Scholarly article on topic 'In vitro and in vivo screening of essential oils for the control of wet bubble disease of Agaricus bisporus'

In vitro and in vivo screening of essential oils for the control of wet bubble disease of Agaricus bisporus Academic research paper on "Biological sciences"

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{" Agaricus bisporus " / "Essential oils" / Mushrooms / " Mycogone perniciosa "}

Abstract of research paper on Biological sciences, author of scientific article — T. Regnier, S. Combrinck

Abstract Proliferation of fungal pathogens, such as Mycogone perniciosa, can severely affect the yields of cultivated mushrooms, including that of the button mushroom, Agaricus bisporus. A reduction in the number of fungicidal products approved for commercial application is currently providing new challenges to the mushroom industry. Forty essential oils, seven pure terpenoids and one phenylpropanoid were screened in vitro to determine the abilities of these substances to inhibit the growth of M. perniciosa. The fungal growth medium of both A. bisporus and M. perniciosa was supplemented with each test substance at a concentration of 50μL/L. Ten essential oils were further investigated at lower concentrations ranging from 5 to 40μL/L. The main components of these oils were determined by GC–FID and GC–MS. Lemon verbena (Lippia citriodora), lemongrass (Cymbopogon citratus) and thyme (Thymus vulgaris) oils were found to substantially inhibit the growth of the pathogen, while demonstrating lower toxicity towards A. bisporus than any of the other oils tested. A preliminary in vivo trial using M. perniciosa-inoculated casings revealed that the preventative use of lemon verbena or thyme oils was able to control the development of the disease. A commercial trial using these oils, as well as two of their main components (nerol and thymol), at a concentration of 40μL/L, revealed that none of these treatments were detrimental to the growth of the A. bisporus and an overall yield similar to that following application of a commercial fungicide (Chronos 450 SC) was obtained. These results suggest that essential oils or mixtures of selected pure components of essential oils may in future find application in button mushroom production, either as a substitute for synthetic fungicides or as an additional protective measure.

Academic research paper on topic "In vitro and in vivo screening of essential oils for the control of wet bubble disease of Agaricus bisporus"

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South African Journal of Botany 76 (2010) 681 - 685

www.elsevier.com/locate/sajb

In vitro and in vivo screening of essential oils for the control of wet bubble

disease of Agaricus bisporus

T. Regnier*, S. Combrinck

Department of Chemistry, Tshwane University of Technology, P.O. Box 56208, Arcadia 0007, South Africa Received 1 April 2010; received in revised form 26 July 2010; accepted 26 July 2010

Abstract

Proliferation of fungal pathogens, such as Mycogone perniciosa, can severely affect the yields of cultivated mushrooms, including that of the button mushroom, Agaricus bisporus. A reduction in the number of fungicidal products approved for commercial application is currently providing new challenges to the mushroom industry. Forty essential oils, seven pure terpenoids and one phenylpropanoid were screened in vitro to determine the abilities of these substances to inhibit the growth of M. perniciosa. The fungal growth medium of both A. bisporus and M. perniciosa was supplemented with each test substance at a concentration of 50 ||L/L. Ten essential oils were further investigated at lower concentrations ranging from 5 to 40 |L/L. The main components of these oils were determined by GC-FID and GC-MS. Lemon verbena (Lippia citriodora), lemongrass (Cymbopogon citratus) and thyme (Thymus vulgaris) oils were found to substantially inhibit the growth of the pathogen, while demonstrating lower toxicity towards A. bisporus than any of the other oils tested. A preliminary in vivo trial using M. perniciosa-inocuAated casings revealed that the preventative use of lemon verbena or thyme oils was able to control the development of the disease. A commercial trial using these oils, as well as two of their main components (nerol and thymol), at a concentration of 40 |L/L, revealed that none of these treatments were detrimental to the growth of the A. bisporus and an overall yield similar to that following application of a commercial fungicide (Chronos 450 SC) was obtained. These results suggest that essential oils or mixtures of selected pure components of essential oils may in future find application in button mushroom production, either as a substitute for synthetic fungicides or as an additional protective measure. © 2010 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Agaricus bisporus; Essential oils; Mushrooms; Mycogone perniciosa

1. Introduction

Wet bubble disease caused by Mycogone perniciosa (Magnus) Delacroix, is not species-specific (Holland and Cooke, 1990) and frequently leads to a substantial reduction in the yields of cultivated mushrooms, particularly of the button mushroom, Agaricus bisporus (J. Lange) Imbach (Fletcher et al., 1995; Umar et al., 2000). It is well established that the development of M. perniciosa is usually associated with the use of infected soil or spawn (Gandy and Spencer, 1978). However, the presence of spores in the ventilation system is frequently a source of contamination (personal communication with Dr M. Van Greuning, Sylvan Incorporated, Pretoria, South Africa).

