The Multifunctional Role of Chitosan in Horticultural Crops; A Review

Chitosan is a naturally occurring compound and is commercially produced from seafood shells. It has been utilized in the induction of the defense system in both pre and post-harvest fruits and vegetables against fungi, bacteria, viruses, and other abiotic stresses. In addition to that, chitosan effectively improves the physiological properties of plants and also enhances the shelf life of post-harvest produces. Moreover, chitosan treatment regulates several genes in plants, particularly the activation of plant defense signaling pathways. That includes the elicitation of phytoalexins and pathogenesis-related (PR) protein. Besides that, chitosan has been employed in soil as a plant nutrient and has shown great efficacy in combination with other industrial fertilizers without affecting the soil’s beneficial microbes. Furthermore, it is helpful in reducing the fertilizer losses due to its coating ability, which is important in keeping the environmental pollution under check. Based on exhibiting such excellent properties, there is a striking interest in using chitosan biopolymers in agriculture systems. Therefore, our current review has been centered upon the multiple roles of chitosan in horticultural crops that could be useful in future crop improvement programs.


Introduction
Recently, chitosan has been one of the most preferred biopolymers due to its biocompatibility, antioxidant, anticancer, biodegradability, antimicrobial, and non-toxic properties as well as being an economical material, produced from waste resources such as seafood shells [1][2][3]. Structurally, chitosan is a linear polymer, composed of two sub-units as D-glucosamine and N-acetyl-D-glucosamine, linked with each other through 1,4-glycosidic bonds [4,5]. Since the last decade, chitosan research is increasing due to its significant diverse uses in several fields of life i.e., plant sciences [6][7][8][9] and medical sciences [10]. About 8700 chitosan-related records are available on Scopus. On a commercial scale, chitin has been extracted by following a classic, well-known acid-base method. The samples are

Antimicrobial Activity of Chitosan
The increase in the number of research papers related to chitosan's antimicrobial activity prove it as a versatile biostimulant in the horticulture sector. The elicitation of the defense system in both pre-and post-harvest fruits and vegetables is highly promising. Therefore, regarding the antimicrobial mechanism of chitosan, several researchers have presented their practical point of view. For example, Goy et al. suggested three antibacterial mechanisms of chitosan; firstly, ionic surface interaction resulting in cell wall leakage; secondly, permeation of chitosan into microorganism nuclei inhibits their protein and mRNA synthesis, and thirdly, formation of an external film over the plant surface, limiting the nutrient availability for microorganisms [55]. Liang et al. [56] stated that chitosan is responsible for the destruction of the bacterial cell membrane which causes death due to the leakage of intracellular substances [56]. It was also found to be involved in altering the growth of fungi and reduced toxin production [57]. However, in recent times, it has been reported that chitosan is responsible for the hydrolysis of peptidoglycans (cell wall component), increasing electrolyte leakage and potentially causing the death of the pathogen ( Figure 2) [58].

Chitosan Effect on Fungi, Bacteria, and Nematodes
Since 1979, when chitosan was reported as a bio fungicide by Allan and Hadwiger [15], it has attracted an ample amount of attention in terms of research in the field of plant protection. In addition, it is also well documented in controlling plant disease caused by bacteria. However, in nematodes, not a great amount of research material is available and therefore further study is required to assess the role of chitosan as a potential nematocide. The effectiveness of chitosan against different pests and pathogens has covered a large number of research articles across different fruits and vegetable. Therefore, from the big collection of chitosan research, we enlisted recent studies (Table 2) reporting the fungicidal, bactericidal, and nematocidal capacity of chitosan in various fruits and vegetables. This body of research suggests that chitosan can be use a bio-stimulant of the plant immune system to tackle diverse unfriendly environments.

Effect of Chitosan on Viral Diseases
The induction of plant resistance against viruses has been reported widely [75]. Generally, viruses that affect plants systematically are more threatening. Therefore, utilization of chitosan as a virucide was reported as the most feasible approach to limit viral infection [76]. Chirkov et al. [77] reported that application of chitosan on potato plants infected with potato virus X (PVX) showed resistance to PVX virus. Moreover, tomato plants treated with chitosan not only showed resistance to tomato mosaic virus but also their vegetative growth was improved [78]. Similarly, chitosan in formulation with plant growth promoting rhizobacteria (PGRP) conferred resistance to leaf curl virus in tomato plant [79]. Also, chitosan was found effective against squash mosaic virus (SMV) [80,81]. Furthermore, Chirkov et al. [18] hypothesized that there might be some peculiar properties of the host plant which initiate the antiviral reaction(s) after chitosan treatment. In line with that, chitosan oligosaccharide induces resistance against tobacco mosaic virus (TMV) by activation of the salicylic acid signaling pathway [82].

