Download Biodiversity of Bacillus subtilis group and beneficial traits of Bacillus

Romanian Biotechnological Letters
Copyright © 2015 University of Bucharest
Vol. 20, No. 5, 2015
Printed in Romania. All rights reserved
REVIEW
Biodiversity of Bacillus subtilis group
and beneficial traits of Bacillus species useful
in plant protection
Received for publication, June 10, 2015
Accepted, September 10, 2015
SICUIA OANA ALINA1,2, FLORICA CONSTANTINSCU2,
CORNEA CĂLINA PETRUŢA1,*
1
Faculty of Biotechnologies, University of Agronomic Sciences and Veterinary Medicine
Bucharest, 59 Mărăşti Blvd, 011464 Bucharest, Romania
2
Research and Development Institute for Plant Protection, 8 Ion Ionescu de la Brad Blvd.,
013813 Bucharest, Romania
*Corresponding author: Călina Petruţa Cornea, Faculty of Biotechnologies, University
of Agronomic Sciences and Veterinary Medicine Bucharest, 59 Mărăşti Blvd, 011464
Bucharest, Romania, phone. 004-021-318.36.40, fax. 004-021-318.25.88, e-mail:
[email protected]
Abstract
Biological control of plant pathogens and plant growth promotion by biological means is the late
tendency in biotechnological approaches for agricultural improvement. Such an approach is
considering the environmental protection issues without neglecting crop needs. In the present study, we
are reviewing Bacillus spp. biocontrol and plant growth promoting activity. As Bacillus genus is a
large bacterial taxon, with grate physiological biodiversity, we are describing some inter-grouping,
differences and similarities between Bacillus species, especially related to Bacillus subtilis.
Keywords: Bacillus subtilis group, beneficial bacteria
1. Introduction
Bacillus genus is a heterogeneous taxon, with ubiquitous spread in nature. Bacillus
species, such as Bacillus cereus, B. megaterium, B. subtilis, B.circulans and B. brevis group
are widely exploited for biotechnological and industrial applications [1, 2]. Their beneficial
traits for plant protection and growth promotion comprise the synthesis of broad-spectrum
active metabolites, easily adaptation in various environmental conditions, benefic plantbacterial interaction and advantageous formulation process [3].
As plants roots exudates and lysates attracts and stimulate microbial activity in the rootsurrounding soil, the rhizosphere became a highly populated area [4]. Root-microbial interaction
involves many forms of coexistence, such as commensalisms, where microorganisms live on
naturally occurring compounds released from the plant; endophytism, where microorganisms
grow inside plant tissue without adversely affect the host; symbiosis, where plant and microbial
organism are creating favorable conditions for mutual conviviality; parasitism, by which the host
plant is suffering due to microbial attack; and saprophytism, by which the organisms can nourish
with the decaying organic matter [5]. Beneficial Bacillus spp. strains can compete other microbes
that could adversely affect crops; they can inhibit phytopathogenic attacks, or induce host-plant
defense system against potential pathogenic attacks, stimulate plant growth, improve nutrient
uptake and reduce some negative environmental traits [3].
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2.
Bacillus genus
The Bacillus genus Cohn (1872) includes Gram positive, rod shaped bacterial cells of 0.32.2 to 1.2-7.0 m, most of them motile, with peritrich flagella. These bacteria are
chemoheterotrophes that can use various nutritional substrates. They can exhibit either
fermentative or both respiratory and fermentative pathway to produce energy. As respiration,
Bacillus species can be obligate aerobe or facultative anaerobe. The terminal electron acceptor
is the molecular oxygen (O2), replaced at some species by nitrate (NO3) in special conditions
[6]. The morphology and colony size are highly variable characteristics, depending on the
environmental conditions. In the presence of specific nutrients, some species can produce
pigments. Most species are widespread in nature. Many of the Bacillus spp. form resistant
endospores, with no more than one endospore in the sporangia cell. The endospores can be
distinguished from vegetative cells, as they are refractile and less colored, containing dipicolinic
acid, 5 to 15% in dry weight. These endospores are dormant structures, non-reproductive, they
can survive without nutrients and resist to extreme physical and chemical agents [7].
