Exploitation of Plant Growth Promoting Bacteria for Sustainable Agriculture: Hierarchical Approach to Link Laboratory and Field Experiments

Abstract

To feed a world population, which will reach 9.7 billion in 2050, agricultural production will have to increase by 35–56%. Therefore, more food is urgently needed. Yield improvements for any given crop would require adequate fertilizer, water, and plant protection from pests and disease, but their further abuse will be economically disadvantageous and will have a negative impact on the environment. Using even more agricultural inputs is simply not possible, and the availability of arable land will be increasingly reduced due to climate changes. To improve agricultural production without further consumption of natural resources, farmers have a powerful ally: the beneficial microorganisms inhabiting the rhizosphere. However, to fully exploit the benefits of these microorganisms and therefore to widely market microbial-based products, there are still gaps that need to be filled, and here we will describe some critical issues that should be better addressed.

Full text

Citation: Massa, F.; Defez, R.; Bianco,
C. Exploitation of Plant Growth
Promoting Bacteria for Sustainable
Agriculture: Hierarchical Approach
to Link Laboratory and Field
Experiments. Microorganisms 2022,10,
865. https://doi.org/10.3390/
microorganisms10050865
Academic Editor: Elisa Gamalero
Received: 29 March 2022
Accepted: 19 April 2022
Published: 21 April 2022
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microorganisms
Perspective
Exploitation of Plant Growth Promoting Bacteria for
Sustainable Agriculture: Hierarchical Approach to Link
Laboratory and Field Experiments
Federica Massa , Roberto Defez and Carmen Bianco *
Institute of Biosciences and BioResources, Via P. Castellino 111, 80131 Naples, Italy;
federica.massa@ibbr.cnr.it (F.M.); roberto.defez@ibbr.cnr.it (R.D.)
*Correspondence: carmen.bianco@ibbr.cnr.it; Tel.: +39-081-613-2610
Abstract:
To feed a world population, which will reach 9.7 billion in 2050, agricultural production
will have to increase by 35–56%. Therefore, more food is urgently needed. Yield improvements for
any given crop would require adequate fertilizer, water, and plant protection from pests and disease,
but their further abuse will be economically disadvantageous and will have a negative impact on the
environment. Using even more agricultural inputs is simply not possible, and the availability of arable
land will be increasingly reduced due to climate changes. To improve agricultural production without
further consumption of natural resources, farmers have a powerful ally: the beneficial microorganisms
inhabiting the rhizosphere. However, to fully exploit the benefits of these microorganisms and
therefore to widely market microbial-based products, there are still gaps that need to be filled,
and here we will describe some critical issues that should be better addressed.
Keywords:
climate change; plant and microbial communities biodiversity; PGPB; microbial competi-
tiveness and persistence; field application
1. Introduction
The beneficial microorganisms interacting with plants support their growth through
various mechanisms: (i) improvement of nutrient availability; (ii) enhancement of root
development; (iii) reduction of toxic compounds levels in the soil; (iv) improvement of
resistance to both biotic and abiotic stresses [
1
]. Plants can grow around the Yellowstone’s
hot springs, in deserts, under flooding, and in salty and contaminated soils thanks to
their genetic plasticity but also to the contribution of microbial communities helping
them overcome these challenges [
2
–
4
]. In addition, certain plant diseases seemed to
be rarer in some plants, leading to hypothesize that the disease suppression could also
be due to living microorganisms [
5
,
6
]. Studies carried out to date suggest that plants
completely free of microorganisms represent an exception. Furthermore, in recent years,
new beneficial microorganisms potentially applicable for agricultural production have
been identified [
7
]. The best known and most ancient beneficial microorganisms are the
Rhizobia. These microorganisms, described for the first time by Martin Beijerinck in 1888 [
8
],
inhabit the roots of leguminous plants and provide them with fixed nitrogen. In the last
few years, the knowledge of microorganisms’ diversity and molecular mechanisms that
regulate plant–microorganism interactions has improved considerably [
9
]. The number
of recognized microbes interacting with plants, including the beneficial ones, is large
and continuously growing, and these microbes can be classified in different ways [
10
].
