Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in, causal agent of citrus black spot

Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in,...

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Biocontrol Science and Technology

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Antimicrobial volatile organic compounds affect morphogenesisrelated enzymes in Guignardia citricarpa, causal agent of citrus black spot

Mauricio Batista Fialho a , Luiz Fernando Romanholo Ferreira b

Regina Teresa Rosim Monteiro b & Sérgio Florentino Pascholati a Department of Plant Pathology and Nematology, ‘Luiz de

Queiroz College of Agriculture’, University of São Paulo, CP 09,

CEP 13418-900, Piracicaba, SP, Brazil b Center for Nuclear Energy in Agriculture, University of São

Paulo, CP 96, CEP 13400-970, Piracicaba, SP, Brazil

Available online: 01 Jun 2011

To cite this article: Mauricio Batista Fialho, Luiz Fernando Romanholo Ferreira, Regina Teresa Rosim Monteiro & Sérgio Florentino Pascholati (2011): Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in Guignardia citricarpa, causal agent of citrus black spot, Biocontrol Science and Technology, 21:7, 797-807

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Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in Guignardia citricarpa, causal agent of citrus black spot

Mauricio Batista Fialhoa, Luiz Fernando Romanholo Ferreirab, Regina Teresa Rosim Monteirob and Sergio Florentino Pascholatia* aDepartment of Plant Pathology and Nematology, ‘Luiz de Queiroz College of Agriculture’, University of Sao Paulo, CP 09, CEP 13418-900, Piracicaba, SP, Brazil; bCenter for Nuclear Energy in Agriculture, University of Sao Paulo, CP 96, CEP 13400-970, Piracicaba, SP, Brazil

Although non-volatile substances toxic to plant pathogenic microorganisms have been extensively studied over the years, few studies have focused on microbial volatile organic compounds (VOCs). The VOCs produced by the yeast Saccharomyces cerevisiae strain CR-1, used in fermentative processes for fuel ethanol production, are ablet oi nhibit the vegetatived evelopment of thef ungus Guignardia citricarpa, causal agent of the disease citrus black spot. How microbial VOCs affect the development of fungi is not known. Thus, the objective of the present work was to study the effect of the artificial mixture of VOCs identified from S. cerevisiae on intracellularenzymes involved in the mycelial morphogenesis in G. citricarpa.T he phytopathogenic fungus was exposed to artificial mixture of VOCs constituted by alcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol) and esters (ethyl acetate and ethyl octanoate) in the proportions naturally found in theatmosphere produced by theyeast.The VOCs inhibitedconsiderably the mycelial development and interfered negatively with the production of the morphogenesisrelated enzymes. After 72 h of exposure to the VOCs the laccase and tyrosinase activitiesdecreased 46 and 32%, respectively, however, the effect on the chitinase and b-1,3-glucanaseactivities was lower, 17 and 13% of inhibition, respectively. Therefore, the exposure of the fungus to the antimicrobial volatiles can influence both fungal mycelial growth rate and activity of enzymes implicated in morphogenesis. This knowledge is important to understand the microbial interactions mediated by VOCs in nature and to develop new strategies to control plant pathogens as G. citricarpa in postharvest.

Keywords: antimicrobial activity; biocontrol; Citrus;m orphogenesis;p lant disease


Citrus black spot, a fungal disease caused by Guignardia citricarpa Kiely (anamorphic stage: Phyllosticta citricarpa McAlpine) [Ascomycetes: Dothideales], is one of the most important diseases of citrus worldwide. It has high economic importance and affects the most important commercial citrus varieties in many producing areas of Africa, Asia, Australia, and South America (OEPP/EPPO 2009). Several fruit symptoms are associated to the disease and although not showing apparent symptoms, the infected fruits can develop them at postharvest during

*Corresponding author. Email:

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transport or storage. The lesions are restricted to the fruit rind, but the fruits become aesthetically damaged, making them undesirable to the fresh fruit market. In addition, it is considered an A1 quarentenary disease and infected fruits cannot be exported especially to the European Community due to phytosanitary restrictions (OEPP/EPPO 2009).

