Biocontrol Activity of Bacillus subtilis Isolated from Agaricus bisporus


Jun 6, 2015 - Bacterial fermentation was carried out in a gyratory shaker (150 rpm/min) at 30 °C. Antifungal activity was detected after incubating t...
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Biocontrol activity of Bacillus subtilis isolated from Agaricus bisporus mushroom compost against pathogenic fungi Can Liu, Jiping Sheng, Lin Chen, Yanyan Zheng, David Y.W. Lee, Yang Yang, Mingshuang Xu, and Lin Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02218 • Publication Date (Web): 06 Jun 2015 Downloaded from http://pubs.acs.org on June 12, 2015

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Biocontrol Activity of Bacillus subtilis Isolated from Agaricus bisporus Mushroom

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Compost Against Pathogenic Fungi

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Can Liu†,§, Jiping Sheng‡,§, Lin Chen†, Yanyan Zheng†, David Yue Wei Lee§, Yang

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Yang†, Mingshuang Xu†, Lin Shen*,†

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† College of Food Science and Nutritional Engineering, China Agricultural University,

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17 Qinghua East Road, Haidian District, Beijing 100083, China

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‡ School of Agricultural Economics and Rural Development, Renmin University of

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China, 59 Zhong Guancun Street, Haidian District, Beijing 100872, China

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§ Bioorganic and Natural Products Laboratory, McLean Hospital, Harvard Medical

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School, 115 Mill Street, Belmont, MA 02478, USA

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ABSTRACT: Bacillus subtilis strain B154, isolated from Agaricus bisporus mushroom

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compost infected by red bread mold, exhibited antagonistic activities against Neurospora

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sitophila. Antifungal activity against phytopathogenic fungi was also observed. The

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maximum antifungal activity was reached during the stationary phase. This antifungal

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activity was stable over a wide pH and temperature range, and was not affected by

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proteases. Assay of antifungal activity in vitro indicated that a purified antifungal

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substance could strongly inhibit mycelia growth and spore germination of N. sitophila. In

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addition, treatment with strain B154 in A. bisporus mushroom compost infected with N.

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sitophila significantly increased the yield of bisporus mushrooms. Ultraviolet scan

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spectroscopy, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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(tricine-SDS-PAGE), matrix-associated laser desorption ionization time of flight mass

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spectrometry (MALDI-TOF-MS), and electrospray ionization tandem mass spectrometry

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(ESI-MS/MS) analyses revealed a molecular weight consistent with 1498.7633 Da. The

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antifungal compound might belong to a new type of lipopeptide fengycin.

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KEYWORDS: Bacillus subtilis, biological control, antifungal activity, Agaricus

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bisporus, fengycin

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Journal of Agricultural and Food Chemistry



INTRODUCTION

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White button mushroom [Agaricus bisporus (Lange) Imbach] is the most widely

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cultivated mushroom in the world, accounting for 35-45% of total mushroom

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production.1 In addition to its cultivation for food, the white button mushroom is a

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potential source of healthy, medicinal molecules.2

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The commercial production of mushrooms is currently threatened by mold disease

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worldwide.3 Various molds (Neurospora sitophila and Trichoderma aggressivum) can

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develop during the mushroom spawn run, affect mycelial growth and, when extensive in

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compost, can reduce yields.3,

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substrates. Standardization of compost composition and the composting process has

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considerably reduced the occurrence of these molds in mushroom crops.5 Nevertheless,

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mold is still a major disease in mushroom-growing regions worldwide. Chemical

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treatments with formaldehyde and fungicides have been used for suppression of potential

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antagonists in the substrates,6 but this method may harm the operator and the environment.

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Resistance to fungicides has already been reported, leaving the crop very vulnerable to

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fungal infection.7

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Such diseases become established in poor quality

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Microorganisms could be used to alleviate the mold problem without harm to the

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environment, food security, or human health.8 The use of bacterial agents is an essential

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alternative to chemicals.9 Bacillus strains have multiple advantages for industrial

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application; for instance, these strains have shorter growth cycles and can secrete 3

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enzymes and antibiotics into the extracellular medium. FDA stated that the non-toxigenic

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and non-pathogenic strains of Bacillus subtilis were widely available and had been used

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safely in a variety of foods.10 Therefore, Bacillus species have become an attractive,

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alternative industrial treatment to reduce mold in mushrooms.11

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Biological control by Bacillus sp. involves a number of mechanisms, such as

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competition, antagonism, systemic resistance induction, and promotion of plant growth.

