ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2022, 27(2): 59–65 DOI: 10.53541/jams.vol27iss2pp59-65 Received 16 June 2021 Accepted 04 Dec 2021 Antifungal Activity of Shirazi Thyme (Zataria multiflora Boiss.) Essential Oil against Hypomyces perniciosus, a causal agent of wet bubble disease of Agaricus bisporus Yumna Juma Rashid Al-Balushi1, Abdullah Mohammed Al-Sadi1, Issa Hashil Al-Mahmooli1, Majida Mohammed Ali Al-Harrasi1, Jamal Nasser Al-Sabahi2, Alaa Khamis Sulaiman Al-Alawi1, Khalid Al-Farsi3 and Rethinasamy Velazhahan1,* Rethinasamy Velazhahan( ) velazhahan@squ.edu.om, 1Department of Plant Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khoud, Muscat 123, Sultanate of Oman, 2Central Instrumentation Laboratory, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khoud, Muscat 123, Sultanate of Oman, 3Oman Botanic Garden, Muscat, Oman. Introduction Mushroom farming is becoming a popular agro-based business worldwide. Over 8.99 million tons of mushrooms are produced annually worldwide (Kumla et al., 2020). White button mush- room [Agaricus bisporus (Lange) Imbach] is the most popular and widely cultivated edible mushroom glob- ally (Sanchez, 2004). Diseases are serious constraints in the commercial production of button mushrooms. Several fungal diseases including wet bubble (Hypomy- ces perniciosus), dry bubble (Lecanicillium fungicola), cobweb (Cladobotryum mycophilum) and green mold (Trichoderma aggressivum) are reported to significantly reduce the yield and quality of mushroom crops (Gea et al., 2021). Wet bubble disease (WBD) caused by the ascomycetes fungus Hypomyces perniciosus Magnus [Mycogone perniciosa (Magnus) Delacroix] was report- ed to cause yield reductions ranging from 15 to 30% in مضادات نشاط فطر Hypomyces perniciosus املسبب ملرض الفقاعة الرطبة على فطر األبيض )املشروم( ) Zataria multiflora Boiss.( إبستخدام الزيت العطري من الزعرت الشريازي Agaricus bisporus ميىن بنت مجعة بن راشد البلوشي1 و عبدهللا بن حممد السعدي1 و عيسى بن هاشل املهمويل1 و ماجدة بنت حممد بن علي احلراصي1 و مجال بن انصر الصباحي2 واآلء بنت مخيس بن سليمان العلوي1 وخالد الفارسي3 و ريثيناسامي فيالزهاهن1 Abstract. Wet bubble disease (WBD) caused by Hypomyces perniciosus is a major constraint of button mushroom (Agaricus bisporus) cultivated worldwide. A few synthetic chemical fungicides are used to control WBD. In our study, the potential of essential oil (EO) from Zataria multiflora in inhibition of H. perniciosus was evaluated as an alternative to chemical fungicides. An isolate of H. perniciosus was isolated from wet bubble diseased A. bisporus and pathogenici- ty of the mycoparasite was determined under artificially inoculated conditions. The mycoparasitic fungus was identified using sequences of the internal transcribed spacer (ITS) region of ribosomal DNA. The EO was extracted from the aerial parts of Z. multiflora by microwave extraction method and evaluated in vitro for its antifungal activity against H. perni- ciosus. The EO of Z. multiflora (ZEO) at the tested concentrations (50% and 100%) inhibited the growth of H. pernicio- sus in the agar diffusion test. The minimum inhibitory concentration (MIC) of ZEO was 0.04% as assessed by the poi- soned food technique. The chemical composition of ZEO was determined by gas chromatography-mass spectrometry analysis. A total of 23 compounds were identified. Among them, the most abundant compounds were Linalool (20.3%) and Bornyl acetate (15.5%). Linalool at the tested concentrations of 0.25% and 0.125% completely inhibited the myce- lial growth of H. perniciosus in an in vitro assay. These results suggest that ZEO can be exploited