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How Do Endophytes Function to Enhance Host Plant Growth and Survival?
James White Kate Kingsley Marcos Soares Kurt Kowalski Qiang Chen Ivelisse Irizarry April Micci Camille English Monica Torres Hai-Yan Li Surendra Gond Marshall Bergen Department of Plant Biology and Pathology Rutgers University, New Jersey *U.S. Geological Survey, Great Lakes Science Center, Ann Arbor, Michigan, USA .
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What are Endophytes? Endophytic/endosymbioti c non-pathogenic microbes (usually fungi or bacteria) are present asymptomatically in tissues of all plants Epichloe endophytes Fungal hyphae of endophyte in stem (culm) tissue of tall fescue grass.
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Defensive mutualism concept: See K
Fungal Endophytes of Grasses: A Defensive Mutualism between Plants and Fungi Keith Clay Chris Schardl
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Epichloe Endophyte Benefits
Increased insect resistance Mammalian feeding deterrence Increased drought tolerance Increased heavy metal tolerance Increased salt stress tolerance Increased persistence
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Endophytes Increase Abiotic Stress Tolerance in Plants
Endophytes have been shown to increase tolerance of hosts to high temperatures, drought and salt stress, heavy metals Stress tolerance enhancing endophytes may be used to enhance the capacity of crops to tolerate stresses of marginal lands
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Regina Redman, Rusty Rodriguez, et al. (2002)
Endophyte Curvularia protuberata imparts thermotolerance to Dichanthelium lanuginosum (Panic Grass) plants in geothermal soils of Yellowstone Nat. Park. Science 298: 1581
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Study of Invasive Phragmites australis
Does invasive Phragmites use microbes (endophytic fungi) to adapt it to hypersaline soils? We examined fungal endophytes from high salt and low salt sites (Soares et al. 2016, Biological Invasions DOI: /s z). USGS Collaborator: Kurt Kowalski Marcos Soares
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Hypothesized mechanisms of increased salt/drought tolerance
Increase in oxidative stress tolerance Up-regulation of antioxidants Up-regulation of aquaporin genes
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How do endophytes increase abiotic stress tolerance?
Need for metabolomic approaches to ‘listen in’ on endophyte-plant communication/interaction.
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Bacterial Endophytes Have similar effects on host plants
Common in diverse plants Seed transmitted May become intracellular Have their own defensive chemistries
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Intracellular bacteria
Bacteria (Burkholderia sp.) in root hairs of switchgrass seedling Intracellular bacteria 10 μm
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TEM of Bacillus subtilis L-forms
Photo by Mark Leaver, New Castle University, UK Jeff Errington New Castle University, UK L-forms in bacteria L-forms are bacterial cells that do not form cell walls (also called ‘cell wall deficient bacteria’). L-forms typically are seen inside eukaryotic cells. They are thought to be a mechanism to evade host defense response. L-form bacteria are typically variable in size.
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‘Cadushy’ cactus: Subpilocereus repandus in Bonaire
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Fruit with seeds
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Cadushy seedling
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Bacteria in root hairs showing recently divided pairs
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Oxidized bacteria in root hair
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Consume Microbes to obtain nutrients?
Figure 1. Roots of axenically grown Arabidopsis and tomato were incubated with E coli or yeast expressing green fluorescent protein (GFPE. coli or GFPyeast). ‘Turning the Table: Plants Consume Microbes as a Source of Nutrients’ “Rhizophagy” Do plant roots Consume Microbes to obtain nutrients? Chany Paungfoo-Lonhienne Bartosz Adamczyk et al Proteins as nitrogen source for plants: A short story about exudation of proteases by plant roots. Plant signaling & behavior 07/2010; 5(7):817-9. Paungfoo-Lonhienne C et al Turning theTable: Plants Consume Microbes as a Source of Nutrients. PLoS ONE 5(7): e11915, doi: /journal.pone
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Do nutrients move from microbes to plants?
Agave tequilana experiments Miguel Beltrán García (Autonomous University of Guadalajara) Paolo di Mascio University of Sao Paulo
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Intracellular colonization: Bacterial endophytes appear to become intracellular. The photo to the left is the root tip of a blue agave seedling showing Bacillus tequilensis (toluidine blue stained) within cells. Beltran-Garcia, M.J. et al. Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria. Sci. Rep. 4, 6938; DOI: /srep06938 (2014). The photo above shows Bacillus tequilensis from agar cultures (toluidine blue).