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Bacteria that remain in the mushroom cultivation substrate after the composting process

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can act directly as mold antagonists.4 Because a heat treatment was applied to the straw

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after partial composting, sporulating bacteria Bacillus spp. were encouraged and, thus,

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may play an important role in the inhibition of fungal pathogens. However, the antifungal

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mechanism and application of Bacillus spp. during mushroom cultivation have not been

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studied in detail. Meanwhile, B. subtilis is well known for the production of antibiotics

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with an amazing variety of structures.12, 13 Most of the research only focused on the

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exploitation of the biocontrol efficiency of bacteria from soil on plant fungal pathogens,14

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and few antimicrobial activities of B. subtilis have been detected against mushroom

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fungal pathogens, especially N. sitophila. Thus, exploration of natural bacteria from

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mushroom compost that inhibit fungal pathogens, and studying their potential

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antagonistic mechanism, can satisfy long-term concerns over food safety and the

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environment in the mushroom industry.

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The objectives of this study were to search for a new biological control agent against

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mushroom red bread mold disease from the A. bisporus mushroom cultivation substrate

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obtained from a commercial mushroom operation, and to isolate and investigate the

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biological properties of the antimicrobial lipopeptide from the antagonistic Bacillus sp.

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strain.

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MATERIALS AND METHODS

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Fungi. The pathogenic fungus strain M21 (N. sitophila) was isolated from a

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contaminated A. bisporus compost-based cultivation substrate in a commercial Agricus

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mushroom

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TCCGTAGGTGAACCTGCGC-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3')

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were used to amplify the internal transcribed space regions (ITS) of 18S rDNA sequences

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of M21.15 PCR sequences were aligned and compared with those available in the

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GenBank database using the BLAST program.16 Then the homology of the sequence was

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determined. A phylogenetic tree was constructed according to the neighbor-joining

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method using MEGA 4.0 based on the 18S rDNA sequence. Nucleotide sequence data for

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the pathogenic fungus have been submitted to GenBank database under accession number

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KM588213.

factory

in

Shandong,

China.

Primers

ITS1

(5'-

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The fungi Trichoderma harzianum (ACCC30371), Fusarium solani (ACCC36223),

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F. graminearum (ACCC37686), Botrytis cinerea (ACCC36028), and F. incarnatum

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(CFCC 84580) were supplied by the Agricultural Culture Collection of China and the 5

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China Forestry Culture Collection Center.

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Bacteria. A total of 600 colonies of bacteria were separated from A. bisporus

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mushroom composts by the serial dilution method.17 Bacteria thus isolated were tested for

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their antifungal activity against the pathogenic fungus N. sitophila M21.

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The bacterium B154, one of the antifungal strains, was characterized using the API

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50 CHB system (BioMerieux, Marcy l'Etoile, France). Microscopic observation and

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biochemical features were tested as described previously.18, 19 To confirm biochemical

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identification, a partial sequence analysis of the 16S rDNA was performed. PCR

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amplification of the 16S rDNA gene of strain B154 with universal forward

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(5′-AGAGTTTGATCCTGGCTCAG-3′)

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(5′-GGCTACCTTGTTACGACTT-3′) primers was carried out.20 The 16S sequence for

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B154 was deposited in GenBank (accession no. KM588212). A neighbor-joining tree was

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constructed based on the 16S rDNA sequences of bacterial strains. A gyrA fragment

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corresponding to Bacillus subtilis gyrA positions 42 to 1066 was PCR amplified by using

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primers gyrA-forward (5′-CAGTCAGGAAATGCGTACGTCCTT-3′) and gyrA-reverse

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(5′-CAAGGTAATGCTCC AGGCATTGCT-3′).21 The gyrA sequence for B154 was

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deposited in GenBank (accession no. KR296983).

and

reverse

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In Vitro Antagonism Experiments. The ability of the B154 strain to inhibit the

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growth of various fungal pathogens was tested in Petri dishes containing a potato

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dextrose agar (PDA) medium. The mycelial plugs of N. sitophila, T. harzianum, F. 6

