Antifungal Activity of Microbiological Chitosan and Coating Treatment on Cherry Tomato

13 Pages·2002·0.25 MB·English

Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
ISSN: 2319-7706 Volume 4 Number 9 (2015) pp. 228-240
http://www.ijcmas.com
Original Research Article
Antifungal Activity of Microbiological Chitosan and Coating Treatment on
Cherry Tomato (Solanum lycopersicum var. cerasiforme) to Post-Harvest
Protection
Adamares M. Silva1,3, Thayza C. M. Stamford2, Patrícia M. Souza3, Lucia R.R. Berger1,3,
Marcela V. Leite1,3, Aline E. Nascimento3 and Galba M. Campos-Takaki3*
1Center of Biological Sciences, Federal University of Pernambuco, 50670-420,
Recife, Pernambuco, Brazil;
2Department of Tropical Medicine, Federal University of Pernambuco, 50670-901,
Recife, Pernambuco, Brazil;
3Nucleus of Research in Environmental Sciences and Biotechnology, Catholic University of
Pernambuco, 50050-590, Recife, Pernambuco, Brazil
*Corresponding author
A B S T R A C T
Chitosan is naturally found in the cell wall of fungi (Zygomycetes) and can be
obtained from chitin by deacetylation to form a higher proportion of polymer chain
units 1,4-2-amino-2-deoxy-D-glucose, and a smaller number of units 1,4-2-
Keywords
acetamido-2-deoxy-D-glucose. In this study aimed to evaluate the effect of
microbial activity of chitosan (Ch), and bioactive coatings on cherry tomato to
Microbiological
protect against to Botrytis cinerea infection. The chitosan was obtained from R.
chitosan,
arrhizus showed degree of deacetylation (DD) of 68% and molecular weights of
Botrytis cinerea,
1.40 x 104 g / mol. The spores viability studies with the pathogen B. cinerea by
Antifungal
light microscopy showed 70% of germ tubes emergency formed in 16 hours of
activity,
incubation. Investigations were carried out with chitosan (Ch) evaluating its
Postharvest
minimal inhibitory concentration (MIC) and minimum fungicide concentration
protection
(MFC) of 100 g / ml against B. cinerea. Bioactive coatings on cherry tomato by
chitosan (Ch) at concentration 100 g / mL showed higher protection to B. cinerea
infection. The promising results showed the potential of microbiological chitosan
(Ch) to pathogen B. cinerea, suggesting to be an important crop protection.
Introduction
Chitosan forming a greater proportion of crustaceans and insects and cell walls of
polymer chain 1,4-2-amino-2-deoxy-D- fungi, in particular Zygomycetes. This
glucose units is a polymer derived from biopolymer has great biotechnological
chitin deacetylation process 1,4-2- importance considering its properties, such
acetamido-2-deoxy-D- glucose, may be as biocompatibility, biodegradability,
found in large amounts in the shells of antimicrobial, anti-inflammatory and healing
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
action. It has immense structural becoming less desirable from an ecological
possibilities for chemical modifications to point of view and public health (Williamson
generate novel properties, functions, and et al., 2007; Badawy and Rabea, 2011).
applications especially in agricultural area
(Ali et al., 2012; Fai et al., 2011; The cherry tomato (Solanum lycopersicum
Yogeshkumar et al., 2013; Freddo, 2014). var. cerasiforme), one of the most important
fruit in the world, is highly susceptible to
In addition, the advances in fermentation necrotrophic pathogenic fungi as Botrytis
technologies suggest that the cultivation of cinerea and Alternaria solani. Alternative
Mucor alean fungi can provide an treatments such as the use of plant extracts,
alternative source of chitin and chitosan. essential oils and biopolymers such as
The amount of these polysaccharides chitosan have been studied to reduce the use
depends on the fungi species, carbono and of synthetic fungicides which do not bring
nitrogen sources, and cultural conditions. benefits to health of living beings and the
Fungal mycelia from Zygomycetes are environment (Smith, 2014).
