From the Lab to the Farm: An Industrial Perspective of Plant Beneficial Microorganisms

Abstract

Any successful strategy aimed at enhancing crop productivity with microbial products ultimately relies on the ability to scale at regional to global levels. Microorganisms that show promise in the lab may lack key characteristics for widespread adoption in sustainable and productive agricultural systems. This paper provides an overview of critical considerations involved with taking a strain from discovery to the farmer’s field. In addition, we review some of the most effective microbial products on the market today, explore the reasons for their success and outline some of the major challenges involved in industrial production and commercialization of beneficial strains for widespread agricultural application. General processes associated with commercializing viable microbial products are discussed in two broad categories, biofertility inoculants and biocontrol products. Specifically, we address what farmers desire in potential microbial products, how mode of action informs decisions on product applications, the influence of variation in laboratory and field study data, challenges with scaling for mass production, and the importance of consistent efficacy, product stability and quality. In order to make a significant impact on global sustainable agriculture, the implementation of plant beneficial microorganisms will require a more seamless transition between laboratory and farm application. Early attention to the challenges presented here will improve the likelihood of developing effective microbial products to improve crop yields, decrease disease severity, and help to feed an increasingly hungry planet.

Full text

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REVIEW
published: 04 August 2016
doi: 10.3389/fpls.2016.01110
Edited by:
Laurent Laplaze,
Institut de Recherche pour le
Développement, France
Reviewed by:
Robin Duponnois,
Institut de Recherche pour le
Développement, France
Louis S. Tisa,
University of New Hampshire, USA
Philippe Normand,
Centre National de la Recherche
Scientifique, France
*Correspondence:
J. Jacob Parnell
JAP@novozymes.com
Specialty section:
This article was submitted to
Crop Science and Horticulture,
a section of the journal
Frontiers in Plant Science
Received: 12 May 2016
Accepted: 12 July 2016
Published: 04 August 2016
Citation:
Parnell JJ, Berka R, Young HA,
Sturino JM, Kang Y, Barnhart DM
and DiLeo MV (2016) From the Lab
to the Farm: An Industrial Perspective
of Plant Beneficial Microorganisms.
Front. Plant Sci. 7:1110.
doi: 10.3389/fpls.2016.01110
From the Lab to the Farm: An
Industrial Perspective of Plant
Beneficial Microorganisms
J. Jacob Parnell*, Randy Berka, Hugh A. Young, Joseph M. Sturino, Yaowei Kang,
D. M. Barnhart and Matthew V. DiLeo
BioAg, Novozymes, Durham, NC, USA
Any successful strategy aimed at enhancing crop productivity with microbial products
ultimately relies on the ability to scale at regional to global levels. Microorganisms
that show promise in the lab may lack key characteristics for widespread adoption in
sustainable and productive agricultural systems. This paper provides an overview of
critical considerations involved with taking a strain from discovery to the farmer’s field. In
addition, we review some of the most effective microbial products on the market today,
explore the reasons for their success and outline some of the major challenges involved
in industrial production and commercialization of beneficial strains for widespread
agricultural application. General processes associated with commercializing viable
microbial products are discussed in two broad categories, biofertility inoculants and
biocontrol products. Specifically, we address what farmers desire in potential microbial
products, how mode of action informs decisions on product applications, the influence
of variation in laboratory and field study data, challenges with scaling for mass
production, and the importance of consistent efficacy, product stability and quality. In
order to make a significant impact on global sustainable agriculture, the implementation
of plant beneficial microorganisms will require a more seamless transition between
laboratory and farm application. Early attention to the challenges presented here will
improve the likelihood of developing effective microbial products to improve crop yields,
decrease disease severity, and help to feed an increasingly hungry planet.
Keywords: biofertility, biocontrol, commercialization, agricultural products, food security
INTRODUCTION
The alarm cry of impending global food shortages is not new. Over the centuries figures such as
Tertullian, Townsend, Malthus, and Ehrlich (Hardin, 1998;Alexandratos and Bruinsma, 2012)
have warned of dire consequences of the inability of Earth’s capacity to sustain its growing
population (Ehrlich and Ehrlich, 1990). Each time, crisis has been averted due to technological
advances in plant breeding, fertilization, crop protection and agronomic management. For
example, over the past 50 years the human population of our planet has doubled, and the
need for increased food production was met by the application of new technologies, such as
the discovery of the Haber-Bosch process (Erisman et al., 2008), and agronomic management
strategies. Although they contributed to staving widespread famine and saving billions of lives,
novel and complementary solutions are needed to continue to improve crop yield. As we face our
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next challenge, it is critical that we continue to discover new
sustainable cropping system solutions to produce more with
fewer resources.
By the year 2050, the global population is expected to reach
9.6 billion which has been estimated as our planet’s maximum
capacity (Wilson, 2003). This increase in population will require
at least double our current agricultural production, despite the
challenges with current resource requirements and a decline in
arable land (Bruinsma, 2009). Similar to the green revolution, in
order to ensure global food security for a growing population
we need to devise enhanced cropping systems that maximize
productivity while minimizing the resources required. In most
agricultural lands, maximizing yield requires additional inputs to
maintain productivity and crop yields. These additions include
both phosphorus and nitrogen as fertilizer, as well as pesticides
that help control invasive weeds, pathogens and insects. Farmers
could benefit from new sustainable products to boost or maintain
yields, often under increasing environmental stresses (Baulcombe
et al., 2009). While chemistries and trait development remain
critical in developing stress tolerance and pathogen resistance
programs of agriculture, the application of microbial products
is now considered a valuable addition to precision agriculture
(Berg, 2009;Bhattacharyya and Jha, 2012).
