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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
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
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
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.
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))
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..
Types of inoculantsTypes of inoculants
Bashan et al. 2014.
Plant Soil 378:1–33
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
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
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).
Peat inoculant:Peat inoculant:
ProductProduct
GERMINATING
EMBRYO
INOCULANT
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
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
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
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
Peat inoculant for cereals: FinalPeat inoculant for cereals: Final
preparations, experimental fieldpreparations, experimental field
SLURRY INOCULANTSLURRY INOCULANT
INOCULANT READY FOR SPRAYINOCULANT READY FOR SPRAY
Peat inoculant: Inoculation on a small scalePeat inoculant: Inoculation on a small scale
MICROPLOT
GRAVITY DRIP IRRIGATIONGRAVITY DRIP IRRIGATION
ONE FIELD EXPERIMENTONE FIELD EXPERIMENT
Peat inoculation on a larger scalePeat inoculation on a larger scale
MACROPLOT
Inoculation during sowingInoculation during sowing
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?
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
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
Are “synthetic” inoculants theAre “synthetic” inoculants the
future of plant inoculation with PGPBfuture of plant inoculation with PGPB
??
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
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
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
Improving
shelf life of
inoculants
Bashan et al.
2014.
Plant and Soil
378:1–33
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
Surface of wet alginate inoculant containingSurface of wet alginate inoculant containing
AzospirillumAzospirillum
BEAD SURFACE
BACTERIAL MICROCOLONIES
10 µ
SURFACE OF WET ALGINATESURFACE OF WET ALGINATE
INOCULANT CONTAININGINOCULANT CONTAINING AzospirillumAzospirillum
10 µ
BACTERIA
SURFACE OF WET ALGINATESURFACE OF WET ALGINATE
INOCULANT CONTAININGINOCULANT CONTAINING AzospirillumAzospirillum
BACTERIA
10 µ
DRY ALGINATE INOCULANTDRY ALGINATE INOCULANT
1 mm
SURFACE OF DRY ALGINATE INOCULANTSURFACE OF DRY ALGINATE INOCULANT
DRY MICROCOLONIES
(Azospirillum)
CROSS SECTION OF DRY ALGINATECROSS SECTION OF DRY ALGINATE
INOCULANTINOCULANT
DRYDRY
ALGINATEALGINATE
LAYERSLAYERS
(layers)(layers)
1 mm
CenterOutsideOutside
Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry
alginate inoculantalginate inoculant
Degradation by soil microbes
Release of PGPB
SoilSoil
SoilSoil
Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry
alginate inoculantalginate inoculant
Degradation by soil microbes
Release of PGPB
SoilSoil
SoilSoil
Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry
alginate inoculantalginate inoculant
Degradation by soil microbes
Release of PGPB
SoilSoil
SoilSoil
Dry alginate inoculant mixed with wheatDry alginate inoculant mixed with wheat
seeds before sowingseeds before sowing
BEADS
SEEDS
Alginate inoculant immediately afterAlginate inoculant immediately after
sowing of wheatsowing of wheat
BEADSBEADS SEEDSSEEDS
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
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
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
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)
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
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
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
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
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
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
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
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
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
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
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
Alginate micro-beads attached to wheat seedsAlginate micro-beads attached to wheat seeds
Micro-beads
Effect of micro-bead inoculation on plantsEffect of micro-bead inoculation on plants
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
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
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
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
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)
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
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
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
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
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.
Abandoned area (for 25 years) before restorationAbandoned area (for 25 years) before restoration
Cardon natural forest
Bare area
Invasive buffel grass
Field experiments -Field experiments - planted 2004planted 2004
Anti-
herbivore
fence
Planting holes
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
One of 1500 planted treesOne of 1500 planted trees
Planting hole belowPlanting hole below
soil surfacesoil surface
Sterile StrawInoculum mixedInoculum mixed
with soilwith soil
High-density planting of cardon & palo verdeHigh-density planting of cardon & palo verde
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….
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.
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
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
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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Yoav Bashan - Inoculantes para cereales

  • 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. 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. 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. 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. 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. 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. Types of inoculantsTypes of inoculants Bashan et al. 2014. Plant Soil 378:1–33
  • 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. 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. 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).
  • 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. 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. 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. 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. 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. 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. Peat inoculation on a larger scalePeat inoculation on a larger scale MACROPLOT Inoculation during sowingInoculation during sowing
  • 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. 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. 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. Are “synthetic” inoculants theAre “synthetic” inoculants the future of plant inoculation with PGPBfuture of plant inoculation with PGPB ??
  • 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. 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. 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. Improving shelf life of inoculants Bashan et al. 2014. Plant and Soil 378:1–33
  • 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. Surface of wet alginate inoculant containingSurface of wet alginate inoculant containing AzospirillumAzospirillum BEAD SURFACE BACTERIAL MICROCOLONIES 10 µ
  • 29. SURFACE OF WET ALGINATESURFACE OF WET ALGINATE INOCULANT CONTAININGINOCULANT CONTAINING AzospirillumAzospirillum 10 µ BACTERIA
  • 30. SURFACE OF WET ALGINATESURFACE OF WET ALGINATE INOCULANT CONTAININGINOCULANT CONTAINING AzospirillumAzospirillum BACTERIA 10 µ
  • 31. DRY ALGINATE INOCULANTDRY ALGINATE INOCULANT 1 mm
  • 32. SURFACE OF DRY ALGINATE INOCULANTSURFACE OF DRY ALGINATE INOCULANT DRY MICROCOLONIES (Azospirillum)
  • 33. CROSS SECTION OF DRY ALGINATECROSS SECTION OF DRY ALGINATE INOCULANTINOCULANT DRYDRY ALGINATEALGINATE LAYERSLAYERS (layers)(layers) 1 mm CenterOutsideOutside
  • 34. Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry alginate inoculantalginate inoculant Degradation by soil microbes Release of PGPB SoilSoil SoilSoil
  • 35. Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry alginate inoculantalginate inoculant Degradation by soil microbes Release of PGPB SoilSoil SoilSoil
  • 36. Slow release ofSlow release of AzospirillumAzospirillum from dryfrom dry alginate inoculantalginate inoculant Degradation by soil microbes Release of PGPB SoilSoil SoilSoil
  • 37. Dry alginate inoculant mixed with wheatDry alginate inoculant mixed with wheat seeds before sowingseeds before sowing BEADS SEEDS
  • 38. Alginate inoculant immediately afterAlginate inoculant immediately after sowing of wheatsowing of wheat BEADSBEADS SEEDSSEEDS
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Alginate micro-beads attached to wheat seedsAlginate micro-beads attached to wheat seeds Micro-beads
  • 55. Effect of micro-bead inoculation on plantsEffect of micro-bead inoculation on plants
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Abandoned area (for 25 years) before restorationAbandoned area (for 25 years) before restoration Cardon natural forest Bare area Invasive buffel grass
  • 67. Field experiments -Field experiments - planted 2004planted 2004 Anti- herbivore fence Planting holes
  • 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. 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. High-density planting of cardon & palo verdeHigh-density planting of cardon & palo verde
  • 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. 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. 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. 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. 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