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EFFECT of SELECTED NATURAL ESSENTIAL OILS on in Vitro DEVELOPMENT of FUNGUS Trichoderma Harzianum FOUND in COMMON MUSHROOM (Agaricus Bisporus ) CULTIVATION

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EFFECT of SELECTED NATURAL ESSENTIAL OILS on in Vitro DEVELOPMENT of FUNGUS Trichoderma Harzianum FOUND in COMMON MUSHROOM (Agaricus Bisporus ) CULTIVATION ECOLOGICAL CHEMISTRY AND ENGINEERING S Vol. 17, No. 2 2010 Romuald GÓRSKI 1*, Krzysztof SOBIERALSKI 2, Marek SIWULSKI 2 and Katarzyna GÓRA 1 EFFECT OF SELECTED NATURAL ESSENTIAL OILS ON in vitro DEVELOPMENT OF FUNGUS Trichoderma harzianum FOUND IN COMMON MUSHROOM (Agaricus bisporus ) CULTIVATION WPŁYW WYBRANYCH NATURALNYCH OLEJKÓW ETERYCZNYCH NA ROZWÓJ IN VITRO GRZYBA Trichoderma harzianum WYST ĘPUJ ĄCEGO W UPRAWIE PIECZARKI DWUZARODNIKOWEJ ( Agaricus bisporus ) Abstract: Investigations on the effect of selected natural essential oils on the development (mycelium growth and sporulation) of fungus Trichoderma harzianum found in common mushroom ( Agaricus bisporus ) growing were conducted in 2008 at the Department of Plant Protection Methods, the Poznan University of Live Sciences. The following natural essential oils were analyzed: coriander oil ( Coriandrum sativum ), manuka oil ( Leptospermum scoparium ), lavender oil ( Lavandula officinalis ), peppermint oil ( Mentha piperita ) applied at concentrations of 250, 500 and 1000 µg/cm 3 of potato dextrose agar (PDA medium produced by Merck Co.). All essential oils used in the analyses were produced by “Pollena Aroma” Co. in Warsaw. The control combination was potato dextrose agar with no oils added. A strong inhibitory action reducing in vitro growth of Trichoderma harzianum was recorded in case of manuka oil at a concentration of 500 and 1000 µg/cm 3 PDA. Inhibition of fungal growth was statistically significant and 4 days after medium inoculation it amounted to 67.4 and 64.7% in relation to the control, respectively. After application of all tested natural essential oils sporulation of the fungus was significantly reduced at 6 days after medium inoculation, by 50.2÷99.9%. Keywords: common mushroom ( Agaricus bisporus ), Trichoderma harzianum , development, inhibition, natural essential oils Within the last twenty years the production of common mushroom has been growing. However, for several years now considerable loses have been recorded in the growing of edible mushrooms, caused by the appearance of Trichoderma fungi. In plant protection these saprotrophic soil fungi were known before as agents of biological control of numerous pathogenic fungi [1-7]. They are used to produce biopreparations applied against pathogenic fungi found on plants [8-11]. 1 Department of Plant Protection Methods, Poznan University of Life Sciences, ul. Zgorzelecka 4, 60-198 Pozna ń 2 Department of Vegetable Crops, Poznan University of Life Sciences, ul. Dąbrowskiego 159, 60-594 Pozna ń * Corresponding author: [email protected] 178 Romuald Górski, Krzysztof Sobieralski, Marek Siwulski and Katarzyna Góra In edible mushroom crops fungi Trichoderma spp. cause green moulds attacking both the substrate with the developing mycelium of common mushroom, oyster mushroom and shiitake, and their fruiting bodies [12-19]. So far, effective methods of protecting mushrooms against green moulds have not been developed. Available chemicals are not very efficient and, at the same time, they pose a risk of toxic substance accumulation in fruiting bodies. Studies aiming at the development of an effective protection program of cultivated mushrooms using natural preparations are gaining in importance. The aim of this study was to evaluate the effect of selected natural essential oils on the development of Trichoderma harzianum found in the common mushroom ( Agaricus bisporus ) crop. Materials and methods Investigations on the effect of selected natural essential oils on the development of Trichoderma harzianum were conducted in 2008 at the Department of Plant Protection Methods, Poznan University of Live Sciences, Poland. Table 1 A list of major chemical compounds found in natural essential oils used in the study and their contents (%) [36-40] Essential Monoter- Monoterpenes Sesquiterpenes Esters Cinnamate Ketones Oxides oil penols Alpha-selinene 7.7 Beta- caryophyllene 5.7 Alpha-pinene Alpha- Trans Manuka 4.9 Geranyl 1.8 cineole Linalool 3.1 humulene 5.6 Methyl - oil Myrcene 2.1 acetate 1.4 2.1 Beta-elemene cinnamate 5 Beta-pinene 1.6 2.9 Gamma- ylangene +Alpha- copaene 2.6 Menthol Menthyl Menthone Pepper- Alpha-pinene 30-45 - acetate - 17-31 1.8 cineole 5 mint oil 1-4 Linalool 0-2 2-10 Pulegone 1 Gamma- terpinene 0.3-11.2 Geranyl Coriander Linalool 58- Camphor Alpha-pinene - acetate - - oil 80.3 3-5.1 0.2-10.9 8-12 p-cymene 0.1-8.1 Myrcene 1-2 Beta-pinene Linalool Beta Lavender 0.8-2.6 18-42 Linyl acetate 1.8 cineole caryophyllene - - oil Alpha-pinene Terpinen- 20-40 28-34.9 0.5-1.9 0.6-1.9 4-ol 3 Ocimene 0.5 The effect of selected natural essential oils on