ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2020, 25(2): 22–29 DOI: 10.24200/jams.vol25iss2pp22-29 Received 30 March 2020 Accepted 19 May 2020 In vitro Antagonistic Potential, Plant Growth-promoting Activity and Indole-3-acetic Acid Producing Trait of Bacterial Isolates from Button Mushroom (Agaricus bisporus) Spent Substrate Shima Nasser Hamed Al-Mamari1, Abdullah Mohammed Al-Sadi1, S. P. Sathish Babu2, Issa Hashil Al-Mahmooli1, Rethinasamy Velazhahan1,* التأثري املضاد، والنشاط املعزز لنمو النبات وميزة إنتاج األندول 3- حمض األسيتيك يف املخترب من العزالت البكتريية من السامد املستهلك )كمبوست( )Agaricus bisporus( بعد إنتاج فطر املرشوم الدائري شيامء بنت نارصبن حمد املعمرية1، عبدالله بن محمد السعدي1، ساثيش بابو2، عيىس بن هاشل املهمويل1 و راثيناسمي فيالزهاهن1* abstRact. Spent mushroom substrate (SMS) is widely used as a fertilizer and to control plant diseases. The microor- ganisms surviving in SMS play a crucial role in plant growth promotion and biocontrol activity. In this study, an effort was made to isolate and characterize the bacterial species present in the SMS of Agaricus bisporus and to study their antagonistic potential, plant growth-promoting ability and indole-3-acetic acid (IAA) producing trait. Six different bacterial isolates exhibiting morphological variabilities were obtained from the SMS by serial dilution technique. On the basis of 16S rRNA gene sequences, these isolates were identified as Staphylococcus epidermidis (Sh1 and Sh3), S. aureus (Sh2), Bacillus albus (Sh4), Delftia lacustris (Sh6) and Comamonas aquatica (Sh7). These bacterial strains were assayed for their antagonism against Pythium aphanidermatum, a phytopathogenic oomycete. The results of in vitro dual culture assay revealed that all the 6 bacterial isolates showed low levels of suppression of P. aphanidermatum and recorded less than 5 mm inhibition zone. Among the bacterial isolates, S. epidermidis Sh3 recorded the maximum inhibition zone of 4.2 ± 0.5 mm. Plant growth promotion test using roll paper towel method revealed that C. aquatica Sh7, B. albus Sh4, D. lacustris Sh6 and S. epidermidis Sh3 caused a significant increase in seedling vigour of cucumber compared to control. The seeds treated with the bacterial isolate C. aquatica Sh7 showed the maximum seedling vigor (2018 ± 255). Assessment of in vitro production of IAA by the bacterial isolates revealed that the bacterial isolates highly varied (ranging from 0.28 to 9.25 mg L-1) in their potential for production of IAA. The maximum amount of IAA was produced by C. aquatica Sh7 (9.25 ± 0.02 mg L-1). Further studies are required to assess the possibility of using the IAA-producing bacterial isolates identified in this study or their metabolites to promote plant growth or to enhance growth and yield of mushrooms. KeywoRds: Button mushroom; spent compost; IAA production; Agaricus bisporus; antagonistic activity; plant growth promotion. املســتخلص:خالصة: يســتخدم الســامد املســتهلك )كمبوســت( بعــد إنتــاج فطــر املــرشوم بشــكل واســع كســامد وأيضــا يف مكافحــة الفطريــات املمرضــة للنباتــات. ــف ــزل وتوصي ــا بع ــة قمن ــذه الدراس ــي. يف ه ــة البيولوج ــاط املكافح ــات ونش ــو النب ــز من ــاًم يف تعزي ــامد دوًرا حاس ــذا الس ــش يف ه ــي تعي ــة الت ــة الدقيق ــات الحي ــب الكائن تلع ــو ــيط من ــات وتنش ــض الفطري ــو بع ــط من ــى تثبي ــا ع ــا قدرته ــة أيض ــر Agaricus bisporus ولدراس ــاج فط ــتخدم يف إنت ــذي يس ــت ال ــذا الكمبوس ــودة يف ه ــا املوج البكتريي ــي ــف التسلس ــة التخفي ــتخدام تقني ــا بإس ــول عليه ــم الحص ــة ت ــة مختلف ــات ظاهري ــا ذات صف ــن البكتريي ــزالت م ــت ع ــيتيك. س ــض األس ــدول 3- حم ــاج اإلن ــات وإنت النبات ــى ــك )S1٦( ع ــض الريبونيوكلي ــى حم ــودة ع ــومات املوج ــدة الريبوس ــل وح ــي يف تسلس ــف الجين ــتخدام التصني ــا بإس ــف البكتريي ــم تصني ــت. ت ــز الكمبوس ــوى