1. Microbial Endophytes Katie Fishburn and Jared Bodecker
Phytoremediation
Microbial Endophytes
Katie Fishburn and Jared Bodecker

2. Endophytes
Microbes that live in plant tissue, that increase plant biomass and resistance to pathogens
Of the 300,000 known species of plants, there is at least one plant-associated endophyte (Strobel G. et al)
Can remediate pollutants just as good as rhizospheric microbes, in some cases better
There are clear advantages of endophytes compared to the closely associated rhizospheric cousins

3. Known endophytes in PR
Highlight the endophytes were discussing

4. Endophyte vs. Rhizospheric
Population control is easier than soil microbes, due to lack of competition (protected inside plant tissue)
Degradation of pollutants will be localized to inside the plant, being contained from leaking in the soil and air environment.
Plant species-specific selection of necessary bacteria for a specific pollutant
Microbial sampling is easier. (plant tissue)

5. Applications endophyte-phytoremediation
The diagram of four major applications

6. Phytoremediation: Organics
2,4-D (herbicide) degradation
Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D
Control: high levels of 2,4-D remain
Inoculated plants: no 2,4-D remains in test soils
PAH tolerance in willow and poplar
Used also as carbon source by microbes
TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar
Mineralized 60% of RDX and HMX in 2 months
2,4-D (herbicide) degradation
Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D
Control: high levels of 2,4-D remain
Inoculated plants: no 2,4-D remains in test soils

7. 2, 4-D Degradation Root tissue Stem tissue
Psuedomonas putida mini Tn5 (GFP)

8. Phytoremediation: Organics
2,4-D (herbicide) degradation
Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D
Control: high levels of 2,4-D remain
Inoculated plants: no 2,4-D remains in test soils
PAH tolerance in willow and poplar
Used also as carbon source by microbes
TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar
Mineralized 60% of RDX and HMX in 2 months
PAH tolerance in willow and poplar
Used also as carbon source by microbes

9. Poly Aromatic Hydrocarbons
Backdrop picture courtesy of NASA

10. Phytoremediation: Organics
2,4-D (herbicide) degradation
Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D
Control: high levels of 2,4-D remain
Inoculated plants: no 2,4-D remains in test soils
PAH tolerance in willow and poplar
Used also as carbon source by microbes
TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar
Mineralized 60% of RDX and HMX in 2 months
TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar
Mineralized 60% of RDX and HMX in 2 months

11. Methylobacterium spp. Strain BJ001

12. Phytoremediation: Inorganics

13. Brassica chinensis Brassica chinensis Mucor isolate
Cd and Pb tolerance
Cd and Pb more water soluble; more bioavailable

14. Thlaspi goesingense Thlaspi goesingense
Methylobacterium and Shpingomonas
Ni accumulation and tolerance siderophores and ethylene relieve

15. Plant Yield and Growth Promotion
Enhanced nutrient/mineral availability and uptake
Phosphate, nitrogen
Phosphate solubilization
Siderophore production
Minerals made more bioavailable; solubilized by siderophore
Osmotic adjustment, stomatal regulation

16. Plant Health and Protection
Lessen/prevent effects of pathogens on plants
Fungal, bacterial, viral diseases less harmful after inoculation of endophytes
Endophytes trigger induced systemic resistance (ISR)
Immune-like response by plants

17. Industrial/Medical Applications
Pseudomonas, Burkholderia, Bacillus
Antibiotics, anti-cancer compounds, antifungal, antiviral, insecticidal, immunosuppressant agents

18. Making a (6) Million $ Endophyte
Engineering plant-associated microbes
Horizontal Gene Transfer
Improving already efficient phytoremediation
By-products of degradation can even more toxic when released into the air
When in planta, you have natural reduction of volatile toxins

19. Improving Toluene Degradation
Normal plant degradation of toluene creates toxic by-products that are volatized into the environment.
Burkholderia cepacia G4 (rhizoshperic) with toluene-degrading qualities from pTOM plasmid (toluene-ortho-monooxygenase)
B. cepacia VM1330 (endophyte), with the pMON plasmid

20. Enhanced Toluene-resistance
Growth Index (Fresh Weight)
Control- Yellow Lupine (no inoculate)
BU0072- YL endophyte strain
G4- YL rhizospheric strain (pTOM)
VM1330- YL transformed endophyte strain (pTOM)
Total Weight (grams)

