This document summarizes the keynote presentation by José-Miguel Barea on beneficial microbes for agriculture and biosphere protection. It discusses: 1) The challenges of intensive agriculture including increased resource use, environmental contamination, and ecosystem degradation. Microbes help plants thrive in stressful environments and can be harnessed to improve plant health and soil quality. 2) Research approaches exploiting plant-microbe interactions like mycorrhizal fungi and rhizobacteria. Microbes provide beneficial services like nutrient cycling, plant protection, and bioremediation. 3) Future opportunities include establishing sustainable agriculture practices using microbial inoculants, understanding plant-microbiome interactions with new techniques, and engineering the

1. Thanks !!!
Beneficial Microbes
for Agriculture
and Biosphere Protection
B
A
B
A
B
José-Miguel Barea

2. Estación Experimental del Zaidín (CSIC),
Granada, Spain
Dpto. Soil Microbiology
and Symbiotic Systems
The Mycorrhiza Group

3. Closing notes:
The era of Microbial Biotechnology in
Agriculture and Biosphere Protection
José-Miguel Barea

4. 1. Problematic and Challenges
2. Research Approaches and Achievements
3. The Future: Challenges and Opportunities
Presentation content:

5. 1. Problematic and Challenges

6. The problem:
more people to feed !!!
The challenge:
to produce more foods
while preserving the
biosphere !!!

7. An intensive agricultural production
is essentially necessary to satisfy
food requirements for the growing
world population, thus modern
agriculture is nowadays being
implemented at a global scale

8. A tremendous problem: intensive agriculture is
associated to the mass consumption of:
 Energy
 Fossil fuel
 Water
 Forested areas
 Topsoil
 Rock phosphate reserves
...and with the emission
of greenhouse gases
generating global (climate)
changes

9. Human population
(increase in resource use)
Human activities
(Industry, Energy, Mining, Agriculture, Leisure, etc)
Land transformations Changes in the biota
(invassions, hanting, fishing)
Biogeochemical cycles
(C, N, P…)
Climatic change
(Global warming)
Biodiversity losses
(species extintion,
ecosystem degradation)
At a glance, the causes and consequencies Global Change:
F. Sapiña, adapted from Vitousek et al. Science, 1977

10. G l o b a l c h a n g e s
S u s t a i n a b l e s y s t e m s
Population
dinamics
Biodiversity
Landscape
dinamics
Ecosystem
functioning
AnthropicactivityInteractions: Biodiversity - Ecosystems functioning
– Global change -
(Kennedy & Smith, 1995)

11. Fertile soil
Degradaded agrosystem
Economic development
Intensive Agriculture:
Induces perturbations and unbalances, which give way to
a spiral of stress situations for the agro-systems
A human activity which
generates Global change and
degraded the agro-systems

12. As a consecuency of Intensive Agriculture, diverse types of stress
situations are generated, all of then impacting on agro-ecosystems
functionallity/ productivity:
Salinity, Drought,
Contamination
Diseases, Pest
Plant invassions
Soil erosion
Nutrient deficit
Intensive Agriculture
Global change
Climatic
change
Stress
factors
Agrochemicals are used
to control disease and
pests and to provide
plant with available
nutrients, but this is not
ecologically acceptable

13.  Agricultural and forestal
productivity losses
 Soil erosion
 Biodiversity losses
 Ecosystem degradation
 Landscape fragmentation
In summary: intensive agriculture, environmental contamination and
deforestation cause an increase in the production of “greenhouse
gases”, thereby increasing Earth´s temperature, known as the
anthropogenic global warming, impacting on biosphere stability
Consequently, a number of
ecological constraits impacts
on agro-ecosystems causing:

14. We need to produce more food, feed, fibre and bio-energy at low environmental
costs, for the increasing world population, but preserving the biosphere, thus the
challenge is to envisage sustainable alternatives and research approaches to meet
these aims
Unless the effects of agriculture are carefully managed through sustainable
development, both agricultural systems and remaining natural ecosystems, will suffer
degradation, increasing the lost of diversity and further limiting the ecosystem
services they are capable of providing
The triangle of sustainability
Zancarini et al., 2013

