TIBTEC 1733 No.

of Pages 12

Opinion

The Inherent Conflicts in Developing Soil

Microbial Inoculants
Laura M. Kaminsky,1,@ Ryan V. Trexler,1,@ Rondy J. Malik,1,2 Kevin L. Hockett,1 and
Terrence H. Bell1,3,*,@

Potentially beneficial microorganisms have been inoculated into agricultural soils Highlights
for years. However, concurrent with sequencing advances and successful manip- Certain soil microorganisms can per-
form agriculturally valuable functions
ulation of host-associated microbiomes, industry and academia have recently
such as ethylene reduction, plant
boosted investments into microbial inoculants, convinced they can increase crop pathogen suppression, and soil nutri-
yield and reduce fertilizer and pesticide requirements. The efficacy of soil micro- ent solubilization.

bial inoculants remains unreliable, and unlike crop breeding, in which target traits Interest and investment in developing
(e.g., yield) have long been considered alongside environmental compatibility, soil microbial inoculants to enhance
microbial inoculant ecology is not sufficiently integrated into microbial selection these functions has recently surged,
but in-field product success remains
and production. We propose a holistic temporal model of the shifting constraints unpredictable and unreliable.
on inoculants at five stages of product development and application, and high-
Microbial inoculants tend to be chosen
light potential conflicts between stages. We question the feasibility of developing
based on their activity in controlled
ideal soil microbial inoculants with current approaches. laboratory screenings and for ease of
mass cultivation, with minimal regard
A Renewed Interest in Soil Microbial Inoculants for ecologically relevant traits that will
Soil microbial inoculants (see Glossary) have been sold since at least the early 20th century both allow them to survive in the field
during a target functional period and
(Box 1), but there is a recent surge in investment in these products by agricultural biotech
prevent excessive persistence.
companies [1,2]. There is demand for effective inoculants with agriculturally-relevant traits (e.
g., nutrient mineralization), as food needs will increase for decades [3], and it is widely We highlight the conflicting roles of
acknowledged that synthetic fertilizer and pesticide use must be reduced [4,5]. Commercially microbial inoculant traits at each pro-
duct stage, and how this may compli-
useful microbes can be rapidly characterized and identified with new omics tools, while cate selection for microorganisms that
demonstrations of microbial consortia as disease treatments in humans and other organisms function as desired in the field.
[6,7] should energize an industry that has long battled comparisons to ‘snake oils’ [8].

Despite decades of research into soil microbial inoculants, there are considerable barriers to
their widespread use. These include poor reliability and difficulties assessing in-field success
[9–13]. In crop breeding, both target traits (e.g., yield) and environmental compatibility have
long been considered side-by-side during product development [14], and a similar integrated
view is needed for screening and developing inoculants. The importance of ecology in inoculant
survival has been noted [15–17], but continued focus on target functional traits, rather than 1
Department of Plant Pathology and
establishment/survival traits, in (at least) the academic literature, suggests that such an Environmental Microbiology, The
integrated view is still not sufficiently considered. Pennsylvania State University,
University Park, PA, USA
2
Department of Ecosystem Science
The true challenge in developing effective inoculants, however, is the inherent conflict created and Management, The Pennsylvania
by the long and varied journeys of these products. Inoculants must progress through at least State University, University Park, PA,
USA
five product stages, which demand different, and sometimes opposing, microbial traits: (i) 3
https://microbiomemanipulationlab.
capture and refinement, (ii) production, (iii) establishment, (iv) function, and (v) downstream weebly.com
impacts (Figure 1). Before a target trait (e.g., nutrient solubilization) can be expressed in the
*Correspondence: @Twitters:
field, an inoculant must be cultivated and mass produced (e.g., in a liquid bioreactor). In @LmKaminsky, ryan_trexler,
contrast to metabolite additives, which lack viable organisms, microbial inoculants must then thb15@psu.edu

Trends in Biotechnology, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.tibtech.2018.11.011 1