Chitosan as an Insecticide
Chitosan shows strong resistance to microbial diseases and insecticidal activity against various plant pests. However, chitosan derivatives have been found to be potentially more harmful than the pest. Due to these reasons, more chitosan derivatives have been developed in the recent past [83]. In line with that, chitin derivative (N-2-chloro-6-fluorobenzyl-chitosan) was found to be lethal against the oleander aphid (Aphis nerii) and larvae of leaf-worm (Spodoptera littoralis) of cotton crops [83].
In addition, nano-chitosan (CSg PAA) was also identified as a potential insecticide against the insects of soybean i.e., Aphis gossypii and Callosobruchus maculatus, as it significantly reduces the number of eggs deposited by the female [84]. More recently, a new chitosan derivative named Avermectin-grafted-N,O-carboxymethyl chitosan (NOCC) was obtained and has shown excellent insecticidal activity against armyworms, carmine spider mites, black bean aphids, and brown plant hoppers [85]. Despite having a strong killing impact against the above-mentioned insects, it might have the same impact on its family members as well. According to the reported studies, the above mentioned insects are involved in causing irreversible damage to tomato [86,87], cucumber [88], eggplant, potato, and chili [89]. Moreover, some insects exhibit chitin in their exoskeleton which can result in resistance to chitosan-based insecticides. To overcome such problems, chitosan has been fed to reared carnivorous insects in order to use them as biological controllers of chitinous pests [90]. Therefore, it would valuable to utilize chitosan as a bio-insecticide for horticultural crops.

Effect on Drought Stress
Drought stress is one of the most important multidimensional environmental stressors that damage plants' physiology, biochemical properties, and molecular traits [91]. For example, in apples, young seedlings were foliar sprayed with chitosan, which enhanced antioxidant activity, reduced electrolyte leakage, and restored moisture content under continuous drought stress for 35 days [92]. It was also reported to induce resistance against drought stress in potato, moth orchid, rice, white clover, and grapevine by means of induced antioxidant activities, increased endogenous H 2 O 2 content, enhanced endogenous chitosan activities, and root system development [93][94][95][96][97]. Moreover, chitosan induced ABA activity, which plays a key role in the regulation of stomatal aperture and reduced the rate of transpiration when the plant is going through stress phase [98,99]. Therefore, we suggest that chitosan might be a potential antitranspirant that helps horticulture crops cope with drought stress.

Effect on Heat Stress
Heat stress is often a complicated issue in agricultural species as it usually occurs simultaneously with drought stress. This makes it hard for the plant researcher to distinguish between the two stresses [100]. It has been reported that in dry bean production under heat stress, chitosan treatment could be the best approach to escape heat stress in late-sown plants [101]. To the best of our knowledge, there is not a large amount of research available on chitosan involvement in response to heat stress. However, the participation of ABA in coping with heat stress [102] via its involvement in inducing HS-related genes is reported in many published notes [103,104]. Overexpression of ABF3 (abscisic acid responsive element-binding factor 3) confers tolerance to heat stress [104]. Therefore, the use of chitosan might be effective for overcoming high-temperature stress by triggering ABA activity, which further induces the expression of defense-related ABA-responsive genes in horticultural species.