Regarding the nomenclature, there is no official classification of prokaryotes [8],
however there are such systems used at this time. The widely accepted classification of
"Taxonomic Outline of the Prokaryotes" is found in Bergey's Manual of Systematic
Bacteriology. Lately it was published the "Taxonomic Outline of the Bacteria and Archaea"
[9]. According to the List of Prokaryotic names with Standing in Nomenclature (LPSN) in
2014 there were 301 species mentioned to be included in Bacillus genus, three of them having
subspecies [http://www.bacterio.net/bacillus.html]. Registered bacterial species, not only that
they are found in international microbial collections, but they were previously analyzed
through molecular and biochemical analysis in order to be validate as novel species. Bacillus
DNA composition is 32-62% G+C mole [10]. Molecular diversity within populations in order
to determine genetic variation between Bacillus species, with their similarities and diversity at
genetic level are the most accurate when using the molecular techniques. However, gene
expression, biochemical and morphological aspects must also be considered.
3. Bacillus subtilis and related species
The phylogenetic studies, based on 16S rRNA sequence, suggest five groups of closely
related species, Bacillus cereus, B. megaterium, B. subtilis, B. circulans and B. brevis group.
There is also mentioned a group of closely related bacteria, referred to as Bacillus pumilus
subgroup, as it is included in Bacillus subtilis group [11].
Bacillus subtilis is a well studied bacterial species, ubiquitous in nature with valuable
treats useful for biotechnological, industrial and agricultural applications [2]. This specie was
first described by Christian Gottfried Ehrenberg in 1835, who entitled it Vibrio subtilhe [12].
However, the Bacillus genus was established by Ferdinand Cohn in 1872, and Bacillus
subtilis was defined as the type species by Soule in 1932 [13]. This has three subspecies,
Bacillus subtilis subsp. subtilis, subsp. spizizenii and subsp. inaquosorum [2]. Along with B.
subtilis there were described other closely related species with high genetic and/or
biochemical similarities. The species that we referred to are Bacillus amyloliquefaciens, B.
atrophaeus, B. axarquiensis, B. licheniformis, B. malacitensis, B. mojavensis, B. pumilus, B.
sonorensis, B. tequilensis, B. vallismortis and B. velezensis. Such species were first called
"Bacillus subtilis spectrum" [14], much later they were grouped into "Bacillus subtilis species
complex" [2], and now are clustered in "Bacillus subtilis group" [15].
Molecular analyzes are providing the most accurate information regarding phylogeny
and evolution. In bacteria, the 16S rRNA molecules are highly conserved, and therefore
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useful in plant protection
suitable for bacterial identification and classification. The comparative analysis of these
sequences can establish phylogenetic distance between the microorganisms studied and the
degree of relatedness between strains and species. The degree of similarity between Bacillus
subtilis and its closely related species is ≥ 99% for 16S rRNA sequence level. With DNADNA hybridization, the complementarity’s percentage at genome level is up to 70%, between
the species from Bacillus subtilis group [16]. This explains the genetic variability when
analyzing the entire genome compared to highly conserved sequence.
The phenotypic and biochemical differentiation between closely related species is
difficult to achieve [2]. For example, B. amyloliquefaciens could be distinguished from B.
subtilis by the fact that it produces acid from inulin, cells tend to be enchained and the
endospores are located in the center of the cell [17].
4. Molecular and microbiological differences between species of Bacillus
subtilis group
Bacillus amyloliquefaciens was first described by Fukomoto in 1943, but has been validated
and registered in the "Lists of Bacterial Names" only in 1980. Due to the similarities with Bacillus
subtilis, B. amyloliquefaciens has received a subspecies status, being classified as "B. subtilis
subsp. amyloliquefaciens", as a variety that produces large quantities of extracellular enzymes [14].
Moreover, this specie is the highest producing α-amylase and protease, between bacterial species.
