Supplementary material

Toxicity effects of africanus L. essential oil against some molecularly identified phytopathogenic bacterial strains Said I. Behirya, Mervat EL-Hefnyb, Mohamed Z.M. Salemc,* a Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Egypt b Department of Floriculture, Ornamental Horticulture and Garden Design, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt c Forestry and Wood Technology Department, Faculty of Agriculture (EL-Shatby), Alexandria University, Alexandria, Egypt

ORCID Mohamed Z.M. Salem: http://orcid.org/0000-0002-3961-7935 Mervat EL-Hefny: https://orcid.org/0000-0001-9760-3956

*Corresponding author. Mohamed Z.M. Salem E-mail addresses: [email protected], [email protected]

1

Abstract

Essential oil (EO) from L. was evaluated against the growth of some phytopathogenic bacteria including Agrobacerium tumifaciens, Dickeya solani, Erwinia amylovora, Pseudomonas cichorii and Serratia pulmithica using the disc diffusion method and minimum inhibitory concentration (MIC) evaluation. Ten compounds in the EO with dominance of Artemisia ketone (2,5,5-trimethyl-2,6-heptadien-4-one) (77.92%) and ledol (19.92%) were revealed. The antibacterial activity indicated efficacy of essential oil against majority of strains isolated. The most effective action was recorded against D. solani, by 7.5 and 10 µL of oil, with 18.33 mm and 100 μg/mL as zone inhibition and MIC, respectively, whereas the lowest activity was exhibited against P. cichorii (diameter inhibition=6.66 mm at 10 µL of oil, MIC=100 μg/mL). The strain S. pulmithica appears to be resistant to the oil when the activity is measured by 10 µL of oil but its growth inhibition was reported with a MIC of 100 μg/mL.

Keywords: Antibacterial activity; Eriocephalus africanus; Essential oil; Phytopathogenic bacteria

2. Experimental 2.1. Chemicals Saline, sodium hypochlorite, Sucrose Nutrient Agar (SNA), glycerol nutrient agar, TE buffer (10 mM Tris-HCl, 1 mM E DTA, pH 8.0), Tris EDTA, Sodium Dodecyl Sulphate (SDS), proteinase, K, NaCl, CTAB, Phenol, chloroform, isopropanol, 70% ethanol, Tris HCL, KCl, MgCl2, dNTP, DMSO and Tween 20 were used. All chemicals were of analytical grade and were used as received without any further purification and were obtained from Sigma-Aldrich. 2.2. Bacterial isolation Isolation trials were carried out from infected pear leaves, cabbage leaves, guava root galls with well developed symptoms collected from Beheira Governorate, during 2018 in Egypt. materials were washed with tap water; soaked in 1% sodium hypochlorite for 3 min, rinsed three times in sterile distilled water and gently blotted dry on sterilized tissue paper. Samples taken from the internal tissues of plant materials were macerated in 3 mL sterile saline solution (0.8%) in a sterile mortar. A loop full of the resulting suspension was streaked on 5% Sucrose Nutrient Agar (SNA) for pear leaves bacteria and the glycerol nutrient agar for other bacteria (Billing et al. 1960). Single colonies observed after 48 h incubation at 28°C were isolated and consequently

2 purified. Two potato soft rot bacterial isolates (Dickeya solani and Serratia pulmithica), used in this study, were obtained from Dr. Said Behiry, Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria, Egypt (Table S1). All isolates were kept in 2% glycerol nutrient agar slants for later use. 2.3. Phenotypic identification The morphology of bacterial isolates (cell and colony morphology, motility) was investigated by light microscopy. The physiological and biochemical characteristics were carried out according to protocols described by Jones and Geider (2001). 2.4. DNA extraction protocol Bacterial isolates were grown overnight in LB medium at 28 °C with constant shaking at 200 rpm. Cells from 3 mL culture were pelleted by centrifugation at 6000 × g for 5 min. Culture cells were washed in TE buffer (10 mM Tris-HCl, 1 mM E DTA, pH 8.0), then resuspended in a mixture of 567 μL Tris EDTA, 30 μL of 10% Sodium Dodecyl Sulphate (SDS) and 3 μL proteinase K (20 mg/mL). After incubation at 37 °C for 1 h, 100 μL of 5M NaCl and 80 μL of CTAB/NaCl solution were added and the tubes were inverted well before incubation for 10 min in a water bath at 65 °C. Phenol/chloroform (24:1) mixture (0.8 mL) was then added, mixed thoroughly and centrifuged at 11000 × g for 5 min. The aqueous supernatant was then taken and the phenol/chloroform step was repeated one more time. DNA was precipitated by adding equal volume of isopropanol and washed with 70% ethanol. DNA pellets were suspended in 100 μL sterilized distilled water (Ausubel et al. 1995). 2.5. PCR analyses Full length of 16S rRNA gene (1550 bp) was amplified for all bacterial isolates using two universal primers P0 (5`-GAAGAGTTTGATCCTGGCTCAG-3`) and P6 (5`- CTACGGCTACCTTGTTACGA-3`). The PCR reaction mixtures (25 μL total volume) contained 10mM Tris HCL (pH=8.8), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 10 pM each primer, 0.5 U of DNA polymerase (Dynazyme TM II) and 2 μL DNA template (at 20 ng). PCR analysis was performed with the following thermal program: 1 cycle of 5 min at 95 °C, 34 cycles (45s min at 94 °C, 1 min at 50 °C, 2 min at 72 °C) and an additional cycle of 10 min at 72 °C in water bath. PCR products were subjected for following analyzes procedure. 2.6. Sequencing of 16S rRNA gene and alignment

