Biological Control 65 (2013) 293–301 Contents lists available at SciVerse ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon Action mechanisms of the yeast Meyerozyma caribbica for the control of the phytopathogen Colletotrichum gloeosporioides in mangoes Pedro Ulises Bautista-Rosales a, Montserrat Calderon-Santoyo b,⇑, Rosalía Servín-Villegas a, Norma Angélica Ochoa-Álvarez a, Juan Arturo Ragazzo-Sánchez b a b Centro de Investigaciones Biológicas del Noroeste, S.C., Mar Bermejo #195, Col. Playa Palo de Santa Rita, La Paz C.P. 23090, BCS, Mexico Laboratorio Integral de Investigación en Alimentos, Instituto Tecnológico de Tepic, Av. Tecnológico #2595, Col. Lagos del Country, Tepic C.P. 63175, Nayarit, Mexico h i g h l i g h t s g r a p h i c a l a b s t r a c t  The evaluated yeast is capable of reducing the degree of anthracnose symptoms.  The combination of different action mechanisms of yeast ensures a good biocontrol.  Yeast cells are necessary in the formula to carrying out biological control. Isolation of microorganisms from mangoes fruit Colletotrichum gloeosporioides Meyerozyma caribbica Biological control tests DCgH BFF Biofilms formation (BFF) by M. caribbica. This yeast competes for nutrients and space and cause deformation of C. gloeosporioides hyphae (DCgH) due to enzyme production (parasitism). a r t i c l e i n f o Article history: Received 20 September 2012 Accepted 19 March 2013 Available online 29 March 2013 Keywords: Meyerozyma caribbica Colletotrichum gloeosporioides Biocontrol Action mechanisms a b s t r a c t The yeast Meyerozyma caribbica was evaluated for their effectiveness against Colletotrichum gloeosporioides in the mango (Mangifera indica L.) cv. ‘‘Ataulfo’’ and to identify the possible mechanisms of action involved in the inhibition. M. caribbica showed a high antagonistic potential in vivo, with significant inhibition of 86.7% of anthracnose. M. caribbica competed for the nutrients sucrose and fructose (p < 0.05). Electron microscopy showed that the yeast produces a biofilm adhering to the fruit and to C. gloeosporioides hyphae. M. caribbica showed competition for space and parasitism to the phytopathogen, furthermore it produces hydrolytic enzymes such as chitinase, N-acetyl-b-D-glucosaminidase and b-1, 3-glucanase. These enzymes caused notched and non-lethal deformations on the fungal hyphae through this specific action mechanism. According to the results obtained here, the combination of the different action mechanisms of the yeast increases their ability to control C. gloeosporioides. The use of biological agents to control C. gloeosporioides may contribute to the integrated management of disease caused by this pathogen. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Colletotrichum gloeosporioides ((Penz.) Penz. & Sacc.) is a pathogenic fungus that attacks mangoes (Mangifera indica L.) and causes a disease known as anthracnose, which occurs in fruit and vegeta⇑ Corresponding author. Fax: +52 3112119401. E-mail address: montserratcalder@gmail.com (M. Calderon-Santoyo). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.03.010 ble crops worldwide. This pathogen is capable of destroying up to 60% of the fruit produced (Arias and Carrizales, 2007; Bally, 2006; León, 2007; Vega-Piña, 2006). Despite this problem, in 2010, Mexico was the fifth largest producer and the largest exporter of mangoes in the world with fruit exported primarily to the USA, Canada and Japan (FAO, 2012). Chemical fungicides are used for the management of anthracnose; however, their indiscriminate use has caused severe environ- 294 P.U. Bautista-Rosales et al. / Biological Control 65 (2013) 293–301 mental pollution problems and resistance to various agronomic products for the management of mango crops, a fact that has resulted in the cancelation of some of the most effective fungicides in Europe (Regulation 1107/2009 and Directive 2009/128) and in the United States (Food Quality Protection Act) (Lopez-Reyes et al. 2010). Thus, there is a need for environmentally friendly alternatives to manage this disease, in this context, the use of biological agents is one of the strategies of interest (Hernández-Luzardo et al., 2007; Ibarra et al., 2006). Fungi, yeasts and/or bacteria have the ability to reduce pest populations and diseases, which can minimize economic losses (Charlet et al., 2002; Hernández-Luzardo et al., 2007). As a result of its ability for suppressing postharvest diseases, yeasts have been recommended for controlling fruit rots (Sharma et al., 2009). The current knowledge has aided in the biological control of the fungi of the genus Colletotrichum among various host plants. Pichia guilliermondii (Wick.), Candida musae ((Nakase) S.A. Mey & Yarrow), Issatchenkia orientalis (Kudryavtsev) and Candida quercitrusa (S.A. Mey & Phaff) are used to inhibit the development of Colletotrichum capsici ((Syd.) E.J. Butler & Bisby) in chili pepper (Capsicum annum L.) in Thailand (Chanchaichaovivat et al., 2007), Torulaspora globosa ((Klocker) Van der Walt & Johannsen) is used to inhibit Colletotrichum sublineolum (Henn. ex Sacc. & Trotter) in sorghum (Sorghum bicolor (L.) Moench) in Brazil (Rosa et al., 2010), and Candida membranifaciens ((Lodder & Kreger) Wick. & Burton) is used against C. gloeosporioides in mango (Mangifera indica L.) crops in Ethiopia (Kefialew and Ayalew, 2008). The ability to use yeasts to biologically control various pathogens depends on antagonistic action mechanisms, of which the most important are