Upregulation of ACC deaminase gene in Bacillus velezensis UTB96 improved yield and shelf Life of Agaricus bisporus

Abstract

Agaricus bisporus is globally a most extensively consumed species of edible mushrooms. Ethylene secreted by A. bisporus mycelium suppress the initiation of fructification. 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing bacteria decrease the content of ethylene by cleaving ACC produced by the A. bisporus hyphae to α-ketobutyrate and ammonia. This study evaluated the effect of various salts on the population of Bacillus velezensis UTB96 with ACC deaminase activity on button mushroom. The results indicated that CaCl₂, MnSO₄ and MgSO₄ salts increased significantly the bacterial population compared by control, among which the medium supplemented with CaCl₂ showed the highest number of living cells in the culture. Evaluation of ACC deaminase gene expression indicated that the highest level belonged to bacteria grown in medium containing CaCl₂ in comparison with control as the expression level of ACC deaminase was 20.9-fold upregulated comparing to the control. The assessment of the effect of each salt lonely, UTB96 cultured in LB and LB containing salts of mushroom indicated that yield was increased 23% and 18% in plots treated with UTB96 cultured in media supplemented, respectively, with CaCl₂ and MnSO₄, in comparison with control. Therefore, UTB96 grown in media supplemented with CaCl₂ has a potential use in mushroom production since the induction of ACC deaminase which results in decrease the level of ethylene and promote mushroom growth.

Introduction

Cultivated mushrooms are important and noteworthy food products, among which Agaricus bisporus is globally a most extensively produced and consumed species^1,2. Edible mushrooms are generally used because of their high protein and fiber contents as well as low fat and energy density. Moreover, their proteins contain all vital amino acids required for humans³.

The casing soil affects on quantity, quality and uniformity of commercial edible mushroom⁴. It has also a critical role in the development of A. bisporus from mycelium-impregnated phase into primordial and harvesting phases⁵. Various volatile organic compounds (VOCs) and microorganisms stimulate sporophore formation in the casing soil which is essential for primordium initiation of A. bisporus^6–9. The A. bisporus mycelium mainly secrete VOCs include 1-Octen-3-ol and ethylene which involve in the suppression of the fructification initiation^9–15. Casing soil microorganisms such as Pseudomonas putida consumes produced VOCs by attaching to the fungal hyphae and consequently stimulates and promotes hyphal growth and primordium formation^{5,7–9,16–18}. Furthermore, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing bacteria inhabiting the casing soil reduce the content of ethylene surrounding mushroom hyphae by cleaving ACC produced by the A. bisporus hyphae to α-ketobutyrate and ammonia which results in primordium initiation by ethylene inhibition^5,18.

ACC deaminase enzyme contribute substantially in facilitating growth by reducing the content of ethylene which is an important decay factor in edible mushrooms, especially in harsh environmental conditions. On the other hand, various techniques were studied to improve the quality of fresh mushrooms and to prolong shelf-life, among which irrigation with water supplemented with calcium chloride solely or containing other agents including stabilized chlorine dioxide, EDTA, or sodium selenite was established to increase dry weight and quality, prolong shelf life^19–21 and reduce postharvest browning²².

However, to the best of our knowledge, no information is available for the effect of chemicals on the expression of ACC deaminase and subsequently on yield and shelf life of A. bisporus. Hence, the main goal of this study was to evaluate the effect of various salts including calcium chloride, manganese sulfate, and magnesium sulfate on Bacillus velezensis UTB96, as a plant growth-promoting bacterium (PGPB) growth and consequently on the yield, dry matter content and shelf life of button mushroom. Moreover, the effect of these treatments was evaluated on the expression of ACC deaminase and its putative contribution in the biosynthesis of ethylene and the improvement of button mushroom quality.

Results

Bacillus velezensis UTB96 and culture media

Bacillus velezensis UTB96 was grown on the DF (Dworkin and Foster) minimal salt medium complemented with 3 mM ACC as the lone nitrogen source which indicate the activity of ACC deaminase. Moreover, UTB96 was able to grow in the DF medium supplemented with 0.2% (NH₄)₂SO₄ as a positive control.

