Introduction
It is well known that a gram of soil contains thousands of individual microbial taxa
including bacteria, fungi, protists, oomycetes and viruses. Many of them play the
main role in ecosystem functioning determining soil fertility and provide plant growth
promotion and disease suppression, (van der Heijden et al., 2008; Glick, 2012; Serna-Chavez
et al., 2013; Maron et al., 2018). However, after many years of chemical fertilization,
soils lost their natural fertility, plant diversity and microbial richness (Huang
et al., 2019). In addition, an increasing number of stress factors are observed such
as salinity, alkalinity/acidity, contamination, nutrient deficiency or overload of
chemical fertilizers, drought, soil erosion due to climate change, and various biotic
factors (Fitzpatrick et al., 2019). The use of plant beneficial microorganisms (PBM)
to mitigate these 0problems in cultivated crop production is now a common practice
particularly in the modern, sustainable agriculture and in the context of increasing
world population and environmental and climate concerns (Shilev et al., 2019). During
the last 20–30 years, a large number of microorganisms have been isolated, characterized
and tested as biofertilizers and biocontrol agents in controlled and natural conditions.
The results confirmed the beneficial effect of the selected microorganisms on plant
growth and health, enhancing nutrient content and improving soil properties. Now,
the emphasis of the scientific activity in the field of microbial inoculants is on
developing environmentally friendly and efficient microbial formulations and analyse
how the introduced microorganisms affect microbial community, diversity, and the specific
plant–microorganisms interactions, which determine the plant holobiome functioning
(Berg et al., 2017). Therefore, at this moment, at least two major lines of research
can be distinguished: the first one deals with holobiome/hologenome studies including
molecular mechanisms and genetic regulation (and epigenetic mechanisms) of beneficial
microbiota (Corbin et al., 2020) and, another important line of research on the process
of establishing a plant beneficial microbiome includes development of efficient single
or multiple microbial inoculants. A combination of pro- and postbiotics could be applied
to manage and stimulate the existing beneficial microbiome.
What is Important to Know Before Selecting a PBM?
There are many interrelated points in our understanding of the role of PBM that should
be taken into consideration when designing inocula of PBM and applying them in the
field. Firstly, the coexistence of all multicellular eukaryotes and microorganisms
forming a holobiome and hologenome was evolutionary proved. The vast majority of recent
studies including in the field of plant–microbe interactions, have confirmed the role
of beneficial microorganisms in host development, metabolism, stress adaptation, and
health. It appears that hosts can attract microorganisms with specific plant-beneficial
characteristics (Rodrigo et al., 2017). Secondly, due to chemicalization of soils,
climate and environmental changes, there is a clear decline in the soil microbial
diversity and in the number of PBM: plants are less able to attract, select, and outsource
their colonizers as the link between them is broken (Hardoim et al., 2015). Therefore,
based on previous physical, chemical, and biological/biochemical analysis of the soil–plant
system and microenvironment, we should introduce microbial inoculants composed by
a single or multiple microorganism(s) (Qiu et al., 2019). Thirdly, in some cases,
microbial formulated products demonstrated excellent plant growth promoting or plant
protection effects under greenhouse-controlled conditions, but showed unsatisfactory
results in field conditions. Moreover, some studies demonstrated reduced plant growth
and increased microbial phytopathogenicity as a result of soil–plant systems inoculation
with potentially beneficial microorganisms in conditions of nutrient saturation, changes
in the microbial community, or environmental and plant genotype effects (Rayan and
Graham, 2002; van der Heijden et al., 2008; Serna-Chavez et al., 2013; Fitzpatrick
et al., 2018).
Prebiotics, Probiotics, and Postbiotics
Based on the above considerations, three strategies for microbial management of soil–plant
systems could be selected based on prebiotics, probiotics, and postbiotics (
Figure 1
).
Figure 1
Diagram showing the three strategies for microbial management of soil–plant based
on prebiotics, probiotics, and postbiotics approaches. Full lines show the direct
effect, dashed lines show the interactions, dotted lines—the formulation/production
processes.
