Yoav Bashan - Inoculantes para cereales

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Los días 20 y 21 de mayo de 2014, la Fundación Ramón Areces organizó el Simposio Internacional 'Microorganismos beneficiosos para la agricultura y la protección de la biosfera' dentro de su programa de Ciencias de la Vida y de la Materia.

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Yoav Bashan - Inoculantes para cereales

  1. 1. Inoculants for cerealsInoculants for cereals Yoav Bashan, Luz de-Bashan and Francisco J. Choix Environmental Microbiology Group, The Northwestern Center for Biological Research (CIBNOR), La Paz, B.C.S., Mexico and The Bashan Foundation, USAand The Bashan Foundation, USA
  2. 2. Reasons for producing microbialReasons for producing microbial inoculantsinoculants • The ultimate goal of any study ofThe ultimate goal of any study of PGPB/PGPR-plant inoculation is toPGPB/PGPR-plant inoculation is to improve the performance of the plantimprove the performance of the plant • No-formulation inoculants (culture medium, water and oil cell suspensions) perform poorly in real life field condition. • Formulation isFormulation is thethe crucial issue forcrucial issue for commercial inoculants.commercial inoculants. Bashan et al. 2014. Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives. Plant Soil 378:1–33
  3. 3. Main goal of any plant inoculationMain goal of any plant inoculation • Provide sufficient numbers ofProvide sufficient numbers of viable andviable and effective bacteriaeffective bacteria to induce rapidto induce rapid colonization of the rhizosphere as sooncolonization of the rhizosphere as soon as possible after germinationas possible after germination
  4. 4. Peat inoculant: most common inoculant todayPeat inoculant: most common inoculant today • Used for Rhizobia inoculationUsed for Rhizobia inoculation • Used for many PGPB, includingUsed for many PGPB, including AzospirillumAzospirillum in many cerealsin many cereals • There are technological proceduresThere are technological procedures • ReliableReliable • InexpensiveInexpensive • Can be used for yet-to-discover PGPBCan be used for yet-to-discover PGPB Deaker et al. 2004. Legume seed inoculation technology. Soil Biology and Biochemistry 36: 1275-1288 Stephens and Rask. 2000. Inoculant production and formulation. Field Crops Research 65: 249-258.
  5. 5. Production of inoculantProduction of inoculant • Culturable bacteria grown in fermenterCulturable bacteria grown in fermenter • Main issues:Main issues: Cheap ingredientsCheap ingredients Simple procedure for growthSimple procedure for growth Production of large population ofProduction of large population of bacteriabacteria Choice of a carrier for the PGPB:Choice of a carrier for the PGPB: traditional (traditional (peatpeat) or advanced) or advanced ((syntheticsynthetic))
  6. 6. Main concerns of end-users ofMain concerns of end-users of inoculantsinoculants • Primary concern is with crop productivity, notPrimary concern is with crop productivity, not bacterial physiology or ecologybacterial physiology or ecology • Producers of inoculants must ensure that theProducers of inoculants must ensure that the probability of inoculation is maximizedprobability of inoculation is maximized • The right microbial strain should be selectedThe right microbial strain should be selected • Formulation must be user-friendly (better if noFormulation must be user-friendly (better if no extra work is needed)extra work is needed) • Strict quality assurance (Strict quality assurance (every step in productionevery step in production process must be met or surpassedprocess must be met or surpassed).). Stephens and Rask. 2000. Inoculant production and formulation. Field Crops Research 65: 249-258..