Beneficial microorganisms can act as free-living bacteria, symbiotic (that establish specific
symbiotic relationships with plants, i.e., Rhizobia and Frankia), and endophytes (that
colonize the interior plant tissues). Within them, a group of soil bacteria called plant growth-
promoting bacteria (PGPB) stimulates the growth and health of the plant through different
mechanisms [
1
]. These mechanisms include: (i) production of plant hormones; (ii) nitrogen
Microorganisms 2022,10, 865. https://doi.org/10.3390/microorganisms10050865 https://www.mdpi.com/journal/microorganisms

Microorganisms 2022,10, 865 2 of 12
fixation; (iii) solubilization of inorganic phosphate and mineralization of organic phosphate;
(iv) antagonism against pathogens by production of antibiotics, enzymes, and fungicidal
compounds; and (v) competition with detrimental microorganisms causing disease to crops.
The effects of human-caused climate changes are becoming more and more evident as we
see more record-breaking heat waves, intense droughts, shifts in rainfall patterns and an
increase in average temperatures. These environmental changes touch every step of crop
production [
11
,
12
]. Studies concerning the effects of climate changes on the living organisms
inhabiting our planet have highlighted that in the future the strong acceleration of these
changes will impact more and more on the growth and productivity of plants [
13
,
14
].
Furthermore, to cope with the increasing world population, an improved food production
is required. However, this objective cannot be achieved by intensifying the exploitation
of natural resources, which will be increasingly limited [
15
]. The application of efficient
and low-cost PGPB, which need minimum external energy and chemicals, could be a valid
strategy to improve the response of plants to stress and to make the best use of available
natural resources [
16
,
17
]. Although considerable efforts have been made in recent years to
clarify the mechanisms of action of these microorganisms, several aspects still remain to be
clarified. Several factors can limit the success of PGPB and some of them are the following:
(i) less than 2% of microbes interacting with plants are cultivable in the laboratory; (ii) PGPB
products are often crop-specific; (iii) the effectiveness of PGPB is generally assessed using
single microorganisms instead of an appropriate consortium and without clarifying the
roles and relationships between the various microorganisms; (iv) PGPB performances are
not evaluated under different climatic and agronomic conditions; (v) the stability and
persistence of PGPB in the host plants are often time-dependent. The latter has many
similarities with human gut microbial communities, which are closely interconnected with
human health. Indeed, several strategic therapies to restore and/or maintain the eubiotic
state of the microbial intestinal ecosystem are under investigation. In this perspective paper,
we discuss and highlight some of the gaps preventing full exploitation of PGPB abilities for
sustainable crop production.
2. Linking Plant Diversity with Microbial Diversity
The microbial community structure is modulated by various factors, whose influ-
ence varies according to the ecosystem in which the host plant grows [
18
]. The main
drivers of microbial community diversity in agricultural ecosystems are: farming practices,
environmental conditions, soil type, and plant species; whereas in natural ecosystems,
these communities are mainly modulated by biotic interactions, plant species, and plant
diversity [
19
]. Microbial communities and their host plants have closely linked identities:
plant’s genotypic differences strongly influence the structure of plant microbial communi-
ties. To better explain this relationship and to analyse the effect of reduced plant genetic
diversity on soil microbial abundance, the identification of plant candidate genes regulating
the interaction with beneficial microbial communities could be an efficient strategy [
20
].
