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Article

Altering Microbial Communities in Substrate to Stimulate the Growth of Healthy Button Mushrooms

by
Svetlana Milijašević-Marčić
1,
Ljiljana Šantrić
1,
Jelena Luković
1,
Ivana Potočnik
1,*,
Nikola Grujić
2,
Tanja Drobnjaković
1 and
Dejan Marčić
1
1
Institute of Pesticides and Environmental Protection, Banatska 31b, 11080 Belgrade, Serbia
2
Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(7), 1152; https://doi.org/10.3390/agriculture14071152
Submission received: 14 June 2024 / Revised: 4 July 2024 / Accepted: 10 July 2024 / Published: 16 July 2024
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
Green mould, caused by Trichoderma aggressivum, is one of the major fungal diseases of button mushrooms. The main problems in chemical disease control include a lack of effective agents, the occurrence of pathogen resistance to pesticides, and the harmful impact on the environment. In an attempt to find a solution, the interaction between two beneficial microorganisms, Bacillus amyloliquefaciens B-241 (an antifungal agent) and Streptomyces flavovirens A06 (a yield stimulant), was investigated in vivo. The synergy factor (SF) was calculated as a ratio between the observed and expected impact on the yield or efficacy of disease suppression after artificial inoculation with T. aggressivum. The highest control of T. aggressivum was achieved by joint application of the two beneficial microorganisms. The additive interaction between microorganisms in efficacy against the pathogen was revealed. The largest yield was obtained in mushroom beds sprayed with the two beneficial microorganisms combined (B-241 80% and A06 20%). Regarding the impact on the yield, synergistic interaction between the two microorganisms was confirmed (SFs were 1.62 or 1.52). The introduction of optimized microbial combinations could create new possibilities for biorational edible mushroom protection, with improved yield and quality and reduced risks to human health and the environment.

Graphical Abstract

1. Introduction

Cultivation of the button mushroom [Agaricus bisporus (Lange) Imbach] is a demanding technology. The major cost inputs in the mushroom industry include the preparation of selective substrates and pest and disease management [1,2]. Commercial button mushroom strains are sensitive to various fungal pathogens. Since the mid-1980s, green mould (the causal agent, Trichoderma aggressivum Samuels & W. Gams) has been a constant threat to mushroom production worldwide, leading to yield losses of up to 100% [3,4]. This fungal pathogen rapidly spreads on the substrate through the force of its cellulolytic activities. It accommodates growing conditions that are similar to those of edible mushrooms [5], especially because the pathogen and host belong to the same kingdom of fungi. Moreover, the intensive production of substrate components, straw and poultry manure, often results in a disturbed genuine microbiome that is no longer able to compete with pathogenic organisms.
Synthetic chemical fungicides for disease control damage the mycelia of edible mushrooms, causing decrease in yield [6]. Their residues are present in harvested mushrooms as well [7]. The major problem nowadays is the absence of effective fungicides as pathogens become resistant to chemicals [8,9,10]. Furthermore, the harmful effects of chemical fungicides on the ecosystem demand a different approach to disease management, which includes biopesticides. Bacillus-based crop protection products have been predominantly microbial biopesticides due to the ability of Bacillus species to survive under adverse conditions and their different modes of action in the suppression of phyto- and mycopathogens [11,12,13,14]. Members of actinobacteria are also involved in favourable interactions with different cultivated plants, promoting growth and reducing disease incidence [15,16,17,18,19]. As disease control agents, they are particularly interesting due to their capacities for wide-ranging and abundant production of antimicrobial compounds [18,19]. Streptomycetes are also designated as plant growth-promoting (PGP) actinobacteria, owing to their important feature of being able to produce various enzymes during their metabolic processes [20].
Commercial mushroom production is closely linked to a controlled succession of fermentation stages, where microorganisms decompose raw materials, diminishing competitive fungi and promoting fructification [2,21,22]. In our previous studies, selective effects on green mould disease control and yield improvement have been confirmed by the bacterium Bacillus amyloliquefaciens, strain B-241, and actinobacterium Streptomyces flavovirens, strain A06, both indigenous and originating from mushroom compost [14,18,23]. The commercial cultivation of A. bisporus relies on complex relationships among a variety of microorganisms inhabiting the substrate ecosystem [2]. Modifying the microbiome in compost and/or casing materials seems to be a promising solution for stimulating healthy mushroom production. Therefore, this study aimed to investigate the mutual relationships between two beneficial microorganisms after their simultaneous application and their overall effect on disease occurrence and mushroom yield.

