Chitosan as alternative treatment to control postharvest losses of tropical and subtropical fruits

P. Gutierrez-Martinez1,* A. Ramos-Guerrero1, H. Cabanillas-Beltran1, J. Romero-Islas1 and A. Cruz-Hernandez2 1 Instituto Tecnológico de Tepic. Laboratorio Integral de Investigación en Alimentos. Av. Tecnológico 2595, Lagos de

Country, Tepic,Nayarit. C.P. 63175. México. 2 Escuela de Agronomía, Universidad De La Salle Bajío. Av Universidad 602. Col. Lomas del Campestre, León Gto.

México. C.P. 37150. * Corresponding author: email: pgutierrez@ittepic.edu.mx

Chitosan, a chitin derivative is a natural product obtained mainly from crustaceous. This natural, biodegradable and nontoxic compound has a broad potential in food, biotechnology, and agriculture industries. This review summarizes recently published literature describing experiments in which Chitosan have been tested on mycelia growth, germination of spores, enzyme activity and gene expression for different postharvest fungi and fruit tissues under in vitro and in vivo conditions. It has been found that chitosan controls postharvest infections by Colletotrichum gloeosporioides, Alternaria alternata, Rhizopus stolonifer and Fusarium oxisporum. Furthermore, the semipermeable characteristic of chitosan originates favourable physic-chemical changes on fruits metabolism, extending their storage life and the effects also include changes in enzymatic activity and genetic expression. Efforts on the evaluation of chitosan alone or in combination with some other compounds, must be done as alternative treatments on a wide scale to control postharvest losses of tropical and subtropical fruits.

Keywords: Alternaria; Antifungal activity; Colletotrichum; gene expression

1. Introduction

1.1 Post-harvest losses in tropical fruits

Globally, more than 63 million tons of tropical fruits are produced, producing almost 100% in developing countries [1]; [2]. Mexico is one of the main producers and exporters of tropical and subtropical fruits. However, these fruits are affected in the post-harvest stage by various pathogens, including Colletotrichum gloeosporioides, Alternaria alternata, Fusarium, Rhizopus stolonifer among others causing significant damage to the fruits, affecting the Post-harvest quality and preventing their commercialization, generating economic losses. Losses in developing countries range from 40 to 50 %[2, 3, 4, 5, 6]. Fungicides are the only method of control to date, with a high cost for the producer and the environment [6, 7, 8]. Chitosan is a natural, biodegradable and non-toxic biopolymer, various antifungal properties and as a resistance inducer, activating enzymes and genes that encode proteins related to the fruit defense system before the pathogen attack [9, 10]. It is necessary to explore other alternatives such as the use of compounds that occur naturally in plants and animals with fungicidal characteristics and with properties that induce defense mechanisms such as chitosan, a commercially viable alternative [11].

1.2 Post-harvest disease control systems for fruits

Due to the great losses of horticultural products in the post-harvest stage caused by pathogens, it is necessary to apply control methods to reduce these losses. Disease control has become difficult due to pathogen resistance to increasingly stringent fungicides and regulations. The great interest in human health and the environment has led to the study of alternative methods to the usual products that guarantee a similar or superior effectiveness to the synthetic fungicides, without the problems that they generate. The methods are classified according to their nature in: physical, including heat, such as Hot water treatment, steam, dry heat, forced air; Irradiation, low temperatures, modified and controlled hypobaric atmospheres, electrolyzed water and biological control, GRAS compounds such as volatile compounds, plant extracts, essential oils, Ethanol, Sodium bicarbonate, peptides and proteins. Finally Resistance Inducers: Focus on improving the individual potential of the host to respond to the attack of pathogens by activating defense mechanisms at the biochemical and molecular level. Products such as jasmonates, salicylic acid, and Chitosan. These control systems, individually are not 100% effective, when combined their effectiveness in controlling pathogens increases significantly [13, 14, 15, 16] . Chitosan is a natural, biodegradable, non-toxic, bioactive polymer with fungicidal effects on the most important post-harvest pathogens affecting the quality of tropical and subtropical fruits as well as an increase in the losses of these products. In addition, chitosan is an inducer of defense mechanisms in plant tissues, including fruits [17, 18, 19]. In the post-harvest stage is one of the most promising products for the control of various fungi [20, 21, 22, 23].