15. 2. Research Approaches and Achievements

16. Different research approaches are being undertaken addressed
to meet environmental and economical sustainability issues, trying
to save at most as possible usage of non-renewable natural
resources, but without compromising on yields
New crops and cropping systems are needed, but production methods
need to focus on efficient recycling of nutrients and effective control of
pests and pathogens
Soil is a non-renewable resource that perform key environmental,
social, and cultural functions which are vital to human life and
for the sustainability of global ecosystems, defined as ecosystem
services. These, in part, result from soil microorganisms

17. A feasible and effective approach toward developing sustainable
practices in modern agriculture is that based on exploiting the
interactions between rhizosphere microbial communities and crops
Most studies on rhizosphere microbial
communities focuss on bacteria and
fungi, either beneficial or pathogens
Angus & Hirsch, 2013
Paungfoo et al, 2013

18. Many belowground interactions are
known to affect plant health and
productivity
Zolla et al., 2013
Understanding root-microbiome interactions
Environmental characteristics
impacting plant performance
are shown in the central triangle
Root exudates potentially play major
roles in attracting and maintaining
beneficial soil communities
The environmental quality of plant
life is defined by interacting
factors, including, soil, microbial
activity, and outcomes of the plant´s
own activity
109 cells,106 taxa, per g of soil,
most of them are “unculturable”

19. Stimulation of seed germination and rooting
Increasing soil nutrient availability
Biocontrol of diseases and pests
Plant protection against abiotic stresses
Improvement of soil structure
 Microorganisms carry out well-known activities able
to improve plant growth and health, and soil quality
 They can be manipulated as crops inoculants
 Were crucial for plants to evolve on Earth

Beneficial services afforded by the soil microbiome
Barea et al., 2013
Soil microbes can be
threfore considered as
“anti-stress” agents

20. Microorganisms played a
fundamental role to facilitate
plant origin, terrestrialization
and evolution on Earth
It is a fascinant history which
demonstrates that microbes were
crucial to help plants to thrive in
a hostile and extremely stressed
environment
Microorganisms have to do the
same now … and we have to
increse our knowledge to exploit
the ad hoc opportunities in
manipulating their capabilities

21. Fossil and extant:
Unicellular
Green
Fossil Extant
Fossil Extant
Cyanobacteria were
found integrating
stromatolites dated to
be 3 300 M years old,
the oldest living entity…
… being able to fix
atmospheric N and C
Stromatolites:
only 4 places in the
world have fossilised
representatives:
Shark Bay, Australia
AlgaeCyanobacteria
Salar Llamara, Chile
2.000 M years old
Lessons from fossils !!!

22. Earth
formation
Life
origin
ARCHAEAN PROTEROZOIC
FANEROZOICPRECAMBRIAN
Years
(x106)
Time/
Era
Period
Cyanobacteria Fungi
Scale of the Geological Time
4500 4000 3500 3000 2500 2000 1500 1000 500 0
PHOTOSYNTHESIS NITROGENASE
Biological C Fixation Biological N Fixation
Unicellular Algae
Plants
“soil” appear
Green Algae
A mechanism for P
supply was needed …
… but we have evidences
to think in AM for that !!!

23. Fossil AM fungal spores
in the Rhynie Chert
(about 410 M year-old)
Dotzler et al, Mycol Progress, 2009
Fossil spores in a dolomite rock in
Wisconsin (Ordivician, 460 MY)
Redecker, Science (2000)
Extant
Arbuscules in early Devonian
bryophyte–like plants (liverworts)
and in early vascular plants
(Rhyniophyta) Taylor, 2004
Smith & Read, 2008
Fossil like-AM fungal
structures in early
liverworts (Ordovician)
Extant
More than 450 MY
of a common history
of plants and their
mycorrhiza
Presence of AM structures in earlier “plants” suggests AM as a mechanism for P supply

24. Equisetes
Licopodia
“Rhynie”
Liverworts
Arbuscules in the stems
Arbuscules is rhizomes
Ferns AM in root, similar
to those extant
Cycadins AM extant
Ginkgoals AM
Araucariaceae AM
Conifers
N hemipher AM/ECM
Cesalpinoideae
Mimosaceae
Salicaceae
ECM y AM
Ericals Ericoid
Orquids Orquidoid
Angiosperms
ECMPinaceae
ECM o AM
Fagals ECM (+ AM)
AM a key tool for plant origin and evolution