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Agave tequilana experiment
Plantlets treated with 15N-labeled Bacillus Chlorophylls extracted and measured for 15N content using mass spec.
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20 40 60 80 100 872 873 874 875 876 20x 100x 875.2 875.6 876.0 876.4 876.8 . A B C D Relative Abundance of Pheophytin Isotopomers (%) m/z 50 150 871.57 872.57 873.57 874.57 875.57 876.57 -50 Relative Abundance of Phaeophytin Isotopomers This study demonstrates that 15N-labeled nitrogen in the Bacillus tequilensis moves from the endophyte into plant chlorophyll. From Beltran-Garcia, M.J. et al Sci. Rep. 4, 6938; DOI: /srep06938 (2014).
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Agave tequilana experiment
Agave plantlets treated weekly for two months with living and heat-killed 15N- labeled Bacillus tequilensis Using living bacteria resulted in 2X nitrogen movement into plants compared to heat-killed bacteria From Beltran-Garcia, M.J. et al Sci. Rep. 4, 6938; DOI: /srep06938 (2014).
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Endophytes often show plant growth stimulation
Auxins or other growth regulators Increased nutrient supply Phosphate solubilization Nitrogen acquisition Iron chelation (siderophores)
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Is there a microbe-mediated nitrogen scavenging mechanism in plants?
Endophytic bacteria colonize root surfaces and facilitate transfer of nutrients to the host plant.
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Microbe-mediated nitrogen scavenging Phragmites australis study
Isotopic 15N assimilation into Plants Marcos Soares Microbacterium oxydans is an endophyte of Phragmites (isolated from shoots) that colonizes all surfaces of roots. In this experiment plants were placed in gas chambers with 15N-labeled gasses for 10 days. Then plants dissected and 15N measured using mass spec analysis. Enhanced assimilation into plants is not necessarily N2 fixation because gas contained other nitrogenous compounds, including nitrous oxides, nitrates, nitrites, etc…
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Microbacterium oxydans B1 (from Phragmites) stimulates root hair development in sterile burmuda grass seedlings but colonizes the rhizoplane rather than entering into the root cells themselves. The bacteria in the rhizoplane evenly colonize the root hairs giving them a granular appearance (arrow), but do not elicit a strong reactive oxygen response in the grass roothair, thus roothairs stain lightly.
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Note the granular appearance due to bacteria on the rhizoplane (arrow)
Note the granular appearance due to bacteria on the rhizoplane (arrow). In a previous study by Marcos Soares Microbacterium oxydans was found to promote the growth of Phragmites australis in greenhouse experiments and was also found to enhance nitrogen assimilation into roots. Full coverage of the rhizoplane of root hairs may enhance microbe-mediated nitrogen scavenging and growth promotion effects.
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Roothairs (arrow) pulled from the agarose remove all the bacteria in the rhizoplane, demonstrating that the bacteria exclusively colonized the rhizoplane rather than enter into the root cells themselves.
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Root hairs without internal bacterial colonization pulled from agarose plates.
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Maximum 15N assimilation into roots translates to maximum growth promotion efficiency!
Application of Microbacterium oxydans to plants more than doubles the growth compared to controls where bacteria were not applied.
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Some plants require seed vectored (or recruited
Some plants require seed vectored (or recruited?) microbes/endophytes for proper root development! Example: fescue grass (Festuca arundinacea) Microbes carried on seed surfaces on adherent paleas and lemmas Removal of microbes from seeds by rigorous surface disinfection (3% NaOCl for 40 mins) results in loss of root hair formation in agarose Root hair formation can be initiated by incorporating 0.01% proteins in agarose
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Root hairs do not form on seedling roots without bacteria!
To conduct studies on turf grasses, seedlings are used. Seeds can be surface sterilized to remove surface microbes and seedlings germinated on sterile 0.7% water agarose media to conduct experiments. Roots that penetrate into the agarose are assessed for root hairs. These are tall fescue seedlings showing two types of roots: the blue arrow shows the initial surface root that bears many long root hairs but does not penetrate into agarose. The black arrow indicates the downward oriented seedling root that penetrates into the agarose. These seedlings bear symbiotic bacteria. If bacteria are not present on roots the initial horizontal root continues to elongate but the second soil penetrating root does not form.
