Diversification of B. subtilis during experimental evolution on A. thaliana leads to synergism in root colonization of evolved subpopulations

SummaryThe soil bacterium Bacillus subtilis is known to suppress pathogens as well as promote plant growth. However, in order to fully exploit the potential as natural fertilizer, we need a better understanding of the interactions between B. subtilis and plants. Here, B. subtilis was examined for root colonization through experimental evolution on Arabidopsis thaliana. The populations evolved rapidly, improved in root colonization and diversified into three distinct morphotypes. In order to better understand the adaptation that had taken place, single evolved isolates from the final transfer were randomly selected for further characterization, revealing changes in growth and pellicle formation in medium supplemented with plant polysaccharides. Intriguingly, certain evolved isolates showed improved root colonization only on the plant species they evolved on, but not on another plant species, namely tomato, suggesting A. thaliana specific adaption paths. Finally, synergism in plant root colonization was observed for a mix of all three morphotypes, as the mix performed better than the sum of its constituents in monoculture. Our results suggest, that genetic diversification occurs in an ecological relevant setting on plant roots and proves to be a stable strategy for root colonization.Significance StatementUnderstanding how plant-growth-promoting rhizobacteria colonize plant roots is crucial to fully utilize their potential for agricultural applications. Here, we utilized experimental evolution of the PGPR Bacillus subtilis on Arabidopsis thaliana to study root colonization. We revealed that evolving populations rapidly improve in root colonization and diversify into distinct morphotypes. Notably, improved root colonization by evolved isolates was observed on A. thaliana, not on tomato. Moreover, isolates of distinct morphotypes interacted during root colonization and the mixture of morphotypes showed higher productivity then predicted. These findings suggest that genetic diversification might be a stable strategy to maximize root colonization.

Download Full-text

Experimental evolution of Bacillus subtilis on Arabidopsis thaliana roots reveals fast adaptation and improved root colonization in the presence of soil microbes

10.1101/2021.07.09.451762 ◽

2021 ◽

Author(s):

Mathilde Nordgaard ◽

Christopher Blake ◽

Gergely Maroti ◽

Mikael L. Strube ◽

Akos T. Kovacs

Keyword(s):

Arabidopsis Thaliana ◽

Bacillus Subtilis ◽

Experimental Evolution ◽

Soil Microbes ◽

Root Colonization ◽

Plant Root ◽

Phenotypic Characterization ◽

Plant Interactions ◽

Plant Polysaccharide ◽

Selective Environment

The soil ubiquitous Bacillus subtilis is known to promote plant growth and protect plants against disease. These characteristics make B. subtilis highly relevant in an agricultural perspective, fueling the interest in studying B. subtilis-plant interactions. Here, we employ an experimental evolution approach to explore adaptation of B. subtilis to Arabidopsis thaliana roots. B. subtilis rapidly adapts to the plant root environment, as evidenced by improved root colonizers observed already after 12 consecutive transfers between seedlings in a hydroponic setup. Further phenotypic characterization of evolved isolates from transfer 30 revealed that increased root colonization was associated with robust biofilm formation in response to the plant polysaccharide xylan. Additionally, several evolved isolates across independent populations were impaired in motility, a redundant trait in the selective environment. Interestingly, two evolved isolates outcompeted the ancestor during competition on the root but suffered a fitness disadvantage in non-selective environment, demonstrating an evolutionary cost of adaptation to the plant root. Finally, increased root colonization by a selected evolved isolate was also demonstrated in the presence of resident soil microbes. Our findings provide novel insights into how a well-known PGPR rapidly adapts to an ecologically relevant environment and reveal evolutionary consequences that are fundamental to consider when evolving strains for biocontrol purposes.