Microbial products have been used commercially in global
agriculture for over 120 years (Nobbe and Hiltner, 1896;Deaker
et al., 2004), but have recently received increased attention. There
are currently over 149 registered microbial strains for agricultural
products (Copping, 2009). A recent special publication by the
American Society for Microbiology suggested that microbes may
be, at least in part, a sustainable solution to increasing agricultural
production and outlined current shortcomings of microbes in
helping to feed the world (Reid and Greene, 2013). The market
for commercial biofertility inoculant and biocontrol products
in 2012 was valued at over $1 billion US dollars (USD) and
is expected to exceed $7 billion USD by 2019, increasing at a
double digit compound annual growth rate (CAGR) between
2013 and 2019 (Transparency Market Research, 2014). Major
growth drivers include growing consumer interest in organic
crops, reducing synthetic products, and the economic potential in
emerging markets such as China (Transparency Market Research,
2014). Despite the benefits and potential of agricultural microbial
products, a recent spotlight on plant yield promoting bacteria
pointed out that “The scientific literature abounds with many
potentially highly useful strains that did not appear on the
commercial market” (Bashan et al., 2014). In a 30 year span
ending in 2002, an estimated 72% of biocontrol business ventures
failed (Glare et al., 2012). Most often, failures result from
underestimating costs associated with developing and marketing
microbial products (CPL, 2006, Biopesticides). The incongruence
between effective microbial strains and successful agricultural
products suggests a need to address obstacles that may not be
anticipated.
Microbes will certainly play a role in revolutionizing
agriculture over the next several decades to help meet the
demands of a growing population. Promising agricultural
products include organisms that increase crop yield through
enhanced nutrient update by plants (inoculants), and organisms
that reduce crop loss due to pests (biocontrol). While timely and
extremely valuable, the American Society for Microbiology report
(Reid and Greene, 2013) focuses primarily on what Bashan et al.
(2014) call the ‘research facility’ side of product development
and omits important characteristics of the ‘industry’ role. This
review provides an industrial perspective on the current state
of these types of microbial products. Also, in an effort to help
maximize the number of strains that make a practical impact
on agriculture, some of the challenges involved with taking a
successful laboratory strain and making a viable commercial
product are discussed.
BIOFERTILITY INOCULANTS
Deployment of microbes to enhance crop productivity by
boosting the availability of key nutrients is a concept widely
referred to as biofertility. Biofertility inoculants as defined
above is not a new concept, and the commercial application
of inoculants dates from the launch of a bacterial product for
legumes called “Nitrogin” by Nobbe and Hiltner (1896) and
Sahoo et al. (2013). In the late 1940s, Timonin (1948) disclosed
bacterial products termed “Alnit” to augment the productivity
of non-legume crops. The market for commercial biofertility
inoculants in 2012 was valued at $440 million USD and is
expected to exceed $1 billion USD by 2019, growing at a CAGR
of 13% between 2013 and 2019 (Transparency Market Research,
2014).
The most limiting soil nutrients for plant growth are nitrogen
and phosphorus (Schachtman et al., 1998). Although many soils
contain ample quantities of these nutrients, most are not readily
accessible for plant growth (Rai, 2006). Consequently, microbial
products have been developed to increase the availability
of nitrogen or phosphorus to crops (Vance, 2001), thereby
maximizing the efficient, sustainable use of nutrients.
Nitrogen-Fixing Microbes
Nitrogen is the most critical nutrient for plant growth, and
perhaps the most recognizable example of biofertility inoculants
are the rhizobia which fix atmospheric nitrogen in nodules of
legume crops. This diverse group of bacteria comprises some of
the most intensely investigated microbes owing to their value
as inoculants. Despite their taxonomic diversity, all rhizobia
establish symbiotic interactions with their host plant via highly
conserved mechanisms which have been reviewed extensively
(Alexander, 1984;Weidner et al., 2003;Zahran, 2009;Terpolilli
et al., 2012). Legume crops are grown on an estimated 250
million hectares globally and fix roughly 90 million metric tons
of atmospheric nitrogen annually (Zahran, 2009).
Effective rhizobial products exhibit high rates of nitrogen
fixation and compete successfully with less efficient indigenous
rhizobia populations to colonize and form nodules on target host
plants. Successful commercial production of rhizobia required
the ability to produce the organisms in large quantities and enable
a long-term shelf life. Unfortunately, many microbial products
fall short in the latter specification leading to overall poor
performance in the field. In the 1980s and 1990s many rhizobial
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products showed poor efficacy (Catroux et al., 2001). However,
over the past decade both quality standards and performance of
these products have improved substantially, and several marketed
products have been shown to affect consistent improvements
in yields of legume crops. Nitrogen-fixing products sold today
contain substantially higher numbers of viable organisms per
gram than those from earlier decades. Additionally, improved
product formulations have resulted in enhanced stability
(Grooms, 2008). In tests with inoculated soybeans, Beuerlein
(2008) reported yield improvements averaging approximately
120 kg per hectare.
Soybeans contain 37–45% protein by weight, and thus, a
3600 kg ha−1crop requires 136 kg of nitrogen (Beuerlein,
2008). To illustrate the impact of rhizobial products on
soybean yields, products sold by the Monsanto BioAg Alliance
(Optimize R
) (Monsanto BioAg Alliance, 2015e), BASF (Vault R
),
ABM (ExcalibreTM), and MycoGoldTM are discussed. In
addition to live Bradyrhizobium cells, Optimize R
for soybeans
contains lipochitooligosaccharide, a molecule that enhances
the soil microbial environment1. Seeds treated with Optimize R

consistently show an increase in yield over untreated controls
(Figure 1). Similarly, Vault R
is a seed treatment consisting
of Bradyrhizobium and a patented rhizobial enhancer (Basf-
Corporation, 2015). ExcalibreSATM is a blend of Bradyrhizobia2,
and MycoGoldTM blends Bradyrhizobium with biostimulants and
other microbes3. The use of bioinoculants on soybean crops
consistently provides a 4:1 return on investment.