relatively consistent in composition and are
not associated with inorganic materials; Therefore, the objective of the present study
therefore, no demineralization treatment is was the production of chitosan by Rhizopus
required to recover fungal chitin and arrhizus and evaluates the activity of coating
chitosan (Chandy and Sharma, 1990; chitosan on cherry tomato (Solanum
Rinaudo et al. 1992; Feofilova et al., 1996; lycopersicum var. cerasiforme) against
Tan et al., 1996; Teng et al., 2001; Andrade Botrytis cinerea infection. Previous
et al., 2000; Pochanavanich and Suntornsuk, experiments were done to evaluate the
2002; Pochanavanich and Suntornsuk, spores germination, radial growth of B.
2002; Synowiecki and Al-Khateeb, 2003; cinerea, and established the minimum
Andrade et al., 2003; Hu et al., 2004; inhibitory concentration (MIC) and the
Campos-Takaki, 2005; Santos et al., 2013; minimum fungicide concentration (MFC) of
Berger et al., 20014). chitosan solution.
Microorganisms plant pathogens are Materials and Methods
considered economically a problem to
agriculture around the world. They induce Microorganisms
decay on a large number of agricultural
crops during the growing season, in The Mucoralean of Rhizopus arrhizus
particular postharvest. They induce decay on UCP/WFCC 1295 was isolated from
a large number of agricultural crops during caatinga soil of Pernambuco, PE, Brazil, and
the growing season and postharvest. In belongs to UCP (Universidade católica de
particular, the fungus Botrytis cinerea Pernambuco) Collection and is registered in
infects some fruit and cause huge economic World Federation Culture for Collection
losses in various stages of harvest and post- (WFCC). The fungus was mantained in
harvest. The agent responsible for the Sabouraud Dextrose agar at 5° C, and was
infection is difficult to control due to their used to production chitosan.
saprophytic activity and forming resistance
structures. One way to control this The fungus Botrytis cinerea URM 2802 was
pathogen is to use chemicals, however, kindly obtained from Micoteca of the
tolerance for chemical residues in food, it is Mycology Department of the Federal
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
University of Pernambuco, Recife-PE, and tem discs was transferred to
Brazil. The strain was isolated from plant Erlenmeyers flasks of 250 mL capacity,
pathogen in tomato fruits (Solanum containing 50 mL of medium, with pH
lycopersicum var. Cerasiforme), and was adjusted to 5.8, containing corn steep liquor
maintained on PDA (potato dextrose agar) at 8.82% glycerin and 6%, incubated at 28° C
5° C. for 96 h in orbital shaker at 150 rpm.
Agroindustrial substrates The chitosan was obtained from biomass if
R. arrhizus by methodology according to Hu
Glycerin was kindly supplied by CETENE (1999) which involves deproteinization by
(Center of Tecnologies of Northeast, Recife, 1M sodium hydroxide (w/v), is followed by
PE, Brazil), and corn steep liquor from Corn centrifugation, acid hydrolysis with 2%
Products (Cabo de Santo Agostinho- acetic acid (3v/w), centrifugation to
PE,Brazil). obtaining chitosan by neutralization to pH
10.
