Journal of Soil Science and Plant Nutrition, 2015, 15 (2), 261-282

REVIEW

Future challenges and perspectives for applying microbial

biotechnology in sustainable agriculture based on a better
understanding of plant-microbiome interactions

J. M. Barea
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC.
Prof. Albareda 1, 18008 Granada, Spain. Corresponding author: [email protected]

Abstract

An intensive agricultural production is necessary to satisfy food requirements for the growing world population.
However, its realization is associated with the mass consumption of non-renewable natural resources and
with the emission of greenhouse gases causing climate changes. The research challenge is to meet sustainable
environmental and economical issues without compromising yields. In this context, exploiting the agro-
ecosystem services of soil microbial communities appears as a promising effective approach. This chapter
reviews the research efforts aimed at improving a sustainable and healthy agricultural production through the
appropriate management of soil microorganisms. First, the plant-associated microbiome is briefly described.
Then, the current research technologies for formulation and application of inocula based on specific beneficial
plant-associated microbes are summarized. Finally, the perspectives and opportunities to manage naturally
existing microbial populations, including those non-culturable, are analyzed. This analysis concerns: (i) a
description of the already available, culture-independent, molecular techniques addressed at increasing our
understanding of root-microbiome interactions; (ii) how to improve the ability of soil microbes for alleviating
the negative impacts of stress factors on crop productivity; and (iii) whether plants can structure their root-
associated microbial communities and, leading on from this, whether the rhizosphere can be engineered (biased)
to encourage beneficial organisms, while prevent presence of pathogens.
Keywords: Sustainable food production, microbial services, root-microbiome interactions, “omics”- driven
microbial ecology, biased rhizospheres

1. Introduction

According to information from specialized sources, agricultural practices are fundamental to meet
demand for agricultural production is expected the future world’s agricultural demands (Altieri,
to increase by at least 70% by 2050. At the same 2004). This is why modern agriculture is being
time, people are becoming aware that sustainable implemented on a global scale and diverse research

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approaches are being undertaken addressed to 2. Problems, challenges and opportunities

meet environmental and economical sustainability
issues, trying to save at most as possible usage of Scientists are aware on addressing their research efforts
non-renewable natural resources. A recommended to face a critical problem derived from the need to
approach is that based on exploiting the role of soil feed a growing, and more and more urbanized, world
microbial communities for a sustainable and healthy population. Actually, 10 billion people are expected
crop production, while preserving the biosphere. to inhabit the Planet by 2050, with a particular
Actually, soil microorganisms play fundamental incidence in developing countries. Consequently,
roles (microbial services) in agriculture mainly by many challenges arise, basically, the need to produce
improving plant nutrition and health, as well as soil more food, fiber and bio-energy, while preserving
quality (Barea et al., 2013a; Lugtenberg, 2015). the biosphere. An intensive agricultural production
Accordingly, several strategies for a more effective appears necessary, however these practices imply
exploitation of beneficial microbial services, as a low- the mass consumption of non-renewable natural
input biotechnology, to help sustain environmentally resources, such as fossil fuel and other energy
friendly agro-technological practices have been, and sources, water, agricultural soil, rock phosphate
are being, proposed. The final goal is to optimize the reserves, etc. In addition, both the energy intensive
role of the root-associated microbiome in nutrient industrial processes for the production of fertilizers,
supply and plant protection (Raaijmakers and and the runoff or leaching of soluble nutrients from
Lugtenberg, 2013). Since the interactions between the applied agrochemicals into the aquatic systems,
microbial communities and crops are influenced are sources of environmental contamination (Browne
by diverse ecological factors and agronomic et al., 2013).
managements, the impact of environmental stress Besides, intensive agriculture is known to cause
factors must be considered, particularly in the current an increase in the production of “greenhouse
scenario of global change, as they affect a proper gases”, thereby rising Earth´s temperature, thus
management of the crop-microbiome interactions affecting biosphere stability (Duarte et al., 2006).
(Zolla et al., 2013). Consequently, diverse types of stress situations are
This article is an overview of those strategies addressed generated by intensive agricultural practices, all of
to an effective exploitation of beneficial microbial them impacting on the functionality/productivity of
services in sustainable agriculture. After analyzing the both agricultural systems and natural ecosystems,
problematic, challenges and opportunities, this study and limit the services that these are able to provide.
focuses on describing the role of the plant-associated The responsible stress factors include salinity,
microbiome and their feasible managements. Finally, drought, nutrient deficits, contamination, soil erosion,
a fundamental part of this review is devoted to discuss diseases, pests, plant invasions, etc. In addition, the
the future perspectives and opportunities related to: (i) application of agrochemicals to control biotic stresses
improving our understanding of the plant-microbiome and nutrient deficiencies provokes environmental
interactions; (ii) enhancing the ability of soil microbes contamination and may threat human health. In
for stress alleviation in crops; (iii) learning whether summary, the above indicated ecological constraints
the rhizosphere can be breaded or engineered to enrich impact on agro-ecosystems and cause agricultural and
beneficial microbial functions, leading to the concept/ forest productivity losses, soil erosion, water deficit,
action of a “biased” rhizosphere.