In species-rich plant communities, the aboveground litterfall and the belowground fine
root mortality lead to the availability of greater amounts of carbon and nutrient resources
for soil microorganisms. Microbial diversity and biomass are also stimulated by diverse
root exudates, whose production is influenced by plant genotypes and development stages
(seedling, vegetative, bolting, and flowering) [
21
]. The exudates released by roots might
have a double effect: they stimulate the interaction with some particular microorgan-
isms (i.e., beneficial microorganisms) and act as antimicrobial agents for other ones (i.e.,
pathogenic microorganisms). Therefore, the diversity and quantity of exudates released can
influence the type of interaction that plants establish with the microorganisms with which
they come into contact. On the other hand, interaction with PGPB can induce changes in the
type of exudates, which in turn modulates the response of the host plant to various external
stimuli [
22
]. In addition, different plant tissues can host distinct microbial communities [
23
].
For instance, since shoot tissues are exposed to challenging environmental factors as com-
pared to roots, a high degree of adaptation is required for microbial colonization, and only

Microorganisms 2022,10, 865 3 of 12
a few microbial groups adapted to environmental variations will be successful. Moreover,
it was observed that microorganisms, such as PGPB, containing key genes responsible for
the health status of the host plant, are common in different plant species [
24
]. However,
there is a knowledge gap concerning the taxonomy of PGPB, and this is probably due to
the following reasons: (i) these microorganisms belongs to many different phyla, and most
of their phylogenetic analyses have been carried out within their specific genus and not
within a group; (ii) the microbial classification is initially carried out only evaluating the
morphological, biochemical, and functional characteristics through culture-based methods,
without considering the genetic features, thus leading to incorrect classifications of many
taxa [
24
]. Therefore, a careful classification of microbial community associated with a given
plant genotype is essential to recognize the efficiency of PGPB as bio-inoculants under
various environmental conditions.
3. Competence and Persistence of Microorganisms in the Field: Effect of Inoculant
Strains on the Resident Microflora
Various critical factors can influence the efficacy of PGPB-based bioinoculants,
including soil health, colonization efficiency, and persistence in the soil [
25
]. One of
the main factors influencing soil health is the carbon transformation [
26
]. The decom-
position of plant residues and other organic matter, the cultivation intensity, and tillage
lead to carbon transformation, which in turn directly affects the composition of microbial
communities [
27
]. The efficiency of PGPB strongly depends on their ability to compete
with autochthonous microorganisms present in the soil, and to adhere and colonize the
external and/or internal part of plant tissues [
28
]. In these processes, the formation of
biofilms by PGPB can play an important role. To assess the ability of PGPB to colonize
roots and persist in the soils, several techniques can be used, including microbial enumera-
tions by culture-based methods, DNA-based methods, and microscopy-based techniques.
To ensure good results in microbiological analyses and to obtain representative samples for
each treatment to be analysed, correct soil sampling in the laboratory, greenhouse trials,
and field experiments is a critical step [
22
]. Temporal and spatial aspects could also be
considered during sampling. To reduce the environmental impact, the cultivation of a
specific crop over a consistent number of years and in the same soils is the most promi-
nent factor [
29
]. The simplest approach used to overcome spatial variables is to collect
the soil samples at random, ensuring that each sample has the same opportunity to be
selected. For rhizosphere studies, soil attached to the roots should be carefully removed
and collected. To evaluate external and internal root colonization, plant roots should be
washed in sterile water or phosphate buffered saline and then homogenized in the same
buffer. To study microbial endophytes, sterilization of the roots’ surface is necessary before
carrying out the homogenization procedure. The procedure widely used to evaluate the per-
sistence of inoculated microorganisms in soil and/or rhizosphere is the culture-dependent
method [
22
]. However, with this method, it is not possible to evaluate the totality of
microorganisms present in the soil and/or rhizosphere, because only 0.1 to 1.0% of soil
microorganisms are culturable [
30
]. Moreover, this method is useful if the experiments are
carried out in sterile conditions, without the interference of soil autochthonous microbial
populations. Culture-based methods may be complemented with culture-independent
approaches, such as PCR-based methods and next-generation sequencing, to examine
the variations in the microbial community after inoculation treatment [
22
,
31
]. Therefore,
a combination of culture-dependent methods and molecular approaches could be used
to track inoculated strains or microbial consortia in natural habitats. The limiting step
in both direct and indirect methods is the DNA extraction: due to the adhesion of DNA
to soil particles, the extracted DNA will mostly represent the dominant species. It has
been observed that to fully exploit the abilities of PGPB, it is very important to reach and
maintain a high-density population on the roots of the host plants [32].
