plant immunity. In general, the plant has an innate immune system against adverse environmental factors, including abiotic and biotic stresses, which differs depending on the cultivar and the adverse factors. The plant’s immune system can be artificially induced by an elicitor. Elicitors are biotic or abiotic compounds that activate defense mechanisms and innate immunity in plants against pathogens and stress conditions. The elicitors may be chemical, microbial, chitosan (CS), plant extracts, algal extracts, composts, or biochar. As such, applying appropriate elicitors could aid plants against pathogens as well as cassava leaf spot disease [
9,
10].
In recent years, nanotechnology has been applied to many fields in agriculture, including nanofertilizers, nanobiotechnology, nanomaterials, nanosensors, nanopesticides, nanoelicitors, and nanoherbicides, to enhance plants’ tolerance to biotic and abiotic stress, improve crop yield and quality, and especially to build sustainable agriculture [
11,
12,
13,
14]. Nanoparticles (NPs) are used as protectants (silver, gold, copper, titanium dioxide, and CS) or carriers (CS, silica, solid lipid, and layered double hydroxide) of active compounds (insecticides, fungicides, herbicides, and RNA-interference) to protect plants against bacteria, fungi, viruses, and insects. The advantages of applying pesticide-based nanoparticles in agriculture include improved shelf-life, target site-specific uptake, increased solubility, and reduced soil leaching and toxicity [
15]. NPs in the form of nutrients and non-nutrients are provided to plants through leaves or roots to improve plant health as well as control plant diseases. In many recent reviews, NPs are considered a biosafe technique. On the indirect side, the amount of chemical pesticides or fertilizers used for crop production is reduced because it is replaced by NPs (nano fertilizers, nano pesticides, and nano elicitors). From a direct perspective, applying NPs to soil may have a negative impact on microbial communities, but to a low degree when compared to chemical application. Although the risk is low, the potential toxicity and hazardous effects also deserve attention. Usually, the safety-by-design principle is applied to screen the potential risks of materials and methods of synthesis to NP formulation [
16,
17,
18,
19]. CS is a natural polysaccharide with superior characteristics, including low toxicity, affordability, biodegradability, biocompatibility, environmental non-toxicity, and absorption abilities that have been applied in crop production for plant disease management and enhancing crop yields [
20,
21]. The biogenic Ag-NPs are environmentally safer, with more interest as high-potential antifungal and antibacterial agents [
22]. Foliar spray of Ag-NPs at 50–70 ppm on tomato plants reduced the severity of diseases caused by
Tomato mosaic virus and
Potato virus Y 3.9–4.8- and 2.2–4.5-fold, respectively. It also increased the chlorophyll content, total soluble protein, activities of peroxidase (POD), and polyphenol oxidase (PPO) [
23]. Also, CS-NP-loaded salicylic acid (SA) at 0.01–0.16% can inhibit
Fusarium verticillioides mycelium growth by 62.2–100% and spore germination by 48.3–60.5% in in vitro conditions. In addition, it acted as an elicitor and was able to activate the defense system of the maize plants to reduce post-flowering stalk rot disease by 40.5 to 59.47% and increase yields 1.3–1.5-fold when compared with the control in field conditions [
24]. CS-NP-loaded Cu at 0.1% has been able to inhibit mycelium growth of
Alternaria alternata,
Macrophomina phaseolina, and
Rhizoctonia solani by 89.5, 63.0, and 60.1%, which was higher than CS-NP-loaded saponin and CS-NP was 80.9, 66.2, 27.7% and 82.2, 87.6, 34.4%, respectively. In addition, both CS-NP, CS-NP-loaded Cu, and CS-NP-loaded saponin at 0.06% inhibited the germination of
A. alternata by 84.4, 83.3, and 78.3%, respectively [
25]. There are two approaches to synthesizing NPs, including top-down and bottom-up methods, which result in NPs with different sizes, shapes, and functions. Some synthetic nanoparticles used as a pesticide are not too difficult to implement, such as sol-gel processes, green synthesis by microorganisms, or plant extracts [
26]. The ionic gelation technique for the production of micro-particles or NPs is based on the electrostatic interaction between ions with different charges; it was discovered by Calvo et al. in 1997 [
27,
28]. This system can load additional macromolecules or drugs as a delivery system to improve biological activity or efficiency. The method is simple, fast, economical, easy to implement, and does not use organic solvents, but it is important to select materials and optimize the process to produce suitably effective NPs [
29,
30,
31,
32,
33,
34].
