Eur Plan: Patho DOL 10.10071810658.016.1107-3 Biological control of green mould on Agaricus bisporus by a native Bacillus subtilis strain from mushroom compost ‘Svetlana Milija’evié-Mardié - Milo Stepanovié « Biljana Todorovié - Bojan Duduk - Jelena Stepanovié« Emil Rekanovig « Ivana Potognik Accept 17 November 2016 © Koninklike Nederandse Plentcrcktenkundige Vereniging 2016 ‘Abstract Fifty bacterial isolates obtained from compost Were tested in vitro against the causal agents of green ‘ould in Agaricus bisporus Isolate B-38 which induced 48.08% in vitro growth inhibition of 7: harziamum TS4 and 52.25% of T. ageressivum £. ewopaeum T1T was ident- fied as Bacillus subtilis, based on 16S rDNA sequence and used in mushroom growing room experiments, B, subtilis B38 did not decrease mycelial growth mte of Agaricus bisporus A$ in mushroom compost in glass tubes. After applying prochloraz-manganese, B. subilis B-38 and B. subtilis QST 713, no significant differences in BE values among treatments were found conceming both total yield and the weight of healthy mushrooms. Statistical analyses showed that only inoculation significantly influ. enced the healtay mushroom yield. In plots inoculated with 7: harzianun TS4 disease incidence was significantly lower after treatments with prochloraz-manganese (11.81%), B. subilis QST 713 (12.269) and B. subtilis B.38 (14.199) compared to the contol (28,16%), as wel as in plo inoculated with 7 aggressivum £. europacum ‘TTT 11.88%, 12.2% and 15.03%, respectively, in compar- ison with the contol (23.47%). Stastially significant differences were not found among the efficacy values of tested bio-fungicides based on B, subtilis and the commer- ial fimgicide prochloraz-manganese suggesting the use of| S.Milijsevig-Martié (&2)--M. Stepanov» B. Todorov - B, Duduk J. Stepanovié« E, Rekanovié «|. Pototik lnstiue of Pesticides and Environmental Protection, Bansska 31D, Belgrade, Zemun 11080, Serbia ‘email: svelana miljasevie@ pesting orgs Published online: 22 November 2016 2B, subtilis B-38 and B. subils QST 713 as good altema- tives to chemical fungicides. Keywords Biofungicide- Efficacy- Mushroom cultivation - Biological efficiency: Trichoderma spp. Introduetion Green mould, caused by various Trichoderma spe- cies, is the most serious disease in mushroom [Agaricus bisporus (Lange) Imbach] farms worldwide, Substantial crop losses have been induced by 7 aggressivum f aggressivum Samuels & W. Gams in North America and T. aggressivum £. europaeum Samuels & W. Gams in Europe (Samuels et al. 2002). In addition, many Trichoderma species with lower abilities to cause disease symptoms have also been isolated from mushroom beds, namely, Tharzianum Rife, T:virens (Miller, Giddens & Foster) Arx, 7: atroviride P. Karsten, T. Koniiigti Oudem, ete. (Kosanovié et al. 2013). Disease control in mush- room units is regularly provided by fungicide treatments. Generally, only @ few fungicides have been per- mitted for use in mushrooms, i.e. prochloraz. and ‘metrafenone in Europe and chlorothalonil, thiabenda- zole and thiophanate-methyl in North America (Grogan and Gaze 2000; EFSA 2013). Pathogen resistance 10 benzimidazoles applied to spawn was noted a decade ‘ago (Romaine etal. 2005). Decreased sensitivity of the mycopathogenic fungi from genera Lecanicillium and CCladobotryum to prochlorsz has been reported in Spein and Great Britain (Gea et al. 2005; Grogan 2008). D SpringerBesides the resistance development in pathogens, the restriction inthe use of chemical fungicides due to concern for the impact on environment and human health is i creasing rapidly (Saha et al. 2012). As an altemative, a varity of natural compounds and beneficial microorgan- isms have been tested against Trichoderma and other pathogens of edible fungi (Sokovié and Van Griensven, 2006; Regnier and Combrinck 2010; Kosanovié etal 2013), The use of microbal-hased bio-fungicides has been considered as a rational and safe altemative to chemical fungicides especially for managing soil-bore pathogens. Recently, many studies have been focused on the use of ‘microbial inoculants in biological control due to their ability to antagonize the pathogen by multiple modes of action and also to effectively colonize the thizo- sphere (Chitihunsa et al. 2007; Saha et al, 2012; Solanki et al. 2013; Khabbaz et al. 2015; Gupta and Vakhlu 2015), Beneficial and pathogenic microorganisms in rmush- oom compos, i. bacteria, atinomycetes and fungi, al temate during substrate changes from the early stage of ‘wheat straw and chicken manure as a feedstock until maturity. A succession of microbial communities is of {rcat importance in bioconversion of cellulosic biomass to a nutrienttich substrate for mushroom production (Zhang et al, 2014). Two phases are distinguished in ‘mushroom composting: Phase I, which includes changes and mineralization