Nevertheless, it is also possible that microorganisms never colonize the plant despite
their high density. Furthermore, there are also cases in which microorganisms induce the

Microorganisms 2022,10, 865 4 of 12
plant to synthesize molecules necessary for their nutritional needs. A particular example
concerns the really useful pathogen Agrobacterium. Through a rare interkingdom DNA
transfer, this bacterium moves some of its genes into its host’s genome, thereby inducing
the host cells to proliferate. The result is uncontrolled cell growth leading to a tumour
or excessive production of roots. The proliferating plant tissues produce opines, which
are compounds that Agrobacterium and a few other organisms can use as a source of nu-
trients [
33
,
34
]. The most commonly used method to detect bacteria inside plant tissues
involves the labelling of the cells with fluorescent systems, such as GFP, and their detection
by fluorescence microscopy [
22
]. However, the use of GFP-tagged microbial strains is not
applicable in field trials since the tagged microbial cells could be released into the envi-
ronment. In addition, the autofluorescence of the plant cell walls makes the visualization
of labelled microorganisms difficult in situ. Therefore, efforts should be made to find a
suitable system to track PGPB interacting with plants under field conditions.
4. Plant Phenotyping to Analyse the Effects of Beneficial Microbes
PGPB efficiency is also greatly affected by plant age and developmental stage [33].
These parameters have greater effects on microorganisms present in the plant com-
partments than in the soil [
35
]. However, the mechanisms that regulate the distribution and
differentiation of microbial communities during the various phases of plant development in
the field are not yet well known. The microbial differentiation may be influenced by plant
factors (such as root growth, physiology, architecture, morphology, and exudates), environ-
mental factors (such as air, dust, rainfall, and temperature), edaphic factors, and fertilization
regimes [
36
]. Plant phenotyping technologies based on non-destructive image analyses
are useful instruments for addressing and understanding the complex plant-environment
dynamics represented. The information resulting from the application of these technologies
can be exploited for the genetic improvement of crops or for the analyses of the beneficial
effects of microbial communities’ presence [
37
–
43
]. The development of new innovative
approaches (visible imaging, fluorescence imaging, thermal infrared imaging, imaging
spectroscopy, and other techniques) allowed to reach accuracy and precision in phenotyp-
ing analyses [
35
,
37
,
39
,
43
]. In particular, visible imaging provides information regarding
plant growth morphology and allows the analysis of changes in phenotypic characteristics
and the plant’s biomass. Another technology commonly used for plant phenotyping is
spectroscopy imaging [
35
,
38
,
43
]. One of the parameters measured with this technology is
the vegetation indices, the most popular of which is the normalized difference vegetation
index, which is used to assess the general health status of crops. Early stress symptoms
of plant diseases or the plant water status can be monitored through the use of thermal
imaging, which is the most commonly used system for the analysis of stomatal activity, as
the stomatal conductance increases with rising temperature [
38
,
43
]. Fluorescence imaging,
consisting of the imaging of fluorescence signals obtained by illuminating samples with
visible or UV (ultraviolet) light, is primarily used to study the effect of environmental
conditions on photosynthesis and its associated metabolism [
43
]. Finally, modern optical
3D structural tomography and functional imaging techniques, such as Nuclear Magnetic
Resonance Imaging and Positron Emission Tomography, have greatly improved living
plant visualization in a non-destructive manner. Phenotyping can take place under labo-
ratory, greenhouse or field conditions. In laboratory experiments, environmental factors
can be controlled and varied during the experiments, but only a limited number of envi-
ronmental factors can be investigated [42]. Anyway, laboratory experiments contribute to
understanding specific plant dynamics in detail. This information should be then related
to field conditions, which are more complex, highly variable and fluctuating in time and
space, and therefore could better support scientists and breeders in the analyses of crop
features [42,44–46].