25. Conclusions:
AM fungi and other microorganisms helped plant to thrive (terrestrialization)
in a very hostile environment
They co-evolved with the plant and are currently continuing playing such a
role in environments suffering from any type of stress
As stress situations impact negatively both on crops and plant communities,
and on their associated root-microbiome, we need to restore beneficial
microorganisms by means of microbial inoculation or by harnessing the
remaining microbiome to improve plant nutrition and health

26. Possible effects of disrupting plant-microbiome adaptation
on sensitivity to subsequent disease outbreaks
Bakker et al. Plant Soil 360:1-13 (2012)
Longstanding and specific relationships between microbes and
plants appear to have led to a co-evolution between the two

27. Sustainable quality production/
Ecological revegetation
To maximize the benefical role of rhizosphere microbes
Modern agriculture/
Ecosystem restoration
Microbial inoculum application or transplanting of microbized plants
GLOMYGEL®

28. Sapropytes
N2–fixing and
others bacteria
Rhizobacteria
Rhizofungi
(Trichoderma)
Endophytic
symbionts Mycorrhizal and
other fungi
Beneficial microorganisms used as crop inoculants
Endo- Ectendo- Ecto-
Seedling establishment
Nutrient cycling
Plant protection
Bioremediation
Soil conservation
Plant succession
Mycoparasitism,
Antibiotic production,
Induced Resistance

29. Plant protection
against osmotic
stress: drought,
salinity etc.
Quorum sensing
Rhizobacteria (PGPR): benefiting plant growth and
health, and improving soil quality
Biocontrol of
plant pathogens
Plant growth
promotion
Antibiosis
Antagonism
Antioxidants
Induced resistance
Siderophores
Plant hormones
N2-fixation
P-solubilization
ACC deaminase
Siderophores
Improvement of soil
quality: soil structure,
organic matter cycling
and fitoremediation
Biodiversity regulation
and eco-dynamics of
microbial populations
Barea, Azcón, Pozo & Azcón-Aguilar, 2013

30. Rhizosphere
Roots
* Plant establishment and development (Phytostimulation)
* Nutrient cycling and supply (Biofertilization)
* Plant protection, Induced resistance (Biocontrol)
* Phytoremediation (Bioremediation)
* Soil conservation (Agro-ecosystems restoration)
Tailored Mycorrhizosphere
Co-inoculation with
target bacteria
Microbe-Microbe
interactions
Mycorrhiza
Mycorrhizosphere
Mycorrhizosphere tailoring
to improve plant fitness
and soil quality through
key ecological processes
Barea, Azcón, Pozo &
Azcón-Aguilar, 2013
MHB

31. Defense
A
B
C
D
EA
B
Synergistic interactions among
antagonistic microorganisms
and mycorrhiza for the biological
control of plant pathogens:
A model including plant roots,
mycorrhizas, bacteria, fungi,
nematodes, etc.
Antagonistic bacteria on the root
surface (A) or associated to spores
or mycelia (B) from mycorrhizal
plants (C) and Trichoderma spp, a
mycoparasitic fungus (D).
These microorganisms synergistically
interact and protect the plant
against pathogens (E) through the
combination of different mechanisms
Barea, Azcón, Pozo &
Azcón-Aguilar, 2013

32. 3. The Future: Challenges and Opportunities

33. The future
The present
Beneficial Microbes for Agriculture and Biosphere Protection
 Establishing action/concepts & scenarios for the application of
beneficial microbes through implementing the production of high quality
microbial inoculants
 Understanding root-microbiome interactions based on using the already
available culture-independent molecular techniques
 Engineering the rhizosphere to optimize their role in nutrient supply and
plant protection: the “biased rhizosphere” concept/action
Challenges and opportunities in the nearest future
The past

34.  Agroecology
 Sustainable agriculture
 Inducing resistance to
environmental stresses
 Improvement of the quality
of agro-derived foods
 Ecosystem restoration
(& recovery of endangered flora)
 Enhancing resilience of plant
communities
 Adaptive strategies for
biodiversity conservation
Action/concepts & scenarios for applying eneficial microbes
to increase stability/productivity of agro-ecosystems while
preserving the biosphere (in the current scenario of climatic change)