3

The amplified products of 16S rRNA gene (1550 bp) were purified using Centri-Sep spin columns. The products were sequenced by using Big Dye terminator cycle sequencing kit and resolved on an ABI PRISM model 310 automated DNA sequencer at Microgen Company. Comparisons with sequences in the GenBank database were achieved in BLASTN searches at the National Center for Biotechnology Information site (http://www.ncbi. nlm.nih.gov). The 16S rDNA sequences have been deposited in the GenBank database under the accession numbers (Table S1). 2.7. Preparation of essential oil Eriocephalus africanus L. plant was supplied from Nursery of Floriculture, Ornamental Horticulture and Garden Design, Faculty of Agriculture during 2018 at Alexandria, Egypt. The plant was identified by Dr. Mervat EL-Hefny and vouchered (MERV003) at the Department of Forestry and Wood Technology, Alexandria University, Alexandria, Egypt. Leaves were separated from the branches then cut into small pieces and 100 g were hydrodistilled in a Clevenger apparatus for 3-4 h to obtain the essential oil (yellow oil with 1.3 mL/100 g fresh weight).

2.8. GC/MS analysis of essential oil The essential oil was analyzed for their chemical composition using Focus GC-DSQ Mass Spectrometer (Thermo Scientific, Austin, TX, USA) apparatus at Atomic and Molecular Physics Unit, Experimental Nuclear Physics Department, Nuclear Research Centre, Egyptian Atomic Energy Authority, Inshas, Cairo, Egypt. The GC-MS with a direct capillary column TG–5MS (30 m x 0.25 µm x 0.25 µm film thickness). The column oven temperature was initially held at 45°C and then increased by 5°C/min to 250°C hold 2 min then increased to 280 °C at the rate of 10 °C/min. The injector and detector (MS transfer line) temperatures were kept at 250°C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 2 min and diluted samples of 1 µL were injected automatically using Autosampler AS3000 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40–550 in full scan mode. The ion source was set at 200. The components were identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database (Adams 2007 and 2009) (Fig. S1). 2.9. Antibacterial activity of E. africanus essential oil

4

The antibacterial evaluation was measured using the disc diffusion method (Bauer et al. 1966). Autoclaved filter paper discs (5 mm in diameter) were used and each has received 1.25, 2.5, 5, 7.5, and 10 µL of oil. Negative control (100% DMSO and Tween 20) was used, and all tests were performed in triplicates. The minimum inhibitory concentrations (MICs) were determined by serial dilution of the oil (50-2000 μg/mL) (Eloff et al. 1998). 2.9. Statistical analysis The values of antibacterial activity (inhibition zones) are presented as mean±SD. Analysis of variance (ANOVA) was used to evaluate the significant difference among various treatments, with a significance criterion of p = 0.05 using SAS system (2001). References Adams, R.P., 2007. Identification of essential oil components by gas chromatography/mass spectroscopy, 4th Edn., Allured Publishing, Carol Stream, IL., ISBN:13- 9781932633214, p 804. Adams, R.P., 2009. Geographic variation in the leaf essential oils of Hesperocyparis (cupressus) abramsiana, H. goveniana and H. macrocarpa: Systematic implications. Phytologia. 91 (1), 226- 243. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1995. Preparation of Genomic DNA from Bacteria. In: Current Protocols in Molecular Biology, Ausubel, F.A., R.E. Brent, D.D. Kingston, J.G. Moore and J.A. Seidman (Eds.). John Wiley and Sons, New York. Bauer, A.W., Kirby, W.M., Sherris, J.C., Turck, M., 1996. Antibiotic susceptibility testing by a standardized single disk method. Amer. J. Clin. Pathol. 45, 49-496. Billing, E., Crosse, J.E. Garrett, C.M., 1960. Laboratory diagnosis of fire blight and bacterial blossom blight of pear. Plant Pathol. 9, 19-25. Eloff, J.N., 1998. A sensitive and quick method to determine the minimum inhibitory concentration of plant extracts for bacteria. Planta Med. 60, 1-8. Jones, A.L., Geider, K., 2001. Gram-Negative Bacteria, In: Laboratory Guide for Identification of Plant Pathogenic Bacteria, Schaad, N.W., J.B. Jones and W. Chun (Eds.). 3rd Edn., APS Press, St Paul, MN., USA. SAS, 2001. Users Guide: Statistics (Release 8.02), SAS Institute, Cary, NC.