In addition to the nodule-forming rhizobia which establish
nitrogen-fixing symbioses in legumes, there are numerous
species of non-legume nitrogen fixing bacteria that associate
with agriculturally important crops (Rai, 2006). Among
these, members of the genera Azospirillum MicroAZ-STTM
(TerraMax4), and Mazospirflo-2 (Soilgro; Owen et al.,
2015), Azotobacter Bio-NTM (Agriculture Solutions5), and
Gluconacetobacter have attracted interest, because they are root-
colonizing and exhibit the potential to transfer fixed nitrogen
to the plants with which they associate. Non-legume nitrogen
fixing bacteria have been shown to increase yield of various
crops including sunflower, carrot, oak, sugar beet, sugar cane,
tomato, eggplant, pepper, cotton, wheat, and rice (Bashan et al.,
1988;Bashan and Holguin, 1997). In a review summarizing
20 years of global field trials, Okon and Labandera-Gonzales
(1994) reported that in 60–70% of the trials, inoculation with
various Azospirillum strains increased crop yields by 5–30%.
Another extensive, multi-year study conducted by Diaz-Zorita
et al. (2012) showed that on-seed inoculation increased wheat
and maize yields by 244 kg ha−1(3.9 bu ac−1) and 514 kg ha−1
(8.2 bu ac−1), respectively. In addition to nitrogen fixation,
some Azospirillum species are capable of producing plant
growth-promoting compounds which may play a role in their
1http://www.monsantobioag.com/global/us/Products/Documents/Labels/
Optimize_200_LiquidSoybean_Extended_Label.pdf
2http://www.abm1st.com/crops-products/soybeans/excalibre- sa/
3http://www.mycogold.com/
4http://www.terramaxag.com/products/micro-az- st-dry/
5http://www.agriculturesolutions.ca/bio-n- azotobacter-inoculants
mode of action (Okon et al., 2015). Non-leguminous nitrogen
fixing bacteria also manifest other plant-beneficial traits such
as remediation of soils polluted with heavy metals (Ullah et al.,
2015) and confer enhanced tolerance in plants to abiotic stresses
such as drought (Vargas et al., 2014).
Phosphate Solubilizing Microbes
Compared with other soil nutrients, phosphorus is the least
mobile and is usually in a relatively unavailable form for
plant uptake. Next to nitrogen, this nutrient is the second
most important nutrient in crop production and is traditionally
applied in the form of chemical fertilizers or manure. The world’s
supply of rock phosphate is expected to be largely depleted in the
next few decades (Gilbert, 2009;Scholz et al., 2013). With China,
India, and the US as the major users of rock phosphate and 70%
of known deposits located in China, Russia, Morocco, and the
US, the long term sustainability of current phosphate resources
is debated. To ensure the most efficient use of limited supplies
of rock phosphate fertilizer and circumvent future shortages,
phosphorus-solubilizing microorganisms have been developed to
enhance the nutrition of crops in a sustainable manner.
Ironically, the total amount of phosphorus in soils may be
high, but it is usually present in forms that are unavailable for
plant growth. These comprise both organic and inorganic pools,
of which 20–80% can be found in organic forms that include
phytic acid (inositol hexaphosphate) as a major component
(Richardson, 1994). The largest fraction of inorganic phosphate
in soil resides in complexes with metals (particularly Ca,
Al, and Fe) (Richardson, 2001). Soil microbes that liberate
phosphate from organic and inorganic pools have been promoted
as products that effectively mobilize phosphate from poorly
available sources in soil and reduce the application of rock
phosphate fertilizer. Products such as these are expected to show
rapid commercial growth over the next few years. While the
genetic and biochemical components underlying the mechanisms
of phosphate-liberation by these organisms have not been as
extensively studied as nitrogen fixation, excretion of organic acids
and synthesis of phosphate-scavenging enzymes such as phytases
have been implicated in their modes of action (Richardson, 2001).
Pools of insoluble phosphate in metal complexes can be
made available to plants through the action of phosphorus-
solubilizing microorganisms. Improved crop yields resulting
from the application of phosphorus-solubilizing organisms in
the field have been reported (Pradhan and Sukla, 2005), notably
Bacillus (Symbion-P R
) and Pseudomonas among bacterial genera,
and Aspergillus and Penicillium are among the most important
fungal taxa. In comparing characteristics of phosphorus-
solubilizing bacteria and fungi, it has been reported that fungi
exhibit greater solubilizing activity than bacteria (Nahas, 1996).
Penicillium bilaiae is a fungus present in the commercial product
Jumpstart R
marketed by the Monsanto BioAg Alliance (2015a).
The organism solubilizes soil phosphorus by a mechanism that
involves secretion of citric and oxalic acids (Cunningham and
Kuiack, 1992). A recent publication by Leggett et al. (2015)
summarized the findings of a large multi-year field study to assess
the yield responses of maize to inoculation with JumpStart R
.