* Corresponding author. Tel.: +27 12 3826126; fax: +27 12 3826286. E-mail address: regniert@tut.ac.za (T. Regnier).

Mushrooms are generally infected in the early developmental stages. On commercial farms, wet bubble disease is controlled by fumigation, spraying with formalin or lysol, soil sterilisation (Smith, 1924) or by applying a mixture of prochloraz-manganese (Eicker, 1987). Immediate application of salt to the infected areas, followed by covering has been reported to provide significant control of the disease (Pieterse, 2005).

The number of approved fungicides for disease control in commercial mushroom production has been severely restricted, thereby placing immense pressure on producers. For example, the European community recently withdrew carbendazim for use in mushroom production, thereby banning all benzimid-azole-based fungicides conventionally applied for the control of pathogens such as M. perniciosa (Grogan, 2008). It is imperative that alternative disease control measures be identified for commercial application. The antifungal properties

0254-6299/$ - see front matter © 2010 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2010.07.018

of plant extracts and essential oils are well documented (Ríos and Recio, 2005; Sokovic et al., 2009). Glamoclija et al. (2006) demonstrated the in vitro antifungal activities of Satureja thymbra and Salvia pomífera volatiles against M. perniciosa. The essential oil of Critmum maritimum, as well as two of the major components of the oil, limonene and a-pinene, was also found to exhibit inhibitory activities against the pathogen (Glamoclija et al., 2009). In this study, 40 essential oils, some of their major terpenoid components and a phenylpropanoid (eugenol) were screened to identify oils and compounds that have the ability to inhibit the growth of M. perniciosa, without affecting the development and yield of the edible mushroom, A. bisporus.

2. Material and methods

Purified A. bisporus and M. perniciosa isolates were kindly supplied by Sylvan Incorporated (Pretoria, South Africa). Essential oils of Lippia scaberrima and Lippia rehmannii were obtained by steam distillation of wild plants as described in Combrinck et al. (2006) and in Linde et al. (2010), respectively. Lippia javanica essential oil was kindly supplied by Scatters (Johannesburg, South Africa). Apelium graveolens, Carum carvi, Cinnamomum zeylanicum and Eucalyptus dives oils were obtained from Essentia Products, South Africa, Eucalyptus citriodora was obtained from NatureSun Aroma, Eucalyptus citriodora, Eucalyptus globulus, Eucalyptus radiata, Lavendula angustifolia, Mentha piperita and Thymus vulgaris essential oils from Burgess and Finch (distributed by Vital Health Foods, Kuilsriver, South Africa), while the remaining essential oils (Table 1) were purchased from Holistic Emporium (Johannesburg, South Africa). All pure terpenoids and eugenol were obtained from Sigma Aldrich Pty Ltd (Johannesburg, South Africa).

Autoclaved potato dextrose agar (PDA) was aseptically supplemented with each of the essential oils or compounds, together with 200 pL/L of the surfactant Triton X-100 (Ajax Laboratory Chemicals, Philadelphia, USA), to obtain a final concentration of 50 pL/L for each test substance (Table 1). Ten replicates per test concentration and per isolate were inoculated and incubated as described by Regnier et al. (2008). Growth inhibition was calculated as percentage of mycelial growth on each plate relative to the mycelial growth of the controls, prepared using PDA supplemented only with Triton X-100. The ten essential oils, three terpenoids and eugenol, that demonstrated the ability to inhibit the pathogen, were further screened in the same way, at concentrations ranging from 5 to 40 pL/L. These oils were analysed by GC-FID and GC-MS to determine the concentrations of the main components as described elsewhere in this issue by Kamatou et al. (2010-this issue).