Nowadays, B. amyloliquefaciens is self-standing specie with two approved varieties, subsp.
amyloliquefaciens and subsp. plantarum [18].
The molecular analysis of Bacillaceae species, using 16S rRNA and the 16S–23S ITS
(internally transcribed spacer), to differentiate, in clusters, this bacterial family, based on their
phylogeny, included B. atrophaeus in the Bacillus group VI, as related with Bacillus
amyloliquefaciens, Bacillus mojavensis and Bacillus subtilis [1]. Regarding the genetic
similarity at genome level, Bacillus atrophaeus and B. subtilis type species show only 32%
resemblance when analyzing by DNA-DNA hybridization [19]. According to the literature,
Bacillus atrophaeus was also referred as “B. subtilis var. subtilis”, “B. globigii”, “B. subtilis var.
niger -red strain”, “B. niger” or “B. atrophaeus subsp. globigii [20, 21, 13]. It should be noted
that the reference strains DSM 675 and DSM 2277, previously identified as B. subtilis were
reclassified as B. atrophaeus [19]. Phenotypic, Bacillus atrophaeus can be distinguished from
the type culture of Bacillus subtilis only in certain nutrient substrates, containing tyrosine, on
which B. atrophaeus produces a dark brown pigment [20]. The pigment production is also used
to differentiate this specie from Bacillus mojavensis and B. vallismortis [21].
Bacillus axarquiensis and B. malacitensis were proposed as new species by Ruiz-García
et al. (2005). However, Wang et al. (2008) [22] revealed these two species to be heterotypic
synonyms of Bacillus mojavensis. The similarity at genome level, between the B. mojavensis
and some of the species from B. subtilis group is 12 to 40% by DNA-DNA hybridization with
B. amyloliquefaciens, B. atrophaeus, B. licheniformis and B. subtilis. Regarding phenotypic
differentiation between of B. mojavensis and B. subtilis was possible only by analyzing the
composition of fatty acids [22].
Bacillus licheniformis was shown to be closely related to B. subtilis and B.
amyloliquefaciens after rigorous taxonomic studies using comparative sequencing of 16S rRNA
and 16S-23S internal transcribed spacer [1]. It is estimated that 80% of the B.licheniformis
genome sequence contains orthologous genes of B. subtilis. However, this two species differ in
the amount and position of prophage and insertion sequence elements, operons of the secondary
metabolic pathway, antibiotic synthases, extracellular enzymes, and metabolic activities that are
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CORNEA CĂLINA PETRUŢA
not present in B.subtilis [23].
For the differentiation of Bacillus pumilus from the other species belonging to "Bacillus
subtilis group", the ARDRA method, using 16S rRNA gene amplification and digestion with
the restriction enzymes Rsa I, Cfo I and Hinf I, could be successfully used. An exception is
for B. pumilus and B. amyloliquefacins separation. In this case, ITS-RFLP analysis method
could be used, and the PCR products from ITS amplification of 16S-23S rRNA enzymatic
digested with Cfo I manage a good differentiation between these two species [15].
B. safensis, B. stratosphericus, B. altitudinis and B. aerophilus, are having a high
similarity, of 99.5%, at the 16S rRNA sequence with Bacillus pumilus type species [24].
Because of this, these species were designated to constitute the complex of related species
called "B. pumilus group". However, given that B. pumilus is included in "B. subtilis group",
the highly related species with B. pumilus represent a subgroup. The latest studies, however,
indicate that in this subgroup should be included seven species B. pumilus, B. safensis, B.
stratosphericus, B. altitudinis, B. aerophilus, B. xiamenensis and B. invictae [25], all of them
with high similarity at molecular level. The diversity and evolution of the species belonging
to this group/subgroup was analyzed considering the highly conserved 16S rRNA gene and
the multilocus analysis (MLSA) of seven housekeeping genes, gyr B, rpo B, aro E, mut L, pyc
A, pyr E and trp B, involved in providing the essential life functions of the cells [24].