Download Full-text

Bacterial Community Members Increase Bacillus subtilis Maintenance on the Roots of Arabidopsis thaliana

Phytobiomes Journal ◽

10.1094/pbiomes-02-20-0019-r ◽

2020 ◽

Vol 4 (4) ◽

pp. 303-313

Author(s):

Noam Eckshtain-Levi ◽

Susanna Leigh Harris ◽

Reizo Quilat Roscios ◽

Elizabeth Anne Shank

Keyword(s):

Arabidopsis Thaliana ◽

Bacillus Subtilis ◽

Crop Production ◽

Crop Yields ◽

Plant Root ◽

Plant Roots ◽

Plant Growth Promoting Bacteria ◽

Bacterial Strains ◽

Root Microbiome ◽

Over Time

Plant-growth-promoting bacteria (PGPB) are used to improve plant health and promote crop production. However, because some PGPB (including Bacillus subtilis) do not maintain substantial colonization on plant roots over time, it is unclear how effective PGPB are throughout the plant growing cycle. A better understanding of the dynamics of plant root community assembly is needed to develop and harness the potential of PGPB. Although B. subtilis is often a member of the root microbiome, it does not efficiently monoassociate with plant roots. We hypothesized that B. subtilis may require other primary colonizers to efficiently associate with plant roots. We utilized a previously designed hydroponic system to add bacteria to Arabidopsis thaliana roots and monitor their attachment over time. We inoculated seedlings with B. subtilis and individual bacterial isolates from the native A. thaliana root microbiome either alone or together. We then measured how the coinoculum affected the ability of B. subtilis to colonize and maintain on A. thaliana roots. We screened 96 fully genome-sequenced strains and identified five bacterial strains that were able to significantly improve the maintenance of B. subtilis. Three of these rhizobacteria also increased the maintenance of two strains of B. amyloliquefaciens commonly used in commercially available bioadditives. These results not only illustrate the utility of this model system to address questions about plant–microbe interactions and how other bacteria affect the ability of PGPB to maintain their relationships with plant roots but also may help inform future agricultural interventions to increase crop yields. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Download Full-text

Deletion of Rap‐Phr systems in Bacillus subtilis influences in vitro biofilm formation and plant root colonization

MicrobiologyOpen ◽

10.1002/mbo3.1212 ◽

2021 ◽

Vol 10 (3) ◽

Author(s):

Mathilde Nordgaard ◽

Rasmus Møller Rosenbek Mortensen ◽

Nikolaj Kaae Kirk ◽

Ramses Gallegos‐Monterrosa ◽

Ákos T. Kovács

Keyword(s):

Bacillus Subtilis ◽

Biofilm Formation ◽

Root Colonization ◽

Plant Root

Download Full-text

Root-colonizing and soil-borne communities of arbuscular mycorrhizal fungi in a temperate forest understorey

Botany ◽

10.1139/cjb-2013-0058 ◽

2014 ◽

Vol 92 (4) ◽

pp. 277-285 ◽

Cited By ~ 67

Author(s):

Ülle Saks ◽

John Davison ◽

Maarja Öpik ◽

Martti Vasar ◽

Mari Moora ◽

...

Keyword(s):

Plant Species ◽

Mycorrhizal Fungi ◽

Plant Root ◽

Arbuscular Mycorrhizal ◽

Small Subunit ◽

Plant Roots ◽

Individual Plant ◽

Amf Communities ◽

Arbuscular Mycorrhizal Fungal ◽

Phylogenetic Dispersion

We analyzed arbuscular mycorrhizal fungal (AMF) communities in plant root samples from a natural forest ecosystem — a primeval forest in Järvselja, Estonia. AMF small-subunit (SSU) ribosomal RNA genes were subjected to 454-pyrosequencing and BLAST-based taxonomic identification. Seventy-six AMF sequence groups (virtual taxa, VT) were identified from plant roots. Taken together with seven additional VT recorded in an earlier investigation of soil AMF communities at the site, this represents the highest number of AMF reported from a single ecosystem to date. The six study plant species hosted similar AMF communities. However, AMF community composition in plant roots was significantly different from that in soil and considerably more VT were retrieved from roots than from soil. AMF VT identified from plant roots as a whole and from individual plant species were frequently phylogenetically clustered compared with local and global taxon pools, suggesting that nonrandom assembly processes, notably habitat filtering, may have shaped fungal assemblages. In contrast, the phylogenetic dispersion of AMF communities in soil did not differ from random subsets of the local or global taxon pools.

