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OF PAGES 7
Please cite this article in press as: Pe rez-Garca A, et al. Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture, Curr Opin Biotechnol
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Available online at www.sciencedirect.com
Plant protection and growth stimulation by microorganisms:
biotechnological applications of Bacilli in agriculture
Alejandro Pe rez-Garca, Diego Romero and Antonio de Vicente
The increasing demand for a steady, healthy food supply
requires an efcient control of the major pests and plant
diseases. Current management practices are based largely on
the application of synthetic pesticides. The excessive use of
agrochemicals has caused serious environmental and health
problems. Therefore, there is a growing demand for new and
safer methods to replace or at least supplement the existing
control strategies. Biological control, that is, the use of natural
antagonists to combat pests or plant diseases has emerged as
a promising alternative to chemical pesticides. The Bacilli offer
a number of advantages for their application in agricultural
biotechnology. Several Bacillus-based products have been
marketed as microbial pesticides, fungicides or fertilisers.
Bacillus-based biopesticides are widely used in conventional
agriculture, by contrast, implementation of Bacillus-based
biofungicides and biofertilizers is still a pending issue.
Address
Departamento de Microbiologa, Facultad de Ciencias, Universidad de
Ma laga, Instituto de Hortofruticultura Subtropical y Mediterra nea,
Boulevard Louis Pateur-Campus Universitario de Teatinos s/n, 29071
Ma laga, Spain
Corresponding author: Pe rez-Garca, Alejandro (aperez@uma.es)
Current Opinion in Biotechnology 2011, 22:17
This review comes from a themed issue on
Food biotechnology
Edited by Oscar Kuipers and Tjakko Abee
0958-1669/$ see front matter
# 2010 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2010.12.003
Introduction
Insects and fungi affecting crops and post-harvested
fruits and vegetables are major threats to food production.
They have led to important economic losses worldwide,
particularly over the past few decades as agricultural
production has intensied. To face these problems, pro-
ducers have become increasingly dependent on agro-
chemicals. However, intensive use of these compounds
in conventional crop management has led to the emer-
gence of frequent problems of pesticide resistance in
insect pests and microbial pathogens and has also caused
serious problems affecting not only human health but also
the quality of the environment. Therefore, there is an
increasing demand by growers and consumers for new
environmentally friendly methods to replace, or at least
supplement, the existing chemical-based strategies
thereby achieving safer and more effective pest and
disease control.
Biological control, that is, the use of natural antagonistic
organisms to combat pests or suppress plant diseases,
offers an interesting alternative to the use of chemicals
[1

,2

]. Some aerobic spore-forming bacteria possess sev-

eral advantages that make them good candidates for use
as biological control agents. Firstly, some of these bacteria
produce several different types of insecticidal and anti-
microbial compounds. Secondly, they induce growth and
defence responses in the host plant. Furthermore, Bacillus
species are able to produce spores that allowthemto resist
adverse environmental conditions and permit easy for-
mulation and storage of the commercial products [3,4].
Members of the genus Bacillus are among the benecial
bacteria exploited as microbial pesticides, fungicides or
fertilizers. Bacillus-based products represent the most
important class of microbial products for phytosanitary
use commercially available [5,6]. In this article, we pro-
vide a short overview about the current biotechnological
applications of bacilli in agriculture (Figure 1), and we
discuss the potential of these microorganisms for promot-
ing plant health in 21st century agriculture.
Bacillus thuringiensis, the rst biopesticide
Since its discovery in 1901 as a microbial insecticide,
Bacillus thuringiensis has been widely used to control insect
pests important in agriculture, forestry and medicine. Its
principal characteristic is the synthesis, during sporulation,
of a crystalline inclusion containing proteins known as d-
endotoxins or Cry proteins, which have insecticidal proper-
ties. To date, over one hundred B. thuringiensis-based
bioinsecticides have been developed, which are mostly
usedagainst lepidopteran, dipteranandcoleopteranlarvae.
In addition, the genes that code for the insecticidal crystal
proteins have been successfully transferred into different
crops plants, whichhas ledtosignicant economic benets.
Because of their high specicity and their safety in the
environment, B. thuringiensis and Cry proteins are efcient,
safe and sustainable alternatives to chemical pesticides for
the control of insect pests [7

,8].
The toxicity of the Cry proteins have traditionally been
explained by the formation of transmembrane pores or ion
channels that lead to osmotic cell lysis [7

