Biocontrol and plant stimulating potential of novel strain Bacillus sp. PPM3 isolated from marine sediment

Microbial Pathogenesis 120 (2018) 71–78

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Biocontrol and plant stimulating potential of novel strain Bacillus sp. PPM3 isolated from marine sediment

T

Neda Radovanovića, Milica Milutinovićb,∗, Katarina Mihajlovskib, Jelena Jovićb, Branislav Nastasijevićc, Mirjana Rajilić-Stojanovićb, Suzana Dimitrijević-Brankovićb a University of Belgrade, Innovation Center of Faculty of Technology and Metallurgy, Department of Biochemical Engineering and Biotechnology, Karnegijeva 4, Belgrade, Serbia b University of Belgrade, Faculty of Technology and Metallurgy, Department of Biochemical Engineering and Biotechnology, Karnegijeva 4, Belgrade, Serbia c University of Belgrade, Vinča Institute of Nuclear Sciences, P.O. Box 522, 11001 Belgrade, Serbia

A R T I C LE I N FO

A B S T R A C T

Keywords: Marine Bacillus sp. Biological control Antifungal Plant stimulation

In the current study, the biocontrol potential of a novel strain Bacillus sp. PPM3 isolated from marine sediment from the Red Sea in Hurghada, Egypt is recognized. This novel strain was selected out of 32 isolates based on its ability to suppress the growth of four plant pathogenic fungi: Aspergillus flavus, Fusarium graminearum, Mucor sp. and Alternaria sp. The new marine strain was identified and characterized by phenotypic and molecular approaches. The culture filtrate of Bacillus sp. PPM3 suppressed the growth and spore germination of all tested fungi in vitro with the highest value of inhibition reported for Mucor sp. (97.5%). The antifungal effect of the culture filtrate from the strain PPM3 was due to production of highly stable secondary metabolites resistant to extreme pH, temperature and enzymatic treatments. A PCR analysis confirmed the expression of genes involved in the synthesis of antifungal lipopeptides: iturin, bacillomycin D, mycosubtilin and surfactin. In a greenhouse experiment strain PPM3 effectively reduced disease incidence of F. graminearum in maize plants and displayed additional plant growth stimulating effect. The results show that novel marine strain PPM3 could have a potential in commercial application as biocontrol agent for treatment of various plant diseases caused by soil-borne and postharvest pathogenic fungi.

1. Introduction Phytopathogenic fungi that infect crops and vegetables present a major threat to food production, causing great economic losses worldwide [1]. Agrochemicals have been traditionally used for the prevention and the control of various plant diseases. However, the detrimental effects that these chemicals have on human health and ecosystem, have raised a concern in consumers and environmentalists, generating a demand for the development of natural, ecofriendly alternatives that would establish safe and sustainable production of crops [2]. Alternative strategies to control pests and diseases include biological control. Utilization of antagonistic microorganisms in suppressing pathogens has numerous advantages over chemicals. Products based on microorganisms are rapidly degraded in the environment and can become a part of the natural element cycles. Microorganisms used as biocontrol agents (BCA) are characterized by a high specificity against the targeted pathogen and low mass production cost [3]. Among antagonistic microorganisms, Bacillus species are the most studied and there are

numerous reports about their effectiveness against plant diseases caused by soil-borne or postharvest pathogens [4–6]. The mechanisms involved in biological control are diverse and they include: competition for nutrients and niche, antibiosis, production of extracellular lytic enzymes, immunization of the host plants and plant growth promotion [4,7–9]. Great genetic diversity, numerous literature data, GRAS (Generally Regarded As Safe) status, and ability to survive very harsh conditions by forming resistant spores, qualify Bacillus species as excellent candidates for the development of efficient biocontrol products. The majority of microbial biocontrol agents and plant growth promoters are isolated from fruit surface, plant tissue and the rhizosphere. Although not widely assessed for this purpose marine environments also harbor diverse taxonomic groups of bacteria with unique morphophysiological characteristics. Among other features, marine microorganisms can produce metabolites that could be potent biocontrol agents. In recent years, the aquatic environment has become increasingly appreciated as a rich source of novel biocontrol agents, and the steps towards investigating the suitability of marine Bacillus strains for

∗ Corresponding author. Faculty of Technology and Metallurgy, University of Belgrade, Department of Biochemical Engineering and Biotechnology, Karnegijeva 4, 11000 Belgrade, Serbia. E-mail address: [email protected] (M. Milutinović).

https://doi.org/10.1016/j.micpath.2018.04.056 Received 4 October 2017; Received in revised form 26 April 2018; Accepted 26 April 2018 Available online 27 April 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.

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2.3. Identification and characterization of novel strain PPM3

application in biocontrol of plant diseases have been initiated [10–12]. The objectives of this study were to find beneficial marine strains that can be used as biocontrol agents by: (a) identifying, potential beneficial strains, based on their genetic and phenotypic characteristics; (b) evaluating inhibiting ability of the cells and culture filtrates of the selected strains in vitro; (c) investigating the potential of different treatments of microbial inoculants for potential application.