Quantification of ACC deaminase activity

To measurement the ACC deaminase activity of UTB96, the production of α-ketobutyrate was quantified by catalyzing the deamination reaction of lone nitrogen source, ACC in DF minimal salt broth media at 540 nm. The results indicated that ACC deaminase activity of UTB96 was 180 nmol α-ketobutyrate per mg of cellular protein per hour.

Effect of ca, mn and mg salts on bacterial growth and ACC deaminase

Population of UTB96 was quantified in the Luria Bertani (LB) broth medium containing CaCl₂, MnSO₄ and MgSO₄ salts each of which with two concentrations, 20 and 40 mM. The results demonstrated that all salts increased the OD₆₀₀ values (Data not shown) and CFU mL^− 1 of UTB96 compared by LB medium as control. Medium supplemented with 20 mM CaCl₂ showed the highest number of living cells in the culture as shown in Fig. 1. The population of UTB96 in medium containing 20 mM CaCl₂ was more than that with 40 mM CaCl₂ (P-value < 0.01) after culturing for 48 h. Media containing 20 mM MnSO₄ and 40 mM MgSO₄ showed the higher population in comparison with those of 40 mM MnSO₄ and 20 mM MgSO₄, respectively (Fig. 1). Therefore, salts including 20 mM CaCl₂, 20 mM MnSO₄, and 40 mM MgSO₄ were selected for more studies.

Fig. 1.

Effect of LB medium containing different salts on the population of Bacillus velezensis UTB96. Population of B. velezensis UTB96 quantified in LB medium as control and LB containing 20 mM CaCl₂, 20 mM MnSO₄, and 40 mM MgSO₄.

ACC deaminase

To investigate the putative involvement of ACC deaminase in the proliferation of UTB96, the expression of ACC deaminase gene was quantified in UTB96 grown in LB medium and media containing salts including 20 mM CaCl₂, 20 mM MnSO₄, 40 mM MgSO₄. Significant difference was observed in ACC deaminase expression in UTB96 cultured on media containing different salts in comparison with that of control culture. The highest expression level difference belongs to bacteria grown in medium containing 20 mM CaCl₂ in comparison with control as the expression level of ACC deaminase was 20.9-fold upregulated comparing to the control (Fig. 2). Moreover, the expression rates of ACC deaminase were higher in the UTB96 grown in media supplemented with 20 mM MnSO₄ comparing to the medium containing 40 mM MgSO₄. Statistically, ACC deaminase produced by the UTB96 cultured in the media containing Ca, Mn and Mg salts showed values different from the control (P-value < 0.01).

Fig. 2.

Comparative expression analysis of the Acc deaminase gene in the Bacillus velezensis UTB96 cultured in LB medium and LB media supplemented with 20 mM CaCl₂, 20 mM MnSO₄, and 40 mM MgSO₄. The standard deviation is indicated as the lines on each bar and Duncan test was performed to mean comparison of each gene in control and treatments which are marked by ‘A’, ‘B’, ‘C’ and ‘D’. The experiment was performed with three technical replicates. The B. velezensis UTB96 cultured in LB medium was used as control and the expression level of ACC deaminase gene in UTB96 cultured in LB media containing 20 mM CaCl₂, 20 mM MnSO₄, and 20 mM MnSO₄ was relatively compared with the expression of that in UTB96 cultured in LB medium. The comparison of means was completed using Duncan’s method at the level of 1%.

Mushroom cropping trials and determination of yield, size and mushroom solids

Yield, dried weight and size of mushroom harvested from substrates treated with UTB96 cultured in LB and LB containing 20 mM CaCl₂, 20 mM MnSO₄, and 40 mM MgSO₄ were measured, among which UTB96 regardless to media significantly influenced the yield and dried weight. The highest mushroom yield (9.7 Kg/m²) and dried weight (16.4 g/100 g) were obtained from the substrates treated with UTB96 cultured in medium complemented with CaCl₂. Subsequently, yield (9.3 Kg/m²) and dried weight (16 g/100 g) were significantly higher in substrate treated with UTB96 cultured in medium supplemented with MnSO₄ rather than other treatments (Fig. 3). In other word, yield was increased 23% and 18% in plots treated with UTB96 cultured in media supplemented, respectively, with CaCl₂ and MnSO₄, compared by control. UTB96 was able to increase the yield and dried wight 15 and 11%, respectively. CaCl₂ and UTB96 cultured in medium supplemented with MgSO₄ increased the yield 11% in comparison with the control. Yield and dried weight of mushroom harvested from substrate treated with MgSO₄ and MnSO₄ salts was same as those of control (Fig. 3). However, different substrate had no significant effect on mushroom cap diameter (Data not shown).

