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Phytoremediation

Phytoremediation. Microbial Endophytes Katie Fishburn and Jared Bodecker. 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)

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Phytoremediation

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  1. 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

  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

  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 Psuedomonasputidamini 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. Brassicachinensis Brassicachinensis • Mucor isolate • Cd and Pb tolerance • Cd and Pb more water soluble; more bioavailable

  14. Thlaspigoesingense • Thlaspigoesingense • Methylobacterium and Shpingomonas • Ni accumulation and tolerancesiderophores 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. • BurkholderiacepaciaG4 (rhizoshperic) with toluene-degrading qualities from pTOM plasmid (toluene-ortho-monooxygenase) • B. cepaciaVM1330 (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

  24. References Kieran J. Germaine, Xuemei Liu, Guiomar Garcia Cabellos, Jill P. Hogan, David Ryan & David N. Dowling. (2006). Bacterialendophyte-enhancedphytoremediationoftheorganochlorine 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 PhytosymbioticMethylobacterium sp. Associated with Poplar Tissues (Populusdeltoides􏰈nigra DN34) APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2004, p. 508–517 Vol. 70, No. 1 ZujunDenga,b, LixiangCaoc, HaiweiHuanga, XinyuJianga, WenfengWangc, Yang Shia, RenduoZhanga,∗ (2010) Characterization of Cd- and Pb-resistant fungal endophyteMucor sp. CBRF59 isolated from rapes (Brassicachinensis) 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 HyperaccumulatorThlaspigoesingense. 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 ApplMicrobiol 84: 937–944. Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M & van derLelie 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 derLelie 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 derLelie 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) Methylobacteriumpopuli sp. nov., a novel aerobic, pink- pigmented, facultativelymethylotrophic, methane-ultilising bacterium isolated from poplar trees (Populusdeltoidesxnigra DN34). EvolMicrobiol 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.

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