Pesticide Biochemistry and Physiology 99 (2011) 250–255

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Pesticide Biochemistry and Physiology

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Cytological and biochemical changes induced by chitosan in the pathosystemAlternaria alternata–tomato

D. Sánchez-Domínguez a, M.Y. Ríos b, P. Castillo-Ocampo c, G. Zavala-Padilla d, M. Ramos-García e,S. Bautista-Baños e,⇑a Centro de Bachillerato Tecnológico, Agropecuario No. 8, Km. 8.5 Carretera, Alpuyeca-Jojutla, Xoxocotla, Morelos, C.P. 62780, Mexicob Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos, C.P. 62209, Mexicoc Universidad Autónoma Metropolitana-Iztapalapa, Laboratorio de Microscopía Electrónica, Av. San Rafael Atlixco # 186, Colonia Vicentina, Delegación Iztapalapa, C.P. 09340, D.F.,Mexicod Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico, Av. Universidad 1001, Chamilpa, Cuernavaca, Morelos, C.P. 62209, Mexicoe Instituto Politécnico Nacional, Centro de Desarrollo de Productos Bióticos, Km. 6 Carr, Yautepec-Jojutla, Col. San Isidro, CEPROBI 8, Yautepec, Morelos, C.P. 62731, Mexico

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 May 2010Accepted 9 January 2011Available online 12 January 2011

Keywords:Black moldChemical profileConidiaLycopersicon esculentum L.Mycelium

0048-3575/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.pestbp.2011.01.003

⇑ Corresponding author.E-mail address: sbautis@ipn.mx (S. Bautista-Baños

The cytological and biochemical response of the fungus Alternaria alternata to chitosan application intomato fruits was evaluated. The research was developed in the following stages: microscopically toobserve the degree of damage that chitosan causes over the conidia and hyphae of the fungus at thestructural level and during the infection process in tomato tissue. Biochemically we tried to identifythe elicitation of the phytoalexin rhisitin and other compounds involved in resistance. At the microscopiclevel, mycelium and conidia of chitosan-treated of A. alternata showed cell wall disintegration, plasmamembrane retraction, cellular distortion, release of the apical portion of the conidia and lysis of fungalcells. Hyphae and conidia were susceptible to chitosan application. Infection always took place in chito-san treated and inoculated tomatoes and it was difficult to observe ultrastructural alterations due tochitosan application. The phytoalexin rhisitin was not isolated from any of the treatments but other com-pounds such as alkenes, fatty acids and vitamin E whose antimicrobial effects have been reported weredetected. The elicitation of precursor compounds in the pathosystem A. alternata–tomato was more asso-ciated with the infection process than with the chitosan application. Further studies are necessary to con-firm these findings.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

The incidence of disease is a major factor limiting the posthar-vest life of tomato fruits. Among others, the black mould rot dis-ease caused by Alternaria alternata (Fr.:Fr.) Keissl grows over thesurface of the fruits, affecting all stages of ripening [1]. For the to-mato industry in Mexico, black mould rot has become the most sig-nificant disease since most tomato genotypes are quite susceptibleto A. alternata.

Chitosan, a chitin derivative is a natural biodegradable com-pound derived from crustaceous shells such as crabs and shrimps,has been extensively mentioned as a natural fungicide. In numer-ous in vitro and in situ investigations the inhibitory effect of chito-san on important phytopathogenic genera has been demonstrated[2,3]. In previous studies, it was reported that chitosan affectedthe spore viability of A. alternata [4], whilst in other findings the de-gree of inhibition of the mycelial growth and sporulation of this

ll rights reserved.

