Effect of chitosan on the in vitro control of Colletotrichum sp., and its influence on post-harvest quality in Hass avocado fruits

L. A. Xoca-Orozco1; S. Aguilera-Aguirre1; U. M. López-García1; P. Gutiérrez-Martínez1; A. Chacón-López1*

Correspondence: *. Corresponding Author: Chacón-López, A. Tecnológico Nacional de México-Instituto Tecnológico de Tepic. LIIA. Laboratorio de Biotecnología. Av. Tecnológico #2595 Lagos del Country. Tepic, Nayarit; México. E-mail: E-mail: , E-mail:


Abstract

Avocado fruit cv Hass, is from an economic point of view, one of the most important crops for Mexico, because Mexico is the main producer of avocado in the world. However, the production is often reduced due to large postharvest losses because the fruit is susceptible to attack by Colletotrichum sp., the causal agent of anthracnose. In recent years it has been explored new alternatives to combat postharvest fungi because of the risk that involve the use of harmful chemical fungicides on the environment and consumer health. One alternative is the application of chitosan films, which has an antifungal effect. In this study the in vitro antifungal activity of low and medium molecular weight chitosan on two strains of Colletotrichum sp., isolated from Hass avocado fruits whit anthracnose symptoms was evaluated. Additionally, the chitosan effect on the post-harvest quality of the avocado fruit was also evaluated. Chitosan solutions at 0.1, 0.5, 1.0, 1.5 and 2.0 % (w/v) were used to assess inhibition of mycelial growth, sporulation and germination of spores recording measurements every 24 h for 9 days. In vivo trials were performed by inoculating avocado fruits with a suspension of Colletotrichum sp (1x106 spores·mL-1). The fruits were immersed in low molecular weight chitosan solution (1.5 % w/v). The incidence of disease, physiological weight loss, firmness and percent of dry matter of the treated fruits were evaluated after 12 days of storage at 25 ºC. The in vitro results demonstrated that low and medium molecular weight chitosan solution at 1 % inhibited more than 90 % of mycelial growth and significantly reduced sporulation and germination of conidia of Colletotrichum sp. The in vivo results showed that the application of chitosan on avocado fruits reduced physiological weight loss, kept the firmness and decreased disease incidence. According to our results, the application of chitosan films on avocado fruits could be a plausible alternative to control of anthracnose and preserve the Hass avocado quality during its storage at room temperature.

Received: 2017 September 28; Accepted: 2018 January 29

revbio. 2020 May 30; 5: e355
doi: 10.15741/revbio.05.e355

Keywords: Keywords: Avocado, chitosan, antifungal, anthracnosis, in vitro, in vivo.

Introduction

Avocado is one of the main agricultural products in Mexico; its production represents an important source of income for local, national and international markets because Mexico ranks first in worldwide production of avocado (SENASICA, 2017) Avocado is particularly susceptible to damage caused by Colletotrichum sp., a pathogen responsible for approximately 20 % of postharvest losses (Rodríguez-López et al., 2009). Chemical fungicides reduce these losses but may have a negative effect on the environment and cause consumer health problems if residues remain in the fruit. Therefore, fungicides of biological origin are attractive alternatives (Ochoa-Ascencio, 2009; Tamayo, 2007; Lárez, 2008).

Chitosan is a biological fungicide that does not pollute the environment and does not presents risks to the health (Martínez-Camacho et al., 2010, Gutiérrez-Martínez et al., 2012). Chitosan, poly [ß (1-4) 2-amino-2-deoxy-D-glucopyranose], is a copolymer of D-glucosamine units of N-acetyl-D-glucosamine derived from the chitin deacetylation in alkaline medium (Monarul et al. 2011). The fungicidal activity of chitosan is partially explained by its cationic character. Positively charged free amino groups in acid media interact with the negative residues from the macromolecules of the exposed fungal wall, inducing changes in the permeability of the plasma membrane and thus alteration of its functions (Benhamou, 1992). Chitosan may also inhibit the synthesis of some essential enzymes for the fungus (El-Ghaouth et al. 1992) and cause cytological disorders, such as blistering or lack of cell cytoplasm, as reported for spores of A. alternata, Colletotrichum sp. and Fusarium sp., subjected to 1-2 % chitosan solutions (Sánchez-Domínguez et al., 2007; López-Mora et al., 2013; Bautista-Baños et al., 2012).