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incarnatum, F. solani, F. graminearum, and B. cinerea were cultivated in the center of

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the plates at 28 °C, and the bacteria were inoculated on the edges. The slow growth fungi

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were inoculated on the plate 24-48 h prior to the bacteria. The inhibition of fungal growth

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was evaluated by the percentage of reduction of mycelium expansion compared to control

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plates without bacteria. The mycelial growth inhibition (MGI) percentage was calculated

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according to the equation: MGI=(A0-A1)/A0 × 100, where A0 is the area of mycelium on

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control plates, and A1 is the area of mycelium on plates inoculated with B154.22 The

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antagonism assay was conducted three times, and all treatments in each assay contained

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three replicates.

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Selection of the Best Medium for Production of Antifungal Activity. To select the

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best culture medium for optimal production of antifungal activity, several broth media

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were tested: tryptic soya broth (TSB), Luria-Bertani broth (LB), nutrient broth (NB), and

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tryptic yeast (TY).23 Incubation was performed at 30 °C with shaking at 150 rpm/min for

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24 h. Cell growth was monitored by optical density measurement at 600 nm, and any

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secreted antifungal activity found in the culture supernatant was described by the

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diameter of the clear zone.

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Fermentation and Kinetic Production of Antifungal Compounds. Bacterial

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fermentation was carried out in a gyratory shaker (150 rpm/min) at 30 °C. Antifungal

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activity was detected after incubating the producer strain in TSB medium for 56 h. We

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collected 10 mL of the culture at an interval of 4 h for the determination of cell growth, 7

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and the anti-N. sitophila activity in the supernatant was obtained by measuring the

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diameter of the clear zone. The pH of the medium was monitored regularly throughout

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the culture period.24

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Antifungal Activity of the Compound. The activity of the antifungal compound

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produced by B154 was measured by the disc diffusion assay on all purification extracts

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and by using high-performance liquid chromatography (HPLC) fractions to determine

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where the antifungal compounds were eluted.25 Arbitrary units (AU) of antifungal

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activity were calculated by the reciprocal of the highest dilution showing inhibition per

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milliliter of antifungal compound fraction. The antagonism assay was conducted three

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times, and all treatments in each assay contained three replicates.

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Isolation and Purification of Antifungal Compound by Ion Exchange

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Chromatography. The antifungal compound was recovered by 20% ammonium sulfate

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precipitation of the B154 culture supernatant.12 The precipitate was collected, and

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dialyzed by distilled water in 3.5 kDa dialysis tubing. The resulting solution was

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concentrated by lyophilization, and the solid was dissolved in sterile milli-Q water to

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obtain the crude bioactive substance (1.0 mg/mL).

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The crude extract (10 mL) was loaded onto a DEAE-Sephadex A50 (1.5 cm × 15 cm

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i.d.) column (GE Healthcare, Uppsala, Sweden) previously equilibrated with Tris-HCl

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buffer (25 mM, pH 8.0). A gradient of 0-1 M NaCl in buffer was run through the column

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at 0.5 mL/min.26 The eluent was monitored at 280 nm using a HD5 spectrophotometer 8

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(Huxi Analysis, Shanghai, China) and fractions were collected every 5 min. All samples

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were assayed for antifungal activity, and the antifungal fractions were collected and

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concentrated by ultrafiltration with an Ultra-15 centrifugal filter concentrator (Millipore,

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Darmstadt, Germany).

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Ultraviolet Spectrum. The ultraviolet absorption spectrum of the antibiotic collected

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from the ion exchange column was measured using a UV-1800 recording

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spectrophotometer (Shimadzu, Kyoto, Japan) from 200 to 600 nm.27

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Detection of Antifungal Activity in Tricine-SDS-PAGE Gel. Tricine-SDS-PAGE

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was performed with active fractions from each purification step using a Mini Protean

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tetra cell vertical gel electrophoresis unit (Bio-Rad, Hercules, CA).25 TSB medium,

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culture supernatant, dialyzed 20% ammonium sulfate fraction, and the antifungal fraction

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that had been purified by ion exchange chromatography were loaded onto the gel; ultra

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low molecular weight protein markers (Sigma-Aldrich, St Louis, MO) were used for size

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reference.