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fungus isolated from tomato fruits relied on the molecular weightof chitosan [5]. These authors also reported in observations carriedout by scanning electron microscopy, that chitosan modified themycelia morphology of A. alternata causing deformation, swollen-ness and nodulations. At the ultrastructural level, it has beenmentioned that chitosan affects the integrity and structure of thecell wall of the hyphae and conidia of Fusarium oxysporum f. sp.radici-lyicopersici and Rhizopus stolonifer as well as the hyphae ofPhythophthora capsici; in some cases the cellular organelles suchas vacuoles and mitochondria of these microorganisms were seri-ously damaged [6–10]. In addition to this fungicidal potential,chitosan is mentioned as inducing host–defense responses thatmay restrict the pathogen entrance. In earlier reports carried outat ultrastructural level, chitosan caused considerable cellular re-sponses from the infected host such as depositions of electronopaque material along the primary cell walls and intercellularspaces, deposition of fibrillar material, and formation of papillaeand bubble-like structures in xylem vessels of roots and leaves ofchitosan-treated tomatoes infected by F. oxysporum f. sp. radicis-licopersici, and in roots of chitosan-treated cucumbers, inoculated

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with Phytium aphanidermatum [7,11,12]. In further studies, similarresponses were observed in the tissues of chitosan-treated bell pep-per infected by Botrtytis cinerea [13].

Another defense response associated with chitosan is the elici-tation of antimicrobial compounds such as phytoalexins. One ofthe first studies demonstrating the chitosan capacity to induce de-fence responses was carried out by Walker-Simons et al. [14]. Theyfound that among four different compounds tested, chitosanshowed the highest elicitation of proteinase on tomato leavesand pisatin in pea pods. Additional studies confirmed the abilityof chitosan to elicit this phytoalexin in immature pea pods. Chito-san fragments of high molecular weight induced the synthesis ofpisatin and inhibited Fusarium solani [15]. As shown by Rodríguezet al. [16], rice seeds previously treated with chitosan induced theenzyme phenylalanine ammonia lyase at the seedling stage thatmay eventually lead to the formation of phytoalexins. In otherstudies, the highest quantities of the phytoalexins rhisitin in thechitosan-treated tomato fruits and inoculated with A. alternata oc-curred after 21 days storage [17].

In order to gather more information about the basic aspects inthe relationship between chitosan and the pathosystem tomato-A. alternata, we carried out observations at the ultrastructural levelon chitosan-treated hyphae and conidia of A. alternata. Moreover,during the infection process, we also identified various chemicalcompounds of infected and noninfected chitosan–tomato probablyinvolved in resistance mechanisms.

2. Materials and methods

2.1. General experimental procedures

2.1.1. Microorganism isolationThe fungus A. alternata was isolated from infected tomatoes

presenting the usual symptoms of the disease (sunken lesions lightto dark brown at the epidermal tissue). Identification of this funguswas according to Barnett and Hunter [18]. Further confirmationsconsisted of re-inoculating A. alternata in healthy tomatoes,observing disease development and re-isolating the fungus to con-firm A. alternata conidia. Culture was maintained in Petri platescontaining PDA and incubated at 26 �C. Continuous re-inoculationsand re-isolations on tomato fruits were carried out to maintainpathogenicity of the inoculum.

2.1.2. Chitosan preparation and treatmentsStock solution of chitosan medium molecular weight. (Mw =

2.38 � 104 Da, 75–85% degree of deacetylation; 200–800 cps), (Sig-ma Aldrich, St. Louis, MO, USA) [19] weighing 2.5 g was preparedand then dissolved in 100 ml of distilled water with 1 ml of aceticacid and then heated while constantly agitated for 24 h. pH solu-tion was adjusted to 5.6 by adding sodium hydroxide 1 N and0.1 ml. The chitosan solution was sterilised and adjusted to theconcentration of 1.5% w/v for both in vitro and in situ studies.

2.1.3. Samples processing for studies in transmission electronmicroscopy (TEM)

Previously fixed samples of mycelium, conidia and treated to-mato fruits were dehydrated in a graded ethanol series and embed-ded 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, washedand examined in a Zeiss EM900 transmission electron microscopeat 60 kV.

2.1.4. Gas chromatography analysisGC–MS analyses of the components and rhisitin of tomato fruits

were obtained using an Agilent 6890 GC/5973 N MSD chromato-graph, equipped with a HP-5MS capillary column (5% phen-ylmetylpolysiloxane, length 30 m, id 0.25 mm, ft 0.25 lm). Thecarrier gas was helium and the linear gas velocity was1 mm min�1. The injector temperature was 250 �C and the columntemperature, initially at 40 �C for 2 min, was gradually increased ata rate of 10 �C/min up to 250 �C, and kept at 250 �C for 10 min,splitless mode. For detection, a flame ionization detector at280 �C, IE (70 Ev, Scan 30–550 uma) was used. The identificationof each component was based on a comparison of its mass spec-trum with those contained in the NIST–MS Library.