Prebiotics and Synbiotics
Prebiotics are products, which improve microbial diversity and soil microbial health
by promoting the growth of soil microorganisms already present within the soil–plant
system. Prebiotics are natural products, normally agro-industrial wastes, including
biochar, sewage sludge, compost, humus, animal manure, and chitin-bearing wastes,
among others, which ameliorate (particularly in degraded soils) the soil structure,
biochemical activity, and increase microbial population and diversity (Baker et al.,
2011; Vassilev et al., 2013; Strachel et al., 2017). Compost and animal manure, however,
can be considered as synbiotic products (Adam et al., 2016) as they contain microorganisms
(some of them with beneficial properties); PBM could be additionally inoculated into
the compost. Solid-state fermentation (SSF) based inoculants can also be defined as
synbionts. The final SSF products are multifunctional mixtures of mineralized organic
matter (with both prebiotic and carrier functions) and plant beneficial microorganism(s)
(with probiotic plant growth promoting or biocontrol functions) (Vassilev and Mendes,
2018). When the probiotic microorganism is a P-solubilizing agent, the synbiotic mixture
could additionally be enriched with plant available P (Shilev et al., 2019). Similar
synbiotic characteristics can be observed in microbial inoculants encapsulated in
natural gels in the presence of additives with beneficial microbial stimulating action
(Vassilev et al., 2020).
Probiotics
In the field of soil–plant science, probiotics are accepted as beneficial microorganisms,
which exert health promoting and nutrient-mobilizing properties, as defined by Haas
and Keel (2003). Particularly attractive are bacteria with high enzyme (ACC-deaminase)
activity, production of phytohormones (auxins, cytokinins, gibberellins), osmolytic
metabolites (e.g. trehalose, glycin betaine) (Schilev et al., 2019). These microorganisms
can be found at best on the surface or within the plants (Mendes et al., 2013; Hardoim
et al., 2015). Once introduced into soil, probiotics should develop a critical biomass
level to exert their plant beneficial traits. As this process is highly dependent
on the soil–plant characteristics and environmental conditions, it seems difficult
for a given single microorganism or a microbial consortium to reach this critical
cell number (Woo and Pepe, 2018). Therefore, after a long period of studies on isolation,
selection, and characterization of PBM, research scientists are focused on development
of economic biotechnological processes for biomass/spores production and formulation
that will solve the above problems (Bashan et al., 2016; Parnell et al., 2016; Vassilev
and Mendes, 2018). Formulated products can be liquid or solid and should fulfil a
number of requirements, the most important of which are to demonstrate high colonizing
effectiveness and competitiveness, and increase plant nutrition and health status
(Malusa and Vassilev, 2014). One of the most promising formulation techniques is the
encapsulation in macro- and micro-beads of polysaccharides which guarantees a continuous
deliver of the inoculant into soil preventing the effect of soil and environmental
stress factors including indigenous microbial community (Bashan et al., 2016; Qiu
et al., 2019). However, a simple gel-entrapment is not sufficient to ensure economical
advantages and desired agronomic impact of the formulates (Vassilev et al., 2020).
Double/multiple inoculants combined with biostimulants and other additives including
seeds (all-in-one smart bio-formulates) should be developed to complete with the traditional
chemical fertilizers (Vassilev et al., 2015; Trivedi et al., 2017). Another option,
to avoid problems during each phase within production, formulation, storage, and establishment/action
of the PBM in soil, is to use their plant beneficial metabolites (postbiotics).
Postbiotics
Postbiotics are metabolic derivatives of PBM, which exert specific, growth promoting
and/or biocontrol, effects on plants thus avoiding the risks associated with applying
microbial cells. Specific examples of such metabolite include phytohormones, volatiles,
and quorum-sensing compounds (Schikora et al., 2016). Which are the risks of using
microorganisms in soil–plant systems? Wrong formulation procedures without osmoprotectants,
UV-protectors, fillers with nutrient value, and other plant benefiting additives usually
provoke inconsistent results under field conditions (Bashan et al., 2016; Vassilev
et al., 2020). Further risks include various abiotic and biotic factors, which affect
the rate of microbial colonization, the presence of other, more competent, components
of the microbial population, the level of plant needs and capacity to attract and
feed beneficial microorganism (Fierer, 2017). It is important to note that the protocols
for field applications of PBM are not assuring that they will find their niche of
establishing and function. Moreover, it is yet not clearly known what kind of metabolites
the introduced microorganisms will release in the soil–plant system. This complex
set of conditions determines the rate of survival of the inoculants and the performance
of their target functions (Kaminsky et al., 2019). Analysing all these aspects, it
appears that endophytic microorganisms are better protected from adverse environmental
conditions and, in addition, more efficient functionally (Santoyo et al., 2017).
