In Vitro and in Vivo Screening of Essential Oils for the Control of Wet Bubble Disease of Agaricus Bisporus ⁎ T

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 μL/L. Ten essential oils were further investigated at lower concentrations ranging from 5 to 40 μL/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 40 μL/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 Mushrooms are generally infected in the early developmental stages. On commercial farms, wet bubble disease is controlled Wet bubble disease caused by Mycogone perniciosa by fumigation, spraying with formalin or lysol, soil sterilisation (Magnus) Delacroix, is not species-specific (Holland and (Smith, 1924) or by applying a mixture of prochloraz– Cooke, 1990) and frequently leads to a substantial reduction manganese (Eicker, 1987). Immediate application of salt to in the yields of cultivated mushrooms, particularly of the button the infected areas, followed by covering has been reported to mushroom, Agaricus bisporus (J. Lange) Imbach (Fletcher et provide significant control of the disease (Pieterse, 2005). al., 1995; Umar et al., 2000). It is well established that the The number of approved fungicides for disease control in development of M. perniciosa is usually associated with the use commercial mushroom production has been severely restricted, of infected soil or spawn (Gandy and Spencer, 1978). However, thereby placing immense pressure on producers. For example, the presence of spores in the ventilation system is frequently a the European community recently withdrew carbendazim for source of contamination (personal communication with Dr M. use in mushroom production, thereby banning all benzimid- Van Greuning, Sylvan Incorporated, Pretoria, South Africa). azole-based fungicides conventionally applied for the control of pathogens such as M. perniciosa (Grogan, 2008). It is ⁎ Corresponding author. Tel.: +27 12 3826126; fax: +27 12 3826286. imperative that alternative disease control measures be E-mail address: [email protected] (T. Regnier). 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.2010.07.018 682 T. Regnier, S. Combrinck / South African Journal of Botany 76 (2010) 681–685 of plant extracts and essential oils are well documented (Ríos day, when a solution of thyme (Thymus vulgaris)orlemon and Recio, 2005; Soković et al., 2009). Glamoclija et al. (2006) verbena (Lippia citriodora) oil (600 mL) was applied by spraying demonstrated the in vitro antifungal activities of Satureja at a concentration of 40 μL/L. The other four uninoculated thymbra and Salvia pomifera volatiles against M. perniciosa. containers were used for the preventative treatment, which The essential oil of Critmum maritimum, as well as two of the entailed the spray application of 40 μL/L solutions of thyme or major components of the oil, limonene and α-pinene, was also lemon verbena oil on the first day, followed by the inoculation of found to exhibit inhibitory activities against the pathogen the casing with the M. perniciosa suspension on the second day. (Glamoclija et al., 2009). In this study, 40 essential oils, some of All the containers were enclosed by a black plastic bag and their major terpenoid components and a phenylpropanoid incubated at 18 to 21 °C. The contents of the containers were (eugenol) were screened to identify oils and compounds that sprayed every alternative day with distilled water. Following the have the ability to inhibit the growth of M. perniciosa, without initial inoculation, the presence or absence of disease was affecting the development and yield of the edible mushroom, recorded weekly for two weeks. The production of healthy A. A. bisporus. bisporus fruiting bodies was visually evaluated. An exploratory commercial trial was conducted at the 2. Material and methods Highveld Mushrooms (Johannesburg, South Africa) farm to establish if the essential oil application reduced button mushroom Purified A. bisporus and M. perniciosa isolates were kindly yield. Trays filled with compost, spawned with A. bisporus,were supplied by Sylvan Incorporated (Pretoria, South Africa). cased with a 40–50 mm layer of peat/lime soil in a container with Essential oils of Lippia scaberrima and Lippia rehmannii a total area of 1 m2. Thyme and lemon verbena essential oils, were obtained by steam distillation of wild plants as described as well as thymol and nerol, were individually applied at a in Combrinck et al. (2006) and in Linde et al. (2010), concentration of 40 μL/L on the second day. A one liter solution respectively. Lippia javanica essential oil was kindly supplied of each test mixture was sprayed onto the surface of each tray and by Scatters (Johannesburg, South Africa). Apelium graveolens, experiments were conducted in duplicate. Two additional trays of Carum carvi, Cinnamomum zeylanicum and Eucalyptus dives cultured mushrooms, treated in the conventional manner with oils were obtained from Essentia Products, South