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GM biofer*lizers – experimental releases of transgenic rhizobia Penny Hirsch Rothamsted Research Harpenden Her0ordshire AL5 2JQ UK Why regulate GMOs? Heracleum mantegazzianum Importance of soil microbes plant nutri*on gaseous fluxes soil gene pool plant health/disease bioindicators nutrient cycling pes*cide/pollutant soil fer*lity decomposi*on soil structure Soil bacterial targets for improvement Biopes*cides Insec*cides Bacillus thuringiensis kills insect pests Leaf & shoot colonisers Bacillus, Burkolderia, Erwinia, Pseudomonas -‐ inhibit fungal pathogens, prevent ice damage Biofer*lizers Root nodule bacteria Rhizobium, Sinorhizobium, Bradyrhizobium – induce nodules, fix nitrogen Root colonisers Azospirillum, Bacillus, Burkolderia, Pseudomonas, Agrobacterium -‐ promote root growth, inhibit pathogens • Phytohormones (auxins, giberellins) produced by bacteria and fungi can modify root growth to increase branching &/or length and thus effecLve area in contact with soil • Microbial components may sLmulate systemic plant disease resistance • BioremediaLon -‐ degraders of recalcitrant organic compounds Soil microbiology & heterogeneous • Soil is structurally complex • Soil microorganisms are numerous & diverse • Many belong to novel groups, minority culturable, archaea largely unknown • • • • • Each g soil contains 109 bacterial cells 107 archaeal cells 108 culturable bacteria 104 bacterial species 105 fungal propagules In comparison, rhizosphere has • more culturable bacteria • fewer bacterial species • more specialist fungi? The rhizobia-‐legume symbiosis Root nodules contain the nitrogen-‐fixing bacterial species Rhizobium leguminosarum Pisum sativum Why rhizobia? • ULlity N-‐fixing symbiont of legumes • Familiarity > 100 years use, > 40 years geneLcs • Innocuous harmless to animals, plant or environment • Ease of idenLficaLon isolaLon via root nodules • GeneLc tools Rhizobium leguminosarum strains contain several large plasmids, 80-‐800 kb, one carrying symbioLc genes (pSym) Why rhizobia? Reliable inocula*on response with compaLble rhizobia 1895 UK patent (Nobbe & Hiltner) Modern soybean inoculant Comericalised by 1910 (Nitragin soybean inoculant) Rhizobial gene*cs bacteriophage DNA 50-100 kb bacterial cell transformation chromosomal DNA 5000 kb Plasmid 80-800 kb tra, oriT (transfer) IS (insertion element) 2-10 kb Group II intron 1-2 kb repC (replication) Rhizobial gene*cs Plasmid profiles Kb 800 80 R. leguminosarum field isolates Bacteriophages PRL1RES temperate (λ-‐like) PRL2RES virulent, generalised TransducLon (T4-‐like) Transmissible symbioLc plasmids Biotechnological improvements Inoculant Year Loca*on S. meliloA 19891 spray inoculant1 19902 alfalfa seed coaLng2 1995-‐64 USA, 1 site1 4 sites2 1 site4 S. meliloA populaLon low B.japonicum Soybean seed coaLng1 USA, 1 site1 1989 Reason Result Nodule occupancy >80% (3 sites), <20% (1 site)1,2 Yield increase where low soil N2, site of transgene inserLon responsible3 No dispersal from site at inoculaLon1 Soil microbial diversity – small increase in spore-‐formers only4 Up-‐regulate N-‐fixaLon (nifA) Increase C supply (dctABD) Seek improved symbioLc efficiency, yield increases Nodule occupancy 17% – 59% Compare soil microflora4 No significant yield increase VerLcal dispersal by roots S. meliloA alfalfa seed coaLng5 1991 Ireland, 1 site Test plasmid maintenance No naLve system based on -‐ use thy+ S. meliloA and thyA strains S. meliloA 1999 alfalfa seed coaLng6 Spain, 1 site, Inoculant populaLon stablised in Up-‐regulate putA to increase indigenous rhizosphere, dropped in soil, no clear nodulaLon compeLLveness S. meliloA advantage for nodulaLon Plasmid maintained in > 90% thyA, lost in <30% thy+ nodule isolates 1Ronson CW et al. 1990 In: Nitrogen FixaAon: Achievements and ObjecAves, Chapman and Hall, New York. 2Bosworth AH et al. 1994 Appl. Environ. Microbiol. 