of organic material in thermophilic conditions, and Phase I, which consists of pasteurization ‘and subsequent conditioning. where beneficial and patho- genie microbes are treated by beat. Afterwards, beneficial ‘microbiota colonize the subsrate, and coexist with mush room mycelia while decomposing the substrate (Fergus 1964; Zhang etal. 2014), ‘Thermophilic bacteria with strong antagonistic abili- ty against pathogenic microorganisms, used as bio-pes- tices, mostly belong to Bacillus species (Fravel 2005). Members of Bacillus spp. are generally soil-inhabiting bacteria or rhizosphere epiphytes and endophytes able to antagonize pathogens by producing specific sub- stances that affect pathogen cell membranes, by induc ing resistance, and partially as competitors to pathogenic fungi for nutrient sources and space. Strong antagonistic activites of Bacillus species are due to their production of antibiotics (bacilysin, bacillomyein, iturin A, surfactin, fengycin),siderophores, volatile compounds [pyrazine and 2, 3-dimethyl-5-(1-methylpropy)], lytic ‘glucanase, chitinase, protease, lipase, amylase), and plant-growth promotion effects by D Springer EurJ Plan Patho Phytohormones (salicylic acid and indol-3-acetie acid) (Saha etal. 2012; Khabbaz et al. 2015), Attempts of the use of antagonistic bacteria ftom Bacillus genera have been made for several mycopathogenic fungi: Chaetomium olivaceum on Agaricus bisporus; (Tautorus and Townsley 1983) Trichoderma sp. on Pleurotus sajor-caju (oyster mush- room) and Lentinula edades(shitake) (Chitthunsa etal 2007); and Trichoderma pleuronum on oyster mushroom (Nagy etal. 2012), However, data on biological contol agents against Trichoderma spp. on Agaricus bisporus, especially those conceming their efficacy and impact on crop yield are generally scarce. The bio-fungicide Serenade®, based on Bacillus subtilis (Ehrenberg) Cohn, strain QST 713 and registered for use against many plant pathogens (Lahiali et al. 2013; Serrano et al. 2013; Kosanovié et al. 2013) was permited for treatment of ‘mushroom compost against 7 ageresivum f. europaeuim in France after extensive testing (Védie and Rousseau 2008). A basic principle for the success of biological ‘control program is the ability of biological contol agent to adapt tothe environment in which itis intended to be used. Therefore, the aim of this study was to identify favorable indigenous microorganisms in mushroom com- Post, and subsequently evaluate their antagonistic activity in vitro and efficacy in mushroom growing rooms against ‘wo most common and most aggressive green mould causal agents: T. aggressivum f, ewropaewm and T.harcianum. It is noteworthy, that these two pathogens affect mushrooms in different ways. T. ageressivum £ ‘eropacum is @ compost mould that can reduce crop yields significantly as well as causing spots on mushrooms while, T harzianum is a casing mould associated largely with similar blemishes on mushroom fuiting bodies. Our inter est in this study was to compare the efficacy of bio! flngicides and their impact on mushroom yield, Materials and methods Fungal species and culture conditions ‘The pathogenic fungi used in this study were obtained from a culture collection of the Institute of Pesticides and Environmental Protection, Belgrade, Serbia Trichoderma harzianum T4 was isolated from diseased fruiting bodies of Agaricus bisporus in 2008, and Trichoderma aggressivum £, europaeum T77 from mushroom compost in Serbian mushroom farms inEur Plant Patol 2010. Both fungi were previously identified based on ‘morpho-physiological characteristics and ITSI/ITS4 se- uence analyses (Kosanovié et al. 2013). Fungal cul- tures were maintained on potato dextrose agar (PDA) at 4 °C. For inoculum preparation, agar discs of fungal isolates were grown on PDA for three deys at 22°C. For in vivo trials, conidia from three-day old cultures of both fungi were flooded with 10 ml of sterile distilled water and Tween 20 (v'v 0.01%), followed by filtration through double layers of cheesecloth. Conidia concen- ‘ration used in @ mushroom growing room as inoculum was set at 10° conidiav/ml and the vol- lume used in the trials was 143 milm? of casing soil for each pathogenic fungi [bolation and identification of antagonistic bacteria. For the compost preparation, raw wheat straw was mixed with manure and gypsum, wetted and stacked in a heap for 21 days ~ aerobic fermentation (phase 1) The heep was mixed and wetted on the 3rd, Sth and 14th day with the inside temperature rising up to 60 °C after 7 days. On day 15, the partially composted substrate was ‘moved into a facility for heat treatment (phase I) that included warming up (20 h), pasteurization (66 °C for 10h) and conditioning (50 °C for approximately 6 days). The substrate was then cooled and spawned with A. bisporus. Characteristics of the compost