تركي يف محت و Delftia lacustris )Shو)٦ Bacillus albus )S4h(Hو S.aureus )Sh2( و Staphylococcus epidermidis )Sh 3 و 1Sh( إنهــا ــرت ــرضPythium aphanidermatum . أظه ــكاذب املم ــر ال ــو الفط ــط من ــى تثبي ــا ع ــم تجربته ــة ت ــالالت البكتريي ــذه الس )Comamonas aquatica )sh٧. ه النتائــج التــي أجريــت يف املختــرب بعــد وضــع كل مــن أوميســيتس P. aphanidermatum مــع جميــع العــزالت البكترييــة الســتة مســتويات تثبيــط منخفضة وســجلت منطقة تثبيــط أقــل مــن ٥ مــم. مــن بــن العــزالت البكترييــة، ســجلت B. و C. aquatica shــة أن ٧ ــل الورقي ــات املنادي ــة لفاف ــتخدام طريق ــات باس ــو النب ــار من ــف إختب ــم. كش ــط 4,2 ± ٠,٥ م ــة تثبي ــى منطق S. epidermidis Sh3 أق albus Sh4 و D. lacustris Sh٦ و. epidermidis Sh 3 تســببو يف زيــادة معنويــة كبــرية يف قــوة إنبــات بــادرات الخيــار مقارنــة بالشــاهد. أظهــرت البــذور املعالجــة البكتريية بالعزلة C. aquatica sh٧ قــوة قصــوى للشــتالت )2٠1٨ ± 2٥٥(. كشــف تقييــم إنتــاج اإلنــدول 3- حمــض األســيتيك IAA يف املختــرب مــن قبــل العــزالت البكترييــة أن C. aquatica ــطة ــن IAA بواس ــدر م ــرب ق ــاج أك ــم إنت ــاج IAA. ت ــى إنت ــا ع ــر( يف قدرته ــرام / ل ــن ٠,2٨ إىل ٩,2٥ ملج ــراوح م ــوع )ت ــديدة التن ــة ش ــزالت البكتريي الع ــة أو ــذه الدراس ــددة يف ه ــة لـــ IAA املح ــة املنتج ــزالت البكتريي ــتخدام الع ــة إس ــم إمكاني ــات لتقيي ــن الدراس ــد م ــة إىل مزي ــاك حاج ــر(. هن ــرام /ل sh٧ )٩,2٥ ± ٠,٠2 ملج نواتجهــم لتعزيــز منــو النبــات أو لتعزيــز منو وإنتــاج الفطر. 23ReseaRch PaPeR Al-Mamari, Al-Sadi, Babu, Al-Mahmooli, Velazhahan Rethinasamy Velazhahan1,*( ) velazhahan@squ.edu.om, 1Depart- ment of Plant Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khoud, Muscat 123, Sultan- ate of Oman. 2 Central Analytical and Applied Research Unit, College of Science, Sultan Qaboos University, Al-Khoud, Muscat 123, Sultanate of Oman. Introduction M ushroom farming has gained recognition in the recent years and has emerged as a prom-ising agro-based business. Malaysia, China, India and Ireland are the world’s leading edible mush- rooms producers (Hanafi et al., 2018). Several edible mushrooms including button mushroom (Agaricus bis- porus), shiitake mushroom (Lentinula edodes), paddy straw mushroom (Volvariella volvacea), oyster mush- room (Pleurotus spp.) and enoki mushroom (Flammu- lina ostreatus) are being cultivated commercially world- wide (Feeney et al., 2014). Agaricus bisporus is cultivated commercially in Oman. Mixtures of agricultural/poul- try/industrial wastes are commonly used as substrates for mushroom cultivation. The mushroom industry dis- charges huge quantities of spent mushroom substrate (SMS) after harvest. The SMS usually contains mycelia and remnants of fruiting bodies of mushrooms, and the substrate used for cultivation of mushrooms (Kang et al., 2017). A wide variety of biologically active com- pounds such as extracellular enzymes, antimicrobial compounds and secondary metabolites that are mainly produced by mushrooms are present in the SMS (Kwak et al., 2015). The potential of SMS in large-scale enzymes production, plant diseases control, bioremediation, fer- tilizer, vermicomposting and for feeding animals has been documented (Inagaki and Yamaguchi, 2009; Ahl- awat et al., 2011; Parada et al., 2011; Parada et al., 2012; Kwak et al., 2015; Roy et al., 2015). Several reports in- dicated the effectiveness of SMS in plant disease man- agement (Yohalem et al., 1996; Uzun, 2004; Goonani et al., 2011; Riahi et al., 2012). Riahi et al. (2012) demon- strated that the extract of SMS inhibited the growth of Lecanicillium fungicola, the causal fungus of dry bubble disease of A. bisporus. Kang et al. (2017) reported that aqueous extract prepared from SMS of Lentinula edodes suppressed the growth of Phytophthora capsici, reduced the Phytophthora blight and enhanced the growth of