© Mexican Journal of Scientific Research, Vol. 1, No. 1, Jul-Dec 2012, pp. 2-9.
Parasitism and Substrate Competitions Effect of Antagonistic Yeasts for Biocontrol of Colletotrichum gloeosporioides in papaya (Carica papaya L.) var Maradol Magallon-Andalon C.G.1, Luna-Solano G.2, Ragazzo-Sanchez J. A. 1, Calderon-Santoyo M.1 1 Laboratorio Integral de Investigación en Alimentos, Instituto Tecnológico de Tepic, México. 2 Instituto Tecnologico de Orizaba, México.
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Abstract. Biocontrol has emerged as a promising alternative in the control of postharvest diseases as anthracnose in papaya caused by Colletotrichum gloeosporioides. The antagonistic action of Rhodotorula mucilaginosa 2 and Candida famata before this fungus has been demonstrated. The objective was to elucidate the antagonistic mechanisms for the biocontrol of anthracnose in papaya in order to produce adequate formulas for postharvest. Microscopic observation reveled C. famata and R. mucilaginosa 2 to have the ability to fix to the pathogen. Both yeasts produced hydrolytic enzymes responsible for fungi wall degradation. The sterile mycelium addition increased the enzymatic activity, being this more evident for R. mucilaginosa 2. A higher diameter growth of C. gloeosporioides inoculated in the presence of C. famata when external nutrients were added, but not in the R. mucilaginosa 2 presence. Both yeast used parasitism for controlling pathogen in papaya fruits, but only C. famata use additionally substrate competition. Keywords: Colletotrichum, biocontrol, substrate competition, parasitism.
1. Introduction The anthracnose disease has been responsible among other diseases for the rapid decline in papaya production in Nayarit, Mexico, by almost 60% since its introduction [1]. Recently the fungus Colletotrichum gloeosporioides has posed serious problems to Mexican producers because of favorable environmental conditions for this pathogen and resistance of this fungi to synthetic fungicides as chlorotalonil, cuprum hydroxide, mancozeb, prochlorazol or propioconazol [2]. Another alternative to control Colletotrichum gloeosporioides has been hot water immersions at 43-49 °C for 20 min, but physiological alterations and deficient ripening are producing by [3]. Nevertheless, when processing papaya fruit, a fungicide is added to the water bath in order to increase effectiveness. Even if fungicides can only be used if the fruit will not be consumed for a minimum of 15 days after treatment, this condition is rarely fulfilled [4]. Additionally, laws concerning fungicides used are nowadays more restrictive conducting to the search for new alternatives [5]. Biological control has emerged as a promising alternative in the control of post-harvest diseases of fruit applied or in preharvest or in postharvest. Recently, a variety of successful cases of biocontrol have been published for pre or postharvest: control of Fusarium root in sorghum using rhizobacteria [6], application of Trichoderma in compost soil for control of strawberry roots [7], control of postharvest diseases of citrus fruit by preharvest applications of Pantoea agglomerans CPA-2 [8] or application of Torulaspora globosa to control Colletotrichum sublineolum in sorgo [9]. In addition, some examples of treatments with biological control agents in pack house trials are the application of fungal strains for control of Lasiodiplodia theobromae in banana fruit [10], use of Candida membranifaciens F-58-22 for controlling anthracnose in mango [11], use of Penicillium frequentans conidia to inhibit Monilinia laxa in peach [12], etc. Moreover, biological control has demonstrated an efficient pathogens inhibition without causing negative effects on physiological or sensorial attributes of fruits [13, 14]. Some commercial formulations containing biological control agents have been applied as Serenade or Kodiak containing Bacillus subtilis, BlightBan containing Pseudomonas fluorescens [15] or Aspire containing Candida oleophila [16].
Received Jan 20, 2012 / Accepted Jan 27, 2012 Editorial Académica Dragón Azteca (EDITADA.ORG)
Magallon-Andalon / Parasitism and Substrate Competitions Effect of Antagonistic Yeasts for Biocontrol of Colletotrichum gloeosporioides in papaya (Carica papaya L.) var Maradol. MJSR, Vol. 1, No. 1, Jul-Dec 2012, pp. 2-9. EDITADA. Among the mechanisms used by the biological control agents, parasitism, antibiosis and competition for nutrients are the most important. Yeasts deserve particular attention, as their activity does not generally depend on the production of toxic metabolites [17, 18]. The main mode of action of yeast biocontrol agents is believed to be competition for nutrients and space [17, 19]. The complex process of parasitism consists of several events, including recognition of the host, attack and subsequent penetration and killing. Trichoderma, a biocontrol agent exerting mycoparasitism, secretes a variety of lytic enzymes: cellulases, chitinases, glucanases, etc. Trichoderma is recognized as secreting these enzymes in a constitutive level, but this secretion is enhanced when the presence of another fungus is detected by sensing the molecules released by the host by enzymatic degradation [20]. Candida famata is a biological control agent of Colletotrichum gloeosporioides, the causal agent of anthracnose in papaya [21]. This biological agent has been isolated from papaya from the peripheral zone of C. gloeosporioides infected tissue and its antagonistic action before this fungus has been demonstrated [21]. It is necessary to determinate the action mechanism used by these yeasts to control C. gloeosporioides in order to produce adequate formulas for application in postharvest. Moreover this information is precious when formulations need to be registered in order to approve their use as biofungicide for the control of postharvest pathogens. The aim of this work was to study the mechanism nutrient competition, parasitism by adhesion to mycelium or hydrolytic enzymes produced as antagonistic mechanisms for the biocontrol of anthracnose in papaya.