Africa, synthetic fungicide, were used as positive controls. The synthetic Eucalyptus citriodora was obtained from NatureSun Aroma, fungicide Chronos 450 SC (Makhteshim-Agan SA (Pty) Ltd, Eucalyptus citriodora, Eucalyptus globulus, Eucalyptus Israel) was sprayed at an application rate of 3.4 mL/m2. radiata, Lavendula angustifolia, Mentha piperita and Thymus Mushrooms were harvested by hand in three successive breaks. vulgaris essential oils from Burgess and Finch (distributed by The effects of the treatments on the total yields obtained were Vital Health Foods, Kuilsriver, South Africa), while the recorded (kg/m2) as the cumulative total of three successive remaining essential oils (Table 1) were purchased from Holistic breaks. These values were compared to those obtained for the Emporium (Johannesburg, South Africa). All pure terpenoids commercial treatment. and eugenol were obtained from Sigma Aldrich Pty Ltd A small taste panel, consisting of five food technologists and (Johannesburg, South Africa). mushroom growers from Highveld Mushrooms, was formed to Autoclaved potato dextrose agar (PDA) was aseptically evaluate the experimental mushrooms and controls. Mushrooms supplemented with each of the essential oils or compounds, were halved, placed on white plates and subsequently evaluated together with 200 μL/L of the surfactant Triton X-100 (Ajax blindly by panel members. Any differences in flavour, firmness Laboratory Chemicals, Philadelphia, USA), to obtain a final or colour were recorded. concentration of 50 μL/L for each test substance (Table 1). Ten In vitro data were analysed using SPSS Version 16 statistical replicates per test concentration and per isolate were inoculated software. The Kolmogorov–Smirnov and Levine tests (Carver and incubated as described by Regnier et al. (2008). Growth and Nash, 2009) were applied to evaluate the normality and inhibition was calculated as percentage of mycelial growth on homogeneity of each data set. Data were found to be normally each plate relative to the mycelial growth of the controls, distributed because the obtained P-values exceeded α=0.05. prepared using PDA supplemented only with Triton X-100. The One-way ANOVA (single factor) was used to determine ten essential oils, three terpenoids and eugenol, that demon- significant differences between concentrations tested in vitro. strated the ability to inhibit the pathogen, were further screened Analysis results obtained were considered to differ significantly in the same way, at concentrations ranging from 5 to 40 μL/L. if P≤0.05. These oils were analysed by GC–FID and GC–MS to determine the concentrations of the main components as described 3. Results elsewhere in this issue by Kamatou et al. (2010-this issue). Ten plastic containers (0.6 m2) were each filled with 4 kg of The results from the in vitro screening trials, incorporating Phase III compost, spawned with A. bisporus and cased with a test substances into the fungal growth medium at a concentra- 40–50 mm layer of peat/lime soil. Six containers were inoculated tion of 50 μL/L, indicated that all of the essential oils and pure on the first day with 100 mL each of a spore suspension of substances totally inhibited the mycelial growth of A. bisporus M. perniciosa (±104 spores/mL). Two of these containers served (Table 1). Only ten of the essential oils, three terpenoids and the as controls, while four of the remaining containers (two for each phenylpropanoid, eugenol, were able to completely inhibit the treatment) were used for the curative application on the second Mycogone isolate and these substances were tested further at T. Regnier, S. Combrinck / South African Journal of Botany 76 (2010) 681–685 683

Table 1 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 (μL/L) Concentration (μL/L) 5 10 20 30 40 50 5 10 20 30 40 50 Anetum graveolens ––––100 A a –––––63 DE b Cananga odorata 0 D e 0 F e 38 E b 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 C h 5 D g 21 G f 61 D c 100 A a 100 A a 0 G i 5 H g 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 MN b Citrus sinensis ––– – – 100 A a –––––43 L b Citrus tangerine ––– – – 100 A a –––––41 LM b CItrus vulgaris ––– – – 100 A a –––––56 FG b Cymbopogon citratus 0 D h 0 F h 10 J g 26 J e 41 F d 100 A a 0 G h 15 D f 45 G c 69 F b 100 A a 100 A a Cymbopogon martinii 0 D h 0 F h 19 H d 36 G b 46 D a 100 A a 0 G h 0 I h 18 J d 26 K c 46 F a 100 A a Cymbopogon nardus ––– – – 100 A a –––––71 B b Eucalyptus citriodora 0 D e 4 E d 100 A a 100 A a 100 A a 100 A a 6 D c 8 G b 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 Q b Eucalyptus radiata ––– – – 100 A a –––––39 MN b Lavendula augustifolia ––– – – 100 A a –––––44 KL b Lippia citriodora 0 D g 0 F g 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 E g 51 E a 85 D c 91 B h 100 A a Lippia rehmannii 0Df 0Ff 0Lf 21Ke 73Cb 100Aa 0G 0If 33Hd 48Hc 85Ca 100Aa 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 D i 5 D g 53 C c 100 