Chitosan Effects on Plant Growth, Yield Attributes, and Physiological Activities
The chitosan effects on fruit physiology and agronomic traits have been highly studied using different concentrations of chitosan over a variety of fruits. In line with that, foliar spraying of 5 mL L −1 chitosan over mango trees improved the number of fruits tree −1 , weight and size of fruit, and vegetative growth [105]. In grapes, chitosan was sprayed at 500 L per hectare during the pre-bunch closure and veraison stage and effectively increased the POX and PAL activities, polyphenol content, and SOD activities [106][107][108]. Moreover, an increase in the weight of fresh fruit of the kiwi plant was observed after spraying with chitosan in field conditions [109]. Furtherly, Gayed et al. reported that chitosan in combination with calcium chloride reduced the early swelling of peach trees, maintained freshness and firmness of fruits, and decreased the weight loss percentage [110]. In nectarine, chitosan improved the soluble solid content and also helped maintained the post-harvest firmness of the fruit [111].
Like in fruits, chitosan also positively affects the agronomic traits of vegetables. Tomato plants were subjected to chitosan treatment, resulting in high phenolic compound and PPO activities, production of phytoalexins, and improvement in fruit weight and overall yield [71,112,113]. Tsugita et al. suggested that application of chitosan in daikon radish triggered the growth of roots and shoots [114]. Similarly, it was reported in sweet basil, grapevine, Gerbera, Dendrobium orchids, and in cabbage, that chitosan treated plants showed better growth than that of controls [115][116][117][118][119]. Moreover, the cucumber plant is highly susceptible to low-temperature stress. However, when treated with chitosan, the plants had reduced reactive oxygen species, improved photosynthetic capacity, and the membrane system was strengthened to alleviate cold stress [120]. In chili, seeds treated with chitosan had significantly improved germination rates, germination index, mean germination time, and germination after accelerated aging, as well as high seed quality and enhanced storage life [121]. Furthermore, we also enlisted some crops on which the impact of chitosan was highly effective in terms of enhancing agronomic traits (Table 3). In the context of the above discussion, chitosan use is recommended to enhance the photosynthetic activity, vegetative growth, antioxidant activities, fruit quality attributes, and overall growth and yield of the crop. Table 3. Reported studies on chitosan effects on the agronomic traits of horticultural crops.

Crop Functions Reference
Artichoke Improved seed germination and plant growth [122] Chili Leaf area, canopy diameter and plant height [123] Cucumber Triggered vegetative growth and quality of cucumber fruits [124] Coffee Plant height and leaf area [125] Chinese cabbage Uniform seed germination and enhanced seedling growth [126] Dendrobium formosum orchid Enhanced seed germination [127] Eggplant Improved antioxidant activity and total phenolic content [64] Grapevine Increased number of internode and improved rooting [97] Okra Plant height, leaf number and fruit yield [128] Potato Increased the fresh weight of tuber and overall yield [129] Peach Induced antioxidant activity and defense-related enzymes [70] Radish Enhanced the nutrient uptake efficiency and mimic cadmium stress [130] Strawberry Produced fruits with an increased shelf life [131] Tomato Improved fruit and productivity [71,72] Tea Enhanced the phenolic content up to 9% [132] Watermelon Increased in weight of fresh and dry seedlings, Stimulated the growth of the primary stems, the root system, and an increase in stomatal width. [19,133]

Chitosan Effects on Post-Harvest Fruits and Vegetables
The diverse beneficial effects of chitosan in fruit shows great value. Furthermore, a coating of post-harvest mango with edible chitosan showed a reduction in the percentage of rotten tissue, increased ascorbic acid content, increased shelf-life, and maintained freshness [134][135][136]. Likewise, in pomegranate, edible chitosan coating enhanced the shelf life and improved freshness for up to 16 days with good chemical and sensory characteristics during post-harvest cold storage, and kept the surface microbial growth under check [137,138]. Petriccione et al. [139] reported upregulated anthocyanins activity, delay in color changes, and preservation of water content in chitosan-coated sweet cherries [139]. Similarly, in post-harvested strawberries, chitosan significantly prolonged the anthocyanins, polyphenol, and antioxidant activities, while inhibiting flesh browning under cold storage conditions [140]. Furthermore, thin-peel rose apples showed a reduction in disease severity, sustained fruit firmness, and a reduction in the weight loss percentage during post-harvest storage when treated with 2% chitosan [141]. The significance of chitosan under cold storage has also been reported for post-harvest apricots, where it has shown improvement in antioxidant enzyme activities and elevating the total phenolic content [142]. Bananas are a highly perishable fruit and deteriorate faster than other fruits. Edible chitosan can be used to delay the ripening and to enhance the shelf life of bananas. Further, it also improves the antioxidant activity and vitamin C content [143]. Green mold disease has a substantial effect during post-harvest storage and transportation of citrus. Coating with edible chitosan maintained fruit firmness, surface color, juice content, and other quality attributes [144]. Ripening and fruit senescence in peach is a major issue that affects their economic value. However, treatment with chitosan resulted in a significant increase in antioxidant enzyme activity, senescence arrest, delayed fruit ripeness, and maintenance of shape and color [70,110]. In kiwi fruit, the high molecular weight of chitosan was found to be effective in increasing the shelf life, fruit firmness, and other quality parameters [145]. It is clear from the above references that chitosan is involved in improving the physical structure and shelf life and maintaining the quality of post-harvest stored fruits.
Refrigeration of tomatoes during summer causes a considerable amount of loss. However, use of chitosan significantly enhanced the shelf life, visual appearance, and other quality attributes of the refrigerated tomatoes [146]. Likewise in carrot, edible chitosan was applied to carrot slices, which reduced the ripening process, reduced sugar, and increased the total phenolic content [147]. As in broccoli, chitosan treatment along with mild heat shock improved the shelf life and also maintained the sensory attributes [148]. Further, chitosan was used to induce chill tolerance, enhanced shelf life, preserve fruit quality, and improve antioxidant activities in post-harvest cucumber and cantaloupe melon [149,150]. Nowadays, people are more concerned about food safety and quality, and due to this reason chitosan has potential to be utilized in different methods in order to enhance the innate immunity of post-harvest products.