competition for nutrients and space, antibiotic production, direct parasitism and enzyme secretion (Chanchaichaovivat et al., 2008; Ge et al., 2010; Li et al., 2011; Liu et al., 2010; Sharma et al., 2009; Zhao et al., 2008). However, in the case of M. caribbica ((Vaughan-Mart. et al.) Kurtzman & Susuki), to date, no reports have been found on its effectiveness as a biological control agent. In Mexico, several studies related to C. gloeosporioides have been conducted on various crops such as mango (Mangifera indica L.), avocado (Persea Americana Mill.), papaya (Carica Papaya L.) and apple (Malus domestica Borkh.) due to the significant losses caused by this fungus (Becerra-Leor, 1995; Casarrubias-Carrillo et al., 2002; Huerta-Palacios et al., 2009; Kader, 2011; Rodríguez-López et al., 2009). Fungicides including benomyl, maneb, thiabendazole and copper oxychloride are mainly used to control C. gloeosporioides (León, 2007). In addition to generating a significant economic cost, the use of fungicides may also has environmental effects. Therefore, the objective of this study was to evaluate the yeast M. caribbica for the possible biological control of anthracnose caused by C. gloeosporioides in the mango variety ‘‘Ataulfo’’ and to determine the mechanisms of antagonistic action that are involved. 2. Materials and methods 2.1. Phytopathogen inoculum The pathogen C. gloeosporioides was isolated from ‘‘Ataulfo’’ mangoes obtained from commercial organic orchards of the municipality of San Blas, Nayarit, Mexico (Chand-Goyal and Spotts, 1996). This region is recognized for its importance in the production of mango. This town is located at 21°280 N, 105°100 W and has annual average rainfall 1316.3 mm. C. gloeosporioides was cultivated in potato dextrose agar (PDA), incubated at 28 °C for 7 days and subsequently maintained at 4 °C for storage. A fresh culture was prepared on PDA plate prior to its use in each bioassay. For the bioassays, a suspension of fungus spores was obtained by picking up mycelium portions with a loop and placing then in 10 mL of sterile distilled water, which were subsequently filtered with gauze and adjusted to a concentration of 1  105 spores/mL using a hemocytometer. 2.2. Phytopathogen identification An isolate of C. gloeosporioides was identified by microscopy and by molecular tools. DNA was extracted (from an isolate grown on PDA for 7 days) using the Sambrook and Russell (2001) technique. The rDNA region ITS1-5.8S-ITS2 was symmetrically amplified using the primers ITS1 (50 -TCCGTAGGTGAACCCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ), following the protocols described by Ochoa et al. (2007). PCR amplifications were performed in a thermal cycler (System 9700 GeneAmpÒ) with a denaturalization period of 2 min at 95 °C, followed by 30 cycles (which included a denaturalization at 95 °C for 1 min, alignment for 30 s at 50 °C and an extension for 2 min at 72 °C), with a final extension of 10 min at 72 °C. The amplification products were separated by gel electrophoresis in agarose (SIGMAÒ) at 1% and were stained with ethidium bromide (0.2 lg/ mL), then they were visualized on a transilluminator (BioDoc-IT system image, UVPÒ) (Ochoa et al., 2007). The extracted amplicons were sequenced by Genewize Inc., and the sequences were aligned with the NCBI online database using the Basic Local Alignment Search Tool (NCBI BLAST) program. 2.3. Isolation and preparation of the biocontrol yeast The yeast strain L6A2 (Bautista-Rosales et al., 2011), previously was isolated from ‘‘Ataulfo’’ mango fruit, obtained from an organic orchard in Nayarit, México. The isolated yeast was grown in potato dextrose broth (PDB) on a rotary shaker at 28 °C and 110 rpm for 72 h. The culture was maintained at 4 °C. The concentration was adjusted to 1  107 cells/mL using a haemocytometer. 2.4. Identification of the biocontrol yeast DNA was extracted (from an isolate of yeast grown on PDB on a rotary shaker at 28 °C and 110 rpm for 72 h) using the rapid isolation of yeast DNA technique described by Sambrook and Russell (2001). The D1/D2 region of the large subunit of the ribosomal DNA, 26S rDNA subunit, was amplified using the primers NL-1 (50 -GCATATCAATAAGCGGAGGAAAAG-30 ) and NL-4 (50 -GGTCCGTGTTTCAAGACGG30 ) according to the protocol of Ochoa et al. (2007). PCR amplifications were performed in a thermal cycler (System 9700 GeneAmpÒ) with a denaturalization period of 2 min at 95 °C, followed by 30 cycles (which included a denaturalization at 95 °C for 1 min, alignment for 30 s at 50 °C and an extension for 2 min at 72 °C), with a final extension of 10 min at 72 °C. The amplification products were separated by gel electrophoresis in agarose (SIGMAÒ) at 1% and were stained with ethidium bromide (0.2 lg/ mL), then they were visualized on a transilluminator (BioDoc-IT system image, UVPÒ). The obtained DNA sequence was sent to Genewize Inc., and the sequence was aligned and compared with the NCBI online database using the Basic Local Alignment Search Tool (NCBI BLAST) program. 2.5. Collection and preparation of the fruit ‘‘Ataulfo’’ mangoes were harvested at physiological maturity and with no apparent mechanical or microbiological damage from 20 year-old-trees cultivated using organic practices in Nayarit, México. The harvested fruits were taken to the laboratory and immediately disinfected with 1.5% sodium hypochlorite for P.U. Bautista-Rosales et al. / Biological Control 65 (2013) 293–301 2 min, washed with tap water, allowed to dry at room temperature (28 °C) and were finally placed in a plastic container (CasarrubiasCarrillo et al., 2002). 