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After electrophoresis, three duplicate gels were fixed in 500 mL of 50% (v/v)

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methanol/10% (v/v) acetic acid for 30 min, and stained with 0.025% Coomassie brilliant

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blue G-250 (Bio-Rad, Hercules, CA) in 10% acetic acid / 45% ethanol.

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pH and Heat Stability. The concentrated antifungal fractions collected from ion

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exchange column were filter-sterilized using 0.22 µm filters (Millipore, Darmstadt,

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Germany), and lyophilized. Each aliquot was dissolved in 200 µL of buffer within the 9

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range of pH 2-12 adjusted with diluted HCl or NaOH. After incubation for 2 h at 25 °C

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and neutralization to pH 7, residual activity against N. sitophila was tested.

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We evaluated thermal stability after incubation of antifungal fractions at different

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temperatures for 30 min or after autoclaving at 121 °C for 20 min. After cooling at room

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temperature, we quantitated residual inhibitory antifungal activity.

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Effect of Proteolytic Enzymes. The effect of the proteolytic enzymes proteinase K,

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pepsin, trypsin, and papain on antifungal activity was investigated. Enzymes were

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dissolved in Tris-HCl buffer (50 mM, pH 7.8) and were added to the antifungal fraction

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at a mass ratio of 1:10 and 1:5. After incubation at 37 °C for 2 h, solutions were

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incubated at 90 °C for 5 min, and cooled at room temperature. The residual antifungal

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activity in the supernatant was tested. The untreated antifungal fraction and the enzymes

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alone in buffer were used as controls respectively.

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Conidial Germination Inhibition Assays. We examined the inhibitory effect of the

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purified antifungal fractions on conidial spore germination of N. sitophila.28,

29

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antifungal fractions were serially diluted with PDA to a final concentration of 30, 20, 10

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µg/mL. Sterile phosphate buffer (20 mM, pH 6.8) was used as a control. Ten mL of the

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mixture was added into Petri dishes, and the N. sitophila spore suspension at a

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concentration of 105 cfu/mL was prepared and inoculated by spreading in the Petri dishes.

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After 6 h of incubation at 25 °C, the plates were observed by optical microscopy. When

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the length of the germ tube was longer than the radius of the spore, the spore was rated as 10

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germinated. At least 200 spores were counted on each plate. Each treatment was

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conducted in triplicate and the experiments were repeated twice. The relative germination

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inhibition (RI) of the treatment compared to the control was calculated by percentage,

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using the following formula:

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RI = (N0-N) × 100/N0

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where RI = Relative Inhibition; N0 = number of germinated spores on the control

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plates; N = number of germinated spores on the treatment plates. Slices of pathogenic fungal spores were dyed by lactophenol cotton blue, and observed using B203 LED microscopy (Optec instrument, Chongqing, China).

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SEM Analyses. The effect of antifungal extracts from B154 on N. sitophila hyphae

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and spores was examined by SEM.10 The antifungal fractions were diluted with potato

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dextrose broth (PDB) to a final concentration of 30 µg/mL. Spore suspension (105 cfu/mL)

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and the hyphae were added to the PDB medium. After incubation at 28 °C for 24 h in a

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shaker at 50 rpm/min, the spore and hyphae were harvested for SEM. Samples were

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sputter coated with gold palladium in a IB-3 nanotech sputter coating apparatus (EIKO,

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Osaka, Japan), and then examined using a S-3400N scanning electron microscopy (SEM)

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(Hitachi, Tokyo, Japan).

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Antifungal Activity of Bacteria B154 on A. bisporus Mushrooms. We studied the

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in vivo effect of the antifungal bacterium B154 against the pathogen fungus N. sitophila

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in the mushroom beds and recorded the associated yields of A. bisporus .The 11

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experimental cultivation trials of A. bisporus were conducted at the JiuFa Mushroom Test

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Demonstration Factory in cooperation with China Agricultural University. Suspensions

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of the fungus N. sitophila (103 cfu/mL) were mixed with the spawned compost (1L

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suspension/m2 of compost) on mushroom beds. One liter each of B154 fermentation

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broth (fermented for 24h, 48h, or 72h respectively) was mixed thoroughly with compost

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to inhibit the fungal pathogen. For comparison, there were two controls. In one control,

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only the pathogen fungus was mixed with compost (negative control); in the other

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(positive control), neither the pathogen nor the antagonistic bacteria B154 was used.