Chitosan Effects on Gene Expression
Gene expression is a biological process which changes according to changing environment, and allows the cells to respond to environmental stimuli. Different exogenous gene regulators have been reported in recent studies. Here, we present some of the recent research done on chitosan involvement in the regulation of gene expression. Hadwiger [151] reported that chitosan activates genes and inhibits RNA synthesis in fungi. The chitosan activates several genes and increases production of proteins and phenolic compounds through the phenylpropanoid pathway, which in turn increases tolerance against pathogens [151]. In Dendrobium, an ornamental flowering plant, chitosan treatmentdown-regulated the expression of the ycf2 gene in young leaves, conferring enlarged chloroplasts [152]. This suggests the important role of chitosan in increasing the flowering ability of ornamental plants through gene pathway regulation. In addition, Hadwiger stated that PR genes are induced by chitosan [153]. Chitinase and a β-1,3 glucanase, two PR proteins, contribute to plant protection against fungal infection by degrading the fungal cell wall [154]. In tomato plants, chitosan extracts have been used to alleviate the effect of two tomato pathogens, namely Alternaria solani and Xanthomonas vesicatoria. The result suggested that chitosan significantly upregulated the expression of the PINII marker, which is responsible for activating the defense signaling pathways [155]. Similarly, in pepper, raspberry, and strawberry plants, chitosan was found to be effective in inducing plant defense mechanisms due to the higher expression level of chitinase (EC 3.2.1.14) and β-1,3 glucanase (EC 3.2.1.39) genes [156][157][158][159]. Moreover, in grapes, the plants pre-treated with chitosan from three different commercially-produced formulations showed higher endochitinase activity and two of the chitosan formulations triggered exo-chitinase activity [160]. However, there is still a lack of research examining the role of chitosan treatment in PR genes under different stresses. Furthermore, the MLO clade V genes in vegetables are a gateway for the pathogen of powdery mildew disease, which has been confirmed in the recent report by Berg et al., to restore the susceptibility to powdery mildew in cucumber [161]. In contrast to that, chitosan is effective against widespread fungal diseases. Therefore, research is required to evaluate the impact of chitosan on MLO clade V gene expression and how much it affects the disease severity index. Below, we tabulated (Table 4) some of the recent studies demonstrating the role of Chitosan in regulating gene expression. ↑ showing up-regulation and ↓ down-regulation of the respective genes presented in the table.