used in the study were as follows: nitrogen content 2.06%; ammonia content 7.5%; PH value 7.5; and relative humidity 66°. Isolation was performed from a sample of mushroom compost, provided by Uta & Co (Vranovo, Serbia), during its phase II (on 21st day), before spawning. A sample of 10 g of compost from the second phase of composting ~ heat treatment (including warming up, pasteurization and conditioning) was mixed with 100 mi of sterile distilled water and several serial dilu- tions were spread onto nutrient agar (NA) plates and incubated at 30 °C for 48 h, The subcultured single colonies were subjected to preliminery characterization by microscopic appearance, Gram reaction and catalase test. The bacterial strains were identified based on the analyses ofthe 16S rDNA sequence. A PCR assay with the universal primers 27F (S'- AGA GTT TGA TCC TGG CTC AG-3') and 1541R (5-AAG GAG GTG ATC CAG CCG CA-3') was performed for amplifica- tion of the bacterial 16S rDNA (Edwards et a. 1989; Lane 1991), Each 25 uL reaction mixture consisted of 1 uL of 20-fold diluted colony suspension, 1 x PCR Master Mix (Fermentas, Vilnius, Lithuania) and 0.4 4M of each primer. A DNA-free reaction mixture was employed asa negative contol. Amplification was dane by initial denaturation at 95 °C for 7 min, followed by 35 cycles of denaturation at 95 °C for 1'min, primer annealing at 37 °C for 2 min and extension at 72 °C for 6 min wit the final extension at 72.°C for 7 min. Six microliters of PCR products were separated by electro: phoresis in 1% agarose gel, stained with ethidium bro- ide and visualized with a UV transilluminator. amplicons were sequenced in forward direction with the 27F primer. Sequencing was performed by @ commercial service (Macrogen Inc., Korea). The obtain- ed sequences were assembled using Pregap4 fom the Staden program package (Staden etal, 2000). In vitro antagonistic activity Dual culture assay, described by Sakthivel and Granamanickam (1986), was used to test the antagonis- tic activity of numerous bacterial strains agsinst two Trichoderma species. Fungal mycelial plugs (10 mm diameter) from three-day old cultures were placed on one side of PDA plates, and « loopful of bacteria roman ovemight culture was streaked 3 em away from the edge of the same plate, Plates inoculated only withthe test pathogens served as controls. All plates were incubated at 22 °C for three days. The assay was conducted with three replicates per treatment and repeated twice. The zone of inhibition was recorded as the distance between the fungal pathogen and bacterial antagonist after three days. The percentage growth inhibition (PGI) was cel- culated using the formula: PGI\%6) = (KR-R1)/KR x 100 where: KR is the distance (measured in mm) from the Point of inoculation to the colony margin on contol dishes, and R7 is the distance of fungal growth from the point of inoculation tothe colony margin on treated dishes in the antagonist’s direction (Korsten and De Jager 1995). Influence of Bacillus subrilis sain B-38 on mushroom ‘mycelial culture in compost ‘Ten spawn grains covered with A. bisporus AIS (Sylvan, Hungary, 2R,) were placed at the bottom of D Springerach sterilized culture tube (160 x 25 mm) and then filled with 30 g of substrate (pasteurized - second phase ‘compost) previously treated with 150 il of B. subtilis strain B-38 suspension adjusted t0 10" CFU ml'', by spraying, The tubes were closed with sterile cotton plugs and incubated upright at 24 °C in the datk. Test tubes filled with untreated compost served as a control (Salar and Aneja 2007). The experiment was conducted twice in three replicates. To estimate the influence of 2. subtilis strain B-38 on the growth rate of mushroom mycelia, growth of A. bisporus mycelia was recorded daily (fortwo weeks) after the intial phase of 7 days and expressed in millimeters per day. Tests in mushroom growing room Crop details Mushroom substrate was provided by the compost pro- ducer Usa & Co,, Vranovo, Serbia, Paste boxes sized 0.340 > 0.215 « 0.130 m (1x w* A) were filled with 2 kg of compost spawned with 0.7% A. bisporus AIS (Sylvan, Hungary, 2R;). The boxes were placed in an experimental mushroom growing room and incubated at 24°C. After 18 days, compost was cased with a 40 mm thick layer consisting of black peat casing soil Terahum (Treset 4.0.0, Veliko Gradite, Serbia) amended with limestone (1.4%, Tara, Dobanovei, Serbia) and stri- ized by peracetic acid 1.2% (15% Pera S, MidraEko, Belgrade, Serbia). Mushroom bed area was disinfected with peracetic acid solution 0.09 Lm”? (0.13 Lin 10 L H,0), Casing was regarded as day 0. The substrate was then incubated at 21°C for 8 days (case-run), and ait temperature was after tht reduced 10 17 °C Inoculation Trichoderma harzianum 54 isolate was obtained from casing soil while T. ageressivum £. europaeum TTT isolate