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Pesticide Biochemistry and Physiology 99 (2011) 250255

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Pesticide Biochemistry and Physiology


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Cytological and biochemical changes induced by chitosan in the pathosystem Alternaria alternatatomato
D. Snchez-Domnguez a, M.Y. Ros b, P. Castillo-Ocampo c, G. Zavala-Padilla d, M. Ramos-Garca e, S. Bautista-Baos e,
Centro de Bachillerato Tecnolgico, Agropecuario No. 8, Km. 8.5 Carretera, Alpuyeca-Jojutla, Xoxocotla, Morelos, C.P. 62780, Mexico Centro de Investigaciones Qumicas, Universidad Autnoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos, C.P. 62209, Mexico Universidad Autnoma Metropolitana-Iztapalapa, Laboratorio de Microscopa Electrnica, Av. San Rafael Atlixco # 186, Colonia Vicentina, Delegacin Iztapalapa, C.P. 09340, D.F., Mexico d Instituto de Biotecnologa, Universidad Nacional Autnoma de Mexico, Av. Universidad 1001, Chamilpa, Cuernavaca, Morelos, C.P. 62209, Mexico e Instituto Politcnico Nacional, Centro de Desarrollo de Productos Biticos, Km. 6 Carr, Yautepec-Jojutla, Col. San Isidro, CEPROBI 8, Yautepec, Morelos, C.P. 62731, Mexico
b c a

a r t i c l e

i n f o

a b s t r a c t
The cytological and biochemical response of the fungus Alternaria alternata to chitosan application in tomato fruits was evaluated. The research was developed in the following stages: microscopically to observe the degree of damage that chitosan causes over the conidia and hyphae of the fungus at the structural level and during the infection process in tomato tissue. Biochemically we tried to identify the elicitation of the phytoalexin rhisitin and other compounds involved in resistance. At the microscopic level, mycelium and conidia of chitosan-treated of A. alternata showed cell wall disintegration, plasma membrane retraction, cellular distortion, release of the apical portion of the conidia and lysis of fungal cells. Hyphae and conidia were susceptible to chitosan application. Infection always took place in chitosan treated and inoculated tomatoes and it was difcult to observe ultrastructural alterations due to chitosan application. The phytoalexin rhisitin was not isolated from any of the treatments but other compounds such as alkenes, fatty acids and vitamin E whose antimicrobial effects have been reported were detected. The elicitation of precursor compounds in the pathosystem A. alternatatomato was more associated with the infection process than with the chitosan application. Further studies are necessary to conrm these ndings. 2011 Elsevier Inc. All rights reserved.

Article history: Received 9 May 2010 Accepted 9 January 2011 Available online 12 January 2011 Keywords: Black mold Chemical prole Conidia Lycopersicon esculentum L. Mycelium

1. Introduction The incidence of disease is a major factor limiting the postharvest life of tomato fruits. Among others, the black mould rot disease caused by Alternaria alternata (Fr.:Fr.) Keissl grows over the surface of the fruits, affecting all stages of ripening [1]. For the tomato industry in Mexico, black mould rot has become the most signicant disease since most tomato genotypes are quite susceptible to A. alternata. Chitosan, a chitin derivative is a natural biodegradable compound derived from crustaceous shells such as crabs and shrimps, has been extensively mentioned as a natural fungicide. In numerous in vitro and in situ investigations the inhibitory effect of chitosan on important phytopathogenic genera has been demonstrated [2,3]. In previous studies, it was reported that chitosan affected the spore viability of A. alternata [4], whilst in other ndings the degree of inhibition of the mycelial growth and sporulation of this
Corresponding author.
E-mail address: sbautis@ipn.mx (S. Bautista-Baos). 0048-3575/$ - see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2011.01.003

fungus isolated from tomato fruits relied on the molecular weight of chitosan [5]. These authors also reported in observations carried out by scanning electron microscopy, that chitosan modied the mycelia morphology of A. alternata causing deformation, swollenness and nodulations. At the ultrastructural level, it has been mentioned that chitosan affects the integrity and structure of the cell wall of the hyphae and conidia of Fusarium oxysporum f. sp. radici-lyicopersici and Rhizopus stolonifer as well as the hyphae of Phythophthora capsici; in some cases the cellular organelles such as vacuoles and mitochondria of these microorganisms were seriously damaged [610]. In addition to this fungicidal potential, chitosan is mentioned as inducing hostdefense responses that may restrict the pathogen entrance. In earlier reports carried out at ultrastructural level, chitosan caused considerable cellular responses from the infected host such as depositions of electron opaque material along the primary cell walls and intercellular spaces, deposition of brillar material, and formation of papillae and bubble-like structures in xylem vessels of roots and leaves of chitosan-treated tomatoes infected by F. oxysporum f. sp. radicislicopersici, and in roots of chitosan-treated cucumbers, inoculated