Control studies of treating cassava brown leaf spot with synthetic pesticides were performed. However, the application of NPs on cassava plants to control or manage cassava diseases in general, and cassava leaf spot in particular, is not yet available. Moreover, ionic gelation is an easy and environmentally friendly method of NP production if the materials are properly selected and the process is optimized. In this study, CS-NP-loaded SA or Ag was prepared by ionic gelation method. Then, their effectiveness as nanoelicitors to reduce leaf spot disease and enhance the growth of cassava plants was evaluated in net-house conditions. More specifically, this study is approached with a focus on the reverse research model of preparing elicitors, toxicity tests, screening formulation effectiveness to concentration effectiveness, and characterizing effective elicitors.
4. Discussion
The hydrodynamic diameter of CS-NP-loaded SA (N3) was 89.86 ± 9.04 nm, which is smaller than the size of CS-NP-loaded SA in the study of [
24], which was 368.7 nm. But the PDI and zeta potential of N3 are larger and smaller, respectively. Previously, CS-NP-loaded Ag was synthesized by the ionic gelation method with a size of 90.29 nm and a zeta potential of +92.05 mV with antibacterial properties that apply in medical (pharmaceutical) applications [
29]. In plant disease management, CS-NP-loaded metals are usually Cu and Zn. In these studies, the DLS of CS-NP-loaded Cu was 295.4 nm, PDI 0.28, 19.6 mV [
45]; 361.3 nm, PDI 0.2, 22.1 mV [
46]; 314 nm, PDI 0.48, 19.5 mV [
47]; 374.3 nm, PDI 0.33, 22.6 mV [
48]; and 196.4 nm, PDI 0.5, +88 mV [
25]. In addition, the DLS of CS-NP-loaded Zn was 387 nm, PDI 0.22, 34 mV [
39]. NPs in the studies of [
39,
46,
47,
48] were used as elicitors. The CS-NP-loaded Ag (N6) with a size of 249.67 ± 23.97 nm was smaller than the size of NP in these studies. But the PDI and zeta potential of N3 are larger and smaller, respectively. This is also the first case study in plant disease management. The FTIR test showed the interaction of a primary amide, an amide group, and an anhydro glycoside group in CS-NP-loaded SA (N3) and CS-NP-loaded Ag (N6) formulations compared with CS bulk. The peaks 3422 (NH
2 stretch), 1656 (CO-NH
2), 1597 (NH
2 bend), and 897 (Anhydro glycoside) of bulk CS shifted to 3421, 1640, 1540, and 895 cm
−1 in N3 and 3423, 1643, 1542, and 894 cm
−1 in N6, respectively. The shift to 1314 cm
−1 in N3 showed an interaction of COOH and NH
2. The FTIR peaks are different from previous studies, but still in the range that confirms successful synthesis [
24,
39,
49].
Before an application on the cassava plant, the CS-NP-loaded SA and Ag were tested for phytotoxicity with cassava leaves by the leaf disk assay method. Previously, this method has also been used to determine the dose threshold for the toxicity of 8-methoxynaphthalen-1-ol—an antifungal compound on tomato—and (±)-botryodiplodin—a phytotoxin produced by
M. phaseolina that causes charcoal rot disease on soybeans [
40,
41]. The results show that the CS-NPs formulations were not the cause of the necrotic spots or the browning around leaves’ disks’ margins. But the SN treatment turned the leaves’ disks’ margins brown (
Figure 5). This confirmed that NP formulations have a potential non-toxicity to the cassava plant when compared with using a single chemical (SN). This allowed that NP formulations could be treated on the cassava plants. Recent studies also show that metal NPs (Ag, MgO, and ZnO) can directly affect a fungal pathogen, a bacterial pathogen, or both under in vitro, greenhouse, and field conditions [
50,
51,
52]. Interestingly, the effectiveness of reducing the incidence of soft rot disease and enhancing sugar beet growth and sucrose content by Ag NP treatment was higher than that of
Bacillus subtilis or algal extract [
50]. Furthermore, MgO NP was able to inhibit spore germination, sporangium formation, and hyphal development of
Phytophthora nicotianae and
Thielaviopsis basicola, as well as reduce tobacco black shank and black root rot disease with an efficacy control reaching 50.20% and 62.10%, respectively [
51]. These CS-NPs continue to further research on the following experiments in net-house conditions.