Bacillus sonorensis is mentioned to be phenotypically and genotypically similar to B.
licheniformis [26]. However, they are ecologically distinct species. This two closely related
species can be distinguished based on a few phenotypic traits, such as colony pigmentation on
tyrosine agar medium, growth characteristics on glicerol/glutamat-agar, or at pH of 5-6, or in
various salt concentrations (5%, 7% and 10% NaCl) and sensitivity to different level of
clindamycin [27]. Some differences can be also observed at genetic level by multilocus
enzyme electrophoresis (MLEE), genomic DNA reassociation and sequencing of 16S rRNA
or secY and rpoB protein-coding genes [27].
Bacillus tequilensis was first mentioned by Gatson et al. in 2006. Initially, it was
identified as B. subtilis based on conventional biochemical techniques. Further analysis
revealed 99% similarity with B. subtilis in the 16S rRNA sequence, but significantly different
from B. subtilis and related species when using pulsed-field gel electrophoresis (PFGE).
DNA-DNA hybridization studies showed less than 70% homology between B. tequilensis and
other species of B. subtilis group [28].
Roberts et al. (1996) isolated and identified the first strains of Bacillus vallismortis.
This specie is referred to be similar with B. subtilis, but differences can be seen at gyrA, polC
and rpoB genes digestion with restriction enzymes [29].
Bacillus velezensis, as it was first mentioned by Ruiz-García et al. (2005) was later
found to be a heterotypic synonym of the Bacillus amyloliquefaciens [22].
Among the molecular methods reliable and commonly used for the differentiation of
Bacillus spp. and strains are the Amplified Rhibosomal DNA Restriction Analysis (ARDRA)
method; Internal Transcribed Spacer (ITS) – PCR; the Restriction Fragment Length
Polymorphism (RFLP), the Random Amplified Polymorphic DNA; Repetitive SequenceBased PCR (rep - PCR) including Repetitive Extragenic Palindromic PCR (REP-PCR),
Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), box elements (BOXPCR) or other repetitive elements (figure 1); the comparison of gyrB gene and the Multiplex
PCR method using specie specific primers.
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useful in plant protection
Bacillus spp. strains included in Bacillus subtilis group
Figure 1. The BOX-PCR
electrophoresis pattern of thirteen
Bacillus spp. strains using BOX
A1R primer.
(Sicuia et. al., unpublished data)
5. Beneficial traits with agricultural purposes in Bacillus subtilis and related
species
The species of Bacillus subtilis group, particularly B. subtilis, B.licheniformis,
B.amyloliquefaciens and B.pumilus are intensively studied for their importance in
biotechnological industry and agriculture, as phytopathogenic antagonists or plant growth
promoters. The terms of "Plant Growth Promoting rhizobacteria" – PGPR, were introduced by
Kloepper and Schroth in 1978. They described the PGPR as naturally occurring soil bacteria
that have the ability to colonize the roots and stimulate plant growth. Various species of
Bacillus were than reported as PGPR [30].
The PGPR improve plant physiological state by phytohormones production or by releasing
beneficial organic compounds [30]. Polyamines, for example, have important physiological role,
being involved in cell division and differentiation, protein synthesis, and membrane stability,
moreover they have protective role against various abiotic stresses [31]. The PGPR can also
improve the mineral uptake of low-soluble or difficultly to be assimilated minerals such as
phosphorus, zinc or silica [32, 33, 34]. Beneficial soil bacteria also have important functions in
rhizoremediation and alleviating negative effects of soil stresses on plants [35].
Beside plant growth stimulation, Bacillus subtilis and related species are involved in plant
protection against phytopathogenic attacks. The mechanisms of action are various. They could act
directly against pathogens by producing extracellular lytic enzymes and secondary metabolites
with inhibitory growth action [36] or interfere by quorum quenching to disturb cell-to-cell
communication of the infectious expression in pathogenic bacteria [37]. They could also compete
plant pathogens for the available nutrients and niche [3]. Another important role is the reduction
of the infection process by inducing defense responses in the host plant [38].
5.1. Plant growth promotion induced by Bacillus spp. strains
Phytohormone production by beneficial Bacillus spp. strain involves auxins, cytokinins
and gibberellins synthesis. Plant growth regulators with inhibitory action, such as ethylene
and abscisic acid in some concentration, could be influenced by Bacillus activity [35].