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biodiversity losses, landscape fragmentation, etc. the environmental quality needed for a sustainable
(Vitousek et al., 1997). healthy food production. Microorganisms are attracted
Agricultural practices are currently implemented on to, and maintained at, rhizosphere microhabitats by
a global scale and different approaches are being the rhizodeposit pools (Hirsch et al., 2013b). The soil
addressed to meet sustainable environmental and microbiome comprises diverse types of organisms, but
economical developments with the final aims of bacteria, fungi, and archaea are those receiving by far
maintaining yield while preserving the biosphere. more attention in soil microbiology studies (Spence
Altieri (2004) defines “sustainable development” as and Bais, 2013). Around 109 microbial cells per g
the result of the intersections among three primary of soil have been recognized. These exhibit a great
factors: environment, society and economy, which in diversity level, reaching about 106 taxa. However,
turn interact between each two of them. Therefore, the only 1 % of microorganisms living in the bulk soil,
intersection economy-environment (agro-ecology), and 10 % of those inhabiting plant-influenced zones,
environment-society (environmental awareness), and are able to grow in standard culture media while the
society-economy (life standard), finally determines rest remains as unculturable microbes, but detectable
the concept/action of “sustainable development”. In using molecular-based approaches, as discussed later
a sustainability context, a key issue is maintaining the (Barret et al., 2013).
quality of the soil, a non-renewable resource, which The plant-associated prokaryotic bacteria and the
exerts many environmental and social functions some eukaryotic fungi have a great variety of trophic/living
of them are driven by soil microbes (Zacarini et al., habits whose saprophytic or symbiotic relationships
2013). A target in sustainability is to find out efficient with the plant could be either detrimental or beneficial.
methods for recycling nutrients, controlling pest and Most of these microbes remain in the rhizospheric
pathogens, and for alleviating the negative impact of soil or rhizoplane, but a small subpopulation of them,
abiotic stress factors, fundamental issues for human designated as “endophytes”, is able to penetrate and
life and for the sustainability of global ecosystems. live within plant tissues (Porras-Alfaro and Bayman,
These activities are typical microbial services, which 2011; Hardoim et al., 2013; Nafalnova et al., 2013;
can be exploited after an appropriate management of Brader et al., 2014; Mercado-Blanco, 2015). The
beneficial microorganisms and their functions (Zolla endophytes escape from immune plant responses
et al., 2013). Accordingly, the role and management and colonize, without causing symptoms of disease,
of the root-associated microbiome, essential to meet different plant parts (roots, shoots, leaves or fruits),
both economically and ecologically sustainable in different compartments of the plant apoplast
issues, is analyzed first in this article. (intercellular spaces and xylem vessels) and, in cases,
inside the plant cells. Some endophytes affect plant
3. Types of root-associated microorganisms and growth and plant responses to pathogens, herbivores,
strategies for their management and environmental changes, or produce important
secondary metabolites. Most endophytes are
3.1. The plant-associated microbiome unculturable, therefore the analysis of their diversity
and the molecular basis of their interactions with the
Soil microbes are recognized as a relevant component plant are revealed by using molecular approaches.
within the diverse interacting factors responsible for Strictly speaking, other microbial groups that colonize

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plant tissues, i. e. mycorrhizal fungi, rhizobia, some (Smith and Read, 2008; van der Heijden et al., 2015).
pathogens and other, are actually endophytes, but They belong to the phylum Glomeromycota (Schüßler
they are considered separately from the core group of et al., 2001).
“endophytes”, as involved in either nutrient transfer AM formation can be considered as an adaptive
from sources outside the root, i. e. soil or atmosphere, strategy, which provides the plant with an increased
or cause disease symptoms in their host plant. ability for nutrient capture and cycling in soils with
low nutrient availability. They are known to induce
3.2. Beneficial rhizosphere microorganisms an increased tolerance to environmental stresses
either biotic (pathogen attack) or abiotic (drought,
Beneficial saprophytic rhizosphere microbes improve salinity, heavy metals, organic pollutants), and to
plant performance acting as: (i) decomposer of improve soil structure through the formation of
organic substances (detritus); (ii) plant growth aggregates necessary for a good soil tilth (Jeffries et
promoting rhizobacteria (PGPR); or (iii) antagonists al., 2003). Therefore, in sustainable agriculture the
of plant pathogens. The PGPR are known to AM symbiosis plays a key role in helping the plant
participate in many important ecosystem processes, to be productive under adversity (Jeffries and Barea,
such as the biological control of plant pathogens and 2012). Similarly, AM fungi play important roles in
nutrient cycling. The PGPR must have the ability to forest ecosystems (Borie et al., 2010).
survive and multiply in rhizosphere microhabitats, in AM-colonization changes the chemical composition
competition with native microbiota, at least for the of root exudates, while the AM soil mycelium
time needed to express their beneficial plant growth itself introduces physical modifications into the
promotion activities (Mártinez-Viveros et al., 2010). environment surrounding the roots thereby affecting
The processes involved in nutrient cycling by PGPR microbial structure and diversity. These processes
include nitrogen fixation, phosphate mobilization give way to the so-called mycorrhizosphere, where
and the release of other nutrients to soil solution specific microbial interactions occur (Barea et al.,
(Richardson et al., 2009; Barea and Richardson, 2013a). Managing these interactions involving
2015). selected AM fungi and PGPR (mycorrhizosphere
Beneficial plant mutualistic symbionts include the tailoring) is recognized as a feasible biotechnological
N2-fixing bacteria and the multifunctional arbuscular tool in sustainable agriculture. Many co-inoculation
mycorrhizal (AM) fungi. Bacteria belonging to experiments using selected AM fungi and rhizosphere
diverse genera, collectively termed as “rhizobia”, are microorganisms have been reported. These include
able to fix N2 in mutualistic symbiosis with legume interactions related to: (a) symbiotic N2-fixation;
plants (Olivares et al., 2013; de Bruijn, 2015). Other (b) phosphate mobilization; (c) phytoremediation of
bacteria (actinomycetes), belonging to the genus heavy metal contaminated soils; (d) biological control
Frankia, form N2-fixing nodules on the root of the of root pathogens; and (e) improvement of soil quality
so-called “actinorrhizal” plant species, having a great (Barea et al., 2013a).
ecological importance (Normand et al., 2007). The The scenarios for applying microbial technology include
other major group of mutualistic microbial symbionts not only sustainable agriculture but also other eco-
are the AM fungi known to establish mycorrhizal systemic issues. These refer to ecosystem restoration,
associations with the roots of most plant species recovering of endangered flora, enhancing resilience