The marine strain PPM3, which exhibited strong antifungal activity against all four tested pathogenic fungi in the dual culture test, was selected and examined for various phenotypic properties. Morphological, physiological and biochemical characteristics of the strain PPM3 was investigated and compared, as described in Bergey's Manual of Systematic Bacteriology [16]. API Bacillus identification sistems (API50 CHB and API ZYM, Biomereux, France) were also employed according to manufacturer's instructions. After inoculation the API strips were incubated at 30 °C, and the results were red after 48 h. Bacteria were identified by API LAB software. A type strain B. amyloliquefaciens ATCC 23350T was also egzamined for phenotypic properties and used as a reference strain. The molecular identification was carried out by sequencing of the 16S rRNA encoding gene and gyrase A encoding gene (gyrA). Genomic DNA was extracted from overnight culture of PPM3 using modified phenol-chloroform method [17]. The 16S rDNA was amplified by PCR in an automated thermal cycler (QB-24 LKB, Austria) using total DNA as a template with universal primers: 27-FOR (5′-AGAGTTTGATCCTGGC TCAG-3′) and 1492-REV (5′-CGGTTACCTTGTTACGAC-3′). Part of the GyrA sequence was amplified using two oligonucleotide primers gyrAFOR (5′-CAGTCAGGAAATGCGTACGTCCTT-3′) and gyrA-REV (5′-CAA GGTAATGCTCCAGGCATTGCT-3′) [18]. Amplifications were performed in 50 μl reactions with GoTaq Green PCR Master Mix (Promega, USA) according to manufacturer's instructions. The thermal conditions for the PCR reaction were according to the method of Chun and Bae [18]. The PCR products were purified using the QIAquickPCR Purification Kit (Qiagen, USA) and sequenced by Macrogen (Amsterdam, The Netherlands). The obtained sequences were compared to those of the reference species with the GenBank database using the BLAST nucleotide search available at the NCBI website [19].

2. Materials and methods 2.1. Microorganisms and growth conditions The total of 32 bacteria was isolated from the 2 m deep sand sediment of the Red Sea in Hurghada, Egypt (27°5′4.36″N, 33°51′30.47″E) by standard dilution method [13]. One gram of sand sample was serially diluted in standard saline solution, and 0.1 mL of each dilution was spread onto International Streptomyces Project1 (ISP1) agar plates (5 g L-1 casein enzymic hydrolyzate, 3 g L−1yeast extract, 12 g L−1 agar). The plates were incubated at 30 °C for 48 h and colonies with different morphologies were purified by subculturing onto ISP1 agar. The working cultures were obtained by transferring one colony from ISP1 agar plate to 50 mL of ISP1 broth and incubating overnight at 30 °C with constant shaking (150 rpm) until the cultures reached a density of approximately 2 × 108 CFUmL−1 (OD 0.1 at 595 nm = 108 CFU mL−1) [14]. Pure cultures were stored at −20 °C in ISP1 broth containing 25% (v/v) glycerol. Pathogenic fungi: Aspergillus flavus, Fusarium graminearum, Mucor sp. And Alternaria sp. were kindly provided from the laboratory isolate collection of The Maize Research Institute, Zemun Polje, Serbia. Stock culture of each pathogen was maintained on ISP1G agar plates (the same composition as ISP1 agar with additional glucose in a concentration of 10 g L-1) at 4 °C. Working cultures were obtained by transferring a stock agar plug with the mycelium onto ISP1G plates and incubating at 25 °C for 5 days. For the antifungal assay the fungal spore suspensions were prepared following the method of Kim et al. [15] with slight modifications. Namely, the suspensions were made by scraping of the conidia from the surface of a fully growing mycelium with a sterile loop and dissolving in 5 mL of the standard saline solution. Two drops of sterile glycerol were added to the suspensions, after which they were shaken vigorously, to release the spores, and then filtered through sterile gauze. The spores were enumerated microscopically using a hemocytometer and the concentration of the fungal spore suspension was adjusted to 1 × 106 spores mL−1 of standard saline solution.

2.4. In vitro antifungal assay 2.4.1. Effect of culture filtrate of strain PPM3 on growth of pathogenic fungi Antifungal activity of the culture filtrate (CF) of strain PPM3 was evaluated in vitro by using the radial growth inhibition assay. A 5% (v/ v) bacterial suspension of strain PPM3 (concentration of 2 × 108 CFUmL−1) was inoculated into 50 mL of sterile ISP1 broth and incubated at 30 °C for 48 h with constant shaking (150 rpm). The cell culture was centrifuged for 15 min at 9000 rpm, the culture filtrate was collected and filter sterilized trough 0.22 μm pore-size membrane filters (LLG, PES syringe filters). The obtained CF of strain PPM3 was mixed with 5 mL of sterile ISP1G agar medium (at 45 °C) at a concentration of 2% (v/v) and immediately poured into 45 mm Petri plates. After solidification a 5 μL aliquot of fungal spore suspension (1 × 106 spores mL−1) of each pathogenic fungi, respectively, was inoculated on the center of the plates containing CF of strain PPM3 and the control plates containing sterile ISP1 broth. Growth of the fungi was measured after 5 days of incubation at 25 °C in the dark. The percent of growth inhibition (PGI,%) due to the presence of antagonistic bacterial CF was calculated using the following equation: (Rc-R/Rc) x 100, where Rc -is the diameter of the fungal growth in the control plates, R-is the diameter of the fungal growth in the plates containing bacterial CF. Three replicates were used for each treatment and the experiment was repeated twice. The results are expressed as mean values of the replications with standard deviations.