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with Phytium aphanidermatum [7,11,12]. In further studies, similar responses were observed in the tissues of chitosan-treated bell pepper infected by Botrtytis cinerea [13]. Another defense response associated with chitosan is the elicitation of antimicrobial compounds such as phytoalexins. One of the rst studies demonstrating the chitosan capacity to induce defence responses was carried out by Walker-Simons et al. [14]. They found that among four different compounds tested, chitosan showed the highest elicitation of proteinase on tomato leaves and pisatin in pea pods. Additional studies conrmed the ability of chitosan to elicit this phytoalexin in immature pea pods. Chitosan fragments of high molecular weight induced the synthesis of pisatin and inhibited Fusarium solani [15]. As shown by Rodrguez et al. [16], rice seeds previously treated with chitosan induced the enzyme phenylalanine ammonia lyase at the seedling stage that may eventually lead to the formation of phytoalexins. In other studies, the highest quantities of the phytoalexins rhisitin in the chitosan-treated tomato fruits and inoculated with A. alternata occurred after 21 days storage [17]. In order to gather more information about the basic aspects in the relationship between chitosan and the pathosystem tomatoA. alternata, we carried out observations at the ultrastructural level on chitosan-treated hyphae and conidia of A. alternata. Moreover, during the infection process, we also identied various chemical compounds of infected and noninfected chitosantomato probably involved in resistance mechanisms.

2.1.4. Gas chromatography analysis GCMS analyses of the components and rhisitin of tomato fruits were obtained using an Agilent 6890 GC/5973 N MSD chromatograph, equipped with a HP-5MS capillary column (5% phenylmetylpolysiloxane, length 30 m, id 0.25 mm, ft 0.25 lm). The carrier gas was helium and the linear gas velocity was 1 mm min1. The injector temperature was 250 C and the column temperature, initially at 40 C for 2 min, was gradually increased at a rate of 10 C/min up to 250 C, and kept at 250 C for 10 min, splitless mode. For detection, a ame ionization detector at 280 C, IE (70 Ev, Scan 30550 uma) was used. The identication of each component was based on a comparison of its mass spectrum with those contained in the NISTMS Library.

2. Materials and methods 2.1. General experimental procedures 2.1.1. Microorganism isolation The fungus A. alternata was isolated from infected tomatoes presenting the usual symptoms of the disease (sunken lesions light to dark brown at the epidermal tissue). Identication of this fungus was according to Barnett and Hunter [18]. Further conrmations consisted of re-inoculating A. alternata in healthy tomatoes, observing disease development and re-isolating the fungus to conrm A. alternata conidia. Culture was maintained in Petri plates containing PDA and incubated at 26 C. Continuous re-inoculations and re-isolations on tomato fruits were carried out to maintain pathogenicity of the inoculum.

2.1.5. Validation of the extraction method of rhisitin The extraction of rhisitin in tomato fruits was carried out following the methodology of Bhaskara-Reddy et al. [17], with some modications. Tomato pulp (10 g) added with rhisitin standard (2 mL) was macerated for 24 h with methanol (30 mL 3), centrifuged at 1000 rpm (5 min 3) and decanted. Extraction solvent was concentrated to dryness in vacuo and the residue was suspended in 80% aqueous methanol (100 mL) and extracted with dichlorometano (100 mL 3). Organic and aqueous-methanol phases were concentrated to dryness in vacuo and suspended in the same solvent (1.5 mL) for GCMS analysis (rhisitin presence was detected in the organic extract at 20.06 min in GCMS analysis). Rhisitin was identied by comparing with the standard obtained from Dr. Susan McCormick at the National Center for Agricultural Utilization Research, US Department of Agriculture Research Service, Peoria, Ill.