© 2018 Elsevier Ltd. All rights reserved.
 TIBTEC 1733 No. of Pages 12

Box 1. Still Challenging to Implement an Old Idea

Glossary
Although there is now a renewed interest in microbial inoculant production by agricultural industries [1,2], the concept of
Consortium: multiple microbial taxa
enhancing agricultural productivity through microbial amendments has existed for well over a century.
that are co-cultured and/or co-
inoculated to an environment. These
In a speech to the Franklin Institute in 1901 [85], Harvey W. Wiley, the first commissioner of the US Food and Drug may be taxa that are expected to
Administration, spoke of the importance of microorganisms in agricultural productivity. He indicated that, at the time, the perform complementary functions, or
public still largely thought of microorganisms as agents of disease, despite numerous processes (e.g., wine fermenta- that could perform the same function
tion) that were known to depend on microbial activity. He described two commercial products, ‘nitragin’ and ‘alinite’, but under different environmental
which were intended to promote nitrogen fixation in root nodules and soils, respectively. conditions.
Establishment: the successful
Wiley described difficulties in translating experimentally determined phenotypes to the field, but was hopeful about survival and growth of a microbial
future approaches for effective inoculant application. Speaking of challenges that are still relevant today [15–17], he strain or consortium after being
stated, ‘The laws which govern the distribution and multiplication of these organisms in the field . . . are not fully introduced into a novel environment
developed.’ Ongoing challenges in the reliable application of inoculants for agriculture and human health [9–13,86], (e.g., an agricultural soil).
suggest that identifying new approaches for promoting inoculant establishment and functional persistence in situ Formulation: the preparation and
remains a top research priority. stabilization of microbial cells in a
manner that can be stored prior to
application.
Microbial inoculant: bacteria, fungi,
or other microorganisms, typically
isolates, that are intentionally
Life stages of a microbial inoculant introduced to an environment to
enhance a target function. Examples
Produc on Func on include microorganisms used in
biocontrol or to promote plant
N2 growth. May be a single strain or a
consortium.
Microbiome: the complex
assemblage of microorganisms living
NH3 in a defined environment (e.g., soil).
Although often used to refer to
bacteria and/or fungi, it also includes
archaea, viruses, protists, and other
organisms. All of these
microorganisms may impact
Capture Establishment Downstream impacts inoculant establishment.
Persistence: survival over time of a
N2 viable microbial population. Could be
Trait 2 viewed positively (e.g., inoculants
persist during target functional period
in soils) or negatively (e.g., inoculants
persist after defined functional
periods, with unknown downstream
impacts).

? ?
N2
Trait 1 Trait 3 Trade-off: a trait that is beneficial in
one context (e.g., at one stage of
inoculant development) may be
detrimental in another context.
Traits: for microorganisms, traits are
jointly determined by genes and the
environment. They are an observable
Effec ve windows of microbial traits component of the microbial
phenotype, and may contribute to
the survival of a microorganism in the
environment or an agriculturally
Figure 1. Depicted Are the Life Stages of a Soil Microbial Inoculant (Top) and a Conceptualization of Trade-
relevant function (e.g., nutrient
offs in Microbial Traits between Life Stages (Bottom). For example, trait 1 is beneficial during capture and
solubilization).
production, but does not benefit establishment in soil, and has unknown impacts on function and downstream impacts.
An example of such a trait could be efficient planktonic growth in nutrient-rich environments. Trait 2 could be high
persistence, allowing high survival through varied product stages, but complicating downstream monitoring if the inoculant
persists beyond the desired functional period. Trait 3 could be found in a microorganism with a strong competitive
advantage in a particular soil (e.g., permits enemy release), complicating downstream monitoring and control, but this trait
also limits cultivation and mass production of the organism.

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Table 1. Microbial Traits Impacting the Survival and Activity of a Commercial Inoculant
Trait Particularly relevant stages

Grows well/poorly in culture media Capture/refinement

Fast/slow-growing Capture/refinement, establishment, function, downstream impacts

Fast/slow-evolving Capture/refinement, establishment, function, downstream impacts

Gene flexibility/stability (e.g., ease of Capture/refinement, establishment, function, downstream impacts

horizontal gene transfer)

Effective/ineffective at targeted activity (e. Capture/refinement, function

g., ethylene reduction)