2.2. Screening of antagonistic bacteria and their effect on mycelia morphology Screening for potential antagonistic bacteria amongst the 32 marine isolates was conducted using dual culture tests. Briefly, 5 μL of fungal spore suspension of the pathogen was spot inoculated on the center of the ISP1G agar plates, and 5 μL of the overnight bacterial culture was inoculated at a distance of 2.5 cm from the fungal inoculum. Plates without potential antagonistic bacteria served as a control (C). The plates were incubated at 25 °C for 5 days in the dark. The antagonistic activity of the tested 32 bacterial isolates was detected by measuring the diameter of fungal growth in the plates with the bacteria in reference to the diameter of the fungal growth in control plates. The strains, showing inhibition of fungal growth were selected for further analysis. The effect of the selected strains on the mycelia morphology was also examined. The mycelia of each tested fungi were taken from the colony edge of the 5 day old dual culture plate and observed under a light microscope (Zeiss, Axio imager. A1). Images were recorded with the AxioCamMRC Camera and Axio Vision Software (Zeiss).

2.4.2. Effect of culture filtrate of strain PPM3 on spore germination of pathogenic fungi The effect of CF from strain PPM3 on spore germination of each pathogen was tested by mixing an equal volume (5 mL) of bacterial CF and spore suspension (1 × 106 sporesmL−1) of each pathogenic fungi, respectively, and incubating for 24 h at 25 °C in static conditions. As a control CF was replaced with sterile ISP1 broth. After the incubation 72

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ethanol for 5 min, 2% (v/v) NaOCl solution for 7 min, then rinsed three times with autoclaved distiled water. Additionally the inoculum of the strain PPM3 was prepared by incubation in 50 mL of ISP1 broth for 48 h at 30 °C with shaking at 150 rpm. After incubation the cell suspension was centrifuged at 9000 rpm for 15 min, the cell pellets were resuspended in standard saline and the cell suspension was standardized to 106 CFUmL−1. The cell suspension was applied either as a seed coating by immersing the seeds in cell suspension of strain PPM3 for an hour at room temperature (40 mLkg−1 seeds), or as a soil drench (50 mLkg−1 soil). F. graminearum (106 sporesmL−1) served as the targeted pathogen that was applied to the soil to a final concentration of 50 mLkg−1 soil. Prior to sowing, in order to ensure that all seeds to be used in the pot experiment were healthy and viable, the seeds were germinated on Petri plates containing sterile moist filter paper at 20 °C in the dark for three days. The germinated seeds were sown at 1 cm depth in pots containing 1 kg of potting soil. The following treatments were employed: C- germinated untreated maize seeds were seeded to soil amended only with sterile destiled water, indicating no treatment; P-germinated untreated maize seeds were seeded to soil infested with F. graminearum spores; T1-maize seeds were seeded to the soil previously infected with F. graminearum spores and cell sususpension of strain PPM3 was applied as a soil drench; T2-maize seeds coated with cell suspension of strain PPM3 were seeded to the soil previously infected with F. graminearum spores. Following all the inoculations the pots were placed in a greenhouse chamber with 60% humidity at 25 °C with 16 h light/8 h dark cycle. The plants were grown to the two leaf stage for two weeks and watered every other day. The effects of the bacterial treatments on the plants overall apperence and growth responses (disease incidence, total root lenght, stem height, seedlings fresh and dry weight) were recorded after 14 days. Disease incidence was determined on the basis of the percentage of emerged diseased seedlings. For the dry weight the plants were ovendried at 60 °C until the mass was constant. Three seeds per pot and five pots per treatment were used and the whole experiment was repeated twice. The data are presented as mean values of five replications with standard deviations. Tukey's post hoc test was used to determine significance of mean values for multiple comparison at P < 0.05.

period the germination rate was determined by enumerating germinated spores of treatments and the control using a hemocytometer by following the method of Droby et al. [20]. All treatments consisted of three replicates, and the experiment was repeated twice. The results are presented as mean values of the replications with standard deviation. 2.5. Characterization of culture filtrate of strain PPM3 In order to gain an insight into the chemical nature of the antifungal metabolites produced by the strain PPM3, the antifungal activity of its CF was subjected to stability tests. The tests included resistance to: extreme pH (incubation at pH 3, 7 and 10, for 20 min), thermal conditions (incubation at 50, 70 and 121 °C for 20 min) and enzymatic degradation with proteinase K (20 mgmL−1, Sigma, Aldrich) for 60 min at 45 °C [9]. Following all treatments the antifungal activity of the treated CF was evaluated by the radial growth inhibition assay, as described in „2.4“ part of manuscript. Untreated CF served as a control. The results are expressed as residual antifungal activity (RAA, %) calculated by the following formula: RAA (%) = Rt/R x 100, where Rt-is the diameter of the fungal growth in the plates containing treated CF, R-is the diameter of the fungal growth in the plates containing untreated CF. Each experiment for each treatment consisted of three replicates and was repeated twice. The results are presented as mean values of the replications with standard deviation. 2.6. PCR detection of antifungal genes in strain PPM3 Genomic DNA was extracted from an overnight culture of strain PPM3 and the reference strain B. amyloliquefaciens ATCC 23350T as described in „2.3“ part of manuscript. PCR amplifications of the targeted antifungal genes were carried out using total DNA as a template with specific primer pairs: ITUD-F1 (5′-TTGAAYGTCAGYGCSCCTTT-3′) and ITUD-R1 (5′-TGCGMAAATAATGGSGTCGT-3′); SRFA-F1 (5′-AAAG GATCCAGCCGAAGGGTG-3′) and SRFA-R1 (′5-AAAAAGCTTGTTTTTC TCAAAGAAC-3′) and FENB-F1 (5′-CCTGGAGAAAGAATATACCGTACCY-3′) and FENB-R1 (5′-GCTGGTTCAGTTKGATCACAT-3′) following the method of Chung et al. [21]. The PCR products were tested by 1.5% agarose gel electrophoresis followed by ethidium bromide staining and UV visualization.