pepper. The antagonistic microorganisms present in the SMS were attributed to the disease suppression (Riahi et al., 2012). The objectives of the present study were to isolate and characterize the bacterial species present in the spent mushroom substrate of A. bisporus in Oman and to study their in vitro antagonistic potential, plant growth-promoting trait and IAA producing ability. Materials and Methods SMS Collection and Bacterial Isolation Spent mushroom substrate of A. bisporus was obtained from the Department of Plant Sciences, CAMS, Sultan Qaboos University. Bacteria from the SMS were isolated by employing serial dilution plate technique. Briefly, 1 g of SMS was suspended in 99 ml of sterile water and kept on a rotary shaker (150 rpm) for 30 min. Later, the suspension was serially diluted at 1:10 ratio with ster- ile water. An aliquot (100 μl) from 10−4 to 10−7 dilutions was gently spread over the Nutrient agar (NA) (Oxoid, UK) with a sterile spreader and then the Petri plates were incubated at 30 °C for 48 h. The bacterial colonies with varying morphological features were selected and transferred to fresh NA plates. Figure 1. Enhancement of cucumber growth by seed bac- terization with Comamonas aquatica Sh7 isolated from spent mushroom substrate of Agaricus bisporus الكلامت املفتاحية: الفطر الدائري، السامد املستهلك، إنتاج IAA، Agaricus bisporus، نشاط تثبيط، تعزيز منو النبات 24 sQU JoURnal of agRicUltURal and MaRine sciences, 2020, VolUMe 25, issUe 2 In vitro Antagonistic Potential, Plant Growth-promoting Activity and Indole-3-acetic Acid Producing Trait of Bacterial Isolates from Button Mushroom (Agaricus bisporus) Spent Substrate Molecular Identification of Bacterial Isolates The 16S rRNA gene sequence analysis was employed for identification of the bacterial isolates. The bacterial isolates were grown individually on a shaker in nutrient broth (NB) medium (100 ml) at 30°C for 48 h. The bac- terial cultures were centrifuged at 14000 g for 15 min and the bacterial cell pellets were collected. DNA was extracted from the bacterial pellet using a commercial foodproof StarPrep Two DNA extraction kit (BIOTE- CON Diagnostics, Germany). The universal primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1429R (5’-TACGGYTACCTTACGACTT-3’) were used for amplification of bacterial 16S rRNA gene by PCR as described by Al-Hussini et al. (2019). The PCR ampli- fied products were sequenced at Macrogen Inc., Seoul, Korea. A database search of homologous sequences was carried out using National Center for Biotechnology In- formation (NCBI) BLASTN program (http://www.ncbi. nlm.nih.gov). Test pathogen A virulent isolate of Pythium aphanidermatum, the cu- cumber damping-off pathogen (Al-Shibli et al., 2019), was used in this study. The oomycete pathogen was mul- tiplied on potato dextrose agar (PDA) (Oxoid, UK) at 25 ± 2 °C. Bacterial Isolates Screening Against P. aphani- dermatum The bacterial isolates were screened for their inhibito- ry effect on P. aphanidermatum using an in vitro dual culture method as described by Al-Hussini et al. (2019). Briefly, a mycelial plug (7 mm diameter) of P. aphanid- ermatum was placed aseptically on one end of the Petri plate (9 cm diameter) containing PDA. The bacterial iso- late was streaked on the other side of the Petri plate (~ 1 cm away from the margin). The inoculated plate was in- cubated at 27 °C for 3-5 days. After incubation, the inhi- bition zone was measured. Petri plates inoculated with P. aphanidermatum discs alone were used as control. Four replications were maintained for each bacterial isolate. Figure 2. HPLC chromatograms showing IAA produced by bacterial strains from spent mushroom substrate of Agaricus bisporus 25ReseaRch PaPeR Al-Mamari, Al-Sadi, Babu, Al-Mahmooli, Velazhahan Plant Growth Promoting Activity of Bacterial Isolates