Ten plastic containers (0.6 m2) were each filled with 4 kg of Phase III compost, spawned with A. bisporus and cased with a 40-50 mm layer of peat/lime soil. Six containers were inoculated on the first day with 100 mL each of a spore suspension of M. perniciosa (± 104 spores/mL). Two of these containers served as controls, while four of the remaining containers (two for each treatment) were used for the curative application on the second

day, when a solution of thyme (Thymus vulgaris) or lemon verbena (Lippia citriodora) oil (600 mL) was applied by spraying at a concentration of 40 pL/L. The other four uninoculated containers were used for the preventative treatment, which entailed the spray application of 40 pL/L solutions of thyme or lemon verbena oil on the first day, followed by the inoculation of the casing with the M. perniciosa suspension on the second day. All the containers were enclosed by a black plastic bag and incubated at 18 to 21 °C. The contents of the containers were sprayed every alternative day with distilled water. Following the initial inoculation, the presence or absence of disease was recorded weekly for two weeks. The production of healthy A. bisporus fruiting bodies was visually evaluated.

An exploratory commercial trial was conducted at the Highveld Mushrooms (Johannesburg, South Africa) farm to establish if the essential oil application reduced button mushroom yield. Trays filled with compost, spawned with A. bisporus, were cased with a 40-50 mm layer of peat/lime soil in a container with a total area of 1 m2. Thyme and lemon verbena essential oils, as well as thymol and nerol, were individually applied at a concentration of 40 pL/L on the second day. A one liter solution of each test mixture was sprayed onto the surface of each tray and experiments were conducted in duplicate. Two additional trays of cultured mushrooms, treated in the conventional manner with synthetic fungicide, were used as positive controls. The synthetic fungicide Chronos 450 SC (Makhteshim-Agan SA (Pty) Ltd, Israel) was sprayed at an application rate of 3.4mL/m2. Mushrooms were harvested by hand in three successive breaks. The effects of the treatments on the total yields obtained were recorded (kg/m2) as the cumulative total of three successive breaks. These values were compared to those obtained for the commercial treatment.

A small taste panel, consisting of five food technologists and mushroom growers from Highveld Mushrooms, was formed to evaluate the experimental mushrooms and controls. Mushrooms were halved, placed on white plates and subsequently evaluated blindly by panel members. Any differences in flavour, firmness or colour were recorded.

In vitro data were analysed using SPSS Version 16 statistical software. The Kolmogorov-Smirnov and Levine tests (Carver and Nash, 2009) were applied to evaluate the normality and homogeneity of each data set. Data were found to be normally distributed because the obtained P-values exceeded a=0.05. One-way ANOVA (single factor) was used to determine significant differences between concentrations tested in vitro. Analysis results obtained were considered to differ significantly if P < 0.05.

3. Results

The results from the in vitro screening trials, incorporating test substances into the fungal growth medium at a concentration of 50 pL/L, indicated that all of the essential oils and pure substances totally inhibited the mycelial growth of A. bisporus (Table 1). Only ten of the essential oils, three terpenoids and the phenylpropanoid, eugenol, were able to completely inhibit the Mycogone isolate and these substances were tested further at

Percentage inhibition of mycelial growth of Agaricus bisporus and Mycogone perniciosa by 40 essential oils and some of their major components. Bold face indicates those oils and pure compounds that totally inhibited the growth of Mycogone perniciosa with minimal effect on Agaricus bisporus.

Oil or terpenoid Agaricus bisporus Mycogone perniciosa

Concentration (|iL/L) Concentration (|L/L)

5 10 20 30 40 50 5 10 20 30 40 50

Anetum graveolens - - - - 100 A a - - - - - 63 DE b

Cananga odorata 0 De 0 F e 38 Eb 100 A a 100 A a 100 A a 1 F d 0 I e 26 I c 100 A a 100 A a 100 A a

Carum carvi - - - - - 100 A a - - - - - 53 G b

Chamaemelum nobile - - - - - 100 A a - - - - - 54 G b

Cinnamomun camphora - - - - - 100 A a - - - - - 30 P b

Cinnamomun zeylanicum 3 Ch 5Dg 21 Gf 61 D c 100 A a 100 A a 0Gi 5Hg 26 I e 42 I d 69 D b 100 A a