3. Results 3.1. Screening of antagonistic bacteria and their effect on mycelia morphology

2.7. Extracellular enzyme production by strain PPM3 The strain PPM3 was screened for the production of four extracellular lytic enzymes: proteases, cellulases, chitinases and amylases using selective agar media (containing, per liter: K2HPO4 3 g, KH2PO4 1 g, MgSO4 0.5 g, yeast extract 3 g and agar 15 g, amended with: 1% (w/v) skimmed milk for proteolytic activity, or 0.5% (w/v) CMC for cellulolytic activity, 1% (w/v) soluble starch for amylase activity and 0.5% (w/v) colloidal chitin for chitinase activity, respectively. Colloidal chitin was prepared from crab shell chitin using concentrated HCl following the method of Hsu and Lockwood [22]. Selective agar media were spot inoculated with an overnight culture of strain PPM3 and the Petri plates were cultivated for 5 days at 30 °C. A clear zone of hydrolysis around the bacterial growth gave an indication of protease/cellulase/chitinase/amylase production. For better visualization of the zones all agar plates were flooded with Gram's iodine (2 g KI and 1 g iodine in 300 mL distilled water) [23].

In the present work out of 32 bacteria isolated from the sand sediment of the Red Sea, in Hurghada, Egypt, strain designated as PPM3 was able to suppress the growth of all tested fungi in a dual culture test, therefore it was selected for further assay (Fig. 1). Microscopic observation of the mycelia growing in the presence of the strain PPM3 revealed certain morphological changes that are characterized by enlarged and swollen hyphae, extensive vacuolization and empty cells devoid of cytoplasm (Fig. 2B and C). Fungi growing in

2.8. In planta biocontrol assay The experiment was done with maize plants growing in potting soil (pH 7.08; 1,23% CaCO3; 13% humus; 0.65% N; 219 mg P2O5 per 100 g of soil; 244 mg K2O per 100 g of soil) that was artificially infested with F. graminearum spores. Maize seeds (Zea mays, L., organically certified, obtained from a local store) were surface disinfected with 70% (v/v)

Fig. 1. Suppression of growth of pathogenic fungi by cell culture of Bacillus sp. strain PPM3 compared to the control (C) in a dual culture test. The plates were incubated at 25 °C for five days in the dark. 73

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Fig. 2. Effect of Bacillus sp. strain PPM3 on Fusarium graminearum mycelia in a dual culture test. Normal mycelia morphology in the absence of Bacillus sp. strain PPM3 (A). Disrupted mycelia showing lysed cells (B) and swollen hyphal cells (C) of F. graminearum in the presence of Bacillus sp. strain PPM3.

The 16S rRNA encoding gene sequence and partial GyrA sequence of the Bacillus sp. strain PPM3 are deposited under the GenBank accession numbers: KP715855 and MF592262.

the absence of strain PPM3 showed well organized mycelia with normal hyphal cells (Fig. 2 A). 3.2. Identification and characterization of strain PPM3

3.3. Antifungal activity of Bacillus sp. strain PPM3 culture filtrate

Preliminary identification and characterization of the strain PPM3 was made by performing standard morphological, physiological and biochemical tests according to Bergeys' Manual of Systematic Bacteriology [16] and by employing API identification systems as described in materials and methods section of the manuscript. The strain PPM3 was G-positive, aerobic, motile, rod shaped and spore forming bacterium. Optimal growth occurred at 30 °C, pH 7 and with 5% of NaCl in the medium. The strain PPM3 was positive for catalase and oxidase production and was able to hydrolyze gelatin and casein. The major phenotypic characteristics are summarized in Table 1. Based on these results strain PPM3 was identified as the member of the genus Bacillus. The phenotypic properties of strain PPM3 were compared to those of the reference strain B. amyloliquefaciens ATCC 23350T, and suggested that the strain PPM3 could be identified as B. amyloliquefaciens (Table 1). The API identification system and API LAB software allowed identifying PPM3 as B. amyloliquefaciens with the higher percentages than 95.5% to database reference strains (Table A1 and Table A2). To correctly identify strain PPM3 a molecular approach was employed. The analysis of the 16S rRNA encoding gene sequence of strain PPM3 showed 99.86% sequence identity with B. amyloliguefaciens WS-8 (CP018200) and B. velezensis T20E-257 (CP021976). The 16S rRNA sequence was not discriminative enough with these closely related species, thus analysis of the partial GyrA sequence was employed, as it has been shown to be effective in distinguishing closely related species of the B. subtilis group [18]. However the analysis of this protein-encoding gene sequence gave similar result as 16S rRNA analysis, showing a 99.50% homology of strain PPM3 with other B. amyloliquefaciens strains and B. velezensis M75 disabling us to accurately identify strain PPM3 to the species level. Therefore, in the following text we will refer to strain PPM3 as Bacillus sp. strain PPM3.