Each bacterial isolate was cultured in NB medium (100 ml) in 250 ml conical flask on a shaker (200 rpm/min) at 30°C for 48 h, and then the bacterial suspension was cen- trifuged at 3000 rpm for 10 min. The bacterial cell pellet was collected and re-suspended in sterile distilled water and the concentration of the bacterial cells was adjusted to 4×108 CFU ml-1. Cucumber seeds (cv. Jabbar, F1; US Agriseeds, USA) were immersed in the bacterial suspen- sion for 3 h at room temperature (25±2 °C), while the control seeds were soaked in sterile distilled water. The roll paper towel method (Shifa et al., 2015) was used to test the effect of bacterial strains on the growth of cu- cumber. The percentage of cucumber seed germination, seedling shoot length and root length were recorded 12 days after treatment and vigor index was calculated by multiplying the germination percentage of seeds with the total of seedling root length and shoot length. Four replicates of 10 seeds each were used for each treatment. Analysis of IAA Production The bacterial isolates were cultivated in NB medium supplemented with 5 mM Tryptophan in a shaker (200 rpm) for 72 h at 30°C. The cultures were centrifuged at 14000 g for 10 min at 4°C and the culture supernatants were collected. The IAA content in the cell-free bacte- rial culture supernatants was analyzed by High-per- formance liquid chromatography (HPLC) (Szkop and Bielawski, 2013). Analysis of IAA was performed using a HPLC system (Agilent-1200 Infinity Series), equipped with a high performance autosampler (G4226A), qua- ternary pump (G4204A), thermostatted column com- partment (G1316C) and a diode array detector (DAD) (G4212A). The separation was achieved with Waters Symmetry C8 (5 µm, 3.0×150 mm) column. The mobile phases consisted of A (2.5% acetic acid with a pH 3.8) and B (80% acetonitrile). The mobile phase began with eluent A: eluent B at 80:20 and changed to 50:50, 0:100, 80:20 in 15, 16, and 16.5 min, respectively, and main- tained in 80:20 for 1.5 min with a flow rate 1 ml per min. The detection wavelength was set at 280 nm. Peaks in the sample were identified and quantified by comparing with the standard RT. Statistical Analysis The experimental design used was completely random- ized design. The data on mycelial growth inhibition, per- cent seed germination and seedling growth of cucumber and IAA production by bacterial isolates,were analyzed by one-way ANOVA (Minitab 17, State College, PA, USA). The data on % seed germination was analyzed af- ter arcsine transformation of values to ensure homoge- neity of variance. Results Isolation and Characterization of Bacteria from SMS A total of 6 morphologically different bacterial isolates were obtained from the SMS of A. bisporus. On the ba- sis of 16S rRNA gene sequences, these bacterial isolates were identified as Staphylococcus epidermidis (Sh1), S. aureus (Sh2), S. epidermidis (Sh3), Bacillus albus (Sh4), Delftia lacustris (Sh6) and Comamonas aquatica (Sh7) (Table 1). The 16S rRNA gene sequences of these bac- terial isolates were deposited in the GenBank database with the accession numbers MT002750, MT002751, MT002756, MT002776, MT002777 and MT002779. Antagonistic Activity of Bacterial Isolates The antagonistic abilities of these bacterial isolates were determined against P. aphanidermatum using an in vitro dual-culture assay. The results indicated that none of the bacterial isolates showed considerable level of inhibition of mycelial growth of P. aphanidermatum. All the bac- terial isolates recorded less than 5 mm inhibition zone (Table 2). Of the 6 bacterial isolates evaluated, S. epider- midis Sh3 produced the maximum inhibition zone of 4.2 mm. Plant Growth Promoting Activity of Bacterial Isolates The bacterial isolates were tested for plant growth pro- motion effects on cucumber using a roll