5

Table S1. Morphological traits, physiological, biochemical reactions and accession numbers of bacterial isolates obtained from infected samples

Bacterial isolates Characteristic

se

acid

source

lactose

sucrose sucrose

Manitol

Maltose

Motility Gluco Dulcitol

trehalose

Ketolactose

production

-

Shape(rods)

Catalase testCatalase

Gram staining Gram

3

methylglucoside

Mucoid growth

Growth at 37°C

-

Oxidase reaction

Citrate Citrate utilization

Indoleproduction

Accession number Accession

Anaerobic growth

R. substanceR. from

Urease production

Pigmenton kingsB

α

Alkali from tartaric

Growth in 5%NaCl Malonate utilization MH368043 This study + - + + + - + + - nd ------nd a - - a a a a a Erwinia amylovora MH368044 This study + - + + - - + + + nd - - - - + - + - - - a a a a a Agrobacterium tumefaciens MH368045 This study + - + + + + - - + + - - - + - + + - a - a - - a - Pseudomonas cichorii HF569035 Dr.Said Dickeya solani Behiry HF569038 Dr.Said Serratia plymuthica Behiry

+: More than 80% of isolates gave positive reaction, -: Less than 20% of isolates gave negative reaction, a: acid, nd: not determined

6

Table S2. Chemical composition of essential oil from E. africanus leaves

Compound Name RT Area Molecular Molecular Standard Reverse (min) % Formula Weight index standard index Yomogi alcohol 8.17 0.26 C10H18O 154 882 886 Artemisia ketone (2,5,5- 10.61 77.92 C10H16O 152 943 968 Trimethyl-2,6-heptadien-4-one) Limonene dioxide 2 23.6 0.12 C10H16O2 168 756 807 13-Tetradec-11-yn-1-ol 23.84 0.26 C14H24O 208 760 790 Dihydromyrcene 24.69 0.19 C10H18O 166 722 741 Longipinenepoxide 25.24 0.13 C15H24O 220 791 796 β-caryophellene 25.94 0.37 C15H24 204 763 773 α-Farnesol 26.16 0.29 C15H26O 222 725 750 9-(1-Methylethylidene)- 26.32 0.53 C12H20 164 764 827 bicyclo[6.1.0]nonane Ledol 26.84 19.92 C15H26O 222 874 875

RT: Retention time (min)

7

Table S3. Antibacterial activity (IZ mm) of essential oil against five phytopathogenic bacterial isolates

Oil volume (µL) Inhibition zone (mm) against the bacterial isolates Agrobacerium Dickeya solani Erwinia Pseudomonas Serratia tumifaciens amylovora cichorii pulmithica 1.25 5.66f± 0.57 7.00c± 0.00 0.00d 0.00c 0.00 2.5 10.33e± 0.57 7.00c± 0.00 0.00d 0.00c 0.00 5 12.33d± 0.57 11.00b± 0.00 0.00d 0.00c 0.00 7.5 16.33c± 0.57 18.33a± 1.52 6.00c± 0.00 0.00c 0.00 10 18.00b± 1.00 18.33a± 1.52 7.00b± 0.00 6.66b±0.57 0.00

Negative controla 0.00g 0.00d 0.00d 0.00c 0.00 MIC 100 100 2000 100 100 Values are mean ± standard deviation Means with the same letter within the same column are not significantly difference according to LSD0.05 a Negative control (100% DMSO and Tween 20) MIC: Minimum inhibitory concentration (μg/mL)

8

Fig. S1. GC/MS chromatogram of the essential oil from E. africanus leaves

9