for control of WBD. Keywords: : Agaricus bisporus; antifungal; essential oil; Mycogone perniciosa; wet bubble disease; Zataria multiflora. Agaricus( هو أحد املعوقات الرئيسية لفطر األبيض Hypomyces perniciosus الناجم عن )WBD( امللخص:مرض الفقاعة الرطبة bisporus( املــزروع يف مجيــع أحنــاء العــامل. يســتخدم عــدد قليــل مــن مبيــدات الفطــرايت الكيميائيــة األصطناعيــة للتحكــم يف WBD. يف دراســتنا، مت تقييــم إمكانيــة الزيــت العطــري مــن Zataria multiflora يف تثبيــط H. perniciosus كبديــل ملبيــدات الفطــرايت الكيميائيــة. مت عــزل H. perniciosus مــن الفطــر االبيــض A. bisporus مصــاب بـــ الفقاعــة الرطبــة وبعــد ذلــك مت إختبــار هــذه العزلــة و حتديــد اإلمراضيــة للطفيــل الفطــري لتســبب هــذا املــرض علــى فطــر أبيــض ســليم حتــت ظــروف مناخيــة معلومــة. مت تعريــف الطفيــل الفطــري إبســتخدام التصنيــف اجليــي يف تسلســل وحــدة الريبوســومات املوجــودة علــى فواصــل النســخ الداخلــي )ITS( مــن محــض النــووي الريبوســومي .مت إســتخراج الزيــت العطــري EO مــن .H. perniciosus بطريقة اإلستخالص ابمليكروويف ومت تقييمه يف املخترب لنشاطه املضاد للفطرايت ضد Z. multiflora األجزاء العلوية لـ أعــاق EO لـــ ZEO(p( Z. multiflora برتكيــزات خمتــربة )50٪ و 100٪( منــو H. perniciosus يف إختبــار خلــط الزيــت ابألجــار. كان احلــد األدىن للرتكيــز املثبــط )MIC( لـــ ZEOهــو 0.04٪ الــذى مت تقييمــه بواســطة تقنيــة الغــذاء املســموم. مت حتديــد الرتكيــب الكيميائــي لـــ ZEO عــن طريــق حتليــل كروماتوجرافيــا الغــاز لقيــاس الطيــف الكتلــي. كذلــك ، مت حتديــد إمجــايل 23 مركبًــا. مــن بينهــا ، كانــت املركبــات األكثــر وفــرة Linalool )20.3٪( و Bornyl acetate )15.5٪(. قــام Linalool برتكيــزات خمتــربة بنســبة 0.25٪ و 0.125٪ بتثبيــط منــو الغــزل .WBD للتحكــم يف ZEO متاًمــا يف إختبــار يف املختــرب. تشــر هــذه النتائــج إىل أنــه ميكــن إســتغالل H. perniciosus )الفطــري )امليســيليوم الكلمــات املفتاحيــة: Agaricus bisporus، مضــاد للفطــرايت، زيــت العطــري، Mycogone perniciosa، مــرض الفقاعــة الرطبــة، Zataria multiflora 60 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 2 Antifungal Activity of Shirazi Thyme (Zataria multiflora Boiss.) Essential Oil against Hypomyces perniciosus, a causal agent of wet bubble disease of Agaricus bisporus. button mushroom (Wang et al., 2016; Zhou et al., 2016; Li et al., 2019; Shi et al., 2020). Deformation of basidi- ome, appearance of white cottony growth of the myceli- um, exudation of brown coloured liquid and appearance of flocculent mycelia on the substrate are the common symptoms of wet bubble disease (Fletcher et al., 1995; Fu et al., 2016). Since the mycoparasite directly affects the formation of caps the yield losses to the mushroom industry due to this disease is very high. In general veg- etative mycelium of A. bisporus was not affected by H. perniciosus, whereas the morphogenesis of its fruiting bodies was severely affected (Zhang et al., 2017). This fungus is also known to infect Pleurotus citrinopileatus (Zhang et al., 2017). The contaminated casing soil has been reported as the main source of inoculum of the fungus (Fletcher and Gaze, 2008). Pathogen also spreads through air, contami- nated tools and operators (Gea et al., 2021). WBD can be prevented by good hygiene, sanitation and application of fungicides without affecting the growth of mushrooms (Gea et al., 2021). Fungicides such as Benomyl (Bollen and Fuchs, 1970), iprodione and prochloraz-Mn (Gea et al., 2010; Potocnik et al., 2010), thiabendazole, fludiox- onil, diniconazole, fenbuconazole and imazalil (Shi et al., 2020) were found to be effective in controlling H. per- niciosus. The use of natural products for the control of foodborne