Quantification of Endophytic TraitsJack EmeryUW Undergraduate – MicrobiologyDoty Lab

What are endophytes?

• Bacteria living within a plant, in a symbiotic relationship with its host

• Nutrient exchanges• Rhizobia of legume

plants are well known N-fixers

Root Nodules (Cotswold Grass Seeds Direct 2015)

GFP tagged endophytes in vivo (Doty)

Poplar Colonizing Endophytes

• Not localized to root system

• Nutrient poor riparian zones

• Little nitrogen in river

• Potentially ideal symbiotic bacteria Snoqualmie River Site (Doty)

Non-Leguminous Endophytes

• Could greatly reduce fertilizer costs/use

• The goal is to use a consortia that is multifunctional

• Quantifying plant growth promoting factors across different strains

• Can protect host in a variety of ways

Grasses

Crops

Trees

• SNOQ 117.2• WPR 5-2• WPL 5-2• WPL 7-2• WPL 8-1• WP 40• WP 41• WP 42

NF Sol. P IAA Trp+ ug/ml Trp- Sid. Sym. IDWPL 1-2 * * 5.39 0.039 - - WPL 2-1 - ** 6.9 0.12 - - Massilia sp.WPR 2-2 46.1 2.73 + - WPR 2-4 ** *** 19.4 0.06 - - WPR 3-1 ** *** 12 0.2 + + Enterobacter sp.WPR 4-2 * 15 4 + Vibrionaceae sp.WPR 4-3 ** * 10.165 + - Enterobacter sp.WPR 5-1 * * 7.2 6.86 + + / - Mucilaginibacter sp.WPR 5-2 ** - 23.092 1.048 + +++ Pseudomonas sp.WPR 5-2 * - 33.93 - + +++ Pseudomonas sp.WPL 5-2 ** ** 25 2 + +++ Rahnella sp.WPR 6-1 ** * 49.2 2.4885 + - Pseudomonas fluorescensWPL 6-2 ** ** 55.35 8.005 - - Pseudomonas sp.WPR 6-3 * - 22.5 - - + / - WPR 6-4 * 56 1 - Vibrionaceae sp.WPR 7-1 * *** 29.35 14.5 + - Pseudomonas sp.WPL 7-1 ** * 10.29 0.18 + / - + WPL 7-2 ** * 27.57 2.86 ++ ++ WPL 7-3 ** * 3.22 0.39 + + WPL 8-1 ** * 19.2 0.29 +++ +++ WPL 8-2 * * 9.087 0.151 + - WPL 8-3 * * 24.07 0.459 - --- Dickeya dadantii

WP5 ** - 4.4 - - + / -

Fluorescent on MGL

Red Incresed Plant Growth

8 Different Endophytes

Lab Class Results (Doty)

NifH

• NifH gene is a subunit of Nitrogenase, the enzyme that fixes N2 into soluble form.

• If NifH is present a strong band appears at 380 bp

Siderophore Production

EDTA (Chemwiki)

• A siderophore reduces the insoluble form of Fe2+ to Fe3+ making it more bioavailable

• Chelation of the metal makes transport easier

Siderophore Production

• EPS formation• Inconsistent standards• Need to know specific

siderophore to quantify

SNOQ 117.2

WPR 5-2

WPL 5-2 WPL 7-2 WPL 8-1 WP 40 WP 41 WP 420

10

20

30

40

50

60

70

IAA Production/Cell Density

Trp -Trp +

(IAA µg/mL)/ Abs 600 nm

IAA Production

• IAA is part of the plant auxin family and is a rooting hormone

• Tryptophan is precursor

• Trp is generally a difficult AA to produce

Phosphate Solubilization• pH of environment

lowered to solubilize Ca3(PO4)2

Insoluble phosphate agar plate (Doty)

• Endophytes to grew in Pikovskaya broth for three days

• Supernatant with “Reagent C”

• Took absorbance at 820 nm

Phosphate Solubilization

SNOQ 117.2

WPR 5-2 WPL 5-2 WPL 7-2 WPL 8-1 WP 40 WP 41 WP 42 Mock0.000

1.000

2.000

3.000

4.000

5.000

6.000

Soluble Phosphate/Cell Density

t+0t+1t+2t+3

(µg/

mL)

/OD

600

Plans for next year

• Better understand the interactions of multiple strains in vivo

• Better understand effects of multiple strains producing plant growth promoting factors

Acknowledgements

Thank you to:• Sharon Doty• Neil Fleck• SEFS• NIFA