60:3815-‐3832. 3Scupham AJ et al. 1996 Appl. Environ. Microbiol. 62:4260-‐4262. 4Donegan KK et al. 1999 J. Appl. Ecol. 36:920-‐936. 5O'Flaherty S et al. 1995 Appl. Environ. Microbiol. 61:4051-‐4056. 6van Dillewijn P et al., 2001 Appl. Environ. Microbiol 67: 3860-‐3865. Ecology and Biosafety Studies Inoculant Year R. leguminosarum Seed-‐coaLng for peas (UK, Germany) Broadcast granules to 19871 soil wheat (UK) Liquid inoculant (France) R. leguminosarum Seed-‐coaLng for peas (UK) S. meliloA Mixed into soil then placed in lysimeters4 Sprayed onto soil5 19942,3 19944 19955 Loca*on Reason Result UK, France, Germany effecLve naLve populaLons Tn5 inserLon in conjugaLve symbioLc plasmid test survival, spread, nodulaLon compeLLveness, gene movement Survival in France < 2 weeks, Germany 30 weeks UK >15 years, some horizontal and verLcal spread, no evidence for marker gene transfer1,2 UK, site of previous release Chromosomal gusA in strain lacking symbioLc plasmid, seek nodules with GUS acLvity Survival but no evidence for symbioLc plasmid transfer to inoculant or movement of gusA to other rhizobia Germany, few naLve S. meliloL 100-‐fold decline over 2 years, survival similar for recA+ and recA-‐ Some horizontal spread to test survival of recA neighbouring lysimeters, no mutants marked with luc, leaching4 invesLgate impact on soil Horizontal spread of inoculant due microbe populaLons8 to spray drit, inoculant impacted rhizoflora diversity of M.saAva but not non-‐host C. album5 1Hirsch PR & Spokes J D 1994 FEMS Microbiol. Ecol. 15:147-‐159. 2Cullen DW et al. 1998 J. Appl. Microbiol. 84:1025-‐1034. 3Hirsch PR 2000 In: Tracking GeneAcally-‐Engineered Microorganisms. Eds JK Jansson, JD van Elsas & MJ Bailey, Landes Bioscience, USA 4Schwieger F et al. 2000 Soil Biol. Biochem. 32:859-‐868. 5Schwieger F & Tebbe CC 2000 Appl. Environ. Microbiol. 66:3556-‐3565. Assessment of GM rhizobia as agricultural inoculants • Long-‐running series of field experiments funded by EU from 1986-‐2001 • Monitor persistence and dispersal of gene*cally-‐modified rhizobia in the field • Inves*gate gene transfer between rhizobia Sites of GM rhizobia release by members of the TRAFFIC consor*um Rothamsted D ijo n B ra u n s c h w e ig S tra s s G ra n a d a Funded by EU Biotechnology programmes BIO2 CT92-‐0370 and BIO4 CT96-‐0434 Site of field release at Rothamsted track N grass grass 20 m 5 m cereals in 1987 1/28 slope peas in 1987 1/30 slope 1/50 slope Wheat fence key RSM2004 in 1987 CT0370 on peas in 1994 Plan*ng peas inoculated with GM rhizobia Rothamsted Research farm, 1994 GM rhizobium leguminosarum bv. viciae RSM2004 released 1987 • • • • • isolate from Norfolk; conjugaLve symbioLc plasmid (pSym) Rifr marker on chromosome, Tn5 inserLon on pSym (Neor Strr) Select inoculant on Rif + Neo; transconjugants on Neo (Rifs) Defined as GM in 1987; not GM under current UK/EU regulaLons pSym::Tn5 transferred to 75% field isolates tested(10-‐5per parent) CT0370 released 1994 • isolate from Germany cured of symbioLc plasmid • Spcr Strr with gusA inserted between recA and alaS on chromosome • Defined as GM (gusA from E. coli) • Regains nodulaLon ability on transfer of pSym::Tn5 -‐ observed in non-‐sterile soil microcosms with 107 each parent CT0370 Detec*on Viable cells in field soil pSym+ transconjugants in nodules Stain pea root nodules with X-‐gluc or MUG SelecLve agar with streptomycin, specLnomycin & X-‐gluc PCR detec*on of gusA inser*on site rec A forward primer rec F rec A gus A PnptII ala S gus R 506 bp product PnptII