pathogens has been considered a safe and en- vironmentally friendly approach. The inhibitory effect of essential oils (EOs) of a few plant species including Lip- pia citriodora and Thymus vulgaris (Regnier and Com- brinck, 2010), Crithmum maritimum (Glamoclija et al., 2009), Origanum majorana (Tanovic et al., 2009) against H. perniciosus was reported. These essential oils are aro- matic and volatile liquids extracted from plants through steam distillation process. In the course of screening of Omani traditional medicinal plants for in vitro antifun- gal activity, we observed that the essential oil of Zataria multiflora Boiss. (Lamiaceae) completely inhibited the growth of Aspergillus flavus, a common contaminant and major aflatoxin producer in a wide range of agricul- tural commodities (unpublished data). Considering the best knowledge of the authors, the antifungal activity of Z. multiflora essential oil (ZEO) against H. perniciosus has not been studied. In this study, the inhibitory effect of ZEO and its major constituent linalool on H. pernicio- sus was determined. Materials and Methods Plant Material Zataria multiflora Boiss. (Lamiaceae) (Accession num- ber 201100114) plants were obtained from Oman Bo- tanic Garden, Muscat, Sultanate of Oman. Mycoparasite isolation Agaricus bisporus fruiting bodies showing symptoms of wet bubble disease were collected from the mushroom cultivation demonstration trials at the Department of Plant Sciences, Sultan Qaboos University. A small piece of tissue was cut with a sterile surgical scalpel from the infected mushroom and surface ster- ilized with 1% sodium hypochlorite for 1 min. The tis- sue was then rinsed in sterile distilled water (SDW) and placed on potato dextrose agar (PDA) (Oxoid Ltd., UK) medium in a petri dish. The plate was kept at 27°C for 3-5 days. A pure culture of the fungus was obtained by hyphal tip culture method. Molecular Identification Mycelia were collected from 7-day-old PDA culture plate. DNA was extracted from approximately 80 mg of mycelium according to Lee and Taylor (1990). The DNA quantity and quality was checked using a NanoDrop 2000 spectrophotometers (Thermo Scientific, USA). Polymerase chain reaction (PCR) was performed in 25 μl reaction volume using ITS4 and ITS5 primers (White et al., 1990) with PuReTaq Ready-to-Go PCR bead (GE Healthcare, UK) according to Al-Rashdi et al. (2020). An aliquot (5 μl) of the PCR product was analyzed by 1.2% (w/v) agarose gel electrophoresis and amplified product (~600 bp) was sequenced at Macrogen Inc. (Seoul, Ko- rea). The DNA sequence from this study was compared with reference sequences of fungal species in the NCBI database (www.ncbi.nlm.nih.gov) using BLAST search. Testing Pathogenicity Polyethylene bags (25×30 cm) were filled with approx- imately 1 kg of Phase III compost spawned with A. bis- porus (obtained from Gulf Mushroom Products Com- pany, Barka, Oman). Casing soil (obtained from Gulf Mushroom Products Company) was applied as a layer (30-40 mm) on the surface. Ten ml of spore suspension (1×104 spores/ml) of H. perniciosus was mixed with the casing soil. The bags were kept at 25°C for 2-3 weeks and checked for the disease development. Extraction of Essential Oil One kg of Z. multiflora leaves and stem was transferred to a glass reactor followed by the addition of 1.5 L of distilled water. The essential oil (EO) was extracted using ETHOS X microwave extraction system (Milestone Inc., Shelton, CT, USA). The essential oil was stored in small amber glass vials in a freezer at -20°C. Testing Antifungal Activity The antifungal activity of the EO of Z. multiflora