Fig. 3.

Mean yield (Kg/m²) (solid fill) and dried weight (g/100 g fresh tissue) (dotted fill) of Agaricus bisporus A15 grown on substrates treated with sterile deionized water as control, 20 mM CaCl₂, 20 mM MnSO₄, 40 mM MgSO₄ and Bacillus velezensis UTB96 cultured in LB, LB supplemented with 20 mM CaCl₂, 20 mM MnSO₄, and 40 mM MgSO₄.

Evaluation of the harvested mushroom

The acceptability index categorized based on texture, color, and percent open caps after three weeks incubation at 4 ºC was the highest in the mushrooms harvested from substrate treated with UTB96 grown in medium containing CaCl₂ 20 mM (Fig. 4). No brown spot observed on caps after three weeks. The mushrooms harvested from plots treated with CaCl₂, UTB96, and UTB96 cultured in medium supplemented with MnSO₄ with minor brown spots were classified in the next rank (Fig. 4). Brown spots were observed on the mushrooms collected from plots treated with MnSO₄, MgSO₄, as well as UTB96 cultured in media containing MgSO₄ and the acceptability index of these mushrooms was higher than that of control. Brown blotches were observed on the mushrooms harvested from plots treated with SDW as control (Fig. 4).

Fig. 4.

Quality of Agaricus bisporus A15 caps grown on substrates treated with sterile deionized water as control (A), Bacillus velezensis UTB96 cultured in LB (B), 20 mM CaCl₂ (C), UTB96 cultured in LB medium containing 20 mM CaCl₂ (D), 20 mM MnSO₄ (E), UTB96 cultured in LB medium containing 20 mM MnSO₄ (F), 40 mM MgSO₄ (G), UTB96 cultured in LB medium containing 40 mM MgSO₄ (H). Harvested mushrooms were incubated three weeks at 4 °C.

Discussion

In this study, Bacillus velezensis UTB96, as a plant growth promoting bacteria (PGPB), with ACC deaminase activity grown in media supplemented with three salts were used to assess the population of UTB96, the expression of the ACC deaminase gene, as well as growth, yield promotion and post-harvest shelf life of mushroom. The results indicated that UTB96 regardless to media could increase the expression of ACC deaminase gene in UTB96 which results in improvement of quality and quantity of button mushroom.

Ethylene is produced in the 1-aminocyclopropane-1-carboxylic acid (ACC) pathway in button mushroom^18,23. Treatment of harvested button mushrooms using 1-methylcyclopropene (1-MCP), an ethylene receptor inhibitor, and ethephon, an ethylene-releasing plant regulator, indicated that ethylene enhanced the maturation and senescence of mushrooms and upregulated the expression of genes involved in the maturation and senescence, whereas the effect of 1-MCP was in conflict with that of ethephon²⁴. Postharvest treatment of mushroom using 4-methoxy cinnamic acid extended the shelf life and verified the critical role of ethylene in the maturation and senescence of button mushroom²⁵. Consequently, the ethylene level needs to be decreased to prolong the freshness and to reduce browning of the button mushroom. Among numerous strategies to decrease the ethylene level, using PGPB with ACC deaminase activity to decrease the content of ethylene has been improving recently. Degradation of ACC as a direct precursor of ethylene to α-ketobutyrate and ammonia by ACC deaminase results in reducing in the concentration of ethylene in plants and in ameliorating the contrary effects of ethylene on plant growth^26–29.