The net-house experiment was conducted to select one CS-NP-loaded SA and one CS-NP-loaded Ag as effective formulations with effective concentrations to reduce cassava leaf spot disease as an elicitor (pre-treating before pathogen infection). Through three rounds of statistical analysis of DS and/or DI data, N3 at 400 ppm and N6 at 200, 400, and 800 ppm were shown as high-potential elicitors (
Figure 1). Specifically, six formulations with six concentrations were statistically analyzed by two factors, which showed that CS-NP-loaded SA (N3) and CS-NP-loaded Ag (N6) were effective formulations. The results of the second statistical analysis showed that N3 at 400 ppm and N6 at 200, 400, and 800 ppm were effective concentrations to reduce cassava leaf spot disease by 68.9, 72.9, 73.6, and 73.2%, respectively. Therefore, they were selected for further pathogen inoculation at 63 DAP (three weeks after the last spraying treatment). The results showed that they were also effective in reducing disease by 37.0, 37.4, 37.0, and 37.7%, respectively. Commercial fungicides also have the ability to reduce cassava leaf spot disease. Of these, pyraclostrobin (Pyr) was more effective than flutriafol (Flu) at 56 DAP (59.6 and 54.7%, respectively) but lower at 75 DAP (14.7 and 35.4%, respectively). They control cassava brown leaf spot disease by spraying fungicide at 10 DAI. A study by [
5] also showed that flutriafol was more effective than pyraclostrobin in reducing the area under the disease progress curve by 68.7 and 11.3%, respectively. In addition, a commercial NP (ZON) only reduces 55.7 and 14.2% at 56 and 75 DAP, respectively. That is significantly lower than the effective concentrations of N3 and N6. In enhancing cassava growth, the effective concentration usually did not significantly increase the shoot height (−13.4–9.1%), but the opposite was true for the number of leaves (45.1–82.4%) and shoots (38.5–46.2%). These treatments significantly increased the largest leaf area (29.6–41.9%), root length (11.6–29.9%), and root weight (27.6–82.8%). The commercial NP usually has a higher potential to enhance plant growth because the active ingredient is ZnO, which contains an important nutrient element (Zn) for plant growth. Overall, N3 and N6 treatments took the spotlight in reducing leaf spot disease and enhancing plant growth on cassava in this study.
In this study, the CS-NP-loaded SA or Ag formulations were used as elicitors that can induce plants’ defense systems against pathogens. In previous studies, CS-NPs (0.05–0.1%) have been reported to reduce finger millet blast [
53], wheat Fusarium head blight [
54], and rice sheath blight [
55] by inducing POD, PAL, chitinase, ROS, superoxide activity, and H
2O
2 content in plants. In addition, CS-NPs have been loaded with active ingredients, including Harpin protein [
56], Cu [
46], Zn [
39], SA [
24], and thiamine [
57], that can induce plants’ defense systems against infections of
R. solani (tomato),
C. lunata (maize),
F. verticillioides (maize), and
F. oxysporum (chickpea) by inducing CAT, chitinase, PAL, PO, POD, PPO, protease, SOD, 𝛽-1,3-glucanase activity, O
2-, H
2O
2 content, and lignin localization. Moreover, the CS-NP-loaded Cu also reduced bacterial pustule disease in soybeans by 40.6–49.7%, but interestingly, a low concentration (0.06%) was more effective than a high concentration (0.16%) [
47]. In our study, N3 at 400 ppm was more effective than N3 at 800 ppm, and N3 at 25 ppm was equally effective as N3 at 100, 200, and 800 ppm. A mixture of CS-NPs (ionic gelation method) and Cu-NPs (chemical reduction method) can also reduce vascular wilt disease in date palms by 16.2–59.3% by inducing total phenol, phenoloxidase, and POD [
58].
The formulations contain CS, SA, or Ag, so their effectiveness in disease reduction may be due to the synergistic effect of CS and SA or Ag (
Figure 9). This is the direct effect of the NP formulations. NPs can also be good carriers to transport CS, SA, or Ag into plant cells, leading to an indirect effect of increasing plants’ resistance against plant diseases.
The plant’s innate immune system has three stages: perception, signal transduction, and defense response. This process could be induced by an elicitor [
10].
The CS can act as an elicitor that activates a plant’s innate immunity, including stimulating H
2O
2 production, nitric oxide, generating PR protein, oxidative burst, enzymes, callose, and secondary metabolite (phytoalexin, suberin, lignin, and phenolic compound) [
21,
59,
60]. In the [
61] study, CS treatment (2.5 mg/mL) increased plant height (39%), stem girth (44%), and reduced powdery mildew (
Erysiphe cichoracearum) disease (66.6%) on cucumbers. Furthermore, this effect was associated with an increase in the production of benzyl aminopurine, indole acetic acid, 1-napthol acetic acid and lignin, callose and H
2O
2, PPO, PAL, POD, and glucanase in the plant.