In vitro antifungal assay showed that CF of Bacillus sp. strain PPM3 reduced the mycelial growth of all tested pathogenic fungi compared to the control with substantial variation of inhibition rates. Mucor sp. was found to be the most sensitive with its growth inhibited by 97.5 ± 0.2%, followed by Aspergillus flavus (82.7 ± 0.5%), Fusarium graminearum (57.1 ± 0.5%) and Alternaria sp. (41.5 ± 1.0%) (Fig. 3). In addition to reducing the growth of the mycelia in vitro, the culture filtrate of PPM3 also inhibited spore germination of all tested fungi. In control, which did not contain the CF from the strain PPM3, the spore germination rate of the tested fungi ranged from 82.3 ± 0.5–87.7 ± 1.2%. However, in the presence of the CF from Bacillus sp. strain PPM3 the spore germination of A. flavus and Alternaria sp. decreased to approximately 40%. The germination rate of F. graminearum spores was reduced to 36.2 ± 1.2%, while only 10% of Mucor sp. spores germinated (Fig. 4).

3.4. Antifungal characterization of Bacillus sp. strain PPM3 culture filtrate In order to get an insight into the chemical nature of the compounds produced by Bacillus sp. strain PPM3 its CF was subjected to different stability treatments. The CF of Bacillus sp. strain PPM3 proved the antagonistic potential through production of highly stable secondary metabolites that were resistant to high temperatures, including sterilization at 121 °C, pH treatments in the range of 3–10, as well as proteinase K treatment. More than 97% of the antifungal potential was preserved in all treated CF when these were compared to the untreated controls (Table 2).

Table 1 Morphological, physiological and biochemical characteristics of Bacillus sp. strain PPM3 compared with those of reference strain B. amyloliquefaciens ATCC 23350T. Characteristic

Colony type Cell shape and size (μm) Gram stain Endospore Growth at 45 °C pH tolerance NaCl tolerance Vouges-Proskauer test Citrate utilization Indole test Catalase test Oxidase test

Bacteria

Characteristic

Bacillus sp. strain PPM3

B.amyloliquefaciens ATCC 23350

Undulant, rhizoid, white Rod, 1.6 × 3.7 + + + 5–9 < 10% + + – + +

Undulant, rhizoid, cream Rod, 1.5 × 3.0 + + + 5–8.5 < 10% + + – + +

Gelatin liquefaction test Hydrolysis of: Starch Casein Chitin Cellulose Carbon utilization: Glucose Fructose Xylose Raffinose Mannitol

+positive reaction; - negative reaction; +/− weakly positive reaction. 74

Bacteria Bacillus sp. strain PPM3

B.amyloliquefaciens ATCC 23350

±

+

+ + + +

+ + ± +

+ + + + +

+ + + + +

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Table 2 Stability of the culture filtrates of Bacillus sp. strain PPM3 measured by its antifungal activity after the influence of particular temperature, pH and proteinase K treatment. Culture filtrate of Bacillus sp. strain PPM3

Residual antifungal activity (RAA, %) A. flavus

Heat treatments 50 °C 97.5 70 °C 97.3 121 °C 97.3 pH treatments 3 97.6 7 97.8 10 97.7 Enzymatic degradation Proteinase K 97.8

F. graminearum

Mucor sp.

Alternaria sp.

± 0.5a* ± 0.9a ± 0.4a

97.9 ± 1.2a 98.0 ± 0.7a 97.9 ± 0.3a

97.6 ± 0.8a 97.9 ± 0.6a 97.7 ± 1.2a

97.9 ± 0.6a 97.7 ± 0.8a 97.7 ± 1.2a

± 1.3a ± 1.0a ± 0.9a

97.7 ± 0.5a 98.0 ± 0.2a 97.9 ± 0.5a

97.8 ± 0.5a 97.9 ± 0.5a 97.9 ± 0.7a

97.9 ± 0.3a 98.0 ± 0.1a 97.9 ± 0.3a

± 0.5a

98.0 ± 07a

97.9 ± 0.3a

97.6 ± 1.2a

*Data are presented as a residual antifungal activity (%) of the treated CF, relative to the control (untreated CF). Values are means of three replicates ± SE. Data followed by different superscript letters are significantly different (P < 0.05) according to Tukey's test.

Fig. 3. Antifungal activity of culture filtrate of Bacillus sp. strain PPM3 against four pathogenic fungi: 1-A.flavus, 2-F.graminearum, 3-Mucor sp. and 4-Alternaria sp. in radial growth inhibition assay. The plates were incubated for 5 days at 25 °C. Graphs depict antifungal activity of CF from Bacillus sp. strain PPM3 presented as percentage of growth inhibition PGI (%). Each value represents the mean of three replicates ± SE. Different letters above the bars indicate significant differences (P < 0.05) according to Tukey's test. Fig. 5. Agarose gel showing PCR products of lipopeptide biosynthetase genes encoding for: a) iturin, bacillomycin D and mycosubtilin (482 bp) and b) surfactin (1200 bp). The corresponding lanes are as follows: lane 1-Bacillus sp. strain PPM3; lane 2: lypopeptide producing positive control (B. amyloliquefaciens ATCC 23350T); lane M:molecular DNA marker (GeneRuler DNA Ladder Mix, Thermo Fisher Scientific).

genes: ituD, bamD and fenF were detected from strain PPM3 with one primer pair ITU-F1/R1 (Fig. 5,a). These are conserved genes that encode for malonyl-CoA transacylases, and are involved in the biosynthesis of the lipopeptides: iturin, bacillomycin D and mycosubtilin, respectively [24]. Another gene, involved in the synthesis of surfactin, was also detected with the specific primer pair SRFA-F1/R1 indicating that the strain PPM3 can also produce surfactin (Fig. 5,b). The gene fenB for the synthesis of fengicin B was not detected from Bacillus sp. strain PPM3 with the primers used in this study (data not shown).

