Antifungal and synergistic activity of five plant essential oils from Serbia against Trichoderma aggressivum

Jelena Luković1, Rada Đurović-Pejčev1, Tijana Đorđević1, Svetlana Milijašević-Marčić1, Nataša Duduk2, Ivana Vico2 and Ivana Potočnik1* 1 Institute of Pesticides and Environmental Protection, Banatska 31b, 11080 Belgrade, Serbia 2 University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia *Corresponding author: ivana.potocnik@pesting.org.rs Received: 31 December 2020 Accepted: 15 January 2021 SUMMARY


INTRODUCTION
Fungal pathogens have a significant negative effect on button mushroom (Agaricus bisporus (Lange) Imbach) quality and yield (Grogan, 2008). The most important fungal diseases of button mushroom and their causal agents are: wet bubble caused by Mycogone perniciosa (Magnus) Delacroix, dry bubble caused by Lecanicillium fungicola var. fungicola (Preuss) Hassebrauk, cobweb disease caused by Cladobotryum spp. and green mould disease caused by Trichoderma spp. The most devastating disease is green mould, caused by Trichoderma harzianum Rifai, Trichoderma aggressivum f. europaeum Samuels & W. Gams and Trichoderma aggressivum f. aggresssivum Samuels & W. Gams, which account for 60-100% of mushroom yield losses (Seaby, 1996;Kredics et al., 2010). Green mould is characterized by the presence of white mycelia of fast-growing colonies that change their colour to dark green after extensive sporulation on the substrate. Brown necrotic spots and lesions may also appear on mushroom fruiting bodies as accompanying symptoms (Seaby, 1996). The predominant fungal pathogen of button mushroom in Europe is T. aggressivum f. europaeum, which has been transmitted from the British Isles to many countries, including Serbia (Kosanović et al., 2013).
Disease control in button mushroom farms worldwide usually includes a complex of preventive measures: strict hygiene, treatments with disinfectants, and application of fungicides and biofungicides. Only a few fungicides are currently available and officially recommended in mushroom industry: prochloraz and metrafenone in the EU countries, and chlorothalonil and thiabendazol in North America (Beyer & Kremser, 2004;Grogan, 2008;Anonymous, 2020). Recently, metrafenone has been introduced to control the fungal pathogens Cladobotryum spp. and L. fungicola in Spain, France, Belgium and the UK after reports of their decreased sensitivity to prochloraz (Carrasco et al., 2017;Anonymous, 2020). However, there is still no alternative regarding green mould control, and data on Trichoderma sensitivity to fungicides are scarce. Luković et al. (2020) found the fungicide prochloraz to be highly toxic to several Trichoderma species (T. harzianum, T. aggressivum f. europaeum, Trichoderma pleuroti S.H. Yu & M.S. Park and Trichoderma pleuroticola S.H. Yu & M.S. Park) isolated from edible mushrooms (button mushroom, oyster mushroom and shiitake), while the fungicide metrafenone was considerably toxic to the same pathogens. Metrafenone could also be recommended for the control of green mould disease in mushroom farms after additional in vivo trials.
In recent years, special attention has been dedicated to alternative measures, such as the use of microbiological products and various natural substances of biological origin (Potočnik et al., 2015). Antifungal activity of essential oils (EOs) and their components against the causal agents of green mould disease of edible mushrooms has been demonstrated mainly in vitro. Oils showing very strong activity may be promising but they require further extensive research and in vivo testing. The oils of oregano (Origanum vulgare L.), common thyme (Thymus vulgaris L.) and peppermint (Mentha piperita L.) have demonstrated very high in vitro activity against T. aggressivum f. europaeum, T. harzianum, Trichoderma atroviride P. Karsten and Trichoderma viride Tul. Đurović-Pejčev et al., 2014). The addition of tea tree oil (Melaleuca alternifolia [Maiden & Betche]) to oyster mushroom substrate (Angelini et al., 2008) or button mushroom casing (Kosanović et al., 2013) resulted in considerable in vivo inhibition of T. harzianum.
Peppermint, spearmint, thyme, basil and common St. John's wort have been acknowledged as herbs with plenty of pharmacological properties that are used in herbal medicine, as flavoring herbs and antimicrobial agents. The present study focused on testing in vitro the antifungal activity of five selected essential oils originating from Serbia, and their ten combinations, against T. aggressivum f. europaeum, using two distinctive methods: microdilution and fumigant macrodilution methods. Plant samples were air-dried at room temperature in the shade for 20 days and then subjected to hydrodistillation for 2.5 h in a Clevenger type apparatus. The obtained essential oils were dried over anhydrous sodium sulphate and preserved in sealed vials at 4°C until further analysis.