]. In a recent
study, a more precise trimeric building block model for
Cry toxins ion channel formation has been proposed
www.sciencedirect.com Current Opinion in Biotechnology 2011, 22:17
based on sequence conservation and mutagenesis data
[9]. In addition to this, Cry toxin monomers also seem to
promote cell death in insect cells through a mechanism
involving an adenylyl cyclase/PKA signalling pathway
[10]. However, despite this entomopathogenic potential,
controversy has arisen regarding the pathogenic lifestyle
of B. thuringiensis. Recent reports claimthat B. thuringiensis
requires the co-operation of commensal bacteria within
the insect gut to be fully pathogenic [11,12]. In clear
opposition, genomic and proteomic studies have been
argued as the most solid data to convincingly demonstrate
that B. thuringiensis is a primary pathogen rather than a
soil-dwelling saprophyte [13,14,15

]. In any case, what is

certainly not doubtful is that B. thuringiensis is one of the
most successful examples of the use of microorganisms in
agricultural biotechnology, with about 70% of the global
biopesticide market involving products based on B. thur-
ingiensis [16], and will continue to be one of the most
important microbial weapons to defend our crops from
insect pests.
Bacilli as biofungicides
Besides the insecticidal properties of B. thuringiensis, some
bacilli display other characteristics that may directly or
indirectly contribute to crop productivity. Members of the
genus Bacillus are often considered as microbial factories
for the production of a vast array of biologically active
molecules, some of which are potentially inhibitory for
fungal growth [3]. Plant pathogenic fungi and oomycetes
are major threats for crops andplant production. Therefore,
the control of fungal diseases by bacilli represents another
interesting opportunity for agricultural biotechnology.
Indeed, several commercial products based on various
Bacillus species such as B. amyloliquefaciens, B. licheniformis,
B. pumilus and B. subtilis have been marketed as biofungi-
cides (Table 1) [6]. These Bacillus-based products have
beendevelopedespeciallyfor thecontrol of fungal diseases
2 Food biotechnology
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Please cite this article in press as: Pe rez-Garca A, et al. Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture, Curr Opin Biotechnol
(2011), doi:10.1016/j.copbio.2010.12.003
Figure 1
Biofertilizers
Bacillus
B
i
o
f
u
n
g
i
c
i
d
e
s
B
i
o
p
e
s
t
i
c
i
d
e
s
Current Opinion in Biotechnology
The bacilli target triangle. Current agricultural usage of Bacillus-based
products as microbial pesticides, fungicides, and stimulators of plant
growth. Although some Bacillus species also display bactericidal and
nematicidal abilities, these features have not been specifically exploited
yet or their implementation is very limited.
Table 1
Some commercial formulations of Bacillus-based biofungicides.
Trade name Bacillus species Target pathogen/disease Crop Manufacturer
Avogreen B. subtilis Cercospora spot Avocado Stimuplant, South Africa
Ballad Plus B. pumilus Rust, powdery mildew,
cercospora, brown spot
Soybean AgraQuest, USA
Biobest B. subtilis Sheath blight, blast, brown spot Rice Appliedchem, Thailand
Companion B. subtilis Rhizoctonia, Pythium, Fusarium,
Phytophthora, Sclerotinia
Greenhouse, nursery
and ornamental crops
Growth Products,USA
EcoGuard B. licheniformis Dollar spot, anthracnose Turf Novozymes, Denmark
HiStick
a
B. subtilis Fusarium, Rhizoctonia, Aspergillus Soybean and peanuts Becker Underwood, USA
Kodiak B. subtilis Rhizoctonia, Fusarium,
Pythium Aspergillus
Cotton, legumes, soybean
and vegetable crops
Bayer CropScience, USA
Larminar B. subtilis Alternaria, Botryodiplodia,
Colletotrichum, Corticium,
Fusarium, Phytophthora
Vegetables, fruit trees,
ornamentals, rice, and
eld crops
Appliedchem, Thailand
Rhapsody B. subtilis Rhizoctonia, Fusarium,
Pythium, Phytophthora
Turf and ornamental,
vegetable and fruit
greenhouse crops
AgraQuest, USA
Serenade B. subtilis Rusts, powdery mildews, Botrytis,
Sclerotinia
Vegetable, wine,
nut and fruit crops
AgraQuest, USA
Sonata B. pumilus Rusts, powdery and downy mildews Vegetable and fruit crops AgraQuest, USA
Subtilex B. subtilis Rhizoctonia, Fusarium, Aspergillus Field, ornamental and
vegetable crops
Becker Underwood, USA
Taegro B. amyloliquefaciens Rhizoctonia, Fusarium Tree seedlings, ornamentals
and shrubs
Novozymes, Denmark
a
This formulation is composed by B. subtlis and rhizobial cells.
Current Opinion in Biotechnology 2011, 22:17 www.sciencedirect.com
in three main environments: soil, greenhouses and post-
harvest. These situations offer the best conditions for
optimal disease suppression activity because biological
control agents require specic environmental conditions
such as high relative humidity [6].
A high number of reports have described the benecial
effects of several Bacillus species against diseases elicited
by oomycetes and fungal pathogens. Some examples are
the suppression of root diseases (such as avocado root rot,
tomato damping-off and wheat take-all), foliar diseases
(such as cucurbit and strawberry powdery mildews) and
postharvest diseases (such as green, grey and blue
moulds) [1719,20