2.2. Experiment 1. Effect of chitosan on cellular changes of mycelium and conidia of A. alternata Discs of A. alternata of 14 days of age were placed in the centre of Petri plates containing potato dextrose agar (PDA) amended with chitosan of medium molecular weight at 1.5% concentration. Plates were incubated for 6 days at 26 C. Control Petri plates contained only PDA. After the given incubation period, mycelium was taken from the periphery and xed in 4% paraformaldehyde and 2% glutaraldehyde in 0.16 M sodium cacodylate buffer at pH 7.0 for 1 h. For conidia obtainment, mycelium was ooded with a glucose solution, scraped from the plate and ltered twice through layers of sterile cheesecloth and xed as mentioned above. Samples were processed at the Laboratory of Electronic Microscopy of the Autonomous Metropolitan University, campus Iztapalapa in Mexico City.

2.1.2. Chitosan preparation and treatments Stock solution of chitosan medium molecular weight. (Mw = 2.38 104 Da, 7585% degree of deacetylation; 200800 cps), (Sigma Aldrich, St. Louis, MO, USA) [19] weighing 2.5 g was prepared and then dissolved in 100 ml of distilled water with 1 ml of acetic acid and then heated while constantly agitated for 24 h. pH solution was adjusted to 5.6 by adding sodium hydroxide 1 N and 0.1 ml. The chitosan solution was sterilised and adjusted to the concentration of 1.5% w/v for both in vitro and in situ studies.

2.3. Experiment 2. Cellular changes by effect of chitosan application during the interaction tomatoA. alternata Healthy tomato fruits Saladette type (Sun 7705 and Loreto) were obtained from a greenhouse at the ripening stage pink (30% of the surface of the fruits showing pink or red color) [20]. They were transported to the laboratory, quickly washed with common detergent and thoroughly rinsed with distilled water. After drying, fruits were injured with the aid of a needle and immersed in a spore solution (106) of A. alternata for 15 min. Later, they were dipped in chitosan of medium molecular weight at the concentration of 1.5% for 15 min. Samples were taken after 6 days. Control treatment consisted of fruits inoculated but not immersed in chitosan for a similar period of evaluation. Portions of infected fruits were xed as earlier described. Samples were processed at the Institute of Biotechnology of the National University of Mexico in Cuernavaca, Morelos.

2.1.3. Samples processing for studies in transmission electron microscopy (TEM) Previously xed samples of mycelium, conidia and treated tomato fruits were dehydrated in a graded ethanol series and embedded in London resin white (EMS cat. 14380). Ultrathin sections, 60 nm thick were cut with a diamond knife (Ultracut R, Leica) and collected in Formvar-coated copper grids (EMS cat. FCF200). They were contrasted in uranyl acetate and lead citrate, washed and examined in a Zeiss EM900 transmission electron microscope at 60 kV.

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2.4. Experiment 3. Isolation of rhisitin and other compounds of noninoculated and inoculated tomatoes by A. alternata and chitosantreated in healthy and rotten fruits The isolation of rhisitin was carried out following the above methodology. Eighty-four tomatoes fruits were separated in 6 groups of 14 members each, and maintained at room temperature (26 C) in humidied chambers. One tomato from each group was extracted daily during this sampling period. Treatments applied were as follows: (1) nontreated fruits, (2) inoculated and chitosan-treated fruits (nonrotten esh), (3) inoculated fruits (nonrotten esh), (4) chitosan-treated fruits, (5) inoculated and chitosantreated fruits (rotten esh) and (6) inoculated fruits (rotten esh). Treatments were arranged in a randomized experimental design. Experiment was repeated three times. 3. Results 3.1. Experiment 1. Effect of chitosan on the cellular changes of mycelium and conidia of A. alternata Examination of ultrasections of the hyphae and conidia of chitosan- treated A. alternata, revealed marked alterations on the cell wall. The nontreated mycelium showed an even and continuous cell wall showing no fractures along of the cell wall and membrane

(Fig. 1a and b). Compared to these observations, the chitosan-treated mycelium showed predominantly loosened cell walls and in some areas, lysis was observed as well (Fig. 1c and d). Untreated conidia were normal in appearance, germination took place unaltered, cell walls were also continuous and even (Fig. 2a and b) whilst the conidia exposed to chitosan were intensely damaged, usually eroded and broken cell walls were seen (Fig. 2c and d) containing in some cases no cytoplasm.