A a 100 A a 100 A a 2 E h 10 F f 34 H e 39 J d 58 E b 100 A a Thymus vulgaris 0 D h 0 F h 0 L h 32 H e 40 F d 100 A a 3 E g 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 O b Citral 0 D g 0 F g 8 K f 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 A i 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 I g 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 0 D g 0 F g 10 J f 21 K e 30 G d 100 A a 0 G g 0 I g 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 μL/L, lemon and thymol elicited a lower fungitoxic effect against the edible verbena (Lippia citriodora) oil totally inhibited the growth of mushroom than the other pure compounds capable of the pathogen, while reducing the mycelial growth of Agaricus controlling the mycelial growth of M. perniciosa. To evaluate by only 35% (Table 1). The use of thyme (Thymus vulgaris) oil the usefulness of thyme and lemon verbena essential oils as and lemon grass (Cymbopogon citratus) oil at a concentration of button mushroom protective agents against M. perniciosa,a 40 μL/L, yielded comparable results; the growth of M. concentration of 40 μL/L was considered appropriate for in vivo perniciosa was effectively controlled, while 40 and 41% growth screening, since the pathogen was still totally inhibited at this inhibition of the mushroom was observed, respectively. Nerol concentration (Table 1). 684 T. Regnier, S. Combrinck / South African Journal of Botany 76 (2010) 681–685

Chromatographic analysis revealed that three of the oils, Table 3 lemon grass, lemon verbena and Lippia rehmannii, contain Average button mushroom yields (n=2) obtained in a simulated commercial trial, following the application (40 μL/L) of two essential oils and two pure predominantly the geometrical isomers of citral: Z-citral, known compounds. as geranial and E-citral, referred to as neral (Table 2). Although Treatment First break Second break Third break Total yield the combined percentages of geranial (28.6%) and neral 2 2 2 2 (kg/m ) (kg/m ) (kg/m ) (kg/m ) (21.1%) in lemon verbena were lower than that of Lippia rehmannii and lemon grass, the oil was more effective in Commercial 15.6 8.52 3.72 27.8 (conventional) suppressing the growth of M. perniciosa. Thyme oil as Lippia citriodora 15.5 6.38 2.71 24.6 expected, contained predominantly thymol (63.1%). (lemon verbena) oil The in vivo preventative application of thyme and lemon Thymus vulgaris 15.6 7.53 1.89 25.0 verbena essential oils to casings inoculated with the pathogen, (thyme) oil yielded healthy mushrooms with no visible signs of M. Nerol 16.5 10.3 3.08 29.9 Thymol 14.2 11.1 6.62 31.9 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 (Cladobotryum dendroides) of edible mushroom at an applica- developed. In the exploratory commercial trial where lemon č verbena and thyme oils, as well as nerol and thymol, were tion concentration of 1% (Poto nik et al., 2010). In this study, applied at a concentration of 40 μL/L, the yields recorded only a limited number of essential oils from a wide range of available oils screened were found to have the ability to indicated that none of the essential oils or pure compounds effectively inhibit the pathogen (M. perniciosa), while exhibit- severely affected the growth of A. bisporus compared to the ing a minimal effect on the growth of the A. bisporus. The oils control (Table 3). Moreover, no off-flavours were detected and of lemon verbena and thyme were found to have potential no differences in colour or firmness of the mushrooms were application as fungicides against M. perniciosa, without recorded by the taste panel members. retarding the growth of the button mushroom. Thymol, the main component of thyme oil, and nerol were selected as pure 4. Discussion test compounds in the in vivo trials to identify compounds contributing to the antifungal properties of the oils. Citral was Essential oils have been applied to control fungi on fruit excluded since it displayed strong toxicity towards the crops such as citrus (Du Plooy et al., 2009; Plaza et al., 2004). mushroom even at 40 μL/L. Although lemon verbena oil However, the use of such oils to control diseases affecting the contains citral as the predominant component, the oil was production of edible mushroom is problematic as both the further investigated. The lower citral (geranial and neral) antagonist and the crop of interest are fungi. Tea tree oil was concentration of the oil (Table 2), compared to that of previously found to be ineffective against cobweb disease 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) N1% 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%) β-Caryophyllene Cinnamaldehyde α-Humulene (1.3%) Isoeugenol (1.2%) – (cinnamon) (7.6%) (2.3%) Cymbopogon citratus Geranial (42.5%) Neral (31.7%) Limonene (8.9) α-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%) cis-Isopulegol Citronellic acid (7.0%) (4.1%) (2.4%) Lippia citriodora Geranial (28.6%) Neral (21.1%) Geraniol (15.5%) Citronellol (10.6%) Nerylisobutyrate α-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 β-Caryophyllene (3.7%) (3.2%) (2.3%) Syzygium aromaticum Eugenol (88.3%) β-Caryophyllene α-Humulene (2.0%) ––– (clove) (8.1%) Thymus vulgaris (thyme) Thymol (63.1%) Linalool (21.3%) Benzyl alcohol (4.3%) β-Caryophyllene α-Terpineol (1.6%) – (1.7%) a The identities were confirmed by GC–mass spectrometry. T. Regnier, S. Combrinck / South African Journal of Botany 76 (2010) 681–685 685