2.1.5. Validation of the extraction method of rhisitinThe extraction of rhisitin in tomato fruits was carried out fol-

lowing the methodology of Bhaskara-Reddy et al. [17], with somemodifications. Tomato pulp (10 g) added with rhisitin standard(2 mL) was macerated for 24 h with methanol (30 mL � 3), centri-fuged at 1000 rpm (5 min � 3) and decanted. Extraction solventwas concentrated to dryness in vacuo and the residue was sus-pended in 80% aqueous methanol (100 mL) and extracted withdichlorometano (100 mL � 3). Organic and aqueous-methanolphases were concentrated to dryness in vacuo and suspended inthe same solvent (1.5 mL) for GC–MS analysis (rhisitin presencewas detected in the organic extract at 20.06 min in GC–MS analy-sis). Rhisitin was identified by comparing with the standard ob-tained from Dr. Susan McCormick at the National Center forAgricultural Utilization Research, US Department of AgricultureResearch Service, Peoria, Ill.

2.2. Experiment 1. Effect of chitosan on cellular changes of myceliumand conidia of A. alternata

Discs of A. alternata of 14 days of age were placed in the centreof Petri plates containing potato dextrose agar (PDA) amendedwith chitosan of medium molecular weight at 1.5% concentration.Plates were incubated for 6 days at 26 �C. Control Petri plates con-tained only PDA. After the given incubation period, mycelium wastaken from the periphery and fixed in 4% paraformaldehyde and 2%glutaraldehyde in 0.16 M sodium cacodylate buffer at pH 7.0 for1 h. For conidia obtainment, mycelium was flooded with a glucosesolution, scraped from the plate and filtered twice through layersof sterile cheesecloth and fixed as mentioned above. Samples wereprocessed at the Laboratory of Electronic Microscopy of the Auton-omous Metropolitan University, campus Iztapalapa in Mexico City.

2.3. Experiment 2. Cellular changes by effect of chitosan applicationduring the interaction tomato–A. 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 withcommon detergent and thoroughly rinsed with distilled water.After drying, fruits were injured with the aid of a needle and im-mersed in a spore solution (106) of A. alternata for 15 min. Later,they were dipped in chitosan of medium molecular weight at theconcentration of 1.5% for 15 min. Samples were taken after 6 days.Control treatment consisted of fruits inoculated but not immersedin chitosan for a similar period of evaluation. Portions of infectedfruits were fixed as earlier described. Samples were processed atthe Institute of Biotechnology of the National University of Mexicoin Cuernavaca, Morelos.

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2.4. Experiment 3. Isolation of rhisitin and other compounds ofnoninoculated and inoculated tomatoes by A. alternata and chitosan-treated in healthy and rotten fruits

The isolation of rhisitin was carried out following the abovemethodology. Eighty-four tomatoes fruits were separated in 6groups of 14 members each, and maintained at room temperature(26 �C) in humidified chambers. One tomato from each group wasextracted daily during this sampling period. Treatments appliedwere as follows: (1) nontreated fruits, (2) inoculated and chito-san-treated fruits (nonrotten flesh), (3) inoculated fruits (nonrot-ten flesh), (4) chitosan-treated fruits, (5) inoculated and chitosan-treated fruits (rotten flesh) and (6) inoculated fruits (rotten flesh).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 ofmycelium and conidia of A. alternata

Examination of ultrasections of the hyphae and conidia of chito-san- treated A. alternata, revealed marked alterations on the cellwall. The nontreated mycelium showed an even and continuouscell wall showing no fractures along of the cell wall and membrane

Fig. 1. Transmission electron microscopy micrographs of A. alternata hyphae. Chitosan fand even, containing various organelles. Chitosan-treated hyphae (c and d). Uneven cellareas cell wall disintegrated.

(Fig. 1a and b). Compared to these observations, the chitosan-trea-ted mycelium showed predominantly loosened cell walls and insome areas, lysis was observed as well (Fig. 1c and d). Untreatedconidia were normal in appearance, germination took place unal-tered, 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) con-taining in some cases no cytoplasm.