Download Full-text

Investigating the role of root exudates in recruiting Streptomyces bacteria to the Arabidopsis thaliana root microbiome

10.1101/2020.09.09.290742 ◽

2020 ◽

Author(s):

Sarah F. Worsley ◽

Michael Macey ◽

Sam Prudence ◽

Barrie Wilkinson ◽

J. Colin Murrell ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Stable Isotope ◽

Plant Growth ◽

Root Exudates ◽

Soil Bacteria ◽

Stable Isotope Probing ◽

Plant Roots ◽

Plant Metabolites ◽

Complex Organic ◽

Root Microbiome

AbstractStreptomyces species are saprophytic soil bacteria that produce a diverse array of specialised metabolites, including half of all known antibiotics. They are also rhizobacteria and plant endophytes that can promote plant growth and protect against disease. Several studies have shown that streptomycetes are enriched in the rhizosphere and endosphere of the model plant Arabidopsis thaliana. Here, we set out to test the hypothesis that they are attracted to plant roots by root exudates, and specifically by the plant phytohormone salicylate, which they might use as a nutrient source. We confirmed a previously published report that salicylate over-producing cpr5 plants are colonised more readily by streptomycetes but found that salicylate-deficient sid2-2 and pad4 plants had the same levels of root colonisation by Streptomyces bacteria as the wild-type plants. We then tested eight genome sequenced Streptomyces endophyte strains in vitro and found that none were attracted to or could grow on salicylate as a sole carbon source. We next used 13CO2 DNA stable isotope probing to test whether Streptomyces species can feed off a wider range of plant metabolites but found that Streptomyces bacteria were outcompeted by faster growing proteobacteria and did not incorporate photosynthetically fixed carbon into their DNA. We conclude that, given their saprotrophic nature and under conditions of high competition, streptomycetes most likely feed on more complex organic material shed by growing plant roots. Understanding the factors that impact the competitiveness of strains in the plant root microbiome could have consequences for the effective application of biocontrol strains.ImportanceStreptomyces bacteria are ubiquitous in soil but their role as rhizobacteria is less well studied. Our recent work demonstrated that streptomycetes isolated from A. thaliana roots can promote growth and protect against disease across plant species and that plant growth hormones can modulate the production of bioactive specialised metabolites by these strains. Here we used 13CO2 DNA stable isotope probing to identify which bacteria feed on plant metabolites in the A. thaliana rhizosphere and, for the first time, in the endosphere. We found that Streptomyces species are outcompeted for these metabolites by faster growing proteobacteria and instead likely subsist on more complex organic material such as cellulose derived from plant cell material that is shed from the roots. This work thus reveals the “winners and losers” in the battle between soil bacteria for plant metabolites and could inform the development of methods to apply streptomycetes as plant growth-promoting agents.

Download Full-text

Exploring plant root-fungal interactions in a neotropical freshwater wetland

Botanical Sciences ◽

10.17129/botsci.2221 ◽

2019 ◽

Vol 97 (4) ◽

pp. 661-674

Author(s):

Jazmín Santillán-Manjarrez ◽

A. Penelope Solis-Hernández ◽

Patricia Castilla-Hernández ◽

Ignacio E. Maldonado-Mendoza ◽

Gilberto Vela-Correa ◽

...

Keyword(s):

Water Column ◽

Plant Species ◽

Mycorrhizal Fungi ◽

Root Colonization ◽

Arbuscular Mycorrhizal ◽

Plant Roots ◽

Freshwater Wetland ◽

Fungal Colonization ◽

Dark Septate Endophyte ◽

Dry Conditions

Background: Wetlands in Neotropics harbor high fungal diversity, including arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). This study describes the interaction of plant roots with AMF and DSE in a freshwater wetland belonging to a hotspot of biodiversity.Hypothesis: Differential root colonization between arbuscular mycorrhizal and dark septate endophyte fungi is influenced by plant species and abiotic conditions in a freshwater wetland. Studied species: Plant species colonized by arbuscular mycorrhizal and dark septate endophyte fungi.Methods: Properties of soils and the water column, floristic composition, root colonization by AMF and DSE, and molecular identification of AMF inside roots were studied.Results: Soils were Gleysol and flooded during the rainy season. Most of identified plant species were herbaceous, with Cyperus articulatus and Mimosa pigra as the dominant species. Seven of 8 analyzed plant species exhibited differential co-colonization between AMF and DSE. Repeated sampling for one year under flooding/dry conditions demonstrated that C. articulatus and M. pigra were mainly associated with DSE and AMF, respectively. A positive correlation between dissolved O2 in the water column and fungal colonization was observed in C. articulatus. Glomerales and Archaeosporales were molecularly identified inside roots containing arbuscules of M. pigra.Conclusions: Findings highlight differential coexistence between AMF and DSE in plant roots; fungal colonization was influenced by flooding/dry conditions in a neotropical wetland; the community of AMF inside arbusculated roots of M. pigra includes at least four clades.