(15.5%) and citronellol (10.6%) may have resulted in the Du Plooy, W., Regnier, T., Combrinck, S., 2009. Essential oil amended coatings reduced toxicity of lemon verbena oil towards A. bisporus, as alternatives to synthetic fungicides in citrus postharvest management. Postharvest Biology and Technology 53, 117–122. while maintaining its inhibitory effect against the pathogen at a Eicker, A., 1987. A report on the use of prochloraz manganese complex for concentration as low as 20 μL/L. Available data indicate that controlling of major fungal pathogens of the cultivated mushroom (Agaricus the high activities of oxygenated monoterpenes against fungal bisporus) in South Africa. South African Journal of Botany 53, 345–348. pathogens are the consequence of interference of the terpenoids Fletcher, J.T., Jaffe, B., Muthumeenakshi, S., Brown, A.E., Wright, D.M., 1995. with enzyme reactions (Zambonelli et al., 1996)and/or Variations in isolates of Mycogone perniciosa and in disease symptoms in Agaricus bisporus. Plant Pathology 44, 130–140. disruption of cell membranes in the target organism (Inouye Gandy, D.G., Spencer, D.M., 1978. Fungicides for the control of Mycogone et al., 2000). Although oregano oil (Origanum marjorana), perniciosa, the cause of wet bubble on the cultivated mushroom. Scientia characterized by high levels of thymol and carvacrol, has been Horticulturae 8, 307–313. reported as a useful M. perniciosa and Cladobotryum sp Glamoclija, J., Sokovic, M., Vukojevic, J., Milinekovic, I.M., Van Griesven, inhibitor (Tanović et al., 2009), no data are available regarding L.J.L.D., 2006. Chemical composition and antifungal activities of essential oils of Satureja thymbra and Salvia pomifera ssp. Calycina (Sm.)Hayek. the effect of the oil on A. bisporus yield or disease development Journal of Essential Oil Research 18, 115–117. under commercial conditions. Our in vitro study indicated that Glamoclija, J., Sokovic, M., Grubisic, D.J., Vukojevic, J., Milinekovic, I.M., oregano oil was not able to sufficiently suppress the growth of Ristic, M., 2009. Antifungal activity of Critmum maritimum essential oil and M. perniciosa, since 50 μL/L resulted in only 50% inhibition of its components against mushroom pathogen Mycogone perniciosa. – the pathogen (Table 1). Chemistry of Natural Compounds 45, 95 97. Grogan, H.M., 2008. Challenges facing mushroom disease control in the 21st The absence of contamination and the good growth of the A century. In: Lelley, J.I., Buswell, J.A. (Eds.), Proceeding of the Sixth bisporus observed after preventative treatment of the spawn International Conference on Mushroom Biology and Mushroom Products, with the essential oils, encouraged us to test their non-target 29th September–3rd October 2008. WSMBMP, Bonn, Germany, pp. 120–127. effects under commercial conditions. None of the treatments Holland, D.M., Cooke, R.C., 1990. Activation of dormant conidia of the wet drastically decreased the total yield of the button mushroom bubble pathogen Mycogone perniciosa, by Basidiomycotina. Mycological Research 94, 789–792. when compared to those treated with synthetic fungicide Inouye, S., Tsuruoka, T., Watanabe, M., Takeo, K., Akao, M., Nishiyama, Y., (control). In fact, the yields of mushrooms treated with nerol Yamaguchi, H., 2000. Inhibitory effect of essential oils on apical growth of and thymol were slightly higher than that of the control. Aspergillus fumigatus by vapour contact. Mycoses 43, 17–23. However, this data cannot be statistically validated since only Kamatou, GPP., Viljoen, A.M., Özek, T., Başer, KHC, 2010. Chemical composition “ ” two replicates were used for each treatment. In addition, the of the wood and leaf oils from the Clanwilliam Cedar (Widdringtonia cedarbergensis J.A. Marsh): critically endangered species. South African Journal variation in yield for trays prepared in the same way would be of Botany 76, 652–654 (this issue). quite large, since the spawning is not entirely reproducible. This Linde, J.H., Combrinck, S., Regnier, T.J.C., Virijevic, S., 2010. Chemical trial was designed to show up severe effects of test substances composition and antifungal activity of the essential oils of Lippia rehmannii on mushroom yield, which would rule out the future use of these from South Africa. South African Journal of Botany 76, 37–42. substances on mushrooms. Pieterse Z, 2005. Mycogone perniciosa, a pathogen of Agaricus bisporus. 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