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 Future challenges and perspectives for applying microbial biotechnology... 265

of plant communities, adaptive strategies for diversity are producing PGPR inoculum products (Ravensberg,
conservation etc. (Barea et al., 2013b). In this chapter, 2015; Kamilova et al., 2015).
the management of beneficial microbial activities is Concerning production and application of AM fungal
focused on sustainable agriculture. Essentially, there inoculants, the information has been reviewed recently
two major strategies for managing the soil microbiome, (Jeffries and Barea, 2012; Singh et al., 2014). The
these are being based either on the development main points addressed in these review articles can be
of microbial inoculants or on the manipulation of summarized as follows. The difficulty in culturing the
naturally existing microbial populations, including also obligate symbionts AM fungi in the absence of their
non-culturable microorganisms. host plant is a major obstacle for massive inoculum
production. Despite this problem, several companies
3.3. Implementing the technology for the production worldwide are producing AM inoculum products,
and application of high quality microbial inoculants which are being applied in forestry, agriculture
and horticulture. Specific procedures are required
According to J. Sanjuan (pers. comm.) for a to multiply AM-fungi and to produce high quality
successful application of microbial inoculants in inocula. The resulting materials (spores, hyphae,
agriculture we need to implement the following root fragments etc.), from “culturing” AM fungi, are
aspects: (a) to increase the scientific/technological added to different carriers, resulting in a wide range of
bases of inoculum production and application; (b) to formulations, including encapsulation, to be applied
generate specific normative for each inoculant type at an agronomical scale using different techniques.
and its application, either on the seeds or on the soil, Recent developments in AM-inoculum production
or to the plant to be transplanted already microbized; systems include the in vitro monoxenic root organ
(c) to establish quality control protocols; (d) to cultures. Inoculation of seedlings (nursery production)
minimize the variability of the field results; and (e) to is potentially a good method for establishing selected
increase knowledge and dissemination by explicating fungi in the roots before potting on or planting-out
advantages and limitations, and benefits for Society. into the field, as is the case with horticulture and
Recently, Bashan et al. (2014) have published a plantation crops, including fruit farming.
comprehensive review on the formulation and Apart from microbial inoculation, there are other
practical perspectives of inoculant technology for challenging opportunities to exploit the beneficial
PGPR. They recommend a number of top priorities of activities of soil microorganisms. The perspectives
research to implement delivery systems for PGPR and for the successful manipulation of naturally
rhizobia. Among others, these priorities include an in- existing microbial population, towards a sustainable
depth evaluation of carriers, an improvement survival production of healthy foods, are becoming feasible
of microorganisms in the inoculants, to enhance shelf- thank to recent advances in the new system-based
life of the inoculants product, to use of multi-strain strategies to study plant-microbiome interactions.
inoculants, to implement polymeric/encapsulated Particularly, understanding of these interactions is
formulations, to follow low-cost technology, using being facilitated by the already available, culture-
local strains, to practice nursery inoculation for independent, molecular techniques, which are
transplanted crops, etc. Several companies worldwide discussed in the next section.

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4. Molecular ecology approaches for a better of culture-independent molecular techniques are

understanding of root-microbiome interactions becoming available, and are currently being applied
either to decipher the hidden diversity of microorganisms
The lack of appropriate methodologies have constrained inhabiting soil and rhizosphere microenvironments, or
advances for a comprehensive understanding of the to dissect the molecular bases of the plant-microbiome
mechanisms underlying plant-microbe interactions in interactions, as summarized in Figure 1. These techniques,
the rhizosphere, Difficulties rely mainly on the need based on molecular approaches, are also fundamental to
of profiling a great array of processes where the large evaluate the impacts of perturbations provoked by biotic
and diverse microbial communities, predominantly and abiotic stress factors on soil microbiome diversity
constituted by unculturable microorganisms, are and on plant-microbe interactions, in the current scenario
involved (Carvalhais et al., 2013). However, a plethora of global change.

Figure 1. Culture-independent (system-based) molecular techniques currently used to decipher the diversity
of microorganisms inhabiting soil and rhizosphere microenvironments, or to dissect the molecular bases of the
plant-microbiome interactions. Ideas based on Barret et al. (2013); Chauhan et al. (2013).

A great advance in molecular ecology technique extract their DNA/RNA and other biochemical
for analyzing soil microbial diversity relies on markers. As it is well known, the DNA characterizes
that nowadays is not necessary to isolate the the phylogenetic identity and functional capability
microorganisms (those culturable). In fact, the of the microbes, while RNA refers the genes, which
whole soil microbiome, including the unculturable are expressed in a given situation. As an example
components, can be lysed directly in soil to further of biochemical markers, phospholipid fatty acids

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 Future challenges and perspectives for applying microbial biotechnology... 267