Chitosan coatings form a semipermeable film on the fruit surface that slows the respiration rate, reduces weight loss, maintains overall quality and increases the shelf life of the fruit (Romanazzi, et al., 2013). In other fruits, such as mango, chitosan has decreased the incidence of fungal diseases, increased firmness and abated the physiological weight loss (López-Mora et al., 2013). Correa‐Pacheco et al. (2017) applied chitosan nanoparticles in combination with thyme essential oil, finding a reduction in the incidence of C. gloeosporioides on avocado Hass, moreover, fruit firmness was better maintained in comparison with untreated fruit. However, publications about chitosan use in avocado fruits are scarce. For this reason, the objective of this research was to determine the in vitro antifungal effect of low and medium molecular weight chitosan on Colletotrichum sp, which is the causal agent of anthracnose and is considered one of the main postharvest fungi that affects Hass avocado fruit (Persea Americana Mill), and on the other hand, evaluate the fruit quality during storage at room temperature.

Materials and Methods

Isolation of phytopathogens

Hass avocado (Persea Americana Mill) fruits in physiological maturity stage were obtained from an orchard located in the Town of Tepic, Mexico. Fungus development was encouraged by placing fruits in chambers at a relative humidity of 90-95 % at 25 °C for 5 days. Once symptoms developed, injured tissue was disinfected by immersion in sodium hypochlorite solution (2 %) for 2 min, rinsed with sterile water for 2 min and placed on filter paper to eliminate moisture. Moisture-free tissue sections were plated on potato dextrose agar (PDA) (DIFCO, cat:DF0013) and were incubated at 25 °C for 24 to 72 h. The fungus genus was determined considering the taxonomic keys described by Barnett & Hunter (1998).

Pathogenesis test of isolates in avocado fruits

For the evaluation of the pathogenesis of the isolates and verify the Koch’s postulates, avocado fruits were inoculated with the isolated strains. Prior to inoculation, strains were pre-incubated for 8 days. A spore suspension was prepared according to Sellamuthu et al. (2013). Spores were counted using a haemocytometer and were adjusted to 1x106 spores·mL-1. Using an insulin needle, 40 μL of the suspension was inoculated through the skin of healthy fruits previously washed with water. Inoculated fruits were then stored at 25 °C until the onset of symptoms appeared. A comparison of symptoms between the non-inoculated (control) and the inoculated fruits was performed (Morales-García et al., 2009).

Preparation of chitosan solutions and treatments

Chitosan was prepared as follows: low molecular weight chitosan (LMW) (Sigma Aldrich cat: 448869; viscosity 35 cps in 1% chitosan solution; 96.1 % deacetylation) and medium molecular weight chitosan (MMW) (Sigma Aldrich cat: 448877; viscosity 590 cps in 1% chitosan solution; 80 % deacetylation) were used to prepare solutions at 0.1, 0.5, 1.0, 1.5 and 2.0 % (w/v) in 2 % acetic acid. Solutions were placed under constant stirring for 24 h, and then pH was adjusted to 5.6 with 1N NaOH prior to the addition of 0.1 mL of Tween 80.

In-vitro evaluations

Application of treatments

An 8 mm disc of mycelium was taken from the fungal periphery after 8 days of incubation at 26 °C. Discs were placed in the center of petri plates containing the PDA-Chitosan media at the corresponding concentrations (0.1, 0.5, 1.0, 1.5 and 2.0 %). A plate of PDA without chitosan was inoculated with the fungus and used as the control. The inoculated petri plates were incubated at 26 °C, for 9 days.

Evaluations of the antifungal effect

Evaluation of the antifungal effect of chitosan on the isolates strain was determined by mycelial growth in petri plates (MG) and inhibition of mycelial growth (IMG), sporulation and germination of spores, was evaluated according to the methodology proposed by Cordova-Albores et al. (2014).

In vivo evaluations

Inoculation of Colletotrichum sp., in avocado fruits

Avocado fruits in physiological maturity and free of pathogens or insect damage were collected from a local orchard. The fruits were washed with potable water and dried at room temperature. Using an insulin syringe, 40 μL of a spore suspension (1x106 spores·mL-1) was inoculated by penetrating 3 mm into the fruit peel. After 30 min, fruits were treated with both low and medium weight chitosan solutions (López-Mora et al., 2013).