Compatible/incompatible with Production

commercial mass production

Compatible/incompatible with Production, establishment

formulation

Compatible/incompatible with pesticides Production, establishment

in seed coatings

Long/short shelf life Production, establishment

Spore forming or not Production, downstream impacts

Tolerant/intolerant of desiccation Production, establishment

Habitat generalist/specialist Establishment, function, downstream impacts

Metabolically diverse/specialized Establishment, function, downstream impacts

Compatible/incompatible with resident Establishment, function, downstream impacts

soil microbiome

Good/poor disperser Establishment, function, downstream impacts

High/low persistence Establishment, function, downstream impacts

Strong/weak competitor Establishment, function, downstream impacts

Resilient/sensitive to disturbance Establishment, function, downstream impacts

Resistant/susceptible to predation Establishment, function, downstream impacts

Biofilm forming or not Production, establishment, function, downstream impacts

Facultative or obligate biotroph Establishment, function, downstream impacts

Toxicity or medical relevance Downstream impacts

survive formulation and storage, and gain footing and perform under variable environmental
conditions, which likely requires further replication and growth in the environment. Despite
efforts to minimize serial cultivation of isolates, microorganisms surviving this progression will be
generations removed from the isolated ancestor [18], with unknown consequences on product
phenotypes and function. Those that grow rapidly, particularly those suited to mass production,
may replicate numerous times post-application, with products forced to adapt to the varied
biotic and abiotic conditions at each application site. Yet excessive survival could be viewed
negatively from an environmental perspective (e.g., uncontrolled inoculant spread) and an
industry perspective (e.g., disincentive to reapply products annually).

In this opinion article, we highlight certain traits and trade-offs that will impact inoculant
progression through these product stages (Figure 1; Table 1). We question whether developing
the ideal soil microbial inoculant, as currently imagined, is feasible. We focus strictly on
microorganisms captured from the environment and possibly modified through adaptation
to growth conditions in the lab or field, as this approach has led to microbial products from new

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startups quickly reaching market [2]. Although genetic manipulation (e.g., gene insertions)
could benefit product development, controversies surrounding genetically modified crops [19]
indicate substantial social and regulatory hurdles that may be independent of perceived
product risks [20].

Stage 1: Capture and Refinement

Inoculant development typically begins with microbial isolation from the environment through
cultivation, which disproportionately selects microbes from a few lineages [21] that tolerate the
artefacts of cultivation methods [22]. An ideal soil microbial inoculant will be amenable to
cultivation, which facilitates product evaluation and mass production. Most soil microorgan-
isms remain resistant to culture, limiting which are available as source material.

Does bias in microbial capture matter? Microbial traits are conserved at different taxonomic
levels [23], meaning that cultivation biases will differentially impact trait capture, potentially
excluding microbes with useful functions (Stage 4). Without genetic modification, the
function of complex, highly conserved traits (e.g., photosynthesis, N2 fixation) depends
heavily on the cultivable diversity of microorganisms possessing that trait. In contrast, less
conserved traits (e.g., carbon source preferences, environmental variability tolerance) could
be derived through selection pressures imposed during inoculant refinement and produc-
tion [24–26]. However, such traits may be more vulnerable to change through various
product stages.

Many standard culture conditions will preferentially, although not exclusively, capture fast-
growing, mesophilic copiotrophs, as opposed to slow-growing oligotrophs, or microbes with
specialized or undetermined metabolic requirements. A notable exception is Bradyrhizobium,
commonly introduced to promote N-fixation in soybean, isolates of which may grow moder-
ately fast (e.g., high cell numbers in a few days) or quite slowly [27,28]. Systematically selecting
copiotrophs at this stage will capture microbes that thrive in nutrient-rich (high C, N, P)
environments. While N and P are typically abundant in conventional agricultural soils, C
may not be, and production of easily accessed C by roots represents a cost to crops [29].
In fields managed for sustainability (e.g., low nutrient inputs), the nutrient and substrate
preferences of easily cultivated microorganisms may be a particularly poor match for the
environment, negatively impacting microbial establishment and growth (Stages 3–4). Microbial
inoculants are of special interest to growers looking to reduce nutrient inputs, exacerbating the
impact of this trade-off.

Culturing advances increase the diversity of capturable microorganisms, raising the odds of
discovering useful traits. Low nutrient concentrations, humic acids, and longer incubations help
isolate new soil clades [30], as should targeted isolation and single-cell manipulation using high-
throughput microfluidics [31]. Newer methods allow isolates to become enriched in situ (e.g.,
ichip) [32]. However, newly cultivable microorganisms (particularly slow growers) may be less
amenable to rapid and cost-effective production (Stage 2).