Download Full-text

Nonspecific responses in plant growth, yield, and root colonization of noncereal crop plants to inoculation with Azospirillum brasilense Cd

Canadian Journal of Botany ◽

10.1139/b89-175 ◽

1989 ◽

Vol 67 (5) ◽

pp. 1317-1324 ◽

Cited By ~ 53

Author(s):

Y. Bashan ◽

Y. Ream ◽

Hanna Levanony ◽

A. Sade

Keyword(s):

Plant Growth ◽

Plant Species ◽

Azospirillum Brasilense ◽

Root Colonization ◽

Growth Parameters ◽

Growth Yield ◽

Dry Weight ◽

Plant Response ◽

Crop Plants ◽

High Yield

Inoculation of seven different crop plant species by Azospirillum brasilense Cd resulted in an increase in plant yield, as well as in changes in several other plant parameters, in tomato, eggplant, pepper, and cotton plants. Analysis of 56 different experiments revealed that the rate of success (positive plant response) ranged from 71 to 75 %. The dry weight of plants and yield responses ranged from significantly high yield increases to negligible or no response in similarly performed experiments. The average increases in yield in the positive response experiments were 30, 23, 18, and 16% for tomato, eggplant, pepper, and cotton, respectively. Significant earlier maturation was also detected in the four responding plant species. The response of other plant growth parameters varied between plant species. The level of root colonization by A. brasilense Cd was similar in all four plant species, i.e. root population size of 5 × 105 cfu/g fresh weight root. It is suggested that inoculation of noncereal crop plants by the cereal-root originate A. brasilense Cd is nonspecific with inconsistency in plant response to inoculation.

Download Full-text

Investigating the Role of Root Exudates in Recruiting Streptomyces Bacteria to the Arabidopsis thaliana Microbiome

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.686110 ◽

2021 ◽

Vol 8 ◽

Author(s):

Sarah F. Worsley ◽

Michael C. Macey ◽

Samuel M. M. Prudence ◽

Barrie Wilkinson ◽

J. Colin Murrell ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Root Exudates ◽

Plant Root ◽

Stable Isotope Probing ◽

Plant Roots ◽

Plant Metabolites ◽

Published Report ◽

Promote Plant Growth

Streptomyces species are saprophytic soil bacteria that produce a diverse array of specialized metabolites, including half of all known antibiotics. They are also rhizobacteria and plant endophytes that can promote plant growth and protect against disease. Several studies have shown that streptomycetes are enriched in the rhizosphere and endosphere of the model plant Arabidopsis thaliana. Here, we set out to test the hypothesis that they are attracted to plant roots by root exudates, and specifically by the plant phytohormone salicylate, which they might use as a nutrient source. We confirmed a previously published report that salicylate over-producing cpr5 plants are colonized more readily by streptomycetes but found that salicylate-deficient sid2-2 and pad4 plants had the same levels of root colonization by Streptomyces bacteria as the wild-type plants. We then tested eight genome sequenced Streptomyces endophyte strains in vitro and found that none were attracted to or could grow on salicylate as a sole carbon source. We next used 13CO2 DNA stable isotope probing to test whether Streptomyces species can feed off a wider range of plant metabolites but found that Streptomyces bacteria were outcompeted by faster growing proteobacteria and did not incorporate photosynthetically fixed carbon into their DNA. We conclude that, given their saprotrophic nature and under conditions of high competition, streptomycetes most likely feed on more complex organic material shed by growing plant roots. Understanding the factors that impact the competitiveness of strains in the plant root microbiome could have consequences for the effective application of biocontrol strains.