  7. 7. Types of inoculantsTypes of inoculants Bashan et al. 2014. Plant Soil 378:1–33
  8. 8. Inoculation techniques are highlyInoculation techniques are highly variablevariable • Seed inoculationSeed inoculation:: – Seed dusting: peat mixed with seed, no re-wettingSeed dusting: peat mixed with seed, no re-wetting – Slurry coating: water solution of peat with adhesivesSlurry coating: water solution of peat with adhesives – Lime or phosphate pelleting: slurry coating followedLime or phosphate pelleting: slurry coating followed by pelettingby peletting – Vacuum infiltration: direct introduction by vacuumVacuum infiltration: direct introduction by vacuum • Soil inoculationSoil inoculation:: – Liquid inoculation: Applied to seedbed at sowingLiquid inoculation: Applied to seedbed at sowing – Granular inoculation: sown with seeds in seedbedGranular inoculation: sown with seeds in seedbed Deaker et al. 2004. Legume seed inoculation technology.Deaker et al. 2004. Legume seed inoculation technology. Soil Biology and Biochemistry 36: 1275-1288Soil Biology and Biochemistry 36: 1275-1288
  9. 9. Techniques forTechniques for Inoculation ofInoculation of Seeds and soilSeeds and soil Bashan et al. 2014.Bashan et al. 2014. Plant Soil 378:1–33Plant Soil 378:1–33
  10. 10. Preparation of traditional inoculantPreparation of traditional inoculant (peat)(peat) DRY PEAT +DRY PEAT + WATER + CALCIUM CARBONATEWATER + CALCIUM CARBONATE + additives (clays, nutrients, bulk material, etc).+ additives (clays, nutrients, bulk material, etc).
  11. 11. Peat inoculant:Peat inoculant: ProductProduct GERMINATING EMBRYO INOCULANT
  12. 12. Peat inoculant for cereals: ProductsPeat inoculant for cereals: Products For ResearchFor Research AzospirillumAzospirillum sp.sp. (Mexico)(Mexico) Contemporary commercial inoculants from ArgentinaContemporary commercial inoculants from Argentina Pseudomonas fluorescens; Azospirillum brasilensePseudomonas fluorescens; Azospirillum brasilense
  13. 13. Azospirillum commercial inoculantsAzospirillum commercial inoculants from India (from India (out of several dozens)) Greenmax Agro Tech Coimbatore Gujarat State Fertilizers Mani Dharma Biotech T Stances Co. Coimbatore
  14. 14. Additives to improve survival on seedsAdditives to improve survival on seeds • Sugars: sucrose, maltoseSugars: sucrose, maltose • Amino acids: all kindsAmino acids: all kinds • Colloids: starch, aluminum hydroxideColloids: starch, aluminum hydroxide • Adhesives currently used in agriculture: gumAdhesives currently used in agriculture: gum arabic, methylcellulose, polyvinlpyrollidone (PVP)arabic, methylcellulose, polyvinlpyrollidone (PVP) caseinate, polyvinylacetate (PVA), lecithincaseinate, polyvinylacetate (PVA), lecithin • Clays (montmorillonite) and bulk material (talcum)Clays (montmorillonite) and bulk material (talcum) • Polymers: alginate, carageenanPolymers: alginate, carageenan
  15. 15. Quality control of inoculants toQuality control of inoculants to avoid frequent failuresavoid frequent failures • Origin of the peat is important –Origin of the peat is important – Survival of strains isSurvival of strains is highly variablehighly variable • Low pH need amendmentsLow pH need amendments (Ca or Mg carbonate)(Ca or Mg carbonate) • Sterilization (Sterilization (gamma irradiation) allows competition) allows competition with potential contaminantswith potential contaminants • No inhibitory substances produced during peatNo inhibitory substances produced during peat manufacturing/packingmanufacturing/packing • Moisture content of 40-50%Moisture content of 40-50% • Minimum number of cells per seed (Minimum number of cells per seed (species dependent); varies among countries and is); varies among countries and is controlled or uncontrolledcontrolled or uncontrolled Deaker et al. 2004. Legume seed inoculation technology.Deaker et al. 2004. Legume seed inoculation technology. Soil Biology and Biochemistry 36: 1275-1288Soil Biology and Biochemistry 36: 1275-1288
  16. 16. Peat inoculant for cereals: FinalPeat inoculant for cereals: Final preparations, experimental fieldpreparations, experimental field SLURRY INOCULANTSLURRY INOCULANT INOCULANT READY FOR SPRAYINOCULANT READY FOR SPRAY
  17. 17. Peat inoculant: Inoculation on a small scalePeat inoculant: Inoculation on a small scale MICROPLOT GRAVITY DRIP IRRIGATIONGRAVITY DRIP IRRIGATION ONE FIELD EXPERIMENTONE FIELD EXPERIMENT
  18. 18. Peat inoculation on a larger scalePeat inoculation on a larger scale MACROPLOT Inoculation during sowingInoculation during sowing
  19. 19. Moving toward the future:Moving toward the future: Alternatives to peat as a carrierAlternatives to peat as a carrier for inoculantsfor inoculants Solid and liquid formulations: synthetic carriers?Solid and liquid formulations: synthetic carriers?