forward primer Pnpt F rec A PnptII gus A gus R 432 bp product ala S Survival of GM rhizobia in field release site Log cfu or nfu per g soil 6 5 4 3 2 1 Sampling date RSM2004 CT0370 NaLve rhizobia GM rhizobium leguminosarum bv. viciae 5 cells g-‐1 soil 1987 RSM2004: p eas o r c ereals i noculated 1 0 • Stabilized at 102 -‐ 103 cfu g-‐1 soil at Rothamsted, survived at least 15 y • No survival detected at Dijon or Bayreuth • PCR signal consistent with 102 -‐ 103 cells g-‐1 soil • PCR on G+C fracLonated soil DNA: signal only in rhizobial fracLon • Two-‐fold more RSM2004 survived where inoculated on host pea • 4325 nodules screened, no transconjugants detected (<2 x 10-‐4 per RSM2004) • Flanking sequence PCR: no evidence for Tn5 transposiLon • Less than one transposiLon event per 500 RSM2004 in 14 years CT0370: peas inoculated 105 cells g-‐1 soil 1994 • Stabilized at 103 – 104 cfu g-‐1 soil (100 x RSM2004) • Number on cereal & alfalfa roots <RSM2004, on pea roots <<RSM2004 • 20,000 pea nodules screened for GUS, not detected (<5 x 10-‐5 pSym per parent) Hirsch PR & Spokes J D 1994 FEMS Microbiol. Ecol. 15:147-‐159. Hirsch PR 1996 New Phytologist 133:159-‐171 Cullen DW et al. 1998 J. Appl. Microbiol. 84:1025-‐1034. Hirsch PR 2000 In: Tracking GeneAcally-‐Engineered Microorganisms. Eds JK Jansson, JD van Elsas & MJ Bailey, Landes Bioscience, USA GM Sinorhizobium melilo4 Germany • • • • • • firefly luciferase gene luc in chromosome, disrupLng recA Select on streptomycin, luciferase confirms idenLty environmental fitness not affected by lucA No clear difference in survival for recA+ / recA-‐ Survival bewer in clay and loam than sand Survival advantage when host alfalfa grown Selbitschka et al. 1992 Mol Ecol 1:9-‐10; Selbitschka et al. 1995 FEMS Microbiol Ecol 16:223-‐232 Schwieger F et al. 2000 Soil Biol. Biochem. 32:859-‐868. Schwieger F & Tebbe CC 2000 Appl. Environ. Microbiol. 66:3556-‐3565 USA • Nitrogenase acLvity and C supply up-‐regulated • Yield advantage in glasshouse • SpaLally and temporally variable results in field Ronson CW et al. 1990 In: Nitrogen FixaAon: Achievements and ObjecAves, Chapman and Hall, New York. Bosworth AH et al. 1994 Appl. Environ. Microbiol. 60:3815-‐3832. Scupham AJ et al. 1996 Appl. Environ. Microbiol. 62:4260-‐4262 Overall conclusions • Rhizobia persist ater field applicaLon in the absence of their host with a lasLng advantage following host infecLon • Soil and climaLc factors influence survival • No evidence for plasmid movement in 15 y Lmescale of experiment despite evidence for transfer over Lme in soil communiLes • RSM2004 pSym confers survival advantage in rhizosphere, burden in soil (reduces probability of coinciding with pSym-‐ CT0370 on roots) • Gene behaviour confounded by environmental factors (recA did not reduce survival; improved N fixaLon not reproducible) • GM marker technology invaluable for ecological experiments! Why did work cease? • Ater iniLal interest, funding sources became difficult • Lack of interest in (micro)biologicals • Inconsistent results from GM “improved” microorganisms compared to convenLonal agrochemicals due to environmental variability • Focus on GM plants rather than microbial inoculants • Slowly increasing public resistance to GM technology during 1990s • Increased cost of obtaining consent for GM environmental releases Will move to sustainable agriculture offer revival? Can semi-‐controlled uses overcome unreliability? (e.g. glasshouse applicaLons; biofilters for waste stream treatments) Thanks to many colleagues and collaborators Especially Ian Clark; Tom Mendum; Danny Cullen Thanks to funders Especially BBSRC and EU Thanks to you for listen*ng
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