Microorganisms 2022,10, 865 5 of 12
5. Model Plants to Identify Plant and Microbial Candidate Genes Governing
Plant-Microbe Interaction
The genetic features needed for an efficient association between plants and micro-
bial communities are complex and still poorly understood. Even though it was observed
that genotypic differences of the host plants significantly affect root associated microbial
communities, plant breeding programs so far did not pay attention to the analysis of the
microbial communities associated with them. In particular, no genetic loci affecting the
establishment of association with the microbial communities have been identified. Ba-
sic studies have detected few plant molecular pathways so far, only those associated with
shaping the plant-microbe interaction, and only for a few model cultivars [
47
]. It was
shown that the plant-associated microorganisms are able to affect different plant traits,
such as nutrient uptake, flowering time, and stress resistance [
48
]. Indeed, recent studies
with microorganisms isolated from soil suggest that the capacity of plants to mobilize
phosphorus (P) improves when they are associated with specific microbial communi-
ties [
49
]. Moreover, it was discovered that rhizosphere microbial communities influenced
the flowering time of
Arabidopsis thaliana
, suggesting that microorganisms play a key role
in plant functioning [
50
]. In addition, several studies concerning the beneficial effects
of microorganisms on plant response to stresses, and in particular to the abiotic ones
have also been reported
[51–53]
. Therefore, to modify specific plant traits, a combined
approach of breeding plants and engineering the microbial community associated with
them could be an effective approach [
54
,
55
]. However, due to the lack of integration be-
tween physiological data and those concerning the interaction with microbial communities,
the targeted microbiome modification remains an arduous process. To design synthetic
microbial communities for higher crop productivity, it is important to understand how
plants and microbes communicate with each other, and which plant genes allow crops
to shape the rhizosphere microbial community [
56
,
57
]. Modern technologies such as
next-generation sequencing (NGS), omics approaches (metagenomics, transcriptomics, pro-
teomics, metabolomics), and computational tools enable the understanding of molecular
aspects of the plant-microbes interactions governing the plant traits. Recently, several
reports investigated the influence of host genotype on different facets of the microbial com-
munities. Genetic information about these interactions is becoming available for several
crops and associated microbes [
58
,
59
]. In this regard, the CRISPR (clustered regularly inter-
spaced short palindromic repeats)-based genome editing, is an ideal technology to obtain
mutant plants or microbes differing from the parental ones only for a point mutation, with-
out introducing exogenous DNA sequences [
60
–
62
]. The use of complementary sequencing
and transcriptomics techniques currently available can lead to the production of a lot of
sequence data, several mutants, even for the main crops, and to identification of specific
genetic loci involved in plant-microbe interactions under field conditions. The CRISPR
technology could exploit this information to introduce targeted genetic modifications in
both plants and microbes, thus leading to the development of improved plants/microbes
usable for sustainable agricultural practices [
63
–
65
]. However, a major obstacle to the
genetic improvement of plants is represented by the lack of adequate legislation.
6. Application of Stress Conditions to Analyse Microbial Traits and Plant Phenotype
under Conditions as Close as Possible to Those Found in the Open Field
The ultimate action of PGPB on plant growth and health depends on several factors,
including the ability to survive, colonize, and establish interactions with the host plants.
The evaluation of these parameters is especially important for PGPB application in
field conditions, where several variables hindering their success come into play.