Rigorous statistical analyses of both large and small test plots
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FIGURE 1 | Performance of Bradyrhizobium japonicum, a nodulating factor (LCO), and the combination of B. japonicum and nodulating factor
(Optimize R
) in field trials. Field trials occurred in pristine soil (no previous soy; violet), and soils with previous soy crops (green). Error bars represent least
significant difference at 95% (adapted from Smith et al., 2015).
revealed significant yield increases in 66 of 92 (72%) small plots
and 295 of 369 (80%) large plots (Table 1). These results strongly
suggest a significant impact on maize yields as a result of the
fungus P. bilaiae. The lack of successful commercial phosphate-
solubilizing inoculants has been noted (Leggett et al., 2001) and
attributed to plant or environmental incompatibility.
Another group of phosphate solubilizing microorganisms are
arbuscular mycorrhizal fungi (AMF) that are able to form a
network of hyphae that interact with the plant roots to improve
nutrient transport and protect the plant against pathogens
and some forms of abiotic stress (Porcel et al., 2012;Hodge
and Storer, 2015). Most of the vascular plants on Earth form
an association with AMF (Smith and Read, 2008); they are
ubiquitous and ecologically important for soil health. Within
the AMF, the most widely used products in agriculture usually
belong to the phylum Glomeromycota (Owen et al., 2015) and
have been shown to increase P uptake. Some of the examples
of AMF products include Mycormax R
(JH Biotech6), BEI
(BioOrganicsTM7), BioGrow Endo (Mycorrhizal Applications8),
and VAM (Microbesmart9).
Products Containing Multiple Biofertility
Microbes
Interestingly, a few commercial products have emerged that take
advantage of combining different biofertility products. One such
6http://jhbiotech.com/docs/Flyer-Mycormax.pdf
7https://bio-organics.com/product/endomycorrhizal-inoculant/
8http://mycorrhizae.com
9http://www.microbesmart.com.au/index.php/what-is- vam
TABLE 1 | Summary of small and large plot field trials to measure maize
yield response to inoculation with the phosphorus-solubilizing fungus
Penicillium bilaiae (adapted from Leggett et al., 2015).
Trials Sample size, nYield increase (kg/ha ±SE) Increase %
Small plot 92 169 ±2.8 1.8
Large plot 92 369 326 ±1.6 3.5
product, marketed under the trade name QuickRoots R
, is sold
by the Monsanto BioAg Alliance (2015b). This product contains
a patented combination of the Bacillus amyloliquefaciens
and the filamentous fungus Trichoderma virens (Monsanto
BioAg Alliance, 2015c,d). Both of these organisms are known
to liberate bound phosphate making this nutrient more
available to plant roots (Fan et al., 2011;Akladious and
Abbas, 2012;Molla et al., 2012;Lamdan et al., 2015), and
the combination purportedly imparts increased availability
of nitrogen, phosphorus and potassium in soil resulting
in expanded root volume for enhanced yield potential10.
Field trial data with QuickRoots R
applied to corn shows
a positive yield ranging from 220 to 500 kg ha−1increase
representing a 2:1 to 5:1 return on investment (Figure 2)10.
Lastly, the combination of these two organisms may also
enhance favorable interactions of plant roots with mycorrhizal
fungi in the soil (Johnson, 2013, 2015). Other examples
of mixed products include Excalibre-SA (ABM), which
combines Trichoderma with Bradyrhizobium for soy11, and
10http://www.monsantobioag.com/global/us/harvest/pages/corn.aspx
11http://www.abm1st.com/crops- products/soybeans/excalibre-sa/
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FIGURE 2 | Performance of QuickRoots R
(Bacillus amyloliquefaciens
plus Trichoderma virens) product compared with untreated corn
seeds in small plot trials (N=104). Error bars represent Standard Error.
Yield values are significantly different (two-tailed t-test <0.001).
BioGrow Endo (Mycorrhizal Applications) combines AMF and
Trichoderma12.
BIOCONTROL ORGANISMS
Plant diseases and pests are among the largest contributors to
crop losses worldwide, with an estimated 27–42% in production
systems and potential losses of 48–83% in the absence of crop
protection (Oerke and Dehne, 2004). The use of biological
organisms to control plant disease (biocontrol) could potentially
augment the use of synthetic pesticides (e.g., residue and
resistance management). Despite clear enthusiasm around the
potential for biocontrol microbes, challenges still exist in efficacy,
field performance, and cost. In this section, the role of biocontrol
in plant pest management from an industry perspective is
addressed. We focus on both the scientific and production
strategies necessary to bring biocontrol products to market, and
highlight a few examples of commercially available biocontrol
strains.
Biocontrol research has received a lot of attention in recent
years, and there are many well documented examples of
biocontrol microbial activity in scientific literature (Glare et al.,
2012;Junaid et al., 2013;Bardin et al., 2015;Pelizza et al., 2015),
however, synthetic pesticides still dominate the commercial
market (Elad, 2003). Only an estimated 3.5% of the global
pesticide market is represented by biocontrol products (valued
at 1.6 billion USD in 2009) (Lehr, 2010). In North America
and Western Europe, biocontrol markets have been estimated to
be $594.2 million in 2009 and $1.09 billion in 2015 (Frost and
Sullivan, 2009). Although challenged by issues of performance
and cost, it is clear that the biocontrol market is growing rapidly.
Estimates have proposed a 15.6% CAGR, resulting in over 7%
global market shares in 2014 (Lehr, 2010;Glare et al., 2012).
Regardless of how size estimates are made, all indications point
12http://mycorrhizae.com/
to continued growth for the biocontrol market, well beyond that
predicted for the synthetic pesticide market (Glare et al., 2012).
Historically, early sales within the biocontrol market consisted
predominately of a single product type containing Bacillus
thuringiensis (Bt) targeted against lepidopterans (e.g., cabbage
worms and gypsy moth). In 1990, over 90% of biocontrol
sales corresponded to Bt-related products, with a total market
of approximately $120 million USD (Rodgers, 1993), although
other biocontrol products such as entomopathogenic nematodes
have played a key role (Shapiro-Ilan and Gaugler, 2002).