35. Agroecology: a basis for sustainability
A target scenario
to manage the soil
microbiome
Sustainability
Agroecology
EnvironmentSociety
Economy
Life
standard
Environmental
awareness
Sustainable
development

36. Sustinable productivity
Resource conservation
Environmentally-acceptable
Safe and healthy
Reduction of chemical inputs
Residue management
Uses renewable energy sources
Biotechnology (nitrogenase, legumes, cereals)
Crop rotation
Water and soil conservation
Integrated control of pest and diseases
Use of microbes to improve nutrient availability
Sustainable Agriculture
Biodiversity conservative

37. What do we need to implement?
- To increase the scientific/technological bases of inoculum
production and application
- Generation of specific normatives for each inoculant
type and its application, either on the seeds or on the soil,
or to the plant,to be transplanted already microbized
- Quality control protocols
- Minimize the variability of the field results
- Implementation of knowledge dissemination approaches:
advantages & limitations, and benefits for Society/Science
Microbial inoculants in Agriculture
By courtesy of Juan Sanjuan
Taking advantage from co-inoculation

38. Yoav Bashan, Luz E. de Bashan, S. R. Prabhu, J. P. Hernandez Plant and Soil 378:1-33 (2014)
Procedures for developing bacterial inoculants

39. Yoav Bashan, Luz E. de Bashan, S. R. Prabhu, J. P. Hernandez Plant and Soil 378:1-33 (2014)
Advances in plant growth-promoting bacterial inoculant technology:
formulations and practical perspectives
Formulations of inoculants for agricultural and environmental uses

40. Visualization of root-establishment of inoculated microorganisms
Inoculated biocontrol Pseudomonas form biofilms covered by a
mucoid layer (f), which created an ideal condition for QS and the
related synthesis of antibiotic & enzymes (Lugtenberg et al., 2013)
Endophytic bacteria (FISH)
Malfanova et al., 2013 Mercado-Blanco & Prieto, 2013
Endophytic Pseudomonas

41. Developing strategies for using microbes and plant in cutting-edge
application areas such as sustainable agriculture and phytoremediation
• Using a plethora of culture-independent molecular techniques, including genomic
sequencing and metagenomics
• How signaling between plants and microorganisms promotes plant growth and
development as well as nitrogen fixation and mycorrhization
• Biocontrol and disease-suppression approaches
• Properties of bacterial endophytes leading to maximized host fitness
• Applications and implications for ecological studies, decontamination of heavy
metals, and food production in the era of climate change
• How plants structure microbial communities in the rhizosphere to encourage
beneficial organisms and ward off pathogens
• Engineering the rhizosphere: The biased rhizosphere concept
de Bruijn, F.J. (Ed.) Molecular Microbial Ecology of the Rhizosphere (2013)

42. System-based approaches to study the plant-microbial interactions
Omics-driven microbial ecology: the tools are available
Barret et al., 2013
. Pyrosequencing
. 3rd generation sequencing
Comparative genomic information
on plant-associated bacteria

43. Rhizosphere metatranscriptomics, although currently challenging, is providing
microbial activity profiles in the form of expressed functional genes that play
important roles during these and other yet unknown interactions Carvalhais et al., 2013
Research scenarios for dissecting root-microbe relationships
Rhizosphere metatranscriptomics: challenges and opportunities

44. PGPR, through impacting on plant
hormone status, modify root
architecture to capture existing soil
resources, including nutrients (N, P,
Fe) and enhance water acquisition
Ortiz-Castro & López-Bucio, 2013
Transkingdon communications
Small molecule signals that regulate
plant architecture, including the six
classic phytohormones and novel
plant signal (alkamides) bacterially-
produced AHLs and diketopiperacines
Fundamental for understanding
root-microbiome interactions

45. Transkingdon communications
Major signaling events occurring between plant roots and PGPR or between PGPR themselves
Drogue et al., 2013
Plants and microbes use chemical signals to communicate and
this determines which microbes associate with the plant