  20. 20. Alternatives to peat as a carrierAlternatives to peat as a carrier • Liquid inoculants: culture media + additivesLiquid inoculants: culture media + additives • Organic waste: bagasse, composts ofOrganic waste: bagasse, composts of many kinds; wastewater sludgemany kinds; wastewater sludge • Inert materials: perlite, talcum, silica, sterileInert materials: perlite, talcum, silica, sterile soil, sand, vermiculite, clayssoil, sand, vermiculite, clays Albareda et al. 2008. Soil Biology and Biochemistry 40: 2771-2779Albareda et al. 2008. Soil Biology and Biochemistry 40: 2771-2779 Singleton et al. 2002. Inoculants and nitrogen fixation in VietnamSingleton et al. 2002. Inoculants and nitrogen fixation in Vietnam Ben Rebah et al. 2007. Bioresource Technology 98:3535-3546Ben Rebah et al. 2007. Bioresource Technology 98:3535-3546
  21. 21. Optimal characteristics of a carrierOptimal characteristics of a carrier for inoculantsfor inoculants Delivers the right number ofDelivers the right number of viableviable cellscells Has high water-holding capacityHas high water-holding capacity Be nearly sterile or easily sterilizedBe nearly sterile or easily sterilized Chemically and physically uniformChemically and physically uniform Non-toxic, biodegradable, and non-pollutingNon-toxic, biodegradable, and non-polluting Etc….etc….Etc….etc…. Naturally, no carrier has all theseNaturally, no carrier has all these qualitiesqualities Bashan 1998. Biotechnology Advances 16:729-770Bashan 1998. Biotechnology Advances 16:729-770 Albareda et al. 2008. Soil Biology and Biochemistry 40: 2771-2779Albareda et al. 2008. Soil Biology and Biochemistry 40: 2771-2779
  22. 22. Are “synthetic” inoculants theAre “synthetic” inoculants the future of plant inoculation with PGPBfuture of plant inoculation with PGPB ??
  23. 23. WHAT CAN AN ENCAPSULATEDWHAT CAN AN ENCAPSULATED FORMULATION DO?FORMULATION DO? Macro-encapsulation (2-4 mm)Macro-encapsulation (2-4 mm) Micro-encapsulation (<200 micron)Micro-encapsulation (<200 micron) ENCAPSULATE THE LIVING CELLENCAPSULATE THE LIVING CELL PROTECT THE CELLS AGAINST ENVIRONMENTALPROTECT THE CELLS AGAINST ENVIRONMENTAL STRESSESSTRESSES RELEASE BACTERIA TO THE SOILRELEASE BACTERIA TO THE SOIL GRADUALLYGRADUALLY
  24. 24. Main advantages of alginate inoculantsMain advantages of alginate inoculants SLOW RELEASE OF BACTERIA INTO THE SOILSLOW RELEASE OF BACTERIA INTO THE SOIL GOOD DEGRADATION IN SOILGOOD DEGRADATION IN SOIL NON-TOXICNON-TOXIC EASY TO USE BY THE FARMEREASY TO USE BY THE FARMER COST EFFECTIVECOST EFFECTIVE MAIN DISADVANTAGEMAIN DISADVANTAGE NO INDUSTRIAL PROCESS EXISTS, so farNO INDUSTRIAL PROCESS EXISTS, so far
  25. 25. Comparison between encapsulation of bacteria for use in industrial fermentation and as an inoculant for agricultural/environment al use Bashan et al. 2014. Plant and Soil 378:1–33
  26. 26. Improving shelf life of inoculants Bashan et al. 2014. Plant and Soil 378:1–33
  27. 27. Wet alginate inoculant, immediately afterWet alginate inoculant, immediately after formation;formation; First generationFirst generation 1 mm Bashan 1986. Applied and Environmental MicrobiologyBashan 1986. Applied and Environmental Microbiology 51: 1089-109851: 1089-1098
  28. 28. Surface of wet alginate inoculant containingSurface of wet alginate inoculant containing AzospirillumAzospirillum BEAD SURFACE BACTERIAL MICROCOLONIES 10 µ
  29. 29. SURFACE OF WET ALGINATESURFACE OF WET ALGINATE INOCULANT CONTAININGINOCULANT CONTAINING AzospirillumAzospirillum 10 µ BACTERIA
  30. 30. SURFACE OF WET ALGINATESURFACE OF WET ALGINATE INOCULANT CONTAININGINOCULANT CONTAINING AzospirillumAzospirillum BACTERIA 10 µ