After 2 decades, the estimated total sales for microbial-based
biocontrols was close to $400 million USD with just over 50%
of sales corresponding to Bt-related products (Glare et al., 2012).
The geographical distribution of biocontrol sales has changed
dramatically over the last two decades to cover a broader global
market and a greater number of agricultural crops (Rodgers,
1993;Glare et al., 2012). These trends suggest that the geography,
market sectors, major arable crops, and diversity of microbial
strains all continue to expand. Major drivers for growth in
biocontrol use include growing consumer interest for products
in emerging markets such as China and India.
Broad adoption of biocontrol products into mainstream
agriculture requires advances in technology, increased
understanding of the biology and ecology of active organisms,
and cost effective, efficacious products. Industry concerns
generally focus on production, formulation, and delivery when
commercializing a biocontrol product (Fravel, 2005). In addition
to these attributes, industry must consider aspects of product
registration, intellectual property, and an understanding growers
needs. Finally, aspects of efficacy, persistence, and mode of action
(biology) must be considered when developing an effective
biocontrol product.
Biology of Biocontrol
Biocontrol agents are broadly classified as preparations either
derived-from or containing living microorganisms that can
prevent or suppress pests like pathogens, insects, and weeds.
Biocontrol agents can include living microbes (bacteria, fungi,
nematodes, viruses and protozoa), bioactive compounds such
as secondary metabolites (e.g., spinosads and avermectins), or
naturally derived material such as plant extracts (Kiewnick,
2007;van Lenteren, 2012). Pest damage prevention by biocontrol
agents is based on several mechanisms that may involve
antibiosis, competition for space and nutrients, mycoparasitism,
enzymatic activity, and induced resistance (Lo, 1998). These
modes of action are certainly not exclusive, and biocontrol
agents likely enlist a combination of activities when counteracting
disease.
As previously mentioned, industrial application of biocontrol
microbes will require a deeper understanding of the biology of the
microbe, the targeted pest or pathogen, and interactions with host
plants, other microbes, and the environment. Drivers of microbe
communities in the rhizosphere, for example, involve soil type
and plant genotype (Berg and Smalla, 2009;de Bruijn, 2013),
whereas the phyllosphere microbiome is influenced by plant
genotype and environmental factors like humidity, ultraviolet
light, and geographic location (Vorholt, 2012;Rastogi et al.,
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2013). Understanding these ecological differences is critical when
making decisions about product development and commercial
application. In an illustration of abiotic effects, biocontrol
efficacy by nonpathogenic Fusarium oxysporum was significantly
affected by both temperature and light (Larkin and Fravel,
2002). In another example of biological complexity, Erlacher
et al. (2014) found that shifts in lettuce microbe communities
caused by pathogen infection (Rhizoctonia solani) were offset
by the biocontrol agent Bacillus amyloliquefaciens FZB42. Such
selective compensation of pathogen impact by a biocontrol strain
suggests a novel mode of action and highlights the complexity
of biocontrol within a plant–microbe ecosystem. Understanding
how biocontrol microorganisms interact with one another
represents another biological challenge for product development.
Co-inoculation of Trichoderma viride strain GB7 and Serratia
plymuthica strain 3Re4-18 resulted in greater biocontrol efficacy
against R. solani in lettuce, compared to application of single
strains (Grosch et al., 2012). However, combined biocontrol
application also had a more pronounced impact on the microbial
community structure at large (Grosch et al., 2012). These
studies highlight the complex and fluid interactions between
plant, pathogen, biocontrol agent, microbe community, and the
environment. To commercialize effective biocontrol microbes as
products, industries will need to invest in fundamental and early
development research surrounding these biological questions.
This will require deeper partnerships within industry as well as
greater communication with academic (public and private) and
government research organizations.
Screening for Biocontrol
Commercialization of a successful biocontrol product ultimately
depends on the availability and isolation of candidate microbes.
This screening process involves isolation from a particular
environment and early trials to characterize a microbe’s
biocontrol capability. While no single screening method is
optimal for all biocontrol endeavors, a logical strategy should
be followed based upon the pathosystem (plant-pathogen-
environment) of interest (Fravel, 2005). For example, finding
biocontrol agents against foliar-specific pathogens would likely
require screening microbes that can colonize the phyllosphere.
Culturing phyllosphere-associated microbes from tomato (Enya
et al., 2007) and wheat (Yoshida et al., 2012) has resulted
in the identification of potential biocontrol microorganisms
for foliar pathogens. Likewise, screening for biocontrol against
post-harvest diseases would require identifying microbes that
effectively protect the harvested crop (Janisiewicz and Korsten,
2002).
Successful candidate identification starts with a suitable
population of microbes to be evaluated. While screening
processes are becoming more robust and generating higher
throughput, less than 1% of candidate microbes make successful
products (Bailey and Falk, 2011). The generation of large
microbe collections, both through targeted and broad sampling
techniques, is required for identifying biocontrol candidates.
One example, TrichobankTM, is a fungal culture collection of
>2000 isolates of 21 Trichoderma spp. (Stewart et al., 2010).
This collection has been successfully used to develop biocontrol
agents like SentinelTM for the control of gray mold of grapes
caused by Botrytis cinerea. In the case of microbe databases
like TrichobankTM, information on the isolates within the
collection is matched to a desired set of biocontrol capabilities
based on pathogen targets, host plants, mode of action, and
environmental niche (Glare et al., 2012). This subset of selected
isolates is then subjected to a series of standardized bioassays to
establish biocontrol efficacy and field performance capability. It
is worth noting here that biocontrol efficacy in a field setting
is key for adoption and implementation of microbial products.