paper towel Table 1. Identification of bacterial isolates from spent mushroom substrate of Agaricus bisporus by 16S rDNA sequence analysis Bacterial isolate GenBank accession number Hit in the NCBI database % identity Sh1 MT002750 Staphylococcus epidermidis (KX348319.1) 99.87 Sh2 MT002751 Staphylococcus aureus (CP045468.1) 100 Sh3 MT002756 Staphylococcus epidermidis (LC499612.1) 100 Sh4 MT002776 Bacillus albus (MN793202.1) 100 Sh6 MT002777 Delftia lacustris (MF457528.1) 100 Sh7 MT002779 Comamonas aquatica (MN216294.1) 100 26 sQU JoURnal of agRicUltURal and MaRine sciences, 2020, VolUMe 25, issUe 2 In vitro Antagonistic Potential, Plant Growth-promoting Activity and Indole-3-acetic Acid Producing Trait of Bacterial Isolates from Button Mushroom (Agaricus bisporus) Spent Substrate technique. The results revealed that seed bacterization with C. aquatica Sh7, B. albus Sh4, D. lacustris Sh6 and S. epidermidis Sh3 resulted in a significant (F=9.57, df=6, p<0.05) increase in seedling vigour compared to control (Table 3). Among the various treatments, seeds treated with C. aquatica Sh7 showed the highest seedling vig- or (Figure 1). No significant (p<0.05) difference in the % seed germination among the treatments was observed. IAA Production All the 6 isolates of bacteria tested produced IAA between 0.28±0.02 and 9.25±0.02 mg L-1 in trypto- phan-amended growth medium (Table 4; Figure 2). The maximum (9.25 mg L-1) and minimum (0.28 mg L-1) production of IAA was recorded with C. aquatica Sh7 and S. epidermidis Sh1, respectively. Discussion The existence of a broad range of bacterial species in the SMS has been documented (Ntougias et al., 2004; Watabe et al., 2004). Ntougias et al. (2004) reported the presence of bacterial genera Arthrobacter, Brevibacte- rium, Bacillus, Comamonas, Carnobacterium, Desem- zia, Microbacterium, Paenibacillus, Exiguobacterium, Sphingobacterium and Staphylococcus in the spent mushroom compost of Agaricus spp. By using DNA se- quence typing, several bacterial species including, Ba- cillus subtilis, Bacillus licheniformis, Paenibacillus len- timorbus, Pseudomonas mevalonii, Stenotrophomonas sp., Klebsiella/Enterobacter sp., Microbacterium sp. and Sphingobacterium multivorum have been reported in the spent mushroom compost (Watabe et al., 2004). The type of substrates used in the compost preparation and their pasteurization conditions are known to influence the diversity of bacterial communities in SMS (Ntougias et al., 2004). Choudhary (2011) isolated Acinetobacter sp., Pseudomonas sp. and Sphingobacterium sp. from the casing material for Agaricus bisporus. Zhu et al. (2014) found Comamonas serinivorans sp. nov. in wheat straw compost. Silva et al. (2009) reported the presence of Bacillus, Paenibacillus spp. and Streptomyces in a sug- arcane bagasse and Cynodon dactylon straw compost used for A. brasilienses cultivation. Gbolagade (2006) reported the presence of Pseudomonas aeruginosa, En- terobacter aerogenes, Micrococcus roseus, Bacillus subti- lis, B. cereus, B. polymyxa, B. licheniformis, Escherichia coli, Clostridium perfringens and Citrobacter freundii in the compost used for cultivation of Lentinus squarro- sulus and Pleurotus tuber-regium. In the present study, Staphylococcus epidermidis (Sh1 and Sh3), S. aureus (Sh2), Bacillus albus (Sh4), Delftia lacustris (Sh6) and Comamonas aquatica (Sh7) were detected in the SMS of A. bisporus. The primary source of these bacteria might be the casing material or compost or water used for cul- tivation of mushrooms (Rainey et al., 1990; Choudhary, 2011; Kertesz and Thai, 2018; Cao et al., 2019). Several bacteria isolated from compost are reported to have ability to suppress the growth of plant patho- genic fungi (Boulter et al., 2002; Suarez-Estrella et al., 2007; Sreevidya and Gopalakrishnan, 2017) and to