2. Materials and Methods

2.1. Mycopathogenic Organism and Culture Conditions

The pathogenic fungus Trichoderma aggressivum f. europaeum T77 was isolated from a mushroom substrate (Lisovići farm, Serbia) in 2010. Pathogen T. aggressivum f. europaeum T77 was previously identified based on its morpho-physiological characteristics and ITS1/ITS4 sequence analyses [Accession number: KC555186 (accessed on 30 January 2013) in Genbank (https://ncbi.nlm.nih.gov) [24]. Fungal culture was preserved on potato dextrose agar (PDA) at 4 °C in the culture collection of the Institute of Pesticides and Environmental Protection, Belgrade (Zemun). The inoculum was prepared by growing the fungal colony on PDA for four days at 22 °C. The colonies were sprayed with distilled water and Tween 20 (v/v 0.01%), and conidia were filtered through double layers of cheesecloth. The concentration of conidia, determined by counting on a haemocytometer, was adjusted to 106 conidia per m2 of casing soil for in vivo trials.

2.2. Beneficial Microorganisms and Culture Conditions

The beneficial microbial strains were obtained from samples of mushroom compost produced in the Compost Factory Uča d.o.o., Vranovo, Serbia, in 2015. Actinobacterium Streptomyces flavovirens strain A06 (SF A06) was isolated on the third day, while bacterium Bacillus amyloliquefaciens strain B-241 (BA B-241) was isolated from a compost sample on the 14th day of fermentation (Phase I). The bacterial and actinobacterial strains were characterized based on their microscopic and macroscopic appearance and on the analysis of the 16S rDNA sequence [accession numbers of B. amyloliquefaciens B-241: KT692730 (accessed on 1 September 2015) and S. flavovirens A06: MF962586 (accessed on 15 September 2017) in Genbank (https://ncbi.nlm.nih.gov) [18,23]. The bacterial and actinobacterial strains were maintained on nutrient agar (NA) (Torlak, Serbia) and yeast malt agar (YMA) (International Streptomyces Project Medium No. 2; Himedia, India), respectively, at 4 °C in the culture collection of the Institute of Pesticides and Environmental Protection, Belgrade (Zemun). The treatment with B. amyloliquefaciens B-241 was prepared from an overnight culture of the bacterium on NA, followed by inoculation into the nutrient broth and incubation on an orbital shaker for 72 h at 30 °C. Strain S. flavovirens A06 was grown on YMA plates for seven days, inoculated into liquid yeast malt broth (YMB) (Himedia, India) media, and incubated at 30 °C on the orbital shaker for seven days. Bacterial and actinobacterial suspensions were adjusted to approximately 109–108 CFU/mL, respectively, and confirmed by the plate count technique.