Treatment of fruits with chitosan

Briefly, fruits were immersed for 1 min into the best treatment resulting from the in vitro test (LMW chitosan solution at 1.5 %, w/v). Fruits were then allowed to dry for 60 min and were stored at 25 °C for 12 days. Treatments were labeled as FCh (fruits treated with chitosan, 8 fruits), Fc (control fruit without chitosan, 8 fruits), FChP (8 fruits treated with chitosan and inoculated with the fungus) and FP (8 fruits inoculated with the fungus and without chitosan).

Incidence and severity of anthracnose

To evaluate the effectiveness of chitosan to inhibit symptoms and decrease anthracnose incidence, 32 fruits ripened to intermediate stage (firmness between 60 and 80 N) were inoculated with the pathogen (as described above). Half of the samples (16 fruits) were treated with LMW chitosan solution (1.5 %, w/v), and the other half was left untreated. All fruits were stored at room temperature for ripening. In order to evaluate the development of anthracnose symptoms, fruits were cut in the area of inoculation and disease development was measured. Those fruits that had size spots >1 cm2 were considered contaminated (Bill et al., 2014). The % of incidence of disease (ID) was measured using the following equation:

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<mml:mn>100</mml:mn>

For the assessment of severity of the infection, the peels from the mature fruits previously treated, were removed and pictures of the fruits without peel were taken, then, the images were processed using the ImageJ software (Schneider et al., 2012) to determine the percentage of the total damaged area. This parameter was expressed as percentage of affected area with respect to the total area of the fruit.

Evaluation of the quality parameters in avocado fruit

Some quality parameters were evaluated in fruit treated and not treated with chitosan. The parameters were the physiological weight loss (PWL), fruit firmness and percent of dry matter. The PWL was measured every 24 h during 12 days of storage. To determine the PWL, 10 fruits per treatment (FCh, Fc, FChP and FP) were weighed every 24 hours using a digital weight scale (Sartorius model BL 3100, USA), and the results were expressed as suggested by El-Ghaouth et al. (1991) and were calculated as follows:

[Formula ID: e2]
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Determination of fruit firmness was performed on 3 samples using a texture analyzer (Shimpo FGE-50, USA), equipped with a punch probe of 5 x 10 mm diameter. The penetration test was applied to 3 points along the fruit husk (ends and middle). The result was expressed as to the average of the obtained values expressed in Newtons (N) (López-Mora et al., 2013). The determination of dry matter was conducted per each treatment on 3 fruits previously peeled and cut into small pieces (<0.5 cm2) and placed in a microwave oven (General Electric JES832WK, 800 W) until a constant weight was achieved. The initial and final weights of each sample were used for the calculations (Wang et al., 2012). The evaluation of the firmness and dry matter was done on days 0, 3, 6, 7, 8, 9 and 10 of storage.

Statistical Analysis

For the in-vitro evaluation a 2x6 bifactorial design was used. The factors were: chitosan molecular weight (LMW and MMW) and concentration (0.1, 0.5, 1.0, 1.5 and 2.0 %). Each treatment was repeated three times. The in vivo evaluations were conducted using a univariate completely randomized block design; all in vivo assays were performed by duplicate. In both cases (in vitro and in vivo) the results were analyzed statistically by analysis of variance (ANOVA). The comparisons of means were made by Tukey test (p≤0.01) using the statistical package SAS System V9.

Results

Isolation and identification of pathogens and pathogenicity test

The isolated pathogenic strains obtained from the damaged tissue were identified as AG1, AG2 and AG3. Strains AG1 and AG3 exhibited similar characteristics: random mycelial growth and small, white, cottony mycelium (Figure 1a). Strain AG2 showed different characteristics than AG3 and AG1, it exhibited whiter cottony growth compared to the grey colony color of AG1 and AG3 and a white-yellowish coloration was observed on the base of the petri-dish (Figure 1b). According to the identification performed with the taxonomic keys, AG1 and AG3 belong to the Colletotrichum genus, whereas AG2 matches the genus Pestalotia (Figure 1c), which is not considered an important pathogen of avocado fruit and for this reason it was not included in the future evaluations. Fruits inoculated with strains AG1 and AG3 presented the symptoms of anthracnose disease described above. The macroscopic and microscopic characteristics of the strains corresponded to those observed in the initial isolated strains (data not shown), complying with Koch’s postulates.


[Figure ID: f1] Figure 1.

