1. 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 .

2. 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.

3. Defensive mutualism concept: See K
Fungal Endophytes of Grasses: A Defensive Mutualism between Plants and Fungi
Keith Clay
Chris Schardl

4. Epichloe Endophyte Benefits
Increased insect resistance
Mammalian feeding deterrence
Increased drought tolerance
Increased heavy metal tolerance
Increased salt stress tolerance
Increased persistence

5. 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

6. 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

7. 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

9. Hypothesized mechanisms of increased salt/drought tolerance
Increase in oxidative stress tolerance
Up-regulation of antioxidants
Up-regulation of aquaporin genes

10. How do endophytes increase abiotic stress tolerance?
Need for metabolomic approaches to ‘listen in’ on endophyte-plant communication/interaction.

11. Bacterial Endophytes Have similar effects on host plants
Common in diverse plants
Seed transmitted
May become intracellular
Have their own defensive chemistries

12. Intracellular bacteria
Bacteria (Burkholderia sp.) in root hairs of switchgrass seedling
Intracellular bacteria
10 μm

13. 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.

14. ‘Cadushy’ cactus: Subpilocereus repandus in Bonaire

15. Fruit with seeds

16. Cadushy seedling

17. Bacteria in root hairs showing recently divided pairs

18. Oxidized bacteria in root hair

19. 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

20. Do nutrients move from microbes to plants?
Agave tequilana experiments
Miguel Beltrán García (Autonomous University of Guadalajara)
Paolo di Mascio
University of Sao Paulo

21. 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).

22. Agave tequilana experiment
Plantlets treated with 15N-labeled Bacillus
Chlorophylls extracted and measured for 15N content using mass spec.

23. 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).

24. 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).

25. Endophytes often show plant growth stimulation
Auxins or other growth regulators
Increased nutrient supply
Phosphate solubilization
Nitrogen acquisition
Iron chelation (siderophores)

26. Is there a microbe-mediated nitrogen scavenging mechanism in plants?
Endophytic bacteria colonize root surfaces and facilitate transfer of nutrients to the host plant.

27. 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…

28. 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.

29. 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.

30. 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.

31. Root hairs without internal bacterial colonization pulled from agarose plates.

32. 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.

33. 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

34. 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.

35. 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.

36. Do invasive and otherwise highly competitive plants carry endophytic microbes that help them compete?

37. 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

38. 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.

39. Systemic distribution of Bacillus amyloliquefaciens on the English ivy epidermis and within vascular tissues.
Bacillus on epidermis
Bacillus in xylem of vascular tissues

40. 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.

41. 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

42. 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.

43. Fungal Inhibition test with Phragmites endophytic bacteria

44. Sclerotinia homoeocarpa control
‘Dollar spot disease in turf’

45. Sclerotinia homoeocarpa and Sandy LB4

46. Sclerotinia homoeocarpa and West 9

47. Sclerotinia homoeocarpa and Microbacterium

48. Gliocladium Control

49. 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.

50. 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.

51. 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.

52. Fusarium and Sandy LB4

53. 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.

54. 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.

55. 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.

56. 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

57. 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.

58. 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.

59. 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).

60. 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)

61. Do the Phragmites bacteria increase growth of grasses in Soil?

62. How do the Phragmites bacteria associate with grasses?
Seedlings growing on 0.7% agarose and stained with DAB for reactive oxygen

63. Burmuda grass (Cynodon dactylon)

64. 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

65. 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.

66. 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.

67. 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.

68. 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.

69. Cells from the root meristem show internal presence of bacteria (arrows).

70. 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.

71. 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.

72. 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.

73. 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.

74. Poa annua

75. Poa annua seedling root in agarose without bacteria showing absence of root hairs. Stained using DAB to visualize reactive oxygen.

76. Poa annua seedling root bearing strain West 9, showing production of root hairs.

77. Poa annua root hair showing internal bacteria (arrows).

78. 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.

79. 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.

80. Oxidizing bacteria can also be seen in the root parenchyma cells.

81. 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.

82. 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!

83. 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).

84. 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!

85. 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.

86. Normal root hair function involves internal cytoplasmic streaming.
Cytoplasmic streaming cessation in blocked hairs may reduce
nutrient absorption.

87. What happens when Phragmites bacteria colonize dandelion seedlings?

88. 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

89. Dandelion Mortality Experiment Examples

90. 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

91. 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).

92. 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