While many biocontrol agents were identified and/or validated
through in vitro screens, caution should be taken when assuming
correlation between in vitro inhibition and field performance
(Burr et al., 1996;Milus and Rothrock, 1997;Fravel, 2005).
Screening strategies can follow varied approaches, but the desired
outcome is the same in identifying efficacious, environmentally
safe, and cost-effective biocontrol agents (Köhl et al., 2011;
Ravensberg, 2011).
SUCCESS OF A PRODUCT
The success of agricultural microbial products, whether
biofertility or biocontrol, is rarely due to just one attribute, but
instead is generally due to a number of factors (Ravensberg,
2011). Gelernter and Lomer (2000) suggest a framework for
evaluating successful biocontrol products, but here we improve
on these criteria to include all microbial products. Aside from
technical efficacy, or the ability to improve yield or reduce crop
damage, successful products meet two or more of the following
conditions.
Efficacy
The most important factor for a successful product is the
ability to increase or protect yield. This is obviously the
most important goal and a given factor in combination with
other factors mentioned below for overall product success.
However, efficacy in the laboratory and/or greenhouse does not
always translate to field success (Nicot et al., 2011). Whipps
(2001) stated “The key to achieving successful, reproducible
biological control is the gradual appreciation that knowledge
of the ecological interactions taking place in soil and root
environment is required to predict the conditions under which
biocontrol can be achieved.” Nicot et al. (2011) suggests that
the success gap between lab and field efficacy can be improved
by understanding of in-field mode of action. Although they
specifically refer to biocontrol microbes, this same principle
applies to biofertility products as well. Efficacy data that do not
account for ecological interactions in a complex microbe-plant
field ecosystem including at least some of the factors discussed
below risks failure (Fravel, 2005). In many cases biocontrol
microbial products are included as a part of an integrated pest
management program (Chandler et al., 2011).
In addition to in field-efficacy, there are often efficacy
challenges that arise with scaling production for widespread
distribution. Some of the challenges described by Takors (2012)
include the genetic stability of the strain and the impact of
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mutation, viruses, and phase variation, as well as other chemical
and physical factors associated with going from bench-top to
industrial scale bioreactors.
Versatility
Plants may recruit specific microbes based on their development
and environment, responding to stresses or nutrient availability
(Smalla et al., 2006;Hartmann et al., 2009). The effectiveness of
microbial strains colonizing the plant are impacted by a number
of biotic and abiotic factors (Barea, 2015), including the previous
cropping history (Berg and Smalla, 2009;Peiffer et al., 2013),
suggesting that microbial compositions of the soil are modulated
by changes in cropping practices. The ability to colonize is
also impacted by genotype of the plant, showing variations
in community structure between variants in the same species
(Siciliano and Germida, 1999;Briones et al., 2002). Specific plant
exudates in the form of volatile organic compounds, carbon
sources or organic acids encourage colonization and growth
of a relatively narrow group of organisms (Sloan and Lebeis,
2015). For example, studies on Arabidopsis demonstrate not only
bacteria-specific responses to targeted exudates like malic acid
(Rudrappa et al., 2008), but also community responses over time
due to development stage of the plant (Chaparro et al., 2014).
In addition to targeted strains and temporal development of
colonization, strains must also associate with the appropriate root
architecture of the plant, whether by interaction with receptors
on the surface of the roots or by maintenance of cell numbers
within the rhizosphere influenced by the plant (Compant et al.,
2010). These factors allow for selective colonization of specific
microorganisms and promote diversity of the community to fit
the functional needs of the plant (Mendes et al., 2015). Improving
microbial support for a crop requires understanding the needs of
the plant, in combination with the composition of the soil and
the surrounding communities to best determine the products that
will benefit the crop. However, it should be noted that there is
often an ecological trade-off when selecting for a specific trait in
a microbial strain. For example Ehinger et al. (2014) explored the
relationship between Bradyrhizobium and either specialized or
generalized hosts and found a trade-off in host range and efficacy.
Conversely, selecting for or developing strains that are specialists
and highly effective in desired traits such as biocontrol or host
interaction can result in loss of fitness (Kassen, 2002).
Biocontrol microbial strains are often highly targeted to
specific species of pests (Nicot et al., 2011), so farmers may
need to apply different products to control multiple pest
species. Relevant narrowed spectrum, short-lasting, slowing-kill
microbial based products are big hurdles for successful product
commercialization. For example, the fungus Colletotrichum
gloeosporioides f. sp. malvae was discovered to cause seedling
blight on round-leaved mallow plants being grown in weed
control trials in Saskatchewan (Harding and Raizada, 2015).
However, the diversity of weeds in the field combined with
its narrow host range have limited its usage in the market.
Since ecological interactions are so important to in-field efficacy,
organisms that have greater versatility will have improved efficacy
over a number of different field conditions. This versatility
includes interaction with different hosts and different pathogens
and will be an ongoing process as pathogens continuously
evolve to circumvent plant defenses and overcome biocontrol
mechanisms (Brockhurst and Koskella, 2013;Zhan et al., 2014).
Practicality
Another important factor in the success of an inoculant or
biocontrol product is practicality for both the producer and
the consumer. The product must ideally have a low barrier to
adoption and be compatible with the farmer’s equipment and
production practices.
Mass production of the microbe responsible for improving
crop yield is one of the prime requirements for commercialization
(Moosavi and Zare, 2015). Pasteuria is a good case study of
a product that in the past was not very practical from an
industrial perspective due to difficulties with mass production.
