Abstract:

As a result of climate change, salinity has become a major abiotic stress that reduces plant growth and crop productivity worldwide. A variety of endophytic bacteria alleviate salt stress; however, their ecology and biotechnological potential has not been fully realized. To address this gap, a collection of 117 endophytic bacteria were isolated from wild populations of the herb Thymus vulgaris in Sheikh Zuweid and Rafah of North Sinai Province, Egypt, and identified based on their 16S rRNA gene sequences. The endophytes were highly diverse, including 17 genera and 30 species. The number of bacterial species obtained from root tissues was higher (n = 18) compared to stem (n = 14) and leaf (n = 11) tissue. The endophytic bacteria exhibited several plant growth-promoting activities in vitro, including auxin synthesis, diazotrophy, phosphate solubilization, siderophore production, and production of lytic enzymes (i.e., chitinase, cellulase, protease, and lipase). Three endophytes representing Bacillus species associated with T. vulgaris such as EGY05, EGY21, and EGY25 were selected based on their ex-situ activities for growth chamber assays to test for their ability to promote the growth of tomato (Solanum lycopersicum L.) under various NaCl concentrations (50-200 mM). All three strains significantly (P < 0.05) promoted the growth of tomato plants under salt stress, compared to uninoculated controls. In addition, inoculated tomato plants by all tested strains decreased (P < 0.05) the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase). Six strains, representing Bacillus and Enterobacter species EGY01, EGY05, EGY16, EGY21, EGY25, and EGY31 were selected based on in vitro antagonistic activity to F. oxysporum for pot experiments under salt stress. All tested strains reduced the disease severity index (DSI) of tomato plants at all tested salt concentrations. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of B. subtilis (EGY16) showed at least ten compounds were known to have antimicrobial activity, with the major peaks being benzene, 1,3-dimethyl-, p-xylene, dibutyl phthalate, bis (2-ethylhexyl) phthalate, and tetracosane. This study demonstrates that diverse endophytes grow in wild thyme populations and that some are able to alleviate salinity stress and inhibit F. oxysporum pathogenesis, making them promising candidates for biofertilizers and biocontrol agents.

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Beneficial Endophytic Bacterial Populations Associated With Medicinal Plant <em>Thymus vulgaris</em> Alleviate Salt Stress and Confer Resistance to <em>Fusarium oxysporum</em>

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Figure S1

A summary of species present at all sites of 117 culturable endophytes from Thymus vulgaris. based on 16S rRNA gene sequences.

Click here for additional data file.^{(1.7M, jpeg)}

Figure S2

Distribution of endophytic isolates and species isolated from Thymus vulgaris on different media.

Click here for additional data file.^{(1.5M, jpeg)}

Figure S3

Confirmation of plant growth promotion traits by a color change or halo zone on the selective medium (A: Protease; B: Cellulase; C: Lipase; D: Siderophore; E: Chitin; F: Nitrogen). (G) response of endophytic strains to F. oxysporum (H) response of endophytic strains to F. fulva, (I) response of endophytic strains to A. solani.

Click here for additional data file.^{(4.8M, jpeg)}

Figure S4

Effect of endophytic isolates on the growth of tomato (Solanum lycopersicum L.) compared with an uninoculated control without salt (CK^{) and inoculated control with salt (CK}^).

Click here for additional data file.^{(7.7M, jpeg)}

Figure S5

Effect of inculation of endophytic strains on the root of tomato plants under salt stress.

Click here for additional data file.^{(10M, jpeg)}

Table S1

Compositions of the different media used for the isolation of endophytic bacteria from Thymus vulgaris.

Click here for additional data file.^{(37K, doc)}

Table S2

Fungal pathogens used in this study.

Click here for additional data file.^{(31K, doc)}

Table S3

GC-MS identified components of the antibiosis crude extract of EGY16 and F. oxysporum mixture at pH7. (Volatile compounds are listed in ascending order of Retention Time).