against H. perniciosus was tested using agar diffusion assay (Al-Maawali et al., 2021). Briefly, sterile filter paper discs (6-mm diameter) were placed on the surface of PDA me- dium in Petri dishes and 10 μl of EO (50% and 100%) was 61Research Paper Al-Balushi, Al-Sadi, Al-Mahmooli, Al-Harrasi, Al-Sabahi, Al-Alawi, Al-Farsi, Velazhahan applied on the discs. Then a 7-mm mycelial disc obtained from a 7-day-old H. perniciosus culture was placed in the center of the Petri dish and incubated at 27°C for 5-7 days. The formation of inhibition zone around the filter paper discs was observed. The assay was conducted in triplicate. Minimal Inhibitory Concentration (MIC) The MIC of ZEO was determined according to Kiran et al. (2016). Briefly, calculated quantity of the ZEO was di- luted in 0.5 ml of 5% Tween-20 and mixed with 19.5 ml of molten PDA and poured into sterile Petri dishes to obtain the final concentrations of 0.1-1.0 μl/ml. In the center of the Petri dish, a 7-mm mycelial disc obtained from a 7-day-old H. perniciosus culture was placed. The Petri plates were incubated at 27°C for 5-7 days and ob- served for the mycelial growth inhibition and MIC (the lowest concentration that causes no visible growth of the fungus). Petri plate containing PDA amended with 0.5 ml of 5% Tween-20 alone was used as negative control. Four replications were maintained per treatment. The data were analyzed using SAS v8, (SAS Institute, NC, USA) and the values were compared by “Duncan’s mul- tiple range test, DMRT” at P ≤ 0.05. Purified linalool (≥ 95.0%) (Fluka, Sigma-Aldrich Chemie GmbH, Switzer- land) was diluted in ethanol and mixed with PDA medi- um to obtain final concentrations of 0.25% and 0.125% and evaluated for its antifungal activity against H. perni- ciosus as described above. Analysis of Essential Oil Shimadzu GC-2010 Plus gas chromatography machine fitted with Rtx-5MS capillary column (30 m × 0.25 mm; 0.25 μm), coupled to a GCMS-QP2010 ULTRA MS was used for analysis of Z. multiflora essential oil (Hanif et al., 2011). The data were obtained by collecting the full- scan mass spectra with the scan range of 40-550 amu. Figure 1. Agaricus bisporus showing symptoms of wet bubble disease, 14 days after artificial inoculation with Hypomyces perniciosus. Figure 2. Inhibition of mycelial growth of Hypomyces perniciosus by essential oil of Zataria multiflora. 62 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 2 Antifungal Activity of Shirazi Thyme (Zataria multiflora Boiss.) Essential Oil against Hypomyces perniciosus, a causal agent of wet bubble disease of Agaricus bisporus. The total run time was 63.5 min. The National Institute of Standards and Technology (NIST) v.2.3 and Wiley 9th edition mass spectrum libraries were used for identifica- tion of the compounds. Results and Discussion Hypomyces perniciosus was isolated from Agaricus bis- porus showing the symptoms of WBD and pure culture was obtained. The fungus was identified based on the analysis of PCR-amplified ITS regions. The fungal isolate showed 100% identity to sequences of over 30 strains of Hypomyces perniciosus stored in the GenBank data- base. The sequence of the fungus was deposited in the GenBank under the accession number MZ149255. The pathogenicity of the fungus was confirmed by artificial inoculation of casing soil with the spore suspension of H. perniciosus. The mycoparasite induced typical symp- toms of WBD on the emerging mushrooms approxi- mately 14 days after inoculation (Figure 1). The EO extracted from Z. multiflora (ZEO) aerial parts had a very strong antifungal activity and complete- ly stopped