46. Jayaraman et al 2012
Signaling in key processes
involved in nutrient supply
and plant protection follows
similar pathways
PAMP = Pathogen-Associated Molecular Partners
Effectors are strain specific and contribute to
pathogen virulence
How signaling between plants and
microorganisms promotes plant growth
and development as well as nitrogen
fixation and mycorrhization

47. Different root-associated microbes elicit
induced systemic resistance (ISR) via
jasmonic acid- (JA), ethylene- (ET,) salicylic
acid- (SA) or abscisic acid- (ABA) signaling
pathways.
Root-associated microbes are also known to
induce plant growth promotion via auxin-
(AUX), cytokinin- (CK), brassinosteroid- (BR)
and gibberellin- (GA) signaling pathways.
JA is considered the main hormone regulating
the switch from growth to defense through
positive and negative crosstalk with other
plant hormones.
Model of interactions between plant hormones regulating plant
defense and development in microbe–plant–insect interactions
Pangesti, N., Pineda, A., Pieterse, C. Dicke, M., and Van Loon, J. Frontiers in Plant-Microbe Interaction|2013

48. Mycorrhiza Induced Systemic Resistance (MIR) aginst plant pathogens
Priming (JA regulated) of plant defenses: a key for MIR”
AM formation reduced release of
strigolactones (SLs), which minimizes the
risk of infection by root parasitic plants
The primed plant defense restrict the
development of necrotrophic pathogens
and the performance of phytophagous
insects in the abovegrown plant parts.
Indirect defenses, such as the release of
volatiles, are boosted and parasitoids are
efficiently attracted
Priming (JA regulated) of plant defenses by
AM leads to a general reduction of the
incidence and/or damage caused by soil-
borne pathogens, nematodes, and chewing
insects
Pozo, M.J., Jung, S.C., Martínez-Medina, A., López-Ráez, J.A.,
Azcón-Aguilar, C., Barea, J.M. 2013.
In: Symbiotic Endophytes (Ed: R. Aroca). Springer-Verlag

49. Schematic overview showing the different types of plant-endophyte
interactions leading to the synthesis of metabolites
Metabolic potential of endophytic bacteria
Current Opinion in Biotechnology 27 (2014) 30 - 37
Brader, G ., Compant, S., Mitter, B., Trognitz F, Sessitsch, A.

50. Improving phytoremediation by rhizosphere microbes
• Extracellular chelation/precipitation
• Trapping the metal by cell wall components
• Intracelullar chelation
• Intracellular storage
Mechanisms involved in HM tolerance in AM fungi
Several studieshave demonstrated that:
AM fungi can play a key role in phytoremediation of
HM-contaminated soils because they improve plant
establishment and development, and reduce HM
translocation to the shoot, therefore,
AM-PLANTS RESULT MORE TOLERANT TO HM
The molecular mechanisms involved are being studied
N. Ferrol et al. EEZ
Germaine et al.,2013
Afzal et al.,2013
Plants can transform alkanes but only rhizobacteria
and endophytes can completely degrade them

51. Schlaeppi et al.,2013 Achouak and Haichar, 2013
Plants select their own microbiome
Philippot et al.,2013 Zolla et al.,2013
Shaping microbial communities in the rhizosphere as a new opportunity
for linking structure and function of the root-microbiome to increase
nutrient supply and plant protection:
Plants having different life-forms have the capacity
to promote diverse AMF communities based of their
functional characteristics
López-García, Barea & Azcón-Aguilar, 2014
Which are the biotic and abiotic factors
that shape the rhizosphere microbiome ?
Which are the factors that regulate the
expression of plant-beneficial microbial
traits ?