A fundamental consideration is which traits should be the focus of screening. Which traits can
be refined or derived through directed selection, and which depend on the initial microbial pool?
For certain traits, directed selection may force undesirable genetic trade-offs [33], requiring
further screening for unknown trait combinations. There are numerous demonstrations of
bacterial trade-offs that could limit inoculant success at one or more stages (Table 2). To avoid
selecting for traits that will limit downstream inoculant success, cultivation efforts should focus
on traits critical to establishment and/or survival (Stages 3–4), as well as traits expected to

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Table 2. Selected Bacterial Trade-offs That Could Be Relevant to Soil Inoculant Success
Refs

Growth in an assemblage Growth in isolation [69]

Spore quantity Spore quality [41]

Stressor tolerance Growth rate in absence of stressor [95,96]

Growth rate Yield [97,98]

Fitness at low pH Fitness at high pH [60]

Biofilm production Dispersal [99]

Antibiotic resistance Growth and number of resources used [100]

Resource exploitation Growth rate [70]

promote crop and/or soil health, with those not easily optimized through lab selection taking
precedence.

Stage 2: Production
Promising isolates, identified and refined in culture, will be greenhouse- and field-tested, with
those selected for commercial use typically grown in large batch cultures [34]. Accordingly, an
ideal soil microbial inoculant will grow rapidly in liquid culture, sufficient to generate enough
biomass for field applications (accounting for preapplication viability losses), while minimizing
production costs.

Traits that benefit batch production may prove detrimental during inoculant establishment and
function (Stages 3–4). Culture-based growth favors planktonic phenotypes, potentially imped-
ing rapid surface attachment [35] in terrestrial systems (Stage 3). Moreover, certain bacterial
genomes change rapidly in culture [36], and Escherichia coli mutation rates have been shown to
increase when left in stationary phase [37]. At least 10–20 generations of growth from seeded
stock isolates in industrial-scale fermenters are required to achieve marketable biomass [18], so
an ideal soil microbial inoculant would be genetically stable under culture conditions. High
genetic plasticity, high mutation rates, and low plasmid retention may not be apparent during
inoculant screening (Stage 1), and such traits could decrease the predictability of downstream
inoculant functions (Stage 4). However, increased mutation rates can be adaptive for respond-
ing to stress [38], and high genetic plasticity could actually benefit the establishment of
inoculants in variable, potentially stress-inducing, environments (Stage 3).

Following mass production, an ideal soil microbial inoculant will remain viable in storage long-
term. This may require tolerance to adhesives and agrochemicals applied as seed coats, and to
standard formulations that facilitate storage [12]. Microbes compatible with dry formulations (e.
g., seed coatings [12], alginate bioencapsulation [39], peat moss [34]) are often preferred, as
liquid formulations typically have shorter shelf lives, higher contamination risk, and require
refrigeration [34]. Stress-tolerant spores produced by some bacteria (e.g., Bacillus) are par-
ticularly amenable to dry formulation and promote inoculant persistence in the environment

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[40], though this may complicate downstream management (Stage 5). Moreover, conditions
preceding sporulation can impact spore revival in response to nutrients [41], which represents
another way in which production practices could influence inoculant establishment in soil
(Stage 3).

Non-sporulating bacteria, however, can experience large viable cell losses during dry formula-
tion, as desiccation disrupts cell membranes, causing cell leakage or death during rehydration
[42]. Desiccation tolerance traits (e.g., production of stress proteins, osmoprotectants)
increase the chance of inoculants surviving dry formulation in high numbers [42], and could
facilitate in situ survival under drought, but may result in trade-offs with growth/competitiveness
in water-saturated soils (Stages 3–4) [43]. Alternatively, gram-negative genera can enter stress-
tolerant physiological states, referred to as ‘persister cells’ [44], mostly known from antibiotic-
tolerant bacteria [45]; however, there is increasing recognition that these are likely important in
other environments [46,47]. Developing production practices to stimulate this physiological
state could generate opportunities for gram-negative bacteria as inoculants.

Stage 3: Establishment
Agricultural inoculants are applied through several routes (Box 2), and it is the goal that
inoculants quickly proliferate under various abiotic and biotic pressures. An ideal soil microbial
inoculant will effectively and predictably establish in a wide range of environments. Since soils
possess diverse characteristics, even within fields, it is infeasible to develop inoculants specific
to every soil type, as this would greatly increase production costs and complexity (Stages 1–2).
Using diverse consortia as inoculants could be helpful in this context (Box 3).