2. Experimental procedures 2.1 Biological material Papaya fruits were obtained in physiological state from local markets. The pathogen Colletotrichum gloeosporioides and antagonistic yeasts (Candida famata and Rhodotorula mucilaginosa 2) previously isolated from decayed and healthy papayas [21], respectively; were maintained at 4º C in PDA. Each culture was inoculated monthly.
2.2 Preparation of pathogen and yeasts suspensions 50 ml of trypticase soy (TCS) or yeast extract (YE) broth were inoculated with spores pathogen or yeasts cells and incubated at 28º C for 5 or 3 days, respectively. Pathogen spores or yeast cells concentrations were determined using a Neubauer chamber and adjusted at 1 × 105 spores/ml or 1 × 107 CFU/ml, respectively with a 50 mM (pH 7.0) phosphate buffer.
2.3 Evaluation of parasitism action mechanism Parasitism test was conducted by two techniques: Evaluation of yeast fixation and hydrolytic enzymes determination.
2.3.1 Evaluation of parasitism by fixation to the mycelium Fixation to the pathogen mycelium was evaluated by a modification of the Castoria et al. [22] and Spadaro et al. [17] techniques. Colletotrichum gloeosporioides was prepared in microculture and incubated for 72 h or until mycelium observation. 30 µl of a antagonistic yeast (1 × 107 cells/ml) were added on the mycelium margin. Microcultures were washed with distilled water after 12, 24 and 48 h of mixture culture. Washed microcultures were observed at optical microscope (100x) to detect yeast fixation.
2.3.2 Evaluation of parasitism by hydrolytic enzymes determination The enzymes determined were β-1,3-glucanase, nagase and chitinase. In vitro enzymatic tests were performed in crude extracts obtained from a 72 h YE cultures of Candida famata or Rhodotorula mucilaginosa 2. Determinations were conducted in the presence or absence of 10% v/v sterile mycelium from C. gloeosporioides.
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Magallon-Andalon / Parasitism and Substrate Competitions Effect of Antagonistic Yeasts for Biocontrol of Colletotrichum gloeosporioides in papaya (Carica papaya L.) var Maradol. MJSR, Vol. 1, No. 1, Jul-Dec 2012, pp. 2-9. EDITADA. β-1,3-glucanase activity was evaluated by the DNS determination of reducing sugars released from a reaction medium containing 50 mM laminarin in 50 mM (pH 5.0) sodium acetate buffer at 45ºC [23], [22]. Nagase activity was evaluated by the determination at 400 nm of the p-nitrophenol released from a medium reaction containing p-nitrophenil-β-D-N-acetylglucosaminide 100 mM in a 50 mM (pH 6.7) phosphate buffer at 45ºC [24]. Chitinase activity was conducted by the determination at 400 nm of the p-nitrophenol released from a medium reaction containing 100 mM p-nitrophenol, NN¨, diacetyl β-D- chitobioside in Tris-HCl 20 mM (pH 7.5) at 37ºC. Enzymatic activities were expressed as μmol of reducing sugars or p-nitrophenol released for protein mg for minute [25]. Protein determination was performed by the Bradford method [26].
2.4 Evaluation of substrate competition action mechanism Substrate competition was examined by the technique proposed by Spadaro et al. [17]. Papaya fruits were bored aseptically (0.7 cm diameter and 1.0 cm depth). Wounds were inoculated with 25 µl of a biocontrol agent suspension (1 × 107 CFU/ml) and 25 µl of C. gloeosporioides suspension (1 × 105 spores/ml). Pathogen growth diameter in papaya fruits was evaluated in presence of the biocontrol agent with or without external nutrients (2% w/v glucose, fructose, sucrose and 0.3% w/v potassium nitrate) added. Fruits were stored at 28 ºC, relative humidity of 90% during 6 days. 2.5 Statistical analysis Differences in treatments were established by LSD at p