The most commonly used approach for the selection of PGPB is to isolate the different
strains and assess their growth promoting traits under aseptic conditions. However, this ap-
proach is limited since it does not take into account that in real conditions the PGPB
efficiency depends not only on the individual traits analysed but also on their interaction
with other factors, which can be associated with both plants and the environment. There-

Microorganisms 2022,10, 865 6 of 12
fore, to identify efficient PGPB, it is essential to evaluate as many variables as possible
in greenhouse experiments, in order to simulate the real field conditions [
66
–
69
]. More-
over, considering that the environmental conditions have strong influence on the microbial
communities associated with plants, having information about the place of origin of the
tested strains can be helpful for field application [
67
,
68
]. The use of throughput sequencing
of nucleic acids for molecular characterization of PGPB under real field conditions might be
an effective approach to select PGPB candidates [
69
]. Plants living in extreme environments,
such as areas characterized by high temperatures, high salinity, and low level of nutrients,
have adapted to those conditions. This adaptation is the result of their genetic plasticity
but is also due to the action of the microorganisms associated with them. Since these
microorganisms have experienced the same adaptation as their host plants, they could
represent an optimal source for the selection of PGPB usable to improve plant growth
under real field conditions where they are frequently subjected to different abiotic stress
conditions [67,70].
7. Effects of Climatic Conditions on Microbial Inoculants
It is now well recognized that climate changes negatively affect most of the living
organisms of our planet, including plants and microorganisms [
71
]. The ever more sudden
alterations in environmental conditions can induce changes in the physiology of plants,
leading to a different distribution of fundamental elements, such as C and N, in the different
compartments of the plants [
71
]. A different distribution of C and N within plants can
cause alterations in the exudates released by the roots into the environment. The ability
of beneficial bacteria, such as PGPB, to colonize the roots of the host plants is strongly
influenced by root exudates, which have a complex composition and include molecules
acting as chemoattractants. The recognition of root exudates represents the first step of
the recruitment and colonization processes that allow PGPB to colonize the host plants.
Therefore, climate changes negatively impact the composition of microbial communities in
the soil and the activities of microbes interacting with the plants [
72
–
75
]. Moreover, climate
changes can directly influence the microbial composition of soils. In particular, the scarcity
of rain can lead to a reduction in the biomass of microorganisms present in the rhizosphere.
Among them, there are PGPB, which help the host plants to counteract the negative effects
caused by stress conditions (abiotic and biotic). As plant-associated bacteria depend on
root exudates or plant metabolites and are substantially influenced by environmental
parameters, it is plausible that these microbial communities will be increasingly affected by
the extreme conditions associated with climate changes [
76
,
77
]. Therefore, understanding
how climate influences, either directly by altering environmental conditions or indirectly
by changing plant physiology, microbial community composition is a major challenge in
the future, when the use of sustainable agriculture will be imperative.
8. Find the Best Practice for Application of PGPB in the Field
An important parameter that must be evaluated before using bioinoculants in agricul-
tural practices is its vitality during the storage and application steps.
To ensure the activity of bio-inoculants, the use of microbial consortia, instead of
single strains, to inoculate the host plants can be useful. Applying consortia, there will be a
good probability that at least some of the microorganisms contained in the bioinoculants
will survive [
78
]. However, even in the case of microbial consortia, it is important to
evaluate how the environmental stresses can affect the vitality of bioinoculants. To this
aim, various parameters, including bioinoculants formulation and delivery, should be
monitored [
79
–
81
]. The identification of the right procedure for inoculant formulation
is crucial to provide stabilization and protection of consortia during transport, storage,
and application. This procedure should ensure the survival of a sufficient number of
microbial cells able to exert their positive effect in the host plants. Bioinoculants industry
has developed several systems that involve the use of different carriers (solid, slurry and
liquid) and additives (i.e., adhesives and surfactants) [
82
]. Another important factor to

Microorganisms 2022,10, 865 7 of 12
consider is the release procedure, which can consist of the release of microbial suspensions
(made in water, oils, or emulsions) directly into soils or applied on the seeds. Although the
direct application in the soils allows to obtain a higher concentration of PGPB compared
to the treatment on seeds [
83
], it is associated with a higher risk of contamination and to
the loss of metabolic activities of PGPB contained therein [
69
]. Microbial encapsulation
with polymeric hydrogels can be used to overcome these limitations. An alternative
system is represented by solid formulations, in which solid carriers, such as peat, charcoal,
vermiculite, cellulose and polymers, are used.