2.6. Effect of yeast on the severity of anthracnose Five healthy mangoes were wounded with a bodkin to a 1 mm depth with two wounds per mango. Within the wounds, 25 lL from a 3 days old yeast culture (1  107 cells/mL) were inoculated and allowed to incubate for 1 h. After that incubation, 25 lL aliquots of a C. gloeosporioides fungus spore suspension (1  105 spores/mL) from 7 days old cultures were added to the wound. Five fruits were prepared as negative controls by adding 50 lL of sterile distilled water into the wounds. Additionally, to verify the non-pathogenicity of the biocontrol agent on the fruit, five fruits were prepared as treated negative controls by depositing suspensions containing 25 lL of 1  107 yeast cells/mL and 25 lL of sterile distilled water. Besides, five fruits were prepared as positive controls by depositing suspensions containing 25 lL of C. gloeosporioides spore suspension (1  105 mL 1) and 25 lL of sterile distilled water. Four treatments used to determine the effect of yeast on the severity of anthracnose. These treatments included: (1) fruits treated with the strain yeast L6A2 to a concentration of 1  107 cells/mL and C. gloeosporioides to a concentration of 1  105 spores/mL, (2) fruits treated with C. gloeosporioides to a concentration of 1  105 spores/mL, (3) fruits treated with sterile destilled water and (4) fruits treated with the strain yeast L6A2 to a concentration of 1  107 cells/mL. After treatments, the fruits were incubated in plastic chambers (30  40  15 cm) for 6 days at high relative humidity (above 90%) and 28 °C (A box per treatment). Five mangoes were placed in each chamber and high relative humidity was maintained by placing a 250 mL flask containing distilled water into each chamber. At the end of the experiment, we observed and measured the diameter of the lesions produced by the fungus in each fruit to determine the percentage of the inhibition of the disease according to the method of Benbow and Sugar (1999). A univariate design was used where the yeast addition treatment was the factor and the diameter lesion was the dependant variable. The experiment was replicated twice (two wounds per fruit and five fruits per replicate) and the whole experiment was repeated three times. 2.7. Reduction in the incidence of anthracnose Ten liters suspensions of cells of M. caribbica were prepared from 3 days old cultures grown in PDB adjusted to a concentration of 1  107 and 1  108 cells/mL. Twenty-five mangoes cv. Ataulfo were immersed in those suspensions for a minute without wounding. Four treatments used to determine the impact of yeast on anthracnoses. These treatments included: (1) fruits treated with a suspension of the fungicide Benomyl (SIFATECÒ, Funlate 50Ò technical grade) to a concentration of 0.1% was used as chemical control, (2) fruits treated with M. caribbica to a concentration 1  107 cells/mL, (3) fruits treated with M. caribbica to a concentration 1  108 cells/mL and (4) fruits submerged in distilled water were used as non-inoculated controls. Treated mangoes were then stored at 28 °C and high relative humidity (above 90%) in 50  35  16 cm plastic storage chambers. Each plastic chamber used contained 25 mangoes and high relative humidity was maintained by placing a 500 mL flask containing distilled water into each chamber. After 10 days of storage, the percentage of rotten mangoes was recorded (Zhang et al., 2011). A univariate design was used where the yeast or benomyl addition treatment was the factor and the incidence of anthracnose (%) or percentage of diseased fruits was the response variable. The experiment was performed two times. 295 2.8. Assessment of competition for nutrients The determination of the competition for nutrients was performed using the technique described by Spadaro et al. (2002) and modified by Ragazzo-Sánchez et al. (2012). Five healthy mango fruits were washed, disinfected and drilled (0.7 cm in diameter and 1 cm deep) to evaluate each of the substrates: 2% of sucrose, glucose or fructose as carbon sources and 0.3% nitrate potassium as the nitrogen source. For evaluation, the wounds were inoculated with 25 lL of the yeast suspension containing 1  107 cells/mL, 25 lL of a pathogen spore suspension containing 1  105 spores/ mL and 50 lL of the nutrients. For the control, the nutrients were replaced with 50 mM phosphate solution (pH 6.7) to compare the diameter of the lesion caused by the disease against the treatments with added sugars or potassium nitrate. The fruits were stored at 28 °C and high relative humidity (above 90%) for 6 days. At the end of the experiment, the diameters of the lesions produced by the fungus in each fruit were observed and measured to determine the diameter of disease development. 