in vitro development of fungus Trichoderma … 179 The following natural essential oils were analyzed: coriander oil ( Coriandrum sativum ), manuka oil ( Leptospermum scoparium ), lavender oil (Lavandula officinalis ) and peppermint oil ( Mentha piperita ), which were applied at concentrations of 250, 500 and 1000 µg/cm 3 of potato dextrose agar (PDA medium produced by Merck Co.). All essential oils used in the analyses were produced by “Pollena Aroma” Co. in Warsaw. For comparison, effectiveness of an organic fungicide Bravo 500 SC (50% chlorothalonil) at a concentration of 2200 µg/cm 3 PDA was also estimated. Specified amounts of the above-mentioned preparations were added to the medium. The control combination was potato dextrose agar with no preparations added. The experiment was performed in six replications. The strain of Trichoderma harzianum used in the experiment was isolated on 12.05.2005 from the substrate based on straw and poultry dung, in the common mushroom farm in Mikulice near Turek. For the inoculation of media, 5-mm discs collected from the margin of a 7-day-old culture of T. harzianum were used. Petri dishes (85 mm) were incubated at 23°C in the dark. Fungal colonies were measured after 2, 3 and 4 days in two perpendicular directions and next their diameter was calculated. Six days after medium inoculation, the sporulation of fungus T. harzianum was determined using a hemocytometer, and the recorded data were calculated using the following formula: X ⋅1000 S = 0.004 where: S - number of conidia in 1 cm 3 suspension, X - mean number of conidia in suspension over one large area. Efficiency of the preparations was determined on the basis of a comparison of fungal colony diameters and concentrations of conidial suspensions in relation to the control combination. Results were analyzed statistically based on the t-Student’s test at the significance level p = 0.05. Results and discussion In Poland such fungicides as Sporgon 50 WP (prochloraz), Bravo 500 SC (chlorothalonil) and Bravo 75 WG (chlorothalonil) are recommended in the protection of common mushroom crops against diseases: wet bubble ( Mycogene perniciosa ) and verticillium spot and dry bubble ( Verticillium spp.) [20]. However, there are no recommendations concerning chemical control of fungi from genus Trichoderma, causing cluster mould in common mushroom growing. However, chemical protection agents, including fungicides, have a negative effect on mycelium growth in common mushroom, they reduced yields and pose a risk of the accumulation of toxic substances in fruiting bodies. This also pertains to preparations recommended in common mushroom farming [19]. This prompts researchers to search for alternative methods to control fungi from genus Trichoderma , as well as other pathogens found in common mushroom growing. A promising option is connected with the application of natural origin preparations, including essential oils, characterized by a complex chemical composition (Tab. 1), with many of their compounds exhibiting bactericidal and fungicidal properties. The natural manuka oil used in this study at a concentration of 500 and 1000 µg/cm 3 medium strongly inhibited the development of fungus T. harzianum , similarly as the compared organic fungicide Bravo 500 SC (Tab. 2). The inhibition of mycelium growth 180 Romuald Górski, Krzysztof Sobieralski, Marek Siwulski and Katarzyna Góra was statistically significant and at 4 days after inoculation amounted in case of the essential oil to 67.4 and 64.7%, while in the combination with the applied fungicide it was 68.8% in relation to the control. Table 2 Growth of Trichoderma harzianum on PDA medium with an addition of natural essential oils or agent Bravo 500 SC No. of days from the date of medium inoculation Agents and their 2 3 4 concentration in Colony % inhibition Colony % inhibition Colony medium [% 3 diameter or diameter or diameter [µg/cm ] inhibition] [mm] increase (+) [mm] increase (+) [mm] Manuka oil 250 25.6 c * 38.8 45.2 c 37.1 61.2 c 28.0 500 10.0 a 76.1 18.9 a 73.7 27.7 a 67.4 1000 10.9 a 73.2 19.4 a 73.0 30.0 a 64.7 Peppermint oil 250 38.4 e 8.1 71.5 ef 0.4 85.0 d 0 500 38.8 e 7.2 70.0 e 2.5 85.0 d 0 1000 18.6 b 55.5 34.9 b 52.4 52.7 b 38.0 Coriander oil 250 43.0 h + 2.8 75.2 g + 4.5 85.0 d 0 500 40.6 efg 2.9 70.6 e 1.7 83.0 d 2.4 1000 35.2 d 15.8 63.2 d 12.0 84.4 d 0.7 Lavender oil 250 41.6 fgh 0.5 74.6 fg + 3.8 85.0 d 0 500 39.4 ef 5.7 71.8 efg 0 85.0 d 0 1000 38.8 e 7.2 69.4 e 3.3 85.0 d 0 Bravo 500 SC 2200 9.0 a 78.5 20.0 a 72.1 26.5 a 68.8 Control 41.8 gh - 71.8 efg - 85.0 d - * Means in columns denoted with identical letters do not differ significantly (p = 0.05) according to t-Student’s test A significant, although much lower reduction of mycelium growth of the investigated pathogen was observed also on media with an addition of manuka oil applied at a concentration of 500 µg/cm 3 and peppermint oil at 1000 µg/cm 3, which on the last day of observations was 28 and 38%, respectively.
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