3.2. Experiment 2. Cellular changes by effect of chitosan application during the interaction tomatoA. alternata For all treatments (nontreated and chitosan-treated), infection by A. alternata took place. Examinations showed that at the beginning of the infection process (time 0) fungal conidia remained ungerminated (Fig. 3a and d) with no physical damage on the host cells. During the following two periods of incubation (3 and 6 days), in the inoculated and non-chitosan-treated fruits, apparently the hyphae developed intra and intercellulary (Fig. 3b and c), invading the host completely, whilst in chitosan-treated fruits the fungus invaded intercellularly (Fig. 3e and f). It seems that the invasion of the hypahe of A. alternata was faster in the untreated fruits in comparison with the chitosan-treated ones.

Fig. 1. Transmission electron microscopy micrographs of A. alternata hyphae. Chitosan free hyphae (a and b). Cell wall (cw) and membrane (M) well delimited, continuous and even, containing various organelles. Chitosan-treated hyphae (c and d). Uneven cell walls, portions loosened and broken in various areas of the treated hyphae. In some areas cell wall disintegrated.

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Fig. 2. Transmission electron micro scopy micrographs of conidia of A. alternata. Chitosan free conidia (a and b). Unaltered germination septae (s) well formed and delimited. Regular cell wall (cw). Chitosan-treated conidia (c and d). Broken and separated cell wall (cw). Devoid of cytoplasm (cy).

Fig. 3. Transmission electron microscopy micrographs of chitosan-nontreated and treated tomato and infected by A. alternata over 6 days. Infection process of non-chitosantreated tomato. Normal germination of fungal spore (fs), hyphae (h) developing intracellularly invading host cell (hc) (a, b and c). Infection process of treated-chitosan tomato. Germination took place normally. Apparently hyphae growth intercellularly (d, e and f).

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3.3. Experiment. Isolation of rhisitin and other compounds of noninoculated and inoculated tomatoes by A. alternata and chitosantreated in healthy and rotten fruits Rhisitin production was not recorded in fruits from any of the treatments. Overall, the chemical prole during the 14-day storage period was different among treatments. As observed in Table 1, the dichloromethane and aqueous-methanolic extracts showed a total of 19 products. Although a specic pattern of compounds was not observed between the treated and nontreated-chitosan tomatoes, in rotten and non rotten tissues, 11 additional chemical constituents were observed with respect to the nontreated fruits such as palmitic acid, palmitol, and palmitic acid methyl ester. Other compounds detected were linoleic and stearic acids, and palmitic stearic acid isopropyl esters. Oleic, linolenic, and stearic acids were also observed, as well as hentriacontane, vitamin E, and escualene. 4. Discussion In recent years, several potential biological uses of chitosan in agriculture have been identied; thus; interest in its application has increased. Through the TEM studies we could observe serious alterations on the conidia and hyphae of A. alternata at the ultrastructural level. According to our observations, we can establish a relationship between the ultrastructural alterations and the fungicidal effect. In this case, the injuries caused a total breakdown of the fungal cells that doubtless involved A. alternata survival. In this investigation, it is shown that A. alternata is quite susceptible to chitosan treatments. It was demonstrated that the cell wall of the fungal hyphae and conidia was seriously damaged. Cytoplasm disorganization, plasmatic membrane retraction, conidial collapse and plasmolysis were unequivocal signs of the effect of chitosan on A. alternata previously reported for R. stolonifer, F. oxysporum f.sp. radicis-lycopersici and Pochonia chlamidosporia treated with chitosan [6,9]. These damages were frequently observed in conidia of A. alternata, leading to the total loss of cellular material. The presence of the morphological and ultrastructural damages in conidia of A. alternata due to chitosan application might be explained by the recent results reported by Palma-Guerrero et al. [21]. New ndings about the permeabilization of the plasma membrane of different cell types of the fungi Neurospora crassa and the