2.9. Determination of the adhesion of the yeast to the pathogen Microcultures (PDA square of 1 cm2) with the fungal pathogen C. gloeosporioides were prepared and incubated at 28 °C until visible mycelium was formed (72 h), according to the technique proposed by Castoria et al. (2001) and Spadaro et al. (2002) and modified by Ragazzo-Sánchez et al. (2012). After that, 30 lL of M. caribbica or Saccharomyces cerevisiae cells adjusted to a concentration of 1  107 cells/mL was added to the obtained mycelium. The mixed microcultures were washed with sterile distilled water after 12, 24 and 48 h so that only the yeasts that adhered to the mycelium remained to determine a possible parasite mechanism. Microcultures were observed using optical microscopy with 40 and 100 objectives. The yeast S. cerevisiae was used as a negative control. Samples were prepared in vitro and in vivo for Scanning Electron Microscopy. For the in vitro treatments, C. gloeosporioides was cultured for 72 h at 28 °C in PDB before an aliquot of M. caribbica (100 lL/1  107 cells/mL) was introduced. The co-culture was incubated for 72 h in the same conditions, later a piece of mycelium was fixed as outlined below. Treatments in vivo were performed as described in Section 2.6 (reduction in the severity of anthracnose). Five fruits were incubated at 28 °C for 72 h. After that, from treated fruits, a piece of pericarp tissue inoculated with yeast and phytopathogen was excised with a scalpel (1 cm3), then it was halved and fixed. The samples for SEM were fixed in 3% glutaraldehyde in cacodylate buffer 0.1 M (pH 7.4) for 72 h at 4 °C to preserve their structural and morphological characteristics, then the samples were placed in a solution of OsO4 in 2% cacodylate buffer and processed as follows. Samples were rinsed three times in cacodylate buffer and dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 95% and 100%), critical point dried with CO2 and coated with gold-plated for cell interaction assays. The specimens were observed under a scanning electron microscope (Hitachi S3000N, San Jose, CA, USA). (Brown and Brotzman, 1979). 2.10. Secretion of hydrolytic enzymes Stimulation of hydrolytic enzymes in the yeast was tested according to the methods of Castoria et al. (2001), Ragazzo-Sánchez et al. (2012) and Spadaro et al. (2002). C. gloeosporioides was grown in 50 mL of PDB. The culture was maintained under conditions of agitation (110 rpm) at 25 °C for 72 h until the first appearance of mycelial growth. The culture was then autoclaved at 121 °C for 20 min and then was centrifuged at 1400g for 296 P.U. Bautista-Rosales et al. / Biological Control 65 (2013) 293–301 10 min. The precipitate was washed twice with 50 mM phosphate solution (pH 6.7). Finally, it was resuspended in 50 mL of the same solution to stimulate the production of hydrolytic enzymes in yeast according Castoria et al. (2001), Ragazzo-Sánchez et al. (2012) and Spadaro et al. (2002). For in vitro testing of enzymatic activity, the yeast was cultured for 72 h at 28 °C in yeast glucose extract broth in the presence and absence of 10% V/V autoclaved mycelium of C. gloeosporioides prepared as above. The cultures were centrifuged at 1400g for 10 min and filtered through a 0.20 lm nitrocellulose membrane. The supernatants were recovered for the enzymatic analysis of b-1, 3-glucanase, N-acetyl-b-D-glucosaminidase (Nagase) and chitinase (Castoria et al., 2001; Ragazzo-Sánchez et al., 2012; Spadaro et al., 2002). To test the induction of these enzymes in vivo, two wounds were made in five mango fruits (3 cm by 3 cm above and below the center of the fruit), and 100 lL of yeast suspensions containing 1  107 cells/mL were inoculated in the wounds. Additionally, a second treatment consisted of inoculating 100 lL of sterile mycelium of the pathogen into these fruits. The fruits were then incubated at 28 °C in high relative humidity (above 90%) for 72 h. The wounds were washed with 250 lL of buffer, in the case of b-1, 3-glucanase enzyme was used as buffer 50 mM sodium acetate buffer (pH 5.0), to N-acetyl-b-D-glucosaminidase (Nagase) enzyme was used 50 mM phosphate (pH 6.7) and to chitinase enzyme was used tris–HCl (pH 7.5) (five replicates for each treatment). The solutions obtained during the washing of the wounds were centrifuged at 1400g for 10 min and filtered through a nitrocellulose membrane of 0.45 lm. The supernatant or enzymatic extract was recovered, and the enzymatic activity was determined as described below. Controls consisted of mangoes to which the wounds were performed above, with 100 lL of sterile destilled water and the presence and absence of autoclaved mycelium of the pathogen. 2.11. Determination of enzyme activity The b-1, 3-glucanase enzyme activity was measured according to the techniques described by Castoria et al. (2001). The activity was quantified by measuring the nmol of reduced sugars released per mg of protein per min (mU/mg of protein). The N-acetyl-b-Dglucosaminidase (Nagase) activity was determined using the technique described by Tronsmo and Harman (1993) and was measured based on the nmol of p-nitrophenol released per mg of protein per min (mU/mg of protein). Chitinase activity was determined using the technique described by Wu et al. (2001) and was estimated as the nmol of p-nitrophenol released per mg of protein per min (mU/mg of protein). Finally, protein quantification was performed using a protein assay kit (Biorad, Ref. 500-0006) based on the Bradford (1976) method. To analyze the data, a bifactorial statistical design was used, where the first factor was the type of treatment (in vitro, fruits inoculated with yeasts and fruits non-inoculated with yeast) and the second one was the addition or not of mycelium. 2.12. Evaluation of the antibiosis mechanism The antagonistic yeast strain was grown in yeast extract glucose broth (YGB) at 28 °C for 72 h until they reached a concentration of 1  107 cells/mL. Then, the culture was sterilised for 15 min at 121 °C, centrifuged at 5600g for 10 min and filtered through a nitrocellulose membrane of 0.2 lm for the determination of nonpeptide antibiotics. To observe the effect of the enzymes on C. gloeosporioides development, one treatment was performed so that the culture medium was only filtered through the nitrocellulose membrane. An additional treatment was conducted in which cells from the yeast strain was inoculated in order to determine whether they were necessary for the biological control of phytopathogenic fungi. 