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Close examination of bacteria on roots shows that they are often surrounded by denatured proteins. Everything staining blue in the figures below is protein. Bacteria on roots may be secreting enzymes to degrade components of the root cell walls or polysaccharide exudates. The grass roots may denature the secreted bacterial enzymes through secretion of ROS. The plant may further degrade the denatured proteins to smaller peptide or oligopeptide units that may be absorbed. Experiments are needed to evaluate whether these grass roots secrete proteases. Figs Bacteria on root hairs of cool-season grass seedlings; stained with DAB/peroxidase for reactive oxygen (brown) and counter stained with aniline blue/lactophenol for protein. 1. Bacteria (arrow) on surface of root hair of Lolium perenne seedling. 2. Bacteria and bacterial protein (arrows) on surface of root hair of Poa annua seedling. 3. Bacteria (arrows) on surface of root hair of Poa annua seedling. 4. Bacteria (small arrows) and denatured proteins (large arrows) on the surface of root hair of Poa annua seedling.
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Do invasive and otherwise highly competitive plants carry endophytic microbes that help them compete?
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Endophytes for plant defense: English ivy experiment
Marcos Soares English ivy plants harbor a bacterial endophyte (Bacillus amyloliquefaciens) in all populations we have examined. USGS Collaborator: Kurt Kowalski
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Endophyte containing Endophyte free
E+ and E- plants treated with the pathogen Alternaria alternata. The endophyte free plants showed significant spotting and necrosis caused by the pathogen Alternaria alternata. Endophyte infected plants were free of disease symptoms.
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Systemic distribution of Bacillus amyloliquefaciens on the English ivy epidermis and within vascular tissues. Bacillus on epidermis Bacillus in xylem of vascular tissues
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BODYGUARD HYPOTHESIS Endophytes produce antimicrobial compounds and thus may serve as a ‘bodyguard’ for plants. The endophyte may reduce colonization of the exterior and interior of the plant.
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Defensive arsenal of Bacillus endophytes: surfactin lipopeptides
Cyclic peptide head + Fatty acid tail Direct antibiosis effects on fungi and bacteria Cause up-regulation of stress resistance genes in plants Produced in seedlings
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Are endophytes of Phragmites defensive against pathogens?
Kate Kingsley Marcos Soares April Micci Kurt Kowalski USGS Collaborator Goals of study: Evaluate whether Phragmites bacteria inhibit soil borne fungal pathogens.
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Fungal Inhibition test with Phragmites endophytic bacteria
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Sclerotinia homoeocarpa control
‘Dollar spot disease in turf’
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Sclerotinia homoeocarpa and Sandy LB4
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Sclerotinia homoeocarpa and West 9
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Sclerotinia homoeocarpa and Microbacterium
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Gliocladium Control
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Gliocladium and Sandy LB4
Like the control, the fungus has grown over the whole disk. Sandy LB4 changes the behavior of the fungus by inhibiting sporulation; it does not affect the radial growth.
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Gliocladium and West 9 At first glance West 9 does not appear to inhibit as strongly as Sandy LB4 but note how thin the area of sporulation is on the left side of the bacteria. Physical behavior of the fungus changes as seen with Sandy LB4, though less dramatically.
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Gliocladium and Microbacterium
Modest inhibition; sporulation may even have increased. Some physical changes and inhibition are most noteable after the fungus passes through the bacteria.
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Fusarium and Sandy LB4
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Do the bacteria protect Poa annua from damping off disease?
Grass: Poa annua Treatments: 1. no bacteria, no Fusarium 2. no bacteria, Fusarium 3. Sandy LB4, Fusarium 4. West 9, Fusarium 5. Microbacterium, Fusariumå Set up: Fusarium was harvested from petri dish then suspended in 100 ml of sterilized water. 10 ml of suspension was mixed into magenta box soil. Seeds were inoculated with bacteria before being placed into magenta box. Approximately 10 seeds per box were added. Three magenta boxes per treatment.
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Numbers are the sum of seedling in the 3 magenta boxes.
Results Treatment no bacteria + Fusarium showed the highest damping off. While all the bacteria offered some protection, Sandy LB4 protected the seedlings best. Treatment Day 8 Day 15 No bacteria + no Fusarium 20 23 Fusarium 12 Sandy LB4 + Fusarium 15 West 9 + 16 Microbacterium + Fusarium 18 Numbers are the sum of seedling in the 3 magenta boxes.