Fig. 4. The effect of culture filtrate from Bacillus sp. strain PPM3 (pattern bar) on spore germination of: A. flavus, F. graminerum, Mucor sp. and Alternaria sp. in comparison to untreated control (colored bar). The culture filtrate was mixed with a spore suspension of each pathogenic fungi (1:1, v/v) and incubated at 25 °C for five days in the dark. Each value represents the mean of three replicates ± SE. Different letters above the bars indicate significant differences (P < 0.05) according to Tukey's test.

3.6. Hydrolytic enzyme production by Bacillus sp. strain PPM3 In an in vitro qualitative analysis, the marine strain Bacillus sp. PPM3 tested positive for the production of all four tested hydrolytic enzymes: proteases, chitinases, cellulases and amylases as shown in Table 1. The production of these hydrolytic enzymes was indicated by the formation of clear halo zones around the cells of Bacillus sp. strain PPM3 inoculated on the selective agar media (Fig. A1).

3.5. Detection of genes encoding for antifungal metabolites in Bacillus sp. strain PPM3

3.7. In planta biocontrol activity of Bacillus sp. strain PPM3

A PCR analysis was employed using specific primers for the detection of antifungal biosynthetic genes in Bacillus sp. strain PPM3. Three

It was considered of interest to validate the results regarding the 75

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4. Discussion This study demonstrates biocontrol and plant growth stimulating potential of the newly isolated marine bacterium Bacillus sp. strain PPM3. Bacillus species are commonly found in nature and are mostly exploited as biocontrol agents (BCA) for the suppression of various fungal plant pathogens [3,4,7,24]. The majority of these reported biocontrol bacilli are representatives of the plant associated or terrestrial bacteria [5,6,25,26]. Although certain reports that recognize the beneficial potential of the marine representatives of bacilli are available in the literature, the data about their diversity and biocontrol potential is still scarce [11,12]. Characterization of novel antagonistic bacterial strains and evaluation of their antagonistic potential is important for better understanding of the ecological significance of the biocontrol of plant diseases. The current study reports, for the first time the biocontrol potential of a Bacillus strain isolated from the marine sediment from the Red Sea in Hurghada, Egypt. The strong antagonistic activity of cell culture of the marine strain Bacillus sp. PPM3 observed in the dual culture test was also preserved in its culture filtrate (CF) suggesting that the suppression of pathogen growth by strain PPM3 is due to production of antifungal secondary metabolites. Different inhibition rates of the CF from Bacillus sp. strain PPM3 against the tested fungi indicate that compounds produced by this strain could be of diverse chemical composition. The antifungal activity displayed by a marine strain Bacillus sp. PPM3 in the current study is similar to that reported by others for different terrestrial or plant associated bacilli. Palumbo et al. [5] reported a growth inhibition rate of 22–73% of various Bacillus strains isolated from immature almond flowers against Aspergillus flavus while in another study a B. subtilis strain Y-1 isolated from apple tree tissues suppressed different representatives of Fusarium sp. in vitro and in vivo reaching 64% inhibition [27]. To the best of our knowledge this is the first report showing antagonism of a marine Bacillus species against Mucor sp. The culture filtrate of the marine strain Bacillus sp. PPM3 also showed prominent effectivness in suppressing spore germination of four different pathogenic fungi when its concentration in the mixture with spore suspension of the pathogens was 50% (v/v). Recently Kim et al. [15] reported a biocontrol potential of B. amyloliquefaciens AK-0 isolated from the rhizosphere of Korean ginseng, whose increasing concentrations of culture filtrate (10–50%, v/v) gradually inhibited conidial germination of C. destructans. As fungal spores are the most present as agents of reproduction, dispersal and survival, they have the largest potential of infecting plants and contaminating food. The ability of strain Bacillus sp. PPM3 to inhibit spore development indicates that its culture filtrate can be a useful tool in controlling the spread and the establishment of the diseases caused by these pathogenic fungi. Bacillus species are reported to produce a variety of secondary metabolites with biocontrol activity that are stable at different pH conditions, and resistant to high temperatures and enzymatic treatment [6,9,25]. These stable compounds typically produced by Bacillus species are cyclic lipopeptides, belonging to the iturin, fengycin and surfactin group. Certain marine strains of bacilli are also reported to produce

Fig. 6. Illustrative photograph of maize seedlings grown in greenhouse chamber for two weeks after different treatments: C-seedlings grown without Bacillus sp. strain PPM3 or pathogen F. graminearum; P-seedlings grown in the soil infested with F. graminearum without Bacillus sp. strain PPM3; T1-seedlings grown in F. graminearum infested soil, drenched with Bacillus sp. strain PPM3; T2-- seedlings grown in F. graminearum infested soil after seed coating treatment with Bacillus sp. strain PPM3.