2.3. Trials in Mushroom Cultivation Chamber

The effects of the combined application of the two beneficial microbial strains on the yield and suppression of green mould disease were tested in an experimental cultivation chamber. The trials were set up to simulate the bag growth system, which is common in mushroom cultivation facilities in Serbia. The mushroom substrate was provided by the compost producer Uča d.o.o., Vranovo, Serbia. Plastic containers sized 0.285 × 0.2 × 0.140 m (l × w × h) were filled with 1.5 kg of compost and spawned with 1% mycelium of A. bisporus A15 (Sylvan, Hungary, zR1). The boxes were incubated at 24 °C. After 17 and 16 days (trials I and II, respectively), the compost was covered with a 40 mm thick layer consisting of black peat casing soil (Okrywa Torfowa Typ S, Wokas Spόlka Akcyjna, Łosice, Poland) previously sterilized by peracetic acid 1.2% (15% Peral S, MidraEko, Belgrade, Serbia) and amended with limestone. Casing time was regarded as day one. The substrate was than incubated at 21 °C for 8 days (case run), and ambient temperature was reduced to 17 °C to induce fruiting body formation. The inoculation of plots with the mycopathogen T. aggressivum f. europaeum T77 was conducted by applying the fungal conidia suspension (106 conidia per m2 bed area, i.e., 10 mL of the suspension per experimental unit) onto the inside walls of each mushroom cultivation box at spawning time.
To evaluate the efficacy of the beneficial bacterium B. amyloliquefaciens B-241 and actinobacterium S. flavovirens A06 in the suppression of the green mould agent, their impact on mushroom yield, and their relationship, treatments were applied in six split applications on the casing soil at seven-day intervals beginning from the first day of casing time. Experiments contained uninoculated bed areas and artificially inoculated bed areas, with five different treatments in each group. The treatments for inoculated and uninoculated plots were as follows: (1) B. amyloliquefaciens B-241 at 100% of the standard rate, 1 × 109 CFU g−1 in 1 l H2O per m2; (2) B. amyloliquefaciens B-241 at 80% of the standard rate, 8 × 108 CFU g−1 in 1 l H2O per m2; (3) S. flavovirens A06 at 20% of the standard rate, 2 × 107 CFU g−1 in 1 l H2O per m2; (4) B. amyloliquefaciens B-241 at 80% of the standard rate (8 × 108 CFU g−1) supplemented with S. flavovirens A06 at 20% of the standard rate (2 × 107 CFU g−1) in 1 l H2O per m2); (5) S. flavovirens A06 at 100% of the standard rate, 1 × 108 CFU g−1 in 1 l H2O per m2. Bed areas sprayed with water, served as the control for each group. The plots were set up in a randomized block system with six replicates per treatment. The experiment was conducted twice. Trial I was conducted from May to July 2023, while trial II was carried out from September to November 2023.
The effects of different treatments and their combinations were assessed for efficacy in controlling the green mould agent (based on disease incidence, I) and for mushroom productivity (biological efficiency, BE). The mushrooms were collected until the end of the second flush, classified as healthy or diseased and weighed. Disease incidence was represented as the percent of diseased fruiting bodies out of the total fruiting bodies. The efficacy (E%) of beneficial microorganisms in controlling of T. aggressivum f. europaeum T77 was calculated by Abbott’s formula, E = [(Ic − It)/Ic] × 100, where Ic represents the disease incidence in inoculated untreated plots, and It signifies the disease incidence in inoculated treated plots [25]. Mushrooms were hand-picked for 35 days in the first in vivo trial, while harvesting lasted 32 days in the second trial. The influence of treatments on button mushroom yield (biological efficiency, BE) was calculated as the ratio of fresh weight to total fruiting body yield (healthy and diseased) and weight of the dry spawned substrate and was expressed as percentages [25,26]. The weight of the dry spawned substrate was determined by the oven-dry method [27].
Synergism between the beneficial bacterium B. amyloliquefaciens B-241 and actinobacterium S. flavovirens A06 was estimated based on data from in vivo trials. The synergy factor (SF), as a ratio between the observed and expected effects, was calculated using Limpel’s formula [28], Ee = (X + Y) − (XY)/100, where Ee signifies the expected effect from the additive responses of two agents (bacterium and actinobacterium), while X and Y express the percentage of inhibition caused by the bacterium or actinobacterium. Accordingly, SF > 1 signifies a synergistic reaction, SF < 1 points to an antagonistic reaction, and SF = 1 indicates an additive reaction.

2.4. Statistical Analyses

The data obtained from in vivo trials in a mushroom cultivation chamber were submitted to a one-way factor analysis of variance (ANOVA). The F-test was used for the mean separation to compare the significance of differences among data on the average efficacy of different single application rates or combinations of two beneficial microorganisms (bacterium B. amyloliquefaciens B-241 and actinobacterium S. flavovirens A06) against the pathogen (T. aggressivum f. europaeum T77) and their impact on yield (biological efficiency). The level of significance was evaluated at p < 0.05 in all tests [29]. The software Statistica for Windows 6.0 [30] was used for the analysis of the statistical data.