Shall we apply cell-free liquids containing specific or complex metabolites produced
by the PBM during fermentation under controlled conditions? There are two options
in developing such kind of biotechnological products. Using cell-free fermentation
broth liquids without further downstream operations for separation/purification of
specific metabolites is the most economic option and, in some cases mixtures of different
microbial cultures demonstrate higher potential even after autoclaving (Mendes et al.,
2017; Hussain et al., 2020). Well-established and easy to perform immobilized cell
technology methods can be applied to repeatedly/continuously use the metabolic activity
of the microorganisms (Kautola et al., 1990), producing plant growth promoting or
biocontrol compounds in repeated-batch or continuous fermentation mode thus making
the whole process more attractive economically (Vassilev et al., 2017; Mishra and
Arora, 2018).
Another approach includes operations such as fragmentation and further use of extracts
of the microbial mass or isolation of specific metabolites from the fermentation liquid.
However, the application of specific metabolites in soil should be assessed carefully,
bearing in mind that in the rhizosphere there is a great variety of microbial and
plant metabolites involved in a wide number of interrelated cooperative or antagonistic
actions (Besset-Manzoni et al., 2018). Therefore, before applying plant beneficial
metabolites directly after the fermentation production process or in purified form,
formulation operations should be performed to ensure their efficient release into
soil. Encapsulation and nano-encapsulation of microbial metabolites was reported as
an effective tool in enhancing proliferation of shoots and rooting (Pour et al., 2019).
In this case, the inclusion of carbon nanotubes and SiO nanoparticles in the alginate-gelatin
nanocapsules increased the overall beneficial effect of the formulated cell-free product.
Nano-formulations by encapsulation are expected to enhance the metabolic stability
of the microbial metabolites but their cost-effectiveness can be increased if the
principles of the precision agriculture are applied (Duhan et al., 2017).
Concluding Remarks
Production and application of PBM is now one of the most promising fields of research.
The period of searching for easy to cultivate soil microorganisms, their characterization,
and testing in controlled conditions was replaced by another one with studies on novel,
more efficient and economic fermentation mode of production and formulations. Co-cultivation
and formulation of compatible PBM and inclusion of various additives in the formulations
become fundamental part of the overall production technology (Vassilev et al., 2014;
Vassilev et al., 2015; Vassilev et al., 2020). Another, pivotal point of the new approach
to understand and manage the functional and genetic role of soil microorganisms in
the soil–plant systems, is the comparison between human gut microbiome and plant microbiome
(Adam et al., 2016). Following the human gut example, new strategies for exploitation
of PBM appeared based on prebiotic, probiotic, synbiotic, and postbiotic products.
A previous analysis of soil physical/chemical characteristics, microbial community
dynamics along the plant growth and depending on the climatic specificity is a part
of the overall assessment on which approach will be most efficient. Here, we consciously
do not discuss, but should mention, other important issues such as how to control
the plant capability of attracting useful microorganisms, the role of core and hub
microbiota (Toju et al., 2018), and development of multi-omics tools and interdisciplinary
(or artificial intelligence) approaches of management of all soil–microbe spatio-temporal
complex data (Aleklett et al., 2017). The advancement in the field of PBM is substantial
but there are still largely unexplored options for “biotics” therapeutic treatment
of soils and biotechnological optimization of microbiome functioning in agro-soil
systems bearing in mind their extreme complexity (Fierer, 2017).
Author Contributions
MV and NV designed and drafted the work. EM and EF-P contributed to the revision of
the manuscript.
Funding
This work was supported by the project EXCALIBUR funded from the European Union’s
Horizon 2020 research and innovation programme under grant agreement No. 817946.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.