Even though their effectiveness is limited, the use of fungicides is the main control method used at pre- and post-harvest. However, the acquisition of resistance by the pathogen and the consumer perception about the potential impact of traditional control practices on health and on environment led to an increased demand for residue-free chemical products. Therefore, farmers and researchers started to consider the use of alternative methods to control diseases (Punja and Utkhede 2003).

During a plant pathogen interaction, microbial antagonists may interrupt some stage of the disease or the pathogen’s life cycle. This may occur by several mechanisms such as parasitism, competition for nutrients and colonization niches, production of hydrolytic enzymes and antibiotic compounds (Sharma, Singh, and Singh 2009), including volatiles (Strobel 2006). Volatile organic compounds (VOCs) produced by one microorganism could enhance its status by affecting the physiology of other competitor organisms causing them disadvantage (Mackie and Wheatley 1999; Wheatley 2002). Typically, such compounds have low molecular weight, high vapor pressure, are active at very low concentrations and belong to several chemical groups (Wheatley 2002). The antagonism caused by these compounds has received limited attention in comparison to medium-diffusible compounds (Chaurasia et al. 2005), but recently new findings have focused attention on these volatile metabolism products. Most of the studies about production of antimicrobial VOCs are related to Muscodor spp., Trichoderma spp., and Bacillus spp. to control phytopathogenic and wood decay fungi (Humphris, Bruce, Buultjens, and Wheatley 2002; Grimme, Zidack, Sikora, Strobel, and Jacobsen 2007; Leelasuphakul, Hemmaneea, and Chuenchitt 2008).

M. albus, an endophytic fungus isolated from cinnamon tree, is a well known volatile antimicrobial producer. The fungus emits a complex mixture of about 30 VOCs and it has been tested to control several pathogens in infested soils, fruits and seeds in storage (Strobel 2006). The use of artificial mixtures showed that the presence of naphthalene, propanoic acid, and 3-methyl-1-butanol was necessary to keep the inhibitory activity against the pathogens Pythium ultimum, Rhizoctonia solani, and Sclerotinia sclerotiorum (Ezra, Hess, and Strobel 2004).

ThesaprophyticfungiTrichodermaspp.havemanyantagonisticmechanismswhich have contributed to their success as biological control agents. Wheatley, Hackett, Bruce, and Kundzewicz (1997) demonstrated the production of 2-propanone, 2-methyl-1-butanol, decanal, heptanal, and octanal by T. pseudokoningii and T. viride as responsible for the antimicrobial activity against wood decay fungi (Wheatley et al. 1997).

The action mechanisms of antimicrobial volatiles are not fully understood until now and the discussions have been merely speculatory. It is likely that volatiles act by changing protein expression (Humphris et al. 2002) and affecting the activity of specific enzymes (Mackie and Wheatley 1999). The knowledge about how this mechanisms works is essential to improve the biocontrol effectiveness as well as to develop innovative control strategies.

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Fungal polyphenol oxidases like tyrosinases and laccases are enzymes linked to mycelial growth. Tyrosinases are directly involved in the melanin biosynthesis. Melanin is a pigment implicated in the resistance to stress factors such as free radicals, UV radiation and contributes to the cell wall resistance against hydrolytic enzymes (Henson, Butler, and Day 1999). The laccases are involved in the morphogenesis, protection against stress, resistance to fungicides, lignin degradation, and plant pathogen interaction (Baldrian 2006). In fungi, the shape and cell integrity are dependent of the cell wall, a complex structure that typically has as main components the polysaccharides chitin and 1,3-band 1,6-b- glucan. During the normal growth, chitinases degrades the chitin present in the hypha tip, with concomitant insertion of chitin oligomers by chitin synthases. In similar way, b-1,3-glucanases and b-glucan synthases act together removing and inserting glucan oligomers in the cell wall. Therefore, chitinases and b-1,3-glucanases have important role in the break and polymer reconstruction leading to cell wall remodeling during cell division and morphogenesis processes, such as growth and hyphal branching, differentiation and germination of spores as well as autolytic processes (Adams 2004). Potential applications for biological fumigation by microbial antagonists or their artificial mixtures of VOCs in closed chambers are currently being investigated and include the control of a wide range of storage pathogens in fresh fruits as well as other commodities, such as seeds, grains, and nuts. This process does not require direct contact with the product and minimizes product handling. Another promising option includes its use to replace methyl bromide fumigation as a means to control soil-borne plant diseases (Strobel 2006). The yeast Saccharomyces cerevisiae strain CR-1, isolated from fermentative processes for fuel ethanol production, is able to inhibit the mycelial growth of G. citricarpa. The antagonism was attributed to production of a mixture of VOCs composed mainlyof ethanol, constituting 85% of the headspace, the aliphatic alcohols 3-methyl-1-butanol and 2-methyl-1-butanol, the aromatic alcohol phenylethyl alcohol and the esters ethyl acetate and ethyl octanoate (Fialho et al. 2010). The biological fumigation of fruits using S. cerevisiae or artificial mixtures of

