Revista Mexicana de Fitopatología

ISSN: 0185-3309

mrlegarreta@prodigy.net.mx

Sociedad Mexicana de Fitopatología, A.C.

México

Bautista Baños, Silvia; Hernández López, Mónica; Bosquez Molina, Elsa

Growth Inhibition of Selected Fungi by Chitosan and Plant Extracts

Revista Mexicana de Fitopatología, vol. 22, núm. 2, julio-diciembre, 2004, pp. 178-186

Sociedad Mexicana de Fitopatología, A.C.

Texcoco, México

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Growth Inhibition of Selected Fungi by Chitosan and PlantExtracts

Silvia Bautista-Baños, Mónica Hernández-López, Instituto Politécnico Nacional, Centrode Desarrollo de Productos Bióticos, km 8.5 Carr. Yautepec-Jojutla, San Isidro Yautepec,Morelos, México CP 62731; and Elsa Bosquez-Molina, Universidad AutónomaMetropolitana-Iztapalapa, Av. San Rafael Atlixco 186 Michoacán y la Purísima, Col.Vicentina, Mexico, D.F. CP 09340. Correspondence to: sbautis@ipn.mx

Bautista-Baños, S., Hernández-López, M., and Bosquez-Molina, E. 2004. Growth inhibition of selected fungi bychitosan and plant extracts. Revista Mexicana deFitopatología 22:178-186.Abstract. Chitosan and plant aqueous extracts were evaluatedfor growth inhibition of Fusarium oxysporum, Penicilliumdigitatum and Rhizopus stolonifer in vitro. Except for P.digitatum sporulation, chitosan at 1.5% onwards inhibitedboth mycelial growth and sporulation as compared with plantextracts. F. oxysporum was the most sensitive to chitosan.Aqueous extracts had a slightly effect on mycelial inhibition,while papaya seed extract had effect only on R. stolonifersporulation. In most treatments, growth rate was differentamong the three fungi. A synergistic effect was observed onlyon Penicillium treated with the combination of chitosan andaqueous papaya seed extract. Conidia morphology of the threefungi varied within each organism when treated with chitosan.The area, length and conidia morphology were differentaccording to the fungus, treatment or/and incubation time, ascompared to the control.

Additional keywords: Fusarium oxysporum, Penicilliumdigitatum, Rhizopus stolonifer, natural compounds.

Resumen. Se evaluó in vitro el efecto inhibidor del quitosanoy extractos acuosos de plantas sobre el crecimiento deFusarium oxysporum, Penicillium digitatum y Rhizopusstolonifer. Con la excepción de la esporulación de P.digitatum, el quitosano al 1.5% en adelante inhibió elcrecimiento micelial y la esporulación en comparación conlos extractos. F. oxysporum fue el más sensible al quitosano.Los extractos acuosos tuvieron un ligero efecto sobre lainhibición micelial, mientras que los extractos de semilla depapaya tuvieron efecto solamente en la esporulación de R.stolonifer. En la mayoría de los tratamientos, la tasa decrecimiento fue diferente entre los tres hongos. Se observóun efecto sinergístico únicamente sobre Penicillium tratadocon la combinación de quitosano y extracto acuoso de semillade papaya. La morfología de los conidios en los tres hongosvarió cuando se trataron con quitosano. El área, longitud y

morfología de los conidios fue diferente de acuerdo al hongo,el tratamiento y/o tiempo de incubación, en comparacióncon el testigo.

Palabras clave adicionales: Fusarium oxysporum, Penicilliumdigitatum, Rhizopus stolonifer, compuestos naturales.

Chitosan is a natural, biodegradable, high molecular weightpolymer with a wide range of uses in cosmetology, the foodindustry, biotechnology, medicine and agriculture (Hirano1989; Sandford 1989). For the agricultural industry, chitosanhas been reported to be useful as a soil modifier, and toimprove seed and flower quality, as well as increasing cropyields (Bhaskara-Reddy et al., 1999; Ohta et al., 1999; Renet al., 2001). Another important attribute of this naturalcompound is associated with its fungistatic or fungicidalproperties against pathogens of various fruits (Bautista-Bañoset al., 2003b; Li and Yu, 2000; Zhang and Quantick, 1997,1998). Growth of important postharvest fungi such asAlternaria alternata (Fries) Keissler, Colletotrichumgloeosporioides (Penz.) Penz. and Sacc., Fusariumoxysporum Schlecht., Rhizopus stolonifer (Ehrenb. Fr.) Lind.,and Penicillium spp. was inhibited on nutrient media amendedwith various concentrations of chitosan (Bautista et al., 1999;Benhamou, 1992; Bhaskara-Reddy et al., 1997; Hirano andNagao, 1989). The antimicrobial properties of plant extractsfrom various species have proven to control fungaldevelopment either in vitro or in vivo. In in vitro studies,Bautista-Baños et al. (2000) reported that among 19 differentbotanical species tested, the aqueous leaf extracts of custardapple (Annona reticulata L.) and papaya (Carica papaya L.)among others, inhibited germination and spore formation ofR. stolonifer. Same conidial inhibition was observed whenC. gloeosporioides was grown on these two extracts (Bautista-Baños et al., 2002). Fusarium oxysporum, Penicilliumdigitatum Link. and Rhizopus stolonifer are importantpostharvest fungi causing severe diseases during storage offruits and vegetables (Snowdon, 1990). The objective of thisresearch was to evaluate the effect of chitosan, aqueous leafextracts of custard apple and papaya, and seeds extract of