Click here for additional data file.^{(54K, doc)}

^{CAS Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Urumqi, China,}

^{Department of Biological, Marine Sciences, and Environmental Agriculture, Institute for Post Graduate Environmental Studies, Arish University, Al-Arish, Egypt,}

^{Department of Environmental Protection, Faculty of Environmental Agricultural Sciences, Arish University, Al-Arish, Egypt,}

^{School of Life Sciences, University of Nevada, Las Vegas, NV, United States,}

^{State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China,}

Edited by: Massimiliano Morelli, Italian National Research Council, Italy

Reviewed by: Zhansheng Wu, Shihezi University, China; Petra Lovecka, University of Chemistry and Technology in Prague, Czechia

*Correspondence: Osama Abdalla Abdelshafy Mohamad, gE.udE.urA@amasO; Wen-Jun Li, nc.ude.usys.liam@3nujnewil; Li Li, moc.kooltuo@obob.ilil

†These authors have contributed equally to this work

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science

Edited by: Massimiliano Morelli, Italian National Research Council, Italy

Reviewed by: Zhansheng Wu, Shihezi University, China; Petra Lovecka, University of Chemistry and Technology in Prague, Czechia

Received 2019 Jul 13; Accepted 2020 Jan 14.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Abstract

As a result of climate change, salinity has become a major abiotic stress that reduces plant growth and crop productivity worldwide. A variety of endophytic bacteria alleviate salt stress; however, their ecology and biotechnological potential has not been fully realized. To address this gap, a collection of 117 endophytic bacteria were isolated from wild populations of the herb Thymus vulgaris in Sheikh Zuweid and Rafah of North Sinai Province, Egypt, and identified based on their 16S rRNA gene sequences. The endophytes were highly diverse, including 17 genera and 30 species. The number of bacterial species obtained from root tissues was higher (n = 18) compared to stem (n = 14) and leaf (n = 11) tissue. The endophytic bacteria exhibited several plant growth-promoting activities in vitro, including auxin synthesis, diazotrophy, phosphate solubilization, siderophore production, and production of lytic enzymes (i.e., chitinase, cellulase, protease, and lipase). Three endophytes representing Bacillus species associated with T. vulgaris such as EGY05, EGY21, and EGY25 were selected based on their ex-situ activities for growth chamber assays to test for their ability to promote the growth of tomato (Solanum lycopersicum L.) under various NaCl concentrations (50–200 mM). All three strains significantly (P < 0.05) promoted the growth of tomato plants under salt stress, compared to uninoculated controls. In addition, inoculated tomato plants by all tested strains decreased (P < 0.05) the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase). Six strains, representing Bacillus and Enterobacter species EGY01, EGY05, EGY16, EGY21, EGY25, and EGY31 were selected based on in vitro antagonistic activity to F. oxysporum for pot experiments under salt stress. All tested strains reduced the disease severity index (DSI) of tomato plants at all tested salt concentrations. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of B. subtilis (EGY16) showed at least ten compounds were known to have antimicrobial activity, with the major peaks being benzene, 1,3-dimethyl-, p-xylene, dibutyl phthalate, bis (2-ethylhexyl) phthalate, and tetracosane. This study demonstrates that diverse endophytes grow in wild thyme populations and that some are able to alleviate salinity stress and inhibit F. oxysporum pathogenesis, making them promising candidates for biofertilizers and biocontrol agents.

Keywords: environmental microbiology, medicinal plants, endophytes, biofertilizer, biocontrol, Fusarium oxysporum, Bacillus subtilis, Thymus vulgaris

Abstract

Acknowledgments

We would like to thank Dr. Chad L. Cross, School of Medicine, University of Nevada for helping on detailed statistical analysis.

Acknowledgments

Click here for additional data file.^{(1.7M, jpeg)}Click here for additional data file.^{(1.5M, jpeg)}Click here for additional data file.^{(4.8M, jpeg)}Click here for additional data file.^{(7.7M, jpeg)}Click here for additional data file.^{(10M, jpeg)}Click here for additional data file.^{(37K, doc)}Click here for additional data file.^{(31K, doc)}Click here for additional data file.^{(54K, doc)}

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