The use of alginate microbeads to encapsulate PGPB has yielded very promising
results [
84
,
85
], as it is a reproducible method. However, even if this method is effective in
laboratory experiments, it is economically disadvantageous when used on a large scale,
such as open field applications. An alternative system, to increase inoculant survival
rate and reduce contamination, could be the reduction of the moisture content of PGPB
formulation by using a fluidized bed dryer (FBD) equipment. This system has a high
drying rate, and the material is dried in a very short time and remains free-flowing and
uniform [
86
,
87
]. One of the main disadvantages of this process is that it operates at about
37
◦
C to 40
◦
C, and therefore is more suitable for mesophilic organisms. To ensure a fair cell
density during storage and to increase formulation efficiency, the alginate or FBD methods
could be promising systems. To date, one of the main obstacles to the use of microbial-based
bioinoculants is the lack of data regarding their efficacy across a range of agricultural field
settings. Moreover, systems to evaluate the effective colonization of the host plants and the
persistence of PGPB in soils over time have not yet been developed.
9. Functional Collaboration Combining Expertise in Basic Science, Development,
Testing, and Marketing
To fully exploit the potentiality of PGPB as bio-inoculants for sustainable agriculture
productions, the integration of biology, chemistry, physics, engineering, microbiology,
biotechnology, genomics, computer science, and many other disciplines is a promising
strategy. Using this approach, the selection of most suitable and effective strains can be
performed for the production of high-quality bio-inoculants having higher performance
and wide applicability. For example, in an area where phosphate utilization is the major
concern, it is relevant to select the inoculant having the property of phosphate-solubilizing
activity [
88
]. Moreover, the solid substrate-based bio-inoculants usually have a short shelf
life (about 6 months) and are highly sensitive to direct sunlight, thus requiring specific
storage conditions [
10
]. Therefore, the selection of bio-inoculants that maintain their
potential efficacy beyond the period of 6 months and do not require temperature-controlled
storage is desirable. Furthermore, since the lack of awareness regarding the use and role
of bio-inoculants has hindered their mass spreading among the different stakeholders,
the use of appropriate market information could be a successful strategy to overcome
this gap [
89
]. To date, the demand for bio-inoculants is still not very high, and this is
probably due to the implementation of policies that are either too restrictive or improper,
especially in the countries of the European Community [
90
,
91
]. In the USA, APHIS regulates
living organisms that could impact animal and plant health and that can be biocontrol
organisms or have biopesticidal properties (https://www.aphis.usda.gov/aphis/home
(accessed on 14 April 2022)). In Latin America, public policies are encouraging the use
of alternatives to agrochemicals (pesticides and fertilisers) through the development of
regulatory frameworks for product evaluation and approval. Colombia is the only country
with specific legislation on biological inputs, while Argentina and Brazil have both recently
developed a national programme to promote bio-inputs [
92
]. Therefore, the implementation
of proper and uniform policies could favour the production of bio-inoculants and help to
overcome the difficulties in registering new products.

Microorganisms 2022,10, 865 8 of 12
10. Conclusions
To enhance plant protection and agriculture production in a sustainable way, there
is a need for new tools to increase our knowledge on the mechanisms that regulate plant-
microbiome interactions. In this context, an in-depth analysis, also based on model plants
and microorganisms, will allow to better understand how microbes can contribute to the
well-being of plants and how these beneficial effects can be harnessed for agricultural
application. Furthermore, the development of specific and easy methodologies for the
evaluation of PGP activities that inoculated strains have on the soil could help to clarify
the processes that take place during plant-soil-microbe interactions. The potential appli-
cation of beneficial microbes in agriculture seems unlimited, but more attention to the
transfer of discoveries from lab to field, as well as technical, regulatory, and marketing
issues, is mandatory. An integrated approach to address the multi-faceted nature of these
challenges is most likely to succeed (Figure 1).