The most studied auxin in Bacillus spp. is indole-3-acetic acid (IAA). Its synthesis was
detected in large spectrum of Bacillus spp. strains. The IAA production is being increased in the
presence of its precursors and specific pH conditions [33]. The most important IAA synthesis
pathway in Bacillus spp. is tryptophan dependent, and involves indole-3-pyruvic acid (IPA).
However, IAA could also be derived through Trp-independent pathways, from other aromatic
amino acids, such as praline, cysteine, phenylalanine, alanine and methionine [33].
Gibberellins production was confirmed in Bacillus pumilus and B. licheniformis, The
quantitative analysis of gibberellic acid (GA), by gas chromatography coupled with mass spectro10741
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CORNEA CĂLINA PETRUŢA
metry, determined 130-150 ng AG1/ml, 50-60 ng AG3/ml, 8-12 ng AG4/ ml and 2-3 ng AG20/ml
bacterial culture. B.pumilus and B.licheniformis GA-producers were able to replace the lack of
this phytohormone induced in the dwarf phenotype of alder seedlings (Alnus glutinosa) pretreated
with paclobutrazol growth retardant, that inhibit GA biosynthesis and plant elongation [39].
Bacterial cytokinins synthesis has been demonstrated in Bacillus subtilis and B.
licheniformis. In Bacillus subtilis culture was quantified 0.8-1.2 μg zeatin riboside/ml [40].
Cytokinins production was also quantified in Bacillus licheniformis Am2 culture, were 521 ng
zeatin riboside/ml and 1091.9 ng trans zeatin/ml were registered after four to five days of
growth in M9 minimal medium supplemented with 0.2% casamino acids, 0.01% thiamine,
and 0.2% biotin [41].
Beside plant hormones with stimulatory action, there are also other plant growth
regulators with inhibitory activity such as ethylene in high levels. Increased ethylene acts as a
stress hormone, and negatively affect plant growth and yield. Improper growth conditions
created by soil salinity, drought, water logging, heavy metals or pathogens increase the amount
of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production. PGPR,
including Bacillus spp. strains, express ACC-deaminase, decreasing plant ethylene levels
expressed in deficient growth conditions improving plant growth and development [35].
Regarding ABA regulation in plants, it was shown that Bacillus subtilis GB03 inoculation
influence plant sugar sensing and photosynthesis. Volatile organic compounds (VOCs) produced
by B. subtilis GB03 decrease ABA level in shoots of Arabidopsis bacterial treated plants. As
hexokinase-dependent sugar signaling is inhibit in reduced ABA levels, and ABA reduction
mediated by B.subtilis GB03 repress plant sugar sensing. This way, B.subtilis GB03 increase seed
germination and repress hypocotyl elongation inhibited by glucose signaling. Moreover, it was
showed that exogenous applied ABA reduces the chlorophyll content, photosynthetic efficiency
and growth of Arabidopsis plants, and negatively affects B.subtilis GB03 activity on plants. In
these respect, reduced ABA levels are necessary for GB03 to be able to express its PGPR
potential and enhance photosynthetic activity and chlorophyll content [42].
Nutrients uptake improvement of low-soluble minerals enhances plant growth.
Phosphorus is one of those minerals; its concentration in soil solution is very low as soluble
phosphate. However, its role is particularly important for plant growth and development.
Adequate amounts of phosphorus uptake in plants leads to a good development of the root
system, an increased productivity, a stronger natural defense mechanism towards the
phytopathogenic agents, an improved yield quality and sometime an early crop maturation [43].
In soil, phosphorus can be found as mineral phosphates and phytates. Bio-fertilization with
PGPR strains, having extracellular phytase activity, leads to plant growth promotion, even under
limited phosphate conditions [44]. Phytase activity and genes encoding for such enzymes have
been determined in several Bacillus spp. isolated from rhizosphere [36]. Beside their ability to
improve phytate hydrolysis and phosphorus assimilation, the uptake of other nutritionally
important minerals (Zn2+, Fe2+ and Ca2+) is enhanced, by limiting chelate-forming phytate [45].