Detail of isolates; AG1: Colletotrichum sp. AG2: Pestalotia sp. AG3: Colletotrichum sp. (a) Top view of the growth plate. (b) Back view of the growth plate. (c) Detail of the conidia of the isolates (100x).


Antifungal effect of chitosan

In vitro evaluation of MG and % IMG of the strains AG1 and AG3 in the presence of both LMW as MMW chitosan solutions at different concentrations, demonstrated that the lowest MG occurred at the highest concentrations (1.5 and 2.0 % w/v) of LMW (Figure 2) and that >90 % IMG was observed under these treatments irrespective of the fungus strain (Figure 3). Similar results were obtained with the MMW chitosan solutions (p<0.05) (data not shown). The data displayed an inverse relationship between the chitosan concentration and sporulation. LMW chitosan reduced the sporulation of strains AG1 and AG3 by 29 and 51 %, respectively, whereas MMW chitosan reduced sporulation by 47 and 45 %, (Table 1). Chitosan concentrations of 0.1, 0.5 and 1.0 % (w/v) decreased spore germination (Figure 4); this parameter was completely inhibited in both strains AG3 and AG1 when 1.5 % LMW and MMW chitosan solutions were used (Table 1).


[Figure ID: f2] Figure 2.

Effect of LMW chitosan at different concentrations on mycelia growth of AG1. Note: a-d means with the same letter are not significantly different (p<0.05).



[Figure ID: f3] Figure 3.

Effect of LMW chitosan at different concentrations on % inhibition of mycelial growth of AG1. Note: a-d means with the same letter are not significantly different (p<0.05).



[Figure ID: f4] Figure 4.

Development of the germination of Colleotrichum spores treated with different concentration of LMW chitosan, during 24 hours of incubation at 27 °C.


Table 1.

Final sporulation and germination of LMW and MMW chitosan treatments for AG1 strains and AG3.


Sporulation (x107 spore·mL-1) % Germination
Treatment LMW MMW LMW MMW
% w/v AG1 AG3 AG1 AG3 AG1 AG3 AG1 AG3
Untreated 3.83 ± 1.54 3.54 ± 0.01 4.05 ± 1.29 3.65 ± 0.41 100 100 100 100
0.1 3.25 ± 0.01Aa* 3.42 ± 0.06Aa 3.93 ± 0.10Aa 3.54 ± 0.03Aa 60Aa* 100Aa 50Aa 50Aa
0.5 3.14 ± 0.2Aa 3.31 ± 0.52Aa 3.22 ± 1.16Ba 3.04 ± 0.79Ba 50Aa 50Ba 50Aa 33Aa
1.0 2.96 ± 0.25Ba 1.77 ± 0.01Ba 3.14 ± 0.02Ba 2.60 ± 0.08Ca 25Ba 50Ba 50Aa 25Ba
1.5 2.73 ± 0.05Ca 1.71 ± 0.13Ba 2.13 ± 0.16Ca 1.99 ± 0.83Da 0Ca 0Ca 0Ba 0Ca
2.0 2.62 ± 0.4Ca 1.47 ± 0.13Ca 1.71 ± 0.01Ca 1.80 ± 0.48Da 0Ca 0Ca 0Ba 0Ca
**Reduction 29 % 51 % 47 % 45 % 100 % 100 % 100 % 100 %

TFN1Note: *Equal capital letters do not represent significant difference between the different concentrations (rows). Equal lower letters do not represent significant difference between the different strains (columns) (p<0.05). Mean ± SD. (n = 3). **Reduction of sporulation and % germination compared to control and the treatment of 1.5 % w/v of chitosan.


Effect of chitosan on the incidence and severity of anthracnose in avocado fruits

LMW chitosan treatment resulted in a decrease in the incidence of anthracnose reducing it by more than 80 % compared to untreated fruits. The results of this study showed that in fruits treated with chitosan the severity of infection decreased by more than 95 % (Figure 5).


[Figure ID: f5] Figure 5.

Effect of chitosan in the incidence and severity of anthracnose in avocado fruits. Different letters means are significantly different between treated and untreated fruits (p<0.05).


Effect of chitosan on avocado fruit

During the 12 days of storage, PWL of untreated (Fc) and inoculated fruits (FP) was of 19.6 and 20.9 %, respectively. Chitosan treated fruits (FCh and QChP) exhibited weight loss of 15.5 and 16.2 %, respectively (Figure 6).