3. Results

3.1. Efficacy in Disease Control

Symptoms associated with green mould disease (brown discolouration on mushroom fruiting bodies) were recorded on the 16th day in trial I and on the 17th day in trial II in plots inoculated with T. aggressivum f. europaeum T77. Several days later, colonies of green mould appeared in the inoculated plots in both trials.
The efficacy values (E%) of different treatments for suppressing symptoms of green mould disease are given in Figure 1 and Figure 2. The highest control of the mycopathogen T. aggressivum f. europaeum T77 was achieved during the joint application of two beneficial microorganisms in both trials (61.93 and 69.37%, respectively). Significantly lower efficacy against the fungal pathogen was found after individual applications of beneficial microorganisms at full standard rates, either B. amyloliquefaciens B-241 (trial I 53.07; trial II 56.7%) or actinobacterium S. flavovirens A06 (trial I 51.93; trial II 60.13%). The least effective treatments in preventing disease symptoms were recorded at reduced rates of singular applications of beneficial bacterium 80% (trial I 37.19; trial II 41.68%) and actinobacterium 20% (trial I 34.88; trial II 41.6%).
The results based on SF calculations (Table 1) revealed additive interactions in efficacy against the pathogen as the SF values were approximately equal to 1 in both trials. The relationship between bacterium and actinobacterium was therefore designated as an additive.

3.2. Mushroom Productivity

The influence of biological agents on productivity is shown in Figure 3 and Figure 4. The weight of dry spawned substrate was 475.5 g in trial I and 483.3 g in trial II. In trial I, the biological efficiency values ranged from 127.38 to 145.76, while they were between 125.66 and 143.43 in trial II. Maximum yield was achieved in plots treated with the two microorganisms combined (B. amyloliquefaciens B-241 and S. flavovirens A06), both uninoculated and inoculated, in both trials (Figure 3 and Figure 4). It is noteworthy that mushroom yields did not differ between different treatments. The individual application of the actinobacterium S. flavovirens A06 improved mushroom yield by 0.1–2.2% in plots without pathogenic fungi and by 1.6–3.6% in inoculated plots. The mixture of both beneficial microorganisms enhanced button mushroom production by 4–5.5% in uninoculated plots, while the yield was improved by 5.1–5.2% in inoculated plots. Lower productivity was noted in uninoculated units when treated with bacterium B-241 at the rates of 80 or 100% in both trials and in inoculated plots treated with the bacterium at the rate of 80% in trial II. Regardless of the differences in mean values of biological efficiency, no statistically significant difference was found in total mushroom yield between the treatments or variants.
Regarding the impact on yield, SFs between the beneficial bacterium B. amyloliquefaciens B-241 and S. flavovirens A06 were 1.62 or 1.52 in respective trials I and II, suggesting a synergistic interaction between the two microorganisms (SF > 1).