Zinc solubilizing rhizobacteria improve plant growth and yield, and increase Zn
concentration of wheat grains. Zn biofortification with PGPR can overcome Zn deficiency in
an economical and organic way [32]. Zn mobilizing ability has been reported in different
bacterial taxa, including in Bacillus genus (B.thuringiensis, B.megaterium) [32, 46].
Some beneficial bacteria could produce organic compounds, such as polyamines, with
positive influence on plant growth promotion, senescence delay and protective role against
abiotic stresses [47, 48, 49]. The polyamines, putrescine, spermine, spermidine and
cadaverine, are organic compounds that contain several amino acids. They are largely spread
and could be found in bacteria and along with other microorganisms, plants, higher animals
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useful in plant protection
and mammals. Beside their key role in the genetic material stability, they also have regulatory
functions [48]. Regarding polyamines role in bacteria, Wortham et al. (2007) speak about the
resistance to acidic environments and protection against reactive oxygen species toxicity
conferred by these compounds [49]. In Bacillus subtilis, polyamines serve as a signal
molecule for bacterial swarming motility and biofilm formation [50]. In plants, the
polyamines and their biosynthesis enzymes are involved in many metabolic processes, such as
cell division, organogenesis, embryogenesis, floral induction and development, leafs aging,
fruits growth and ripening, or in response to certain biotic and abiotic stress factors [48, 50].
5.2. Plant protection activity triggered by Bacillus spp. strains
Bacillus spp. strains could act as biological control agents (BCAs) to provide plant
protection against microbial and insect pathogens. BCAs are a promising alternative to
chemical pesticides. Therefore, many studies are focused on their interaction with plants,
pests, pathogenic and beneficial microorganisms, and understanding their impact to the
environment, and implication on animals and humans. Important traits, such as efficacy,
formulation, stability and viability were also intensively studied. All these in order to select
most efficient and suitable BCAs for plant protection and, in the same time, to promote only
the strains neutral to non-target organisms.
The various mechanisms involved in plant protection refer to pathogen inhibitors and
extracellular lytic enzymes production, antagonism (figure 2), nutrients and niche
competition, quorum quenching and induced defense responses in host plants.
Figure 2. Antifungal activity
expressed by different Bacillus spp.
strains.
A – Bacillus spp. antagonistic activity against
Fusarium solani;
B – Fungal cell wall degradation, cell lysis and
cytoplasm bleeding due to Bacillus spp.
extracellular enzymes.
5.2.1. Cellulolytic enzymes
Cellulolytic enzymes synthesized by the BCAs can be involved in two plant defense
mechanisms against phytopathogenic fungi. One of these mechanisms triggers elicited biotic
defense in plants, preventing them from phytopathogenic attacks The second course action
involves the lytic action of cellulases. Cellulolytic enzymes hydrolyze β-1,4 glycoside bonds,
and break down the cell wall cellulose from Pythium and Phytophthora pathogens. Cellulase
activity (figure 3) was found in B.subtilis [51], B.amyloliquefaciens [52], B.licheniformis [53]
or B.pumilus [54].
Figure 3. Cellulase activity exposed on Luria
Bertani medium supplemented with carboxy-methyl
cellulose (CMC), reveal a clear halo of CMC
degradation, after two days of Bacillus spp. strains
incubation and Congo red stain.
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5.2.2. Chitinase activity
Chitin is a difficult biodegradable matter. For its hydrolysis, β-1,4 N-glycosidic linkages
are cutted by the chitinolytic enzymes [55]. Chitinases are synthesized by numerous species
of the Bacillus genus, including B.subtilis, B.amyloliquefaciens, B.licheniformis [55], or
B.thuringiensis [56]. Among the potential applications of chitinases are bioremediation and
bioconversion of chitin food waste, namely N-acetylglucosamines (NAG) and chitooligosaccharides [55]. However, they can also be used for their anti-fungal properties and
insect biological control [55, 56].