Plant samples and preparation of essential oil
Combinations were prepared by mixing each essential oil with each other at 1:1 ratio. Out of the selected five EOs, a total of ten combinations were obtained and tested: spearmint-peppermint, spearmintthyme, spearmint-basil, spearmint-St. John's wort, peppermint-thyme, peppermint-basil, peppermint-St John's wort, thyme-basil, thyme-St. John's wort and basil-St. John's wort.

Test organism and inoculum preparation
The strain of Trichoderma aggressivum f. europaeum T77 used in the study was obtained from button mushroom compost containing mycelia characteristic for green mould (2010, Barajevo-Lisovići, Serbia), previously identified by Kosanović et al. (2013). The isolate was maintained on potato dextrose agar (PDA) medium at 20 o C for 72 hours. Conidia were harvested by flooding the plates with 10 ml of sterile distilled water and Tween 20 (v/v 0.01%), followed by filtration through a double layer of cheesecloth. Conidial suspension was prepared daily in sterile distilled water and adjusted to a concentration of approximately 10 6 conidia ml -1 .

Screening of antifungal activity of essential oils in vitro
Antifungal activity was tested using two methods: fumigant macrodilution and microdilution. Five concentrations of the tested EOs were applied. The same volume range of the five EOs was used for macrodilution and microdilution methods: 1.56, 3.12, 6.25, 12.5 and 25 µl. The range of volumes of the selected oil combinations using the microdilution method was: 1.76, 3.75, 7.5, 15 and 30 µl. Respective concentrations were calculated when these volumes of oils were added to air phase (fumigant macrodilution) or microtitar wells (microdilution). The macrodilution test was repeated three, while microdilution test was repeated five times.
In the fumigant macrodilution test, antifungal activity was tested on PDA medium in glass Petri plates (R=90 mm) inoculated with mycelial fragments (R=6 mm) of the investigated strain placed at the plate center. The isolate was exposed to the volatile phase of essential oils for three days at 22°C. The selected oils were pipetted onto the inner side of plate covers on filter paper cuttings in a range of oil volumes: 1.56, 3.12, 6.25, 12.5 and 25 µl. The EO concentrations were calculated by considering the volumes evaporated in the air phase volume of Petri plates above PDA medium (78 ml). The final oil concentrations obtained in the air phase were (volume of essential oil divided by volume of air phase in Petri plate): 0.02, 0.04, 0.08, 0.16 and 0.32 µl ml -1 of air. Plate bottoms were immediately placed on top of the covers. The plates were left upside down and sealed with parafilm to prevent gas exchange with the outside environment. Control plates were without essential oils added. Inhibition of the mycelial growth was estimated three days after treatment by measuring the radial growth of the isolate treated with different oil concentrations and comparison with control plates. Fungal growth (colony diameter) was measured and the percentage growth inhibition (PGI) was calculated using the formula: where C is colony diameter (mm) in control plates, and T is colony diameter (mm) in tested plates (Kaiser et al., 2005).
Concentrations of EOs which completely inhibited mycelial growth after three-day-exposure at 22°C were considered to be fungistatic and the lowest of these concentrations was determined as the minimum inhibitory concentration (MIC). Afterwards, mycelial fragments without visible growth were transferred to PDA medium and incubated for three days at 22°C. The lowest concentration with fungicidal effect was defined as the minimum fungicidal concentration (MFC). Three replicates per treatment were used for all concentrations of each oil.
Antifungal activity was tested on a malt-extractbroth (MEB) medium in microtiter plates with 96 wells, using the microdilution method. Conidial suspension of the test fungus was added by pipetting 10 μl of conidial suspension into a total volume of 100 µl. A negative control was made by mixing 90 μl MEB medium and 10 μl conidial suspension, while a positive control included 80 μl MEB medium, 10 μl control fungicide prochloraz solution (adjusted to final concentration of 10 µl ml -1 ) and 10 μl conidial suspension, and antifungal tests were performed with 80 μl MEB medium, 10 μl solution of selected oils and 10 μl of conidial suspension. Stock solution of each essential oil was prepared by solubilizing 5 µl of essential oil in 15 µl of Tween 20, while stock solution of a combination of two oils was prepared by adding 6 µl of mixed essential oil (3 µl per oil) to 14 µl of Tween 20. Stock solution was further diluted with Tween 20 (1:1) to achieve a final range of concentrations of 1.56, 3.12, 6.25, 12.5 and 25 µl ml -1 for each oil, and 1.76, 3.75, 7.5, 15 and 30 µl ml -1 for oil mixtures. Inhibition of mycelial growth was estimated seven days after treatment by visual inspection of fungal growth. Oil concentrations which completely inhibited mycelial growth after seven-day exposure at 22°C were considered to be fungistatic, and the lowest of these concentrations was determined as the minimum inhibitory concentration (MIC). Minimum fungicidal concentration (MFC) was determined by sub-cultivation of 2 μl of suspension without visible growth in 100 μl of MEB medium in microtiter plates and further incubation for three days. The lowest concentration without any visible growth was defined as MFC, indicating a 99.5% inhibition of spore germination, compared to the original inoculum . Five replicates per treatment were used for all oil concentrations.