pro- mote plant growth (Chin et al., 2017; Sreevidya and Go- Table 2. Inhibition of mycelial growth of Pythium aphanider- matum by bacterial isolates from spent mushroom substrate of Agaricus bisporus Bacterial Isolate Inhibition zone (mm) Staphylococcus epidermidis Sh1 3.0 ± 0.8abc Staphylococcus aureus Sh2 4.0 ± 0.8ab Staphylococcus epidermidis Sh3 4.2 ± 0.5a Bacillus albus Sh4 2.0 ± 0.8c Delftia lacustris Sh6 3.0 ± 0.0abc Comamonas aquatica Sh7 2.5 ± 0.6c Data are mean of four replications ± standard deviation. Values in the column with the same letter are not significantly different from each other at P<0.05 Table 3. Effect of bacterial isolates from spent mushroom substrate of Agaricus bisporus on cucumber seed germination and seedling vigor Bacterial Isolate % germination* Shoot length (cm) Root length (cm) Vigour Index** Staphylococcus epidermidis Sh1 75.0 ± 5.8 6.3 ± 1.5b 14.8 ± 3.8ab 1583 ± 293bc Staphylococcus aureus Sh2 72.5 ± 5.0 6.1 ± 1.5b 16.1 ± 2.1a 1612 ± 156bc Staphylococcus epidermidis Sh3 75.0 ± 5.8 7.0 ± 1.8b 15.3 ± 1.9ab 1671 ± 185b Bacillus albus Sh4 75.0 ± 5.8 8.0 ± 1.2ab 16.6 ± 3.2a 1844 ± 294ab Delftia lacustris Sh6 75.0 ± 5.8 8.0 ± 0.9ab 16.1 ± 1.9a 1805 ± 152ab Comamonas aquatica Sh7 77.5 ± 5.0 9.1 ± 1.6a 17.0 ± 2.5a 2018 ± 255a Control 72.5 ± 5.0 6.4 ± 1.5b 12.1 ± 2.0b 1343 ± 160c * Non-significant (P<0.05) . **Vigor index was calculated by multiplying the % germination of seeds with the sum of shoot length and root length. Data are mean of three replications ± standard deviation. Values in the column with the same letter are not significantly different from each other at P<0.05 27ReseaRch PaPeR Al-Mamari, Al-Sadi, Babu, Al-Mahmooli, Velazhahan palakrishnan, 2017). Riahi et al. (2012) identified three bacterial species viz, Bacillus subtilis, B. licheniformis and B. amyloliquefaciens from the extract of leached spent mushroom compost that showed antagonistic ef- fect towards Lecanicillium fungicola, the causal agent of dry bubble disease of button mushroom. In the present study, none of the bacterial isolates showed substantial level of suppression of growth of P. aphanidermatum and all the bacterial isolates recorded less than 5 mm in- hibition zone. However, plant growth promoting effect of these bacterial isolates was observed. Although no sig- nificant difference in % seed germination was observed, seed bacterization with C. aquatica Sh7, B. albus Sh4, D. lacustris Sh6 and S. epidermidis Sh3 resulted in a signif- icant increase in seedling vigor of cucumber compared to control and C. aquatica Sh7 treated seeds showed the maximum seedling vigour. Several reports indicate the beneficial effects of bacteria present in the substrates used for cultivation of mushrooms (Rainey et al., 1990; Straatsma et al., 1994; Ahlawat and Vijay, 2010). The bacteria such as Alcaligenes faecalis and Pseudomonas putida which are surviving in casing layer are reported to influence the growth and morphogenesis of A. bispo- rus by producing growth inducing compounds, which stimulate initiation of pinheads (Rainey et al., 1990). Straatsma et al. (1994) demonstrated that the thermo- philic fungi present in mushroom compost enhanced the growth rate of Agaricus mycelium up to two fold. Inoculation with Bacillus megaterium or Staphylococcus has been shown to enhance mushroom production and early cropping (Ahlawat and Vijay, 2010). The increase in seedling vigor of cucumber in the present study could be as a result of production and release of growth pro- moting compounds like IAA by the bacterial isolates. IAA is a common auxin and is a product of L-trypto- phan metabolism of microorganisms. In bacteria, IAA is primarily synthesized via the indole-3-pyruvic acid pathway (Gomes et al., 2017). IAA produced by plant growth-promoting rhizobacteria (PGPR) is known to enhance root