5.2.3. Antibiotic compounds
The most effective mechanism in biological control of plant pathogens is the antibiotic
synthesis from beneficial microorganisms. In Bacillus spp., such compounds are released
during sporulation, and in the stationary growth stage. Among the antibiotic compounds
synthesized by the Bacillus bacteria, kanosamine, zwittermicin A, iturins, bacitracin,
gramicidin, fengycin or plipastatin, kurstakin and surfactins are mentioned [3].
Kanosamine (3-amino-3-deoxy-D-glucose) has a strong inhibitory activity against
Oomycetes fungal pathogens and moderate activity against various other fungi from Ascomycetes
(Aspergillus flavus, Botrytis cinerea, Sclerotinia spp., Venturia spp.), Basidiomycetes
(Rhizoctonia solani, Ustilago maydis) and Deuteromycetes fam. (Alternaria spp., Colletotrichum
spp., Helminthosporium spp., Fusarium spp., Phomopsis obscurans, Verticillium spp) and some
bacteria [57]. Kanosamine synthesis was evidenced in several species of Bacillus spp., such as
B.pumilus and B.subtilis [58], or B.cereus [57].
Zwittermicina A is a linear aminopolyol with a broad spectrum of inhibitory activity
against certain gram-positive and gram-negative bacteria, or eukaryotic organisms, including
Oomycetes [58]. It also enhances the insecticidal activity of the toxin protein from
B.thuringiensis. However, zwittermicina A synthesis has not been evidenced in Bacillus
subtilis and related species.
Iturins is a large family of antibiotic compounds, which includes bacillomycin D and F,
bacillopeptin (or bacillomycin L), ituin A and C, and mycosubtilin [59]. Iturins have strong
antibiotic effect and moderate activity as surfactants, and they are mentioned to enhance
swarming motility [59, 60]. Iturin synthesis have been shown in various B.amyloliquefaciens,
B. licheniformis, B. pumilus and B. subtilis strains [60].
Bacitracin is an antibiotic compound with bactericidal activity, synthesized by some
strains of B.licheniformis and B. subtilis [61, 62].
Fengicin A and B, also called plipastatinare lipopeptide antibiotics. These compounds
were found to be useful in the biological control of mosquito larvae [63] and plant pathogens
degrading their cell structure and permeability [64, 65]. They are also mentioned to induce
systemic resistance (ISR) in plant [66]. Used as biosurfactants, they can inhibit biofilm
formation on some bacteria [67], and degrade polycyclic aromatic hydrocarbons (PAHs) [68].
These metabolites are produced by various species of the Bacillus genus, such as B.subtilis
[60], B.amyloliquefaciens [69], or B.licheniformis [70].
Kurstakins are non-ribosomal lipopeptides produced by Bacillus thuringiensis. Further
investigation revealed kurstakin production in other more species, but only from "Bacillus
cereus group" except for B.anthracis and B.cytotoxicus. For these reason kurstakin is
considered a biomarker for this group of species [71].
Surfactins include bamylocin A, esperin, lichenysin, pumilacidin and surfactin [59].
Surfactins have antimicrobial [72, 73], antiviral [74], anti-mycoplasmatic [75]. These
lipopeptides have an amphiphilic character. The most popular uses are as biosurfactant and
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useful in plant protection
antibiotic. Because of these properties, surfactins are good candidates in solving some of the
global issues in medicine, industry and environmental protection [76]. They are also among
the best biosurfactants, for biotechnological applications and environmental protection,
mainly used for emulsifying, foaming, and oil recovery [77], remediation of heavy metals
contaminated soils [78, 79] and biological control of plant diseases [80, 81] and pests [82].
5.2.4. Antifungal volatile compounds
Microbial volatile compounds, like aldehydes, ketones, alcohols, aliphatic alkenes,
organic acids, sulphides [83, 84], can interfere as biological control mechanisms against
fungal phytopathogens. Among the organic volatile compounds synthesized by some strains
of Bacillus spp. bacteria include iturin, ammonia, acetoin, indole, hydrogen sulfide may occur
in the biocontrol mechanism, and can trigger the defense response in plants, by inducting
phytoalexin formation [85].