originated from compost (Kosanovié et al. 2013) and therefore inoculations of plots with these ‘two fungi were at different stages: ( Taggressivum f. europeaum isolate T77 was added ‘one day after the spawned compost was placed in boxes by pipetting its suspension (10° conidiaim! Per m* ie. 10:ml of the conidia suspension per plot) ‘on the inside walls at the edges of mushroom beds (Helen Grogan, personal communication). D springer Eur Pant Patol (i) T. harzianum T54 was artificially added by spraying (10° conidiaiml per m?) the casing sur- face by a hand sprayer 48 b after biofungicide! fungicide application on day 6. Each plot was inoculated with 10 ml of appropriate conidial sus- pension (Kosanovie etal. 2013), Anuifengal treatments Efficacy and biological efficiency (BE) of a potential antifungal agent B, subtilis strain B-38 against T. harzianum and 7. aggressivum f. europacum was. evaluated in comparison with the commercial fungicide prochloraz-manganese and the biofungicide based on B. subtilis (Table 1.) Treatment with the B-38 strain was prepared from an overnight culture ofthe bacterium inoculated into nutrient broth and incubated at 30°C on orbital shaker for 72 h. The concentration of bacterial suspension was adjusted to $x 10" CFU mI” and confirmed by the plate count technique. The inoculated and uninoculated boxes that received no bio-fungicide/ fungicide treatment were used as control treatments ‘Two trials with three replicates per treatment were con- ducted at the same time, The plots were arranged in a ‘completely randomized block design. Due to notable variability of replicates in both trials, the results were analyzed as one tral with six replicates. The treatments used in the experiments are given in Table 1 Impact on yield and disease control ‘The efficacy evaluation of the bio-fungicidesfungicides was based on green mould disease incidence and on productivity data obtained during all three flushes. The ‘mushrooms were hand-picked in three Ashes over a 48-day period and examined for green mould symp- toms, spots and lesions on fruiting bodies of A. bisporus. The first flush was harvested from day 18-22, the second flush from day 23-36, and the third from day 37 until day 48. The harvested mushrooms ‘were classified into two groups based on the presence of green mould symptoms as either healthy or infected with Trichoderma spp., and then weighed. Disease in- ccidence was expressed as a percentage value of fruiting bodies with symptoms compared to those without symptoms. Bio-fungicideifingicide efficacy (E) in the control of Trichoderma spp. was calculated by AbbottsEur J Pant Patho! Table1 Fungicidevatitungal agents and application rates ‘Trade name’ Manuficrarer Octave WP? Bayer, Germany Procloraz-manganes: 00 mg L* Serenade® WP/AgmQuest, Aacilus subs QST 713 15.7% Canada G.13 * 10! CRU ge} Tested srain B38 Concentration of active ingredient Applicaton rate* Time of application 12 gin L810 Four days aftr casing and ater fir sh (on 20th day) 8% 10°CFU mm" in 11:0 Four days afer casing actus subtils B38 10° CFU mL‘ 5 * 10 CFU min 110. Four days afer easing “perm? of mushroom bed aca formula: E (%) = [(le-ItV1e] « 100; le- disease incidence 'm inoculated control; It - disease incidence in treated samples (Gea et al. 2010). The effects of bio-fungicides’ fungicide on mushroom productivity were evaluated as biological efficiency (BE), calculated as the ratio of fresh weight of total fruiting body yield (healthy and diseased) and the weight of dry spawned substrate, and expressed as %: BE = (fresh total fruiting body yield/dry spawned substrate mass) « 100 (Chrysay-Tokousbalides tal. 2007). In addition, impact of both inoculation and treatments on the yield of healthy mushrooms was expressed as @ ratio of healthy mushroom mass and fresh total fruiting body yield, Statistical analyses ‘Data were subjected to the one-way analysis of variance (ANOVA), with a comparision of means by Fest. The ‘est was used to compare the significance of differences ‘among data showing the average values of disease inci- dence, efficacy in disease contol and biological efficiency (Gmmpact on mushroom total yield) of diferent bio-fungi- cide/fungicide treatments against T. aggressivum £, ewropaeum 177 and T: harzianum TS4 in the mushroom growing room, In addition, two-way ANOVA was con- ducted to estimate the impact of both factors - inoculation and treatments and any interaction effects of these factors on the mass of healthy mushrooms. In all analyses, the level of significance was atleast P< 0.05 (Sokal and Rohlf 199), Statistical data analysis was performed using the software Statistica for Windows 6.0 (Stat Soft Italia, 1997), Results Isolation and identification of antagonistic bacteria Approximatelly 50 bacterial isolates were obtained from Phase Il of compost preparation process and