the growth of H. perniciosus at the tested concentrations (50% and 100%) in agar diffusion test, whereas the control recorded 4.8 cm diameter growth after 7 days of incubation (Figure 2). The MIC value of ZEO was 0.4 μl/ml as assessed by poisoned food tech- nique (Table 1). Antifungal activities of EOs of a few plants against M. perniciosa have been reported in pre- vious studies (Regnier and Combrinck, 2010; Glamoclija et al., 2009). Glamoclija et al. (2009) demonstrated that the EO of Critmum maritimum and its major compo- nents viz., limonene and α-pinene effectively inhibited the growth of M. perniciosa. Preventive application of EOs of Lippia citriodora and Thymus vulgaris at a con- centration of 40 μl/L was demonstrated to control the development of WBD in a simulated commercial trial (Regnier and Combrinck, 2010). Different modes of action of EOs on fungi have been reported. Cox et al. (2000) while studying the mode of action of essential oil on yeast (Candida albicans) and bacteria (Staphylococcus aureus and Escherichia coli) re- ported that the EO of Melaleuca alternifolia suppressed the respiration and augmented the permeability of plas- ma membranes of yeast and cytoplasmic membranes of bacteria. Tian et al. (2012) demonstrated that EO of Anethum graveolens induced morphological alterations in Aspergillus flavus cells and reduced the ergosterol content and activities of ATPase and dehydrogenase and increased the mitochondrial membrane potential and production of reactive oxygen species. Chaudhari et al. (2020) reported that the EO of Pimenta dioica complete- ly inhibited the growth of A. flavus and the production of aflatoxin B1. The oil caused reduction of methylgly- oxal, a signaling molecule that can trigger aflatoxin bio- synthesis gene aflR, increased ions leakage from the cells and ergosterol content of fungal plasma membrane, sug- gesting plasma membrane of fungi as the action site. The inhibition of mycelial growth of M. perniciosa by ZEO as observed in this study might be due to the presence of antifungal compounds in ZEO (Nazzaro et al., 2017). Phytochemical profile of ZEO by GC-MS analysis in Table 1. Minimum inhibitory concentration of Zataria mul- tiflora essential oil against Hypomyces perniciosus. Concentration of ZEO (μl/ml) Diameter of mycelial growth (cm) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 5.3 a 2.5 b 1.8 c 1.2 d 0 e 0 e 0 e 0 e 0 e 0 e 0 e Values followed by the same alphabetical letter, do not differ significantly (P = 0.05; Duncan’s multiple range test). Figure 3. Gas chromatogram of Zataria multiflora essential oil. 63Research Paper Al-Balushi, Al-Sadi, Al-Mahmooli, Al-Harrasi, Al-Sabahi, Al-Alawi, Al-Farsi, Velazhahan this study identified 23 compounds (Figure 3). Among these, linalool (20.3%) and bornyl acetate (15.5%) were the major components (Table 2). Antimicrobial effect of linalool, an acyclic monoterpene (Park et al., 2012) has been reported earlier (Peana et al., 2002). Bornyl acetate has been identified as the major component of EO of Tetraclinis articulata that showed antibacterial activities (Rabib et al., 2020). Several reports indicated the chemical composition of EO of Z. multiflora (Shafiee and Javidnia, 1997; Moosavy et al. 2008; Mahboubi and Bidgoli, 2010; Raeisi et al., 2016). Carvacrol (71.20%), γ-terpinene (7.34%) and α-pinene (4.26%) were report- ed as the major components of ZEO (Moosavy et al. 2008). Mahboubi and Bidgoli (2010) reported thymol (38.7%), carvacrol (15.3%) and p-cymene (10.2%) as the major components in ZEO. In another study, Carvacrol (63.2%) and thymol (15.1%) were reported as the