3.2. Experiment 2. Cellular changes by effect of chitosan applicationduring the interaction tomato–A. alternata

For all treatments (nontreated and chitosan-treated), infectionby A. alternata took place. Examinations showed that at the begin-ning of the infection process (time 0) fungal conidia remainedungerminated (Fig. 3a and d) with no physical damage on the hostcells. During the following two periods of incubation (3 and6 days), in the inoculated and non-chitosan-treated fruits, appar-ently the hyphae developed intra and intercellulary (Fig. 3b andc), invading the host completely, whilst in chitosan-treated fruitsthe fungus invaded intercellularly (Fig. 3e and f). It seems thatthe invasion of the hypahe of A. alternata was faster in the un-treated fruits in comparison with the chitosan-treated ones.

ree hyphae (a and b). Cell wall (cw) and membrane (M) well delimited, continuouswalls, portions loosened and broken in various areas of the treated hyphae. In some

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-chitosan-treated 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 ofnoninoculated and inoculated tomatoes by A. alternata and chitosan-treated in healthy and rotten fruits

Rhisitin production was not recorded in fruits from any of thetreatments. Overall, the chemical profile during the 14-day storageperiod was different among treatments. As observed in Table 1, thedichloromethane and aqueous-methanolic extracts showed a totalof 19 products. Although a specific pattern of compounds was notobserved between the treated and nontreated-chitosan tomatoes,in rotten and non rotten tissues, 11 additional chemical constitu-ents were observed with respect to the nontreated fruits such aspalmitic acid, palmitol, and palmitic acid methyl ester. Other com-pounds detected were linoleic and stearic acids, and palmitic stea-ric acid isopropyl esters. Oleic, linolenic, and stearic acids were alsoobserved, as well as hentriacontane, vitamin E, and escualene.

4. Discussion

In recent years, several potential biological uses of chitosan inagriculture have been identified; thus; interest in its applicationhas increased. Through the TEM studies we could observe seriousalterations on the conidia and hyphae of A. alternata at the ultra-structural level. According to our observations, we can establish arelationship between the ultrastructural alterations and the fungi-cidal effect. In this case, the injuries caused a total breakdown ofthe fungal cells that doubtless involved A. alternata survival. In thisinvestigation, it is shown that A. alternata is quite susceptible tochitosan treatments. It was demonstrated that the cell wall ofthe fungal hyphae and conidia was seriously damaged. Cytoplasmdisorganization, plasmatic membrane retraction, conidial collapseand plasmolysis were unequivocal signs of the effect of chitosanon A. alternata previously reported for R. stolonifer, F. oxysporumf.sp. radicis-lycopersici and Pochonia chlamidosporia treated withchitosan [6,9]. These damages were frequently observed in conidiaof A. alternata, leading to the total loss of cellular material. Thepresence of the morphological and ultrastructural damages in con-idia of A. alternata due to chitosan application might be explainedby the recent results reported by Palma-Guerrero et al. [21]. Newfindings about the permeabilization of the plasma membraneof different cell types of the fungi Neurospora crassa and the

Table 1Summary of the chemical profile of chitosan-treated tomato fruits by A. alternata and chit

Compounda rt (min) Tr

T1

2,3-Butanediol 5.6 x4-Hydroxy-2,5-dimethyl-3(2H)furanone 8.9 x2,3-Dihydro-3,5-dihydroxy-6-methyl 4-piranone 11.2 x5-Hydroxymethyl-2-furancarboxaldehyde 11.9 xPalmitic acid 19.4 –Palmitol 20.1 –Palmitic acid methyl ester 20.6 –Linoleic acid 20.9 –Stearic acid 21.1 xPalmitic acid isopropyl ester 21.5 xLinoleic acid methyl ester 22.3 –Oleic acid methyl ester 22.4 –Stearic acid methyl ester 22.5 –Oleic acid 22.7 xLinolenic acid 22.8 –Stearic acid 22.9 xHentriacontane 24.3 –Vitamin E 25.7 –Escualene 35.8 –

a GC–MS analysis of the dichloromethane and aqueous-methanolic extracts. Method:compound. Treatments: T1 = nontreated, T2 = chitosan immersed and inoculated, nonrimmersed and inoculated, rotten fruit, and T6 = inoculated, rotten fruit.

membrane composition among various resistant and nonresis-tant–chitosan fungi seem to be the key factors that involve themode of action of chitosan [21,22]. In further studies [23], it wasalso demonstrated that chitosan application to R. stolonifer affectedpotassium efflux, pH of the media and H+-ATP-ase of the plasmamembrane activity.