tested in vitro against T: harzianum and T. aggressivum f ‘europaeum. Three strains: B-34, B-37 and B-38, exhib- ited significant activity in Trichoderma spp. growth inhibition. These isolates were proved to be Gram- positive and catalase positive in preliminary tests and were identified as Bacillus subtilis (Ehrenberg) Cohn according to their 16S rDNA sequences deposited in the NCBI GenBank under the accession numbers: KT948078, KT948079 and KT948080 (for strains B-34, B-37 and B.38, respectively). In vitro antagonistic activity Values of mycelial growth inhibition of 7 aggressivum £ ewopaeum 77 induced by strains B-34, B-37 and B38 were $0.98, 51.89 and 52.25%, respectively. The same strains induced 47.22, 47.27 and 48.08% groweh inhibition of 1 harcianum TS4, The stain B-38 was slcted for futher in vivo investigation, Inhibition zones of mycelile growth of 7 harzianum TS4 and of T. aggressivum £. europacum T77 were 10 and 12 mm, respectively. Influence of Bacillus subrilis strain B-38 on mushroom ‘mycelial culture in compost Impact on the growth rates of A. bisporus mycelia was recorded daily. After thirteen days, the growth rate of A. bisporus Sylvan A15 in substrate supplemented with 2B. subtilis B-38 was 7.95 mm day” compared to the ‘growth rate in control tubes (7.59 mm day“), However, no statistically significant differences were recorded between the B. subtilis treatment and control (F = 0.62; P-value = 0.47), ‘Tests in mushroom growing room Small brown spots of « few millimeters were found on A bisporus ftng bodies on day 16 (after easing), in plots D springerinoculated with 7 harzianum TS4 and 7 aggressivum £ europaeum 77, (11 and 31 days after inoculation, respec. tively). Larger spots and necrotic lesions ofa few centime ters were found several days later. Small white colonies were noted on the casing surface 29 days after casing in plots inoculated with both fungal species. A few days later, colonies became intensive green after a profise sporula tion. The productivity in untreated uninoculated plots was, 61.4%. Biological efficiency values (BE) in uninoculated treated plots were 60.5%, 59.2% and 55.5% ater applying prochloraz-manganese, B. subtilis B-38 and B. subtilis QST 713, respectively. n untreated plots inoculated with T-harzianum TSA BE value was 58.9% while BE values in the treatments were 63.2%, 57.4% and $8.9, respective- ly. Untreated plots inoculated with 7 aggressivum eurepaeum 177 had BE value 54.9% while in the teat- ‘ments values were 54.9%, $1.0% and 63.2%, respectively. However, no significant differences in BE values among all weatments were found (Fig. 1). Analyzing the influence of both factors (inoculation ‘and treatments) on healthy mushroom yield, only treat- ‘ment with prochloraz-manganese in plots inoculated with T. aggressivum f europaeum displayed significant differences compared to inoculated control plots provid- ing the highest yield of healthy mushrooms. No signif ‘cant differences in BE values among all treatments, were found conceming both total yield and the weight ‘ofhealthy mushrooms (Figs. 1, 2 and 3). Expectedly, the highest symptom rates were found in the control treatments inoculated with T: harcianum TS4 and T. ageressivum f, europaeum T77. Statistically sig- nificant differences were found between the inoculated control and all three bio/fungicide treatments (Table 2) ‘The respective efficacy values for prochloraz-man- sganese, B. subtilis QST 713 and B. subrils B-38 were: 58.1%, 56.5% and 49.6% in the contol of 7: harzianum TS4. Efficacy results for T. ageressivum f. ewopacum TIT were: 49.4%, 48.0% and 35.9% for these treat- ments, respectively. No statistically significant differ. ences were found among the efficacy values of al tested bio/fungicides. Discussion Previuos research had shown that Bacillus sp. recovered from mushroom compost exhibited strong activity against some mushroom pathogens such as Chaetomium olivaceum Cooke & Ells, causing green olive mould on D springer Eur Plane Patol ‘mushrooms (Tautorus and Townsley 1983) and fom ive stock manure and cotton-waste composts (Kim et al 2008). These authors tested Bacillus sp. against T harcianum, 7. koningii and T: viridescens and found respective inhibition zones of 8 12 and 6 mm, similar to the B-38 strain tested in the present study against T harsiamum and T. aggressivum f. ewopaeum (10 and 12 mm, respeetively, Tested B. subtilis strain B-38 did not inhibit the growth of mycelia of A. bisporus. Likewise, Nagy etal. (2012) found that B. subs exhibited a positive impact on the spawn run of Pleuronisostreaus in oyster ‘mushroom substrate, Contrarily, Kim et al. (2008) reported that several tested Bacillus strains slighty inhibited mycelial growth of the edible mushrooms Flammulina velutipes, Lentinus edodes, and P. ostreatus in Korea In regard to the tests in a