Tomato
Characterization of chitosan
Samples of sherry tomatoes (S. lycopersicum
var. cerasiforme) were used for Determination of degree of deacetylation
experimental purposes in the test against of chitosan fungal
Botrytis cinerea. The tomatoes fruits were
obtained from informal plantation from The chitosan was submitted to infrared
Cabo de Santo Agostinho Pernambuco, spectroscopy, using Fourier Transform
PE, Brazil. The tomatoes were pretreated spectrophotometer (FTIR), IFS 66 Bruker
with 1% of sodium hypochlorite solution, Mod (Analytical Center - Fundamental
followed wash in sterile distilled water. Chemistry Department - DQF - UFPE). The
degree of deacetylation was determined
Microbiological chitosan production according to the equation described by
Roberts (1992):
The fungus A. arrhizus was grown in Petri
dishes (9cm diameter) containing YMA DD% = 100- [(A1655 / A3450) 100 / 1.33]
[yeast malt agar], and the plates were
incubated at 28° C for 120h until Molecular Weight
sporulation. Spores were collected and
transferred to Erlenmeyers flasks with The molar mass was determined by
125mL capacity, containing 50 ml of viscosity, according to the methodology
buffered saline with 0.9% [sodium proposed by Santos et al. (2003). The
phosphate monobasic 0.45 g sodium intrinsic viscosity measurements were
phosphate dibasic 5.8125 g, sodium chloride performed using a glass capillary Cannon-
2.25 g water 250mL distilled], counted in Fenske type (dinterno = 1,01mm)
hematocitociometer to a concentration of thermostated at (25 ± 0.01) ° C, in a AVS
107 spores.mL-1, as pre-inoculum. 350-Schott-Geräte viscometer. To determine
the intrinsic viscosity [h], chitosan solutions
The R. arrhizus spores suspension was were prepared (using acetic acid buffer plus
spread on Petri dishes containing YMA and acetate as solvent) at concentrations ranging
incubated at 28º C for 24h, and discs of from 1.0 x 10-3 to 8.0 x 10-3 g. mL-1. The
6mm diameter was cut using corker borer flow times in seconds were determined.
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
Samples were prepared in triplicate and the minimum fungicide concentration (MFC) to
average value was calculated. microbiological chitosan against to B.
cinerea tested were performed using the
Scanning electron microscopy broth microdillution method as described by
Ahmed et al. (2008). The assay was carried
In order to observe the microstructures of out using 20µL of the inoculum of B.
chitosan used scanning electron microscopy cinerea (105 spores/mL) in wells containing
analyze was performed. Sample of chitosan potato juice with chitosan concentrations
produced by R. arrhizus was ground and between 100 to 600 g.mL. The system was
dried under vacuum using a lyophilizer and incubated at 28° C for 24 hours.
its surface was observed using a scanning
electron microscope XL 30 series (Umax) The MIC was defined as the lowest
ESEM (Env. Scan. Electron Micros, Jeol, concentration of chitosan capable of
Tokyo, Japan) with tungsten filament, 20 kV inhibiting the cellular multiplication. The
accelerating voltage. CFM was determined based on the
methodology of Santurio et al. (2007).
Botrytes cinerea Spores Viability From the wells in which there was no visible
fungal growth pulled out a 10µL aliquot,
Rates of 0.5 ml suspension of 105 which was inoculated on the surface of PDA
spores/mL-1 of B. cinerea URM 2802, were medium. The plates were incubated at 28°
transferred to Erlenmeyer flasks containing C, and after 24 h, was determined as the
100 ml of medium potato dextrose, lowest concentration CFM chitosan able to
incubated orbital shaker at 28°C. Aliquots of inhibit the growth or cause the death of
2.5 mL were collected in triplicate at fungus spore. MIC and MFC assays were
intervals: 0h, 4h, 8h, 12h, 20h and 24h, and performed in triplicate.
was observed under light microscopy at
40X to determine the emergency tubes Treatment of Tomatoes with chitosan
formation, and number of cells viability. solution
Effect of Chitosan on Radial Growth of B. To evaluate the direct effect of chitosan on
cinerea the development of B. cinerea, the tomatoes
were inoculated with a conidial suspension
Petri dishes of 6cm of diameter containing at a concentration of 105 conidia/mL-1,
PDA cultivation medium, PDA plus 1% determined by hemacytometer count,
acetic acid, PDA added chitosan in the according to methodology described by
concentrations of 100 g.mL-1 (MIC), Camili et al. (2007) after 4pm immersed in
1000 g.mL-1 and 2000 g.mL-1. After chitosan solution at CIM concentration then
solidification the medium discs of 6mm of arranged on sterile petri dish on a tray
B. cinerea grown in PDA was done in the covered with plastic wrap at 28°C.
center of the plate, incubated at 28°C during
72h. The effect of different concentrations The treatment was accompanied by the loss
of chitosan on radial growth of B. cinerea weight of tomatoes control without chitosan
was determined. with acetic 1% and coated with chitosan
acid, the results are monitoring each 24h of
Determination MIC and MFC intervals. The weight lost was calculated as
a percentage relative to the initial weight of
Minimal inhibitory concentration (MIC) and the samples Meng et al. (2008).