(PLFAs) extracted from cell membranes are used RNA (rRNA) is the most frequent molecular method
as indicators of soil microbial community structure used for microbial identification. Actually, rRNA
(Hirsch et al., 2013a). gene is a universal marker as encoded in nearly
The culture-independent techniques currently used all microbial genomes. The more highly conserved
to analyze the genetic and functional diversity of regions in the rRNA gene sequence can be used
microbial communities in the bulk of soil and in the to construct “universal” primers to amplify this
rhizosphere have been recently reviewed (Barret et gene from the DNA extracted from environmental
al., 2013; Chauhan et al., 2013; Hirsch et al., 2013a; samples. The sequence analysis of cloned 16S/18S
Schreiter et al., 2015). Key information from these rRNA genes is the basis to compare, composition,
comprehensive and extended chapters is briefly and richness, evenness, and structure of microbial
pragmatically summarized below. The start point is communities. The PCR products (amplicons) sharing
the processes for extracting DNA/RNA from soil similar or identical variable region are considered as
samples. The nucleic acids can be isotopically labeled operational taxonomic units (OTUs).
prior to their extraction from the environmental Assessing the diversity of PCR products can be
samples for further monitoring. The collective performed by well-known molecular typing methods,
genome of microbes, i. e. the DNA isolated from which allow for a molecular fingerprint for the
microbial communities, constitutes an entity termed structure of a target microbial community. The
as a metagenome. Hence, metagenomics refers to the amplicon diversity can also be assessed by other
isolation and cloning of large intact DNA fragments, methods involving cloning and sequencing. These
which included several genes and operons. The total approaches are being nowadays facilitated by the
DNA extracted from environmental samples can be high throughput next-generation sequencing methods
submitted to different techniques based on cloning able to assess directly the sequence of the 16S/18S
approaches, PCR amplification, high throughput rDNA amplicons. Moreover, the 3rd generation
sequencing, or microarray hybridization. Cloning sequencing technology, SMRT (“single molecule
based approaches are allowing for the construction real time sequencing”), does not need a previous
of metagenomic libraries that can be screened either PCR process as is based on a single DNA molecule.
for structural and functional genes or for phenotypic Alternatively, to the fingerprinting strategies other
traits related to proteins, including enzymes, and approaches are being used to monitor the abundance
secondary metabolites profiling. Bioinformatic of specific taxonomic group in the communities such
based approaches are always involved in soil as the functional gene microarray-based GeoChip
microbial metagenomics studies. and PhytoChip methods. Other high throughput
The application of the PCR technique, and its sequencing techniques, such as the pyrosequencing of
derived quantitative approach (qPCR), to microbial 16S/18S rDNA amplicons, is being used to monitor
DNA extracted from soil communities have resulted the taxonomic identity (from the phylum to the genus
in a major break-through for deciphering microbial level) of microbial communities in different soils and
diversity. The small ribosomal subunit sequences (16S biomes.
for bacteria and 18S for fungi) are target molecular The functional diversity of the microbial community
markers of microbial communities. Particularly, the can be assessed by amplifying specific functional
comparative gene analysis of 16S/18S ribosomal genes (functional markers) responsible for specific

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268 Barea

metabolic processes. Since in functional diversity microbial communities in the rhizosphere. A basic
studies some insights on microbial activity are concept is that plant specific rhizodeposition (carbon-
needed, strategies based on transcript abundance have containing materials of plant origin), including root
been employed as monitored by qRT-PCR. Functional exudation, drives the selection of microbial diversity
gene arrays have been developed to evaluate the that the target plant recruit in its rhizosphere (Hirsch et
expression of transcripts from different genes and this al., 2013b). Since the root-associated microorganisms,
has been employed to assess the activity of specific stimulated by rhizodeposition, carry out specific
functional microbial activities. In this context, activities impacting on plant nutrition and health, a
shotgun sequencing involving DNA microarrays feedback loop between plant and microorganisms is
containing environmental cDNA has been used to generated (Zancarini et al., 2013). These authors point
compare the response of soil microbiome to external out that plant functioning, as affected by the activities
impacts at a transcriptional level. Metatranscriptome of microbial communities, can be analyzed thanks to
analysis of RNA from environmental samples is being high throughput plant phenotyping, while the effects
used to compare the effect of environmental factors of plant genotype on the diversity and functioning
on the transcriptomes of microbial communities. of microbial communities can be analyzed thanks to
Diverse approaches are currently used to understand molecular ecology tools (sequencing, meta- “omics”,
the molecular basis of interactions among plants and etc.), as illustrated in Figure 2.

Figure 2. Feedback activities during plant-microbe interactions in the rhizosphere. Ideas based on Zankarini et al. (2013)

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 Future challenges and perspectives for applying microbial biotechnology... 269

Application of novel technologies has allow for colonization. Other approaches are RNA-based and
detection and functioning of signal molecules rely on monitoring bacterial gene expression by either
and signaling processes in the trans-kingdom detecting active promoter or evaluating the abundance
communications leading to the establishment of of the mRNAs. Actually, the relative abundance
PGPR-plant assemblages. Drogue et al. (2013) of mRNAs in contrasting situations (comparative
divided these signal molecules according to whether transcriptome) can be evaluated by differential gene
they are produced by: (i) the plant, which affects expression studies based on microarrays. Carvalhais et
gene expression by PGPR, (ii) the PGPR, that affect al. (2013) proposed rhizosphere metatranscriptomics
plant nutrition and health, and (iii) PGPR, which as a challenging approach to provide microbial
affect gene expression by other PGPR. The primary activity profiles by assessing the expressed functional
metabolites of plant origin include amino acids, genes responsible for key rhizosphere interactions.
organic acids, vitamins, proteins, sugars, etc., while Another aspect addressed in the review chapter of
the secondary ones include flavonoids, phenol, Barret et al., (2013) is the identification and prospection
phytohormones, etc. The small signal molecules of the spatiotemporal production of signal molecules
produced by PGPR able to affect directly plant growth in the rhizosphere affecting microbial communities.
and or stress alleviation include the different classic Among these molecules are fundamental those
phytohormones, acyl homoserine lactones (AHLs) involved in the microbial quorum sensing (QS) and
and diacetylphloroglucinol (DAPG). These molecules the plant-derived quorum quenching (QQ). The QS
are involved in the control of root architecture, system is also known to modulate biofilm formation
phytostimulation, induction of systemic resistance and the production of antibiotic, siderophores and
(ISR), stimulation of root exudation, etc. (Ortiz- secondary metabolites. Microbial QS molecules, like
Castro and López-Bucio, 2013; Drogue et al., 2013). AHLs, seem to be involved also in the induction of
Interestingly, signaling processes in plant-microbe systemic resistance against leaf pathogens.
symbioses related to nutrient supply, i. e. nodulation New analytical techniques are currently being
and mycorrhization follow similar pathways, as also proposed to help understanding the dynamics of
occurs in plant-pathogen interactions (Jayaraman et rhizosphere colonization where many interactions
al., 2012; Bonfante and Desirò, 2015). are taken place (Barret et al., 2013). One of the most
System-based strategies addressed to decipher the commonly used techniques is the fluorescence in
molecular basis of plant microbiome interactions at the situ hybridization (FISH), where DNA/RNA probes
genomic, transcriptomic, proteomic and metabolomic label microbes containing homologous sequences and
levels, based on functional genomics analysis of model enable localization of individuals. Variants of the FISH
soil microbes, have recently been reviewed (Barret et like the confocal laser scanning microscopy (CLSM)-
al., 2013). Consequently, only a brief summary of FISH, among others, are improving the sensitivity of
this information is given below. Some approaches are the technique. A new alternative approach, termed
DNA-based and one of them relies on sequencing the secondary ion mass spectrometry (SIMS) is enabling
genome. Bioinformatic studies of different bacterial the analysis of the structure of microbial communities.
genomes are used to establish the distribution and Another tool to follow microbial functioning dynamics
abundance of functional traits within the genomes, in the rhizosphere is that based on the characterization
for example, the traits involved in rhizosphere of nutrient availability. These include the use of stable