main constituents of ZEO (Raeisi et al., 2016). Saleem et al. (2004) reported higher thymol concentration in fresh plant (73.21%) and carvacrol in dry plant (62.87%) tis- sues. The difference in the composition of ZEO might be due to plant samples collected at varying growth stages, geographical locations, prevailing climatic conditions and habitat (Ruiz-Navajas et al., 2012; Abd-ElGawad et al., 2019). The results of this study also revealed that linalool, one of the major components of ZEO also in- hibited the growth of H. perniciosus even at a concen- tration of 0.125% under laboratory conditions (Figure 4). The inhibitory effect of ZEO on H. perniciosus in this study could be attributed to the presence of linalool. Figure 4. Inhibition of mycelial growth of Hypomyces perniciosus by linalool. Table 2. Chemical composition of essential oil of Zataria multiflora. S.No. Name of the compound Rt (min) Area % Calculated KI NIST KI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Camphene trans-.beta.-Ocimene .beta.-Pinene 3-Octanone .(-)-Limonene .gamma.-Terpinene Linalool 1,5,7-Octatrien-3-ol, 3,7-dimethyl- 2,6-Dimethyl-1,3,5,7-octatetraene, E,E- Isoborneol Terpinen-4-ol .alpha.-Terpineol 2,6-Dimethyl-3,5,7-octatriene-2-ol, ,E,E- Linalyl acetate Bornyl acetate Thymol (-)-.beta.-Elemene Caryophyllene .gamma.-Elemene Humulene Germacren D-4-ol .alpha.-Cadinol Shyobunol 7.83 8.24 8.99 9.23 10.41 11.24 12.53 12.57 12.96 14.26 14.49 14.84 15.07 16.43 17.31 17.37 19.53 20.00 20.54 22.28 23.98 25.62 26.43 6.195 10.703 1.454 0.727 1.593 0.954 20.332 2.567 2.169 10.696 1.209 2.518 4.368 3.043 15.564 2.269 0.858 0.720 2.611 1.393 1.289 1.323 5.434 941 956 984 993 1036 1066 1113 1114 1129 1177 1185 1198 1207 1260 1294 1296 1385 1404 1428 1504 1581 1659 1793 935 958 970 962 1020 1047 1081 1115 1134 1146 1161 1172 1187 1236 1269 1262 1377 1424 1425 1456 1570 1641 1709 64 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 2 Antifungal Activity of Shirazi Thyme (Zataria multiflora Boiss.) Essential Oil against Hypomyces perniciosus, a causal agent of wet bubble disease of Agaricus bisporus. Conclusion Z. multiflora is commonly used as a flavor ingredient in foods and has a wide range of biological and medicinal properties including antibacterial, antiseptic, anesthet- ic, antioxidant and immunomodulatory activities. This study demonstrated the antifungal activities of ZEO and its major constituent linalool against H. perniciosus. The results of this study suggest that ZEO is a safe, environ- mentally friendly natural product for control of WBD. However, further research is required to investigate the inhibitory effect of ZEO on A. bisporus. Acknowledgement We thank the Gulf Mushroom Products Company, Bar- ka, Oman for providing Phase III compost and casing soil and Oman Botanic Garden for providing Zataria multiflora plants. References Abd-ElGawad AM, Elshamy AI, Al-Rowaily SL, El-Ami- er YA. (2019). Habitat affects the chemical profile, al- lelopathy, and antioxidant properties of essential oils and phenolic enriched extracts of the invasive plant Heliotropium curassavicum. Plants 8: 482. Al-Maawali SS, Al-Sadi AM, Sathish Babu SP, Velazha- han R. (2021). In vitro tolerance to antifungal gly- coalkaloids and biofilm forming ability of the an- tagonistic yeast Meyerozyma guilliermondii strain SQUCC-33Y. Indian Phytopathology 74: 817-821. Al-Rashdi FKH, Al-Sadi AM, Al-Riyamy BZ, Maharac- hchikumbura SSN, Al-Ruqaishi HK, Velazhahan R. (2020). Alternaria alternata and Neocosmospora sp. from the medicinal plant Euphorbia larica exhib- it antagonistic activity against Fusarium sp., a