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Closed mushrooms were harvested, counted, and weighed daily. At the end of the third

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flush, yields were determined and expressed as kg/m2. Each treatment was replicated

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three times, and these experiments were repeated twice.

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Purification of Antifungal Active Compounds. The antifungal compounds

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collected through the DEAE-Sephadex A50 column from B. subtilis B154 were purified

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by a 1200 series HPLC system (Agilent Technologies, Wilmington, DE) using a ODS-A

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reverse phase (RP) C18 column, 4.6 mm × 250 mm i.d., 120 A (YMC, Allentown, PA)

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with a linear gradient elution of deionized water and acetonitrile.30 Mobile phase: 0.05%

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trifluoroacetic acid water solution (A), acetonitrile (B). Flow rate: 0.8 mL/min. Gradient

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program: 30% B in 0-5 min; 30-45% B in 5-10 min; 45-80% B in 10-60 min; 80-100% B

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in 60-80 min; 100% B in 80-90 min. This step was repeated several times, and peaks with

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the same retention time were collected for determination of activity. The final active

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collection was dried, weighed, and used for studies of chemical characterization.

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Analysis of Antifungal Conpound by MALDI-TOF MS Analysis. The

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HPLC-purified compound was analyzed on a 4800 Plus MALDI-TOF-MS system

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(Applied Biosystems, Foster, CA).31, 32 For MALDI-TOF-MS analysis, HPLC fractions

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extracted from B. subtilis B154 were mixed with α-cyano-4-hydroxycinnamic acid

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(α-CHCA) saturated in 50% (v/v) acetonitrile containing 0.1% trifluoroacetic acid in

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equal amounts, applied to the MALDI sample plate, and left to air dry.

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ESI-MS/MS Analysis. ESI-MS/MS analysis of antifungal compound was performed

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on a SYNAPT G2-Si quadrupole time-of-flight (Q-TOF) mass spectrometry system

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(Waters MS Technologies, Manchester, UK).31, 33 MS survey scans in a mass range of m/z

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100-2000 and of 30 s duration were run. The electrospray source was operated at a spray

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cone voltage of 80 V, a capillary voltage of 3 kV, and a source temperature of 100 °C.

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For the ESI experiment, the nanoflow gas pressure was set at the 0.5 bar, and the purge

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gas flow was set at 10 mL/min. Helium was used as the collision gas.

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Statistical Analysis. All data were presented as the mean ± standard deviation (SD).

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One-way analysis of variance (ANOVA) and Duncan’s multiple range tests were used to

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determine significant differences (p < 0.05) between the means using the statistical

244

software SPSS 18.0 (IBM Corp., Armonk, NY).

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RESULTS AND DISCUSSION Identification of Pathogenic Fungus M21 and Antifungal Bacterium B154. The

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BLAST results based on the ITS sequence showed M21 had the highest similarity (100%)

248

with Neurospora sitophila (GU192459.1) The phylogenetic tree constructed based on the

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ITS sequence indicated M21 closely clustered with N. sitophila as well. Mycelium of

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M21 was white and villi form (Figure 1A). After growing on PDA medium for 3 d,

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several orange spores formed (Figure 1B). M21 mycelium amplified 40 times showed

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that the diameter of its hypha was about 10-12 µm, and many hypha branches formed

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(Figure 1C). The combination of colony traits, mycelium morphological traits, and

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molecular characteristics allowed us to identify pathogenic fungus M21 as N. sitophila.

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The red bread mold (N. sitophila) is one of the major diseases threatening mushroom

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production.3, 34 N. sitophila became epidemic rapidly, when the room temperature and

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humidity were relatively high, leading to inhibition of primordial formation and reduction

258

in yield.

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Strain B154 isolated from the A. bisporus mushroom compost had the highest

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activity against the strain M21 (Figure 2A). Therefore, strain B154 was used in the

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subsequent investigation. The colonies of bacterial strain B154 were white and its

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morphology was irregular (Figure 2B). A preliminary identification of this strain through

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phenotypical and biochemical characteristics showed that this isolate belonged to the

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genus Bacillus. It is a Gram-positive bacillus, motile spore-forming organism, and 14

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catalase- and oxidase-positive. The tests of hydrogen sulfide, indole production, and the

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Voges-Proskauer were positive. The use of an API 50 CHB kit with the APILAB Plus

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software indicated 99.6% identity with B. subtilis.