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isotope labeling or biosensors. Using stable isotopes The analysis of the genome of the AM fungi (Gianinazzi-
(nutrients) in combination with FISHs approaches Pearson et al., 2012) and AM functioning, when
and oligonucleotide probes makes possible to identify symbiotically associated to plants (Franken et al., 2012;
the microbial or plant cells, which have taken up the Bonfante and Desiró, 2015), has demanded particular
labeled nutrient. research approaches based on using molecular tools.

Figure 3. The impact of environmental stress challenges on plant-microbe interactions. Ideas based on Zolla et al. (2013).

5. Improving the ability of soil microbes for signal output, which enables plants to respond to these
stress alleviation in crops based on a better environmental constraints. As plants are exposed to
understanding of plant-microbiome interactions multiple stresses simultaneously, appropriate meta-
analyses reveal a complex regulation of plant growth
Diverse types of stress factors, including salinity, and immunity. Understanding how phytohormones
drought, nutrient deficits, contamination, diseases and interact in the signaling network is fundamental
pests, etc. can alter plant-microbe interactions in the to learn how plant-microbiome systems thrive and
rhizosphere (Figure 3). Recent research is evidencing survive in stressed environments. This understanding
that plant perception of environmental stress cues is relevant to design biotechnological strategies to
triggers the activation of signaling molecules, optimize plant adaptation mechanisms and to improve
phytohormones play a key role. This signal input the ability of soil microbes for stress alleviation in
is followed by a signal processing, and finally by a crops (Pozo et al., 2015). Mechanisms involved in

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plant-microbe interactions under stress situations boosting the plant ability to respond to pathogen
are poorly understood. However, ongoing research attack, where jasmonic acid (JA) plays a key role.
is evidencing the involvement of changes in plant ISR in AM symbiosis is termed Mycorrhiza Induced
morphology, physiology, transporter activity and root Resistance (MIR). The identification of defense
exudation profiles, changes that can induce the plant regulatory elements coordinating AM development
to recruit microbes with stress-alleviating capacities, and MIR is a major challenge for research since
a strategy able to help crop productivity under stress can facilitate the development of biotechnological
(Zolla et al., 2013). strategies for improving the use of AM fungi in
As stress factors cause detrimental impacts on the the integrated management of pests and diseases
functionality/productivity of agricultural systems, (Pozo and Azcón-Aguilar, 2007; Jung et al., 2012;
the role of rhizosphere microorganisms in helping Pozo et al., 2013). In addition, PGPR, Trichoderma
plants to thrive in adverse conditions has recently spp, and non-pathogenic Fusarium strains also
been discussed (Barea et al., 2013b) The aim of this prime local resistance and ISR, as they produce
section is to analyze how we can improve the ability microbe-associated molecular patterns (MAMPs),
of soil microorganisms for stress alleviation in crops, which trigger immune responses. The priming
as aided by a better understanding of plant-microbe effects of MAMPs rely on that they activate the JA
interaction based on the already available meta signaling pathway, which regulates the inducible
“omic” and sequencing approaches. plant defenses (Pozo et al., 2015). Indeed, in the
plant hormone signaling crosstalk, which regulates
5.1 Improving the ability of soil microorganisms for plant defense and development in microbe-plant-
the biocontrol of pathogens (diseases, pests…) insect interactions, JA results in the main hormone
at switching from growth to defense responses
Rhizospheric and root endophytic microbes, including (Pangesti et al., 2013).
PGPR, Trichoderma spp. and AM fungi, protect Because the effectiveness of biocontrol practices is
plants against pathogens by competition for space and affected by the prevailing environmental conditions,
nutrients, antibiosis (for PGPR and Trichoderma), biocontrol-related research has to envisage the
mycoparasitism (for Trichoderma) and by inducing challenge of finding appropriate screening procedures
plant defense mechanisms (Barea et al., 2013a). to select microorganisms able to be highly effective
Defense priming is the preconditioning of immunity under the current changing scenarios. Understanding
induced by microbial colonization, fundamental for the impact of the environment on the biocontrol
an efficient protection against pathogens. Priming agent performance will help to predict the resulting
also acts systemically on distant parts of the root and output and to develop effective combinations of
shoots thereby inducing systemic resistance (ISR) to antagonistic microorganisms. A major challenge
protect efficiently plants against both roots and foliar in rhizosphere biotechnology is to exploit the
pathogens (Selosse et al., 2014). prophylactic ability of AM fungi in association with
The AM symbiosis protects plants against antagonist microorganisms. The final aim is to find
deleterious organisms including microbial out an enhanced capacity for bioprotection achieved
pathogens, herbivorous insects and parasitic plants. by the combination of the mechanisms used by each
AM colonization can prime plant immunity by organism individually. While experimental evidence