Download Full-text

Cyclic di-AMP Acts as an Extracellular Signal That ImpactsBacillus subtilisBiofilm Formation and Plant Attachment

mBio ◽

10.1128/mbio.00341-18 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 26

Author(s):

Loni Townsley ◽

Sarah M. Yannarell ◽

Tuanh Ngoc Huynh ◽

Joshua J. Woodward ◽

Elizabeth A. Shank

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Intracellular Signaling ◽

Biofilm Formation ◽

Plant Root ◽

Second Messenger ◽

Pathogen Resistance ◽

Plant Roots ◽

Extracellular Signal ◽

The Impact

ABSTRACTThere is a growing appreciation for the impact that bacteria have on higher organisms. Plant roots often harbor beneficial microbes, such as the Gram-positive rhizobacteriumBacillus subtilis, that influence their growth and susceptibility to disease. The ability to form surface-attached microbial communities called biofilms is crucial for the ability ofB. subtilisto adhere to and protect plant roots. In this study, strains harboring deletions of theB. subtilisgenes known to synthesize and degrade the second messenger cyclic di-adenylate monophosphate (c-di-AMP) were examined for their involvement in biofilm formation and plant attachment. We found that intracellular production of c-di-AMP impacts colony biofilm architecture, biofilm gene expression, and plant attachment inB. subtilis. We also show thatB. subtilissecretes c-di-AMP and that putative c-di-AMP transporters impact biofilm formation and plant root colonization. Taken together, our data describe a new role for c-di-AMP as a chemical signal that affects important cellular processes in the environmentally and agriculturally important soil bacteriumB. subtilis. These results suggest that the “intracellular” signaling molecule c-di-AMP may also play a previously unappreciated role in interbacterial cell-cell communication within plant microbiomes.IMPORTANCEPlants harbor bacterial communities on their roots that can significantly impact their growth and pathogen resistance. In most cases, however, the signals that mediate host-microbe and microbe-microbe interactions within these communities are unknown. A detailed understanding of these interaction mechanisms could facilitate the manipulation of these communities for agricultural or environmental purposes.Bacillus subtilisis a plant-growth-promoting bacterium that adheres to roots by forming biofilms. We therefore began by exploring signals that might impact its biofilm formation. We found thatB. subtilissecretes c-di-AMP and that the ability to produce, degrade, or transport cyclic di-adenylate monophosphate (c-di-AMP; a common bacterial second messenger) affectsB. subtilisbiofilm gene expression and plant attachment. To our knowledge, this is the first demonstration of c-di-AMP impacting a mutualist host-microbe association and suggests that c-di-AMP may function as a previously unappreciated extracellular signal able to mediate interactions within plant microbiomes.

Download Full-text

Plant Growth Promotion by Spermidine-Producing Bacillus subtilis OKB105

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-14-0010-r ◽

2014 ◽

Vol 27 (7) ◽

pp. 655-663 ◽

Cited By ~ 67

Author(s):

Shan-Shan Xie ◽

Hui-Jun Wu ◽

Hao-Yu Zang ◽

Li-Ming Wu ◽

Qing-Qing Zhu ◽

...

Keyword(s):

Bacillus Subtilis ◽

Plant Growth ◽

High Performance ◽

Growth Promotion ◽

Plant Root ◽

Enzyme Linked Immunosorbent Assay ◽

Plant Interactions ◽

Root Cells ◽

Plant Growth Promoting ◽

Growth Promoting

The interaction between plants and plant-growth-promoting rhizobacteria (PGPR) is a complex, reciprocal process. On the one hand, plant compounds such as carbohydrates and amino acids serve as energy sources for PGPR. On the other hand, PGPR promote plant growth by synthesizing plant hormones and increasing mineral availability in the soil. Here, we evaluated the growth-promoting activity of Bacillus subtilis OKB105 and identified genes associated with this activity. The genes yecA (encoding a putative amino acid/polyamine permease) and speB (encoding agmatinase) are involved in the secretion or synthesis of polyamine in B. subtilis OKB105. Disruption of either gene abolished the growth-promoting activity of the bacterium, which was restored when polyamine synthesis was complemented. Moreover, high-performance liquid chromatography analysis of culture filtrates of OKB105 and its derivatives demonstrated that spermidine, a common polyamine, is the pivotal plant-growth-promoting compound. In addition, real-time polymerase chain reaction analysis revealed that treatment with B. subtilis OKB105 induced expansin gene (Nt-EXPA1 and Nt-EXPA2) expression and inhibited the expression of the ethylene biosynthesis gene ACO1. Furthermore, enzyme-linked immunosorbent assay analysis showed that the ethylene content in plant root cells decreased in response to spermidine produced by OKB105. Therefore, during plant interactions, OKB105 may produce and secrete spermidine, which induces expansin production and lowers ethylene levels.

Download Full-text