25 lL of filtrates or suspension cells were placed in a filter paper disk (Whatman #1) and was deposited on a PDA plate that had been previously inoculated with 500 lL of a solution of 105 spores/mL of C. gloeosporioides. The plate was incubated at 25 °C for 72 h. After 72 h, the presence of a pathogen inhibition halo around the disk caused by the extracts and/or the yeast cells was checked. Simultaneously, a control plate was incubated in which a solution of 50 mM phosphate (pH 6.7) was applied instead of the sterile centrifuged yeast culture. The treatments were performed in triplicate in accordance with Ragazzo-Sánchez et al. (2012). 2.13. Statistical analysis Data were analyzed using an analysis of variance (ANOVA) with the plastic chambers as blocks with the SAS Statistical Software version 9.2 for Windows, and the least significant differences (LSD) test was used to separate differences among the means. Statistical significance was considered as p < 0.05. 3. Results 3.1. Identification of microorganisms The strain of the pathogen was identified as C. gloeosporioides ((Penz.) Penz. and Sacc.) and was called MA2 (GenBank ID: JQ366003), while the biocontrol yeast was identified as Meyerozyma caribbica ((Vaughan-Mart. et al.) Kurtzman & Susuki), also known as Pichia caribbica (Vaughan-Mart. et al.) (anamorph: Candida fermentati (Saito)), and assigned the code to this strains was L6A2 (GenBank ID: JQ398674). 3.2. Yeast effectiveness as potential antagonists for anthracnose management M. Caribbica reduced significantly the injury caused by the fungus in wounded fruits by 86.7% compared to control (p < 0.05), from an average diameter of the lesion of 2.86 cm in the control fruit to 0.38 cm in the treated fruits. Furthermore, when compared to M. caribbica in two concentrations against a commercial chemical fungicide and control, there were no statistically significant differences in the incidence of disease among fruit treated with yeast at different concentrations and chemical fungicide (Table 1). 3.3. Competition for nutrients After 6 days incubation of treated fruits, the diameter of the disease area caused by the fungus was measured (Table 2), it was Table 1 Incidence of anthracnose (%) on Ataulfo mangoes naturally infected upon immersion treatments in suspensions containing M. caribbica in comparison with immersion in benomyl fungicide and a non-treated control. Treatments Control M. caribbica 107 cells/mL M. caribbica 108 cells/mL Benomyl 0.1% a % Anthracnose incidence 10 daysa 13 days 28.66ab 14.00b 12.66b 9.33b 47.33a 17.33b 20.00b 19.33b Days after immersion treatment. Values are the means of incidence (%) of two replicates of 25 fruits per replicate after storage at 28 °C and relative humidy >90%. Means within the column that are followed by different letters are significantly different according to LSD (p < 0.05). b P.U. Bautista-Rosales et al. / Biological Control 65 (2013) 293–301 Table 2 Severity of anthracnose in wounded fruits of Ataulfo mangoes treated with M. caribbica with addition or not of exogenous nutrients and inoculated with the fungal pathogen C. gloeosporioides. Treatments Diameter of anthracnose development (cm) 13 days of storage M. caribbica P A+P A+P+S A+P+F A+P+G A+P+K 1.90 ± 0.31aa 0.58 ± 0.06c 1.93 ± 0.34a 1.61 ± 0.58ab 0.76 ± 0.03bc 0.82 ± 0.06bc P = pathogen, A = antagonist, S = sucrose, F = fructose, G = glucose, and K = potassium nitrate. a Values are the means of severity of three replicates of five fruits per replicate and two wounds per fruit, after storage at 28 °C and relative humidy >90%. Means within the column that are followed by different letters are significantly different according to LSD (p < 0.05). found that, under in vivo conditions, M. caribbica manifest different levels of competition for the substrate. When sucrose or fructose was added in excess to the fruits inoculated with yeast and the pathogen, there was an increase in the development of the disease by 232% and 177% respectively in relation to the control (p < 0.05). 297 washed by sterile water. The scanning in electron microscopy of in vitro mixed culture shown that cells of M. caribbica produced notches in C. gloeosporioides hyphaes, causing non-lethal strain under these conditions (Fig. 2a and b). It was also shown that M. caribbica was able to form biofilms in vitro and in vivo cultures (Fig. 2c). 3.5. Yeast enzymatic activities 3.5.1. b-1, 3-glucanase production The enzymatic activity of b-1, 3-glucanase was found in the whole treatments performed. In the fruits without addition of yeast and in vitro treatments, a significant increase in the enzymatic activity was observed when the sterile mycelium was added (p < 0.05) (Table 3). However, in fruits inoculated with yeast treatments, there were no significant differences with and without the addition of sterile mycelium (p > 0.05). On the other hand, the fruits non inoculated with the yeasts and without addition of sterile mycelium did not show statistically significant differences with respect to fruits inoculated with yeasts without the addition of sterile mycelium. However, the fruits inoculated with yeast and with addition of sterile mycelium showed a significant decrease in enzyme activity with respect to the fruits no inoculated with yeast and with addition of sterile mycelium (p < 0.05) (Table 3). 