Pasteuria was originally described from water fleas over 100 years
ago, however, cultivation efforts were unsuccessful (Metchnikoff,
1888). Nearly two decades later, Cobb (1906) discovered
these organisms infecting a nematode. Pasteuria species are
able to effectively parasitize different developmental stages of
nematodes (Chen and Dickson, 1998), but for over a century
commercialization of Pasteuria was limited due to the inability
to mass produce spores as a product. Pasteuria penetrans is
an obligate parasite of Meloidogyne species, which are obligate
plant parasites (Davies, 2009). Until recently, harvesting spores
for commercial product required extracting spores from infected
nematodes extracted from infected plants and was not an ideal
system for mass production. It is currently a commercial product
Clarivar R
(Syngenta R
13).
Many farmers perceive inoculants and biocontrol microbial
products as more costly and less effective than traditional
agrochemicals. For example, microbial biocontrol strains are not
always a quick acting option: they often work by suppressing
pest populations through slower processes rather than killing
on contact which may allow crop damage to continue for
some amount of time. In some cases, to use biocontrol strains
effectively, growers need to identify and know a great deal about
the lifecycle of the pest or pathogen they are trying to control and
understand the timing and appropriate conditions for application
of the product. More outreach is needed between industrial
or technical specialists and the agricultural community to help
growers accustomed to broad-spectrum agrochemicals integrate
inoculants and biocontrol microbial products into their cropping
systems.
Delivery
Appropriate formulation is required for a high quality product.
Since microbial products are often stored under less than
optimum conditions (e.g., high temperature, light exposure,
high humidity), they must have an extended shelf life and the
microorganism needs to be either robust or well protected to
be able to survive under harsh conditions. Good formulation
will also provide optimal conditions to enhance microorganism
life on roots or on leaves to obtain optimal benefits after
13http://www.syngentacropprotection.com/clariva-complete-beans-seed-
treatment
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application to the target plants. To be widely adopted by farmers,
an inoculant or biocontrol product must be cost effective and
easy to apply, ensuring that the microorganisms are delivered
to the target plant in the most appropriate manner and form.
Formulation of inoculants and biocontrol products is a crucial
issue but little research has been conducted on this subject.
For some strains, particularly gram positive spore formers,
formulation and long-term stability methods are much more
developed than for gram negative strains. A literature survey by
Xavier et al. (2004) showed that since the 1980s, most rhizobial
research focused on the bacterial genetics and physiology and
less than 1% of research articles on rhizobia have focused on
formulation aspects of products. However, there is a real need for
improved formulations of products, to create and commercialize
new microbial products that will be more effective, stable, and
higher quality to meet farmers’ needs.
Formulation of products by adding compounds to active
ingredients can improve field performance, shelf life, and
stability (Warrior et al., 2002;Leggett et al., 2011;Ravensberg,
2011), ultimately reducing variability. Formulation allows for
several functional goals including safety, effective application,
and enhanced persistence (Ravensberg, 2011). A lack of
published research in this area is likely indicative of protection
through intellectual property, like trade secrets, which is often
necessary to protect investments in product development.
Industry investments in current and future technologies will
be critical in formulating novel products. One example of
formulation utility is around microbes that do not form
spores (e.g., gram negatives) or microbes that are highly
sensitive to desiccation and temperature extremes. Serratia
entomophila is the active ingredient in BioShieldR
, an insect
biocontrol agent (Glare et al., 2012). New formulation techniques
have reportedly allowed for stabilization of BioShieldR
to
extend shelf life to more than 6 months without loss
of viability (Swaminathan and Jackson, 2011). In addition,
formulation additives like diluents and oils have been used
successfully for Metarhizium acridium products, enhancing
fungal spore attachment and infection in target insects (Hunter,
2010).
The microbial ecology of biocontrol agents has been shown
to indicate whether they are rhizosphere or phyllosphere
competent (Kamilova et al., 2005;Bruck, 2010;Vorholt,
2012). Formulation technologies can therefore be used to
improve delivery, colonization, germination, and establishment
of microbes in those particular zones. Seed coating with
microbes can provide an inexpensive option for targeted
delivery, but improvements still need to be made in coating
materials, microbe and chemistry compatibility and application
technology, especially when considering the diverse requirements
of biological organisms (Glare et al., 2012). One of these
requirements is water availability, which can have profound
influence on survival of bio-products (Connick et al., 1996).
Dry or desiccated products weigh less, are more cost effective
to ship, and have a lower risk of contamination. This type of
formulation may be amenable to microbes that produce stable
storage structures like spores, but non-spore producers likely
require different formulation strategies.
Closely tied to formulation parameters is the actual delivery
system used to apply beneficial microbes in an agriculture setting.
A delivery system targeting precise timing and specific sites
can greatly improve both bioinoculant and biocontrol product
efficacy, persistence, and cost-effectiveness. Delivery presents a
major challenge to industry in part because it requires mass
production, formulation, and application of biocontrol microbes
and/or their bioactive compounds (Ravensberg, 2011;Glare et al.,
2012). As previously mentioned, the biology of the microbe
may dictate the best avenue for delivery, leading to decisions of
application site (e.g., seed, foliar, root) and timing. Researchers
are looking beyond traditional seed coats or foliar sprays and
investigating aspects of timing and treatment location. Varied
spray schedules of Trichoderma biocontrol strains were used to
control gray mold and anthracnose in strawberries (Freeman
et al., 2004). Continuous application of the Pseudomonas putida
in low concentrations through irrigation water resulted in soil
populations similar to a single application at a 10-fold higher
concentration (Steddom and Menge, 2001). This suggests that
targeted delivery systems (site and timing) can result in field
efficacy in a cost-saving manner.