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
Results and Discussion arrhizus in medium containing corn steep
liquor and glycerin from biodiesel
Chitosan characterization production corresponded to lower molecular
weight (1.40 x 104g. mol-1). The results
Deacetylation Degree obtained from of R. arrhizus is similar to
reported by Pochanavanich and Suntornsuk
Spectroscopy analysis of the infrared region (2002) and Berguer et al. (2014) for
of the chitosan produced by R. arrhizus was molecular weight from fungal chitosan
performed to determine the degree of which ranging from 5.00 × 103 to 9.4 × 105
deacetylation (DD%), with the purpose of (Table 1).
characterization, according to Roberts
(1992). Scanning electron microscopy
The infrared spectrum absorption of the The figure 2 (A and B) showed the
chitosan showed bands regarding OH-axial morphology of chitosan produced by R.
stretching between 3396 and 2931cm-1, arrhizus and chitosan from crustaceous by
superimposed on the NH stretching band scanning electron microscopy. The chitosan
with axial deformation of amide C = O at from fungus also exhibited an amorphous
about 1650cm-1, NH angular deformation at structure that was more compact and dense,
approximately 1575cm-1; axial deformation and nor porous was observed. This
of amide-CN at around 1415cm-1, biopolymer also showed crumbling layers of
symmetrical angular deformation in CH at flakes was detected as similar morphology
3
1379 cm-1, -CN axial deformation of amino was observed in crustacean chitosan and few
groups from 1078 to 1250cm-1 and aggregated flakes formed a dense and firm
polysaccharide structures bands in the range structure, without porosity, as described to
of between 597 and 1150 cm-1 (Figure 1). fungal chitosan by the literature (Yen and
Mau, 2007; Yen et al., 2009; Berger et al.,
However, the same results were observed by 2014).
Mario et al. (2008) who described the N-
deacetylation is associated with a Biochemical studies with Botrytis cinerea
progressive weakening of the band
occurring at 1,655 cm 1(amide I vibrational Spore viability
mode), and the disappearance of the band at
1,550 cm 1 (amide II vibrational mode). Our Previous studies of cell viability by light
results showed deacetylation degree of microscopy showed that the culture of B.
microbiological chitosan of 68%, and it can cinerea grown on potato broth at
be seen that in the fingerprint region. The concentration of 105 conidia/mL-1 appeared
DD% is an important parameter that to be feasible for use as a phytopathogenic
determines the physicochemical properties of fungus. After 8 to 12h of cultivation 100%
the chitosan because it is linked to the of the spores increase in the size was
cationic properties of chitosan (Fai et al., observed.
2011).
The emergency of the germinated tubes
Molecular weight were formed corresponding to 70% of
viability at 16h. However, at the end of
The viscosimetry evaluation of chitosan cultivation the spores (30%) germinated
obtained from submerged fermentation of R. between 20 and 24 h of cultivation.