Various abiotic factors will influence soil colonization, including soil pH, moisture, texture,
nutrients, and salinity [48], but inoculants are likely best adapted to conditions resembling the
soil they were isolated from (Stage 1) [13]. Inoculants may require traits allowing quick
adherence to surfaces (e.g., clay particles) or colonization of germinating seeds and growing
roots. Interestingly, inoculant addition routes (Box 2) may impact inoculant success [49,50].

Constituents of the soil microbiome could alter inoculant establishment through mechanisms
such as niche overlap, priority effects, and facilitation, or lack thereof [13,17]. If the metabolic
niche of an inoculant is filled by resident microbes, establishment success declines [51–53].
Bacteriophage, microbial predators, and other higher organisms may also shape soil microbial
populations [54–56]. The suitability of an inoculant for particular biotic contexts should depend
on the organisms present in its soil of origin (Stage 1).

An additional challenge for plant-associative inoculants is colonization of developing tissues.

Germination presents a physically, chemically, and biologically dynamic environment to inoc-
ulants, in which they compete with soil microorganisms responding to these same changes.
Inoculants may need to associate with higher organisms that move them to appropriate
locations [57], or require stress response genes, such as superoxide dismutase, to tolerate
the plant interior [58].

Inoculants could potentially be conditioned to particular soils or plant secretions through

directed selection during product refinement and production (Stages 1–2), although this could
lead to trade-offs in production cost and efficiency. Others have proposed enhancing plant–
microbe relationships during plant breeding [59], which could provide an advantage to inoc-
ulants that are co-bred with a plant. Whether inoculants are selected for superior establish-
ment/survival under ephemeral (e.g., plant secretions at specific developmental stage) or

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Box 2. Application of Agricultural Inoculants

Industry has explored a number of approaches for introducing microbial inoculants to agricultural fields. The most
common methods of introducing inoculants into soil systems fall into the following categories.

In furrow: microorganisms are added directly to soil, and will contend with established soil microorganisms, as well as
abiotic soil characteristics. An advantage to this approach is that the inoculants can be maintained in either ‘dry’ or ‘wet’
formulations preapplication, and applications can consist of either a liquid or powder inoculant. In addition, depending
on the application approach, microorganisms could have the opportunity to establish throughout the field, whereas
inoculants that are adhered to seed surfaces, for example, are introduced only at planting sites. Inoculants could also be
applied at different points through the growing season with this method.

On seeds: microorganisms are added to seed surfaces, and, in theory, will be quickly accessible to germinating plants.
In this model, the added microorganisms are also required to tolerate any additional seed coatings that producers apply,
including a variety of insecticides and fungicides that can negatively impact inoculant survival and activity [12]. Seed-
added microbes should also tolerate rapid drying following inoculation of the seed. Rapid drying is beneficial economic-
ally, but results in a greater decrease in recoverable cells than slow drying [12]. Microbial traits that could enhance
desiccation tolerance include increased biofilm formation and increased synthesis of compatible solutes. Inoculants
should be targeted to the needs of a plant at germination. In general, seed inoculation is less expensive than in-furrow
inoculation, especially for small seeds [12]. In addition to selecting or engineering strains that are well adapted to seed
application, the choice of culturing approach can influence post-seed inoculation survival. For example, rhizobia
cultured on peat or crude peat extract exhibited greater desiccation tolerance than the same organisms cultured
on more traditional liquid media [87].

In seeds: Mitter and colleagues [88] describe an interesting approach, in which inoculants are added to flowers, leading
to modification of the microbiome in the resulting seeds. An advantage of endophytes is that they can be sheltered to
some extent from the biotic and abiotic components of soil by plant root structures. Relative to seed surface
applications, microbial inoculants could also be less impacted by seed coating treatments.

constant (e.g., pH) field conditions might impact their long-term fate (Stage 5). Another potential
trade-off between establishment and function (Stage 4), is that introduced inoculants may
disrupt the abundance of established microorganisms [50,52] that benefit plant or soil health. It
is not clear whether this would be widely relevant in practice, but represents another consid-
eration for screening.

Stage 4: Function
Ultimately, soil microbial inoculants should perform target functions effectively. Agriculturally
relevant functions identified through screening (Stage 1) are broad, and include solubilizing soil

Box 3. Single-Strain Inoculants or Diverse Consortia?