Persistence
Some of the issues associated with failure of microbial products
involve the timing of the application of the product in the field
(Chutia et al., 2007). Microbial products tend to act on more
specific targets and have a shorter shelf and sometimes active
life than chemical fertilizer/pesticides (van Lenteren, 2012). The
combination of selectivity of the microbial strain to host or
target and lack of persistence often results in inconsistent field
data (Nicot et al., 2011). For example, Bt toxin proteins are
degraded very quickly when they are exposed to sunlight. Bt-
based microbial products often need multiple applications and
result in high cost. In other cases, the efficacy of a product
presents a tradeoff between immediate short-lived impact and
persistence in the environment (Barea, 2015). The persistence
of strains varies greatly in the environment. Some strains such
as Trichoderma harzianum and Bacillus amyloliquefaciens FZB42
decrease below detectable limits within a few weeks of application
(Papavizas, 1982;Kröber et al., 2014), whereas other strains
such as Rhizobium phaseoli and Bradyrhizobium japonicum will
persist indefinitely, but at a lower abundance than is required for
efficacy (Robert and Schmidt, 1983;Naro˙
zna et al., 2015). Some
products may be formulated to successfully enable persistence of
the product long enough to show activity due to compatibility
between a strain and the environment if they can occupy a niche
or colonize before competitors show up (Verbruggen et al., 2012)
impacting community assembly in the rhizosphere (Nemergut
et al., 2013). In cases where the biological product does not readily
colonize the rhizo/phyllosphere, compatibility and niche space in
the environment will severely impact efficacy.
Commercial Viability
High cost associated with production is another obstacle for
success of developing a biological product. For example, AMF
products generally contain spores, colonized roots, hyphae
segments, or a mixture of the three (Dalpé and Monreal, 2004),
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and a wide range of carriers can be used (peat, compost,
vermiculite, perlite, sand). Because AMF are obligate symbionts
in nature, their proliferation and high-scale production require
more specific skills and infrastructure. First attempts in AMF
cultures used the pot-culture methods. Colonized root segments
or spores of well-known AMF species are used to inoculate
young seeds in a fresh sterile substrate. Plants are grown in
pots, bags, or beds and the AMF colonize roots and substrate
as the host develops, leading to a high concentration of AMF
spores and colonized roots. Spores and roots obtained can then
be used for commercial product preparation or to inoculate a
new batch of sterile substrate. This kind of production method
faced some difficulties such as uniformity of spores from batch
to batch, production space requirements, and quality variation.
In addition to production costs and return on investment for
farmers, economic aspects of agricultural microbial products
include market size and value (Nicot et al., 2011).
Regulations
Regulatory frameworks and product registrations are used
worldwide to guide the commercial development of microbial
products. When developing new microbial products, the
requisite regulatory framework varies by country, the product’s
characteristics, and its intended usage. These national and
international regulations must be taken into account during every
part of the product development cycle, including its earliest
stages, as certain regulations also outline where natural microbes
can and cannot be harvested. Interestingly, the regulations
pertaining to inoculants and biocontrol strains, while similar,
may differ in certain parts of the world. Nevertheless, regulatory
cycles for the development of new bioinoculants and biocontrol
products are generally streamlined and well-articulated. As a
result, microbial products are an appealing and cost-effective
choice when taking an integrated, systems-level approach toward
crop productivity and agricultural pest management.
CONCLUSION
Microbial products to improve crop yields and health are readily
available commercially, and their quality as well as efficacy
has improved considerably over the past decade. The field
performance of these products continues to be enhanced as major
agricultural companies commit substantial research revenues
to discovery and development of new products. Determining
the appropriate microbial products for the functional needs of
each crop will require input from both farmers and researchers.
Soil type, microbiome, environmental conditions, pest presence
and cropping system are all factors that could influence the
benefit that a microbe may provide. The crop being planted is
another key consideration, as many plants colonized by specific
bacteria are unable to maintain high populations when other
crops are planted. Further exploration into the mechanisms and
specificity of plant growth promotion from key microorganisms
will refine their specific use and maximize the potential inherent
in the microbiome of plants and soils. In this regard, recently
published studies (Agler et al., 2016;van der Heijden and
Hartmann, 2016) revealed that the complex, interconnected
microbial communities associated with plants harbor discrete
keystone species, termed “microbial hubs” that play a critical
role in mediating communications between the plant and its
microbiome. Clearly, the ability to influence these functions for
more efficacious biofertility and biocontrol applications is an area
that will receive much attention.
Increased understanding of the impact microorganisms play
in the growth and development of crops is key to future
development of microbial products. In-depth studies into the
effects of consortia and bacterial community structure on crop
development will continue to expand our knowledge of the
necessary effects the microbial community has on plants. Further
examination of responses between target crops and microbes
will better determine the specific signals that recruit or prevent
colonizing microorganisms of critical food crops. These areas
of research will result in a better understanding of the complex
associations between the microbes in the soil and critical crops,
a necessary step in providing farmers the tools necessary to
continue feeding the planet in a sustainable manner.
AUTHOR CONTRIBUTIONS
All authors listed, have made substantial, direct and intellectual
contribution to the work, and approved it for publication.
ACKNOWLEDGMENTS
We are grateful to Todd Sladek, Shawn Semones, Michael
Frodyma, John Sedivy, Thomas Schafer, and Sharon Inch for
helpful suggestions in preparation of this manuscript.
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Conflict of Interest Statement: The authors are employed by Novozymes.
The reviewer RD and handling Editor declared their shared affiliation, and the
handling Editor states that the process nevertheless met the standards of a fair and
objective review.
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