[Figure ID: f6] Figure 6.

Effect of LMW chitosan 1.5 % in physiological weight loss during storage at 25 °C. FCh: fruits with chitosan. Fc: fruits untreated (control). FChP: fruits treated with chitosan and inoculated with pathogen. FP: fruits inoculated with pathogen without chitosan.


Chitosan treatment (FCh and FChP) maintained fruit firmness during 10 days of evaluation at values of 30.8 and 29.5 N, respectively. Untreated samples (Fc and FP) exhibited a decrease in firmness up to values of 19.8 and 11.8 N, respectively (Figure 7).


[Figure ID: f7] Figure 7.

Effect of LMW chitosan at 1.5 % on firmness fruit. FCh: fruits with chitosan. N: Newton. Fc: fruits untreated (control). FChP: fruits treated with chitosan and inoculated with pathogen. FP: fruits inoculated with pathogen without chitosan.


Chitosan treated fruits exhibited lower dry matter content until 9 days of storage compared to untreated samples. At the end of the evaluated period, the percent of dry matter of fruits treated with chitosan (FCh and FChP) was of 30 to 33 %, and of untreated fruits (Fc and FP) was of 33 and 28 %, respectively (Figure 8).


[Figure ID: f8] Figure 8.

Effect of LMW chitosan 1.5 % in dry matter of avocado fruits. FCh: fruits with chitosan. Fc: fruits untreated (control). FChP: fruits treated with chitosan and inoculated with pathogen. FP: fruits inoculated with pathogen without chitosan.


Discussion

Beside of two strains of Colletotrichum, colonies identified as Pestalotia sp. were also isolated from lesions similar to anthracnose. This fungus is found mainly in the leaves of avocado trees and causes dead of branches and leaf spots (Morales-Garcia et al., 2009). Some authors do not consider this fungus as an important postharvest pathogen due to its low occurrence in avocado orchards (Tamayo, 2007). However, it is commonly confused with C. gloeosporioides because it causes similar symptoms in fruits, although Pestalotia sp develops only in the peel, does not damage the pulp and is less apparent in mature avocado (Morales-Garcia et al., 2009). In contrast, Colletotrichum causes the anthracnose disease in avocado fruits forming dark, sunken, circular or ellipsoidal lesions with large numbers of spores that form compact salmon, orange or pink masses. In Mexico, C. gloeosporioides is the main cause of anthracnose in avocado, but also C. acutatum has been isolated from orchards in the state of Michoacán (Guillén-Andrade et al., 2007). Among C. gloeosporioides fungus a morphological genetic and pathogenic diversity has been identified using isozyme and fragments of randomly amplified polymorphic DNA (Montero-Tavera et al., 2010). Genetic relationships between different monoconidial strains are influenced by the geographic origin and the symptoms they produce. The strains isolated in our study named AG1 and AG3 had different coloration at the base of the petri dish but share the same shape and size of spores. The color difference may be attributed to this genetic variability observed between isolates.