  31. 31. DRY ALGINATE INOCULANTDRY ALGINATE INOCULANT 1 mm
  32. 32. SURFACE OF DRY ALGINATE INOCULANTSURFACE OF DRY ALGINATE INOCULANT DRY MICROCOLONIES (Azospirillum)
  33. 33. CROSS SECTION OF DRY ALGINATECROSS SECTION OF DRY ALGINATE INOCULANTINOCULANT DRYDRY ALGINATEALGINATE LAYERSLAYERS (layers)(layers) 1 mm CenterOutsideOutside
  34. 34. Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry alginate inoculantalginate inoculant Degradation by soil microbes Release of PGPB SoilSoil SoilSoil
  35. 35. Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry alginate inoculantalginate inoculant Degradation by soil microbes Release of PGPB SoilSoil SoilSoil
  36. 36. Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry alginate inoculantalginate inoculant Degradation by soil microbes Release of PGPB SoilSoil SoilSoil
  37. 37. Dry alginate inoculant mixed with wheatDry alginate inoculant mixed with wheat seeds before sowingseeds before sowing BEADS SEEDS
  38. 38. Alginate inoculant immediately afterAlginate inoculant immediately after sowing of wheatsowing of wheat BEADSBEADS SEEDSSEEDS
  39. 39. DEGRADATION OF MACROBEADS IN SOILDEGRADATION OF MACROBEADS IN SOIL 0 20 40 60 80 100 120 1 2 3 4 5 WEEKS AFTER INOCULATION PERCENTAGEOF DEGRADATION ALGINATE BEADS ALGINATE + SKIM MILK Bashan et al. 1987. Canadian Journal of Microbiology 33: 1074-1079
  40. 40. SLOW RELEASE OF BACTERIA FROMSLOW RELEASE OF BACTERIA FROM BEADS OF ALGINATEBEADS OF ALGINATE 0 2 4 6 8 1 2 3 4 WEEKS AFTER INOCULATION LOGNUMBEROF BACTERIA ALGINATE BEADS ALGINATE BEADS WITH SKIM MILK Bashan et al. 1987. Canadian Journal of MicrobiologyBashan et al. 1987. Canadian Journal of Microbiology 33:33: 1074-10791074-1079
  41. 41. Wheat root colonization ofWheat root colonization of AzospirillumAzospirillum in peatin peat and alginate carriersand alginate carriers 0 2 4 6 8 1 2 3 4 5 6 WEEKS AFTER INOCULATION LOGNO.BACTERIA/g ROOTS PEAT INOCULANT ALGINATE INOCULANT Bashan et al. 1987. Canadian Journal of MicrobiologyBashan et al. 1987. Canadian Journal of Microbiology 33:33: 1074-10791074-1079
  42. 42. SURVIVAL OF PGPB IN INOCULANTSSURVIVAL OF PGPB IN INOCULANTS In peatIn peat: One year mandatory, two years: One year mandatory, two years desirabledesirable In alginateIn alginate: 1-3 years has been: 1-3 years has been demonstrated in many studiesdemonstrated in many studies Record in alginateRecord in alginate: 14 years without: 14 years without losing activitylosing activity (Bashan and Gonzalez, 1999. Applied Microbiology and Biotechnology 51: 262-266)(Bashan and Gonzalez, 1999. Applied Microbiology and Biotechnology 51: 262-266)
  43. 43. Main use of macro-inoculants madeMain use of macro-inoculants made of alginate todayof alginate today • Water bio-remediation using microalgaeWater bio-remediation using microalgae and “microalgae growth-promoting bacteria”and “microalgae growth-promoting bacteria” • Many experimental agricultural inoculationMany experimental agricultural inoculation studies mainly of cerealsstudies mainly of cereals • Biological control of insectsBiological control of insects
  44. 44. Main deficiencies of macro-Main deficiencies of macro- inoculants made of alginateinoculants made of alginate • Requires extra field work by the growerRequires extra field work by the grower • The PGPB needs to move fromThe PGPB needs to move from inoculation site to the growing rootsinoculation site to the growing roots • The technique is partly affected by the soilThe technique is partly affected by the soil type; less efficient in heavy soilstype; less efficient in heavy soils