plant pathogenic fungus. All Life 13: 223-232. Bollen GJ, Fuchs A. (1970). On the specificity of the in vitro and in vivo antifungal activity of benomyl. Netherlands Journal of Plant Pathology 76: 299-312. Chaudhari AK, Singh VK, Dwivedy AK, Das S, Upad- hyay N, Singh A, Dkhar MS, Kayang H, Prakash B, Dubey NK. (2020). Chemically characterised Pimen- ta dioica (L.) Merr. essential oil as a novel plant based antimicrobial against fungal and aflatoxin B1 con- tamination of stored maize and its possible mode of action. Natural Product Research 34: 745-749. Cox SD, Mann CM, Markham JL, Bell HC, Gustafson JE, Warmington JR, Wyllie SG. (2000). The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). Journal of Applied Microbi- ology 88: 170-175. Fletcher JT, Gaze RH. (2008). Mushroom Pest and Dis- ease Control: A Colour Handbook, 1st ed. Manson Publishing Ltd. Academic Press: San Diego, CA, USA, p.192. Fletcher JT, Jaffe B, Muthumeenakshi S, Brown AE, Wright DM. (1995). Variations in isolates of My- cogone perniciosa and in disease symptoms in Agari- cus bisporus. Plant Pathology 44: 130-140. Fu Y, Wang X, Li D, Liu Y, Song B, Zhang C, Wang Q, Chen M, Zhang Z, Li Y. (2016). Identification of re- sistance to wet bubble disease and genetic diversity in wild and cultivated strains of Agaricus bisporus. International Journal of Molecular Sciences 17: 1568. Gea FJ, Navarro MJ, Santos M, Diánez F, Carrasco J. (2021). Control of fungal diseases in mushroom crops while dealing with fungicide resistance: A re- view. Microorganisms 9: 585. Gea FJ, Tello JC, Navarro MJ. (2010). Efficacy and effects on yield of different fungicides for control of wet bub- ble disease of mushroom caused by the mycoparasite Mycogone perniciosa. Crop Protection 29: 1021-1025. Glamoclija J, Sokovic M, Grubisic DJ, Vukojevic J, Mil- inekovic IM, Ristic M. (2009). Antifungal activity of Critmum maritimum essential oil and its compo- nents against mushroom pathogen Mycogone perni- ciosa. Chemistry of Natural Compounds 45: 95-97. Hanif MA, Al-Maskari MY, Al-Maskari A, Al-Shukaili A, Al-Maskari AY, Al-Sabahi JN. (2011). Essential oil composition, antimicrobial and antioxidant activi- ties of unexplored Omani basil. Journal of Medicinal Plants Research 5: 751-757. Kiran S, Kujur A, Prakash B. (2016). Assessment of preservative potential of Cinnamomum zeylanicum Blume essential oil against food borne molds, aflatox- in B1 synthesis, its functional properties and mode of action. Innovative Food Science and Emerging Tech- nologies 37: 184-191. Kumla J, Suwannarach N, Sujarit K, Penkhrue W, Ka- kumyan P, Jatuwong K, Vadthanarat S, Lumyong S. (2020). Cultivation of mushrooms and their lignocel- lulolytic enzyme production through the utilization of agro-industrial waste. Molecules 25: 2811. Lee SB, Taylor JW. (1990). Isolation of DNA from fun- gal mycelia and single spores. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, p. 282-287. Li D, Sossah FL, Sun L, Fu Y, Li Y. (2019). Genome anal- ysis of Hypomyces perniciosus, the causal agent of wet bubble disease of button mushroom (Agaricus bispo- rus). Genes 10: 417. Mahboubi M, Bidgoli FG. (2010). Anti-staphylococcal activity of Zataria multiflora essential oil and its syn- ergy with vancomycin. Phytomedicine 17: 548-550. Moosavy MH, Basti AA, Misaghi A, Salehi TZ, Abbasifar R, Mousavi HAE, Alipour M, Razavi NE, Gandomi H, Noori N. (2008). Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium 65Research Paper Al-Balushi, Al-Sadi, Al-Mahmooli, Al-Harrasi, Al-Sabahi, Al-Alawi, Al-Farsi, Velazhahan and Staphylococcus aureus in a food model system and on the bacterial cell membranes. Food Research International 41: 1050-1057. Nazzaro F, Fratianni F, Coppola R, Feo VD. (2017). Essen- tial oils and antifungal activity. Pharmaceuticals 10: 86. Park SN, Lim YK, Freire MO, Cho E, Jin D, Kook JK. (2012). Antimicrobial effect of linalool and α-terpin- eol against periodontopathic and cariogenic bacteria. Anaerobe 18: 369-372. Peana AT, D’Aquila PS, Panin F, Serra G, Pippia P, Moretti MDL. (2002). Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils. Phytomedicine 9: 721-726. Potocnik I, Vukojević J, Stajic M, Tanovic B, Rekanovic E. (2010). Sensitivity of Mycogone perniciosa, patho- gen of culinary-medicinal button mushroom Agari- cus bisporus (J. Lge) Imbach (Agaricomycetideae), to selected fungicides and essential oils. International Journal of Medicinal Mushrooms 12: 91-98. Rabib H, Elagdi C, Hsaine M, Fougrach H, Koussa T, Badri W. (2020). Antioxidant and antibacterial activi- ties of the essential oil of Moroccan Tetraclinis artic- ulata (Vahl) Masters. Biochemistry Research Inter- national 2020: 1-6. Raeisi M, Tajik H, Rohani SMR, Tepe B, Kiani H, Khosh- bakht R, Aski HS, Tadrisi H. (2016). Inhibitory effect of Zataria multiflora Boiss. essential oil, alone and in combination with monolaurin, on Listeria monocyto- genes. Veterinary Research Forum 7: 7-11. Regnier T, Combrinck S. (2010). In vitro and in vivo screening of essential oils for the control of wet bub- ble disease of Agaricus bisporus. South African Jour- nal of Botany 76: 681-685. Ruiz-Navajas Y, Viuda-Martos M, Sendra E, Perez-Al- varez JA, Fernandez-Lopez J. (2012). Chemical char- acterization and antibacterial activity of Thymus moroderi and Thymus piperella essential oils, two Thymus endemic species from southeast of Spain. Food Control 27: 294-299. Saleem M, Nazli R, Afza N, Sami A, Shaiq Ali M. (2004). Biological significance of essential oil of Zataria mul- tiflora Boiss. Natural Product Research 18: 493-497. Sanchez C. (2004). Modern aspects of mushroom cul- ture technology. Applied Microbiology and Biotech- nology 64: 756-762. Shafiee A, Javidnia K. (1997). Composition of essential oil of Zataria multiflora. Planta Medica 63: 371-372. Shi N, Ruan H, Jie Y, Chen F, Du Y. (2020). Sensitivity and efficacy of fungicides against wet bubble disease of Agaricus bisporus caused by Mycogone perniciosa. European Journal of Plant Pathology. 157: 873-885. Tanovic B, Potocnik I, Delibasic G, Ristic M, Kostic M, Markovic M. (2009). In vitro effect of essential oils from aromatic and medicinal plants on mushroom pathogens: Verticillium fungicola var. fungicola, My- cogone perniciosa, and Cladobotryum sp. Archives of Biological Sciences Belgrade 61: 231-237. Tian J, Ban X, Zeng H, He J, Chen Y, Wang Y. (2012). The mechanism of antifungal action of essential oil from dill (Anethum graveolens L.) on Aspergillus flavus. PLoS One 7: 1-10. Wang W, Li X, Chen B, Wang S, Li C, Wen Z. (2016). Analysis of genetic diversity and development of SCAR Markers in a Mycogone Perniciosa Population. Current Microbiology 73: 9-14. White TJ, Bruns TD, Lee SB, Taylor JW. (1990). Ampli- fication and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: A Guide to Methods and Applications. Academic Press, New York, p. 315-322. Zhang CL, Xu JZ, Kakishima M, Li Y. (2017). First report of wet bubble disease caused by Hypomyces pernicio- sus on Pleurotus citrinopileatus in China. Plant Dis- ease 101(7): 1321. Zhou C, Li D, Chen L, Li Y. (2016). Genetic diversity analysis of Mycogone perniciosa causing wet bubble disease of Agaricus bisporus in china using SRAP. Journal of Phytopathology 164: 271-275.