3.4. Adhesion of the yeast to the pathogen To establish the mixed culture of the fungus and M. caribbica, after 48 h it was observed by optical microscopy that the yeast cells were attached to the hyphae of C. gloeosporioides (Fig. 1b), while in the case of the negative control yeast (S. cerevisiae) (Fig. 1c and d) this phenomenon was not observed, since by performing the microcultures washes, cells of S. cerevisiae were 3.5.2. Production of N-acetyl-b-D-glucosaminidase (Nagase) In vitro treatments did no present a significant increment in the nagase enzyme activity with the addition of sterile mycelium of C. gloeospoioides (p < 0.05). On the other hand, the fruits no inoculated and inoculated with yeast showed a significant increase with the addition of sterile mycelium of the pathogen (p < 0.05). Nevertheless, a significant decrease in the activity of this enzyme was Fig. 1. Interaction between C. gloeosporioides and yeast after 48 h. Adhesion is observed by M. caribbica on C. gloeosporioides 40 (a) and 100 (b). No adhesion is observed by the negative control S. cerevisiae on C. gloeosporioides at 40 (c) and 100 (d). 298 P.U. Bautista-Rosales et al. / Biological Control 65 (2013) 293–301 Fig. 2. Scanning electron micrographs (SEM) of the interaction between M. caribbica and C. gloeosporioides hyphae inoculated by direct pipetting on the Ataulfo mango wounds (a and b) and inoculated in PDB (c) after 36 h. It is observed the biofilms formation (BFF) and deformation of C. gloeosporioides hyphae (DCgH). Magnification of 900 (a), 3200 (b) and 4000 (c). Table 3 b-1, 3-glucanase activity of M. caribbica grown in PDB (in vitro) or in Ataulfo mango wounds (in vivo) with addition or not of C. gloeosporioides sterile mycelium after 3 days of storage. Treatments Fruits non inoculated with yeasts Fruits inoculated with yeasts In vitro Enzymatic activity (mU/mg of protein)a Without mycelium With mycelium 3.65 ± 0.03ea,b 4.47 ± 0.22de 22.50 ± 0.20bc 9.38 ± 1.24c 5.24 ± 0.32d 26.58 ± 0.30a a Means that are followed by different letters are significantly different according to LSD (p < 0.05). b Values are the means of enzymatic activity of three replicates of five fruits per replicate (in vivo) and two wounds per fruit, after 3 days of storage at 28 °C and relative humidy >90%. c Values are the means of enzymatic activity of three replicates (in vitro), after incubation at 28 °C. Table 4 N-acetyl-b-D-glucosaminidase (nagase) activity of M. caribbica grown in PDB (in vitro) or in Ataulfo mango wounds (in vivo) with addition or not of C. gloeosporioides sterile mycelium after 3 days of storage. Treatments Fruits non inoculated with yeasts Fruits inoculated with yeasts In vitro Enzymatic activity (mU/mg of protein)a Without mycelium With mycelium 247.79 ± 8.04ba,b 57.98 ± 9.69d 161.49 ± 14.26cc 304.20 ± 31.95a 125.56 ± 21.01c 159.77 ± 10.83c a Means that are followed by different letters are significantly different according to LSD (p < 0.05). b Values are the means of enzymatic activity of three replicates of five fruits per replicate (in vivo) and two wounds per fruit, after 3 days of storage at 28 °C and relative humidy >90%. c Values are the means of enzymatic activity of three replicates (in vitro), after incubation at 28 °C. observed in the fruits treated with yeasts with respect to the fruits no treated with yeasts, in the treatments either with addition or not of sterile mycelium (Table 4). 3.5.3. Production of chitinase All treatments showed chitinase enzyme activity, but the addition of sterile mycelium did not produce a significant increase in the chitinase activity in treatments with M. caribbica, neither in vitro nor in fruits inoculated with yeasts (p < 0.05). In the case of fruits no inoculated with mycelium, the activity of this enzyme is significantly increased with the addition of mycelium (p < 0.05). A significant decrease in chitinase activity was detected in fruits inoculated with yeasts with respect to the fruits no inoculated with yeasts, these in the treatments either with addition or not of sterile mycelium (Table 5). 3.6. Production of antifungal compounds M. caribbica did not produce inhibition zone neither in the nonpeptide antibiotics test (filtered and autoclaved extracts) nor in the enzymatic extracts (filtered) because there was no inhibition halos of the pathogen (data not shown). However, when the inoculation was performed with yeast cells, a clear mycelial collapse of the pathogen was shown. 4. Discussion M. caribbica can be considered as an effective antagonist of C. gloeosporioides when it is applied in concentration of 107 or 108 cells/mL. This effectiveness can be compared with that obtained with a chemical fungicide (benomyl 0.1%) because there is no statistically difference at p < 0.05. It has several action mechanisms; among them, the competition for nutrients, particularly sucrose and fructose at different rates. This competition was detected based on the increase in fruit rot caused by C. gloeosporioides on treatments with addition of the nutrients evaluated comparing with the control. This behavior occurs because both microorganisms were subjected to an environment containing an excess of carbohydrates with sufficient amounts of these substrates for the free development of both the pathogen and the yeast. Competition for these nutrients can be associated