biocontrol activity of Bacillus sp. strain PPM3 with an in planta experiment. The strain Bacillus sp. PPM3 was tested for its ability to control F. graminearum infection in maize plants grown in a potting soil in greenhouse conditions. A significant difference in the plants' overall appearance was observed between the control and the treatments employed in the experiment. Both, the soil drench treatment (T1) and seed coating treatment (T2) showed a complete protection of the seedlings, while untreated, but infected control (P) showed severe decay (Fig. 6). The seed coating treatment with Bacillus sp. strain PPM3 was more effective than the soil drench treatment leading to the reduction of disease incidence of 2.5 ± 0.3% compared to the 81.5 ± 1.1% in untreated infected control (Table 3). The effect on the plant growth responses was also better with T2 treatment when compared to T1 treatment. When Bacillus sp. strain PPM3 was applied as a seed coating, the total root length and stem height of the seedlings increased approximately four times and the fresh and dry weight tripled, compared to untreated but infected seedlings (Table 3). Compared to control without the pathogen, the fresh and dry weight of the maize seedlings, as well as the root and stem height significantly increased after both T1 and T2 treatments with strain PPM3. The total root length and stem height of maize seedlings increased by approximately 50% and 20%, respectively, after soil drench treatment with strain PPM3 (Fig. 6, Table 3). Seed coating treatment was again slightly more effective than soil drench treatment by almost tripling the total root length, from 21.0 ± 1.3 mm to 59.0 ± 1.3 mm, and increasing the stem height by approximately 30% compared to untreated sterile control C (Fig. 6, Table 3). Both treatments increased fresh and dry weight of maize seedlings by 20% and 50%, respectively (Table 3).

Table 3 Disease incidence and growth responses of maize (Zea mays, L.) growing in potting soil in a greenhouse conditions after different treatments. Treatment

Disease incidence (%)

Plant growth responses Total root length (mm)

C P T1 T2

/ 81.5 ± 1.1c* 3.5 ± 0.5b 2.5 ± 0.3a

21.0 15.5 31.3 59.0

± ± ± ±

1.3c 1.7d 1.6b 1.3a

Stem height (mm)

Fresh seedling weight (g)

120.0 ± 1.4c 40.0 ± 1.3d 142.0 ± 1.7b 165.0 ± 1.1a

0.5 0.2 0.6 0.6

± ± ± ±

0.03b 0.04c 0.03a 0.01a

Dry seedling weight (g) 0.2 0.1 0.3 0.3

± ± ± ±

0.03b 0.02c 0.01a 0.02a

*Values are means of five replicates ± SE. Values with different superscript letters (a-d) within one column are significantly different (P < 0.05) according to Tukey's test. 76

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potentially relevant for soil fertility [34]. Additional work is needed to elucidate possible biocontrol mechanisms of Bacillus sp. strain PPM3.

lipopeptides fengycin A and surfactin [11,12]. Lipopeptides act on fungal cell wall influencing membrane permeability. The underlying mechanism includes osmotic perturbation owing to the production of ion-conducting pores caused by iturin and membrane disruption and solubilization, caused by surfactin [28]. Abnormal mycelia morphology (reflected by distorted, swelling and lysed hyphal cells) of the fungi was observed in this study in the presence of Bacillus sp. strain PPM3. This feature, along with fully retained antifungal activity of the CF from strain PPM3 after proteolysis, autoclaving and extreme pH treatment, indicate that some of these lipopeptide compounds are produced by marine strain PPM3. Similar traits, attributed to the production of lipopeptides, have been reported by other authors [9,28]. A PCR analysis conducted in the current study confirmed the production of lipopeptides: iturin, bacillomycin D, mycosubtilin and surfactin by Bacillus sp. strain PPM3. The use of genetic markers such as lypopeptide synthetases genes has been used previously for the identification of new biocontrol agents from the environment [25,29,30]. It is known that the genome of Bacillus species harbours huge gene clusters associated with production of secondary metabolites [24,29]. Kim et al. [15] detected genes involved in the synthesis of: iturin, surfactin, bacillomycin and dificidin in B. amyloliquefaciens AK-O, while another study reported a B. subtilis strain YB-05 with four antifungal genes [30]. Novel marine strain Bacillus sp. PPM3 was similar with at least four antifungal biosynthetase genes. Other antifungal genes may also be present in strain PPM3 but they were not detectable with the primers used in this study. The results of this study indicate that antibiosis could be the main mechanism of action in disease control by Bacillus sp. strain PPM3, with lipopeptides as major compounds responsible for its antagonism against fungi. However, future work on gene expression will be of importance for determining involvement of lipopeptide synthetase genes in biocontrol of the strain PPM3. Novel strain Bacillus sp. PPM3 also tested positive for the production of proteases, chitinases, cellulases and amylases. According to BasurtoCadena et al. [31], protease production by a B. subtilis strain may be responsible for the antagonistic activity against pathogenic bacteria and fungi in vitro. Gowtham et al. [32] reported two cellulase and chitinase producing Bacillus amyloliquefaciens strains which inhibited growth of F. oxysporum f. sp. lycopersici by about 30% in an in vitro assay. Production of hydrolytic enzymes which act on the fungal cell walls containing chitins, glucans and mannoproteins is another mechanism by which phytopathogenic fungi can be inhibited, and could have also contributed to the strong antifungal activity of Bacillus sp. strain PPM3 tested in this study. The high antifungal activity of Bacillus sp. strain PPM3 was also apparent in greenhouse conditions. The biocontrol of maize seedlings was reflected in a curative effect of the cell culture of strain PPM3. In addition to the biocontrol effect (primary effect), the marine strain Bacillus sp. PPM3 also demonstrated the stimulation of maize seedlings (secondary effect) (Fig. 6). The seed bacterization method has been proved to be effective as the biocontrol agent can rapidly grow, covering the surface of the seeds and thus protecting the plants from soil borne pathogens [26,30,33]. Kulimushi et al. [26] have recently reported that pre-treatment of seeds with Bacillus strain S499 demonstrated almost full protection of maize seedlings against various fungal pathogens with additional positive effect on plant growth. Yang et al. [30] found that the treatment of wheat seeds with B. subtilis YB-05 protected the plant more effectively than soil drench treatment. Our results are in accordance with these findings. The beneficial effect of Bacillus sp. strain PPM3 to maize plants in the terms of disease suppression and growth promotion could be due to a combination of direct inhibition of pathogen through production of secondary metabolites like iturins and induction of systemic resistance in plants with surfactin as an elicitor [4] or due to production of primary metabolites like hydrolytic enzymes (cellulases, proteases, chitinases and amylases) which act on the fungal cell walls and are