Serbia
Pathogen growth was inhibited by four of the five essential oils applied in a concentration range from 0.02 to 0.32 µl ml -1 of air using the fumigant macrodilution method, and from 1.56 to 25 µl ml -1 using the microdilution method (Table 1). Growth inhibition of the test pathogen after three days was achieved by spearmint, thyme, peppermint and basil oils using .25 ml ml -1 when microdilution was applied and 0.08 ml ml -1 of air when the fumigant macrodilution method was used. Also, medium antifungal activity of basil and peppermint oils was recorded under both methods. None of the selected oils exhibited a fungicidal effect, having МFCs of over 25 ml ml -1 (microdilution) or 0.32 ml ml -1 of air (fumigant macrodilution). The percentage of mycelial growth inhibition of the test pathogen caused by five tested essential oils using the fumigant macrodilution method is shown in Figure 1.

In vitro antifungal activity of ten combinations of selected essential oils from Serbia
Pathogen growth was inhibited by all ten tested essential oil combinations which were applied in a concentration range from 1.76 to 30 ml ml -1 using

DISCUSSION
The inhibitory and fungicidal activity of five selected EOs (peppermint, spearmint, thyme, basil and common St. John's wort), isolated from plants originating from Serbia, against T. aggresssivum f. europaeum was tested using two distinctive methods: microdilution and fumigant macrodilution, while the antifungal activity of ten combinations (spearmintpeppermint, spearmint-thyme, spearmint-basil, spearmint-St. John's wort, peppermint-thyme, peppermint-basil, peppermint-St John's wort, thymebasil, thyme-St. Johns' wort and basil-St. John's wort) was tested using only microdilution. Four of the five tested EOs (all except common St. John's wort) and their ten combinations inhibited the growth of T. aggresssivum f. europaeum, while seven of the ten EO combinations (all except spearmint-St. John's wort, peppermint-St. John's wort and basil-St. John's wort) showed fungicidal effects on the pathogen.
Antimicrobial effects of essential oils against myco-and phytopathogens (especially fungi and bacteria) have been evaluated in many studies. Analysing 22 EOs from Germany and Albania by fumigant macrodilution, Todorović et al. (2016) found that wintergreen, lemongrass and oregano demonstrated the strongest activity against three bacteria (Xanthomonas campestris pv. phaseoli, Clavibacter michiganensis subsp. michiganensis and Pseudomonas tolaasii) that are pathogens of common bean, tomato and cultivated mushroom. On the other hand, two mint oil samples showed the strongest activity at 0.02 µl ml -1 of air (followed by eucalyptus, black pine and cade) against Verticillium dahliae, a pathogenic fungus of pepper (Luković et al., 2019a), and against Chryphonecria parasitica (followed by black pine, eucalyptus, cade, sage and silver fir), a pathogenic fungus of chestnut (Luković et al., 2019b). Many publications have reported significant antifungal activity of various EOs against important button mushroom pathogens: L. fungicola var. fungicola, M. perniciosa and Cladobotryum spp. (Tanović et al., 2006;Glamočlija et al., 2006, Džamić et al., 2008Soković et al., 2009;Luković et al., 2018). Testing 18 EOs, Tanović et al. (2006) found that thyme, cinnamon, clove and tea tree oils had the highest antifungal activity against these mycopathogenic fungi, while Luković et al. (2018) found that clove and cinnamon completely inhibithed the growth of L. fungicola and Cladobotryum dendroides in tests that used three distinctive methods. Various EOs have been tested against various Trichoderma species, pathogens of cultivated mushrooms, and they demonstrated inhibitory effects in vitro and in vivo. Oils of oregano, common thyme and peppermint have shown very high in vitro activity against T. aggressivum f. europaeum, T. harzianum, T. atroviride and T. viride Đurović-Pejčev et al., 2014), while tea tree oil