4. Discussion

Based on previous studies [18,23], strains B. amyloliquefaciens B-241 and S. flavovirens A06, both isolated from phase I (fermentation) of the composting process, were selected for research to provide a practice-based understanding of the microbial community–pathogen relationships. The influence of two beneficial microorganisms on efficacy in green mould disease control and mushroom productivity (biological efficiency) was investigated when each of them was individually applied at standard application rates (100%) or mutually combined at a proportion of 80:20%. The effects of their respective lower application rates (80 or 20%) in singular treatments were examined to calculate and determine their possible mutual relationships (additive, synergistic, or antagonistic). The highest efficacy against T. aggressivum was achieved by mixing the beneficial bacterium B. amyloliquefaciens B-241 and actinobacterium S. flavovirens A06, followed by the effects of their single applications. As expected, the lowest suppression of the pathogen was recorded when reduced application rates of both microorganisms were used. So far, interactions between bacteria and cultivated mushrooms have been reported to have either positive or negative effects depending on the bacterial strain and mushroom maturity, as both could influence the microbiome in which they coexist [31]. Many strains of Bacillus spp. have been reported so far to exhibit remarkable activities in suppressing various fungal pathogens of edible mushrooms, such as Chaetomium olivaceum Cooke & Ellis (green olive mould disease of the button mushroom) [32], T. harzianum, T. koningii, T. viridescens (green mould disease) [33], T. pleurotum (green mould disease of the oyster mushroom) [34], T. harzianum (casing green mould of the button mushroom) [24], T. aggressivum [35] and both T. aggressivum and Lecanicilium fungicola (dry bubble disease agent of the button mushroom) [14]. A previous study by Milijašević-Marčić et al. [35] showed that the native B. subtilis B-38 strain was effective in green mould disease suppression (both against T. harzianum and T. aggressivum). Also, no statistically significant differences between the efficacies of this native strain and the commercial strain B. amyloliquefaciens QST 713 or the chemical fungicide prochloraz-manganese were recorded. In addition, Stanojević et al. [14] noted that the best control of T. aggressivum f. europaeum T77 was obtained after treatment with prochloraz-manganese (80.1–88.7%), followed by B. amyloliquefaciens B-241 (51.7–67.6%). Furthermore, Kosanović et al. [24] concluded that a commercial product based on B. amyloliquefaciens QST 713 was less effective than prochloraz-manganese in T. harzianum T91 control. Moreover, this strain exhibited higher disease control compared to a biofungicide based on tea tree (Melaleuca alternifolia L.) oil. Similarly, in the current study, beneficial microorganisms used at full standard rates, B. amyloliquefaciens B-241 and the actinobacterium S. flavovirens A06, had satisfactory efficacy against T. aggressivum f. europaeum T77 when applied individually. However, when compared to their joint application at the ratio 80:20 (in favour of B. amyloliquefaciens), their singular efficacy in controlling T. aggressivum f. europaeum 77 was significantly lower.
Data on the synergy between the biofungicides and fungicides in Agaricus production system are scarce. In the current study, values of the synergy factor in assessments of disease control efficacy showed additive effects of the tested beneficial microorganisms when the two were applied simultaneously. This means that the values of individual efficacy against green mould symptoms of both beneficial microorganisms, the bacterium B. amyloliquefaciens B-241 and actinobacterium S. flavovirens A06, are simply added up in their joint application. On the other hand, the simultaneous application of both microorganisms upgraded mushroom production much more than the simple summation of both yields reached by a single application of the bacterium and actinobacterium. Kosanović et al. [24] evaluated the interactions between commercially available biofungicides, one based on B. amyloliquefaciens QST 713, another based on tea tree oil, and the synthetic pesticide prochloraz-manganese. The authors reported higher SF values for the