2. Effects on Post-harvest Decay of fruits

Results obtained by López Mora et al. [21] in 2013 and Berumen et al. [11] in 2015 in mango Tommy Atkins, Coronado-Partida et al. [24] in 2017 in Jackfruit and Ramos-Guerrero et al. [25] in 2017 in Soursop fruits, and Xoca et al. [26] 2017 in Hass avocado, indicated that when applying chitosan at 1%, infection by C. gloeosporioides, Fusarium oxisporum, Rhizopus stolonifer was controlled (Fig.1). These results mentioned, chitosan individually or in some cases combined with another alternative control system, organic salts, hydrothermal treatment, salicylic acid, jasmonic acid, ethanol, etc. By combining chitosan with another system it is possible to use lower concentrations, which results in a more effective and economical treatment [27].

Fig. 1 Effect of chitosan and chitosan combine with Potasium sorbate on Colletotrichum gloeosporioides and Rhizopus stolonifer postharvest control in mango. Avocado and Jackfruit 72 h after applying treatments, stored at 25°C.

2.1 Mechanisms of action.

Chitosan has an excellent fungicidal potential against the major fungi that cause damage to tropical fruits such as Colletotrichum gloeosporioides [28, 29, 30]. Some mechanisms of action proposed to explain fungicidal ability of chitosan, propose that the electrostatic interaction between the NH3 + groups of chitosan and the phosphoryl groups of the phospholipids present in the cell membrane of the fungi causes damage in this, causing an increase in permeability [31, 32, 33, 34]. In addition, it is possible that short chains of chitosan can pass through the wall and membrane and interact with the DNA and the RNA interfering with their function, losing functionality of the fungal structure[35, 36]. Its chelating effect could decrease the availability of some metals needed in enzymatic processes, inhibiting the process of the pathogenesis of fungi [37]. Alterations in the internal and external morphology of the conidia and mycelium caused by chitosan would provoke a biochemical-physiological stress [25].

2.2 Antimicrobial Properties

Before application of chitosan in fruits with the aim of reducing or inhibiting the growth of post-harvest fungi, it is necessary to determine the optimal concentrations that will decrease or stop mycelial growth as well as the concentration of chitosan capable of inhibiting the process of spore germination. López-Mora et al. [21] 2013, applied chitosan to the A. alternata fungus, isolated from mango fruits and observed a gradual control of the growth and development of the fungus as the concentration of chitosan increased (Figures 2 and 3)(Table 1). Similar results were obtained in the Colletotrichum-Mango interaction system [24], Colletotrichum-Fusarium-Banana [29], Rhizopus-Jackfruit [38] Colletotrichum-Rhizopus-Soursop [25], where chitosan control growth, inhibited germination of spores and altered morphology of conidia

Fig. 2 Effect of chitosan on mycelial growth of A. alternata.

Fig. 3 Effect of Chitosan on C. gloeosporioides conidium. Control (A). Chitosan 1.0%B). Scanning Electron Microscopy.

Table 1 In vitro development of two isolates of Colletotrichum chitosan-treated at different concentrations and incubated at 20 ± 2ºC.

Chitosan Concentration (%)

Soursop Mango Rate of growth

(mm día-1) Sporulation

Germination (%)+

Rate of growth (mm día-1)

Sporulation Germination

(%)+ 0.5 3.4c 2.2 x 106c 20b 5.4b 3.5 x 105c 13b

1.0 1.3b 1.3 x 106b 0a 5.0b 1.5 x 105b 0a

1.5 0.9a 1.2 x 106a 0a 4.5a 1.0 x 105a 0a

Control 5.8d 3.3 x 106d 100c 5.8c 7.5 x 105d 100c

Means followed by the same letter are not significantly different (≤ 0.05) as determined by Tukey’s multiple test. Rate of growth after 12 days of incubation. Sporulation and germination after 8 h incubation.