Applying diverse consortia as opposed to single strains may impact inoculant survival and function. Diverse inoculants
may establish more successfully than single strains, as there is a greater chance of at least one strain escaping
competitive exclusion [89,90]. Although there is promise in developing diverse consortia with strains carrying redundant
or complementary functions, it is generally unknown how such consortia would establish across a range of environ-
ments, and whether establishment of certain members could actually limit the function of others under some
circumstances (Stage 4). Generally, traits such as motility, fast growth, and quick dispersal should be useful in
establishing inoculants in a wide range of soils, though these same traits could lead to inoculants that are overly
persistent downstream (Stage 5).

Studies in different systems have demonstrated a positive effect of using consortia (either multiple bacteria or
combinations of bacteria and fungi) for plant growth promotion [91–93]. When a metabolic pathway is too complex
or energy intensive to be executed by a single taxon, there may be advantages to ‘metabolic division of labor’ within a
consortium [94]. However, this is also likely to produce trade-offs, by reducing reaction efficiencies [94]. The reasons for
consortium benefit are not always well known, but complementary functional mechanisms and/or greater chance of
environmental colonization would likely result in more reliable functional outcomes (Stage 4). However, developing
consortia that are synergistic under a wide range of environmental conditions complicates both screening and
production (Stages 1–2).

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nutrients, nitrogen fixation, and suppressing plant pathogens. The ideal soil microbial inoculant
will perform a target function across a wide range of soil types, environmental conditions, and
crop cultivars. This is a substantial challenge, as adaptation to specific conditions (e.g., pH) can
lead to trade-offs that diminish survival under other conditions [60].

Even if an inoculant establishes well (Stage 3), it may not perform as expected. Microbial
recruitment to the rhizosphere is driven by plant root exudates [61], and exudate profiles vary
widely with plant and environmental characteristics [62]. Therefore, applying inoculants at the
wrong time or to inappropriate crops, cultivars, or plant mixtures could limit colonization of the
rhizosphere and result in variable crop responses [48,63,64]. Moreover, microbial expression of
beneficial pathways can be affected by plant and soil conditions [65,66]. For instance, ammonium
can suppress root-hair infection genes, negatively impacting rhizobial nodule formation [67].

It is possible that certain inoculants will rapidly evolve when confronted with a variety of new
environments (as has been shown for other types of organisms [68]), particularly in response to
new biotic interactions [69]. For example, Pseudomonas fluorescens quickly evolved to use
resources underexploited by coexisting microorganisms after its introduction to various micro-
bial communities, but this led to a reduction in growth rate [70]. Selection pressures may force
investment in traits allowing survival, potentially compromising target functions. It has been
proposed that host-benefitting traits in microbiomes are unlikely to be maintained without hosts
exerting selective pressures to promote their persistence [71].

These factors highlight the vast disconnect between in vitro screening conditions (Stage 1) and
in situ success. Potential inoculants should be screened in relevant soil and planting conditions
to understand likely in situ functional ranges and adaptive responses affecting function.
Exposing inoculants to separated components of the environment during screening (e.g.,
sterile soils versus soil microbiomes) will help to characterize the gap between function under
ideal and variable field settings.

Stage 5: Downstream Impacts

Of the stages described, long-term inoculant impacts on the environment undoubtedly receive
the least attention. In part, this is because there is little evidence of negative impacts from
existing products, and strong evidence that effective products could positively impact sustain-
able agriculture by reducing harmful synthetic inputs. However, given our limited understanding
of the ecological and dispersal traits of most soil microorganisms, it is important to continue to
probe existing and future products for unwanted secondary effects [72]. An ideal microbial
inoculant will not spread from sites of application, will persist only through target functional
periods, will not negatively impact human health (e.g., secondary metabolites) or the surround-
ing environment (e.g., reducing populations of indigenous taxa), and should only affect the land
of growers choosing to implement it.

Inoculant application sites cannot feasibly be monitored for all hypothetical outcomes, so
understanding long-term impacts will lag far behind rapid microbial screening and selection
(Stages 1–2). In addition to regulatory compliance, the financial benefits to industry of down-
stream screening might seem limited; however, year-to-year isolate persistence in soil would
impact inoculant application and purchase frequency. Some existing products may survive well
enough to negate the need for reapplication in, at least, the following year [73].