added to oyster mushroom substrate or button mushroom casing resulted in considerable in vivo inhibition of T. harzianum (Angelini et al., 2008;Kosanović et al., 2013). Đurović-Pejčev et al. (2014) analyzed six essential oils originating from Serbia (peppermint, basil, yarrow, walnut, juniper and St. John's wort), using fumigant macrodilution. They found basil and peppermint oils to have the strongest activity against T. aggressivum f. europaem at 0.02 and 0.04 μl ml -1 of air, respectively, and only peppermint oil showed lethal effect at 0.64 μl ml -1 of air. The main components of that peppermint EO were menthone (37.02%), menthol (29.57%) and isomenthone (9.06%). The strongest activity of peppermint and thyme essential oils were confirmed in the current study, but lethal effect was not recorded at the tested concentrations and it was higher than 0.32 μl ml -1 of air. Essential oils of different Mentha species had a satisfactory antimicrobial potential, especially peppermint and spearmint EOs, which inhibited the growth of T. viride at 2.5 μl ml -1 and two other pathogens of cultivated mushrooms: T. harzianum and P. tolaasii Soković et al., 2009). Saroglou et al. (2007) found that St. John's wort EO originating from Serbia exhibited antibacterial activity against P. tolaasii, which causes bacterial blotch on button mushroom, while the current study did not show St. John's wort EO to exhibit antifungal activity against T. aggressivum f. europaeum. Using the macrodilution method, Abdolahi et al. (2010) confirmed the antifungal activity of basil EO against the phytopathogenic fungus Botrytis cinerea at 0.5 µl ml -1 with a mycelial growth inhibition of 42.5%. In the study, which used the fumigant macrodilution method, basil essential oil completely inhibited the growth of tested pathogenic fungi at 0.32 µl ml -1 of air. Comparing different methods, microdilution enables the testing of spore germanation, while macrodilution shows effects on spore germination and mycelial growth. However, the volume of EO spent in a macrodilution test is higher than in microdilution test. Therefore, it is recommended to use the microdilution method in preliminary screening of antimicrobial activity of EOs, while macrodilution is also suitable for antimicrobial assessment and for prediction of further practical applications of EOs as fumigants. In the previous study, Luković et al. (2018) described and compared in detail three distinctive methods, including the two used in the current study.
Although studies of combinations and synergistic antifungal effects of EOs and their components are scarce compared to those focusing on the effects of oils applied individually, they have become more frequent in recent years. Stević et al. (2014) described a synergy between thymol and carvacrol in thyme and oregano EOs against fungi isolated from medicinal plants: Aspergillus niger, Aspergillus flavus, Alternaria alternata and Fusarium spp. A similar synergistic effect of thymol and carvacrol (thyme-oregano EO combination) has been reported against some Aspergillus spp. and Penicillium chrysogenum (Hossain et al., 2016) and against B. cinerea and Penicillium expansum (Nikkhah et al., 2017) as the combined treatments caused a more significant decrease in fungal growth than each oil individually. Similar findings of improved activities of combinated oils were confirmed in the current study, i.e. that antifungal activity of the EO combinations thyme-spearmint and spearmintpeppermint were more efficient than the activity of each oil individually.
The obtained results indicate possible synergistic effects of essential oils and their components. Also, the study showed that four tested essential oils (peppermint, spearmint, thyme and basil) completely inhibited the growth of T. aggressivum f. europaeum, while their combinations had fungicidal effects against it, indicating that they could be considered as eligible candidates for further in vivo experiments.