5. Conclusions In conclusion, this study reports for the first time antagonistic activity of the cells and culture filtrate of novel strain Bacillus sp. PPM3, isolated from marine sediment from the Red Sea in Hurghada, Egypt. Bacillus sp. strain PPM3 exhibited strong antifungal activity in vitro against various plant pathogenic fungi as well as in planta antagonism against F. graminearum infection of maize with additional plant growth stimulating effect. The broad spectrum of action and the stability of antifungal compounds in a wide range of pH and temperature, as well as the genetic potential of Bacillus sp. strain PPM3 to produce antifungal lipopeptides, indicate that this novel marine strain could be a good candidate for development of new biocontrol agents active against soilborn or postharvest pathogenic fungi. Further studies on marine strain Bacillus sp. PPM3 regarding extraction and identification of the antifungal substances contributing to biocontrol activity will be of interest, as well as verification of its effectiveness in field conditions. Conflicts of interest The authors declare no conflict of interest. Acknowledgements The financial support for this investigation given by Ministry of Education, Science and Technological Development of the Republic of Serbia under the project TR 31035 is gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.micpath.2018.04.056. References [1] R.N. Strange, P.R. Scott, Plant disease: a threat to global food security, Annu. Rev. Phytopathol. 43 (2005) 83–116. [2] C.A. Damalas, I.G. Eleftherohorinos, Pesticide exposure, safety issues, and risk assessment indicators, Int. J. Environ. Res. Publ. Health 8 (5) (2011) 1402–1419. [3] B.B. McSpadden Gardener, A. Driks, Overview of the nature and application of biocontrol microbes: Bacillus spp, Phytopathology 94 (11) (2004) 1244–1244. [4] M. Ongena, E. Jourdan, A. Adam, M. Paquot, A. Brans, B. Joris, P. Thonart, Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants, Environ. Microbiol. 9 (4) (2007) 1084–1090. [5] J.D. Palumbo, J.L. Baker, N.E. Mahoney, Isolation of bacterial antagonists of Aspergillus flavus from almonds, Microb. Ecol. 52 (1) (2006) 45–52. [6] S.M. Zhang, Y.X. Wang, L.Q. Meng, J. Li, X.Y. Zhao, X. Cao, X. Chen, A.X. Wang, J.F. Li, Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28, Afr. J. Microbiol. Res. 6 (8) (2012) 1747–1755. [7] H. Cawoy, W. Bettiol, P. Fickers, M. Ongena, Bacillus-based biological control of plant diseases, in: M. Stoytcheva (Ed.), Pesticides in the Modern World-pesticides Use and Management, InTech, Shanghai, 2011, pp. 273–303. [8] J.M. Raaijmakers, M. Vlami, J.T. De Souza, Antibiotic production by bacterial biocontrol agents, Antonie Leeuwenhoek 81 (1) (2002) 537–547. [9] D. Romero, A. de Vicente, R.H. Rakotoaly, S.E. Dufour, J.W. Veening, E. Arrebola, F.M. Cazorla, O.P. Kuipers, M. Paquot, A. Pérez-García, The Iturin and Fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca, Mol Plant Microbiol. 20 (4) (2007) 430–440. [10] J.W. Blunt, B.R. Copp, W.P. Hu, M.H. Munro, P.T. Northcote, M.R. Prinsep, Marine natural products, Nat. Prod. Rep. 26 (2) (2009) 170–244. [11] L. Chen, W. Nan, W. Xuemei, H. Jiangchun, W. Shuin, Characterization of two antifungal lipopeptides produced by Bacillus amyloliquefaciens SH-B10, Bioresour. Technol. 101 (22) (2010) 8822–8827. [12] X. Liu, B. Ren, M. Chen, H. Wang, C.R. Kokare, X. Zhou, J.D. Wang, H. Dai, F. Song, M. Liu, J. Wang, S. Wang, L. Zhang, Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3, Appl. Microbiol. Biotechnol. 87 (5) (2010) 1881–1893. [13] R.A. Pollack, L. Findlay, W. Mondschein, R.R. Modesto, Laboratory Exercises in Microbiology, third ed., John Wiley and Sons, Inc, Hoboken, New Jersey, 2009. [14] M. Webster, G. Dixon, Calcium, pH and inoculum concentration influencing

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