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The BLAST results indicated that strain B154 had 100% similarity with Bacillus

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subtilis (NCDO1769) according to the sequence of the 16S rDNA gene. Phylogenetic

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trees constructed based on the 16S rDNA gene also indicated that B154 closely clustered

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with B. subtilis.

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Since strain B154 identified as B. subtilis group on the basis of 16S rDNA sequence

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analysis is not satisfactory, we applied another method to obtain more reliable species

274

identification. We performed gyrA sequencing preliminarily identified as B. subtilis on

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the basis of 16S rDNA sequencing. Cluster analysis of the gyrA sequence of B154 with

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those from the study of Chun and Bae35 showed that strain B154 was identified as B.

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subtilis subsp. subtilis.

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Antifungal Activity Spectrum. When tested in vitro against two fungal species of

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mushroom pathogens (N. sitophila and T. harzianum) and four phytopathogens (F.

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incarnatum, F. solani, F. graminearum, and B. cinerea), B. subtilis strain B154 could

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inhibit the mycelia growth of all of the fungi (Table 1). The MGI of B154 on N. sitophila

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was 7.5%, and the MGI on T. harzianum was 26.7%. B. subtilis had inhibitory ability

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against various plant fungal pathogens, such as Fusarium oxysporum, Exserohilum

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turcicum,12 and Alternaria alternate.30 Our study indicated that strain B154 isolated from 15

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A. bisporus mushroom compost provided a broad spectrum of antifungal activities against

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both plant and mushroom pathogenic fungi, thus, it would be a potential effective

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alternative to chemical fungicides, especially in mushroom industry.

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Kinetic Production of the Antifungal Activity in the Optimized Medium. The

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antagonism potential of B154 could play a major role in biocontrol, because of the

290

production of antifungal compounds. Variation in the composition of the culture medium

291

was shown previously to affect the production of antimicrobial activity.23,

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production of antifungal activity by strain B154 was tested in various media (Figure 3A).

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The TSB medium allowed maximum biomass accumulation and the highest antifungal

294

activity against N. sitophila. The activity obtained by culturing in LB medium dropped

295

significantly compared to TSB medium (p < 0.05), while the biomass in these two kinds

296

of media was the same.

36

The

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Synthesis of antimicrobial compounds by Bacillus species generally starts at the end

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of the exponential phase and reaches its maximum level during the stationary phase.37, 38

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During the growth period of B154 in TSB medium, antifungal activity against N.

300

sitophila (diameter of inhibition zone was 10.17 mm) was detected during the

301

exponential growth phase (12 h after inoculation), and maximum antifungal activity

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(inhibition zone was 14.33 mm) was attained during the stationary phase (28 h after

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inoculation) (Figure 3B). This meant that the bacterium could produce high levels of the

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bioactive substance within a short period. Medium pH was monitored throughout the

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experiment and increased from 7 to 8.7 at 56 h.

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Ultraviolet Spectrum and Tricine-SDS-PAGE Gel Detection of Antifungal Ion

exchange

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Compound

308

chromatography of B154 culture extract on a DEAE-Sephadex A50 column contained

309

one unadsorbed fraction and one adsorbed fraction (1 and 2). Of these fractions, only

310

fraction 2 showed strong antifungal activity (Figure 4A). The antibiotic was eluted from

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the DEAE-Sephadex matrix in 0.55-0.6 M NaCl.

Purified

by

Ion

Exchange

Chromatography.

312

Purification of the biologically active component from the culture medium is

313

summarized in Table 2. The initial 20% ammonium sulfate precipitation yielded 47.2%

314

of the original activity, and 25.5% of the initial activity was recovered by ion exchange

315

chromatography.