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272 Barea

supports that mycorrhizosphere management is a has been discussed recently (Dimkpa et al., 2009;
promising biotechnological tool to enhance plant Aroca et al., 2012; Dood and Pérez-Alfocea, 2012;
resistance/tolerance to pathogen attack, research on Groppa et al., 2012; Porcel et al. 2012; Ruiz-Lozano
the optimal microbial combinations is essential for et al., 2012a; b; Bal et al., 2013; Calvo-Polanco et al.,
the successful application in sustainable agriculture 2013; Barzana et al., 2014), and is briefly summarized
(Barea et al., 2013a). here.
Recent research on agricultural weed control is The two general mechanisms (maintaining water
revealing strategies focused on the initial steps in the and ROS balance) may be ameliorated by both the
host-parasite interaction. Actually, parasitic weeds establishment of the AM symbiosis and by inoculation
are difficult to control because most of their life with PGPR, which act through diverse specific
cycle occurs underground. The strigolactones, a new mechanisms. These can be summarized as follows:
class of plant hormones are signaling molecules, (i) cell osmoregulation (related to the accumulation
which stimulate germination of root parasitic plant of the compatible solutes such as proline, glycine,
seeds. Besides, it has been shown that strigolactones betaine, soluble sugars, pinitol and mannitol);
are involved in root colonization by the AM fungi. (ii) ionic homeostasis (based on ion balance and
Upon AM colonization, plants reduce the production compartmentalization and related to maintaining
of strigolactones thereby lowering parasitic plant a fine balance of K+:Na+ and Ca2+:Na+ ratios); (iii)
infection, and consequently diminishing the regulation of root water uptake and redistribution
deleterious effect of these weeds on plant fitness along plant tissues by aquaporins (where a
and yield (López-Ráez et al., 2012). The possible phytohormone crosstalk is involved); (iv) antioxidant
applicability of the AM symbiosis in weed control, defense (to compensate the production of harmful
based on AM activities regulating plant production reactive oxygen species (ROS); and (v) maintenance
of strigolactones, as an agricultural practice in the of photosynthetic capacity. Such microbial activities
context of sustainability issues, has been discussed result in a better regulation of plant water status and
recently (Jung et al., 2012; Pozo et al., 2013). contribute to increase plant resistance to osmotic
stress conditions. Finally, the improved water uptake
5.2. Improving the ability of soil microorganisms for capacity of microbiologically inoculated plants
alleviating the negative effects of osmotic stressors allows them to have higher transpiration rates and
(drought, salinity…) hence higher photosynthetic rates under conditions of
water deficit.
The level of aridity in many land areas of the world Particular attention is receiving the role played by
has increased progressively rising thereby drought AM fungi and other rhizosphere microorganisms to
and salinity problems. To cope with such osmotic improve plant water status based on the improvement
stressors plants must develop a number of adaptation of root hydraulic conductance, which ultimately
mechanisms including mainly a fine regulation of their depends on aquaporin functioning (Aroca et al.,
water uptake capacity and transpiration rates, and the 2012; Groppa et al., 2012; Ruiz-Lozano et al., 2012a;
activation of the antioxidant machinery to overcome b; Calvo-Polanco et al., 2013; Barzana et al., 2014).
the overproduction of reactive oxygen species (ROS) Aquaporins are membrane intrinsic proteins that
caused by the stress. The related available information allow for water and other small neutral molecules

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 Future challenges and perspectives for applying microbial biotechnology... 273

to pass across biological membranes following an associated microorganisms have also to be adapted to
osmotic gradient (Chaumont and Tyerman, 2014; the presence of contaminants (Pongrac et al., 2013).
Li et al., 2014). The improvement of the nutritional Most phytoremediation research involving plant-
status of microbial-inoculated plants, together with associated microorganisms concerns heavy metals
the release of volatile organic compounds (VOCs) (HMs) cleaning or organic xenobiotic degradation
by some microorganisms, regulates root aquaporins (Azcón et al., 2013).
expression and/or activity, and thereby root hydraulic The mechanisms underlying the role of plant associated
conductance. The regulation of root aquaporins is bacteria in phytoremediation of environments
based on phytohorme interactions, in which plant contaminated with HMs or organic xenobiotics,
levels of abscisic acid (ABA) appear to play a central in general (Germaine et al., 2013), and alkanes, in
role (Dood and Pérez-Alfocea, 2012; Groppa et al., particular (Afzal et al., 2013), have recently been
2012; Barzana et al., 2014). analyzed. These mechanisms include improvement of
While is becoming clear that AM fungi and other plant growth, nutrient (P and N) supply, production of
rhizosphere microorganisms are able to increase Fe-binding siderophores, plant hormones production,
resistance/tolerance to osmotic stressors, further enhanced ACC-deaminase activity (ethylene
studies are still needed to yield a comprehensive reductions), organic xenobiotic degradation, etc.
analysis of the transfer of this knowledge to natural Another mechanism for improving phytoremediation
ecology. This is fundamental because soil and is the bio-augmentation of plant associated microbial
rhizosphere microorganisms are key factors for communities based on horizontal gene transfer
plant survival under a changing environment where (Germaine et al., 2013). This mechanism, a challenge
plants are going to be exposed to adversity on the of future research, relies on that many resistance
oncoming years, as driven by the climatic change genes involved in HMs bioremediation processes
(Duarte et al., 2006). are located in plasmids that can be transferred within
the bacterial communities. The complete genomes of
5.3. Improving the ability of soil microorganisms for a number of plant-associated bacteria are becoming
the phytoremediation of contaminated soil available. This, together with the genome sequence
of diverse plant host, would facilitate establishing
Plant-associated microorganisms, i.e. AM fungi and the molecular communications between plant and
bacteria can enhance plant abilities for the remediation bacteria, a key step to provide new insights allowing
(phytoremediation) of environments contaminated for design improved strategies in phytoremediation
with heavy metals, radionuclides or organic (Germaine et al., 2013; Afzal et al., 2013).
xenobiotics (including volatile organic compounds, oil The AM fungi have also evolved a series of strategies
derived alkanes or polycyclic aromatic hydrocarbons). to restrict entry of toxic metal species and to keep
Current phytoremediation technologies use plants and intracellular metal homeostasis (Ferrol et al., 2009;
their associated microorganisms to remove, transfer, González-Guerrero et al. 2009; 2010). According
stabilize, decrease, and/or decompose pollutants in to these authors, the mechanisms of HM tolerance
the environment (Azcón et al., 2013). To be successful in AM fungi include reduction of metal uptake and/
for phytoremediation purposes plants must be capable or increased efflux, metal immobilization, e.g. cell-
to thrive in polluted environments while their wall adsorption, extracellular metal sequestration,