  45. 45. The micro-bead conceptThe micro-bead concept If the beads are small enough, Yet, still able to encapsulate sufficientYet, still able to encapsulate sufficient numbers of bacteria,numbers of bacteria, Then, it is possible to produce powder-like formulations This “bead-dust” can be coat seeds in the factory Farmers buy “improved seeds”. Bashan, Y., Hernandez, J.-P., Leyva, L.A. and Bacilio, M. 2002. Alginate microbeads as inoculant carrier for plant growth-promoting bacteria. Biology and Fertility of Soils 35: 359-368
  46. 46. THE MICRO -BEAD MACHINETHE MICRO -BEAD MACHINE Bashan, Y., Hernandez, J.-P., Leyva, L.A. and Bacilio, M. 2002. Alginate microbeads as inoculant carrier for plant growth-promoting bacteria. Biology and Fertility of Soils 35: 359-368
  47. 47. THE MICRO -BEAD: Ho w do e s it lo o k like ?THE MICRO -BEAD: Ho w do e s it lo o k like ? Az Az
  48. 48. Detection and verification of purity of a strainDetection and verification of purity of a strain in the inoculantin the inoculant Fluorescence-Fluorescence-in-situin-situ-hybridization (FISH)-hybridization (FISH) Bashan et al. 2011. Biology and Fertility of Soils 47: 963-969 Probe for domainProbe for domain BacteriaBacteria Probe forProbe for Azospirillum brasilenseAzospirillum brasilense Superimposed images 100% verification Phase microscopyPhase microscopy
  49. 49. Multiplication ofMultiplication of AzospirillumAzospirillum in micro-beadsin micro-beads 0 2 4 6 8 10 12 LOGNO OF BACTERIA / g BEADS 1 2 3 4 DIFFERENT CULTURE MEDIA Bashan, Y., Hernandez, J.-P., Leyva, L.A. and Bacilio, M. 2002. Alginate microbeads as inoculant carrier for plant growth-promoting bacteria. Biology and Fertility of Soils 35: 359-368
  50. 50. Concentration ofConcentration of AzospirillumAzospirillum in dry micro-beadsin dry micro-beads 9 9.1 9.2 9.3 LOGNO BACT. / g BEADS 1 2 HEAT-DRY FREEZED-DRY
  51. 51. Biodegradation of micro-beads in soilBiodegradation of micro-beads in soil • This is the wayThis is the way bacteria are releasedbacteria are released in the soilin the soil • Depends on chemicalDepends on chemical composition of thecomposition of the micro-beadmicro-bead
  52. 52. Slow release ofSlow release of AzospirillumAzospirillum fromfrom alginate micro-spheresalginate micro-spheres 0 1 2 3 4 5 6 7 8 1 7 14 30 Alginate Alginate Skim milk Alginate harden DaysDays LognumberofbacteriaLognumberofbacteria Bashan et al. 2011. Biology and Fertility of Soils 47: 963-969
  53. 53. Alginate micro-beads attached to wheat seedsAlginate micro-beads attached to wheat seeds Micro-beadsMicro-beads Bashan, Y., Hernandez, J.-P., Leyva, L.A. and Bacilio, M. 2002. Alginate microbeads as inoculant carrier for plant growth-promoting bacteria. Biology and Fertility of Soils 35: 359-368
  54. 54. Alginate micro-beads attached to wheat seedsAlginate micro-beads attached to wheat seeds Micro-beads
  55. 55. Effect of micro-bead inoculation on plantsEffect of micro-bead inoculation on plants
  56. 56. Detection of the inoculant in the fieldDetection of the inoculant in the field • The Plant Growth-The Plant Growth- Promoting Bacteria isPromoting Bacteria is tagged with a genetagged with a gene producing a coloredproducing a colored expression, such as:expression, such as: LacZLacZ (blue),(blue), gfpgfp (green),(green), rfprfp (red),(red), yfpyfp (yellow),(yellow), oror bioluminiscencebioluminiscence (light)(light) Bacilio et al. 2004.Bacilio et al. 2004. Biology and Fertility of SoilsBiology and Fertility of Soils 40:40: 188-193188-193 Rodriguez et al. 2006.Rodriguez et al. 2006. FEMS Microbiology EcologyFEMS Microbiology Ecology 5757: 217-225: 217-225 gfp-tagged Azospirillum on wheat root gfpgfp-tagged-tagged AzospirillumAzospirillum