Both LMW and MMW chitosan had a significant effect on the growth of the fungus, and the maximum inhibition achieved was over 90% (at 1.5 and 2.0 % chitosan) (Figure 3b). Salvador et al. (1999) reported 100 % inhibition of Colletotrichum sp and Diplodia sp., isolated from Hass avocado with anthracnose symptoms, using chitosan solutions of 0.1 to 0.5 % w/v, but the study did not specify the chitosan characteristics. Chitosan concentration and characteristics (molecular weight and % of deacetylation) are responsible for the inhibition of fungus growth, mainly through its polycationic nature that interacts electrostatically with negatively charged phospholipids in the membrane, forming spaces through which the chitosan can penetrate and reach the cytosol (Palma-Guerrero et al., 2010). The interaction of the free amino groups, which are positively charged in acid media, with the negative residues of exposed macromolecules in the wall of the fungi, change the permeability of the plasmatic membrane, resulting in an alteration of its principal functions, such as the output of wastes and intake of nutrients (Benhamou, 1992). In other studies it was demonstrated that chitosan influences the output of potassium, the pH of the medium and the activity of the ATPase in the cell membrane of the fungus R. stolonifer (García-Rincón et al., 2010). The spaces formed in the membrane may also allow the free passage of calcium, causing an imbalance gradient that destabilizes the cell (Bautista-Baños et al., 2005, Palma-Guerrero et al., 2010). Chitosan altered the outer and nuclear membrane permeability and may also inhibit RNA and protein synthesis when it entering the cell and can be able to interact with the DNA, interfering the transcription process, in addition to the polycationic nature (Möller et al., 2004; Rodríguez et al., 2005). Furthermore, there is evidence that chitosan induces marked morphological changes such as cell disruption and structural alterations (López-Mora et al., 2013; El-Ghaouth et al., 1992). Evidence of the in vitro effectiveness of chitosan is described by López-Mora et al. (2013), who used 1 % LMW solution achieved inhibition of approximately 80 % for A. alternata isolated from mango; by the other hand Liu et al. (2007), achieved 100 % and 90 % of inhibition of mycelial growth of Botrytris cinerea and Penicillium expansum, respectively, using 1% (w/v) chitosan solution. Our results were more effective compared to those described by Bautista-Baños et al. (2005), who reported only 10 % of IMG for C. gloeosporioides, isolated from papaya using 1 and 2 % (w/v) LMW chitosan. Some authors suggest that the molecular weight of chitosan influences its action mechanism, so that, higher molecular weight reduces permeability of the double nuclear membrane (Liu et al., 2001; Aranaz et al., 2009). However, our results did not show significant differences between the LMW and MMW treatments. The contrasting results may be partially explained by the genetic and morphological variability between different monoconidial strains of C. gloeosporioides (Ramos-García et al., 2010). Both chitosan solutions (LMW and MMW) had a % IMG >90 %. Previuos studies reported similar % IMG with low and medium molecular weight chitosan (Kong et al., 2010).

For its part, sporulation is important during fungal growth. The chitosan concentration affects sporulation and alters the cell differentiation processes and the spores production. All the chitosan treatments reduced sporulation up to 50 % compared to the control. Strain AG3 was more susceptible to chitosan treatment, whereas AG1 showed more resistance. However, neither strain (AG1 and AG3) showed significant difference. Bautista-Baños et al. (2005) studied the behavior of two C. gloeosporioides strains isolated from papaya (Veracruz and Guerrero, Mexico) subjected to LMW, MMW and high molecular weight chitosan (HMW) and found a reduction in sporulation greater than 50 %, but there was no effect of polymer weight on this parameter. We found that spore germination was reduced by chitosan regardless of the type. Spore germination of 0 % was achieved with 1.5 % chitosan solutions (Table 2). According to Ocampo et al. (2009) the development, growth, sporulation, germination, differentiation and virulence in a variety of fungi is mediated by signal transduction, specifically by protein kinase A which, which regulates, the asexual sporulation of the fungus Mucor circinelloides (Rodríguez-López et al., 2009). This process may be affected in mutants that lack the regulatory subunit of this protein. Chitosan treatments may affect signal transduction of the protein kinase A pathway and affect spore development by impairing the structure, physiological and reproductive functions or by differentially activating signaling pathways. According to our results, spore germination was decreased by the effect of chitosan and according to our results, it occurs independently of the type used but is affected by chitosan concentration. As mentioned previously chitosan affects the integrity of the fungi cell membrane, which can cause losses of intracellular material (Zakrzewska et al., 2005). This could be a signal to inhibit the germination of those spores that were formed.

Since that in vitro tests we did not find significant differences between LMW and MMW, for the in vivo experiments we used LMW only. LMW treatment reduced disease incidence and severity in comparison to untreated fruits. This result may be attributed to the effectiveness of the chitosan elicitor-like substance that may activate the fruit defense system (Benigne-Ernest et al., 2008). In previous studies, we analyzed the global gene expression of interaction of the Hass avocado-Colletotrichum-chitosan system, the transcriptomic analysis showed a broad expression for genes participating in fruit defense response (Xoca-Orozco et al., 2017; Gutiérrez-Martínez et al., 2016). In addition to this, we also observed a high expression in some genes related to epicatechin and AFD synthesis, compounds that are related to the defense system of the avocado fruit against the attack of Colletotrichum (Wang et al., 2004a; 2004b; 2006).

Chitosan films also act as barriers for oxygen and carbon dioxide, which reduce the incidence of the disease (Miranda et al., 2003) by ensuring water permeability, slowing gas exchange limiting the development of the pathogen due to low oxygen and ethylene production (Xaun-Zhu et al., 2008). This coating effect also reduced the physiological weight loss of chitosan-treated fruits during storage (Figure 4a). Salvador et al. (1999) evaluated different chitosan films and observed that fruits lost 10 % of their physiological weight after 6 days of storage, which was similar to our results.