combination of prochloraz-manganese 80% and B. amyloliquefaciens QST 713 20% (SF = 0.4) in comparison to the combination of prochloraz-manganese 80% and tea tree oil 20% (SF =0.2). Unlike the results in the present study, those relations were designated as antagonistic. However, the authors detected less antagonism when the chemical fungicide was combined with the B. amyloliquefaciens QST 713 than in combination with the tea tree essential oil. A possible explanation for the antagonistic relationship lies in the very fact that the beneficial microorganism B. amyloliquefaciens QST 713 was combined with a chemical (prochloraz-manganese) or biochemical (essential oil) fungicide, which were potentially both toxic to the beneficial bacteria. On the other hand, the synergy detected in our trials could be possibly due to a combination of two beneficial microorganisms joining forces to suppress the disease, especially since both the bacterium and actinobacterium were isolated from the same substrate during the fermentation stage of the composting process.
Regarding mushroom productivity, it was somewhat lower in treatments with B. amyloliquefaciens B-241 than in the control. It is also noteworthy that we applied treatments with the bacterium B. amyloliquefaciens B-241 and actinobacterium S. flavovirens A06 six times at seven-day intervals in the current study after our previous investigations had shown that more a frequent application of the same amount of microbial biofungicide resulted in higher yield [36,37]. However, no statistically significant influence on mushroom productivity was found between the treatments. The application of these beneficial strains implicates no harmful effects on total mushroom yield. Likewise, Milijašević-Marčić et al. [35] did not find any significant difference in mushroom productivity between the treatments with both a native B. subtilis strain B-38 and the commercially available B. amyloliquefaciens strain QST 713. Similarly, Stanojević et al. [14] also find no statistically significant difference in the total weight of healthy and diseased mushrooms between different treatments, including B. subtilis B-233, B. pumilus B-138, B. amyloliquefaciens B-241, and commercial B. amyloliquefaciens QST 713. Increased spawn run and A. bisporus yield were also reported by Potočnik et al. [4,36,37] in experiments where the B. subtilis strain Ch-13 was used to suppress T. agressivum f. europaeum T77. Opposing records were reported by Kosanović et al. [24], stating that the B. amyloliquefaciens strain QST 713 showed a negative impact on productivity compared to untreated controls (inoculated and uninoculated with T. harzianum T91) and the chemical pesticide prochloraz-manganese. Several other authors have reported different results on the effects of Bacillus species on the yield of various edible mushroom species. Namely, Nagy et al. [34] found that a B. amyloliquefaciens strain, which successfully inhibited T. pleurotum (green mould of oyster mushroom), also improved yield by 10%, while Kim et al. [38] noted a slight suppression of the mycelial growth of edible mushrooms Flammulina velutipes (Curt. Ex Fr.) Sing., Lentinula edodes (Berk.) Pegler, Pleurotus ostreatus (Jacq. Ex Fr.) Kumer.
To summarize, the strain used in the current study, B. amyloliquefaciens B-241, did not negatively affect the yield of a white button mushroom (A. bisporus). In addition, the synergy factors in repeated in vivo trials implied a synergistic interaction between the bacterium B. amyloliquefaciens B-241 and the actinobacterium S. flavovirens A06 (SF > 1). The yield elevation is possibly a result of the joint action of bacteria and actinobacteria in decomposing straw in the substrate to a greater degree and increasing nutrients for A. bisporus. Likewise, it is known that microbial biomass and dead bacterial and actinobacterial cells serve as a food source, covering up to 10% of edible mushroom requirements [39], so these two microorganisms may promote each other’s multiplication. Hence, the application of these two beneficial microorganisms into a mushroom substrate has a significant potential as a means to improve both green mould disease control and button mushroom yield. The addition of microbes that are most likely to survive all conditions during mushroom cultivation is a bonus that should also be taken into account.