SA is a plant hormone that plays a role in plant germination, growth, and immunity. In the cell, endogenous SA is produced by the PAL and isochorismate pathways. When the SA concentration is increased, the cellular reduction potential is changed, and the NPR1 structure changes to a monomer that can enter the nucleus. Here, NPR1 binds to specific TGA transcription factors and then expresses defenses against pathogen attacks [
62,
63]. SA treatment can increase a plant’s defense system in chickpeas, including enzymes (POD and PPO), total phenol, H
2O
2, and protein content [
64]. Previously, [
65] reported that exogenous SA treatment (1 mM) was able to reduce bacterial leaf blight in rice by 38% with an increase in superoxide anion production and hypersensitive response, as well as lignin and pectin content in the cell wall. In addition, the formatted SA (Zacha11 at 500 mg/L) has increased stem height, root length, and the number of roots, and reduced root rot disease by 53.33% on cassava [
66].
Silver nitrate also has effects on plants, depending on its concentration. MS medium containing silver nitrate (1 mg/L) could increase rooted shoots (63.6%), plant height (78.6%), number of roots (181.3%), and root length (508%), and reduce bacterial contamination in
Gentiana lutea tissue culture [
67]. Furthermore, MS medium containing silver nitrate (1 mg/L) could improve the quality of shoots and decrease the time required for rooting to earlier than two months in two cultivars of
Anthurium andraeanum under tissue culture [
68]. In addition, [
69] compared the effectiveness of silver nitrate and Ag-NP (green synthesis) on the growth of rice under biotic stress conditions. The results showed that Ag-NP was more effective than silver nitrate at the same 75 mg/L concentration in increasing root length (1.2 and 12.8%), shoot length (21 and 20%), root number (8.1 and 6.8%), fresh weight (6.4 and 5%), dry weight (4.6 and 3.5%), leaf area (58.5 and 57.2%), leaf number (4.3 and 3.7%), leaf fresh weight (1.7 and 1.4%), and leaf dry weight (0.9 and 0.8%) under
Aspergillus infection. Furthermore, the aflatoxins of Ag-NP were 3.5 ± 0.1 µg/kg compared to silver nitrate, which was 3.9 ± 0.3 µg/kg. In [
70]’s study, Ag-NP (60 ppm) also increased common bean and maize growth, including shoot length (47.0 and 27.9%), root length (56.1 and 46.1%), fresh weight (85.9 and 109.2%), dry weight (74.4 and 122.0%), leaf area (56.5 and 70.0%), chlorophyll a (49.0 and 46.0%), chlorophyll b (33.0 and 26.0%), and carbohydrate content (57.0 and 62.0%). However, at higher concentrations (100 ppm), growth was reduced. In addition, Ag-NP (50 ppm) treatment in lilies resulted in increased plant height (7.6%), number of leaves (27.2%), greenness index (17.6%), leaf fresh weight (35.1%), bulb fresh weight (73.4%), and number of scales (24.3%), but at higher concentrations the effect is not equivalent [
71].
Figure 9.
The effect of CS, SA, and Ag on plants’ defenses. Modified from [
21,
59,
60,
61,
62,
63,
64,
65,
66,
69].
Figure 9.
The effect of CS, SA, and Ag on plants’ defenses. Modified from [
21,
59,
60,
61,
62,
63,
64,
65,
66,
69].
Why are NP formulations highly effective in reducing plant diseases and enhancing plant growth? In general, the preeminent characteristics of NPs are their small size, large contact surface area, and high reactivity, leading to their applications in controlling disease and enhancing plant growth [
72]. NPs can be absorbed by plants through foliar, brand, trunk, and root [
73]. CS-NPs with a nano size and a positive charge are able to easily penetrate cells or stick to plant surfaces [
21]. CS-NP can enter the plant via leaves (the stomata and cuticular pathway) and roots (the diffusion and cuticular pathway). The stomata of cassava leaves are from 18.2–24.9 µm × 12.1–16.1 µm [
74]. The hydrodynamic diameters of N3 and N6 were 89.86 ± 9.04 and 249.67 ± 23.97 nm, while the PDIs were 0.36 ± 0.02 and 0.53 ± 0.03 and the zeta potentials were 22.27 ± 1.01 and 13.53 ± 0.74 mV, respectively. Therefore, these NPs can pass through the stomata or be easily absorbed by the cassava plant. CS-NP can adjust osmotic pressure in the cell, resulting in increased uptake and availability of water and nutrients [
75]. In addition, when sticking to plants, the CS-NPs loaded with active ingredients including Hexaconazole, Zn, Cu, SA, Harpin protein, NPK, and silicon can slowly release their active ingredients so that plants can absorb them slowly, as reported in the studies [
24,
39,
46,
56,
76,
77,
78]. CS is commonly used as a carrier due to its solubility in aqueous media and its ability to mix with organic, inorganic, or copolymer compounds to increase solubility [
79]. The main component of CS is nitrogen, so the carrier (CS) can act as a source of nitrogen for plants to absorb, or enhance cell division, cell elongation, enzymatic activation, and synthesis of protein, which leads to increased yields [
21,
80].