During fruit ripening and storage, firmness gradually decreased due to changes at the cell wall level, hydrolysis of peptides, cellulase, pectin methylesterase and polygalacturonase enzyme activity, which in turn degrade high molecular weight polymers such as cellulose and hemicellulose (Silveira, 2007). In fruits treated with LMW chitosan these processes are delayed by the presence of the film resulting in less firmness loss and an extension of the fruit shelf life. Chitosan may contribute to the preservation of pectin links and the distribution of cellulose in the cell wall preventing degradation and maintaining fruit firmness (Elsabee & Abdou, 2013). Bill et al. (2014) applied chitosan films to avocado fruits and reduced the incidence and severity of anthracnose disease, increased resistance to the pathogen and also maintained the firmness, the content of phenolic compounds and the activity of enzymes related to the defense system of the fruit.

Fruit dry matter is an important parameter to determine avocado ripeness and it depends on the period of fruit harvesting. However, as maturity progresses there is a gradual increase in dry matter, and when the fruit ripens in the field it decreases (Wang et al., 2012). Our results showed that dry the matter of fruits treated with LMW did not increased during storage but significantly (p<0.05) decreased when fruit reached maturity. Ozdemir & Cavallazzi (2004) reported that this effect could be due to inactivation of acetyl-CoA carboxylase, a key enzyme in the production of long chain fatty acid from acetate-14C. Chitosan probably affected the function of this enzyme in avocado tissue, which caused this behavior. Further studies are needed to verify the effect of chitosan in inactivation of acetyl-CoA carboxylase in avocado fruits.

Conclusions

The in vitro antifungal effectiveness of chitosan was optimal at concentrations of 1.5 % w/v irrespective of the molecular weight. This treatment allowed a high percent of IMG and reduced sporulation and germination of both strains of Colletotrichum sp. Statistical analysis revealed no significant difference (p<0.05) between LMW and MMW but some differences among the evaluated strains of Colletotrichum sp were observed.

Chitosan treatment maintained the postharvest quality of the fruit, reduced the incidence and severity of disease and physiological weight loss, and maintained the firmness during storage. We propose that chitosan films could be a plausible alternative to preserve the avocado quality and lessen the anthracnose incidence. Additional to quality parameters, could be evaluated others such as CO2 and ethylene production, and enzyme activity in order to determinate the effectiveness of chitosan films during storage of avocado fruits.


fn1Cite this paper: Xoca-Orozco, L. A., Aguilera-Aguirre, S., López-García, U. M., Gutiérrez-Martínez, P., Chacón-López, A. (2018). Effect of chitosan on the in vitro control of Colletotrichum sp., and its influence on post-harvest quality in Hass avocado fruits. Revista Bio Ciencias 5, e355 doi: https://doi.org/10.15741/revbio.05.e355

Acknowledgement

The authors thanks the CONACYT for the fellowship number 262345 awarded to the participating master student and the PROMEP ITTEP-PTC-004 for the support given to this project.

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Revista Bio Ciencias, Año 11, vol. 7,  Enero 2020. Sistema de Publicación Continua editada por la Universidad Autónoma de Nayarit. Ciudad de la Cultura “Amado Nervo”,  Col. Centro,  C.P.: 63000, Tepic, Nayarit, México. Teléfono: (01) 311 211 8800, ext. 8922. E-mail: revistabiociencias@gmail.com, revistabiociencias@yahoo.com.mx, http://revistabiociencias.uan.mx. Editor responsable: Dr. Manuel Iván Girón Pérez. No. de Reserva de derechos al uso exclusivo 04-2010-101509412600-203, ISSN 2007-3380, ambos otorgados por el Instituto Nacional de Derechos de Autor. Responsable de la última actualización de este número Dr. Manuel Iván Girón Pérez. Secretaria de Investigación y Posgrado, edificio Centro Multidisciplinario de Investigación Científica (CEMIC) 03 de la Universidad Autónoma de Nayarit. La opinión expresada en los artículos firmados es responsabilidad del autor. Se autoriza la reproducción total o parcial de los contenidos e imágenes, siempre y cuando se cite la fuente y no sea con fines de lucro.

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