Degradation of ethylene as a volatile self-inhibitor for fruiting in A. bisporus³⁰ either by using microflora inhabit in the casing soil or venting the air in the fruiting chamber is considered as a critical factor involved in formation of primordia by A. bisporus³¹. Inhibition of 1-aminocyclopropane-1-carboxyl synthetase (ACS) and 1-aminocyclopropane-1-carboxyl oxidase (ACO) enzymes activity by using 1-methyl cyclopropane and potassium permanganate in packaging material resulted in removal of scavenges ethylene which could prolong shelf life by postpone the mushrooms softening, and browning³². Two hybrid histidine kinases responsible for ethylene reception, AbETR1 and AbETR2, are involved in the senescence of A. bisporus. Downregulating the expression of these receptors resulted in the inhibition of maturation and senescence of mushroom fruit bodies³³. CO₂-rich modified atmosphere was also stablished to extend shelf life and delay browning of button mushroom by inhibiting ethylene biosynthesis. High CO₂ atmosphere storage inhibited the expression of genes involved in enzyme-catalyzed browning including AbPPO1, AbLAC10, and AbLAC11³⁴. The results of this study indicated that UTB96 increases the expression of ACC deaminase which could decrease the content of ethylene and subsequently improve the yield and prolong the shelf life of harvested mushroom.

Among media used for UTB96 growth that supplemented with CaCl₂ showed the best effect on bacterial growth, yield, dried weight and the acceptability of button mushroom. The effect of CaCl₂ combined with citric acid established to have a beneficial impact on yield, firmness and color of button mushrooms³⁵. These chemicals prevent softening and browning of the button mushrooms during the storage period which is attributed to adhesion of the CaCl₂ to the vacuole to make the cell wall stronger^35,36. The evaluation of the expressions of genes encoding the cell wall degrading enzymes indicated that the post-harvest chemical treatment inhibited cellulase, chitinase, phenylalanine ammonia-lyase and beta-1, 3 glucanase activities which are involved in the degradation of the cell wall during post-harvest period³⁵. As well, adding high concentrations of CaCl₂ into PDA medium significantly inhibited the mycelial growth of Hypsizygus marmoreus³⁷. The finding of this research is consistent with previously published results where the CaCl₂ improve the quantity and shelf life of mushroom.

Taking into account the information above, UTB96 grown in media supplemented with CaCl₂ has a potential use in mushroom production since the induction of ACC deaminase in UTB96 which results in the ethylene content decrease and the mushroom growth promotion.

Conclusion

This study established the significantly increasing effect of CaCl₂, MnSO₄ and MgSO₄ salts on the population and the expression of ACC deaminase gene of Bacillus velezensis UTB96. Among evaluated salts, medium supplemented with 20 mM CaCl₂ showed that the highest number of living cells in the culture and the highest expression level of ACC deaminase gene. Moreover, yield and dried weight of mushroom harvested from substrates treated with UTB96 specially that of cultured in LB containing CaCl₂ significantly was increased. This study established that the mentioned salts induced the expression of ACC deaminase in UTB96 which results in reducing the level of ethylene and promoting mushroom growth. These results are valuable for the production of edible mushroom in improving the quantity and quality of yield.

Methods

Bacillus velezensis UTB96 and culture media

Bacillus velezensis UTB96 was collected from the rhizosphere soil of the pistachio tree in Iran³⁸ and its biocontrol effect was established on bacterial leaf streak³⁹, take-all⁴⁰, and head blight⁴¹ of wheat, green decay of orange⁴², as well as degradation of aflatoxin⁴³. Moreover, it could improve the wheat growth^39,41. This biocontrol agent was developed by Biorun Company (Karaj, Iran) with Probio 96^R product number⁴⁴. Bacterial cells of UTB96 were grown to late-log phase in tryptic soybean broth (TSB) and 50 µl of bacterial suspension was cultured in three culture tubes containing 20 mL DF medium as control, (2) 20 mL DF medium supplemented with 3 mM ACC, (3) 20 mL DF medium containing 0.2% (NH₄)₂SO₄ as positive control. Bacterial growth was measured in optical density (OD) at 600 nm after 48 h incubation at 37 °C and 180 rpm.