Journal of Soil Science and Plant Nutrition, 2015, 15 (2), 261-282

274 Barea

intracellular chelation by e.g. metallothioneins or activity; and (iii) inoculated HM-adapted bacteria
phytochelatins, and metal compartmentalization increased enzymatic activities and plant hormone
into spores and vesicles. Specific metal transporters production in the mycorrhizosphere. Inoculation of
regulate cytosolic metal ion concentrations and autochthonous AM fungi and PGPR, together with
translate the excess of metal within vacuoles, where the application of treated agrowaste residue, changed
they would cause less damage. AM fungi have also the bacterial community structure and enhanced
evolved mechanisms to fight against the oxidative phytoextraction to remediate HM contaminated soils.
stress produced by HMs or to repair the oxidative A challenging topic for future research is to realize
damage. Increased HM tolerance of mycorrhizal the phytoremediation effects of mycorrhizosphere
plants may be related to extensive changes in gene interactions under field conditions.
expression and protein synthesis induced by the
symbiosis itself. Glomalin-related soil proteins 6. Engeneering the rhizosphere to encourage
produced by AM fungi can irreversibly sequester beneficial microbe establishment: a great
HMs (Cornejo et al., 2008), thereby contributing to challenge for the future
metal stabilization in the soil.
Understanding the key molecular determinants of Diverse research approaches are currently addressed
metal homeostasis in AM fungi is challenging. To trying to ascertain whether the rhizosphere can be
get some insights into the underlying mechanisms, engineered to encourage beneficial organisms,
a genome-wide analysis of HMs transporters was while prevent presence of pathogens. The related
undertaken (Tamayo et al., 2014), making use of the research topics offer many challenges because there
recently published whole genome of the AM fungus are many gaps in our understanding on the ad hoc
Rhizophagus irregularis. This in silico analysis research strategies. Undoubtedly, getting biased
allowed identification of 30 open reading frames rhizosphere opens new opportunities for future
in the R. irregularis genome, which potentially agricultural developments based in exploiting the
encode HMs transporters. The authors depict a beneficial microbial services to reduce the inputs
comprehensive scheme of the mechanisms involved. of agrochemicals thereby reaching sustainable
A current challenge is to characterize the functionally environmental and economical goals.
of these transporters and to identify their location and
roles in the AM symbiosis. 6.1. Learning how plants shape microbial community
Interactions between HM-adapted rhizobacteria structure in the rhizosphere
and AM fungi have been investigated in diverse
experiments (Medina and Azcón, 2010). The main Current research is realizing that plants can structure
achievements resulting from these experiments were: their root-associated microbial communities,
(i) the target bacteria accumulated large amounts of concerning both diversity and functions (Achouak
HMs; (ii) co-inoculation enhanced plant establishment and Haichar, 2013; Hirsch et al., 2013b). Particularly,
and growth, and lowered HM concentrations in plants, Achouak and Haichar (2013) used the stable isotope
supporting a phytostabilization-based activity, while probing (SIP) together with fingerprinting approaches
the total HM content in plant shoots was higher in as a molecular detection tool to analyze the impact
dually-inoculated plants, suggesting a phytoextraction of the plant species on their rhizosphere microbiome.

Journal of Soil Science and Plant Nutrition, 2015, 15 (2), 261-282

 Future challenges and perspectives for applying microbial biotechnology... 275

They confirmed the differential impact of each target adapted microbiome, which does not constrain
plant species on the genetic and functional diversity pathogen establishment and therefore becoming
of the plant-associated bacterial communities. susceptible to diseases.
Therefore, such ability of the plants for shaping According to Bakker et al. (2012), there are two
microbial communities in their rhizospheres appears main strategies for manipulating the plant to recruit
as a new opportunity for linking structure and function beneficial microorganisms in its rhizosphere, both of
of the root-microbiome related to nutrient supply them are based on plant breeding, and are addressed
and plant protection. Carbon compounds and signal to foster beneficial microbial services for improving
molecules from root exudates are the main drivers agricultural developments. One of these alternate
of plant specific effects on rhizosphere bacteria and paths relies on develop plants able to shape their
their proteomes. Actually, the identity and quality microbiome by targeting particular taxa for specific
of rhizodeposits varies from plant to plant thereby functions i. e. N2-fixation, P-mobilization, biocontrol,
attracting a specific set of bacteria to the rhizosphere etc. The other approach is based on develop plants able
and providing them with a selective pressure to to shape their microbiome for broad characteristics
stimulate bacteria to compete and persist (Hirsch et related to promotion of plant growth and health. All
al., 2013b), a property which is depending on plant in all, in the nearest future it appears that the more
age (Spence and Bais, 2013). feasible approach to enhance beneficial microbial
Harnessing the rhizosphere microbial communities services in agriculture is the direct manipulation of
through agricultural managements the soil microbiome. Particularly, a target aim is to
In a comprehensive analysis of the available reconstruct a minimal rhizosphere microbiome able
experimental evidence, combined with theoretical to provide a maximized benefit to plant at a minimal
models, Bakker et al. (2012) outlined strategies photosynthetic cost (Raaijmakers, 2015).
to manipulate root exudation for a plant-driven A challenging strategy which offers opportunities
selection of beneficial rhizosphere microbes. The to enable plants to recruit microorganisms targeted
main ideas/conclusions of this review article are for specific functions, is that aimed at engineering
summarized here. In some cases, the ideas are nitrogen-fixing cereals (Rogers and Oldroyd, 2014;
based on speculations but these have a reasonable Oldroyd and Dixon, 2014; Venkateshwaran, 2015).
feasibility in the nearest future.
An example of harnessing the rhizosphere microbiome 6.2. The “biased rhizosphere” concept/action
derives from the existence of plant-microbe co-
adaptation, involving a shared evolution history of The “biased rhizosphere” concept is based on the
interactions between plants and soil microbiome. In possibility of provoking the production by the plant
a co-adapted rhizosphere, pathogens can be present of special compounds that can be catabolized only by
but their activities are controlled. In contrast, when target beneficial bacteria introduced as inoculants, e.g.
an agricultural plant species is moved to other parts PGPR (Savka et al., 2013). These authors have revised
of the world, as happens with the current agricultural the biased rhizosphere concept and provide pioneering
global exchange, the plant will grow in association insights on its origin and significance. The origin of the
with a microbiome, which has not shared evolutionary biased rhizosphere concept derived from an analysis of
history. The target crop will associate with an un- the interactions between rhizobia and plant (generated