VOCs is an attractive alternative method to control the citrus black spot at postharvest during storage and shipment since the traditional control methods has been ineffective due to resistance to the limited spectrum of fungicides permitted for the postharvest management (Adaskaveg, Forster, and Sommer 2002). This process would be safer to human health and environment as the yeast is classified as Biosafety Level 1 by U.S. Office of Health and Safety (CDC/OHS 2009), since it is not a human pathogen, does not produces mycotoxins, antibiotics, or other molecules whose presence is unacceptable in foods. In addition, all VOCs produced by the yeast are generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA 2011). Another advantage is the better acceptance by consumers, who are familiar with S. cerevisiae widely used in the production of foods and drinks. Due to the lack of knowledge about the action mechanisms of antimicrobial

VOCs, this study aimed to investigate the activity of morphogenesis-related enzymes in G. citricarpa exposed to the artificial mixture of VOCs identified from S. cerevisiae.

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Materials and methods Phytopathogenic fungus

Guignardia citricarpa, isolated from orange fruit lesions, was maintained in potato dextrose agar (PDA) at 268C, under fluorescent light and a 12 h L:12 h D photoperiod. The fungus is deposited as isolate IP-92 in the Mycological Culture Collection of the Laboratory of Plant Pathology in the Department of Phytosanity at FCAV/UNESP, in Jaboticabal-SP, Brazil.

Antimicrobial activity of the artificial mixture of VOCs

From the information obtained by Gas Chromatography coupled to Mass Spectrometric Detection (GC MS) analysis of the gaseous atmosphere produced by S. cerevisiae strain CR-1 (Fialho et al. 2010) it was produced an artificial mixture of VOCs, using authentic standard chemicals (9% ACS reagent grade, Sigma/Aldrich Chemical Co., St. Louis, USA). The mixture contained the six compounds positively identified and the proportion of each compound was calculated from the relative peak areas in relation to all other components of the mixture (Table 1). Two section-divided polystyrene plates (BD Falcon, USA) were used to the bioassays as illustrated in the Figure 1. In one side it was added 10 mL of PDA and over the medium a semi-permeable membrane (5 5 cm) was placed. On top of the membrane, a mycelium plug (5 m) of the pathogen was added. The headspace of the polystyrene plate was 50 mL and this was used to calculate the concentration of VOCs per mL of air space. After 5 days of growth, on the opposite side of the plate, 24 mL (0.48 mLm L 1 of air space) of the artificial mixture was added on a piece of sterile cotton wool. The plates were immediately wrapped with parafilm and maintained at 268C under a 12 h L: 12 h D photoperiod. The control consisted of plates containing the pathogen in the absence of the artificial mixture. After 24, 48, and 72 h of exposure to VOCs the membranes containing the mycelium were removed from the medium and the biomass harvested, weighed and stored at 208C. The mycelial growth was also evaluated daily based upon the average between two opposing measurements of the colonies. All experiments were carried out in triplicate.

Table 1. VOCs produced by S. cerevisiae strain CR-1 on PDA.

Compound1 % Relative (v/v)

1 Ethanol 85.3 2 Unidentified 1.5 3 Ethyl acetate 1.8 4 3-Methyl-1-butanol 6.9 5 2-Methyl-1-butanol 2.4 6 Phenylethyl alcohol 0.7 7 Ethyl octanoate 1.4

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