  57. 57. Highly specific molecular detection ofHighly specific molecular detection of AzospirillumAzospirillum on sorghum rootson sorghum roots (confocal laser(confocal laser microscopy)microscopy) Trejo et al. 2012. Environmental and Experimental BotanyTrejo et al. 2012. Environmental and Experimental Botany 7575: 65-73: 65-73 SorghumSorghum Root tipRoot tip AzospirillumAzospirillum Other bacteria
  58. 58. Practical inoculation with inoculants -Practical inoculation with inoculants - Use ofUse of inoculants “out of the box” of agricultureinoculants “out of the box” of agriculture • Prevent soil erosion in desert landsPrevent soil erosion in desert lands • Recycle wastewaterRecycle wastewater • Green-energy, bio-ethanol, and biodieselGreen-energy, bio-ethanol, and biodiesel
  59. 59. Dry beads after wastewater treatment Inoculation of plants, planted in soil with low fertility Application: Increase fertility ofApplication: Increase fertility of eroded desert soilseroded desert soils Trejo et al. 2012. Environmental and Experimental Botany 75: 65-73 Chlo rella Chlor ella Azospirillum Azospirillum
  60. 60. Effect of inoculationEffect of inoculation on sorghum rootson sorghum roots 0 20 40 60 80 100 120 140 160 First cycle Second cycle Third cycle mg(dryweight) Trejo et al. 2012. Environmental and Experimental Botany 75: 65-73 Effect of inoculation onEffect of inoculation on sorghum shootsorghum shoot 0 10 20 30 40 50 60 70 80 mg(dryweight) First cycle Second cycle Third cycle The outcomes:The outcomes: Accumulation of organic matter inAccumulation of organic matter in surghum during cycles of plantingsurghum during cycles of planting Dryweight(mg)
  61. 61. Accumulation of organic matter during cycles of planting 0 1 2 3 4 5 6 7 8 9 10 g/Kgsoil First cycle Second cycle Third cycle Debris Plant alone Eroded soil Trejo et al. 2012. Environmental and Experimental Botany 75: 65-73
  62. 62. Root tip colonization of sorghum after inoculation with debris from wastewater used as inoculant Root tip Other bacteria Trejo et al. 2012. Environmental and Experimental Botany 75: 65-73 Lopez et al. 2013. Biology and Fertility of Soils 49: 1053-1063 Using FISH, fluorescent microscopy and confocal laser microscopy Root hair Elongation zone
  63. 63. S c a t t e r p lo t 2 D F in a l C o n f ig u r a t io n , d im e n s io n 1 v s . d im e n s io n 2 1 2 3 5 6 7 89 1 1 1 2 1 3 - 1 . 2 - 1 . 0 - 0 . 8 - 0 . 6 - 0 . 4 - 0 . 2 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 1 . 4 D im e n s io n 1 - 0 . 8 - 0 . 6 - 0 . 4 - 0 . 2 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 Dimension2 1 2 3 5 6 7 89 1 1 1 2 1 3 No planted soil Beads alone No inoculated plant Plant inoculated with leftover beads Effect of the inoculation of wastewater debris on the rhizosphere bacterial community N on inoculated plant Jointly im m obilized 1.0 1.1 1.2 1.3 1.4 a b c d N o planted soil Alginate beads DiversityindexH Lopez et al. 2013. Biology and Fertility of Soils 49: 1053-1063
  64. 64. CONCLUSIONS (from 10 year of studies on inoculationCONCLUSIONS (from 10 year of studies on inoculation withwith AzospirillumAzospirillum on native desert planton native desert plant VS. Possible additional “helpers” and advantages:Possible additional “helpers” and advantages: - Low level of compost needed- Low level of compost needed - Micorrhizae fungi provides additional support- Micorrhizae fungi provides additional support - Limited amount of water needed- Limited amount of water needed - A small legume tree nearby- A small legume tree nearby Non-inoculatedNon-inoculated Inoculated