Quantification of ACC deaminase activity

In the first step, minimal DF salts media (DF salts per liter: 6.0 g Na₂HPO₄, 2.0 g KH₂PO₄, 2.0 g glucose, 2.0 g gluconic acid, 0.2 g MgSO₄:7H₂O, and 2.0 g citric acid with trace elements: 124.6 mg ZnSO₄:7H₂O, 78.22 mg CuSO₄:5H₂O, 1 mg FeSO₄ 7H₂O, 0.01 mg H₃BO₃, 0.01 mg MnSO₄:7H₂O, 0.01 mg MoO₃, pH 7.2) was prepared^44,46. Bacterial suspension was cultured on (1) DF salt minimal medium, complemented with 3 mM ACC, (2) DF salt minimal medium, supplemented with 3 mM (NH₄)₂SO₄ (3) and DF salt minimal medium, as control, and incubated at 28 °C for 48 h. Bacterial growth was daily checked and colonies size was compared with those cultured in control medium.

In the second step, bacterial cells of UTB96 were re-suspended in TSB minimal medium and re-suspended in 30 mL containing ACC 5 mM and incubated in a shaking incubator at 28 °C and 180 rpm for 24 h. The cells were harvested at 8,000 × g for 10 min at 4 °C and pellets were suspended in 5 mL of 0.1 M Tris-HCl, pH 7.6, and transferred to a 1.5-ml microcentrifuge tube. The cells were harvested by centrifuge at 16,000 g for 5 min at 4 °C. After removing the supernatant, the pellet was suspended in 600 mL 0.1 M Tris-HCl, pH 8.5. Thirty microliters of toluene were added to the cell suspension and mixed by vortexing at the highest setting for 30 s. Then, 1 ml 1 M Tris-HCl was added to tubes and mixed. Centrifugation was performed at 16,000 × g and 25 °C for 5 min. One mL of supernatant plus 800 µL 0.56 M HCl were mixed by vortexing and 300 mL of the 2,4-dinitrophenylhydrazine reagent (0.2% 2,4-dinitrophenyl-hydrazine in 2 M HCl) were added, and the contents were vortexed and incubated at 30 °C for 30 min. The mixture’s absorbance was quantified at a wavelength of 540 nm after adding 2.0 mL 2 M NaOH in order to develop the color of the phenylhydrazone.

To induce of ACC, 15 mL of bacterial suspension with OD 0.5 absorbance at 600 nm, equal to 1 × 10⁹ CFU mL^− 1 were grown in LB media containing 20 and 40 mM CaCl₂, MnSO₄, and MgSO₄ as well as LB medium as a control. Cultures were incubated overnight at 30 °C and 180 rpm. Concentration of cells was measured using a spectrophotometer at OD₆₀₀.

The bacterium was cultured in the DF salt minimal medium, complemented with 3 mM ACC in a shaking incubator at 180 rpm for 48 h to induce ACC deaminase activity of UTB96. The cells were centrifuged at 12,000 × g for 15 min and cell pellet was suspended in 0.03 M MgSO₄ to achieve the concentration of cells (10⁸–10⁹ CFU mL^− 1) at OD₆₀₀.

Effect of ca, mn and mg salts on bacterial growth and ACC deaminase gene expression

Bacterial growth

The quantification of bacterial colony formation of UTB96 was performed according to the protocols as described by Li et al.,⁴⁷ with some modifications. In particular, a single UTB96 colony from LB agar plate inoculated into 10 mL LB broth, which was then cultured at 28 °C and 180 rpm for 24 h. Fresh LB broth medium with salt including CaCl₂, MnSO₄ and MgSO₄ were mixed with 1 mL of 10⁸ CFU mL^− 1 at OD₆₀₀ overnight bacterial culture at the ratio of 100:1. The cultures were incubated at 28 °C and 180 rpm for 48 h. The growth of bacteria was quantified in the number of living cells (CFU mL^− 1). Each treatment included three replications. The experiment was repeated three times independently.