Chitosan as a Bio-Fertilizer and Fertilizer Protectant
Ecological toxicity is at a critical point due to the high-level production and usage of inorganic fertilizers. Therefore, biodegradable biofertilizers, like chitosan, are attracting the research community to avoid the hazards of using inorganic fertilizers. Chitosan gets degraded enzymatically without affecting the soil-borne beneficial rhizosphere biota at low concentrations, and also induces the symbiotic exchange between plant and microbes [73]. In addition, chitosan is a polysaccharide-based biopolymer, which stimulates the activity of plant symbiotic microbes, resulting in the alteration of rhizosphere microbial equilibrium, thus disadvantaging the plant pathogens [173,174]. In recent research, chitosan was used as a biofertilizer to improve crop yield with less environmental contamination. In line with that statement, chitosan in combination with lysozyme has shown beneficial effects, where it significantly reduced the rate of lesions in tomato stems to 14% [175]. In potato, late blight is an important disease that causes economic damage to potato yields. However, after soil inoculation with chitosan as a biofertilizer, a significant reduction in tuber infestation by late blight was detected, a significant increase in plant nutrient uptake was also recorded. The study was done to produce organic potato seeds for an organic grower [176]. Similarly, 1% chitosan mixed with fertilizer improved the nitrogen and phosphorous content in the roots and shoots of Eustoma grandiflorum (Raf) compared with non-chitosan mixed soil grown plants [177]. In Chinese cabbage, plants treated with a chitin-based product showed faster growth than plants treated with a standard mineral fertilizer [178]. In another study, chitosan in combination with N, P, K fertilizer proved to be significant in altering the effect of Botrytis cinerea-caused grey mold disease in Begonia × hiemalis Fotsch, and also increased the antioxidant activities and other commercial traits [179]. Besides that, soil supplemented with chitin enhanced plant growth by improving nutrient uptake [180]. Additionally, inoculation of soil with 0.1% chitin from crustaceans induces defense against root-infecting fungi in chili plants and also improved the nutrient uptake [181]. Likewise, NPKB bio-fertilizer mixed with the chitosan-containing fungi Cunninghamella elegans produced NPKP influenced melon plant growth positively and also improved NPK uptake [182]. Furthermore, tomato plants irrigated with chitosan exhibited decreased multiplication of root-knot nematodes and Pochonia chlamydosporia (a parasitic fungi), resulting in improved root length, weight, and increase shoot growth (Nuria Escudero et al., 2017) [73]. Furthermore, Ornithogalum saundersiae bulbs coated with chitosan produced earlier flowers and had broader leaves (Salachna et al., 2015) [183]. In another report by Salachna et al. 2017, chitooligosaccharide in combination with gellan gum induced vigorous growth, high antioxidant activity, and increased polyphenol content of Ornithogalum saundersiae [184]. This highly suggests its role as a potential bio-fertilizer. However, the use of chitosan as a bio-fertilizer in horticulture crops is quite underestimated and not a great amount of research material is available on it. Therefore, it might be useful to use chitosan bio-fertilizer over different horticulture crops to evaluate its effect on growth parameters against different stresses. Furthermore, chitosan bio-fertilizers are available commercially from different manufacturers. CANADA OCEANIC sells it in the name of Softguard, as a potential biofertilizer that can be used for a variety of purposes (www.canadaoceanic.com/Fertilizers/SoftGuard.shtml).
The other important aspect of using chitosan is to control the release of fertilizer. It is estimated that about 40-70% of nitrogen, 80-90% of phosphorus, and 50-70% of potassium from applied fertilizers is lost to the environment and cannot be absorbed by plants [185]. Due to this, fertilizers are overused with horticultural crops, and vegetables in particular are highly affected by fertilizer toxicity. Nitrate is also considered as an important parameter of water pollution due to its involvement in surface and groundwater contamination [186]. Similarly, application of excessive nitrogen leads to water contamination, as surplus nitrogen is usually carried away by water or leaches down to pollute the water table [187]. Therefore, to minimize the negative effects of agriculture on the surrounding environment, it is important to reduce the use of fertilizer [188]. Chitosan has been used recently to control the release of inorganic fertilizers in order to limit the harmful effects of excessive fertilization [189][190][191]. Wu et al. [189] suggested that the use of chitosan coating on fertilizer improves the efficiency of plant fertilizer uptake and also reduce the production cost. Therefore, in order to minimize the overuse of chemical fertilizers in vegetables and fruit by reducing the leaching and volatilization process, local farmers must consider chitosan as a coating material, according to the suggested model of Hähndel [192].

Conclusions
The plethora of available and ongoing research on chitosan utilization continues to present its efficacy. Chitosan has shown great importance in improving the physiological mechanisms and post-harvest shelf life of fruits and vegetable against biotic and abiotic stress. In addition, chitosan derivatives possess good insecticidal activity, however, to the best of our knowledge, there is no report on chitosan use in a field study against insects in major horticultural crops. Besides that, chitosan plays a role in regulating gene expression and inducing molecular defense systems in plants and post-harvest produce. Moreover, chitosan bio-fertilizer and fertilizer coated in chitosan triggers the plant growth more, compared to synthetic fertilizers. Furthermore, chitosan and its derivatives provide good antifungal and nematocidal activity without disturbing the beneficial microbes in the soil and can be considered as a green approach for soil sterilization [73,193]. Therefore, more research is required to utilize chitosan against heat stress, for the application of chitosan to the root zone, to prevent nematode-borne diseases, and to reduce the overuse of synthetic fertilizers in horticulture crops.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (31572110 and 31501740). Further we apologize to the authors whose works are not cited because of space constraints. We thank Nooral Amin and Naveed Ahmad (Jilin Agricultural University), Izhar Muhammad and Muhammad Ali (Northwest Agriculture and Forestry University) for helping in revising the manuscript, Shah Faisal (Northwest Agriculture and Forestry University) and Aimal Nawaz Khattak (Guizhou University) for revising the English.