Citrus aurantifolia - - - - - 100 A a - - - - - 46 K b

Citrus aurantium - - - - - 100 A a - - - - - 59 EF b

Citrus bergamia - - - - - 100 A a - - - - - 60 E b

Citrus paradisi - - - - - 100 A a - - - - - 60 E b

Citrus reticulata - - - - - 100 A a - - - - - 39 MNb

Citrus sinensis - - - - - 100 A a - - - - - 43 L b

Citrus tangerine - - - - - 100 A a - - - - - 41 LM b

Citrus vulgaris - - - - - 100 A a - - - - - 56 FGb

Cymbopogon citratus 0Dh 0 F h 10 J g 26 J e 41 F d 100 A a 0Gh 15 D f 45 G c 69 F b 100 A a 100 A a

Cymbopogon martinii 0Dh 0Fh 19 H d 36 G b 46 D a 100 A a 0Gh 0Ih 18 Jd 26 K c 46 F a 100 A a

Cymbopogon nardus - - - - - 100 A a - - - - - 71 B b

Eucalyptus citriodora 0 De 4 E d 100 A a 100 A a 100 A a 100 A a 6Dc 8 Gb 100 A a 100 A a 100 A a 100 A a

Eucalyptus dives - - - - - 100 A a - - - - - 52 GH b

Eucalyptus globulus - - - - - 100 A a - - - - - 26 Qb

Eucalyptus radiata - - - - - 100 A a - - - - - 39 MN b

Lavendula augustifolia - - - - - 100 A a - - - - - 44 KL b

Lippia citriodora 0 D g 0Fg 35 F d 51 E c 80 B b 100 A a 13 B f 28 B e 100 A a 100 A a 100 A a 100 A a

Lippia javanica 10 B h 10 C h 47 D f 78 C d 100 A a 100 A a 10 C h 12 Eg 51 E a 85 D c 91 B h 100 A a

Lippia rehmannii 0 Df 0 F f 0 Lf 21 Ke 73 C b 100 A a 0 G 0 I f 33 H d 48 H c 85 C a 100 A a

Melaleuca alternifolia - - - - - 100 A a - - - - - 48 J b

Mentha citrata - - - - - 100 A a - - - - - 37 N b

Mentha piperita - - - - - 100 A a - - - - - 64 D b

Mentha spicata - - - - - 100 A a - - - - - 61 E b

Origanum marjorana - - - - - 100 A a - - - - - 50 I b

Pelargonium graveolens - - - - - 100 A a - - - - - 69 C b

Pelargonium radens - - - - - 100 A a - - - - - 37 N b

Pinus palustris - - - - - 100 A a - - - - - 62 DE b

Piper nigrum - - - - - 100 A a - - - - - 48 J b

Romarinus officinalis - - - - - 100 A a - - - - - 50 I b

Salvia officinalis - - - - - 100 A a - - - - - 48 J b

Syzigium aromaticum 0 Di 5Dg 53 C c 100 A a 100 A a 100 A a 2 Eh 10 F f 34 H e 39 J d 58 E b 100 A a

Thymus vulgaris 0Dh 0Fh 0 L h 32 H e 40 F d 100 A a 3Eg 11 E f 46 G c 64 G b 100 A a 100 A a

Vetiveria aizanoids - - - - - 100 A a - - - - - 66 D b

R-(-)-carvone - - - - - 100 A a - - - - - 54 G b

S-(+)-carvone - - - - - 100 A a - - - - - 58 F b

1,8-Cineole - - - - - 100 A a - - - - - 35 Ob

Citral 0 D g 0Fg 8 Kf 34 H d 100 A a 100 A a 0 G g 29 B e 60 C c 77 E b 100 A a 100 A a

Eugenol 25 Ai 44 A g 65 B e 80 B c 100 A a 100 A a 32 A h 48 A f 77 B d 88 C b 100 A a 100 A a

+ Limonene - - - - - 100 A a - - - - - 41 LM b

Nerol 4 C j 17 B h 16 Ig 29 I e 44 E d 100 A a 11 C i 19 C f 54 D c 90 B b 100 A a 100 A a

Thymol 0Dg 0Fg 10 J f 21 Ke 30 G d 100 A a 0Gg 0Ig 49 F c 86 D b 100 A a 100 A a

Averages (n= 10) followed by the same upper-case letter did not differ significantly within a column.