growth (Persello-Cartieaux et al., 2003) and the growth of root hairs (Desbrosses et al., 2009). Asghar et al. (2002) observed a significant relationship between in vitro auxin production by PGPR and yield of Brassica juncea. Deepa et al. (2010) demonstrated that Enterobacter cloacae and Enterobacter aerogens strains, which produced IAA, exhibited growth-promoting ef- fect in Vigna unguiculata. In addition to the effects of IAA produced by beneficial bacteria on plants, the growth and yield of mushrooms also reported to be in- fluenced by IAA (Maniruzzaman et al., 2008; Ramache- la and Sihlangu, 2016). Maniruzzaman et al. (2008) demonstrated that the culture media amended with IAA (5 ppm) caused rapid proliferation of oyster mushroom mycelia. Ramachela and Sihlangu (2016) reported that auxins promoted the cap size of Pleurotus ostreatus. In the present study, all the 6 bacterial isolates produced IAA in vitro and the production levels varied between 0.28 and 9.25 mg L-1. Among the bacterial isolates test- ed, C. aquatica Sh7 showed the highest production of IAA (9.25 mg L-1). The same bacterial isolate displayed the highest plant growth promoting activity. These re- sults suggest that IAA produced by this bacterial isolate might have involved in enhancing vigor of cucumber seedlings. An interesting observation in our study is that the bacterial isolate B. albus B4, which is producing low amounts of IAA in vitro, enhanced the growth of cucumber. These results suggest that other mechanisms of action might have been involved in plant growth pro- motion by this bacterium. However, Schwachtje et al. (2012) reported that the non-growth promoting bacte- rial strains Pseudomonas sp. WCS417r and G53 isolated from the rhizosphere of Arabidopsis showed the highest levels of IAA production. Conclusion This study demonstrated the existence of different bac- teria in SMS of Agaricus bisporus in Oman. These bac- terial isolates displayed low levels of antagonism against P. aphanidermatum and produced less than 5 mm inhi- bition zone. However, these bacterial isolates enhanced the plant growth as demonstrated by increased seed- ling vigor of cucumber compared to control. The level of production of IAA by these bacterial isolates varied among isolates. Among the bacterial isolates tested, Comamonas aquatica Sh7 showed the highest produc- tion of IAA as well as plant growth promoting activity. Further studies are required to evaluate the potential of these bacterial isolates or their cell free culture filtrates in promoting growth of edible mushrooms and in en- hancing plant growth under in vivo conditions. Acknowledgements This work was supported by the SQU research grants IG/AGR/ CROP/18/01 and RC/RG-AGR/CROP/19/02. We thank the Central Analytical and Applied Research Unit, SQU for HPLC analysis. Table 4. Production of IAA by bacterial isolates from spent mushroom substrate of Agaricus bisporus Bacterial Isolate IAA (mg L-1) Staphylococcus epidermidis Sh1 0.28 ± 0.02e Staphylococcus aureus Sh2 1.07 ± 0.01c Staphylococcus epidermidis Sh3 0.77 ± 0.01d Bacillus albus Sh4 0.33 ± 0.00e Delftia lacustris Sh6 7.57 ± 0.07b Comamonas aquatica Sh7 9.25 ± 0.02a Data are mean of four replications ± standard deviation Values in the column with the same letter are not significantly different from each other at P<0.05 28 sQU JoURnal of agRicUltURal and MaRine sciences, 2020, VolUMe 25, issUe 2 In vitro Antagonistic Potential, Plant Growth-promoting Activity and Indole-3-acetic Acid Producing Trait of Bacterial Isolates from Button Mushroom (Agaricus bisporus) Spent Substrate References Ahlawat OP, Vijay B. (2010). Potential of thermophilic bacteria as microbial inoculant for commercial scale white button mushroom (Agaricus bisporus) com- post production. 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