(Received: November 3, 2003 Accepted: February 27, 2004)

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papaya on mycelial growth, sporulation and conidiamorphology of F. oxysporum, P. digitatum and R. stolonifer.

MATERIALS AND METHODSPlant material and growing media. Custard apple andpapaya leaves were collected from commercial orchardslocated in the state of Morelos, Mexico. Papaya seeds wereobtained from mature papaya fruit cv. Maradol. Chitosan(trade name Fluka) was obtained from BioChemika, Sigma-Aldrich. The growing medium, potato-dextrose-agar (PDA)(trade name Bioxon) was obtained from Becton.Strains and culture conditions. Fusarium oxysporum,Penicillium digitatum and Rhizopus stolonifer strains wereisolated from infected papaya and identified according topublished descriptions (Barnett and Hunter, 1972; Streets,1984). Cultures were maintained in Petri plates containingPDA, and incubated at 20°C.Plant extracts and chitosan preparation. Plant material wasselected discarding the damaged or diseased leaves or seeds.Plant material was disinfected in sodium hypochlorite (10%)and rinsed in distilled water, air dried and macerated with theaid of a blender, and stored at ambient temperature in amberbottles for further use. Aqueous extracts of plant material (2:10w/v) were left at room temperature (25-28°C) for 24 h, filteredand autoclaved. Before sterilization, extracts wereincorporated (10:4 v/v) with PDA. Chitosan solutions wereprepared weighing 0.5, 1.5, 2.5, and 3.0 g of chitosan, andthen dissolved in 100 ml of distilled water with 2 ml of aceticacid, and heated to 35°C, while constantly agitating for 24 h.The pH solution was adjusted to 5.5 adding sodium hydroxide1N and 0.1 ml of Tween 80 (El Ghaouth et al., 1991).Evaluation of the effect of chitosan and/or plant extractson sporulation and mycelial growth. The effect of chitosanand/or the three plant extracts on growth of F. oxysporum, P.digitatum, and R. stolonifer was studied using PDA agarplates. A PDA agar disk of a pure culture of each strain wasplaced onto the center of a PDA plate containing chitosan at0.5, 1.5, 2.5, and 3.0% or aqueous leaf extracts of custardapple and leaves or papaya seeds (10/4 v/v). The effect ofthe combination of each plant extract with chitosan at 2.5%was evaluated as well. Petri plates were inoculated intriplicates and incubated at 25°C for 10, 7 or 4 days for F.oxysporum, P. digitatum, and R. sotolnifer, respectively. Daily,radial growth measurements were taken until fungi coveredthe plate. The number of spores/mL of filtrate was determinedusing a Neubauer haemocytometer. Spores were harvestedby scrapping off the agar with the aid of a glass rod anddistilled water. The mycelium and the spore mixture werethen double filtered through cheesecloth, and the resultingfiltrate was adjusted to 20 mL. Observations were determinedat 40X magnification.Evaluation of the effect of chitosan on conidiamorphology. Conidia of F. oxysporum, P. digitatum and R.stolonifer previously grown on PDA amended with 1.5%chitosan were incubated for 7 h on water agar. The area (µm2),

length (µm), and the elliptical (F. oxysporum) or circular (P.digitatum and R. stolonifer) form were measured at thebeginning, and after the 7 h incubation period. Images ofconidia were obtained using a microscope (Nikon, Alphaphot-2 YS2) with a charged-coupled device camera (Nikon,Coolpix 900). Magnification of the image was 40X. Theimage analysis software was Meta Imaging series, MicrosoftWindows (version 4.0, Universal Imaging Corporation, USA).Forty conidia per treatment were measured.Statistical analysis. Chitosan and/or plant extracts treatmentswere arranged in a completely randomized design, while thespore morphology experiment was designed as a factorial.Means separation by Tukey’s multiple range test (p = 0.05)were carried out for all parameters. For the three fungi tested,and to fulfill one of the ANOVAR assumptions, data frommycelial inhibition was arcsine transformed, while sporulationwas square root transformed. Area, length, and form data werelog transformed. A simple linear regression was carried outwith data of daily mycelial growth.