mushroom growing room, two differen inoculation methods were used, one for cach pathogenic fungi. It is noteworthy, that these two pathogens affect mushrooms in different ways 1 harcianum affects casing and later on produces symp. toms on fruiting bodies influencing their quality and Yield. Therefore, inoculation was performed by spraying ‘the casing asit would occur ina natural infection, On the other hand, T ageressivum firstly infects compost be- sides the fruiting bodies and casing, which occurs late Also, the first symptoms are visible in the compost and largest losses occur in the compost which is why inoc- ulation of the compost was performed instead of spraying he casing. The novel method used inthis study “on the inside walls at the edges of mushroom beds simulated what naturally occurs when T: aggressivien appears in mushroom beds. In addition, statistical anal yses of the influence of inoculation on healthy mush- oom yield showed significant differences between in- oculated and uninoculated conttol plots indicating that the pathogen inoculation methods were suit- able for both fungi. It also needs to be highlighted that the inoculation with the different pathogens occurred at different times. Considering that T. ageressivum is a compost mould, inoculation was conducted shortly after spawning while T. harzianum, being a casing mould was inoculated after casing. For above mentioned reasons, antifungal treatments all o¢- curred after casing, nour tals, no significant differences in yields were found among treatments with fungicide and bio- fungicides implying that bio-fungicides did not haveEur Plane Pathol Fig. 1 Efsc of bi'fungicides on biological efficiency (BES) of ‘Agaricus bisporus in inoculated and uninoculated plots; data ore ‘means of six repliates + SE, standard enor of means; BES = ratio negative influence on the mushroom yield. Nagy etal (2012) found that B. amyloliguefaciens strain, effective in Vivo against 7: pleurotum, the causal agent of green ‘mould on oyster mushroom, improved cop yield in unin- culated bags of oyster mushrooms by 10%. Conta, Kosinovié etal. 2013) found thar & subifis QST 713 Aeereased mashroom yield in comparison with both con- trols (urinoeulated and inoculated with 7! harcianum) and the prochloraz fungicide treatment. As 7. harzianum is ‘mainly found in the casing layer, itis expected that rochloraz manganese aplied through a casing sil would provide the lowest mushroom loss. However, improve- ‘ment of crop yield after application of biocontrol agents ‘has been reported. Tautorus and Townsley (1983) found that Bacillus sp. added to compost inoculated with CChaetomiun oltvacewn significantly entanced the myce- lial development of A, bisporus, mushroom yield and ‘efficacy in olive groen mold conto ‘ofthe fesh weight of total mushroom yield and the weight of dry spawned subsite; SEDs, standard eror of differences = 66.68.cF, degrees of freedom = 24; F = 0.63; Pvalue = 0.79 To highlight the impact of both factors and any interaction effects of inoculation and treatments on the mass of healthy mushrooms, two-way ANOVA was conducted. This analyses showed that only inoculation significantly influenced the healthy mushroom yield. As expected, the highest value of healthy mushroom yield was found in untreated control, and the lowest in inoc- ulated untreated plots with both Trichoderma species. Significant influence of treatments and interaction effect of both factors were not found, All bio/fungicide treatments showed satisfactory ef ficacy in controlling green mould incidence compared to inoculated control. Although, the lowest disease inci- dence was found in the treatment with the fungicide prochloraz-manganese, compared to both bio-fungi- cides, no statistically significant differences between these three treatments were found. These results implied that bio-fungicides could serve as a harmless alternative 2 SpringerTherma ly moteurs) Fig. 2. The percentage of healthy fruiting bodies of Agaricus ‘opores weight inoculated with Trichoderma harsianum TS, Data are means of six replicates; SE, standard err of means, Two-way ANOVA analysis: a within inflence of inoculation SEDs, sandand enor of differences = 0105; df. degrees of fee- dom = 1; F = 5.54; P-value = 0.03; b within influence of to synthetic Fungicides in mushroom production, espe- cially considering rapid cropping in mushroom growing facilities. Moreover, these naturally derived agents could be combined with chemicals in integrated strategy for management of green mould in button mushroom. In addition, Kosanovié etal. (2013) found a higher efficacy of prochloraz-manganese in a mushroom growing room after inoculation with the T: harzianum isolate T91 (72%) compared to the results in our experiments using the other T- harzianum isolate TS4 (58%). This may be attributed to different in