  65. 65. Field experiments for practical use of basic studiesField experiments for practical use of basic studies • Use three legume trees, alone andUse three legume trees, alone and combined, with cardon cactus.combined, with cardon cactus. • Inoculate with local desert mycorrhizae.Inoculate with local desert mycorrhizae. • Inoculate with a mix ofInoculate with a mix of AzospirillumAzospirillum andand phosphate-solubilizing bacteria.phosphate-solubilizing bacteria. • Add low amount of compost.Add low amount of compost. • Add very limited irrigation water.Add very limited irrigation water. • All treatments in dense initial planting,All treatments in dense initial planting, using the trees as shrubs.using the trees as shrubs.
  66. 66. Abandoned area (for 25 years) before restorationAbandoned area (for 25 years) before restoration Cardon natural forest Bare area Invasive buffel grass
  67. 67. Field experiments -Field experiments - planted 2004planted 2004 Anti- herbivore fence Planting holes
  68. 68. Planting design for field experimentsPlanting design for field experiments Legume tree Cardon Sterilized straw Soil surface Barren top soil + inoculants + compost (mixed) 20 cm 30 cm 25 cm 5 cm LegumeLegume treetree cardoncardon Vertical viewVertical view
  69. 69. One of 1500 planted treesOne of 1500 planted trees Planting hole belowPlanting hole below soil surfacesoil surface Sterile StrawInoculum mixedInoculum mixed with soilwith soil
  70. 70. High-density planting of cardon & palo verdeHigh-density planting of cardon & palo verde
  71. 71. Does the transfer of knowledge from theDoes the transfer of knowledge from the laboratory work in the field?laboratory work in the field? == UNDISTURBED AREAUNDISTURBED AREA UNDISTURBED AREAUNDISTURBED AREA OROR Hopefully not….Hopefully not….
  72. 72. Long term effects on legume trees inoculatedLong term effects on legume trees inoculated with synthetic inoculants in the fieldwith synthetic inoculants in the field 2004 2006 20092009 Bashan et al. 2012: Journal of EnvironmentalBashan et al. 2012: Journal of Environmental ManagementManagement 102102: 26-36.: 26-36.
  73. 73. General view of the experimental field fiveGeneral view of the experimental field five years after planting (22 October, 2009)years after planting (22 October, 2009) MesquiteMesquite Bashan et al. 2012. Restoration of eroded soil in the Sonoran Desert with native leguminous trees Journal of Environmental Management 102: 26-36
  74. 74. General view of the field 9 years afterGeneral view of the field 9 years after planting (22 March, 2014)planting (22 March, 2014) ““Giant”Giant” CardonsCardons Bashan et al. 2009.Bashan et al. 2009. Enhanced establishment and growth of giant cardon cactusEnhanced establishment and growth of giant cardon cactus in an eroded field in the Sonoran Desert ….in an eroded field in the Sonoran Desert ….Biology and Fertility of Soils 45:585–594Biology and Fertility of Soils 45:585–594
  75. 75. General conclusionGeneral conclusion Inoculant formulation and more advanced inoculantInoculant formulation and more advanced inoculant are essentialare essential for applications rangingfor applications ranging from agriculture to environmental problemsfrom agriculture to environmental problems Baja California desert All published papers areAll published papers are availableavailable as PDFs at: www.bashanfoundation.orgas PDFs at: www.bashanfoundation.org Muchas gracias

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