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Sandy LB4 on the left and Fusarium only treatment on the right.
In the magenta boxes, we also found that Sandy LB4 and West Yes 9 had less visible hypha present on the soil surface. Sandy LB4 on the left and Fusarium only treatment on the right. Sandy LB4 + Fusarium Fusarium only Sandy LB4’s soil (left photo) appears darker because soil surface hyphae are suppressed.
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Do the Phragmites bacteria move from the plant out into the soil?
We dipped a sterile probe into the soil in between seedlings and then streaked it onto plates to see if the bacteria were going out into the soil. Sandy LB 4 on the left West Yes 9 on the right The images indicate both bacteria colonize Fusarium and spread into the soil
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Summary These few Pseudomonas sp. bacteria we have investigated show evidence of inhibiting and altering the behavior of pathogenic fungi. The magenta box pilot study supports our hypothesis that these endophytic bacteria are capable of providing protection from some pathogens and may influence soil ecology.
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Are endophytes of non-cultivated plants compatible with related species?
Kate Kingsley Marcos Soares April Micci Kurt Kowalski USGS Collaborator Goals of study: Evaluate whether Phragmites bacteria are compatible with other grass species. Explore the patterns of colonization of grass seedlings. Explore the range of grass species that microbes are compatible with.
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Outline of Methods 17 bacteria were obtained from seeds, seedlings or shoots of invasive Phragmites australis and Fallopia japonica (Japanese knotweed). Bacteria were screened for their capacity to induce formation of root hairs in axenic grass seedlings (derived from seeds that were surface disinfected for 40 mins in 4% Na-hypochlorite to remove all surface vectored microbes) and grown in 0.7% agarose. Bacteria were stained in roots by overnight staining of agarose plates bearing seedlings with diaminobenzidine (DAB) to visualize reactive oxygen staining of bacteria and roots. Roots were examined through the reverse of Petri plates using a compound light microscope (Mags = X 10, X20, X40).
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Some microbes shown to induce root hair formation in grasses:
Results Some microbes shown to induce root hair formation in grasses: 1) From invasive Phragmites australis A. Microbacterium oxydans B2 (from sterilized shoot tips) B. Unidentified bacterium strain West 9 (from washed seeds) C. Unidentified bacterium strain Sandy LB4 (from washed seeds). D. TBN (from Japanese knotweed)
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Do the Phragmites bacteria increase growth of grasses in Soil?
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How do the Phragmites bacteria associate with grasses?
Seedlings growing on 0.7% agarose and stained with DAB for reactive oxygen
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Burmuda grass (Cynodon dactylon)
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More developed region of
Burmuda (Cynodon dactylon) grass seedling root in agarose without bacteria showing absence of root hairs More developed region of seedling root Root tip
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Bacterial strain West 9 is a root meristem colonizer: Bacterial strain West 9 colonizes the Burmuda grass seedling root-tip meristem and becomes intracellular in meristem cells then transmits to all parts of the seedling root and root hairs as the meristem cells divide. In these seedlings root hairs form very close to the root tip. West 9 restores root hair development in sterile seedlings. Root stained in agarose using DAB (diaminobenzidine) to visualize reactive oxygen (H2O2) production around bacteria.
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Root hairs (arrow) behind the root-tip are seen to stain brown internally due to production of reactive oxygen in the vicinity of the intracellular bacteria.
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Early developing root hairs show internal presence of the spherical wall-less L-forms (arrows) of bacterial strain West 9. Roots were stained with DAB then counterstained with aniline blue stain.
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Early developing root parenchyma cells also show chains of the spherical L-form bacteria. The root was stained with DAB then counterstained with aniline blue stain.
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Cells from the root meristem show internal presence of bacteria (arrows).
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The bacteria are degraded internally
The bacteria are degraded internally. They are seen to swell and become clear internally (arrows). Clusters of degraded bacteria are seen in these root parenchyma cells.
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More degrading bacteria (arrows) in root parenchyma cells
More degrading bacteria (arrows) in root parenchyma cells. Bacterial L-forms swell as their internal contents vanish due to degradation.
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As the root continues to develop the bacteria within root hairs and root parenchyma cells are seen to disappear, apparently due to complete degradation by the root cells. Here several root hairs are seen to be free of internal bacteria (arrows). Bacteria that were intercellular are not degrade and remain as endophytes in the plant.