vitro susceptibilities of the T. harzianum isolates used in these two studies to prohloraz-manganese. Namely, in vitro experiments of Kosanovié etal. (2013) showed that the isolate T91 had 4 10-fold lower EDso value (effective dose that reduces D springee Eur J Pant Pathol twcatments: SEDs, standard enor of difeences = 001; df degrees of fresdom = 3; F= 0.11; P-value = 0.96; e within influence of both inoculation and different reaments: SEDs, standard eror of slferences = 0.03; df, degrees of freedom = 16; F = 287, Ps 50% of colony growth compared to control) of the fungicide than the isolate T$4 used in the present study. Our study also showed that both tested bio- fungicides and prochloraz-manganese exhibited higher efficacy against 7 harzianum TS4 than against T. aggressivum f. europacum 177, This research also showed that composting material represented a valuable source of antagonistic microor- ganisms with a potential for use in biological control of green mould in button mushroom production, Also, results need to be verified at larger scale, both in com. post facilities and mushroom growing rooms. Further research will focus on discovering other strains of the Bacillus genus that occur in mushroom composting ‘material which could be used as an alternative toEur 1 Pant Patol Fig. 3. The percentage of healthy fruiting bodies of Agaricus bisporus weight inoculated with Trichoderma aggressivum £ ‘ewopacin T77, Data are mens of six replicates; SE, standard ror of means. Two-way ANOVA analysis: a within infucnce of ‘noculation: SEDs, standard eror of differences ~ 0.09 df, degrees of feedom = 1; F = 835; P-value = 001; b within influence of Table 2 Disease incidence on Agaricus bisporus fing bodies inoculated with Trichoderma harzlanum TS4 (SEDs, standard or of itferences = 0.02; df, degress of feedom = 3; F = 2.1; Treats treatments: SEDs standard enor of differences = 0.01; df degrees of fieedom = 3; F = 157; P-value = 0.35; € within influence of both inoculation and different weatmens: SEDS, standard err of uifferences = 0.03; df, degrees of fedom = 16; F = 249; P- value = 003 Pvalue = 0.18) and Trichoderma ageressivum f. europacum 177 (SEDs, standard enor of differences = 001; df, degrees of ie- dom ='3; F= 11.3: P-value = 0003) “Treament ‘Trichoderma harcianum 54 Trichoderma ageressivum f. europacum TT? Mean! SE Mean SE Inoculated contol 28.168 1530 23474 22 Bacillus subiis B38 1419 750 1503 294 Bacillus subtiis QSTH3 12266 508 1226 244 Prochloraz-Mn sib 350 11886 345 "Data are means of six epicaes = SE, standard eror of means * Means within the same column followed by the same leter are not significantly diferent (P < 0.5) D Springer‘commercial fungicide, studies on the application timing, Possible use of mixtures of antagonisis, and combined application of fungicides and biocontrol agents under controlled-environment conditions in mushroom grow- ing rooms. Acknowledgements This study was funded by the Ministry of Education, Science and Technological Development a the Repub lic of Serbia, grant number TR3103. References Chittihunsa, T., Bangeekhan, F., Wongsamitkul, N., & Subsomboon, T2007). Screening of Bacillus sp. suppres ing the infection of Trichoderma sp. in mushioom euliva- tion. KMITL Science and Technology J, ASD. 19-29 CChrysayTokousblides. M., Kasaias, M. ., Philippoussis, A, & Diamantopoutou, P. (2007). Selective fungitoneity of famaxadone, tebuconazole and trfloxysirobin between Vertcillium fungicola and Agaricus bisporus. Crop Protection, 26, 469-415, Edwards, U., RogallT. Bldcker, H., Emde, M., & Batger EC. (1989), solaton and direct complete nuceotide detomina tion of entre genes. Characterization of a gene coding for 16S ribosomal RNA, Nucleic Alls Research 1719) 7943 7855 European Food Safety Authority. Reasoned opinion on the mode ification of the exiting MRLs for metrafenore in various ‘crops. EFSA Joural 2013:1(I}: 3075.30 pp. Ferzus, CL. (1964). Thermophilic and thermetolerant mold and acinomyoetes of mushroom compost during peak heating Mycologia, $62), 267-284 Fravel D. R. (2005), Commercializaion ane implementation of biocontol. Annual Review of Phropathologh 43, 337-389, Gea, FJ, Navarro, M J, & Tello, J.C. (2005), Reduced ses tivity of the mushroom pathogen Veriilium fungicola to prochloraz- manganese in Vio. Mycological Research, 109, 7A1-745, Gea. FJ, Tell, J, & Navarro, M, (2010). Eicacy and effect on ‘eld of different fungicides For contol of wet bubble disease ‘of mushroom caused by the mycoparssite Mivogone pericisa. Crop Prowecion, 29, 1021-1025, Grogan, H. M. (2008), Challenges facing mushroom disease con- tol in the rwenty-first century. IJ. 1 Lelley & J, Buswell (68), Proceeding ofthe sith international conference on ‘mushroom biology and mushroom produets, 2th September « 3rd October 2008 (pp. 120-127). Bonn, Germany WSMBMP. Grogan, H. M. & Gaze, R.H, (2000). Fungicde resistance among ‘Cladobotryum spp, ~ causal agents of cobweb disease ofthe ‘edible mushroom Agaricus bisporus. Mycological Research, 10413), 357-364, Gupta, R, & Vakhlu, J. (2015), Naive bcilasamoliqueficiens 'W2 as a potential biocontrol for Fusarium axysporum Rt ‘using corm rot of Crocus Sativus. European Jounal of Plant Pathology, 143, 123-131 D springer Eur Pant Pathol ba. S. Zhang, L., Ciceres L.A. Sumarah, M., Wang. Abbasi, A: 2015). Characterization of antagonistic Bacillus and Pseudomonas strains or brocontol potential and suppression of damping-off and root rot diseases, “Annals of Applied Biology, 166, 486-471. ‘Kim, W.G., Weon, H.