Traits that are particularly relevant at this stage can be in direct opposition to relevant traits from
earlier stages of the product timeline. As mentioned, resistant spores may favor extended shelf-

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life (Stages 2–3), but could cause undesired persistence in soil [74]. Quick growth and Outstanding Questions
dispersal, and the ability to outcompete numerous soil microorganisms are advantageous If desirable traits have high plasticity,
during microbial capture and production (Stages 1–2), and may be essential for establishment and are quickly modified in production
environments or upon introduction to
(Stage 3). These same traits, however, could indicate microorganisms that are more likely to soils, can stability be enhanced
move off-site and establish in nontarget environments. through changes to production/appli-
cation or through genetic
rearrangements?
Many microorganisms naturally disperse frequently and widely, through mechanisms such as
rain, dust, or movement with invertebrates [75,76]. However, excessive resource pulses,
Like certain crops that require multi-
common to agricultural regions, could both limit the ability of soil microbiomes to resist year time investments (e.g., trees),
introduced microbes [51], and boost inoculant populations, increasing opportunities for off- what are the unexplored production
site invasions [77]. Bell and Tylianakis [78] hypothesize that, because common agricultural models for developing slow-growing
oligotrophic inoculants?
practices promote high concentrations of specific microorganisms, those microbes may
disproportionately impact microbial composition and function in nearby soils.
High inoculum size has been shown to
increase the success of microbial inva-
Continued investigation of the long-term fates of microbial products is warranted. Two applied sion of new environments, which
strains of Bradyrhizobium japonicum remained viable in soil 20 years after application, even incentivizes the large batch production
of microbial inoculants. However, a
though soybean had not been planted for most of that time [79], and viable Beauveria
number of other factors also contribute
brongniartii has been detected 14 years post-inoculation [80]. Persistence does not automati- to the success and proliferation of
cally equate to problematic, but this suggests that some products are likely to outlive the invaders. Are there opportunities pro-
companies that produce them. vided by inoculant capture and pro-
duction in realistic environments (e.
g., within soils or plants) to enhance
Is the Ideal Inoculant a Consortium? invasion potential and reduce inocu-
As discussed in Box 3, the use of consortia as soil inoculants can provide functional benefits lum size requirements?
over single-strain inoculants in some cases, but the identification of synergistic consortia (Stage
1) complicates product screening and development [81]. The use of consortia could help to What is the interaction between land
management and product persis-
compensate for traits that are not ideal for early product survival, by enhancing desiccation
tence? For example, can the use of
tolerance in formulation, for instance [42] (Stage 2), or facilitating establishment of a target strain cover crops or forest buffers affect
(Stage 3). Interestingly, unsuccessful microbial invasions may modify soil receptiveness to the long-term persistence or off-site
future invasions [52], suggesting that pioneer inoculants could widen the window for establish- migration of microbial products?

ing target inoculants. In situ function (Stage 4) could be enhanced by integrating microorgan-
isms with distinct functional roles (e.g., rhizobia and mycorrhizal fungi) to supplement different
plant nutritional needs [82], or by identifying microorganisms that additively enhance a single
function [83].

Added microorganisms may also be able to directly interact with each other in soils through the
construction of novel niches, such as biofilms. For example, multistrain soil surface consortia,
targeted to fix N and C on bare soil surfaces, can generate biofilm mats that favor their own
growth and activity [84]. Although such an approach could help to maintain interactions
between product strains, it is not clear that these interactions would remain constant through
changing environments, although niche construction may help to buffer the impacts of biotic
and abiotic variability between soils.

Concluding Remarks and Future Directions

Inoculants need to thrive in vastly different environments, including liquid culture and varied soil
conditions. This creates conflicts for survival and function across the life of microbial products,
which may extend beyond the intended period of use. Due to the varied roles inoculants must
play, ideal soil microbial inoculants are likely to be diverse. Modified approaches to screening (e.
g., co-selecting for survival and function) and new approaches to commercial production and
application of the many soil microorganisms that do not thrive in nutrient-rich environments,
should help in developing such products (see Outstanding Questions).

Trends in Biotechnology, Month Year, Vol. xx, No. yy 9

 TIBTEC 1733 No. of Pages 12
Acknowledgements
We thank Patrick Dudas and Brennan Dincher from the Huck Institutes for the Life Sciences at Penn State University for
assistance in developing Figure 1. We also thank Idalys Bonet and Emily Grandinette for help in brainstorming ideas for this
project. We thank two anonymous reviewers and the Editor for providing helpful suggestions for revising our text. This work
was supported by the USDA National Institute of Food and Hatch Appropriations under Project #PEN04651 and
Accession #1016233.
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