21. Reduced Toluene-volatization
Improved degradation in VM1330 and Yellow Lupine.
Which lowers phytotoxicity and evapotranspiration
Sub-phytotoxic levels used in experiments (100mg/L)

22. Lupine endophytes in Poplar
Decreased Toluene-volatization
Control- Poplar (no innoculate)
VM1466- P., endophyte (pTOM)
BU61- P., rhizospheric (pTOM)

23. Horizontal Gene Transfer
Opposite of vertical transfer (parental or ancestral)
Genetic material is transferred from another organism to another organism without being an offspring
Common routes of transfer
Transformation: Uptake of endogenous DNA (nature/lab)
Transduction: Delivery via vector (bacteriophage)
Conjugation: a physical connection between two organisms delivers DNA from one to the other

25. References
Kieran J. Germaine, Xuemei Liu, Guiomar Garcia Cabellos, Jill P. Hogan, David Ryan & David N. Dowling. (2006). Bacterialendophyte-enhancedphytoremediationofthe organochlorine herbicide 2,4 - dichlorophenoxyacetic acid. FEMS Benoit Van Aken,* Jong Moon Yoon, and Jerald L. Schnoor. (2003) Biodegradation of Nitro-Substituted Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5-Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a Phytosymbiotic Methylobacterium sp. Associated with Poplar Tissues (Populus deltoides 􏰈 nigra DN34) APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2004, p. 508–517 Vol. 70, No. 1 Zujun Denga,b, Lixiang Caoc, Haiwei Huanga, Xinyu Jianga, Wenfeng Wangc, Yang Shia, Renduo Zhanga,∗ (2010) Characterization of Cd- and Pb-resistant fungal endophyte Mucor sp. CBRF59 isolated from rapes (Brassica chinensis) in a metal- contaminated soil. Journal of Hazardous Materials Rughia Idris,1 Radoslava Trifonova,1 Markus Puschenreiter,2 Walter W. Wenzel,2 and Angela Sessitsch1*. (2004) Bacterial Communities Associated with Flowering Plants of the Ni Hyperaccumulator Thlaspi goesingense. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2004, p. 2667–2677 Robert P. Ryan1, Kieran Germaine2, Ashley Franks1, David J. Ryan2 & David N. Dowling2 (2007) Bacterial endophytes: recent developments and applications. FEMS Germaine K, Keogh E, Borremans B et al. (2004) Colonisation of poplar trees by gfp expressing bacterial endophytes. FEMS Microbiol Ecol 48: 109–118 Germaine K, Liu X, Cabellos G, Hogan J, Ryan D & Dowling DN (2006) Bacterial endophyte-enhanced phyto-remediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid. FEMS Microbiol Ecol 57: 302–310. Miller CM, Miller RV, Garton-Kenny D, Redgrave B, Sears J, Condron MM, Teplow DB & Strobel GA (1998) Ecomycins, unique antimycotics from Pseudomonas viridiflava. J Appl Microbiol 84: 937–944. Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M & van der Lelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21: 583–606. Lodewyckx C, Taghavi S, Mergeay M, Vangronsveld J, Clijsters H & van der Lelie D (2001) The effect of recombinant heavy metal resistant endophytic bacteria in heavy metal uptake by their host plant. Int J Phytoremediation 3: 173–187. Taghavi S, Barac T, Greenberg B, Borremans B, Vangronsveld J & van der Lelie D (2005) Horizontal gene transfer to endogenous endophytic bacteria from poplar improved phyto-remediation of toluene. Appl Environ Microbiol 71: 8500–8505. Strobel G, Daisy B, Castillo U & Harper J (2004) Natural products from endophytic microorganisms. J Nat Prod 67: 257–268. Van Aken B, Peres C, Doty S, Yoon J & Schnoor J (2004) Methylobacterium populi sp. nov., a novel aerobic, pink- pigmented, facultatively methylotrophic, methane-ultilising bacterium isolated from poplar trees (Populus deltoides x nigra DN34). Evol Microbiol 54: 1191–1196. Xi C, Lambrecht M, Vanderleyden J & Michiels J (1999) Bi-functional gfp-and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies. J Microbiol Methods 35: 85–92.