RESULTSMycelial inhibition and sporulation had highly significantdifferences (p = 0.001) among treatments for the three fungistudied (Tables 1 and 2). Chitosan at 1.5% onwards waseffective in partially inhibiting growth of Fusarium, while at1.5% concentration Penicillium and Rhizopus growth wassignificantly less than control treatment (Table 1). Mycelialgrowth inhibition of F. oxysporum and P. digitatum was highercompared to the controls when treated with custard appleleaves or papaya seeds extracts, respectively. The combinationof chitosan and papaya seed extracts significantly affected F.oxysporum and R. stolonifer growth, while the combinationof chitosan and papaya leaves extracts affected P. digitatummycelial growth. Growth of Fusarium at 0.5% began afterthe second day of treatment until the tenth day incubationtime with a growth rate of 0.23 (R2 = 0.94), while the controlfollowed a normal growth rate (0.50, R2 = 0.92) (Fig. 1A).Fusarium treated with plant extract showed similar growthrates compared to the control (Fig. 1B), while the combinationof chitosan and both papaya seed and leaf extracts showedthe least growth rate (Fig. 1C). Mycelial growth of Penicilliumbegan after the second day on chitosan at 1.5% (Fig. 2A).Except for the aqueous papaya seed extract, the remainingaqueous extracts treatments showed similar growth rate (0.52-0.57) compared to the control (Fig. 2B). Growth rate ofPenicillium held on the combinations of chitosan and plantextracts was similar (0.33-0.37) (Fig. 2C). Rhizopus growthbegan after the first day of incubation with chitosan at 1.5%following until the fourth day (0.61, R2 = 0.89) (Fig. 3A).When treated with plant extract, same growth was showncompared to the control (Fig. 3B), while growth began afterthe first day of incubation when treated with chitosan andplant extracts, showing the least growth rate with papaya seedsor custard apple leaf extracts (0.75, R2= 0.93 and 0.79, R2 =0.79, respectively) (Fig. 3C). Sporulation of Fusarium was

Revista Mexicana de FITOPATOLOGIA/ 179

significantly reduced (p = 0.001) when treated with chitosanat 0.5%, while Penicillium showed significantly highersporulation at both chitosan concentrations (0.5 and 1.5%)(p = 0.001) compared to the control. Chitosan at 1.5%significantly reduced sporulation of R. stolonifer (p = 0.001)(Table 2). Sporulation of Fusarium and Penicillium increasedwith plant extracts, while sporulation of Rhizopus was reduced

with extract of papaya seed. Compared to the control, thecombinations of chitosan at 2.5% with plant extracts increasedspore formation of Fusarium as compared to the control, whilethe contrary occurred for Penicillium and Rhizopus. WhenF. oxysporum was incubated for 7 h, the area of spores wassignificantly different from the control (p = 0.001) (Table 3).For the main effect, the form, and length of conidia of P.

Table 1. Effect of chitosan and/or plant extracts on mycelial growth inhibition of Fusariumoxysporum, Penicillium digitatum, and Rhizopus stolonifer. Mycelial growth inhibition (%)z

Treatments F. oxysporum P. digitatum R. stoloniferChitosan (%): P = 0.001 P = 0.001 P = 0.0010.5 45 b 0 c 0 c1.5 100 a 62 b 52 b2.5 100 a 100 a 100 a3.0 100 a 100 a 100 aControl 0 c 0 c 0 cPlant aqueous extracts (w/v): P = 0.001 P = 0.001 NSPapaya leaves 0 c 5 c 0Papaya seeds 7.5 b 25 a 0Custard apple leaves 10 a 10 b 0Control 0 c 0 d 0Chitosan 2.5% + plant aqueous extracts: P = 0.001 P = 0.001 P = 0.001Chitosan + papaya leaves extracts 2.5 b 46 a 36 bChitosan + papaya seeds extracts 39 a 43 b 43 aChitosan + custard apple leaves extracts 2.5 b 43 b 36 bControl 0 0 c 0 c

zAverage of three replicates; means separation within columns by Tukey’s multiple test (p =0.05). NS = nonsignificant. P values after arcsine transformation.