52.  Plants select their associated microbiome, a selection
which is depending on the age of the plant
 Belowgrown microbiome by changing plants´ physiological
and metabolical response, facilitates ISR and predation
strategies above-grown
 The impact of root-associated microbiome on plant fitness
changes with plant age
Spence & Bais (2013)
Temporal dynamics of
AMF colonizing roots of
representative shrub
species in a semi-arid
Mediterranean ecosystem
Sánchez-Castro, Ferrol, Cornejo & Barea, 2012
Microbial diversity in the
rhizosphere may change
with age and season in
both agrosystem and
natural ecosystem
A key protagonist is rhizodeposition

53. Hirsch et al., 2013
The possibilities for manipulating
plant-driven regulation for
intended agricultural,
environmental, ecological, and
food safety outcomes
are now becoming apparent
The rhizodeposits influence soil
microbial community structure
associated to a target plant
Now is becoming feasible that
the pattern of plant host
exudation can be bred or
engineered to establish biased
rhizospheres with bacteria that
can naturally, or by engineering,
to use metabolic resources
produced by the host plant
Origing of the various rhizodeposit pools

54. Alternate paths to realizing
the goal of using plants to
enrich beneficial microbial
functions: targetting
particular microbial taxa or
services (left side),
or a broad microbiome
characteristics that may
promote plant performance
(right side)
Bakker et al. Plant Soil 360:1-13 (2012)
Harnessing the rhizosphere microbiome through
plant breeding and agricultural management
Strategies for more effectively exploiting beneficial microbial services on
agricultural systems

55. Bakker et al. Plant Soil 360:1-13 (2012)
Harnessing the rhizosphere microbiome through
plant breeding and agricultural management
A variety of strategies could be used to promote beneficial services provided by
soil microbial communities, with the aim of reducing chemical inputs while sustaining
or improving crop yields
Manipulating plant traits that are related to interactions with microbes (left
side), or manipulating soil microbial communities directly (right side),could improve
conditions for plant productivity (center mechanisms)
Strategies for more effectively exploiting beneficial microbial services on
agricultural systems

56. Priorities for future research to advance the goal of more fully
exploiting beneficial microbial functions in agriculture (I)
• Make positive interactions with the rhizosphere microbiome an explicit
goal of plant breeding, and expand understanding of the mechanistic
basis for the interactions.
• Understand the impacts of plant genotype on the rhizosphere microbiome
and on the ability of plants to interact with beneficial microbes.
• Develop strategies to selectively enrich for indigenous microbes
performing beneficial functions.
• Identify root exudate components that have the largest/most consistent
effects on shaping microbial communities.
• Clarify the relative importance of exudate identity, quantity, and diversity.
Bakker et al (2012)

57. • Clarify the importance of chemical signaling (vs. resource provision) in
plant-driven structuring of the rhizosphere microbiome.
• Expand study of mechanisms and extent of plant impacts on the bulk
soil microbiome.
• Understand the extent and significance of microbial adaptation to host
plants.
• Expand study of naturally occurring positive plant-soil feedbacks to
draw new insights for agriculture.
• Investigate the importance of broad microbiome characteristics (such
as richness and evenness) in promoting plant health.
Priorities for future research to advance the goal of more fully
exploiting beneficial microbial functions in agriculture (II)
Bakker et al (2012)

58. Feed-back loop in plant–microbe interactions in the rhizosphere
that links plant genotype/functioning and microbial communities
Zancarini et al., 2013
Combining molecular microbial ecology with ecophysiology and plant genetic for
a better understanding of plant-microbiome interactions in the rhizosphere
Exploiting plant genotypic influence to manipulate the functional
capabilities of rhizosphere microbiome, or “direct” plant-soil-microbes
interactions to benefit nutrient supply and plant protection: a key tool
for future sustainable agriculture

59. Impact of stress on plant – microbiome interactions in the rhizosphere
Zolla et al., 2013
• Plant stresses alter plant-microbe interactions in the rhizosphere through a
combination of altered root exudation and shared experience of stress by
soil microbes.
• Plants may direct interactions with microbes to encourage microbial activities
that alleviate plant stress.
• Plant changes in morphology, stress physiology, and transporter activity result
in changed a root exudate profile
The cross talk involved in plant-microbiome
interactions in the rhizosphere is altered
under stress conditions, and may be used by
plants to recruit microbes with stress-alleviating
functions.
Improved and emerging technologies
will allow for more complete characterization
of the rhizosphere microbiome and root exudation
Harnessing beneficial microbial functions
to enhance plant performance under stress
will complement ongoing improvements through
conventional plant breeding and genetic engineering

60. josemiguel.barea@eez.csic.es
Thanks for your attention !!!