2.3 Inducing properties

The use of chitosan as an inducer of resistance may be significant if one considers the systemic and persistent nature of defense proteins in plant tissues in response to the presence of chitosan, which may be important in delaying the resumption of an infection Latent tissue that typically begins to activate when tissue resistance declines [39,17, 20]. One of the current trends in the control of post-harvest diseases is to stimulate the fruit and vegetable product to reactivate its own defense mechanisms [4] [15]. Zhang et al. [18] in 2011, indicated that as an exogenous inducer the chitosan can activate resistance in the host by increasing the activities of various defense-related enzymes, such as chitinase and β-1,3- Glucanase in oranges, Berumen et al. [11] in 2013, reported that it was possible to induce the activity of PFO and POD enzymes, important in the plant defense system, being the concentration of 1% chitosan that obtained Better results being able to induce more the enzymatic activity of both enzymes in mango (Figure 4).

Fig. 4 Effect of chitosan at different concentrations on POD activity in mango fruits cv. ‘Tommy Atkins’: a) uninoculated and b) inoculated with Colletotrichum sp. Bars indicate mean standard error of 5 observations per treatments.

The information that exists in relation to the induction by the chitosan is mainly related to the analysis of the enzymatic activity of proteins related to the defense to pathogens As for the information generated in tropical fruits, considering the inductive capacity of the chitosan, is scarce (Mango, soursop, banana, jackfruit) [11, 25, 29, 38] and even more when referring to the use of molecular biology to evaluate the effect of chitosan at the genetic level, -PCR of the genes involved in the defense of the plant system (fruit or plant). Few reports exist on the evaluation of the effect of chitosan on the pathogen, at the spore or mycelial level [30], considering the expression of genes, in fungi as important from the point of view of post-harvest as is the genus Colletotrichum, agent Pathogen that infects and causes large losses in tropical fruits such as mango, banana, soursop, avocado, papaya, etc. Berumen in 2013 [11] reported that when applying 1% chitosan films to mango fruits, it was observed that there was an increase In early genetic expression of the C. gloeosporioides infection in mango fruits, suggesting that chitosan acts from the contact with the host, through a process of signal transduction, thus achieving the induction and increase of the Gene expression of both enzymes (Polyphenol oxidase (PPO) and Peroxidase (POD), generating the activation of the fruit defense mechanisms [11]. Hernández-Ibañez [29] evaluated the genetic expression of NPR1 (a gene related to defense systems) by applying 1.5% of chitosan in banana fruits inoculated with C. gloeosporioides, observing a high genetic expression in the early Pathogen-fruit-chitosan interaction. Ochoa-Jiménez [30] in 2012, analyzed the expression of the gene encoding polygalacturonase (PG) in spores of C. gloeosporioides, isolated from banana fruits, reporting a decrease in the expression of the polygalacturonase (PG) gene, , As a consequence of the application of chitosan to 1%, which has as consequence the alteration of spore germination. Xoca et al. [40] in 2017 indicate that the chitosan-avocado-Colletotrichum interaction transcriptomic analysis proposes an effect of chitosan at the level of induction of metabolic pathways to eliminate the pathogen Colletotrichum. In conclusion, Chitosan has shown great potential as a natural substance, biodegradable, biocompatible without toxicity or side effects with antifungal activities with a direct effect on the growth and development of the fungus at the level of conidium and mycelium. A very important effect of chitosan is the induction of defense mechanisms at the fruit level. The information generated by transcriptome studies in chitosan-pathogen interaction systems indicates the effect of chitosan on metabolic pathways important for the defense of the fruit against the attack of the pathogen and in the fungus, resulting in morphological and biochemical alterations that Inhibit the growth and germination of the pathogen. Surely with the new technologies omics will have more idea of the mechanism of action of chitosan in interaction with the pathogen and in the interaction of the fruit in the post-harvest stage. It is necessary to perform tests at the level of tropical fruit

packers and thus to have the complete picture from the in vitro, in situ and packing tests that will allow making formulations and to market products based on chitosan, as bio-fungicide.

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