Table 2. Effect of chitosan and/or plant extracts on sporulation of Fusarium oxysporum, Penicilliumdigitatum, and Rhizopus stolonifer. Sporulation (spores/ml)y

Treatments F. oxysporum P. digitatum R. stoloniferChitosan (%): P = 0.001 P = 0.001 P = 0.0010.5 3.5 x 105 a 4.2 x 107 a 1.5 x 106 b1.5 ----------z 2.1 x 107 b 1.3 x 106 c2.5 ---------- ---------- ----------3.0 ---------- ---------- ----------Control 4.0 x 105 b 1.8 x 107 c 1.8 x 106 aPlant aqueous extracts (w/v): P = 0.01 P = 0.01 P = 0.01Papaya leaves 5.6 x 105 b 3.8 x 107 b 4.1 x 106 aPapaya seeds 6.0 x 105 b 8.3 x 107 a 0.83 x 106 cCustard apple leaves 8.3 x105 a 2.3 x107 c 1.0 x 106 bControl 4.4 x 105 c 2.4 x107 c 1.4 x 106 bChitosan 2.5% + plant aqueous extracts: P = 0.001 P = 0.001 P = 0.001Chitosan + papaya leaves extracts 1.8 x 106 a 1.4 x 106 b 7.1 x 105 bChitosan + papaya seeds extracts 1.3 x 106 b 1.1 x 106 d 4.1 x 105 dChitosan + custard apple leaves extracts 3.6 x 106 b 1.3 x 106 c 6.2 x 105 cControl 4.0 x 105 c 1.8 x 107 a 1.8 x 106 a

yAverage of three replicates; means separation within columns by Tukey’s multiple test (p = 0.05).P values after log transformationzSince mycelial growth did not take place, no spores were counted.

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less mycelial growth and spore formation of R. stolonifer andB. cinerea as the chitosan concentration increased (El Ghaouthet al., 1992). Nevertheless, in this study, chitosan stimulatedsporulation of P. digitatum. The same effect was reported byBhaskara-Reddy et al. (1998) on Alternaria alternata (Fr.:Fr.)Keissl. grown at sub-lethal doses of chitosan. Those authorsindicate that this high sporulation might be due to a stressresponse caused by chitosan. Microscopically, studies havedemonstrated cellular and morphological alterations of fungalhyphae in contact with chitosan (Benhamou, 1992; ElGhaouth et al., 1994; 1997). In those studies, a severe cellulardamage was observed on mycelium of fungi treated withchitosan. For more accuracy on the effects of this compoundon conidial morphology of these tested fungi, further studiesare necessary. Contrary to other findings, aqueous extractshad a slightly effect on mycelial development, while papayaseed extract had effect only on R. stolonifer sporulation. Sporestimulation was observed for the remaining fungi when treatedwith plant extracts. In previous in vitro and in situ studies,we have observed similar selective fungistatic effect fromplant extracts. We have found differences in the fungicidalactivity of extracts from the same plant species associated

with various factors such as fungal specie, solvent used, plantorgan, harvesting season, and climatic conditions (Bautista-Baños et al., 2003a; Bermúdez-Torres et al., 2002; Bravo-Luna et al., 2002). In this study, we did not observe asynergistic effect between chitosan and plant extracts. On thecontrary, an antagonistic effect was observed on sporulationof F. oxysporum with all the combinations tested. In previousin situ experiments, we have reported that chitosan reducedimportant postharvest fruit rots (Bautista-Baños et al., 2003b);for example, a 60% control of anthracnose on papaya fruitwas obtained using chitosan coatings. In future experiments,it is highly recommended to evaluate the effect of chitosanon control of pre and postharvest diseases originated by thesethree microorganisms.

Acknowledgments. To the National Polytechnic Institute.Project CGPI (2003-882) for financial support. Specialthanks to the anonymous reviewers.

LITERATURE CITEDBarnett, H.L., and Hunter, B.B. 1972. Illustrated Genera of

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Table 3. Effect of chitosan at 1.5% on Fusarium oxysporum, Penicillium digitatum,and Rhizopus stolonifer on spore area, length, and form after 7 h incubation. Source Level Area Length FormF. oxysporumMain effect NS NS NSTreatment Control 10.2 a 3.7 a 3.7 a

Chitosan 1.5% 10.5 a 3.8 a 3.8 aP = 0.001 NS NS

Time (h) 0 8.7 b 3.7 a 3.8 a7 11.9 a 3.8 a 3.7 a

Interaction:Treatment x time NS NS NSP. digitatumMain effect P = 0.001 P 0.05 NSTreatment Control 3.1 b 2.2 b 0.5 a

Chitosan 1.5% 3.6 a 2.5 a 0.5 aP = 0.001 NS P = 0.001

Time (h) 0 3.2 b 2.2 0.4 a7 3.5 a 2.5 0.6 b

Interaction:Treatment x time P = 0.001 NS NSR. stoloniferMain effect NS P = 0.05 P =0.05Treatment Control 10.9 a 3.9 b 0.5 a

Chitosan 1.5% 10.3 a 4.4 a 0.4 bP = 0.001 P = 0.001 P = 0.001

Time 0 6.3 b 3.0 b 0.3 b(h) 7 14.9 a 5.4 a 0.5 aInteractionTreatment x time P = 0.001 P = 0.001 NSP values and data after log transformation; NS = non significant.

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