Averages (n= 10) followed by the same lower-case letter did not differ significantly within a row for the same pathogen.

The (—) represents test concentrations that were not screened.

lower concentrations. At a concentration of 20 p,L/L, lemon verbena (Lippia citriodora) oil totally inhibited the growth of the pathogen, while reducing the mycelial growth of Agaricus by only 35% (Table 1). The use of thyme (Thymus vulgaris) oil and lemon grass (Cymbopogon citratus) oil at a concentration of 40 p,L/L, yielded comparable results; the growth of M. perniciosa was effectively controlled, while 40 and 41% growth inhibition of the mushroom was observed, respectively. Nerol

and thymol elicited a lower fungitoxic effect against the edible mushroom than the other pure compounds capable of controlling the mycelial growth of M. perniciosa. To evaluate the usefulness of thyme and lemon verbena essential oils as button mushroom protective agents against M. perniciosa, a concentration of 40 |L/L was considered appropriate for in vivo screening, since the pathogen was still totally inhibited at this concentration (Table 1).

Chromatographic analysis revealed that three of the oils, lemon grass, lemon verbena and Lippia rehmannii, contain predominantly the geometrical isomers ofcitral: Z-citral, known as geranial and E-citral, referred to as neral (Table 2). Although the combined percentages of geranial (28.6%) and neral (21.1%) in lemon verbena were lower than that of Lippia rehmannii and lemon grass, the oil was more effective in suppressing the growth of M. perniciosa. Thyme oil as expected, contained predominantly thymol (63.1%).

The in vivo preventative application of thyme and lemon verbena essential oils to casings inoculated with the pathogen, yielded healthy mushrooms with no visible signs of M. perniciosa infection. However, the in vivo curative use of these essential oils was ineffective to control the occurrence of wet bubble disease on the mushrooms and severe infections developed. In the exploratory commercial trial where lemon verbena and thyme oils, as well as nerol and thymol, were applied at a concentration of 40 pL/L, the yields recorded indicated that none of the essential oils or pure compounds severely affected the growth of A. bisporus compared to the control (Table 3). Moreover, no off-flavours were detected and no differences in colour or firmness of the mushrooms were recorded by the taste panel members.

4. Discussion

Essential oils have been applied to control fungi on fruit crops such as citrus (Du Plooy et al., 2009; Plaza et al., 2004). However, the use of such oils to control diseases affecting the production of edible mushroom is problematic as both the antagonist and the crop of interest are fungi. Tea tree oil was previously found to be ineffective against cobweb disease

Table 3

Average button mushroom yields (n=2) obtained in a simulated commercial trial, following the application (40 pL/L) of two essential oils and two pure compounds.

Treatment First break Second break Third break Total yield

(kg/m2) (kg/m2) (kg/m2) (kg/m2)

Commercial 15.6 8.52 3.72 27.8

(conventional)

Lippia citriodora 15.5 6.38 2.71 24.6

(lemon verbena) oil

Thymus vulgaris 15.6 7.53 1.89 25.0

(thyme) oil

Nerol 16.5 10.3 3.08 29.9

Thymol 14.2 11.1 6.62 31.9

(Cladobotryum dendroides) of edible mushroom at an application concentration of 1% (Potocnik et al., 2010). In this study, only a limited number of essential oils from a wide range of available oils screened were found to have the ability to effectively inhibit the pathogen (M. perniciosa), while exhibiting a minimal effect on the growth of the A. bisporus. The oils of lemon verbena and thyme were found to have potential application as fungicides against M. perniciosa, without retarding the growth of the button mushroom. Thymol, the main component of thyme oil, and nerol were selected as pure test compounds in the in vivo trials to identify compounds contributing to the antifungal properties of the oils. Citral was excluded since it displayed strong toxicity towards the mushroom even at 40 pL/L. Although lemon verbena oil contains citral as the predominant component, the oil was further investigated. The lower citral (geranial and neral) concentration of the oil (Table 2), compared to that of lemongrass and L. rehmannii, and the presence of geraniol

Table 2

Chemical profiles determined by gas chromatography with flame ionization detection of the ten most promising essential oils for control of M. perniciosa. Essential oil Six most abundant compounds (GC-FID) > 1%a

Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6

Cananga odorata Benzyl benzoate (24.42%) Benzyl salicylate Geraniol (9.18%) Farnesol (7.06%) Linalool (8.67%) Benzyl acetate