5. Conclusions

Modifying the microbiome of casing soil by the joint addition of native microorganisms exhibited significant effects both in disease control and yield improvement. Both supplemented microorganisms, bacterium B. amyloliquefaciens B-241 and actinobacterium S. flavovirens A06, originating from the mushroom substrate. Maximum values regarding either pathogen suppression or mushroom productivity were found in experimental plots treated simultaneously with both microorganisms. Expectedly, lower efficacy in disease control was achieved when the two beneficial species were applied individually at their full application rates and the least when the agents were applied at reduced rates (80 or 20%). The study on the mutual relationship (synergy factors) in green mould disease control revealed an additive effect of the two beneficial microorganisms when they were applied together. Additionally, synergy factors in assessing the mushroom yield discovered a synergistic reaction between the bacterium and actinobacterium. The future prospects of the revealed results are expected to create new possibilities for biorational edible mushroom protection with improved yield and quality while simultaneously reducing risks to human health and the environment.

Author Contributions

Conceptualization, I.P. and S.M.-M.; methodology, J.L., L.Š. and S.M.-M.; validation, D.M., N.G. and T.D.; formal analyses, I.P. and J.L.; investigation, J.L. and L.Š., writing-original draft preparation, I.P. and S.M.-M.; writing-review and editing, D.M. visualization, I.P., J.L.; supervision, D.M., S.M.-M. and I.P.; funding acquisition D.M. and I.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science Fund of the Republic of Serbia: Green Program of Cooperation between Science and Industry #GRANT No 3/4848 (2023-25) Microbial recipe for edible mushroom production—MICRO-MUSH, and the Ministry of Science, Technological Development and Innovation of the Republic of Serbia: contract Nos 451-03-66/2024-03/200214 and 451-03-65/2024-03/200116.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors are grateful to Milica Hrustić (student, the Faculty of Agriculture, the University of Belgrade) for their great help in setting up the experiment.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 14 01152 g001
Figure 1. Efficacy of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A056 in controlling Trichoderma aggressivum f. europaeum T77 on Agaricus bisporus; data are means of six replicates ± SE, standard error of means; SEDs, standard error of differences = 0.98; df, degrees of freedom = 4; F = 1289.2. * The same letters designate values that are not significantly different according to the F-test (p < 0.05).
Figure 1. Efficacy of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A056 in controlling Trichoderma aggressivum f. europaeum T77 on Agaricus bisporus; data are means of six replicates ± SE, standard error of means; SEDs, standard error of differences = 0.98; df, degrees of freedom = 4; F = 1289.2. * The same letters designate values that are not significantly different according to the F-test (p < 0.05).
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Agriculture 14 01152 g002
Figure 2. Efficacy of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A056 in controlling Trichoderma aggressivum f. europaeum T77 on Agaricus bisporus; data are means of six replicates ±SE, standard error of means; SEDs, standard error of differences = 2.06; df, degrees of freedom = 4; F = 1045.3. * The same letters designate values that are not significantly different according to the F-test (p < 0.05).
Figure 2. Efficacy of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A056 in controlling Trichoderma aggressivum f. europaeum T77 on Agaricus bisporus; data are means of six replicates ±SE, standard error of means; SEDs, standard error of differences = 2.06; df, degrees of freedom = 4; F = 1045.3. * The same letters designate values that are not significantly different according to the F-test (p < 0.05).
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Agriculture 14 01152 g003
Figure 3. Biological efficiency (BE%) of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A06 on Agaricus bisporus artificially inoculated with Trichoderma aggressivum f. europaeum T77; data are means of six replicates ± SE, standard error of means; BE% = ratio of the fresh weight of total mushroom yield and the weight of dry spawned substrate; SEDs, standard error of differences = 4.78; df, degrees of freedom = 11; F = 0.7; p = 0.74.
Figure 3. Biological efficiency (BE%) of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A06 on Agaricus bisporus artificially inoculated with Trichoderma aggressivum f. europaeum T77; data are means of six replicates ± SE, standard error of means; BE% = ratio of the fresh weight of total mushroom yield and the weight of dry spawned substrate; SEDs, standard error of differences = 4.78; df, degrees of freedom = 11; F = 0.7; p = 0.74.
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Figure 4. Biological efficiency (BE%) of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A06 on Agaricus bisporus artificially inoculated with Trichoderma aggressivum f. europaeum T77; data are means of six replicates ± SE, standard error of means, in two trials; BE% = ratio of the fresh weight of total mushroom yield and the weight of dry spawned substrate; SEDs, standard error of differences = 5.42; df, degrees of freedom = 11; F = 56; p = 0.95.
Figure 4. Biological efficiency (BE%) of the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A06 on Agaricus bisporus artificially inoculated with Trichoderma aggressivum f. europaeum T77; data are means of six replicates ± SE, standard error of means, in two trials; BE% = ratio of the fresh weight of total mushroom yield and the weight of dry spawned substrate; SEDs, standard error of differences = 5.42; df, degrees of freedom = 11; F = 56; p = 0.95.
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Table 1. Interaction between beneficial microorganisms in efficacy against Trichoderma aggressivum f. europaeum T77 and impact on mushroom yield a.
Table 1. Interaction between beneficial microorganisms in efficacy against Trichoderma aggressivum f. europaeum T77 and impact on mushroom yield a.
TreatmentTrialBiological Efficiency (BE%) in
Mushroom Productivity		Efficacy (E%) in Suppressing Green Mould Disease Symptoms
Observed BE%Expected BE%Synergy FactorObserved E%Expected E%Synergy Factor
Mean ± SE bMean ± SE
Bacillus amyloliqufaciens
B-241+ Streptomyces
flavovirens A06		I145.76 ± 2.4489.941.6261.93 ± 1.8959.091.05
II140.18 ± 5.8792.221.5269.37 ± 4.5465.941.05
ANOVA Analysis
F 12.32 0.48
p-value 0.04 0.66
SEDs c 1.58 1.9
df 2 2
a Expected values: Exp = (X + Y) − (XY)/100, E—effect from additive responses of two inhibitory agents, X and Y—the percentage of effect caused by the beneficial bacterium Bacillus amyloliqufaciens B-241 and actinobacterium Streptomyces flavovirens A06; Synergy factor (SF) is a ratio between the observed and expected inhibition. b Data are means of six replicates ± SE, standard error of means. c SED, standard error of differences.
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Milijašević-Marčić, S.; Šantrić, L.; Luković, J.; Potočnik, I.; Grujić, N.; Drobnjaković, T.; Marčić, D. Altering Microbial Communities in Substrate to Stimulate the Growth of Healthy Button Mushrooms. Agriculture 2024, 14, 1152. https://doi.org/10.3390/agriculture14071152

AMA Style

Milijašević-Marčić S, Šantrić L, Luković J, Potočnik I, Grujić N, Drobnjaković T, Marčić D. Altering Microbial Communities in Substrate to Stimulate the Growth of Healthy Button Mushrooms. Agriculture. 2024; 14(7):1152. https://doi.org/10.3390/agriculture14071152

Chicago/Turabian Style

Milijašević-Marčić, Svetlana, Ljiljana Šantrić, Jelena Luković, Ivana Potočnik, Nikola Grujić, Tanja Drobnjaković, and Dejan Marčić. 2024. "Altering Microbial Communities in Substrate to Stimulate the Growth of Healthy Button Mushrooms" Agriculture 14, no. 7: 1152. https://doi.org/10.3390/agriculture14071152

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