,2124]. However, the implementa-

tion in conventional agriculture of these Bacillus-based
products is still a pending issue. Most of these reports
highlight the need to integrate these Bacillus agents
mainly with fungicides to optimise disease management
[21,24,25,26]. Unfortunately, very little work has been
done on their integration with other management tools
such as cultural practises, host resistance, natural products
and other biological control agents. The research progress
made in the use of bacilli as biofungicides during the past
two decades has been remarkable; if this pace continues,
the use of Bacillus-based products to combat fungal dis-
eases will be greatly expanded in the future.
Bacillus lipopeptides, key molecules for
biological control of plant diseases
Antagonism to pathogens is the main mechanism of bio-
control that has been exploited to combat plant diseases
withBacillus species. Cell wall-degradingenzymes (suchas
chitinases, glucanases and proteases), peptide antibiotics
and other small molecules (such as volatile organic com-
pounds) are secreted by various species, and many of these
have been shown to contribute to pathogen suppression
[27]. Lipopeptides are among the antibiotic compounds
most frequently produced by Bacillus species and the
Bacillus compounds more extensively studied. These are
amphiphilic compounds that share a common structure
consistingof a lipidtail linkedtoa short cyclic oligopeptide.
Lipopeptides are classied into three families depending
on their amino acid sequence: iturins, fengycins and sur-
factins (Figure 2) [28

]. The surfactins are powerful

biosurfactants, which show antibacterial activity but no
marked fungitoxicity (with some exceptions) [29]. The
Plant protection and growth stimulation by microorganisms Pe rez-Garc a, Romero and de Vicente 3
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Please cite this article in press as: Pe rez-Garca A, et al. Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture, Curr Opin Biotechnol
(2011), doi:10.1016/j.copbio.2010.12.003
Figure 2
surfactin
iturin A
fengycin
H
H
H
H
H
H
H
H
H
H
H
H
H H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Current Opinion in Biotechnology
2D chemical structures of representative members of the three lipopeptide families produced by Bacillus species, iturins (iturin A), fengycins (fengycin)
and surfactins (surfactin). Nitrogen and oxygen atoms are depicted in blue and red, respectively. The 2D chemical structures were taken from
PubChem (http://pubchem.ncbi.nlm.nih.gov/).
www.sciencedirect.com Current Opinion in Biotechnology 2011, 22:17
iturins display strong antifungal action against a wide
variety of yeasts and fungi but only limited antibacterial
activity. Fengycins also show a strong fungitoxic activity,
specically against lamentous fungi [28

]. The ability of
various Bacillus strains to control fungal soilborne, foliar
and postharvest diseases has been attributed mostly to
iturins and fengycins [28

,30

,31]. The amphiphilic struc-

ture of lipopeptides allows them to interact with biological
membranes andinduce the formationof pores. Suchaltera-
tions of plasma membrane integrity promote internal
osmotic imbalance and widespread disorganisation of cyto-
plasm in fungal cells [32

]. Interestingly, iturins have also

been involved in the biological control ability of B. subtilis
strains against gram-negative phytopathogenic bacteria (H
Zeriouh et al., unpublished), thus expanding the range of
plant diseases that couldbepotentiallycontrolledbybacilli
(Figure 3).
Besides antibiosis, lipopeptides may have additional
roles in biocontrol. Selected strains of Bacillus and other
plant growth-promoting rhizobacteria (PGPR) can sup-
press plant diseases caused by both root and foliar patho-
gens, by inducing a resistance response in the host plant
designated as induced systemic resistance or ISR [33

].
Surfactin and fengycin lipopeptides have recently been
identied as bacterial determinants responsible for eli-
citation of ISR in the host plant [34

]. It is yet unclear
whether the induction of the ISR response by lipopep-
tides requires specic receptors in the plant membrane.
It is postulated that some lipopeptides may induce a
disturbance or transient channelling in the plasma mem-
brane, which in turn activates a cascade of molecular
events leading to enhanced defence [35