with the high inhibition exerted by the yeast on the pathogen, in as much as M. caribbica Table 5 Chitinase activity of M. caribbica grown in PDB (in vitro) or in Ataulfo mango wounds (in vivo) with addition or not of C. gloeosporioides sterile mycelium after 3 days of storage. Treatments Enzymatic activity (mU/mg of protein)a Without mycelium Fruits non inoculated with yeasts Fruits inoculated with yeasts In vitro a,b 180.468 ± 14.004a 101.181 ± 10.556bc 259.020 ± 12.669ac With mycelium 273.172 ± 43.799b 150.859 ± 6.134c 276.965 ± 4.926a a Means that are followed by different letters are significantly different according to LSD (p < 0.05). b Values are the means of enzymatic activity of three replicates of five fruits per replicate (in vivo) and two wounds per fruit, after 3 days of storage at 28 °C and relative humidy >90%. c Values are the means of enzymatic activity of three replicates (in vitro), after incubation at 28 °C. P.U. Bautista-Rosales et al. / Biological Control 65 (2013) 293–301 expressed a high degree of pathogen inhibition also competed for the primary carbohydrates present in the Ataulfo mangoes in physiological maturity, which are sucrose and fructose (0.2–0.5 and 0.2–0.9 g/100 g of sample, respectively) (García-Delgado et al., 2010; Montalvo et al., 2009). This finding indicates a high affinity and the rapid assimilation of carbohydrates by M. caribbica. Filonow (1998), Sharma et al. (2009) and Spadaro et al. (2010) mentioned that yeasts generally have the ability to successfully assimilate a wide variety of mono- and di-saccharides, such as sucrose and fructose, making these nutrients unavailable to C. gloeosporioides and allowing it to rapidly proliferate. Therefore, under conditions of starvation of sugars, M. caribbica is able to compete with C. gloeosporioides for nutrients and to inhibit it in an efficient manner. Optical and scanning electron microscopy revealed that the yeast strain was able to adhere to the hyphae of C. gloeosporoides, which gives the yeast an important mechanism for the biocontrol of the fungus. Several previously reported studies confirm this outcome, as in the case of Pichia guilliermondii (Wick.) that parasitizes Botrytis cinerea (Pers.) in apples (Malus domestica Borkh) (Wisniewski et al., 1991); Candida guilliermondii ((Castell) Langeron & Guerra) and Candida oleophila (Montrocher), which have a similar relationship with Botrytis cinerea in tomatoes (Saligkarias et al., 2002); and Pichia guilliermondii, noted for its biological control of Colletotrichum capsici (Syd.) on affected chili pepper (Capsicum annum L.) fields (Chanchaichaovivat et al., 2008). Several studies have suggested that some functional proteins from the antagonists and pathogens are involved in the adhesion process (Chan and Tian, 2005). Other studies have indicated that this mechanism is related to nutrients competition in which yeasts are interposed between the pathogen and the substrate, which allows them to be taken in more easily and thus limiting the growth of pathogens such as C. gloeosporioides (Sharma et al., 2009). Furthermore, in the adhesion process, some notches in the pathogen hyphae made by the yeast cells were detected by scanning electron microscopy (Fig. 2b), thus, it is evident that M. caribbica is able to parasitize C. gloeosporioides, through the production of lytic enzymes such as glucanases, chitinases and proteases degrading the pathogen cell wall. This action causes a distortion of the fungus hyphae and eventually causes cellular lysis (Janisiewicz and Korsten, 2002; Sharma et al., 2009). According to these results, M. caribbica is capable of producing hydrolytic enzymes (glucanase, nagase and chitinase) in vitro and in vivo, although the mango fruit by itself can also produce these enzymes in response to injury or detection of C. gloeosporioides. During the treatments in vitro and fruits non inoculated with yeasts, a statistically significant increase in the enzyme b-1, 3-glucanase activity was observed when M. caribbica or mango fruit come in contact with the sterile mycelium of the pathogen. This situation is consistent with the findings reported by Chan and Tian (2005) and Theis and Stahl (2004), who hypothesized that yeast– pathogen or plant–pathogen interactions are due to a signal recognition system between the yeast cells, the fungal hyphae and fruit cells. In contrast, in the case of fruits inoculated with M. caribbica, no statistically significant difference was observed in the enzyme b-1, 3-glucanase activity with or without the addition of sterile mycelium of the pathogen, although there was a slight increase in the activity of this enzyme. In the case of the enzymes nagase and chitinase, fruits inoculated and not with the yeast showed statistically significant increase when the sterile pathogen mycelium was added, which indicate a plant–pathogen interaction, i.e., the pathogen induce the production of these enzymes. On the other hand, no statistically significant differences in the activity of these enzymes between the treatments with or without the addition of sterile mycelium of the pathogen were observed in in vitro tests. No 299 answer regarding the yeast–pathogen interaction by these enzymes, which indicates that these enzymes are constitutive and are the result of normal yeast metabolism (Theis and Stahl, 2004). Furthermore, in fruits inoculated with the yeast, the activity of these three enzymes (glucanase, nagase and chitinase) decreases with respect to fruits no