The induction of resistance through the formation of certainstructures that impede the entrance and dissemination of fungiinside the host is broadly documented [12,24,25]. In those stud-ies, it is reported that in other pathosystems, chitosan-inducedbiochemical and physical changes limit the penetration anddevelopment of the fungi in the host tissues. In our investiga-tion, we did not conclusively detect the formation of those struc-tures previously reported. This structural induction has beenshown in different organs of much lower water content than to-mato such as roots and leaves of the same fruits. The soft con-sistency of tomato fruits favored their rapid decomposition andhence it was very difficulty to work with this biological materialin the laboratory.

In our investigation rhisitin was not isolated from any of thetreatments. In a previous investigation [17], where rhisitin was iso-lated from chitosan-treated and inoculated tomatoes, fruits wererinsed in sodium hypochlorite during the assay, circumstance thatmight affect the fruits and probably generate the production ofrhisitin. Hong and Gross [26], suggested that application in post-harvest experiments in tomato fruits altered their physiologicaland biochemical responses. In that study firmness, electrolyte leak-age, rate of respiration and ethylene production of sliced fruitswere altered. In addition, it is known that phytoalexins are not onlyproduced after infection of plants but as a result of other chemicalor physical stresses such as cupric chloride application and ultravi-olet radiation [27,28]. Cruiskshank [29], reported that certain fungican tolerate, detoxify, suppress and avoid phytoalexins induction.In our research, the inoculated tomatoes, including those chito-san-treated, showed an advanced infection after 14 days storage,demonstrating the lack of resistant of tomatoes to A. alternatainfection. Lo et al. [30], reported that although various phytoalex-ins may be induced, some of them are induced exclusively in resis-tant interactions; for example, the phytoalexin luteolinidin wasinduced only in resistant sorghum inoculated with Colletotrichumsublineolum.

osan-treated and noninoculated, and inoculated after 14 days storage.

eatment

T2 T3 T4 T5 T6

– – – – –x x x – –x x x – –– – x – –x x x – xx x – x xx x – x xx x x – xx x x x xx – x – –x x x x x– – – – x– – – x xx – x – –– x – x xx x – – xx – – x xx – – x –x x – x x

Splitless 40–250 �C 10 �C/min. rt = retention time; x = present and – = nonpresentotten fruit, T3 = inoculated, nonrotten fruit, T4 = chitosan immersed, T5 = chitosan

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In the inoculated tomatoes, there were a higher quantity andvariety of compounds than in the noninoculated tomatoes. We be-lieve that their production was more associated with infection thanwith chitosan application. Some of the identified compounds be-long to the group of alkanes that according to Kunst and Samuels[31], have different attributes including certain activity againstbacteria and fungi. Other significant compounds detected in ourstudy were the fatty acids (palmitic, linoleic, stearic and oleicacids) which are also associated with antimicrobial activity. It is re-ported that these compounds are converted to secondary metabo-lites such as oxylipines known for stimulating defense genes, andregulating plant growth and development [32]. However, the pro-duction of fatty acids may also be associated with the decomposi-tion of the fruits tissue as a consequence of the enzymatic activityof A. alternata on the fruits. El-Shareb and Malibari [33], reportedthat in addition to macerating enzymes this fungus produces fattyacids. In our study, the presence of the vitamin E only in chitosan-treated and inoculated fruits indicates that this combination prob-ably induced the synthesis of this product. It is reported that thiscompound is one of the first reactions produced in response toinfection by microorganisms which will eventually trigger otherdefensive reactions.

In conclusion this study demonstrates that chitosan affects atthe structural level the mycelium and conidia of A. alternata. Webelieve that the elicitation of precursor compounds in the patho-system A. alternata–tomato was more associated with the infectionprocess than with the chitosan application. Further studies are nec-essary to confirm these findings.

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