membrane composition among various resistant and nonresistantchitosan fungi seem to be the key factors that involve the mode of action of chitosan [21,22]. In further studies [23], it was also demonstrated that chitosan application to R. stolonifer affected potassium efux, pH of the media and H+-ATP-ase of the plasma membrane activity. The induction of resistance through the formation of certain structures that impede the entrance and dissemination of fungi inside the host is broadly documented [12,24,25]. In those studies, it is reported that in other pathosystems, chitosan-induced biochemical and physical changes limit the penetration and development of the fungi in the host tissues. In our investigation, we did not conclusively detect the formation of those structures previously reported. This structural induction has been shown in different organs of much lower water content than tomato such as roots and leaves of the same fruits. The soft consistency of tomato fruits favored their rapid decomposition and hence it was very difculty to work with this biological material in the laboratory. In our investigation rhisitin was not isolated from any of the treatments. In a previous investigation [17], where rhisitin was isolated from chitosan-treated and inoculated tomatoes, fruits were rinsed in sodium hypochlorite during the assay, circumstance that might affect the fruits and probably generate the production of rhisitin. Hong and Gross [26], suggested that application in postharvest experiments in tomato fruits altered their physiological and biochemical responses. In that study rmness, electrolyte leakage, rate of respiration and ethylene production of sliced fruits were altered. In addition, it is known that phytoalexins are not only produced after infection of plants but as a result of other chemical or physical stresses such as cupric chloride application and ultraviolet radiation [27,28]. Cruiskshank [29], reported that certain fungi can tolerate, detoxify, suppress and avoid phytoalexins induction. In our research, the inoculated tomatoes, including those chitosan-treated, showed an advanced infection after 14 days storage, demonstrating the lack of resistant of tomatoes to A. alternata infection. Lo et al. [30], reported that although various phytoalexins may be induced, some of them are induced exclusively in resistant interactions; for example, the phytoalexin luteolinidin was induced only in resistant sorghum inoculated with Colletotrichum sublineolum.

Table 1 Summary of the chemical prole of chitosan-treated tomato fruits by A. alternata and chitosan-treated and noninoculated, and inoculated after 14 days storage. Compounda rt (min) Treatment T1 2,3-Butanediol 4-Hydroxy-2,5-dimethyl-3(2H)furanone 2,3-Dihydro-3,5-dihydroxy-6-methyl 4-piranone 5-Hydroxymethyl-2-furancarboxaldehyde Palmitic acid Palmitol Palmitic acid methyl ester Linoleic acid Stearic acid Palmitic acid isopropyl ester Linoleic acid methyl ester Oleic acid methyl ester Stearic acid methyl ester Oleic acid Linolenic acid Stearic acid Hentriacontane Vitamin E Escualene 5.6 8.9 11.2 11.9 19.4 20.1 20.6 20.9 21.1 21.5 22.3 22.4 22.5 22.7 22.8 22.9 24.3 25.7 35.8 x x x x x x x x T2 x x x x x x x x x x x x x x T3 x x x x x x x x x x x T4 x x x x x x x x x T5 x x x x x x x x x T6 x x x x x x x x x x x x

a GCMS analysis of the dichloromethane and aqueous-methanolic extracts. Method: Splitless 40250 C 10 C/min. rt = retention time; x = present and = nonpresent compound. Treatments: T1 = nontreated, T2 = chitosan immersed and inoculated, nonrotten fruit, T3 = inoculated, nonrotten fruit, T4 = chitosan immersed, T5 = chitosan immersed and inoculated, rotten fruit, and T6 = inoculated, rotten fruit.

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In the inoculated tomatoes, there were a higher quantity and variety of compounds than in the noninoculated tomatoes. We believe that their production was more associated with infection than with chitosan application. Some of the identied compounds belong to the group of alkanes that according to Kunst and Samuels [31], have different attributes including certain activity against bacteria and fungi. Other signicant compounds detected in our study were the fatty acids (palmitic, linoleic, stearic and oleic acids) which are also associated with antimicrobial activity. It is reported that these compounds are converted to secondary metabolites such as oxylipines known for stimulating defense genes, and regulating plant growth and development [32]. However, the production of fatty acids may also be associated with the decomposition of the fruits tissue as a consequence of the enzymatic activity of A. alternata on the fruits. El-Shareb and Malibari [33], reported that in addition to macerating enzymes this fungus produces fatty acids. In our study, the presence of the vitamin E only in chitosantreated and inoculated fruits indicates that this combination probably induced the synthesis of this product. It is reported that this compound is one of the rst reactions produced in response to infection by microorganisms which will eventually trigger other defensive reactions. In conclusion this study demonstrates that chitosan affects at the structural level the mycelium and conidia of A. alternata. We believe that the elicitation of precursor compounds in the pathosystem A. alternatatomato was more associated with the infection process than with the chitosan application. Further studies are necessary to conrm these ndings. References
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