inoculated with M. caribbica, this phenomenon may be due to the presence of biofilms produced by the yeast, which are communities of microorganisms viable and nonviable primary protected by extracellular polymeric substances (EPS) polyanionic fixed to a surface (Chmielewski and Frank, 2003), there have been several studies demonstrating that the EPS of biofilms protect microorganisms to antimicrobial agents, prevent access of biocides, sequestering metals, toxins, prevent dehydration, allow microorganisms to capture nutrients, alter the pattern of gene expression and increase stress resistance of microorganisms and in some cases of the host (Droby et al., 2009; Lazazzera, 2005; Navia et al., 2010; Nobile and Mitchell, 2005; Suntharalingam and Cvitkovitch, 2005; Visick and Fuqua, 2005), which presumably decreases the induction of enzyme production aforementioned, by the same plant and yeast. In this study the biofilm study formation was detected (Fig. 2a and b) or the presence of a mature biofilm (Fig. 2c) which can be associated with mechanisms of competition for space and nutrients, and parasitism against C. gloeosporioides, because the yeast cells as primary colonizers exclude other potential colonizers, either by the production of any metabolite or by competition for space and nutrients (Lasa et al., 2005; San-José and Orgaz, 2010). The ability of microorganisms to adhere to specific surfaces (pathogen, host or both) and biofilm formation is regulated by the phenomenon called quorum sensing (Droby et al., 2009) and starts with the adhesion of cells to the host and, through chemical signals and activation of their genes form microcolonies producing EPS (Fig. 2a–c) (Navia et al., 2010; Visick and Fuqua, 2005). Regarding the fungal inhibition tests performed with filtered and sterilized extracts of the antagonist yeast, pathogen development with no inhibition halos was observed. This observation may indicate that there was no production of antibiotics or the amount produced was not enough to produce a sufficient inhibition. A similar response occurred when the filtered extracts without sterilization containing the previously mentioned hydrolytic enzymes were used. This result suggests that, in the absence of antibiotics and/or enzymes in sufficient quantity, there is no inhibition of the development of C. gloeosporioides. Rosa et al. (2010) found that Torulaspora globosa ((Klocker) Van der Walt & Johannsen) did not produce chitinase or b-1,3-glucanase in sufficient amounts to inhibit the development of Colletotrichum sublineolum (Henn. ex Sacc. & Trotter) in sorghum (Sorghum bicolor (L.) Moench) but in quantities that could cause nonlethal deformities to the pathogen mycelium, which could affect hyphae penetration in the host tissue. Such a situation would indicate that the abovementioned hydrolytic enzymes affect the infective process of C. gloeosporioides, which would have delayed the onset of illness symptoms that were caused by the fungus in the mangoes used in this study. However, when the yeast cells were inoculated in the filter paper discs and placed on PDA with C. gloeosporiodies, there was a clear inhibition of the fungus, i.e., this yeast likely has several mechanisms for inhibiting the development of C. gloeosporioides. The combination of the action mechanisms provides to yeast the antagonistic capacity for this process, such as previously stated by Ippolito et al. (2005) and Janisiewicz and Korsten (2002). The results of this study clearly show that M. caribbica can reduce anthracnose when they are applied directly to the fruit. This specie has been accepted by many researchers because they are nontoxic and therefore, has even been used in the alcoholic beverage industry for the production of tequila (Saucedo-Luna et al., 2011) and tchapalo, a traditional sorghum drink prepared in Ivory 300 P.U. Bautista-Rosales et al. / Biological Control 65 (2013) 293–301 Coast (N’guessan et al., 2011). It has even been found associated microbial communities present in tibico from Brazil (Miguel et al., 2011). This study shows that M. caribbica has several action mechanisms that can be used for the management of mango anthracnose caused by the fungus C. gloeosporioides. The use of this yeast provides an advantage over synthetic fungicides because having multiple ways to attack and/or compete against a pathogen reduces the risk of induced resistance (Guédez et al., 2009; Janisiewicz and Korsten, 2002; Van-Lenteren, 2008). However, further research is required (1) to obtain the technology for increasing the effectiveness of the biocontrol yeast specie studied and (2) to optimize the cultivation medium for obtaining large-scale production that enables field applications according to the demands of the mango cultivators of our country and especially for those engaged in the organic production of this important fruit. 5. Conclusions The yeast M. caribbica L6A2 is an efficient biocontrol for the phytopathogenic C. gloeosporioides, presenting different antagonistic mechanisms of action such as competition for space and nutrients, production of hydrolytic enzymes, parasitism and biofilm formation through quorum sensing. According to the action mechanisms observed, it is assessed that the presence of yeast cells is necessary in the formula to carrying out biological control. This information needs to be taken account for further studies, especially in formulation and large scale production. 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