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The absorbance spectrum for the antibiotic measured in milli-Q water showed

317

absorbance maxima at 236 and 275 nm, and there was no appreciable absorbance above

318

300 nm. B. subtilis is the producer of cyclic lipopeptides, for example, molecules of

319

seven amino acids with a β-fatty acid.26, 39 The ultraviolet spectrum of the antibiotic

320

suggested that our strain might be a producer of cyclic lipopeptides with antifungal

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activity. The absorbance maximum between 220 and 240 nm was due to the presence of

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tyrosine.27

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The active fraction that was purified by ion exchange column, the TSB medium, the

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culture supernatant, and 20% ammonium sulfate precipitate were loaded onto a tricine gel

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containing 16.5% separating gel. The active bands were visualized with Coomassie

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brilliant blue G-250 solution (Figure 4B). Compared to the polypeptide marker, the

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molecular mass of purified peptide (lane 4) was estimated to be approximately 1.4 kDa.

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Tricine-SDS-PAGE confirmed the purity of the antibiotic preparation. The culture

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supernatant (lane 2) produced many bands of stained contaminated material. Evidently,

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almost all of this contaminated material was removed by the purification protocol (lane 4).

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Although ion exchange extraction actually decreased antibiotic activity relative to the

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previous step (ammonium sulfate precipitate) (Table 2), the ion exchange protocol was

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very effective at eliminating colored material contaminating the sample, and it separated

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the antibiotic peak from other peaks seen in the chromatogram.

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pH, Heat Stability, and Effect of Proteolytic Enzymes on Antifungal Peptide.

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The antifungal activity of the purified peptide from the ion exchange column was

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conserved until 90 °C. In addition, it was stable within a wide range of pH (4-10). The

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antifungal activity was severely reduced at pH 2-3, and at a pH higher than 10 (p < 0.5).

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This heat-stable property was also observed in other antimicrobial metabolites produced

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by Bacillus species.40,

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provide greater application for the food and agrochemical industries.

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Temperature and pH stability of the antifungal peptide can

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The treatment of the antifungal peptide with different proteases, such as proteinase

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K, pepsin, papain, and trypsin, in enzyme/peptide mass ratios of 1:10 and 1:5 (wt/wt) for

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2 h at 37 C, did not affect its antifungal activity. The resistance of the antifungal

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peptide to the proteases might be attributed to the cyclic structure of the peptide, which

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inhibits the enzymatic action of proteases on peptides.25

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Effect of B. subtilis B154 Antifungal Compound upon N. sitophila Conidia and

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Hyphae. We evaluated the ability of the B154 antibiotic compound purified by ion

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exchange column on arresting the germination of N. sitophila conidia by an in vitro

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bioassay (Figure 5). The RI rate for conidial germination increased with an increase in

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the antibiotic peptide concentration (Figure 5A). At 10 µg/mL, B. subtilis B154 antibiotic

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peptide reduced the number of germinated spores by 7.7%, at 20 µg/mL by 28.4%, and at

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30 µg/mL by 83.4%. The subsequent development of branched mycelia in untreated

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(Figure 5B, a) or antifungal peptide treated (Figure 5B, b, c, d) discs observed by light

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microscopy showed that the development of N. sitophila conidia was arrested at the stage

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of the germ tube. Conidial germination represents the first step in triggering an asexual

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life cycle of aerial pathogenic fungi and the spread of the disease.29 Therefore, the use of

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micro-organisms that produce antifungal compounds that inhibit conidial germination is a

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potential means of biocontrol for fungal disease.42

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The surface appearance of hyphae and spores observed by SEM showed that the

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control hyphae and conidia of N. sitophila grew normally, and were smooth with no 19

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breakage (Figure 6, A, C), while the structures of hyphae and spores treated with B154

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antifungal peptide were damaged and irregular (Figure 6, B, D). When treated with

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antifungal peptide, the hyphae were deformed, distorted, wilted on the surface, cavitated,

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and the conidia displayed a collapsed morphology characterized by distortion, shrinking,

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and loss of turgor. These findings support the hypothesis that the secretion of antifungal

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compounds, such as lipopeptides, is the primary mechanism contributing to the

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deleterious effect of the B. subtilis strain against conidial germination and hyphae growth

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of fungi.43

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Antifungal Activity of Bacteria B154 on A. bisporus Mushrooms. B154, which

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was effective against the particular pathogen N. sitophila M21 in vitro, was selected to

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test for A. bisporus mushroom yield. B154 notably enhanced the yield of A. bisporus (p