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In older parts of the root all root hairs are seen to be free of internal bacteria and reactive oxygen staining (brown coloration). In the parenchyma cells of the root axis the brown coloration also lessens due to degradation of the internal bacteria.
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Poa annua
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Poa annua seedling root in agarose without bacteria showing absence of root hairs. Stained using DAB to visualize reactive oxygen.
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Poa annua seedling root bearing strain West 9, showing production of root hairs.
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Poa annua root hair showing internal bacteria (arrows).
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Strain Sandy LB4 (also from Phragmites) is also a meristem colonizer
Strain Sandy LB4 (also from Phragmites) is also a meristem colonizer. It is shown here in a Burmuda grass seedling root.
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The bacteria appear to persist longer in Burmuda grass seedling roots than strain West 9. This may mean that the effect of Sandy LB4 in Burmuda grass seedlings is of longer duration than that of West 9.
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Oxidizing bacteria can also be seen in the root parenchyma cells.
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Close-up of Burmuda grass root hair from Petri dish reverse showing irregular colonization or bacterial banding (arrows). The bacterial strain TBN from Japanese knotweed stimulates root hair development but is superficial on the hairs and its coverage is uneven on the hair surface. Plate was stained overnight with DAB. TBN was an intracellular endophyte in Japanese knotweed.
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Endophytes may be specifically adapted to their hosts!
Strain Tiny-Bac-N (from Japanese Knotweed) on Burmuda grass grown on filter paper showing that root hairs are free of internal colonization. A cluster of bacteria (arrow) is seen adjacent a hair. The cluster may have been superficial on hairs prior to slide preparation. Stained with aniline blue stain. TBN is intracellular in its host (Japanese Knotweed; family Polygonaceae). This suggest that it is only partially compatible with grasses. Experiments in soils show that TBN colonizes grasses and stimulates root hairs but does not increase growth of grasses. Endophytes may be specifically adapted to their hosts!
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TBN is intracellular in root hairs of Japanese knotweed
TBN is intracellular in root hairs of Japanese knotweed. Bacteria appear to be located in the periplasmic space (between cell wall and plasma membrane) of root hairs. Bacteria are often seen in recently divided pairs (arrows).
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Phragmites endophytes may show partial compatibility!
What happens when a Phragmites bacterium is put into seedlings of Rumex crispus (Polygonaceae)? Bacteria stimulate root hairs Bacteria enter root cells Bacteria occlude hairs with large masses of bacterial L-forms Phragmites endophytes may show partial compatibility!
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Strain Sandy LB4 from Phragmites in root hair of Rumex crispus
Root hair blockage may affect the absorptive function of root hairs. This may reduce the competitive ability of seedlings.
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Normal root hair function involves internal cytoplasmic streaming.
Cytoplasmic streaming cessation in blocked hairs may reduce nutrient absorption.
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What happens when Phragmites bacteria colonize dandelion seedlings?
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Inoculated with Sandy LB4
Dandelion seedlings (2-wk-old) showing high mortality when colonized by Phragmites bacterium Sandy LB4 No bacteria Inoculated with Sandy LB4
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Dandelion Mortality Experiment Examples
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How do the Phragmites bacteria help Phragmites?
Preliminary studies suggest: Phragmites endophytes increase root and shoot growth of grass hosts Phragmites endophytes colonize soil around roots and suppress growth of soil borne fungal pathogens of plants We hypothesize that through partial compatibility Phragmites endophytes reduce competitiveness of distantly-related competitor plants
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Summary: What do endophytes do?
Growth promotional endophytes may colonize plants inter- or intra-cellularly. Endophytes that colonize roots may enhance nutrient acquisition into plants Endophytes may defend plants from biotic and abiotic stresses Partial compatibility of endophytes to seedlings of distantly-related plants could result in antagonistic effects on those seedlings (thus functioning as bioherbicides).
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Acknowledgements Monica Torres Qiang Chen Camille English Marshall Bergen Chris Zambell Mariusz Tadych Kate Kingsley Mohini Pra Somu Ray Sullivan Haiyan Li Ivelisse Irizarry Marcos Antonio Soares Surendra Gond April Micci For Funding: John and Christina Craighead Foundation; New Jersey Agric. Exp. Sta.; USDA NIFA Multistate 3147; Rutgers Center for Turfgrass Science
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