Microorganisms2022,10,xFORPEERREVIEW8of12


andapproval.Colombiaistheonlycountrywithspecificlegislationonbiologicalinputs,
whileArgentinaandBrazilhavebothrecentlydevelopedanationalprogrammeto
promotebio‐inputs[92].Therefore,theimplementationofproperanduniformpolicies
couldfavourtheproductionofbio‐inoculantsandhelptoovercomethedifficultiesin
registeringnewproducts.
10.Conclusions
Toenhanceplantprotectionandagricultureproductioninasustainableway,there
isaneedfornewtoolstoincreaseourknowledgeonthemechanismsthatregulate
plant‐microbiomeinteractions.Inthiscontext,anin‐depthanalysis,alsobasedonmodel
plantsandmicroorganisms,willallowtobetterunderstandhowmicrobescancontribute
tothewell‐beingofplantsandhowthesebeneficialeffectscanbeharnessedforagricul‐
turalapplication.Furthermore,thedevelopmentofspecificandeasymethodologiesfor
theevaluationofPGPactivitiesthatinoculatedstrainshaveonthesoilcouldhelpto
clarifytheprocessesthattakeplaceduringplant‐soil‐microbeinteractions.Thepotential
applicationofbeneficialmicrobesinagricultureseemsunlimited,butmoreattentionto
thetransferofdiscoveriesfromlabtofield,aswellastechnical,regulatory,andmarket‐
ingissues,ismandatory.Anintegratedapproachtoaddressthemulti‐facetednatureof
thesechallengesismostlikelytosucceed(Figure1).

Figure1.PlantGrowthPromotingBacteria(PGPB)forsustainableagriculture:linkbetweenla‐
boratoryandfieldexperiments.
AuthorContributions:Conceptualization,F.M.andC.B.;writing—originaldraftpreparation,F.M.
andC.B.;writing—reviewandediting,R.D.andC.B.;fundingacquisition,R.D.andC.B.All
authorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:Thisworkwassupportedbythegrant“MICRO4Legumes”(Ilmicrobiomavegetale
simbiontecomestrumentoperilmiglioramentodelleleguminoseforaggere),D.M.n.89267(MIUR,
ItalianMinistryofAgriculture).Thisworkwasalsopartiallysupportedbythegrants“E‐Crops”
(Tecnologieperl’AgricolturaDigitaleSostenibile),ARS01_01136(MIUR,ItalianMinistryof
Agriculture),and“Bio‐Memory”(Laretedellebio‐banchedelCNRperilbio‐monitoraggio,la
conservazionedellabiodiversità,lasostenibilitàagro‐alimentareeambientale,eilbenessere),
SAC.AD002.173(CNR,NationalResearchCouncil).
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Notapplicable.
Figure 1. Plant Growth Promoting Bacteria (PGPB) for sustainable agriculture: link between labora-
tory and field experiments.
Author Contributions:
Conceptualization, F.M. and C.B.; writing—original draft preparation, F.M.
and C.B.; writing—review and editing, R.D. and C.B.; funding acquisition, R.D. and C.B. All authors
have read and agreed to the published version of the manuscript.
Funding:
This work was supported by the grant “MICRO4Legumes” (Il microbioma vegetale sim-
bionte come strumento per il miglioramento delle leguminose foraggere), D.M.n.89267 (MIUR, Italian
Ministry of Agriculture). This work was also partially supported by the grants “E-Crops” (Tecnolo-
gie per l’Agricoltura Digitale Sostenibile), ARS01_01136 (MIUR, Italian Ministry of Agriculture),
and “Bio-Memory” (La rete delle bio-banche del CNR per il bio-monitoraggio, la conservazione della
biodiversità, la sostenibilitàagro-alimentare e ambientale, e il benessere), SAC.AD002.173 (CNR,
National Research Council).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Marco Petruzziello for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.

Microorganisms 2022,10, 865 9 of 12
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