(ylang ylang) (9.912%) (4.58%)

Cinnamomum zeylanicum Eugenol (81.2%) p -Caryophyllene Cinnamaldehyde a-Humulene (1.3%) Isoeugenol (1.2%) -

(cinnamon) (7.6%) (2.3%)

Cymbopogon citratus Geranial (42.5%) Neral (31.7%) Limonene (8.9) a-Terpineol (5.2%) Citronellol (2.8%) Linalool (2.8%)

(lemon grass)

Cymbopogon martinii Geraniol (67.06%) Geranyl acetate Piperitone (2.46%) Linalool (2.14%) Trans-ocimene Geranyl butyrate

(palma rosa) (17.85%) (1.49%) (1.39%)

Eucalyptus citriodora Citronellal (57.8%) trans-Isopulegol Citronellol (6.4%) Unidentified (4.8%) c/s-Isopulegol Citronellic acid

(7.0%) (4.1%) (2.4%)

Lippia citriodora Geranial (28.6%) Neral (21.1%) Geraniol (15.5%) Citronellol (10.6%) Nerylisobutyrate a-Terpineol (5.3%)

(lemon verbena) (5.5%)

Lippia javanica (fever tea) Myrcenone (60.7%) Perillaldehyde Germacrene D (3.8%) p-mentha-8-dien-4-ol Carvone (3.5%) 1,8-Cineol (3,3%)

(6.8%) (3.7%)

Lippia rehmannii Geranial (42.3%) Neral (26.8%) Caryophyllene oxide Camphor (3.3%) Isocaryophyllene p -Caryophyllene

(3.7%) (3.2%) (2.3%)

Syzygium aromaticum Eugenol (88.3%) p -Caryophyllene a-Humulene (2.0%) - - -

(clove) (8.1%)

Thymus vulgaris (thyme) Thymol (63.1%) Linalool (21.3%) Benzyl alcohol (4.3%) p -Caryophyllene a-Terpineol (1.6%) -

(1.7%)

a The identities were confirmed by GC-mass spectrometry.

(15.5%) and citronellol (10.6%) may have resulted in the reduced toxicity of lemon verbena oil towards A. bisporus, while maintaining its inhibitory effect against the pathogen at a concentration as low as 20 |L/L. Available data indicate that the high activities of oxygenated monoterpenes against fungal pathogens are the consequence of interference of the terpenoids with enzyme reactions (Zambonelli et al., 1996) and/or disruption of cell membranes in the target organism (Inouye et al., 2000). Although oregano oil (Origanum marjorana), characterized by high levels of thymol and carvacrol, has been reported as a useful M. perniciosa and Cladobotryum sp inhibitor (Tanovic et al., 2009), no data are available regarding the effect of the oil on A. bisporus yield or disease development under commercial conditions. Our in vitro study indicated that oregano oil was not able to sufficiently suppress the growth of M. perniciosa, since 50 |L/L resulted in only 50% inhibition of the pathogen (Table 1).

The absence of contamination and the good growth of the A bisporus observed after preventative treatment of the spawn with the essential oils, encouraged us to test their non-target effects under commercial conditions. None of the treatments drastically decreased the total yield of the button mushroom when compared to those treated with synthetic fungicide (control). In fact, the yields of mushrooms treated with nerol and thymol were slightly higher than that of the control. However, this data cannot be statistically validated since only two replicates were used for each treatment. In addition, the variation in yield for trays prepared in the same way would be quite large, since the spawning is not entirely reproducible. This trial was designed to show up severe effects of test substances on mushroom yield, which would rule out the future use of these substances on mushrooms.

The taste panel could not detect off-flavours in any of the mushrooms produced from treated casing. This result was expected, since the concentrations of essential oils applied were very low and therefore unlikely to accumulate in the fruiting body to a level where an off-taste would be discernable. Our preliminary trial, involving the preventative application of the oils on M. perniciosa-inoculated casings, validates the potential use of thyme and lemon verbena oil in button mushroom production.

Acknowledgements

The authors thank Sylvan Incorporated (Pretoria, South Africa) for the supply of the A. bisporus and M. perniciosa isolates and the staff of Highveld Mushrooms (Johannesburg) for assistance with the commercial trial.

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