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Table.1 Properties of fungal chitosan and crab shell chitosan standard
Chitosan sample DD* (%) MW**(Da) Reference
Rhizopusarrhizus UCP/WFCC1295 68.00± 0.9 1.4 ×104 This study
Cunninghamella elegans UCP/WFCC 0542 82.24 ± 2.0 5.00 × 103 Berguer et al. (2014)
Pochanavanich and
Crab shell (Sigma) 97.9 ± 0.9 9.4 × 105
Suntornsuk (2002)
Pochanavanich and
Aspergillus niger TISTR3245 90.9 ± 2.1 1.4 × 105
Suntornsuk (2002)
* DD (%) = Degreeofdeacetylation (%), ** MW= Molecular Weight
Figure.1 Infrared absorption spectra of microbiological chitosan
obtained from Rhizopus arrhizus
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
Figure.2 Scanning electron microscopy (SEM) of chitin (A) and (B) chitosan produced by
Rhizopus arrhizus . (500 × magnification 200/1000). The measurement bar = 10 µM
These results are similar to those presented Determination MIC and MFC to
by Gull and Trinci (1997) which reveal that microbiological chitosan
there was spore germination with a germ
tube from Botrytis cinera cultivation, the B. cinerea showed higher inhibition by
germination remaining stable in values of microbiological chitosan and the minimum
50% from 20h to 24h. Tratci and Beettiol inhibitory concentration (MIC) and the
(1997) described in their studies the action minimum fungicide concentration (MFC)
of biofertilizers on mycelial growth and was found 100 g mL-1. Studies carried out
germination of pathogenic fungi, obtained by Oliveira et al. (2014) with chitosan
spore germination of B. cinerea (104 spores obtained from Cunninghamella elegans
mL-1) in water resulting in values of 50% UCP 0542 versus grapes phytopathogen
after 20h of incubation at 20°C. infection, exhibited an MIC value OF 15
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
mg.mL-1 against B. cinerea and Penicillium Evaluation of microbiological chitosan
expansum. However, Coqueiro and Piero coated protection on cherry tomato
(2011) was evaluated the effect of
commercial chitosan isolate from The cherry tomato was treated with
crustaceous with different molecular weights MIC/MFC concentration against to B.
to Alternaria solani showed fungistatic cinerea crop infection and was accompanied
effect inhibiting the mycelial growth and during 19 days at environment temperature
spore germination in the concentrations of (28°C). The coated cherry tomatoes were
0.125 and 0.5 mg mL-1, respectively. The evaluated and control of the tests was done
antimicrobial effect of fungal chitosan to R. contamination with B. cinerea spores (105
arrhizus and Cunninghamella elegans was /mL) during this period and evaluated
observed in the concentrations of 300 µg visually and the lost weight each 24 h.
mL-1.
It is believed that the cationic nature
Recently studies with the MIC and MFC of collaborate for the actuation of the chitosan
chitosan was observed similar values (500 since their positive charges can interact with
µg.mL-1) to inhibit the growth and death of the negatively charged residues of
Salmonella enterica and Staphylococcus macromolecules exposed on the surface of
aureus (Berger et al., 2014). The chitosan pathogen cell causing changes in
from crustaceous with 85% DD showed permeability of the cell membrane,
increasing inhibition rates in amounts Benhamou (1996). Through experiments
ranging from 0.25 to 0.5 gL-1 according to Olsen et al. (1989) reported that the amine
the polymer concentration against groups of chitosan into contact with the
Aspergillus flavus and Aspergillus intracellular fluids are protonated and bind
parasiticus (Souza et al., 2013). strongly to the anionic groups of
microorganisms, which raise the process of
Tests were carried out using potato dextrose bonding between them, preventing their
agar (PDA) added the concentration of development.
chitosan based in the CMI (100 mgmL-1)
and inoculated discs of B. cinerea, Camili et al. (2007) using chitosan
incubated at 28°C and the radial growth was originating from crustaceans against
evaluated in comparison with control infection of the pathogen B. cinerea on
(without chitosan), however, in the solid grapes mentions that chitosan could bind to
medium the fungus was totally inhibited the DNA and mRNA synthesis inhibition occurs
growth. via penetration of the product in the nucleus
of the microorganism also interfere with the
The results obtained with B. cinerea synthesis of proteins. The mechanism of
inhibiton of growth by chitosan are inhibition of growth of pathogens by
corroborated by Liu et al. (2007), Camili et chitosan differs from the used mechanism by
al. (2007), Liu (2007) and Botelho et al., the synthetic fungicides.