5.2.5. Quorum quenching
Bacterial communication inside their population is possible by quorum sensing molecules
or N-acyl homoserine lactone (AHL) and oligopeptide as signaling factors. In pathogenic
bacteria, the infectious activity is triggered by such signaling molecules. The pathogenicity is
expressed only in certain population density, and quorum sensing is the communication
mechanism involved in informing about cell density. AHL lactonase, can hydrolyze quorum
sensing signaling molecule, and inhibit bacterial communication systems. This way, AHL
lactonase producing microorganisms can act as potential biocontrol agents. Such mechanism of
biological control is even more useful if the pathogenic bacteria are multi-drug tolerant [86].
AHL lactonase activity expressed by microorganisms is named quorum quenching effect.
In biocontrol Bacillus spp. strains, such mechanism was analyzed and it has been shown in
Bacillus licheniformis [87], B.cereus [88], B.thuringiensis [89] strains.
5.3. Nutritional and space competition
One of the competitive factors in nutrient uptake is siderophore formation, capable to
specifically bind the ferric iron (Fe3+) into high-affinity iron chelating compounds. This
essential element, although is abundant in nature has a very low solubility. For microorganisms,
ferric iron bioavailability is possible by siderophore formation that makes it soluble from the
mineral phases. The Fe-siderophore complex has high specificity and can be used only by the
issuing microorganisms. The high-affinity Fe-siderophore compounds create a competition
between soil microbials to iron uptake. Siderophore formation in Bacillus sp. is not that well
documented as in other bacterial species, such as Pseudomonads. However, specific iron uptake
was mentioned in plant growth-promoting B.subtilis and B.amyloliquefaciens strains [30].
Swimming and swarming motility enhance the colonization ability of beneficial bacteria,
reflecting in better space competitiveness. Bacillus species are among the most prominent
bacteria found to colonize soil and plant roots. Bacillus root-colonization and biofilm
formation is a prerequisite of phyto-stimulation [30]. The ability to stimulate plant growth,
compete and suppress plant pathogens is enhanced when roots are better colonized by
beneficial bacteria [44].
5.4. Induced systemic resistance in plants
Induced systemic resistance (ISR) is an enhanced state of defensive capacity, elicited by
specific environmental stimuli [38]. Rhizobacteria-mediated ISR enhance the innate defensive
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capacity against a broad spectrum of pests and pathogens attack [90]. In plants, rhizobacteria
induced resistance has been demonstrated to be systemically expressed, by beneficial bacteria
seed inoculation that triggered defensive mechanism against foliar disease [91].
Several Bacillus amyloliquefaciens, B.cereus, B.mycoides, B.pasteurii, B.pumilus,
B.sphaericus, and B.subtilis strains were mentioned to elicit ISR in a wide range of host
plants, where they reduced diseases incidence or severity. Results on Bacillus eliciting ISR
have been reported against systemic viruses, pathogenic bacteria, damping-off, crown rotting,
stem-blight, and leaf-spotting fungal pathogen, blue mold, and late blight diseases, or rootknot nematodes [38]. Therefore, biological controlled strategy using ISR mediating bacteria
are promising to be valuable for agriculture application.
6. Conclusions
Bacillus species demonstrated to have a wide spectrum of plant protection and growth
promoting abilities. They are considered of great importance for biotechnological industry
and agriculture. The greatest advantage of using Bacillus species as biological inoculant in
agriculture and related fields is the ability to produce endospores with prolong viability and
high resistance [92, 13], which makes them easy to be formulated in various types and stored
in simple conditions, marketing them similar to chemical fungicides in a certain manner.
Mixtures of plant-beneficial bacterial-strains with different mechanisms of actions has been
suggested as more reliably than individual strains [93].
7. Acknowledgements
This work was financed by Operational Program Human Resources Development 20072013, project no. POSDRU/159/1.5/S/132765 using European Social Fund.
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