]. Nevertheless,
these ndings open up a new area of research to further
exploit these potential benecial effects of bacilli and
especially to gain more insight on the key structural
features and constituents of lipopeptides involved in
the induction of plant defence responses [36

].
The efcient protection of plants by biocontrol agents
requires their proper establishment in the host plant.
Chemotaxis, motility and growth are essential players
in this process. Lipopeptides can also inuence the eco-
logical tness of the producing strain contributing to plant
colonisation and persistence in the plant environment.
Thus, surfactin seems to be essential for swarming moti-
lity in B. subtilis [37]. In planta, the secretion of surfactin
and the formation of a stable, extensive biolm that
occurs upon root colonisation by B. subtilis has been
shown to be crucial for disease suppression [38]. Similar
results have been obtained with surfactin-decient
mutants of B. subtilis, which show disorganised biolm
formation and reduced biocontrol ability of fungal and
bacterial diseases after application on leaves (H Zeriouh
et al., unpublished). Moreover, a recent study attributes to
surfactin the role of triggering signal molecule for extra-
cellular matrix formation in functional and robust biolms
of B. subtilis [39

]. The lack of knowledge on many of

these crucial aspects of Bacillus ecology should stimulate
more intensive research. In this sense, the isolation of
Bacillus strains with enhanced colonisation capabilities
could be another strategy to improve the performance of
bacilli-based fungicides. Nevertheless, the attempts pub-
lished so far following this strategy have resulted in the
isolation of a remarkable low percentage of endospore
formers, suggesting that Bacillus species are poor com-
petitive colonizers at least for roots [40,41].
Bacilli as biofertilisers
Another interesting and well-documented ability of bacilli
is their capacity to promote plant health by stimulating
nutrition and growth. The mechanisms used by Bacillus
species to achieve this are biofertilisation and direct plant
growth promotion. In many soils, essential mineral nutri-
ents such as inorganic phosphate and ferric iron ions are
largely unavailable to plants because they are xed in
insoluble forms. Through biofertilisation, Bacillus popu-
lations improve the bioavailability of essential compounds
and increase the supply of mineral nutrients to the host
plant. In soil, organic phosphorus is stored mainly as
insoluble myo-inositol hexaphosphate or phytate. Several
Bacillus species, such as B. amyloliquefaciens, contribute to
soil biofertilisation through the production of extracellular
phytases, which are special phosphatases that catalyse the
4 Food biotechnology
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Figure 3
antibacterials motility and
biofilm formation
elicitors for
ISR activation
antifungals
fe
n
, itu
i
t
u
s
r
f
Bacillus
fe
n
, s
rf
Current Opinion in Biotechnology
Main roles of Bacillus lipopeptides regarding the biological control of
plant diseases by bacilli. Fengycin and iturin lipopeptides have been
widely characterised as antifungal compounds against several
phytopathogenic fungi and oomycetes. Iturins may also have additional
antibacterial potential at least against some Gram-negative plant
pathogenic bacteria. Fengycin and surfactin lipopeptides seem to act as
elicitors for activation of induced systemic resistance (ISR) in the roots of
the host plant, leading to suppression or reduction of plant diseases
caused by soil-borne and air-borne plant pathogens. Surfactin
lipopeptides are essential for motility. Surfactin acts as a signalling
molecule for biofilm formation and also seems to be required for
colonisation of root and leaf surfaces by plant-associated bacilli.
Abbreviations are: fen, fengycin lipopeptides; itu, iturin lipopeptides; and
srf, surfactin lipopeptides.
Current Opinion in Biotechnology 2011, 22:17 www.sciencedirect.com
sequential hydrolysis of phytate to less-phosphorylated
myo-inositol derivatives and inorganic phosphate [42

].
In addition, phytases eliminate chelate-forming phytate,
which is known to bind nutritionally important minerals
(such as Zn
2+
, Fe
2+
, Ca
2+
). Something similar happens to
iron. In the soil, the most prevalent form of iron is Fe
3+
,
which is relatively insoluble compared with the more
reduced form Fe
2+
and less readily taken up by plants
andmicroorganisms. Bacillus species, suchas B. megaterium,
can reduce metals, potentially increasing the bioavailabil-
ity of iron [43].
Direct plant growth promotion by Bacillus involves the
modulation of plant development through the production
of phytohormones [44]. Thus, several Bacillus species are
capable of producing auxin that might stimulate root
proliferation and nutrient uptake [45]. For example, in
B. amyloliquefaciens the biosynthesis of indole-3-acetic
acid (IAA) is responsible for plant growth promotion,
which in turn is strictly dependent on the presence of
tryptophan, one of the main compounds present in plant
root exudates [46]. Similarly, the inoculation of plants
with cytokinin-producing B. subtilis or B. megaterium
strains has a benecial effect on plant growth [47,48