(2010). Recently, Freddo et al. (2014)
showed that chitosan at 0.25, 0.5, 1 and 2% Besides being applied alone, there are many
concentrations reduced the mycelial growth reports on applications of chitosan combined
of R. solani in vitro culture, and progressive with other antifungal compounds. Yu et al.,
ecrease of mycelial growth related to the (2007) found that chitosan applied alone or
increase of chitosan concentration. Cryptococcus laurentii can effectively
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
inhibit infection caused by Penicillium (1997) findings which reported that chitosan
expansum on apple fruit after seven days of from R. arrhizus grapes coating, realized by
incubation at 20°C. When applied alone, controlling the weight that the coated fruits
treatment with chitosan at the highest with chitosan had a lower percentage loss
concentration (1%) and low viscosity (12 wet weight than the control samples.
cP) was the most effective. Used in
combination, the treatment of Cryptococcus Similar studies carried out by Pastor et al.,
laurentii with chitosan at a concentration of (2011) found that less weight loss with
0.1% and low viscosity (12 cP) was the most grapes treated with propolis extract and
effective. hydroxypropyl methylcellulose was
evidenced.
As mentioned by Zang et al. (2011), the low
molecular weight chitosan has been studied According to edible coating's ability to
to control the fruit postharvest diseases. The prevent the fruit weight loss is based on their
results indicated that low molecular weight hygroscopic properties, which allows the
chitosan significantly inhibited the formation of a water barriers between the
decomposition of fruits caused by fruit and environment (Morillon et al., 2002;
Penicillium digitatum, Penicillium italicum, Xu et al., 2007).
Botrydiplodia lecanidion and Botrytis
cinerea after 14 days storage at 25°C being The recourse to naturally-occurring products
more effective than the high molecular with interesting antifungal activity as
weight chitosan. chitosan and their derivatives has been
getting more attention in the recent years to
Results presented in Liu et al. (2007) have make profits while guaranteeing a
shown that the application of chitosan of sustainable agriculture. Strategies were used
low molecular weight crustacean showed a for microbiological chitosan production by
greater effect on inhibition of growth of Rhizopus arrhizus and application against
Penicillium expansum and Botrytis cinerea. the pahytopathogenic fungi Botrytes
Lauzardo et al. (2008) also reported a cinerea.
greater inhibitory effect when applied to low
molecular weight chitosan on Rhizopus The results obtained showed that R. arrhizus
stolonifer with modifications in fungal is a chitosan polymer producer with 68% of
reproductive structures. degree deacetylation and lower molecular
weight. The polymer shown antifungal
The monitoring of cherry tomato during 19 activity and the CMI and CMF are similar
days at a temperature of 25°C, was related to (100 mgmL-1). The CMI chitosan solution
the initial weight and showed loss weight of was able to inhibit the mycelial growth of
the control sample (without chitosan and the fungus Botrytis cinerea. The coating
with 1% acetic acid), with values of 3.36% application of chitosan treatment provided
(control) and 3.58% (acetic acid effective protection for post-harvest cherry
treated),respectively. tomato (S. lycopersicum var. cerasiforme) to
infection against B. cinerea. The results
However, were observed that coating obtained provide additional support to crop
treatment to cherry tomatoes with chitosan protection.
the loss weight was very lower (2.75%). Our
results are corroborated by Oliveira et al.
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Int.J.Curr.Microbiol.App.Sci (2015) 4(9): 228-240
Acknowledgments by Cunninghamella elegans. Can. J.
Microbiol., 46: 1042 1045.
This work was financially supported by Arbia, W., Adour, L., Amrane, A., Lounici,
National Council for Scientific and H. 2013. Optimization of medium
Technological Development (CNPq) composition for enhanced chitin
563382/2010-4-SISBIOTA and extraction from Parapenaeus
308245/2010-5 (G.M.C.T.), Foundation for longirostris by Lactobacillus
the Support of Science and Technology of helveticus using response surface
the State of Pernambuco (FACEPE), and methodology. F. Hyd., 31: 392 403.
fellowship to CAPES (Coordination for the Badawy, M.E.I., Rabea, E.I. 2014. A
Improvement of Higher Level Education Biopolymer chitosan and its
Personnel) to A.M.S. The authors are derivatives as promising
grateful to technician S. H. Almeida and to antimicrobial agents against plant
NPCIAMB-Catholic University of pathogens and their applications in
Pernambuco, Brazil, for use of laboratories. crop protection. Inter. J., 2011: 1 29.
Benhamou, N. 1996. Elicitor-induced plant
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