Journal of Soil Science and Plant Nutrition, 2015, 15 (2), 261-282

276 Barea

by rhizopine-like molecules) and Agrobacterium and fungi, either saprophytic or endophytic symbionts
plant (generated by opine-like molecules), specific (with special reference to N2-fixing rhizobia and
compounds able to foster such interactions. Savka et AM fungi) are protagonists of applied microbial
al. (2013) also discuss how the throughput sequencing biotechnology in agriculture. Particular emphasis is
methods have provided comparative genomic being paid to formulation, quality control and modes
information on plant-associated bacteria while the of application of microbial inoculants. Many of the
omics technologies have facilitated further research mechanisms underlying plant-microbe interactions in
of these interactions. For example, the time-course the rhizosphere are still poorly understood. Difficulties
effect of host rhizosphere chemistry can be monitored rely mainly on the need of profiling the great array of
in studying microbial community structure and processes in which are involved the large and diverse
metagenomics. Future studies have to be undertaken microbial communities, predominantly constituted by
to find specific metabolite-plant species-microbe unculturable microorganisms. A plethora of culture-
combinations. Deciphering the biotic and abiotic plant independent molecular techniques is becoming
factors that shape the plant-associated microbiome available, and is currently being applied either to
through biasing the rhizosphere offers many challenges decipher the hidden diversity of microorganisms
that current research is trying to envisage. According to inhabiting soil and rhizosphere microenvironments, or
Savka et al. (2013) future work on plants must focus to define the molecular bases of the plant-microbiome
on reprogramming transport functions, while those on interactions. Diverse types of stress factors, including
microorganisms have to focus on the uptake secreted salinity, drought, nutrient deficits, contamination,
nutrients and the time-course changes in the microbial diseases and pests, cause detrimental impacts on the
community structure. A combination of all of these functionality/productivity of agricultural systems.
approaches can improve our understanding on how to A signaling network orchestrated plant-microbiome
enhance the competitiveness and persistence of bacteria interactions needed to thrive and survive in stressed
in the biased rhizosphere to finally improve plant health environments. Understanding this signal crosstalk is
and agro-ecosystem productivity. fundamental to design biotechnological strategies to
optimize plant adaptation mechanisms and to improve
7. Concluding remarks the ability of soil microbes for stress alleviation in
crops. Several approaches are currently addressed to
Exploiting the interactions between soil microbial ascertain whether the rhizosphere can be engineered
communities and crops is a relevant approach to (biased) to encourage beneficial organisms, while
increase food production for the growing world prevent presence of pathogens. The target research
population at the lowest environmental costs, in topics offer many challenges because there are
the current scenario of global change. Essentially, many gaps in our understanding. As a general
there are two major strategies for managing the conclusion, we can say that many achievements
soil microbiome, these are being based either on have been reached with the application of microbial
the development of microbial inoculants or on biotechnology in agriculture but many challenges
the manipulation of naturally existing microbial as well as opportunities need to be explored for the
populations. Both rhizosphere bacteria (PGPR) and future sustainable agricultural developments.

Journal of Soil Science and Plant Nutrition, 2015, 15 (2), 261-282

 Future challenges and perspectives for applying microbial biotechnology... 277

Molecular Microbial Ecology of the Rhizosphere.

Acknowledgements and Dedicatory
Wiley Blackwell, Hoboken, New Jersey, USA,
pp: 991-1002.
The author of this review chapter thanks the
Spanish National Research Programs AGL2012- Bakker, M.G., Manter, D.K., Sheflin, A.M., Weir,
T.L., Vivanco, J.M. 2012. Harnessing the
39057-C02-02 project and to the PAIDI Program P11-
rhizosphere microbiome through plant breeding
CVI-7640 project for financial support.
and agricultural management. Plant Soil 360,
This chapter is dedicated to Prof. José Olivares
1-13.
Pascual who, during more than half a century (1959-
2015), carried out and coordinated, and still continues Bal, H.B., Nayak, L., Das, S., Adhya, T.K. 2013.
advising on, scientific research of excellence in the Isolation of ACC deaminase producing PGPR
from rice rhizosphere and evaluating their plant
Departamento de Microbiología del Suelo, Estación
growth promoting activity under salt stress. Plant
Experimental del Zaidín, CSIC, Granada, Spain.
Soil 366, 93-105.
The author also thanks Prof. Olivares for its critical
reading of this manuscript. Barea, J.M., Pozo, M.J., Azcón, R., Azcón-
Aguilar, C. 2013a. Microbial interactions in the
rhizosphere. In: F.J. de Bruijn (ed). Molecular
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