ACC deaminase

Bacterial total RNA was extracted using the GeneAll Kit (RiboEX, Korea) and cDNA was produced from 200 ng RNA using the first-strand cDNA synthesis kit (Reverse Transcriptase (SMOBio) (Fermentas, Taiwan) according to the manufacturer’s instructions. Real-time PCR amplification was fulfilled using the ABI StepOne Real-Time PCR (Applied bioscience, USA) and RealQ Plus 2× Master Mix Green, 1 pmol of each oligonucleotide primer and 12.5 ng template cDNA. Two independent biological replicates were performed, each with three technical qPCR replicates. A primer pair ACC-F (5ʹ-TGGAAACAGRTGCTAATACCG-3ʹ) / ACC-R (5ʹ-GTCCATTGTGGAAGATTCCC-3ʹ) were designed according to sequence of JQ995371. Primer pair 338 F (5ʹ-ACTCCTACGGGAGGCAGCAG-3ʹ) / 518R (5ʹ-ATTACCGCGGCTGCTGG − 3ʹ) which target 16 S rRNA was used as internal control⁴⁸. PCR was performed in 15 µl- reactions mixtures containing 2× Taq DNA Polymerase MasterMix (Ampliqon A/S, Odense, Denmark), 10 pmol of each primer and 2 µL of cDNA. The PCR program consisted of an initial denaturation step of 15 min at 95 °C followed by 40 cycles, of 15 s at 94 °C, 40 s at 59 °C and 20 s at 72 °C.

Real-time PCRs were performed in ABI StepOne Real-Time PCR (Applied bioscience, USA), and each 10 µL reaction contained 1× RealQ Plus 2× Master Mix Green High ROX™ (Amplicon, Denmark), 0.1 µM ACC-F and ACC-R primers, 0.1 µM of each probe, and 2 µL cDNA template. PCR and fluorescence detection were completed on a LightCycler480 II instrument (Roche). The PCR program used consisted of 15 min at 95 °C; followed by 40 cycles of 15 s at 94 °C, 40 s at 59 °C, and 20 s at 72 °C. Analysis of relative gene expression was performed based on the 2^−ΔΔct method⁴⁹. Each qRT-PCR analysis was performed in three biological replicates. The experiment was repeated twice. More than two-fold difference at each treatment was regarded as either increase or decrease of ACC deaminase expression. The efficiency of each primer pair was analyzed using the LinRegPCR 11.0. program⁵⁰.

Mushroom cropping trials

The composts were inoculated using mycelium of A. bisporus A15 spent nearly 16 days of the spawn run period. Its temperature was regulated between 24 and 26^°C and relative humidity 67%. Composts were covered using casing soil with a thickness of about 5 cm and the second mycelial development started on the 15th day. UTB96 strain was cultured in LB media containing 20 mM CaCl₂, 20 mM MnSO₄, and 40 mM MgSO₄ for 48 h. The casing soil was soaked with 10 mL of bacterial suspensions. Sterile deionized waster (SDW) used as negative control.

Evaluation of the harvested mushroom

The yield percentage was calculated by weighting the mushrooms collected from each treatment before complete maturation 18 days after soiling using a digital scale. Mushrooms were sliced into thin layers and each sample (100 g) was kept in a paper bag and dried by incubation at 70 °C for 24 h. Fifteen fresh mushrooms were randomly collected from each treated substrate and cap diameter was measured from the widest part using a digital caliper in mm. Harvested mushrooms were weighted and then packed in polypropylene. The acceptability of harvested mushrooms was determined three weeks after incubation at 4 °C. It was judged by four persons based on texture, color, and percent open caps using a four-point scale, where 1 = poor, 2 = fair, 3 = good, and 4 = excellent⁵¹.

Experiments were completed using a factorial design with three replications. Data analysis was performed using one-way ANOVA by SAS 9.4. Mean separations were performed by LSD test (DPS version 6.55) at 1% level.

Author contributions

MD, MasA and NFC conceived and designed the study, with assistance MarA and EMJ. MD carried out the experiments, with assistance from MasA and NFC. MD, MarA and EMJ provided the grant. MD and NFC analyzed and interpreted the data. NFC prepared the paper, with assistance MD and MasA.

Funding

This research was supported by University of Tehran, Razhfadak Mehrshahr Company, and Nature Biotechnology Company (Biorun).

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

This research does not contain any studies with human participants or animals. We confirm that all the methods were carried out in accordance with relevant Institutional guidelines and regulations.