¥.,Seok, S.J, Les, K, H. (2008) fe vito antagonistic characteristics of bacilli isolates against Trichoderma spp. and three species of mushrooms Mbcobiolg: $68), 265-269, Konten .,& De Jager, ES. (1995). Mode of action of Bacillus subtilis for conteol of avocado postharvest pathogens SAAGA Yearbook. 18, 124-130, Kosanovig, D., Pototnk, I, Duduk,B., Vukojevi, J, Sai, M,, Rekanovie, E., & Milijaievié-Martié, 8. (2013), Trichoderma species on Agaricus bisporus farms in Serbia and their biocontol. Annals of Applied Biology, 163, 218 230. Lala, R. Peng. G., Gossen, B.D., MeGregor, La Yu FQ. Hynes, RK. Hoang, SF, MeDonad, MR, & Boyetchka, S. M. Q013), Evidence thatthe biofungivide serenade Bacitus subtilis) suppresses Chubrot on canola via anti sis and induced bos resistance. Phyxopathology. 103,245 254. ‘Lane, D. (1991), 168238 FRNA sequencing. In M. Goodfellow (Ed). Stackebrandt E (pp, L1S-176), New York, NY. Nace sd techniques in bacterial systematics. Join Wiley & Sons, Ine Nagy. A., Manezinger, L.. Tombicz, D, Hatvani, L., Gy, J Ant... Sajben, E. Vaguallgy, C., & Kreis, L. (2012) Biological contol of oyster mushroom green mould disease bby antagonistic Bacilus species. Biological Control of Fungal and Bacterial Plant Pathogens 1OBC-WPRS Bulletin, 78, 289-295. Regsier.T, & Combrinck, S. (2010). fn vivo and in vivo serecning ‘of essential oils forthe contol of wet bubble disease of Agaricus bisporus. South African Journal of Botany, 76, 681-685, Romaine. C. P, Royse, D. J., & Schlagnhaufer, C. (2005) Superpathogente Trichoderma resistant to Topsin M found tm Pennsylvania and Delaware. Mushroom News. $3, 6-9. Saha, D., Purkayastha, G. D., Ghosh, A, Isha, M..& Saha, A (@0i2) Isolation and characterization of two new Bails subs strains fom the shizosphere of eggplant as potential biocontol agents. Journal of Plant Patholog: 941). 109- is. Sakthivel, N., & Gnanamanickam, S. S, (1986). Toxicity of ‘Pseudomonas fuorescens towards rice sheath rt pathogen ‘Acrociindrum oryzae. Current Science 5, 106-10? Sala, RK. & Ancja, K. R. (2007). Sinifeanceofthemnophilic angi in mushroom compost preparation: effect on groweh and yield of Agaricus spars (Lange) sing. Journal of Agriculure and Technology, 2), 241-25 Samuels, G. J, Dodd, S. L, Gams, W, Castebury, Le Ay & Penni, ©. 2002). Trichoderma species associated with tbe ‘green mold epidemic of commercially grown Agaricus Disponss. Mycologia, 94, 146-170, Serrano, L., Manker, D. Brandi, F.& Cali, T 2013). The use of acts subilis QST 713 and Baclus pum QST 280825 Dotectantfingicides in conventional application programs for black leaf steak contol, ISHS Acta Honicaltree 986 VII Intemational Symposium on Banana: ISHS-ProMusaEar Pant Patho Cream on Basen and Panis: TovanisSusuinble Sade, R. Beal, KF. & Bonfield, J. K. (2000), The Staden Global Production and Improved Use Sola, R. Rs & Roblf FJ (1995) Biometry: The Principles and Prucice of Statistics in Biological Research edn. New York, NY: W.H, Freeman and Comper, Sokovig, M.,& Van Griensven, LJ LD. (2006). Antimicrobial activity of essential oils and ther components against the three major pathogens of the cultivated buton mushroom, Agaricus bispores. European Journal of Plant Pathology 116, 211-224. Sola, M. K Singh, R. K,, Srivasava, S, Kuma, 8, Kashyap, P. La de Srivastava, A. K. (2013)” Characterization of antagonisti-potential of two Balls stains und thet bio, contol setivity agains Rhzoctona solani tomato, Journal 2f Basic Microbiology, $3.19, Package, 1998. Methods in Molecular Biology, 132, 11S. 130, Tautorus 7. E., & Townsley, P.M. (1983), Biological conto of olive green mold in Agaricus bisporus culation Applied and Environmental Mirobiology, #50), 311-815, Velie, Ro, & Rousseau, T. (2008). Serenade biofungcide: une innovation mjeure dans tes champignonniees fangaises our later contre Trichoderma aggressiwum, agent de ln ‘moisissure verte du compost. La Let dy CTC: 21, 1-2 ‘Zhang, X. Zhong, Y., Yang, 8. Zhang. W, Xu, M. Ma, A. Zhuang, Gu Chen, Gu & Liv, W. 014). Diversity and dynamics of the microbial community on decomposing ‘wheat straw during mushroom compost production Bioresource Technology, 170, 183-195, D Springer