].
In B. pumilus, however, plant growth promotion has been
associated with production of either gibberellin or ABA
and jasmonic acid [49,50]. Finally, volatile organic com-
pounds from B. subtilis have been shown to trigger growth
promotion in Arabidopsis by regulating auxin homeostasis,
thus providing a new paradigm as to how these bacteria
promote plant growth [51

]. For all of these reasons,

various Bacillus-based products have been launched to
the market under the category of biofertilisers. Further
research on this topic will help to accelerate the devel-
opment and application of new products that improve
crop quality and yields.
Conclusions and perspectives
The need for a continuous supply of food has led conven-
tional agriculture to be strongly dependent on chemicals.
The increasing concern of consumers and governments on
foodsafety has ledgrowers toexplorenewenvironmentally
friendly methods to replace, or at least supplement, the
current chemical-based practices. During the past few
decades, research on agricultural applications of aerobic
endospore-forming bacteria has conducted to the devel-
opment of a variety of Bacillus-based products exploiting
their insecticidal, antifungal and fertilising properties. The
molecular and physiological mechanisms by which Bacillus
species exert these benecial activities are not completely
understood in many cases. Nevertheless, bacilli may have
additional applications. Some Bacillus species have shown
an ability to control plant-parasitic nematodes. The mech-
anisms by which bacilli reduce nematode population and
root infestation are not completely elucidated. The pro-
duction of cuticle-degrading proteases as well as Cry toxins
seems to be responsible for toxicity of Brevibacillus later-
osporus and B. thuringienis, respectively. In other species
such as B. amyloliquefaciens and B. rmus benecial effect is
believed to be achieved through ISR elicitation [52

].
There are a few commercial Bacillus-based bionematicidal
formulations but their implementation is still very limited.
Furthermore, novel functions of bacilli such as quorum
quenching have been demonstrated to restrain bacterial
infections. Many bacterial pathogens have evolved cell
cell communication (quorum-sensing) mechanisms to
regulate expression of virulence factors. Key components
in these regulation systems are N-acyl homoserine lactones
(AHLs), which act as signal molecules. Some Bacillus
species such as B. thuringiensis are able to break down
AHLs (quorumquenching) by production of N-acyl homo-
serine lactone lactonases, which open the lactone ring of
AHLs, signicantly silencing bacterial virulence [53

].
Therefore, exploitation of these activities could expand
the phytoprotection possibilities of bacilli also as microbial
bactericides.
Bacillus-based products have great potential for use in
integrated pest management (IPM) systems; unfortu-
nately, relatively little work has been undertaken on their
integration with other IPM tools such as cultural prac-
tices, host resistance, chemical control, and other bio-
logical control agents. Formulation and application
methods are key issues inuencing the efcacy of com-
mercial products [54

]; research on these topics should be

focused on overcoming environmental restrictions, which
are the main reasons for failure or lack of consistence of
biological control agents in the eld. Genetic engineering
may provide a useful tool for the enhancement of per-
formance although genetically engineered microbes are
not yet allowed to enter the market. Finally, as whole
genome sequences become available for Bacillus species
of commercial interest [55

], high-throughput studies
can be undertaken to gain knowledge about the biocon-
trol competence of these agents. As newprogress on these
topics is made, new and better Bacillus-based formu-
lations will be developed. Therefore, we anticipate a
more relevant role for these microorganisms in promoting
plant health in the 21st century agriculture.
Acknowledgements
The authors gratefully acknowledge past and ongoing support for their work
on the use of bacilli as biocontrol agents from Plan Nacional de I + D + I of
the Ministerio de Ciencia e Innovacio n, Spain (AGL2001-1837; AGL2004-
0656; AGL2007-65340; AGL2010-21848), co-nanced with FEDER funds
(European Union).
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
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Droby S, Wisniewski M, Macarisin D, Wilson C: Twenty years of

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(2011), doi:10.1016/j.copbio.2010.12.003
www.sciencedirect.com Current Opinion in Biotechnology 2011, 22:17
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6 Food biotechnology
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(2011), doi:10.1016/j.copbio.2010.12.003
Current Opinion in Biotechnology 2011, 22:17 www.sciencedirect.com
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Plant protection and growth stimulation by microorganisms Pe rez-Garc a, Romero and de Vicente 7
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